Sample records for cell mutation test
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Zhu, Guanshan; Ye, Xin; Dong, Zhengwei; Lu, Ya Chao; Sun, Yun; Liu, Yi; McCormack, Rose; Gu, Yi; Liu, Xiaoqing
2015-05-01
Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin
2014-02-01
BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.
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Patterson, Melissa N; Maxwell, Patrick H
2014-10-16
Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
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Germline stem cell competition, mutation hot spots, genetic disorders and older dads
Arnheim, Norman; Calabrese, Peter
2016-01-01
Some de novo human mutations arise at frequencies far exceeding the genome average mutation rate. Examples are the common mutations at one or a few sites in the genes causing achondroplasia, Noonan syndrome, multiple endocrine neoplasia 2B and Apert syndrome. These mutations are recurrent, provide a gain of function, are paternally derived and are more likely transmitted as the father ages. Recent experiments tested whether the high mutation frequencies are due to an elevated mutation rate per cell division, as expected, or an advantage of the mutant spermatogonial stem cells over wild-type stem cells. The evidence, which includes the surprising discovery of testis mutation clusters, rejects the former model but not the latter. We propose how the mutations might alter spermatogonial stem cell function and discuss how germline selection contributes to the paternal age effect, the human mutational load and adaptive evolution. PMID:27070266
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Sample features associated with success rates in population-based EGFR mutation testing.
Shiau, Carolyn J; Babwah, Jesse P; da Cunha Santos, Gilda; Sykes, Jenna R; Boerner, Scott L; Geddie, William R; Leighl, Natasha B; Wei, Cuihong; Kamel-Reid, Suzanne; Hwang, David M; Tsao, Ming-Sound
2014-07-01
Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.
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da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R
2010-12-25
The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.
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Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer.
Malapelle, Umberto; Sirera, Rafael; Jantus-Lewintre, Eloísa; Reclusa, Pablo; Calabuig-Fariñas, Silvia; Blasco, Ana; Pisapia, Pasquale; Rolfo, Christian; Camps, Carlos
2017-03-01
The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories' expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required. Areas covered: The EGFR mutation testing landscape is manifold and includes both screening and targeted methods, each with their own pros and cons. Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its liquid-biopsy applications. Expert commentary: The Roche cobas® EGFR mutation test v2, based on real time RT-PCR, is a reliable option for testing EGFR mutations in clinical practice, either using tissue-derived DNA or plasma-derived cfDNA. This application will be valuable for laboratories with whose purpose is purely diagnostic and lacking high-throughput technologies.
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Epidermal growth factor receptor mutations in 510 Finnish non--small-cell lung cancer patients.
Mäki-Nevala, Satu; Rönty, Mikko; Morel, Mike; Gomez, Maria; Dawson, Zoe; Sarhadi, Virinder Kaur; Telaranta-Keerie, Aino; Knuuttila, Aija; Knuutila, Sakari
2014-06-01
Among the driver gene mutations in non-small-cell lung cancer, mutations in epidermal growth factor receptor (EGFR) are the most important because of their predictive role in selecting patients eligible for targeted therapy. Our aim was to study EGFR mutations in a Finnish non-small-cell lung cancer cohort of 528 patients. Mutation testing was conducted on DNA extracted from paraffin-embedded, formalin-fixed tumor material using the following real-time polymerase chain reaction-based kits: Therascreen EGFR PCR Kit and cobas EGFR Mutation Test. EGFR mutation frequency was 11.4% and all positive cases were adenocarcinomas, of which a majority had an acinar predominant pattern. Mutations were seen significantly more often in females and never-smokers than in males and smokers. The most frequent mutations were L858R in exon 21 and deletions in exon 19. Overall survival of the patients, not treated with EGFR inhibitor, did not differ between EGFR mutation-positive and EGFR mutation-negative patients. EGFR mutation profile in this Finnish non-small-cell lung cancer cohort resembles in many respect with that of other Western European cohorts, even though the overall frequency of mutations is slightly higher. We show the occurrence of EGFR mutations in patients with occupational asbestos exposure and also in those diagnosed with chronic obstructive pulmonary disease who have not been often investigated before.
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NASA Astrophysics Data System (ADS)
Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan
2018-03-01
EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.
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Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine
2017-10-01
The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tomkins, D.J.; Wei, L.; Laurie, K.E.
1985-01-01
It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinesemore » hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.« less
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Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells
ERIC Educational Resources Information Center
Szeberenyi, Jozsef
2013-01-01
The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…
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Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog
2013-01-01
Introduction Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Methods Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. Results This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. Conclusions In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients. PMID:23468851
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Choi, Yoon-La; Sun, Jong-Mu; Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog
2013-01-01
Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients.
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Moorchung, Nikhil; Phillip, Joseph; Sarkar, Ravi Shankar; Prasad, Rupesh; Dutta, Vibha
2013-01-01
Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC) is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR)-based allele-specific Amplification Refractory Mutation System (ARMS). Molecular analysis for the sickle cell mutation was done by standard methods. The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23%) of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS) because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.
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Wang, Shu; Yu, Bing; Ng, Chiu Chin; Mercorella, Belinda; Selinger, Christina I.; O’Toole, Sandra A.
2015-01-01
Background Patients with advanced non-small cell lung cancer (NSCLC) benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) when their tumor harbors an activating EGFR mutation. As the majority of NSCLC patients present with advanced disease, cytology and small biopsy specimens are frequently the only tissue available for mutation testing, but can pose challenges due to low tumor content. We aim to better define the suitability of these specimens for mutation testing. Methods NSCLC cases referred to our institution for mutation testing over a 15-month period were retrospectively reviewed. Specimens were tested for mutations including EGFR, KRAS, and BRAF, using a multiplex PCR assay (OncoCarta Panel v1.0) and analyzed on the Agena Bioscience MassARRAY platform. Results A total of 146 specimens were tested, comprising 53 (36.3%) resection specimens (including 28 lung resection specimens), 55 (37.7%) small biopsy specimens and 38 (26%) cytology specimens. Of 142 cases with sufficient DNA for mutation testing, EGFR mutations were detected in 31 specimens (21.8%), KRAS mutations in 31 specimens (21.8%) and BRAF mutations in three specimens (2.1%). There was no significant difference in the EGFR mutation rate between lung resection (10 of 28 cases; 35.7%), small biopsy (9 of 53 cases; 17%), and cytology specimens (8 of 36 cases; 22.2%). Conclusions Our results support the utility of small biopsy and cytology specimens for mutation testing. Careful evaluation of the adequacy of small specimens is required to minimize the risk of false negative or positive results. PMID:25870794
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Ten Broek, Roel W; Bekers, Elise M; de Leng, Wendy W J; Strengman, Eric; Tops, Bastiaan B J; Kutzner, Heinz; Leeuwis, Jan Willem; van Gorp, Joost M; Creytens, David H; Mentzel, Thomas; van Diest, Paul J; Eijkelenboom, Astrid; Flucke, Uta
2017-12-01
Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH. © 2017 Wiley Periodicals, Inc.
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Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.
Powrózek, Tomasz; Krawczyk, Paweł; Pankowski, Juliusz; Reszka, Katarzyna; Jakubiak, Magdalena; Obrochta, Anna; Wojas-Krawczyk, Kamila; Buczkowski, Jarosław; Milanowski, Janusz
2015-11-14
Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.
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Rare Complex Mutational Profile in an ALK Inhibitor-resistant Non-small Cell Lung Cancer.
Azzato, Elizabeth M; Deshpande, Charuhas; Aikawa, Vania; Aggarwal, Charu; Alley, Evan; Jacobs, Benjamin; Morrissette, Jennifer; Daber, Robert
2015-05-01
Testing for somatic alterations, including anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and epidermal growth factor receptor gene (EGFR) mutations, is standard practice in the diagnostic evaluation and therapeutic management of non-small cell lung cancer (NSCLC), where the results of such tests can predict response to targeted-therapy. ALK rearrangements, EGFR mutations and mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) are considered mutually exclusive in NSCLC. Herein we identified a KRAS Q22K mutation and frameshift mutations in the genes encoding serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated serine/threonine kinase (ATM) by next-generation sequencing in a patient with ALK rearrangement-positive oligo-metastatic NSCLC, whose disease progressed while on two ALK-targeted therapies. Such a complex diagnostic genetic profile has not been reported in ALK fusion-positive NSCLC. This case highlights the utility of comprehensive molecular testing in the diagnosis of NSCLC. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop.
Pirker, Robert; Herth, Felix J F; Kerr, Keith M; Filipits, Martin; Taron, Miquel; Gandara, David; Hirsch, Fred R; Grunenwald, Dominique; Popper, Helmut; Smit, Egbert; Dietel, Manfred; Marchetti, Antonio; Manegold, Christian; Schirmacher, Peter; Thomas, Michael; Rosell, Rafael; Cappuzzo, Federico; Stahel, Rolf
2010-10-01
Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations. The "EGFR testing in NSCLC: from biology to clinical practice" International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group. Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests. The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.
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Bahreini, Amir; Li, Zheqi; Wang, Peilu; Levine, Kevin M; Tasdemir, Nilgun; Cao, Lan; Weir, Hazel M; Puhalla, Shannon L; Davidson, Nancy E; Stern, Andrew M; Chu, David; Park, Ben Ho; Lee, Adrian V; Oesterreich, Steffi
2017-05-23
Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect. Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.
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Karachaliou, Niki; Rosell, Rafael
2014-01-01
Deeper understanding of the pathobiology of non-small-cell lung cancer (NSCLC) has led to the development of small molecules that target genetic mutations known to play critical roles in the progression to metastatic disease. The discovery of epidermal growth factor receptor (EGFR) mutations in 15%–20% of lung adenocarcinomas and the associated response to EGFR tyrosine kinase inhibitors have provided a successful avenue of attack in late-stage adenocarcinomas. Use of the EGFR tyrosine kinase inhibitors gefitinib, erlotinib, and afatinib is limited to patients who have adenocarcinomas with known activating EGFR mutations. However, the EGFR mutation testing landscape is varied and includes many screening and targeted methods, each with its own benefits and limitations. These tests can simplify the drug discovery process, make clinical trials more efficient and informative, and individualize cancer therapy. In practice, the choice of method should be determined by the nature of the sample to be tested, the testing laboratory’s expertise and access to equipment, and whether the detection of only known activating EGFR mutations, or of all possible mutations, is required. Development of companion diagnostic tests for this identification is advancing; nevertheless, the use of such tests merits greater attention. PMID:28210145
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Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A
2017-10-26
EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.
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Sheikholeslami, M R; Jilani, I; Keating, M; Uyeji, J; Chen, K; Kantarjian, H; O'Brien, S; Giles, F; Albitar, M
2006-07-15
Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy. (c) 2006 International Society for Analytical Cytology.
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The Lambda Select cII Mutation Detection System.
Besaratinia, Ahmad; Tommasi, Stella
2018-04-26
A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.
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Shackelford, Rodney E.; Whitling, Nicholas A.; McNab, Patricia; Japa, Shanker
2012-01-01
Activating point mutations in codons 12, 13, and 61 of the KRAS proto-oncogene are common in colorectal, non–small cell lung, pancreatic, and thyroid cancers. Constitutively activated KRAS mutations are strongly associated with a resistance to anti–epidermal growth factor receptor (EGFR) therapies, such as panitumumab and cetuximab used for treating metastatic colorectal carcinoma and EGFR tyrosine inhibitors used for advanced non–small cell lung cancers. Since anti-EGFR therapies are costly and may exert deleterious effects on individuals without activating mutations, KRAS mutation testing is recommended prior to the initiation of anti-EGFR therapy for these malignancies. The goal of this review is to summarize the KRAS mutation testing methods. Testing is now routinely requested in the clinical practice to provide data to assign the most appropriate anticancer chemotherapy for each given patient. Review of the most relevant literature was performed. Several areas were considered: ordering of the test, selection of the sample to be tested, and review of the testing methodologies. We found that several different methods are used for clinical KRAS mutation testing. Each of the methodologies is described, and information is provided about their performance, cost, turnaround times, detection limits, sensitivities, and specificities. We also provided “tips” for the appropriate selection and preparation of the sample to be tested. This is an important aspect of KRAS testing for clinical use, as the results of the test will affect clinical decisions with consequences for the patient. PMID:23264846
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Asaka, Shiho; Yoshizawa, Akihiko; Nakata, Rie; Negishi, Tatsuya; Yamamoto, Hiroshi; Shiina, Takayuki; Shigeto, Shohei; Matsuda, Kazuyuki; Kobayashi, Yukihiro; Honda, Takayuki
2018-01-01
The detection of epidermal growth factor receptor (EGFR) mutations is necessary for the selection of suitable patients with non-small cell lung cancer (NSCLC) for treatment with EGFR tyrosine kinase inhibitors. Cytology specimens are known to be suitable for EGFR mutation detection, although tissue specimens should be prioritized; however, there are limited studies that examine the utility of bronchial lavage fluid (BLF) in mutation detection. The purpose of the present study was to investigate the utility of BLF specimens for the detection of EGFR mutations using a conventional quantitative EGFR polymerase chain reaction (PCR) assay. Initially, quantification cycle (Cq) values of cell pellets, cell-free supernatants and cell blocks obtained from three series of 1% EGFR mutation-positive lung cancer cell line samples were compared for mutation detection. In addition, PCR analysis of BLF specimens obtained from 77 consecutive NSCLC patients, detecting EGFR mutations was validated, and these results were compared with those for the corresponding formalin-fixed paraffin-embedded (FFPE) tissue specimens obtained by surgical resection or biopsy of 49 of these patients. The Cq values for mutation detection were significantly lower in the cell pellet group (average, 29.58) compared with the other groups, followed by those in cell-free supernatants (average, 34.15) and in cell blocks (average, 37.12) for all three series (P<0.05). Mutational status was successfully analyzed in 77 BLF specimens, and the results obtained were concordant with those of the 49 matching FFPE tissue specimens. Notably, EGFR mutations were even detected in 10 cytological specimens that contained insufficient tumor cells. EGFR mutation testing with BLF specimens is therefore a useful and reliable method, particularly when sufficient cancer cells are not obtained. PMID:29399190
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Chiu, Chao-Hua; Chou, Teh-Ying; Chiang, Chi-Lu; Tsai, Chun-Ming
2014-10-01
There is no argument over using epidermal growth factor receptor (EGFR) mutation status to guide the frontline treatment for advanced lung adenocarcinoma (LADC); however, the role of the testing in lung squamous cell carcinoma (LSQC) remains controversial. Currently, the guidelines/consensus statements regarding EGFR mutation testing in LSQC are not consistent among different oncology societies. American Society of Clinical Oncology recommends performing EGFR mutation testing in all patients; European Society for Medical Oncology, College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology, and National Comprehensive Cancer Network suggest for some selected group. EGFR mutation is rarely found in LSQC; however, more importantly, it is not a valid predictive biomarker for EGFR tyrosine kinase inhibitors (EGFR-TKI) in LSQC as it has been shown in LADC. Available data showed that the response rate and progression-free survival in EGFR mutant LSQC patients treated with EGFR-TKI are not better than that observed in patients treated with platinum-doublet chemotherapy in the first-line setting. Therefore, in contrast to advanced LADC, EGFR mutation testing may not be necessarily performed upfront in advanced LSQC because not only the mutation rate is low, but also the predictive value is insufficient. For LSQC patients with known sensitizing-EGFR mutations, both conventional chemotherapy and EGFR-TKI are acceptable frontline treatment options.
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Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond
Rogers, Toni-Maree; Fellowes, Andrew; Bell, Anthony; Fox, Stephen
2015-01-01
Somatic mutational profiling in cancer has revolutionized the practice of clinical oncology. The discovery of driver mutations in non-small cell lung cancer (NSCLC) is an example of this. Molecular testing of lung adenocarcinoma is now considered standard of care and part of the diagnostic algorithm. This article provides an overview of the workflow of molecular testing in a clinical diagnostic laboratory discussing in particular novel assays that are currently in use for somatic mutation detection in NSCLC focussing on epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK), ROS1 and RET rearrangements. PMID:25870795
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Ess, S M; Herrmann, C; Frick, H; Krapf, M; Cerny, T; Jochum, W; Früh, M
2017-11-01
In order to improve outcomes, identification of the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes has become crucial in advanced non-small-cell lung cancer (NSCLC). The aim of the present study is to analyse time trends and frequency of testing, factors affecting testing as well as prevalence of mutations in the Swiss population. We analysed EGFR and ALK testing in a cohort of patients with newly diagnosed metastasised non-squamous NSCLC in the catchment area of the cancer registry Eastern Switzerland in the years 2008-2014. We analysed prevalence of mutations and studied clinicopathological characteristics and survival of tested and non-tested patients and of patients with and without mutations. Among 718 patients identified, 11% (51/447) harboured an EGFR mutation in the exons 18, 19 or 21 and further 12% (31/265) showed a positive test result for ALK rearrangements. In non-smokers the proportions of mutations were 31% and 23% respectively. Testing rates increased over time and reached 79% in 2014. We observed significantly lower testing rates and poorer survival in elderly, patients with limited life expectancy and patients treated at hospitals not involved in clinical research. Outcomes can be further improved in a considerable proportion of patients with advanced non-squamous NSCLC. © 2017 John Wiley & Sons Ltd.
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Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D
2015-01-01
Objectives: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Methods: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Results: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Conclusions: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment. PMID:26325104
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Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D
2015-09-29
We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.
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Li, Xuefei; Zhou, Caicun
2017-01-01
Somatic mutations in the gene encoding epidermal growth factor receptor (EGFR) play an important role in determining targeted treatment modalities in non-small cell lung cancer (NSCLC). The EGFR T790M mutation emerges in approximately 50% of cases who acquire resistance to tyrosine kinase inhibitors. Detecting EGFR T790M mutation in tumor tissue is challenging due to heterogeneity of the tumor, low abundance of the mutation and difficulty for re-biopsy in patients with advanced disease. Alternatively, circulating tumor DNA (ctDNA) has been proposed as a non-invasive method for mutational analysis. The presence of EGFR mutations in ctDNA predicts response to the EGFR TKIs in the first-line setting. Molecular testing is now considered a standard care for NSCLC. The advent of standard commercially available kits and targeted mutational analysis has revolutionized the accuracy of mutation detection platforms for detection of EGFR mutations. Our review provides an overview of various commonly used platforms for detecting EGFR T790M mutation in tumor tissue and plasma. PMID:29246024
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Thierry, Alain R
2016-01-01
Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.
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40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.
Code of Federal Regulations, 2010 CFR
2010-07-01
...) The test should be designed to have a predetermined sensitivity and power. The number of cells... cleansed of pre-existing mutant cells. (D) Metabolic activation. Cells shall be exposed to the test...=/− test, colony sizing should be performed on at least one of the test cultures (the highest positive...
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40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.
Code of Federal Regulations, 2013 CFR
2013-07-01
...) The test should be designed to have a predetermined sensitivity and power. The number of cells... cleansed of pre-existing mutant cells. (D) Metabolic activation. Cells shall be exposed to the test...=/− test, colony sizing should be performed on at least one of the test cultures (the highest positive...
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40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.
Code of Federal Regulations, 2012 CFR
2012-07-01
...) The test should be designed to have a predetermined sensitivity and power. The number of cells... cleansed of pre-existing mutant cells. (D) Metabolic activation. Cells shall be exposed to the test...=/− test, colony sizing should be performed on at least one of the test cultures (the highest positive...
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40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.
Code of Federal Regulations, 2014 CFR
2014-07-01
...) The test should be designed to have a predetermined sensitivity and power. The number of cells... cleansed of pre-existing mutant cells. (D) Metabolic activation. Cells shall be exposed to the test...=/− test, colony sizing should be performed on at least one of the test cultures (the highest positive...
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40 CFR 799.9530 - TSCA in vitro mammalian cell gene mutation test.
Code of Federal Regulations, 2011 CFR
2011-07-01
...) The test should be designed to have a predetermined sensitivity and power. The number of cells... cleansed of pre-existing mutant cells. (D) Metabolic activation. Cells shall be exposed to the test...=/− test, colony sizing should be performed on at least one of the test cultures (the highest positive...
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Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population.
Horowitz, M; Pasmanik-Chor, M; Borochowitz, Z; Falik-Zaccai, T; Heldmann, K; Carmi, R; Parvari, R; Beit-Or, H; Goldman, B; Peleg, L; Levy-Lahad, E; Renbaum, P; Legum, S; Shomrat, R; Yeger, H; Benbenisti, D; Navon, R; Dror, V; Shohat, M; Magal, N; Navot, N; Eyal, N
1998-01-01
Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that approximately 1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon.
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Messai, Yosra; Gad, Sophie; Noman, Muhammad Zaeem; Le Teuff, Gwenael; Couve, Sophie; Janji, Bassam; Kammerer, Solenne Florence; Rioux-Leclerc, Nathalie; Hasmim, Meriem; Ferlicot, Sophie; Baud, Véronique; Mejean, Arnaud; Mole, David Robert; Richard, Stéphane; Eggermont, Alexander M M; Albiges, Laurence; Mami-Chouaib, Fathia; Escudier, Bernard; Chouaib, Salem
2016-10-01
Clear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel-Lindau (VHL) gene. To elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. A series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon-intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. Fisher's exact test was used for VHL mutation and Kruskal-Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal-Wallis test was used; otherwise Cochran-Mantel-Haenzsel test was used. We also used the Fligner-Policello test to compare two medians when the distributions had different dispersions. We demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the human PD-L1 proximal promoter in 786-O cells. Our work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. We investigated the relationship between von Hippel-Lindau mutations and programmed death-ligand 1 expression. We demonstrated that von Hippel-Lindau mutation status significantly correlated with programmed death-ligand 1 expression in clear cell renal cell carcinomas. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.
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Walker, Christopher J; Rush, Craig M; Dama, Paola; O'Hern, Matthew J; Cosgrove, Casey M; Gillespie, Jessica L; Zingarelli, Roman A; Smith, Blair; Stein, Maggie E; Mutch, David G; Shakya, Reena; Chang, Chia-Wen; Selvendiran, Karuppaiyah; Song, Jonathan W; Cohn, David E; Goodfellow, Paul J
2018-05-01
Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.
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Detection of BRAF mutations from solid tumors using Tumorplex™ technology
Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah
2015-01-01
Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko
Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important rolemore » of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.« less
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TERT promoter mutation in adult granulosa cell tumor of the ovary.
Pilsworth, Jessica A; Cochrane, Dawn R; Xia, Zhouchunyang; Aubert, Geraldine; Färkkilä, Anniina E M; Horlings, Hugo M; Yanagida, Satoshi; Yang, Winnie; Lim, Jamie L P; Wang, Yi Kan; Bashashati, Ali; Keul, Jacqueline; Wong, Adele; Norris, Kevin; Brucker, Sara Y; Taran, Florin-Andrei; Krämer, Bernhard; Staebler, Annette; Oliva, Esther; Shah, Sohrab P; Kommoss, Stefan; Kommoss, Friedrich; Gilks, C Blake; Baird, Duncan M; Huntsman, David G
2018-02-15
The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, which is a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified as gain-of-function mutations that promote transcriptional activation of TERT in multiple cancers, such as melanoma and glioblastoma. A recent study investigating TERT promoter mutations in ovarian carcinomas found C228T and C250T mutations in 15.9% of clear cell carcinomas. However, it is unknown whether these mutations are frequent in other ovarian cancer subtypes, in particular, sex cord-stromal tumors including adult granulosa cell tumors. We performed whole-genome sequencing on ten adult granulosa cell tumors with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that adult granulosa cell tumors with mutated TERT promoter have increased expression of TERT mRNA and exhibited significantly longer telomeres compared to those with wild-type TERT promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary adult granulosa cell tumors (22%), 24 of 58 recurrent adult granulosa cell tumors (41%), and 1 of 22 other sex cord-stromal tumors (5%). There was a significant difference in overall survival between patients with TERT C228T promoter mutation in the primary tumors and those without it (p = 0.00253, log-rank test). In seven adult granulosa cell tumors, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggest that TERT C228T promoter mutations may have an important role in progression of adult granulosa cell tumors.
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EGFR T790M mutation testing within the osimertinib AURA Phase I study.
Dearden, Simon; Brown, Helen; Jenkins, Suzanne; Thress, Kenneth S; Cantarini, Mireille; Cole, Rebecca; Ranson, Malcolm; Jänne, Pasi A
2017-07-01
Reliable epidermal growth factor receptor (EGFR) mutation testing techniques are required to identify eligible patients with EGFR mutation/T790M positive advanced non-small cell lung cancer (NSCLC), for treatment with osimertinib (AZD9291), an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR-TKI-sensitizing and T790M resistance mutations over wild-type EGFR. There is no current consensus regarding the best method to detect EGFR T790M mutations. The aim of this study was to describe the concordance between local testing, which used a variety of methods, and central testing, using the cobas ® EGFR Mutation Test, for EGFR-sensitizing mutations and the T790M resistance mutation. Tumor samples were obtained from all patients screened for inclusion onto the osimertinib Phase I expansion component of the AURA Phase I/II study (NCT01802632). Samples underwent central laboratory testing for EGFR-sensitizing mutations and T790M resistance mutation using the cobas ® EGFR Mutation Test. Results were compared with local laboratory test results, based on other testing methodologies including Sanger sequencing, therascreen ® , PNAClamp™, and Sequenom MassARRAY ® . Central laboratory testing was successful in 99% of samples passing histopathology review and testing success rates were comparable across the three central laboratories. Concordance between central and local testing for common sensitizing mutations was high (>98%) and concordance for the T790M mutation was also high (>90%). Tumor heterogeneity, along with other technical factors may have influenced this result. Within the osimertinib AURA Phase I study, EGFR mutation testing across three centralized laboratories using the cobas ® EGFR Mutation Test was feasible and successful, with strong concordance between local and central laboratory results, including for T790M. The cobas ® EGFR Mutation Test has subsequently been approved as the companion diagnostic test for osimertinib in the USA and Japan. Copyright © 2017 Elsevier B.V. All rights reserved.
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EGFR Mutation Testing Practices within the Asia Pacific Region
Kerr, Keith M.; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma. Luisa D.; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony
2015-01-01
Introduction: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non–small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. Methods: A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Results: Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%–32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. Conclusions: In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia. PMID:25376513
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Yatabe, Yasushi; Kerr, Keith M; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma Luisa D; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony
2015-03-01
The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%-32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia.
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... Autoantibodies Digoxin Direct Antiglobulin Test Direct LDL Cholesterol Drug Abuse Testing EGFR Mutation Testing Electrolytes Emergency and Overdose Drug Testing Epstein-Barr Virus (EBV) Antibody Tests Erythrocyte ...
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Zhu, Huixia; Zhang, Ye; Chen, Jianfeng; Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin
2017-01-01
Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.
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Martin, Petra; Leighl, Natasha B
2017-06-01
This article considers the use of pretest probability in non-small cell lung cancer (NSCLC) and how its use in EGFR testing has helped establish clinical guidelines on selecting patients for EGFR testing. With an ever-increasing number of molecular abnormalities being identified and often limited tissue available for testing, the use of pretest probability will need to be increasingly considered in the future for selecting investigations and treatments in patients. In addition we review new mutations that have the potential to affect clinical practice.
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Andolfo, Immacolata; Russo, Roberta; Manna, Francesco; De Rosa, Gianluca; Gambale, Antonella; Zouwail, Soha; Detta, Nicola; Pardo, Catia Lo; Alper, Seth L.; Brugnara, Carlo; Sharma, Alok K.; De Franceschi, Lucia; Iolascon, Achille
2016-01-01
Isolated familial pseudohyperkalemia is a dominant red cell trait characterized by cold-induced ‘passive leak’ of red cell potassium ions into plasma. The causative gene of this condition is ABCB6, which encodes an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. In this study analyzing three new families, we report the first functional characterization of ABCB6 mutants, including the homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q (in trans). All these mutations are annotated in public databases, suggesting that familial pseudohyperkalemia could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Four weeks’ storage of heterozygous R276W blood cells resulted in massive loss of potassium compared to that from healthy control red blood cells. Moreover, measurement of cation flux demonstrated greater loss of potassium or rubidium ions from HEK-293 cells expressing ABCB6 mutants than from cells expressing wild-type ABCB6. The R276W/R723Q mutations elicited greater cellular potassium ion efflux than did the other mutants tested. In conclusion, ABCB6 missense mutations in red blood cells from subjects with familial pseudohyperkalemia show elevated potassium ion efflux. The prevalence of such individuals in the blood donor population is moderate. The fact that storage of blood from these subjects leads to significantly increased levels of potassium in the plasma could have serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for familial pseudohyperkalemia could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration. PMID:27151991
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Leary, Alexandra F; Castro, David Gonzalez de; Nicholson, Andrew G; Ashley, Sue; Wotherspoon, Andrew; O'Brien, Mary E R; Popat, Sanjay
2012-01-01
EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (<2 ng/μL) had more test failures (30% versus 3.9% for [DNA]>2.2 ng/μL), the mutation rate was 9.2%. Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Yoshimura, Yasushi; Kurasawa, Mitsue; Yorozu, Keigo; Puig, Oscar; Bordogna, Walter; Harada, Naoki
2016-03-01
Alectinib is a highly selective next-generation anaplastic lymphoma kinase (ALK) inhibitor. Although alectinib shows inhibitory activity against various crizotinib-resistant ALK mutations in studies using cell-free kinase assays and Ba/F3 cell-based assays, it has not been tested for efficacy against non-small cell lung cancer (NSCLC) with the ALK mutations. We conducted in vitro and in vivo investigations into the antitumor activity of alectinib against an ALK-positive NSCLC cell line, SNU-2535, which harbors an ALK G1269A mutation. The clinical efficacy of alectinib against a NSCLC patient harboring ALK G1269A mutation was evaluated in the phase I part of the North American study. Alectinib exhibited antiproliferative activity against SNU-2535 cells in vitro with IC50 of 33.1 nM. Alectinib strongly inhibited phosphorylation of ALK and its downstream signaling molecules ERK1/2, AKT, and STAT3. In a mouse xenograft model, once-daily oral administration of alectinib for 21 days resulted in strong tumor regression. In addition, administration of alectinib for 100 days achieved continuous tumor regression without tumor regrowth in all mice. Notably, eradication of tumor cells was observed in half of the mice. In the clinical study, a patient with ALK G1269A mutation showed partial response to alectinib with a duration of response of 84 days. These results indicated that alectinib has potent antitumor activity against NSCLC cells harboring the crizotinib-resistant mutation ALK G1269A. It is expected that alectinib would provide a valuable therapeutic option for patients with NSCLC having not only native ALK but also crizotinib-resistant ALK mutations.
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Lim, Eun-A; Lee, Haeyoung; Bae, Eunmi; Lim, Jaeok; Shin, Young Kee; Choi, Sang-Eun
2016-01-01
As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR) mutation status compared with a no-testing strategy that is the current clinical practice in South Korea. A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer's perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles). In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs) were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs) increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib. Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy.
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T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus
Shuda, Masahiro; Feng, Huichen; Kwun, Hyun Jin; Rosen, Steven T.; Gjoerup, Ole; Moore, Patrick S.; Chang, Yuan
2008-01-01
Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors. PMID:18812503
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Practical aspects of mutagenicity testing strategy: an industrial perspective.
Gollapudi, B B; Krishna, G
2000-11-20
Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.
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Wang, Wen-Chung; Lee, Ya-Ting; Lai, Yen-Chein
2017-03-27
Granulosa cell tumors are rare ovarian malignancies. Their characteristics include unpredictable indolent growth with malignant potential and late recurrence. Approximately 95% are of adult type. Recent molecular studies have characterized the FOXL2 402C > G mutation in adult granulosa cell tumor. Our previous case report showed that unique FOXL2 402C > G mutation and defective DNA mismatch repair system are associated with the development of adult granulosa cell tumor. In this study, the DNA sequences of four genes, MSH2, MLH1, MSH6, and PMS2, in the DNA mismatch repair system were determined via direct sequencing to elucidate the exact mechanism for the development of this granulosa cell tumor. The results showed that two missense germline mutations, T485K and N775L, inactivate the PMS2 gene. The results of this case study indicated that although FOXL2 402C > G mutation determines the development of granulosa cell tumor, PMS2 mutation may be the initial driver of carcinogenesis. Immunohistochemistry-based tumor testing for mismatch repair gene expression may be necessary for granulosa cell tumors to determine their malignant potential or if they are part of Lynch syndrome.
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HER2 activating mutations are targets for colorectal cancer treatment
Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron
2015-01-01
The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863
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Kim, Juwon; Jung, Jinsei; Lee, Min Goo; Choi, Jae Young; Lee, Kyung-A
2015-06-19
GJB2 alleles containing two cis mutations have been rarely found in non-syndromic hearing loss. Herein, we present a Korean patient with non-syndromic hearing loss caused by the R75Q cis mutation with V37I, which arose de novo in the father and was inherited by the patient. Biochemical coupling and hemichannel permeability assays were performed after molecular cloning and transfection of HEK293T cells. Student's t-tests or analysis of variance followed by Tukey's multiple comparison test was used as statistical analysis. Biochemical coupling was significantly reduced in connexin 26 (Cx26)-R75Q- and Cx26-V37I-transfected cells, with greater extent in Cx26-R75Q and Cx26-R75Q+V37I cells. Interestingly, our patient and his father with the mutations had more residual hearing compared with patients with the dominant mutation alone. Although the difference in hemichannel activity between R75Q alone and R75Q in combination with V37I failed to reach significance, it is of note that there is a possibility that V37I located upstream of R75Q might have the ability to ameliorate R75Q expression. Our study emphasizes the importance of cis mutations with R75Q, as the gene effect of R75Q can be modulated depending on the type of additional mutation.
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Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.
Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S
2017-01-01
Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.
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Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens
Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.
2017-01-01
Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity. PMID:28125707
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HER2 activating mutations are targets for colorectal cancer treatment.
Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron
2015-08-01
The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.
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Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew
2007-09-01
JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.
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Mignot, Cyril; Delarasse, Cécile; Escaich, Séverine; Della Gaspera, Bruno; Noé, Eric; Colucci-Guyon, Emma; Babinet, Charles; Pekny, Milos; Vicart, Patrick; Boespflug-Tanguy, Odile; Dautigny, André; Rodriguez, Diana; Pham-Dinh, Danielle
2007-08-01
Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.
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Martin, Petra; Leighl, Natasha B.
2017-01-01
This article considers the use of pretest probability in non-small cell lung cancer (NSCLC) and how its use in EGFR testing has helped establish clinical guidelines on selecting patients for EGFR testing. With an ever-increasing number of molecular abnormalities being identified and often limited tissue available for testing, the use of pretest probability will need to be increasingly considered in the future for selecting investigations and treatments in patients. In addition we review new mutations that have the potential to affect clinical practice. PMID:28607579
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Kasahara, Norimitsu; Kenmotsu, Hirotsugu; Serizawa, Masakuni; Umehara, Rina; Ono, Akira; Hisamatsu, Yasushi; Wakuda, Kazushige; Omori, Shota; Nakashima, Kazuhisa; Taira, Tetsuhiko; Naito, Tateaki; Murakami, Haruyasu; Koh, Yasuhiro; Mori, Keita; Endo, Masahiro; Nakajima, Takashi; Yamada, Masanobu; Kusuhara, Masatoshi; Takahashi, Toshiaki
2017-04-01
Epidermal growth factor receptor (EGFR) mutation testing is a companion diagnostic to determine eligibility for treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). Recently, plasma-based EGFR testing by digital polymerase chain reaction (dPCR), which enables accurate quantification of target DNA, has shown promise as a minimally invasive diagnostic. Here, we aimed to evaluate the accuracy of a plasma-based EGFR mutation test developed using chip-based dPCR-based detection of 3 EGFR mutations (exon 19 deletions, L858R in exon 21, and T790M in exon 20). Forty-nine patients with NSCLC harboring EGFR-activating mutations were enrolled, and circulating free DNAs (cfDNAs) were extracted from the plasma of 21 and 28 patients before treatment and after progression following EGFR-TKI treatment, respectively. Using reference genomic DNA containing each mutation, the detection limit of each assay was determined to be 0.1%. The sensitivity and specificity of detecting exon 19 deletions and L858R mutations, calculated by comparing the mutation status in the corresponding tumors, were 70.6% and 93.3%, and 66.7% and 100%, respectively, showing similar results compared with previous studies. T790M was detected in 43% of 28 cfDNAs after progression with EGFR-TKI treatment, but in no cfDNAs before the start of the treatment. This chip-based dPCR assay can facilitate detection of EGFR mutations in cfDNA as a minimally invasive method in clinical settings. Copyright © 2017 Elsevier B.V. All rights reserved.
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Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin
2017-01-01
Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098
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Satouchi, Miyako; Tanaka, Hiroshi; Yoshioka, Hiroshige; Shimokawaji, Tadasuke; Mizuno, Keiko; Takeda, Koji; Yoshino, Ichiro; Seto, Takashi; Kurata, Takayasu; Tashiro, Naoki; Hagiwara, Koichi
2017-09-01
Detection of epidermal growth factor receptor (EGFR) gene mutations is essential in deciding therapeutic strategy in non-small cell lung cancer (NSCLC) patients at initial diagnosis. Moreover, in EGFR mutation-positive (EGFRm) NSCLC patients, re-biopsy at disease progression to clarify resistance mechanisms is also important. However, collecting histology samples is often difficult because of inaccessibility and invasiveness. In some cases, only cytology samples can be collected, and studies have reported that cytology samples are appropriate for EGFR gene mutation testing. The cobas ® EGFR Mutation Test (Roche Molecular Systems Inc., Branchburg, New Jersey, USA) is approved as a companion diagnostic for osimertinib, a third-generation EGFR-tyrosine kinase inhibitor approved in Japan. However, it is not clear whether the EGFR T790M mutation can be detected in cytology samples using this test. The primary objective of this study was to assess concordance of EGFR T790M gene mutation detection between histology and matched cytology samples using the cobas ® EGFR Mutation Test. We conducted a multicenter, observational study in Japan. Overall, 41 EGFRm NSCLC patients who had both histology and cytology samples collected at the same time at re-biopsy and with the results of EGFR mutation test using histology samples were enrolled. The EGFR mutation status of both sample types was tested using the cobas ® EGFR Mutation Test and the concordance rates were calculated. The EGFR T790M mutation detection rate in histology and cytology samples was 42.5% and 37.5%, respectively. The overall percent agreement between the histology and cytology samples was 91.7%. These data demonstrate that the cobas ® EGFR Mutation Test can detect the EGFR T790M mutation in both cytology and histology samples. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
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Caroccia, Brasilina; Prisco, Selene; Seccia, Teresa Maria; Piazza, Maria; Maiolino, Giuseppe; Rossi, Gian Paolo
2017-12-01
Aldosterone-producing adenoma (APA), a major subtype of primary hyperaldosteronism, the main curable cause of human endocrine hypertension, involves somatic mutations in the potassium channel Kir3.4 ( KCNJ5 ) in 30% to 70% of cases, typically the more florid phenotypes. Because KCNJ5 mutated channels were reported to be specifically sensitive to inhibition by macrolide antibiotics, which concentration dependently blunts aldosterone production in HAC15 transfected with the G151R and L168R mutated channel, we herein tested the effect of clarithromycin on aldosterone synthesis and secretion in a pure population of aldosterone-secreting cells obtained by immunoseparation (CD56 + cells) from APA tissues with/without the 2 most common KCNJ5 mutations. From a large cohort of patients with an unambiguous APA diagnosis, we recruited those who were wild type (n=3) or had G151R (n=2) and L168R (n=2) mutations. We found that clarithromycin concentration dependently lowered CYP11B2 gene expression (by 60%) and aldosterone secretion (by 70%; P <0.001 for both) in CD56 + cells isolated ex vivo from KCNJ5 mutated APAs, although it was ineffective in CD56 + cells from wild-type APAs. By proving the principle that the oversecretion of aldosterone can be specifically blunted in APA cells ex vivo with G151R and L168R mutations, these results provide compelling evidence of the possibility of specifically correcting aldosterone excess in patients with APA carrying the 2 most common KCNJ5 somatic mutations. © 2017 American Heart Association, Inc.
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Taooka, Yasuyuki; Takeichi, Nobuo; Noso, Yoshihiro; Kawano, Noriyuki; Apsalikov, Kazbek N; Hoshi, Masaharu
2006-02-01
From 1949 to 1989, 488 nuclear explosions were carried out in Semipalatinsk, and the cancer risk is increased in this region. Measuring somatic-cell mutation frequencies may be a useful tool for evaluating cancer risk within radiation-exposed populations. Here, we report the first evidence of increased T-cell receptor (TCR) mutations in peripheral blood from radiation-exposed residents of Semipalatinsk. The TCR mutation frequency in the highly exposed residents (Dolon and Sarzhal) was significantly higher than in the control group (Kokpekti). There was no statistically significant difference between the control group and the weakly exposed group (Kaynar and Semipalatinsk-city). The TCR mutation assay appeared to be a useful biological dosimeter even after a period of 40 years since radiation exposure. This may be the result of specific conditions, such as the presence of internal exposure.
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Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria
2015-02-01
Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.
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Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria
2015-01-01
Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome. PMID:24916645
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Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer
Kriegshäuser, Gernot; Fabjani, Gerhild; Ziegler, Barbara; Zöchbauer-Müller, Sabine; End, Adelheid; Zeillinger, Robert
2011-01-01
This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. PMID:22272089
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BRCA mutation genetic testing implications in the United States.
Bayraktar, Soley; Arun, Banu
2017-02-01
BRCA mutation carriers have a very high risk of breast and ovarian cancer by age 70, in the ranges 47%-66% and 40%-57%, respectively. Additionally, women with BRCA mutation-associated breast cancer also have an elevated risk of other or secondary malignancies. Fortunately, the breast and ovarian cancer outcome for BRCA1/2 mutation carriers is at least as good as for non-carriers with chemoprevention, prophylactic surgeries and appropriate use of therapies. Therefore, identification of those who might have a mutation is important so that genetic counseling, testing, screening and prevention strategies can be applied in a timely manner. This article reviews the impact of genetic testing in general, timing of genetic testing after diagnosis and prior knowledge of mutation status in BRCA carriers with newly diagnosed breast cancer. Additionally, risk-reducing surgeries including the prophylactic contralateral mastectomy, and bilateral salpingo-oophorectomy and the sensitivity of BRCA-defective breast cancer cell lines to differential chemotherapeutic agents will be discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Inhibition of the Growth of Papillary Thyroid Carcinoma Cells by CI-1040
Henderson, Ying C.; Ahn, Soon-Hyun; Clayman, Gary L.
2015-01-01
Background Papillary thyroid carcinoma (PTC), the most common type of thyroid malignancy, usually possesses mutations, either RET/PTC rearrangement or BRAF mutation. Both mutations can activate the mitogen-activated protein kinase kinase/extracellular signal–related kinase signaling transduction pathway, which results in activation of transcription factors that regulate cellular proliferation, differentiation, and apoptosis. Objective To test the effects of CI-1040 (PD184352), a specific MEK1/2 inhibitor, on PTC cells carrying either an RET/PTC1 rearrangement or a BRAF mutation. Design The effects of CI-1040 on PTC cells were evaluated in vitro and in vivo. Main Outcome Measures The effects of CI-1040 on PTC cells were evaluated in vitro using a cell proliferation assay, cell cycle analysis, and immunoblotting. The antitumor effects of CI-1040 in vivo were evaluated in an orthotopic mouse model. Results The concentrations of CI-1040 needed to inhibit 50% cell growth were 0.052μM for PTC cells with a BRAF mutation and 1.1μM for PTC cells with the RET/PTC1 rearrangement. After 3 weeks of oral administration of CI-1040 (300 mg/kg/d) to mice with orthotopic tumor implants of PTC cells, the mean tumor volume of implants bearing the RET/PTC1 rearrangement (n=5) was reduced 47.5% compared with untreated mice (from 701.9 to 368.5 mm3), and the mean volume of implants with a BRAF mutation (n=8) was reduced 31.3% (from 297.3 to 204.2 mm3). Conclusions CI-1040 inhibits PTC cell growth in vitro and in vivo. Because RET/PTC rearrangements are unique to thyroid carcinomas and a high percentage of PTCs possess either mutation, these findings support the clinical evaluation of CI-1040 for patients with PTC. PMID:19380355
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Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.
Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana
2018-03-23
Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.
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Lin, Qiaohong; Mei, Jie; Li, Zhi; Zhang, Xuemei; Zhou, Li; Gui, Jian-Fang
2017-11-01
Spermatogenesis is a fundamental process in male reproductive biology and depends on precise balance between self-renewal and differentiation of male germ cells. However, the regulative factors for controlling the balance are poorly understood. In this study, we examined the roles of amh and dmrt1 in male germ cell development by generating their mutants with Crispr/Cas9 technology in zebrafish. Amh mutant zebrafish displayed a female-biased sex ratio, and both male and female amh mutants developed hypertrophic gonads due to uncontrolled proliferation and impaired differentiation of germ cells. A large number of proliferating spermatogonium-like cells were observed within testicular lobules of the amh -mutated testes, and they were demonstrated to be both Vasa- and PH3-positive. Moreover, the average number of Sycp3- and Vasa-positive cells in the amh mutants was significantly lower than in wild-type testes, suggesting a severely impaired differentiation of male germ cells. Conversely, all the dmrt1 -mutated testes displayed severe testicular developmental defects and gradual loss of all Vasa-positive germ cells by inhibiting their self-renewal and inducing apoptosis. In addition, several germ cell and Sertoli cell marker genes were significantly downregulated, whereas a prominent increase of Insl3-positive Leydig cells was revealed by immunohistochemical analysis in the disorganized dmrt1 -mutated testes. Our data suggest that amh might act as a guardian to control the balance between proliferation and differentiation of male germ cells, whereas dmrt1 might be required for the maintenance, self-renewal, and differentiation of male germ cells. Significantly, this study unravels novel functions of amh gene in fish. Copyright © 2017 by the Genetics Society of America.
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2017-01-01
Specific mutations in epidermal growth factor receptor (EGFR) gene are predictive for response to the EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer patients (NSCLC). According to international guidelines, the molecular testing in patients with advanced NSCLC of a non-squamous subtype is recommended. However, obtain a tissue sample could be challenging. Liquid biopsy allows to determine patients suitable for EGFR-targeted therapy by analysis of circulating-free tumor DNA (cfDNA) in peripheral blood samples and might replace tissue biopsy. It allows to acquire a material in convenient minimally invasive manner, is easily repeatable, could be used for molecular identification and molecular changes monitoring. Many studies show a high concordance rate between tissue and plasma samples testing. When U.S. Food and Drug Administration (FDA) approved the first liquid biopsy test, analysis of driver gene mutation from cfDNA becomes a reality in clinical practice for patients with NSCLC. PMID:28251125
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Adler, Ilse-Dore; Carere, Angelo; Eichenlaub-Ritter, Ursula
2007-05-15
Germ cell mutagenicity testing provides experimental data to quantify genetic risk for exposed human populations. The majority of tests are performed with exposure of males, and female data are relatively rare. The reason for this paucity lies in the differences between male and female germ cell biology. Male germ cells are produced throughout reproductive life and all developmental stages can be ascertained by appropriate breeding schemes. In contrast, the female germ cell pool is limited, meiosis begins during embryogenesis and oocytes are arrested over long periods of time until maturation processes start for small numbers of oocytes during the oestrusmore » cycle in mature females. The literature data are reviewed to point out possible gender differences of germ cells to exogenous agents such as chemicals or ionizing radiation. From the limited information, it can be concluded that male germ cells are more sensitive than female germ cells to the induction of chromosomal aberrations and gene mutations. However, exceptions are described which shed doubt on the extrapolation of experimental data from male rodents to the genetic risk of the human population. Furthermore, the female genome may be more sensitive to mutation induction during peri-conceptional stages compared to the male genome of the zygote. With few exceptions, germ cell experiments have been carried out under high acute exposure to optimize the effects and to compensate for the limited sample size in animal experiments. Human exposure to environmental agents, on the other hand, is usually chronic and involves low doses. Under these conditions, gender differences may become apparent that have not been studied so far. Additionally, data are reviewed that suggest a false impression of safety when responses are negative under high acute exposure of male rodents while a mutational response is induced by low chronic exposure. The classical (morphological) germ cell mutation tests are not performed anymore because they are animal and time consuming. Nevertheless, information is needed to place genetic risk extrapolations on more solid grounds and thereby to prevent an increased genetic burden to future generations. It is pointed out that modern molecular methodologies are available now to experimentally address the open questions.« less
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Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna
2015-01-01
The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806
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Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018.
Bhalla, Amarpreet; Zulfiqar, Muhammad; Bluth, Martin H
2018-06-01
The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenomaecarcinoma sequence, serrated polyp pathway, and microsatellite instability (MSI). The genetic basis for hereditary nonpolyposis colorectal cancer is the detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. Genetic testing for Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in MMR genes. Molecular makers with predictive and prognostic implications include quantitative multigene reverse transcriptase polymerase chain reaction assay and KRAS and BRAF mutation analysis. Mismatch repair-deficient tumors have higher rates of programmed death-ligand 1 expression. Cell-free DNA analysis in fluids are proving beneficial for diagnosis and prognosis in these disease states towards effective patient management. Copyright © 2018 Elsevier Inc. All rights reserved.
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Osawa, Masaki
2018-01-01
It is difficult to target and kill cancer cells. One possible approach is to mutate bacteria to enhance their binding to cancer cells. In the present study, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis were randomly mutated, and then were positively and negatively selected for binding cancer vs normal cells. With repetitive mutation and selection both bacteria successfully evolved to increase affinity to the pancreatic cancer cell line (Mia PaCa-2) but not normal cells (HPDE: immortalized human pancreatic ductal epithelial cells). The mutant E. coli and B. subtilis strains bound to Mia PaCa-2 cells about 10 and 25 times more than to HPDE cells. The selected E. coli strain had mutations in biofilm-related genes and the regulatory region for a type I pilus gene. Consistent with type I pili involvement, mannose could inhibit the binding to cells. The results suggest that weak but specific binding is involved in the initial step of adhesion. To test their ability to kill Mia PaCa-2 cells, hemolysin was expressed in the mutant strain. The hemolysin released from the mutant strain was active and could kill Mia PaCa-2 cells. In the case of B. subtilis, the initial binding to the cells was a weak interaction of the leading pole of the motile bacteria. The frequency of this interaction to Mia PaCa-2 cells dramatically increased in the evolved mutant strain. This mutant strain could also specifically invade beneath Mia PaCa-2 cells and settle there. This type of mutation/selection strategy may be applicable to other combinations of cancer cells and bacterial species.
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Checkpoint Kinase 2 (CHEK2) Mutation in Renal Cell Carcinoma: A Single-Center Experience
Huszno, Joanna; Kołosza, Zofia
2018-01-01
Renal cell carcinoma (RCC) occurs in sporadic and heritable forms. Genetic mutations have been identified as risk factors in 1–2% of RCC. The aim of this study was to evaluate I157T and CHEK2*1100delC mutations of checkpoint kinase 2 (CHEK2) gene in RCC. Medical records of 40 clear cell RCC patients who had genetic tests and consultation at the Genetic Outpatient Clinic, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland, were reviewed retrospectively. Mutation profile was assessed by ASA-PCR and RFLP-PCR techniques. Only three female patients had CHEK2 mutation (I157T). No CHEK2*1100delC was observed in any of the patients. These tumors were N0, and two were Grade 3. One showed capsular infiltration. No blood vessel infiltration or metastases was observed. Overall, RCC from patients with CHEK2 mutation did not display any special characteristics when compared with those without the mutation. While no association between CHEK2 mutation and RCC could be established, all three patients with CHEK2 mutation developed second neoplasms many years after first diagnosis. Further studies, especially regarding CHEK2 mutation as a predictive factor for second neoplasm in RCC patients, are warranted. PMID:29682443
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Checkpoint Kinase 2 (CHEK2) Mutation in Renal Cell Carcinoma: A Single-Center Experience.
Huszno, Joanna; Kołosza, Zofia
2018-01-01
Renal cell carcinoma (RCC) occurs in sporadic and heritable forms. Genetic mutations have been identified as risk factors in 1-2% of RCC. The aim of this study was to evaluate I157T and CHEK2*1100delC mutations of checkpoint kinase 2 (CHEK2) gene in RCC. Medical records of 40 clear cell RCC patients who had genetic tests and consultation at the Genetic Outpatient Clinic, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland, were reviewed retrospectively. Mutation profile was assessed by ASA-PCR and RFLP-PCR techniques. Only three female patients had CHEK2 mutation (I157T). No CHEK2*1100delC was observed in any of the patients. These tumors were N0, and two were Grade 3. One showed capsular infiltration. No blood vessel infiltration or metastases was observed. Overall, RCC from patients with CHEK2 mutation did not display any special characteristics when compared with those without the mutation. While no association between CHEK2 mutation and RCC could be established, all three patients with CHEK2 mutation developed second neoplasms many years after first diagnosis. Further studies, especially regarding CHEK2 mutation as a predictive factor for second neoplasm in RCC patients, are warranted.
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Domingo, Diana; Neco, Patricia; Fernández-Pons, Elena; Zissimopoulos, Spyros; Molina, Pilar; Olagüe, José; Suárez-Mier, M Paz; Lai, F Anthony; Gómez, Ana M; Zorio, Esther
2015-05-01
Catecholaminergic polymorphic ventricular tachycardia is a malignant disease, due to mutations in proteins controlling Ca(2+) homeostasis. While the phenotype is characterized by polymorphic ventricular arrhythmias under stress, supraventricular arrhythmias may occur and are not fully characterized. Twenty-five relatives from a Spanish family with several sudden deaths were evaluated with electrocardiogram, exercise testing, and optional epinephrine challenge. Selective RyR2 sequencing in an affected individual and cascade screening in the rest of the family was offered. The RyR2(R420Q) mutation was generated in HEK-293 cells using site-directed mutagenesis to conduct in vitro functional studies. The exercise testing unmasked catecholaminergic polymorphic ventricular tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%; negative predictive value = 93%), all of them carrying the heterozygous RyR2(R420Q) mutation, which was also present in the proband and a young girl without exercise testing, a 91% penetrance at the end of the follow-up. Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant post-effort U-waves were identified in patients. Upon permeabilization and in intact cells, the RyR2(R420Q) expressing cells showed a smaller peak of Ca(2+) release than RyR2 wild-type cells. However, at physiologic intracellular Ca(2+) concentration, equivalent to the diastolic cytosolic concentration, the RyR2(R420Q) released more Ca(2+) and oscillated faster than RyR2 wild-type cells. The missense RyR2(R420Q) mutation was identified in the N-terminus of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is associated with sinus bradycardia, atrial and junctional arrhythmias, and giant U-waves. Collectively, functional heterologous expression studies suggest that the RyR2(R420Q) behaves as an aberrant channel, as a loss- or gain-of-function mutation depending on cytosolic intracellular Ca(2+) concentration. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.
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Cell-selfish modes of evolution and mutations directed after transcriptional bypass.
Holmquist, Gerald P
2002-12-29
During transcription, prokaryotic and eukaryotic RNA polymerases bypass and misread (transcriptional mutagenesis) several classes of DNA lesions. For example, misreading of 8-OH-dG generates mRNAs containing G to T transversions. After translation, if the mutant protein briefly allowed the cell a growth-DNA replication advantage, then precocious DNA replication would bypass that unrepaired 8-OH-dG and misinsert dA opposite the directing DNA lesion with a higher probability than would be experienced for 8-OH-G lesions at other positions in otherwise identical neighboring cells. Such retromutations would have been tested for their imparted growth advantage as mRNA before they became heritable DNA mutations. The logical properties of a mode of evolution that utilizes directed-retromutagenesis were compared one by one with those of the standard neo-Darwinian mode. The retromutagenesis mode, while minimizing mutational load, is cell-selfish; fitness is for an immediate growth advantage rather than future reproductive potential. In prokaryotes, an evolutionary mode that involves standard Darwinian fitness testing of novel alleles in the genetic background of origin followed by clonal expansion also favors cell-selfish allele combinations when linkage disequilibrium is practiced. For metazoa and plants to have evolved organized tissues, cell-selfish modes of evolution represent systems-poisons that must be totally suppressed. The feedback loops that allow evolution to be cell-serving in prokaryotes are actively blocked in eukaryotes by traits that restrict fitness to future reproductive potential. These traits include (i) delay of fitness testing until after the mutation is made permanently heritable, (ii) diploidy to further delay fitness testing, (iii) segregation of somatic lines from germ lines, (iv) testing of novel alleles against randomized allele combinations constructed by obligate sex, and (v) obligate genetic death to insure that that the most basic systems unit of selfish allele combinatorial uniqueness is the species instead of the cell. The analyses indicate that modes of evolution in addition to our neo-Darwinian one could have existed utilizing known molecular mechanisms. The evolution of multicellularity was as much the discarding of old cell-selfish habits as the acquisition of new altruistic ones.
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The ALCHEMIST Lung Cancer Trials
A collection of material about the ALCHEMIST lung cancer trials that will examine tumor tissue from patients with certain types of early-stage, completely resected non-small cell lung cancer for gene mutations in the EGFR and ALK genes, and assign patients with these gene mutations to treatment trials testing post-surgical use of drugs targeted against these mutations.
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Jeannot, Emmanuelle; Pogribny, Igor P.; Beland, Frederick A.; Rusyn, Ivan
2010-01-01
This study used tissue samples from male B6C3F1 mice treated with ethanol in drinking water (0, 2.5, or 5%) for 4 or 104 weeks. We tested whether chronic alcohol drinking promotes oxidative stress in the liver and characterized the mutation profile of spontaneous and ethanol-induced tumors. We show that ethanol does not cause detectable oxidative stress in the liver at any time point and acts by promoting H-ras mutated cells. PMID:21168264
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Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kukat, Alexandra; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases; Edgar, Daniel
2011-06-10
Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of themore » molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.« less
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Van Bockstaele, Femke; Janssens, Ann; Piette, Anne; Callewaert, Filip; Pede, Valerie; Offner, Fritz; Verhasselt, Bruno; Philippé, Jan
2006-07-15
ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL. (c) 2006 International Society for Analytical Cytology.
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Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui
2015-07-01
DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.
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Mutation Analysis in Cultured Cells of Transgenic Rodents
Zheng, Albert; Bates, Steven E.; Tommasi, Stella
2018-01-01
To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable. PMID:29337872
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Chougule, A; Basak, S
2017-12-01
Lung cancer is one of the major causes of mortality worldwide and is on the rise in India. The identification of epidermal growth factor receptor (EGFR) mutations in nonsmall cell lung cancer (NSCLC) has paved the way for personalized therapy in lung cancer with EGFR-tyrosine kinase inhibitors (TKIs). Despite the proven efficacy of EGFR-TKIs in patients harboring EGFR mutations, their clinical utility is limited by the development of acquired resistance mechanisms by the tumor cells. T790M mutation accounts for 60% of all resistance mechanisms to EGFR TKIs and is responsible for treatment failure with first- and second-generation TKIs. With the development of novel therapeutic agents such as osimertinib to overcome this resistance mechanism, it is essential to detect patients harboring T790M mutation. There are several limitations with the use of tissue biopsy specimens for molecular testing such as poor quality and quantity of sample, tumor heterogeneity, occurrence of complications, and issues with repeat biopsy. Liquid biopsy offers a noninvasive approach that can be used for diagnostic purposes as well as for monitoring treatment response and evaluation of resistance mechanisms. This review focuses on the methods for molecular testing of tissue and liquid biopsy specimens for EGFR mutations, particularly EGFR T790M mutation.
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Selection against Heteroplasmy Explains the Evolution of Uniparental Inheritance of Mitochondria
Christie, Joshua R.; Schaerf, Timothy M.; Beekman, Madeleine
2015-01-01
Why are mitochondria almost always inherited from one parent during sexual reproduction? Current explanations for this evolutionary mystery include conflict avoidance between the nuclear and mitochondrial genomes, clearing of deleterious mutations, and optimization of mitochondrial-nuclear coadaptation. Mathematical models, however, fail to show that uniparental inheritance can replace biparental inheritance under any existing hypothesis. Recent empirical evidence indicates that mixing two different but normal mitochondrial haplotypes within a cell (heteroplasmy) can cause cell and organism dysfunction. Using a mathematical model, we test if selection against heteroplasmy can lead to the evolution of uniparental inheritance. When we assume selection against heteroplasmy and mutations are neither advantageous nor deleterious (neutral mutations), uniparental inheritance replaces biparental inheritance for all tested parameter values. When heteroplasmy involves mutations that are advantageous or deleterious (non-neutral mutations), uniparental inheritance can still replace biparental inheritance. We show that uniparental inheritance can evolve with or without pre-existing mating types. Finally, we show that selection against heteroplasmy can explain why some organisms deviate from strict uniparental inheritance. Thus, we suggest that selection against heteroplasmy explains the evolution of uniparental inheritance. PMID:25880558
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Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek
2012-01-01
Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532
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Lalonde, R; Hayzoun, K; Selimi, F; Mariani, J; Strazielle, C
2003-11-01
Grid2(ho/ho) is a loss of function gene mutation resulting in abnormal dendritic arborizations of Purkinje cells. These mutants were compared in a series of motor coordination tests requiring balance and equilibrium to nonataxic controls (Grid2(ho/+)) and to a double mutant (Grid2(ho/Lc)) with an inserted Lc mutation. The performance of Grid2(ho/ho) mutant mice was poorer than that of controls on stationary beam, coat hanger, unsteady platform, and rotorod tests. Grid2(ho/Lc) did not differ from Grid2(Lc/+) mice. However, the insertion of the Lc mutation in Grid2(ho/Lc) potentiated the deficits found in Grid2(ho/ho) in stationary beam, unsteady platform, and rotorod tests. These results indicate a deleterious effect of the Lc mutation on Grid2-deficient mice.
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Casadio, Chiara; Guarize, Juliana; Donghi, Stefano; Di Tonno, Clementina; Fumagalli, Caterina; Vacirca, Davide; Dell'Orto, Patrizia; De Marinis, Filippo; Spaggiari, Lorenzo; Viale, Giuseppe; Barberis, Massimo
2015-10-01
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive procedure that has revolutionized the diagnosis and staging of lung cancer. The goal of the present study was to investigate the yield and applicability of molecular testing in the specimens obtained by EBUS-TBNA from patients with advanced non-small cell lung cancer (NSCLC), comparing the results with a series of patients who underwent diagnostic surgical procedures in the same institution. The study followed 306 consecutive patients with clinically diagnosed primary lung cancer who had the EBUS-TBNA procedure. EGFR and KRAS mutations were evaluated on cytologic specimens by Sanger sequencing and Cobas real-time polymerase chain reaction, whereas ALK rearrangement was tested by fluorescence in situ hybridization. The results were compared with those obtained from a series of 1,000 NSCLC surgical samples routinely analyzed. Molecular testing was possible in 96.9% of the samples obtained by EBUS-TBNA. EGFR (exons 18-21) mutations were found in 16.9%, KRAS mutation (exons 2-3) in 31.6%, and ALK rearrangement in 3.9% of the cases. In the surgical series, the mutations' distribution were 14.8%, 29.0%, and 3.4%, respectively. There were no statistical differences between the two series. Our study demonstrates that EBUS-TBNA can be effectively used not just for diagnosis but also for complete mutational testing. Copyright© by the American Society for Clinical Pathology.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xing, Joanna; Liu, Ruixin; Xing, Mingzhao
2011-01-28
Research highlights: {yields} Exciting therapeutic potential has been recently reported for the BRAF{sup V600E} inhibitor PLX4032 in melanoma. {yields} We tested the effects of PLX4032 on the growth of thyroid cancer cells which often harbor the BRAF{sup V600E} mutation. {yields} We observed a potent BRAF{sup V600E}-dependent inhibition of thyroid cancer cells by PLX4032. {yields} We thus demonstrated an important therapeutic potential of PLX4032 for thyroid cancer. -- Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF{sup V600E}, as a result of the BRAF{sup T1799A} mutation, plays a fundamental role in thyroid tumorigenesis. This studymore » investigated the therapeutic potential of a BRAF{sup V600E}-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF{sup T1799A} mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF{sup T1799A} mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC{sub 50} values (0.115-1.156 {mu}M) in BRAF{sup V600E} mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC{sub 50} values (56.674-1349.788 {mu}M). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF{sup T1799A} mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF{sup T1799A} mutation-selective therapeutic agent for thyroid cancer.« less
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O'Reilly, L; Roth, M J
2000-01-01
Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.
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O'Reilly, Lucille; Roth, Monica J.
2000-01-01
Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells. PMID:10623753
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Zhao, Jing; Chen, Minjiang; Zhang, Li; Li, Longyun; Wang, Mengzhao
2017-01-01
We evaluated the use of droplet digital PCR (ddPCR) to detect plasma cell-free DNA (cfDNA) epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients. Compared with tumor-tissue-based detection, the sensitivity of ddPCR for detecting plasma cfDNA tyrosine kinase inhibitor (TKI)-sensitizing EGFR mutations was 61.3%, the specificity was 96.7%, and the consistency rate was 81.4% (?=0.605, 95% confidence interval: 0.501-0.706, p <0.0001). The sensitivity declined from 82.6% to 46.7% with decreasing cfDNA inputs (p=0.028). The plasma cfDNA concentration correlated with gender (males vs.females =11.69 ng/mL vs. 9.508 ng/mL; p=0.044), EGFR mutation status (tumor-tissue EGFR mutation-positive (EGFR M+) vs. EGFR mutation-negative (EGFR M-) = 9.61 ng/mL vs. 12.82 ng/mL; p =0.049) and specimen collection time (=2 years vs. >2 years=13.83 ng/mL vs. 6.575 ng/mL; p <0.001), and was greater in tumor-tissue EGFR M+ / plasma EGFR M+ patients than in tumor-tissue EGFR M+/plasma EGFR M- patients (11.61 vs. 7.73 ng/mL, respectively; p=0.003). Thus total cfDNA input crucially influences the sensitivity of plasma cfDNA EGFR mutation testing with ddPCR. Such analysis could be an effective supplemental test for advanced NSCLC patients. PMID:28052016
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Jia, Shiyu; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Gao, Peng; Han, Yanxi; Fu, Yu; Ding, Jiansheng; Wu, Qisheng; Zhang, Kuo; Xie, Jiehong; Li, Jinming
2018-06-01
KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material. © 2018 Wiley Periodicals, Inc.
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Villaruz, Liza C.; Ross, Jeffrey
2016-01-01
Abstract Molecular profiling and the discovery of drugs that target specific activating mutations have allowed the personalization of treatment for non‐small cell lung cancer (NSCLC). The epithelial growth factor receptor (EGFR) is frequently over‐expressed and/or aberrantly activated in different cancers, including NSCLC. The most common activating mutations of EGFR in NSCLC fall within the tyrosine kinase‐binding domain. Three oral EGFR tyrosine kinase inhibitors (TKIs) have been approved by the U.S. Food and Drug Administration (FDA) for first‐line use in patients with EGFR mutation‐positive NSCLC (exon 19 deletions or exon 21 [L858R] substitution mutations), as detected by an FDA‐approved test. However, disease progression is common and is often the result of secondary mutations, of which the EGFR T790M mutation is the most prevalent. Few options were available upon progression until the introduction of osimertinib, a kinase inhibitor that targets the T790M mutation, which was recently approved for use in patients with metastatic EGFR T790M mutation‐positive NSCLC, as detected by an FDA‐approved test, who progressed on or after EGFR TKI therapy. With the introduction of osimertinib, outcomes can now be improved in select patients. Therefore, performing a biopsy at progression to determine the underlying molecular cause of the acquired resistance is important for the enabling of individualized options that may provide the greatest opportunity for improved outcomes. This review discusses the latest updates in molecular testing at progression and outlines treatment options for this difficult‐to‐treat population. Implications for Practice. Although the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs)—gefitinib, erlotinib, and afatinib—have changed the treatment paradigm for non‐small cell lung cancer among those with EGFR mutation positive disease, most patients experience progression after approximately 12 months of treatment. Until recently, options were limited for patients who progressed, but improvements in molecular profiling and the approval of osimertinib, which targets the resistance mutation T790M, afford the opportunity for improved outcomes in many patients with this mutation. This article explains the options available after progression on initial EGFR TKI therapy and the importance of molecular testing at progression in making treatment decisions. PMID:27821794
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Normanno, Nicola; Pinto, Carmine; Taddei, Gianluigi; Gambacorta, Marcello; Castiglione, Francesca; Barberis, Massimo; Clemente, Claudio; Marchetti, Antonio
2013-06-01
The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology organized an external quality assessment (EQA) scheme for EGFR mutation testing in non-small-cell lung cancer. Ten specimens, including three small biopsies with known epidermal growth factor receptor (EGFR) mutation status, were validated in three referral laboratories and provided to 47 participating centers. The participants were requested to perform mutational analysis, using their usual method, and to submit results within a 4-week time frame. According to a predefined scoring system, two points were assigned to correct genotype and zero points to false-negative or false-positive results. The threshold to pass the EQA was set at higher than 18 of 20 points. Two rounds were preplanned. All participating centers submitted the results within the time frame. Polymerase chain reaction (PCR)/sequencing was the main methodology used (n = 37 laboratories), although a few centers did use pyrosequencing (n = 8) or real-time PCR (n = 2). A significant number of analytical errors were observed (n = 20), with a high frequency of false-positive results (n = 16). The lower scores were obtained for the small biopsies. Fourteen of 47 centers (30%) that did not pass the first round, having a score less than or equal to 18 points, used PCR/sequencing, whereas 10 of 10 laboratories, using pyrosequencing or real-time PCR, passed the first round. Eight laboratories passed the second round. Overall, 41of 47 centers (87%) passed the EQA. The results of the EQA for EGFR testing in non-small-cell lung cancer suggest that good quality EGFR mutational analysis is performed in Italian laboratories, although differences between testing methods were observed, especially for small biopsies.
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Comparative Oncogenomics for Peripheral Nerve Sheath Cancer Gene Discovery
2015-06-01
neurofibromas and MPNSTs, establish gene signatures defining distinct tumor subtypes and functionally test the role of selected driver mutations ...allografted tumor cells, and a variety of in vitro functional assays. We will validate the relevance of these mutated mouse genes in human neurofibromas...and MPNSTs by determining whether these same genes are mutated in human tumors. 15. SUBJECT TERMS Nothing listed 16. SECURITY CLASSIFICATION OF: 17
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Ma, MeiLi; Shi, ChunLei; Qian, JiaLin; Teng, JiaJun; Zhong, Hua; Han, BaoHui
2016-10-10
The aim of this study was to assess the effectiveness and accuracy of blood-based circulating-free tumor DNA on testing epidermal growth factor receptor (EGFR) gene mutations. In total, 219 non-small cell lung cancer patients in stages III-IV were enrolled into this study. All patients had tissue samples and matched plasma DNA samples. EGFR gene mutations were detected by the Amplification Refractory Mutation System (ARMS). We compared the mutations in tumor tissue samples with matched plasma samples and determined the correlation between EGFR mutation status and clinical pathologic characteristics. The overall concordance rate of EGFR mutation status between the 219 matched plasma and tissue samples was 82% (179/219). The sensitivity and specificity for the ARMS EGFR mutation test in the plasma compared with tumor tissue were 60% (54/90) and 97% (125/129), respectively. The positive predictive value was 93% (54/58) and the negative predictive value was 78% (125/161). The median overall survival was longer for those with EGFR mutations than for those without EGFR mutations both in tissue samples (23.98 vs. 12.16months; P<0.001) and in plasma (19.96 vs. 13.63months; P=0.009). For the 68 patients treated with EGFR- tyrosine kinase inhibitors (TKIs), the median progression-free survival (PFS) was significantly prolonged in the EGFR mutant group compared to the non-mutation group in tumor tissue samples (12.26months vs. 2.40months, P<0.001). In plasma samples, the PFS of the mutant group was longer than that of the non-mutant group. However, there was no significant difference between the two groups (10.88months vs. 9.89months, P=0.411). The detection of EGFR mutations in plasma using ARMS is relatively sensitive and highly specific. However, EGFR mutation status tested by ARMS in plasma cannot replace a tumor tissue biopsy. Positive EGFR mutation results detected in plasma are fairly reliable, but negative results are hampered by a high rate of false negatives. Copyright © 2016. Published by Elsevier B.V.
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Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo.
Amoyel, Marc; Simons, Benjamin D; Bach, Erika A
2014-10-16
Neutral competition, an emerging feature of stem cell homeostasis, posits that individual stem cells can be lost and replaced by their neighbors stochastically, resulting in chance dominance of a clone at the niche. A single stem cell with an oncogenic mutation could bias this process and clonally spread the mutation throughout the stem cell pool. The Drosophila testis provides an ideal system for testing this model. The niche supports two stem cell populations that compete for niche occupancy. Here, we show that cyst stem cells (CySCs) conform to the paradigm of neutral competition and that clonal deregulation of either the Hedgehog (Hh) or Hippo (Hpo) pathway allows a single CySC to colonize the niche. We find that the driving force behind such behavior is accelerated proliferation. Our results demonstrate that a single stem cell colonizes its niche through oncogenic mutation by co-opting an underlying homeostatic process. © 2014 The Authors.
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Janku, F; Huang, H J; Fujii, T; Shelton, D N; Madwani, K; Fu, S; Tsimberidou, A M; Piha-Paul, S A; Wheler, J J; Zinner, R G; Naing, A; Hong, D S; Karp, D D; Cabrilo, G; Kopetz, E S; Subbiah, V; Luthra, R; Kee, B K; Eng, C; Morris, V K; Karlin-Neumann, G A; Meric-Bernstam, F
2017-03-01
Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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Pfuhler, Stefan; Albertini, Silvio; Fautz, Rolf; Herbold, Bernd; Madle, Stephan; Utesch, Dietmar; Poth, Albrecht
2007-06-01
Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.
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MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB*
Bleymüller, Willem M.; Lämmermann, Nina; Ebbes, Maria; Maynard, Daniel; Geerds, Christina; Niemann, Hartmut H.
2016-01-01
The facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand β2 of the β-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization. PMID:27789707
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Distinct Brca1 Mutations Differentially Reduce Hematopoietic Stem Cell Function.
Mgbemena, Victoria E; Signer, Robert A J; Wijayatunge, Ranjula; Laxson, Travis; Morrison, Sean J; Ross, Theodora S
2017-01-24
BRCA1 is a well-known DNA repair pathway component and a tissue-specific tumor suppressor. However, its role in hematopoiesis is uncertain. Here, we report that a cohort of patients heterozygous for BRCA1 mutations experienced more hematopoietic toxicity from chemotherapy than those with BRCA2 mutations. To test whether this reflects a requirement for BRCA1 in hematopoiesis, we generated mice with Brca1 mutations in hematopoietic cells. Mice homozygous for a null Brca1 mutation in the embryonic hematopoietic system (Vav1-iCre;Brca1 F22-24/F22-24 ) developed hematopoietic defects in early adulthood that included reduced hematopoietic stem cells (HSCs). Although mice homozygous for a huBRCA1 knockin allele (Brca1 BRCA1/BRCA1 ) were normal, mice with a mutant huBRCA1/5382insC allele and a null allele (Mx1-Cre;Brca1 F22-24/5382insC ) had severe hematopoietic defects marked by a complete loss of hematopoietic stem and progenitor cells. Our data show that Brca1 is necessary for HSC maintenance and normal hematopoiesis and that distinct mutations lead to different degrees of hematopoietic dysfunction. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
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Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells.
Bassuk, Alexander G; Zheng, Andrew; Li, Yao; Tsang, Stephen H; Mahajan, Vinit B
2016-01-27
Induced pluripotent stem cells (iPSCs) generated from patient fibroblasts could potentially be used as a source of autologous cells for transplantation in retinal disease. Patient-derived iPSCs, however, would still harbor disease-causing mutations. To generate healthy patient-derived cells, mutations might be repaired with new gene-editing technology based on the bacterial system of clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9, thereby yielding grafts that require no patient immunosuppression. We tested whether CRISPR/Cas9 could be used in patient-specific iPSCs to precisely repair an RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from a skin-punch biopsy of an XLRP patient were transduced to produce iPSCs carrying the patient's c.3070G > T mutation. The iPSCs were transduced with CRISPR guide RNAs, Cas9 endonuclease, and a donor homology template. Despite the gene's repetitive and GC-rich sequences, 13% of RPGR gene copies showed mutation correction and conversion to the wild-type allele. This is the first report using CRISPR to correct a pathogenic mutation in iPSCs derived from a patient with photoreceptor degeneration. This important proof-of-concept finding supports the development of personalized iPSC-based transplantation therapies for retinal disease.
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Farhat, M; Poissonnier, A; Hamze, A; Ouk-Martin, C; Brion, J-D; Alami, M; Feuillard, J; Jayat-Vignoles, C
2014-05-01
Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.
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Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rocchi, P.; Ferreri, A.M.; Capucci, A.
1981-01-01
Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (less than 8 x 10(-6)). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTr) mutants up to 1000-fold. The maximum recovery of DTr mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages.
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Induction of diphtheria toxin-resistant mutants in human cells by ultraviolet light
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rocchi, P.; Ferreri, A.M.; Capucci, A.
1981-01-01
Stable spontaneous mutants resistant to the protein synthesis inhibitor diphtheria toxin (DT) have been selected in human cell line EUE at a very low frequency (< 8 x 10/sup -6/). U.v.-induced mutation has been quantitatively measured: treatment of cells with u.v. light increased the frequencies of diphtheria toxin resistant (DTsup(r)) mutants up to 1000-fold. The maximum recovery of DTsup(r) mutants was observed after a short expression period, for all u.v. doses tested, and was followed by a decrease in mutation frequency on subsequent passages.
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Arrieta, Oscar; Anaya, Pablo; Morales-Oyarvide, Vicente; Ramírez-Tirado, Laura Alejandra; Polanco, Ana C
2016-09-01
Assess the cost-effectiveness of an EGFR-mutation testing strategy for advanced NSCLC in first-line therapy with either gefitinib or carboplatin-paclitaxel in Mexican institutions. Cost-effectiveness analysis using a discrete event simulation (DES) model to simulate two therapeutic strategies in patients with advanced NSCLC. Strategy one included patients tested for EGFR-mutation and therapy given accordingly. Strategy two included chemotherapy for all patients without testing. All results are presented in 2014 US dollars. The analysis was made with data from the Mexican frequency of EGFR-mutation. A univariate sensitivity analysis was conducted on EGFR prevalence. Progression-free survival (PFS) transition probabilities were estimated on data from the IPASS and simulated with a Weibull distribution, run with parallel trials to calculate a probabilistic sensitivity analysis. PFS of patients in the testing strategy was 6.76 months (95 % CI 6.10-7.44) vs 5.85 months (95 % CI 5.43-6.29) in the non-testing group. The one-way sensitivity analysis showed that PFS has a direct relationship with EGFR-mutation prevalence, while the ICER and testing cost have an inverse relationship with EGFR-mutation prevalence. The probabilistic sensitivity analysis showed that all iterations had incremental costs and incremental PFS for strategy 1 in comparison with strategy 2. There is a direct relationship between the ICER and the cost of EGFR testing, with an inverse relationship with the prevalence of EGFR-mutation. When prevalence is >10 % ICER remains constant. This study could impact Mexican and Latin American health policies regarding mutation detection testing and treatment for advanced NSCLC.
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Arriola, E; Paredes-Lario, A; García-Gomez, R; Diz-Tain, P; Constenla, M; García-Girón, C; Márquez, G; Reck, M; López-Vivanco, G
2018-04-05
The analysis of epidermal growth factor receptor (EGFR) mutations in many patients with advanced non-small-cell lung cancer (aNSCLC) has provided the opportunity for successful treatment with specific, targeted EGFR tyrosine kinase inhibitors. However, this therapeutic decision may be challenging when insufficient tumor tissue is available for EGFR mutation testing. Therefore, blood surrogate samples for EGFR mutation analysis have been suggested. Data were collected from the Spanish cohort of patients in the large, non-interventional, diagnostic ASSESS study (NCT01785888) evaluating the utility of circulating free tumor-derived DNA from plasma for EGFR mutation testing. The incidence of EGFR mutation in Spain and the level of concordance between matched tissue/cytology and plasma samples were evaluated. In a cohort of 154 eligible patients, EGFR mutations were identified in 15.1 and 11.0% of tumor and plasma samples, respectively. The most commonly used EGFR mutation testing method for the tumor tissue samples was the QIAGEN Therascreen ® EGFR RGQ PCR kit (52.1%). Fragment Length Analysis + PNA LNA Clamp was used for the plasma samples. The concordance rate for EGFR mutation status between the tissue/cytology and plasma samples was 88.8%; the sensitivity was 45.5%, and the specificity was 96.7%. The high concordance between the different DNA sources for EGFR mutation testing supports the use of plasma samples when tumor tissue is unavailable.
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Vigliar, E; Malapelle, U; Bellevicine, C; de Luca, C; Troncone, G
2015-10-01
Guidelines from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) consider cytology suitable for testing epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma. The guidelines recommend that cytopathologists first discuss the possibility of testing squamous cell carcinomas (SqCC) in multidisciplinary meetings. Second, cell blocks should be analysed rather than smear preparations and, third, specimens should be sent to external molecular laboratories within three working days of receiving requests. This study monitored how these recommendations are met in practice. Our laboratory received 596 requests from cytologists from 13 different institutions. For each case, the cytological diagnosis, cytopreparation type, and time between the request and sample mailing were compared with the recommendations. Of the 596 samples, 32 (5.4%) had been reported as SqCC. Three of these (9.4%) showed EGFR mutation. Cytological slides, either ThinPrep(™) (51.2%) or direct smears (43.2%), were more frequently received than cell blocks (5.7%). The mean time between the oncologist's request and specimen dispatching was 5.8 working days. The occurrence of mutations in samples reported as SqCC was higher than expected. This questions the reliability of the original diagnosis, which reinforced the recommendation to evaluate the opportunity for testing non-adenocarcinoma cytology on a case-by-case basis. In spite of CAP/IASLC/AMP recommendations, cell blocks were underutilized for EGFR testing, but cytological slides were suitable for DNA analyses. Significant efforts are needed to avoid delays in outsourcing cytological samples for EGFR testing. © 2014 John Wiley & Sons Ltd.
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2010-01-01
Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486
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1992-06-01
gene mutation procedures normally is done involving microbes or mammalian cells- most frequently it is the Ames assay which we have already completed...success- fully for the compound Emery 3004. Tier 2 assays for gene mutation utilize in vivo and host mediated tests which usually are considered in...compounds for more complex costly test procedures can be accomplished in this tier, but results should not be used to estimate risk to mammalian systems. Gene
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Deletion of p66Shc in mice increases the frequency of size-change mutations in the lacZ transgene.
Beltrami, Elena; Ruggiero, Antonella; Busuttil, Rita; Migliaccio, Enrica; Pelicci, Pier Giuseppe; Vijg, Jan; Giorgio, Marco
2013-04-01
Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2- to 24-month-old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2 O2 . These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
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Zacharias, Triantafyllos; Kulej, Katarzyna; Wang, Kevin; Torggler, Raffaela; la Cour, Jonas M.
2016-01-01
Calmodulin (CaM) is a Ca2+ binding protein modulating multiple targets, several of which are associated with cardiac pathophysiology. Recently, CaM mutations were linked to heart arrhythmia. CaM is crucial for cell growth and viability, yet the effect of the arrhythmogenic CaM mutations on cell viability, as well as heart rhythm, remains unknown, and only a few targets with relevance for heart physiology have been analyzed for their response to mutant CaM. We show that the arrhythmia-associated CaM mutants support growth and viability of DT40 cells in the absence of WT CaM except for the long QT syndrome mutant CaM D129G. Of the six CaM mutants tested (N53I, F89L, D95V, N97S, D129G, and F141L), three showed a decreased activation of Ca2+/CaM-dependent kinase II, most prominently the D129G CaM mutation, which was incapable of stimulating Thr286 autophosphorylation. Furthermore, the CaM D129G mutation led to bradycardia in zebrafish and an arrhythmic phenotype in a subset of the analyzed zebrafish. PMID:27815504
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Pitfalls in lung cancer molecular pathology: how to limit them in routine practice?
Ilie, M; Hofman, P
2012-01-01
New treatment options in advanced non-small cell lung carcinoma (NSCLC) targeting activating epidermal growth factor receptor (EGFR) gene mutations and other genetic alterations demonstrated the clinical significance of the molecular features of specific subsets of tumors. Therefore, the development of personalized medicine has stimulated the routine integration into pathology departments of somatic mutation testing. However, clinical mutation testing must be optimized and standardized with regard to histological profile, type of samples, pre-analytical steps, methodology and result reporting. Routine molecular testing in NSCLC is currently moving beyond EGFR mutational analysis. Recent progress of targeted therapies will require molecular testing for a wide panel of mutations for a personalized molecular diagnosis. As a consequence, efficient testing of multiple molecular abnormalities is an urgent requirement in thoracic oncology. Moreover, increasingly limited tumor sample becomes a major challenge for molecular pathology. Continuous efforts should be made for safe, effective and specific molecular analyses. This must be based on close collaboration between the departments involved in the management of lung cancer. In this review we explored the practical issues and pitfalls surrounding the routine implementation of molecular testing in NSCLC in a pathology laboratory.
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Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.
Reid, Anna L; Freeman, James B; Millward, Michael; Ziman, Melanie; Gray, Elin S
2015-10-01
Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
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Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki
2018-06-01
Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.
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Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda
2013-01-01
BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872
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Yin, Ji-Ye; Huang, Qiong; Yang, Youyun; Zhang, Jian-Ting; Zhong, Mei-Zuo; Zhou, Hong-Hao; Liu, Zhao-Qian
2009-01-01
Multidrug resistance (MDR) is one of the major obstacles for successful cancer chemotherapy. Over-expression of ATP-binding cassette (ABC) transporters such as MRP1/ABCC1 has been suggested to cause MDR. In this study, we explored the distribution frequencies of four common single nucleotide polymorphisms (SNPs) of MRP1/ABCC1 in a mainland Chinese population and investigated whether these SNPs affect the expression and function of the MRP1/ABCC1. We found that the allelic frequencies of Cys43Ser (128G>C), Thr73Ile (218C>T), Arg723Gln (2168G>A) and Arg1058Gln (3173G>A) in mainland Chinese were 0.5%, 1.4%, 5.8% and 0.5%, respectively. These four SNPs were recreated by site-directed mutagenesis and tested for their effect on MRP1/ABCC1 expression and MDR function in HEK293 and CHO-K1 cells lines. We found that none of these mutations had any effect on MRP1/ABCC1 expression and trafficking, but that Arg723Gln mutation significantly reduced MRP1/ABCC1-mediated resistance to daunorubicin, doxorubicin, etoposide, vinblastine and vincristine. The Cys43Ser mutation did not affect all tested drugs resistance. On the other hand, the Thr73Ile mutation reduced resistance to methotrexate and etoposide while the Arg1058Gln mutation increased the response of two anthracycline drugs and etoposide in HEK293 and CHO-K1 cells as well as vinblastine and methotrexate in CHO-K1 cells. We conclude that the allelic frequency of the Arg723Gln mutation is relatively higher than other SNPs in mainland Chinese population and therefore this mutation significantly reduces MRP1/ABCC1 activity in MDR. PMID:19214144
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Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A
2001-01-01
Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype. PMID:11606538
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Schnorr, J D; Holdcraft, R; Chevalier, B; Berg, C A
2001-10-01
Little is known about the genes that interact with Ras signaling pathways to regulate morphogenesis. The synthesis of dorsal eggshell structures in Drosophila melanogaster requires multiple rounds of Ras signaling followed by dramatic epithelial sheet movements. We took advantage of this process to identify genes that link patterning and morphogenesis; we screened lethal mutations on the second chromosome for those that could enhance a weak Ras1 eggshell phenotype. Of 1618 lethal P-element mutations tested, 13 showed significant enhancement, resulting in forked and fused dorsal appendages. Our genetic and molecular analyses together with information from the Berkeley Drosophila Genome Project reveal that 11 of these lines carry mutations in previously characterized genes. Three mutations disrupt the known Ras1 cell signaling components Star, Egfr, and Blistered, while one mutation disrupts Sec61beta, implicated in ligand secretion. Seven lines represent cell signaling and cytoskeletal components that are new to the Ras1 pathway; these are Chickadee (Profilin), Tec29, Dreadlocks, POSH, Peanut, Smt3, and MESK2, a suppressor of dominant-negative Ksr. A twelfth insertion disrupts two genes, Nrk, a "neurospecific" receptor tyrosine kinase, and Tpp, which encodes a neuropeptidase. These results suggest that Ras1 signaling during oogenesis involves novel components that may be intimately associated with additional signaling processes and with the reorganization of the cytoskeleton. To determine whether these Ras1 Enhancers function upstream or downstream of the Egf receptor, four mutations were tested for their ability to suppress an activated Egfr construct (lambdatop) expressed in oogenesis exclusively in the follicle cells. Mutations in Star and l(2)43Bb had no significant effect upon the lambdatop eggshell defect whereas smt3 and dock alleles significantly suppressed the lambdatop phenotype.
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Polyploidy can drive rapid adaptation in yeast
NASA Astrophysics Data System (ADS)
Selmecki, Anna M.; Maruvka, Yosef E.; Richmond, Phillip A.; Guillet, Marie; Shoresh, Noam; Sorenson, Amber L.; de, Subhajyoti; Kishony, Roy; Michor, Franziska; Dowell, Robin; Pellman, David
2015-03-01
Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.
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The clinical features of squamous cell lung carcinoma with sensitive EGFR mutations.
Taniguchi, Yuri; Matsumoto, Yoko; Furukawa, Ryutaro; Ohara, Sayaka; Usui, Kazuhiro
2018-06-01
The process of selecting patients on the basis of epidermal growth factor receptor (EGFR) mutations would likely result in a patient population with greater sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, EGFR mutation status is not routinely examined in patients with squamous cell lung cancer (Sq) because of the low incidence of EGFR mutations and the poor clinical response to EGFR-TKIs. We retrospectively reviewed the clinical features of patients at our hospital with Sq who carried EGFR-TKI-sensitive EGFR mutations and assessed their responses to EGFR-TKIs. EGFR mutation status was tested in 23 of 441 patients with Sq (5.2%) admitted to our hospital during the study period. An EGFR mutation (exon 19 deletion 3, L858R 2) was identified in five of the 23 patients (21.7%), all of whom were female never-smokers. Of these five patients, four (4/9; 44.4%) were in the normal lung group, one (1/12; 8.3%) was in the emphysematous lung group, and none (0/2; 0%) in the fibrotic lung group. Two of these five patients with the EGFR mutation received gefitinib and two received afatinib. Although the two patients who were treated with gefitinib did not respond well to treatment (stable disease, 1 patient; progressive disease, 1 patient), the two patients who were treated with afatinib showed a good response (partial response, 2 patients). The administration of afatinib to Sq patients after selecting patients using the EGFR mutation test based on their underlying pulmonary disease and smoking status would likely result in a population with a greater sensitivity to afatinib.
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Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers.
Wyatt, Eugene J; Demonbreun, Alexis R; Kim, Ellis Y; Puckelwartz, Megan J; Vo, Andy H; Dellefave-Castillo, Lisa M; Gao, Quan Q; Vainzof, Mariz; Pavanello, Rita C M; Zatz, Mayana; McNally, Elizabeth M
2018-05-03
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
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Translational read-through of a nonsense mutation causing Bartter syndrome.
Cho, Hee Yeon; Lee, Beom Hee; Cheong, Hae Il
2013-06-01
Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.
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Kurt, Habibe; Bueso-Ramos, Carlos E; Khoury, Joseph D; Routbort, Mark J; Kanagal-Shamanna, Rashmi; Patel, Umang V; Jorgensen, Jeffrey L; Wang, Sa A; Ravandi, Farhad; DiNardo, Courtney; Luthra, Rajyalakshmi; Medeiros, L Jeffrey; Patel, Keyur P
2018-05-01
Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations occur in a variety of myeloid neoplasms. Immunohistochemistry (IHC)-based direct visualization of mutant clones of hematopoietic cells can be useful for rapid diagnostic screening and for monitoring treatment response. In this study, we first evaluated the sensitivity and specificity of the IDH1 p.R132H mutation-specific antibody by IHC. All IDH1 wild type cases (n=11) and IDH1 mutant cases with a non-p.R132H mutation (n=30) were negative by IHC, demonstrating 100% antibody specificity. All the initial diagnostic specimens with IDH1 p.R132H mutation including acute myeloid leukemia (n=30), myelodysplastic syndromes (MDS) (n=10), MDS/myeloproliferative neoplasms (MPN) (n=4), and MPN (n=5) were positive by IHC, demonstrating 100% antibody sensitivity. Both immature and mature myeloid cells showed immunoreactivity. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative by IHC. We then evaluated the follow-up specimens with a known IDH1 mutation status including acute myeloid leukemia (n=23), MDS (n=2), MDS/MPN (n=2), and MPN (n=2). Thirty-three IDH1 p.R132H mutant cases were positive by IHC and 12 IDH1 mutation negative cases were negative by IHC. However, IHC reactivity in up to 25% of bone marrow cells was noted in 8 of 20 polymerase chain reaction-negative cases, all from patients with a known history of IDH1 p.R132H mutation indicating sampling error or a sensitivity issue with molecular tests. These data indicate that IHC is a highly specific and sensitive tool to detect IDH1 p.R132H mutation in bone marrow involved by myeloid neoplasms. In addition, IDH1 p.R132H IHC also allows localization and assessment of the maturation stage of the clones carrying the mutation.
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Hendry, Jolyon H.
2017-01-01
There is compelling biological data to suggest that cancer arises from a series of mutations in single target cells, resulting in defects in cell renewal and differentiation processes which lead to malignancy. Because much mutagenic damage is expressed following cell division, more-rapidly renewing tissues could be at higher risk because of the larger number of cell replications. Cairns suggested that renewing tissues may reduce cancer risk by partitioning the dividing cell populations into lineages comprising infrequently-dividing long-lived stem cells and frequently-dividing short-lived daughter transit cells. We develop generalizations of three recent cancer-induction models that account for the joint maintenance and renewal of stem and transit cells, also competing processes of partially transformed cell proliferation and differentiation/apoptosis. We are particularly interested in using these models to separately assess the probabilities of mutation and development of cancer associated with “spontaneous” processes and with those linked to a specific environmental mutagen, specifically ionizing radiation or cigarette smoking. All three models demonstrate substantial variation in cancer risks, by at least 20 orders of magnitude, depending on the assumed number of critical mutations required for cancer, and the stem-cell and transition-cell mutation rates. However, in most cases the conditional probabilities of cancer being mutagen-induced range between 7–96%. The relative risks associated with mutagen exposure compared to background rates are also stable, ranging from 1.0–16.0. Very few cancers, generally <0.5%, arise from mutations occurring solely in stem cells rather than in a combination of stem and transit cells. However, for cancers with 2 or 3 critical mutations, a substantial proportion of cancers, in some cases 100%, have at least one mutation derived from a mutated stem cell. Little difference is made to relative risks if competing processes of proliferation and differentiation in the partially transformed stem and transit cell population are allowed for, nor is any difference made if one assumes that transit cells require an extra mutation to confer malignancy from the number required by stem cells. The probability of a cancer being mutagen-induced correlates across cancer sites with the estimated cumulative number of stem cell divisions in the associated tissue (p<0.05), although in some cases there is sensitivity of findings to removal of high-leverage outliers and in some cases only modest variation in probability, but these issues do not affect the validity of the findings. There are no significant correlations (p>0.3) between lifetime cancer-site specific radiation risk and the probability of that cancer being mutagen-induced. These results do not depend on the assumed critical number of mutations leading to cancer, or on the assumed mutagen-associated mutation rate, within the generally-accepted ranges tested. However, there are borderline significant negative correlations (p = 0.08) between the smoking-associated mortality rate difference (current vs former smokers) and the probability of cancer being mutagen-induced. This is only the case where values of the critical number of mutations leading to cancer, k, is 3 or 4 and not for smaller values (1 or 2), but does not strongly depend on the assumed mutagen-associated mutation rate. PMID:28196079
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Little, Mark P; Hendry, Jolyon H
2017-02-01
There is compelling biological data to suggest that cancer arises from a series of mutations in single target cells, resulting in defects in cell renewal and differentiation processes which lead to malignancy. Because much mutagenic damage is expressed following cell division, more-rapidly renewing tissues could be at higher risk because of the larger number of cell replications. Cairns suggested that renewing tissues may reduce cancer risk by partitioning the dividing cell populations into lineages comprising infrequently-dividing long-lived stem cells and frequently-dividing short-lived daughter transit cells. We develop generalizations of three recent cancer-induction models that account for the joint maintenance and renewal of stem and transit cells, also competing processes of partially transformed cell proliferation and differentiation/apoptosis. We are particularly interested in using these models to separately assess the probabilities of mutation and development of cancer associated with "spontaneous" processes and with those linked to a specific environmental mutagen, specifically ionizing radiation or cigarette smoking. All three models demonstrate substantial variation in cancer risks, by at least 20 orders of magnitude, depending on the assumed number of critical mutations required for cancer, and the stem-cell and transition-cell mutation rates. However, in most cases the conditional probabilities of cancer being mutagen-induced range between 7-96%. The relative risks associated with mutagen exposure compared to background rates are also stable, ranging from 1.0-16.0. Very few cancers, generally <0.5%, arise from mutations occurring solely in stem cells rather than in a combination of stem and transit cells. However, for cancers with 2 or 3 critical mutations, a substantial proportion of cancers, in some cases 100%, have at least one mutation derived from a mutated stem cell. Little difference is made to relative risks if competing processes of proliferation and differentiation in the partially transformed stem and transit cell population are allowed for, nor is any difference made if one assumes that transit cells require an extra mutation to confer malignancy from the number required by stem cells. The probability of a cancer being mutagen-induced correlates across cancer sites with the estimated cumulative number of stem cell divisions in the associated tissue (p<0.05), although in some cases there is sensitivity of findings to removal of high-leverage outliers and in some cases only modest variation in probability, but these issues do not affect the validity of the findings. There are no significant correlations (p>0.3) between lifetime cancer-site specific radiation risk and the probability of that cancer being mutagen-induced. These results do not depend on the assumed critical number of mutations leading to cancer, or on the assumed mutagen-associated mutation rate, within the generally-accepted ranges tested. However, there are borderline significant negative correlations (p = 0.08) between the smoking-associated mortality rate difference (current vs former smokers) and the probability of cancer being mutagen-induced. This is only the case where values of the critical number of mutations leading to cancer, k, is 3 or 4 and not for smaller values (1 or 2), but does not strongly depend on the assumed mutagen-associated mutation rate.
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Osman, Abdullah A.; Monroe, Marcus M.; Ortega Alves, Marcus V.; Patel, Ameeta A.; Katsonis, Panagiotis; Fitzgerald, Alison L.; Neskey, David M.; Frederick, Mitchell J.; Woo, Sang Hyeok; Caulin, Carlos; Hsu, Teng-Kuei; McDonald, Thomas O.; Kimmel, Marek; Meyn, Raymond E.; Lichtarge, Olivier; Myers, Jeffrey N.
2015-01-01
Although cisplatin has played a role in “standard-of-care” multimodality therapy for patients with advanced squamous cell carcinoma of the head and neck (HNSCC), the rate of treatment failure remains particularly high for patients receiving cisplatin whose tumors have mutations in the TP53 gene. We found that cisplatin treatment of HNSCC cells with mutant TP53 leads to arrest of cells in the G2 phase of the cell cycle, leading us to hypothesize that the wee-1 kinase inhibitor MK-1775 would abrogate the cisplatin-induced G2 block and thereby sensitize isogenic HNSCC cells with mutant TP53 or lacking p53 expression to cisplatin. We tested this hypothesis using clonogenic survival assays, flow cytometry, and in vivo tumor growth delay experiments with an orthotopic nude mouse model of oral tongue cancer. We also used a novel TP53 mutation classification scheme to identify which TP53 mutations are associated with limited tumor responses to cisplatin treatment. Clonogenic survival analyses indicate that nanomolar concentration of MK-1775 sensitizes HNSCC cells with high-risk mutant p53 to cisplatin. Consistent with its ability to chemosensitize, MK-1775 abrogated the cisplatin-induced G2 block in p53-defective cells leading to mitotic arrest associated with a senescence-like phenotype. Furthermore, MK-1775 enhanced the efficacy of cisplatin in vivo in tumors harboring TP53 mutations. These results indicate that HNSCC cells expressing high-risk p53 mutations are significantly sensitized to cisplatin therapy by the selective wee-1 kinase inhibitor, supporting the clinical evaluation of MK-1775 in combination with cisplatin for the treatment of patients with TP53 mutant HNSCC. PMID:25504633
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Kang, Hyo Jae; Hwangbo, Bin; Lee, Jin Soo; Kim, Moon Soo; Lee, Jong Mog; Lee, Geon-Kook
2016-01-01
Introduction Although the use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasing for epidermal growth factor receptor (EGFR) testing in lung cancer, the discordance rate in EGFR mutations between lymph node (LN) samples obtained by EBUS-TBNA and primary tumor (PT) is not well known. Thus, we compared the EGFR mutation status of LN samples obtained by EBUS-TBNA and PTs to estimate the efficacy of using EBUS-TBNA specimens for EGFR testing in advanced, non-squamous, non-small cell lung cancer (NSCLC). Materials and Methods Using data of patients from the EBUS-TBNA database (N = 1914) obtained between January 2009 and January 2013, we identified 100 treatment-naïve, advanced, non-squamous NSCLC patients (stage 3 and 4) with matched LN specimens obtained by EBUS-TBNA and PT specimens. Of these, 74 patients with paired specimens were feasible for EGFR mutation analysis, which we performed using a direct sequencing method. Results Of the 74 cases, at least one major [exon 19 deleted (19del) and L858R] or minor (T790M, exon 20 insertion, and other point mutations) EGFR mutation was detected in 31 cases (41.9%), which included PT (n = 31, 41.9%) and LN (n = 28, 37.8%) specimens. Major mutations were detected in 25 PT (33.8%, 19del = 13, L858R = 12) and 22 LN (29.8%, 19del = 11, L858R = 11) specimens. The discordance rate in major mutations between matched PT and LN specimens was 4.1% (3/74). Among minor mutations, T790M was detected in LN specimen only in 2 cases with L858R in PT and LN. The discordance rate major and minor EGFR mutations combined between matched PT and LN specimens was 12% (9/74). Conclusions We observed a high concordance rate of major EGFR mutations between matched LN specimens sampled by EBUS-TBNA and PTs, suggesting that LN samples obtained by EBUS-TBNA from advanced non-squamous NSCLC patients are effective for use in EGFR mutation testing. PMID:27685950
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Albitar, Adam; Ma, Wanlong; DeDios, Ivan; Estella, Jeffrey; Ahn, Inhye; Farooqui, Mohammed; Wiestner, Adrian; Albitar, Maher
2017-03-14
Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.
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Cogliatti, Sergio B; Bertoni, Francesco; Zimmermann, Dieter R; Henz, Samuel; Diss, Tim C; Ghielmini, Michele; Schmid, Ulrico
2005-07-01
Mantle cell lymphoma (MCL) is associated with a very unfavourable clinical course. This is particularly true for mantle cell lymphoma of the blastoid subtype (MCL-b). In order to define prognostic factors, we analysed the impact of immunoglobulin heavy chain variable (IgV H) gene somatic hypermutations on clinical outcome in a series of 21 cases of morphologically, phenotypically, and genotypically well-characterized MCL-b. Testing and estimation were performed using log-rank statistics and displayed on Kaplan-Meier graphs. Thirteen of 21 cases of MCL-b revealed a homology rate of > or = 99% compared to IgV H germ-line sequences in the databases and were scored as non-mutated. Eight of 21 cases (38%) of MCL-b were mutated. In MCL-b the mutation frequency was usually low and the mutation pattern was only rarely antigen-selected, in contrast to a control group of 11 cases with morphologically almost identical, but phenotypically and genotypically clearly distinguishable, diffuse large B cell lymphoma, derived, most likely, from germinal centre B cells. In our series of 21 MCL-b, positive IgV H mutational status, irrespective of varying homology thresholds, had no statistically significant prognostic impact on event-free or overall survival. However, mutated MCL-b tended to present more frequently at an earlier stage and without bone marrow involvement and to show lower rates of relapse and death, resulting in a more favourable clinical outcome. Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Van Der Luut, R.; Khan, P.M.; Van Leeuwen, C.
Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogensis. For the identification of protein truncating mutations of the APC gene, the authors developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectivenessmore » of this method. 9 refs., 2 figs.« less
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Tothova, Zuzana; Krill-Burger, John M; Popova, Katerina D; Landers, Catherine C; Sievers, Quinlan L; Yudovich, David; Belizaire, Roger; Aster, Jon C; Morgan, Elizabeth A; Tsherniak, Aviad; Ebert, Benjamin L
2017-10-05
Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34 + human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.
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Graf, U; Moraga, A A; Castro, R; Díaz Carrillo, E
1994-05-01
Five wines and one brandy of Spanish origin as well as three herbal teas and ordinary black tea were tested for genotoxicity in the wing Somatic Mutation And Recombination Test (SMART) which makes use of the two recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) on the left arm of chromosome 3 of Drosophila melanogaster. 3-day-old larvae trans-heterozygous for these two markers were fed the beverages at different concentrations and for different feeding periods using Drosophila instant medium. Somatic mutations or mitotic recombinations induced in the cells of the wing imaginal discs give rise to mutant single or twin spots on the wing blade of the emerging adult flies showing either the mwh phenotype or/and the flr phenotype. One of the red wines showed a clear genotoxic activity that was not due to its ethanol content. Two herbal teas (Urtica dioica, Achillea millefolium) and black tea (Camellia sinensis) proved to be weakly genotoxic as well. Furthermore, it was shown that quercetin and rutin, two flavonols present in beverages of plant origin, also exhibited weak genotoxic activity in the somatic cells of Drosophila. These results demonstrate that Drosophila in vivo somatic assays can detect the genotoxicity of complex mixtures such as beverages. In particular, it is possible to administer these test materials in the same form as that in which they are normally consumed.
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Schenk, K; Zölzer, F; Kiefer, J
1989-01-01
Mutation induction was investigated in wild-type haploid yeast Saccharomyces cerevisiae after split-dose UV-irradiation. Cells were exposed to fractionated 254 nm-UV-doses separated by intervals from 0 to 6 h with incubation either on non-nutrient or nutrient agar between. The test parameter was resistance to canavanine. If modifications of sensitivity due to incubation are appropriately taken into account there is no change of mutation frequency.
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Understanding genetics in neuroimaging.
Vasquez, Marina Lipkin; Renault, Ilana Zalcberg
2015-02-01
Gene expression is a process of DNA sequence reading into protein synthesis. In cases of problems in DNA repair/apoptosis mechanisms, cells accumulate genomic abnormalities and pass them through generations of cells. The accumulation of mutations causes diseases and even tumors. In addition to cancer, many other neurologic conditions have been associated with genetic mutations. Some trials are testing patients with epigenetic treatments. Epigenetic therapy must be used with caution because epigenetic processes and changes happen constantly in normal cells, giving rise to drug off-target effects. Scientists are making progress in specifically targeting abnormal cells with minimal damage to normal ones. Copyright © 2015. Published by Elsevier Inc.
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Clock-like mutational processes in human somatic cells
Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; ...
2015-11-09
During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutationmore » rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.« less
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Kim, Do-Hee; Kwak, Yeonui; Kim, Nam Doo; Sim, Taebo
2016-01-01
ABSTRACT Aberrant mutational activation of FGFR2 is associated with endometrial cancers (ECs). AP24534 (ponatinib) currently undergoing clinical trials has been known to be an orally available multi-targeted tyrosine kinase inhibitor. Our biochemical kinase assay showed that AP24534 is potent against wild-type FGFR1-4 and 5 mutant FGFRs (V561M-FGFR1, N549H-FGFR2, K650E-FGFR3, G697C-FGFR3, N535K-FGFR4) and possesses the strongest kinase-inhibitory activity on N549H-FGFR2 (IC50 of 0.5 nM) among all FGFRs tested. We therefore investigated the effects of AP24534 on endometrial cancer cells harboring activating FGFR2 mutations and explored the underlying molecular mechanisms. AP24534 significantly inhibited the proliferation of endometrial cancer cells bearing activating FGFR2 mutations (N549K, K310R/N549K, S252W) and mainly induced G1/S cell cycle arrest leading to apoptosis. AP24534 also diminished the kinase activity of immunoprecipitated FGFR2 derived from MFE-296 and MFE-280 cells and reduced the phosphorylation of FGFR2 and FRS2 on MFE-296 and AN3CA cells. AP24534 caused substantial reductions in ERK phosphorylation, PLCγ signaling and STAT5 signal transduction on ECs bearing FGFR2 activating mutations. Akt signaling pathway was also deactivated by AP24534. AP24534 causes the chemotherapeutic effect through mainly the blockade of ERK, PLCγ and STAT5 signal transduction on ECs. Moreover, AP24534 inhibited migration and invasion of endometrial cancer cells with FGFR2 mutations. In addition, AP24534 significantly blocked anchorage-independent growth of endometrial cancer cells. We, for the first time, report the molecular mechanisms by which AP24534 exerts antitumor effects on ECs with FGFR2 activating mutations, which would provide mechanistic insight into ongoing clinical investigations of AP24534 for ECs. PMID:26574622
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Shackelford, David B.; Abt, Evan; Gerken, Laurie; Vasquez, Debbie S.; Seki, Atsuko; Leblanc, Mathias; Wei, Liu; Fishbein, Michael C.; Czernin, Johannes; Mischel, Paul S.; Shaw, Reuben J.
2013-01-01
SUMMARY The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ~20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors. PMID:23352126
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Shackelford, David B; Abt, Evan; Gerken, Laurie; Vasquez, Debbie S; Seki, Atsuko; Leblanc, Mathias; Wei, Liu; Fishbein, Michael C; Czernin, Johannes; Mischel, Paul S; Shaw, Reuben J
2013-02-11
The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ∼20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors. Copyright © 2013 Elsevier Inc. All rights reserved.
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Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer.
Mu, Zhaomei; Benali-Furet, Naoual; Uzan, Georges; Znaty, Anaëlle; Ye, Zhong; Paolillo, Carmela; Wang, Chun; Austin, Laura; Rossi, Giovanna; Fortina, Paolo; Yang, Hushan; Cristofanilli, Massimo
2016-09-30
The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
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Kim, Sung-Su; Choi, Hyun-Jeung; Kim, Jin Ju; Kim, M Sun; Lee, In-Seon; Byun, Bohyun; Jia, Lina; Oh, Myung Ryurl; Moon, Youngho; Park, Sarah; Choi, Joon-Seok; Chae, Seoung Wan; Nam, Byung-Ho; Kim, Jin-Soo; Kim, Jihun; Min, Byung Soh; Lee, Jae Seok; Won, Jae-Kyung; Cho, Soo Youn; Choi, Yoon-La; Shin, Young Kee
2018-01-11
In clinical translational research and molecular in vitro diagnostics, a major challenge in the detection of genetic mutations is overcoming artefactual results caused by the low-quality of formalin-fixed paraffin-embedded tissue (FFPET)-derived DNA (FFPET-DNA). Here, we propose the use of an 'internal quality control (iQC) index' as a criterion for judging the minimum quality of DNA for PCR-based analyses. In a pre-clinical study comparing the results from droplet digital PCR-based EGFR mutation test (ddEGFR test) and qPCR-based EGFR mutation test (cobas EGFR test), iQC index ≥ 0.5 (iQC copies ≥ 500, using 3.3 ng of FFPET-DNA [1,000 genome equivalents]) was established, indicating that more than half of the input DNA was amplifiable. Using this criterion, we conducted a retrospective comparative clinical study of the ddEGFR and cobas EGFR tests for the detection of EGFR mutations in non-small cell lung cancer (NSCLC) FFPET-DNA samples. Compared with the cobas EGFR test, the ddEGFR test exhibited superior analytical performance and equivalent or higher clinical performance. Furthermore, iQC index is a reliable indicator of the quality of FFPET-DNA and could be used to prevent incorrect diagnoses arising from low-quality samples.
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Buttitta, Fiamma; Felicioni, Lara; Del Grammastro, Maela; Filice, Giampaolo; Di Lorito, Alessia; Malatesta, Sara; Viola, Patrizia; Centi, Irene; D'Antuono, Tommaso; Zappacosta, Roberta; Rosini, Sandra; Cuccurullo, Franco; Marchetti, Antonio
2013-02-01
The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells. We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS. In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS. The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.
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Matamoros-Angles, Andreu; Gayosso, Lucía Mayela; Richaud-Patin, Yvonne; di Domenico, Angelique; Vergara, Cristina; Hervera, Arnau; Sousa, Amaya; Fernández-Borges, Natalia; Consiglio, Antonella; Gavín, Rosalina; López de Maturana, Rakel; Ferrer, Isidro; López de Munain, Adolfo; Raya, Ángel; Castilla, Joaquín; Sánchez-Pernaute, Rosario; Del Río, José Antonio
2018-04-01
Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient.
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Clock-like mutational processes in human somatic cells
Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.
2016-01-01
During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell’s genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells. PMID:26551669
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Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.
Ahn, Eun Hyun; Hirohata, Kensen; Kohrn, Brendan F; Fox, Edward J; Chang, Chia-Cheng; Loeb, Lawrence A
2015-01-01
Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.
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Chen, Jieping; Yao, Kai; Li, Zaishang; Deng, Chuangzhong; Wang, Liangjiao; Yu, Xingsu; Liang, Peili; Xie, Qiankun; Chen, Peng; Qin, Zike; Ye, Yunlin; Liu, Zhuowei; Zhou, Fangjian; Zhang, Zhenfeng; Han, Hui
2016-08-09
To establish penile cancer (PeCa) cell lines for the study of molecular mechanisms of carcinogenesis and testing therapeutic reagents. We successfully established two PeCa cell lines from fresh tumor tissues from 21 cases. One cell line named Penl1 was isolated from a lymph node metastasis (LNM) of penile squamous cell carcinoma (PeSCC), usual type and comprehensively characterized here. Our in-depth characterization analysis of the Penl1 cell line included morphology, tumorigenicity, genetic characteristics, protein expression, biology, and chemosensitivity. Penl1 was authenticated by single tandem repeat (STR) DNA typing. Comparative histomorphology, genetic characteristics, and protein expression patterns revealed essential similarities between the cell line and its corresponding LNM. In-depth characterization analysis of Penl1 cell line revealed tumorigenicity in immunodeficient mice, negative human papilloma virus (HPV) and mycoplasma infection, TP53 mutations and sensitivity to cisplatin and epirubicin. STR DNA typing did not match any cell lines within three international cell banks. The limitation of this study is that one patient cannot represent the complete heterogeneity of PeCa, especially primary tumor. We established and characterized an HPV-negative and moderately differentiated PeCa cell model with a TP53 missense mutation from a PeSCC, usual type patient. A preliminarily study of carcinogenesis and chemosensitivity suggests that this cell model carries a tumor suppressor gene mutation and is sensitive to chemotherapy drugs.
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Li, Zaishang; Deng, Chuangzhong; Wang, Liangjiao; Yu, Xingsu; Liang, Peili; Xie, Qiankun; Chen, Peng; Qin, Zike; Ye, Yunlin; Liu, Zhuowei; Zhou, Fangjian; Zhang, Zhenfeng; Han, Hui
2016-01-01
Purpose To establish penile cancer (PeCa) cell lines for the study of molecular mechanisms of carcinogenesis and testing therapeutic reagents. Materials and Methods We successfully established two PeCa cell lines from fresh tumor tissues from 21 cases. One cell line named Penl1 was isolated from a lymph node metastasis (LNM) of penile squamous cell carcinoma (PeSCC), usual type and comprehensively characterized here. Our in-depth characterization analysis of the Penl1 cell line included morphology, tumorigenicity, genetic characteristics, protein expression, biology, and chemosensitivity. Penl1 was authenticated by single tandem repeat (STR) DNA typing. Results Comparative histomorphology, genetic characteristics, and protein expression patterns revealed essential similarities between the cell line and its corresponding LNM. In-depth characterization analysis of Penl1 cell line revealed tumorigenicity in immunodeficient mice, negative human papilloma virus (HPV) and mycoplasma infection, TP53 mutations and sensitivity to cisplatin and epirubicin. STR DNA typing did not match any cell lines within three international cell banks. The limitation of this study is that one patient cannot represent the complete heterogeneity of PeCa, especially primary tumor. Conclusions We established and characterized an HPV-negative and moderately differentiated PeCa cell model with a TP53 missense mutation from a PeSCC, usual type patient. A preliminarily study of carcinogenesis and chemosensitivity suggests that this cell model carries a tumor suppressor gene mutation and is sensitive to chemotherapy drugs. PMID:27351128
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Normanno, Nicola; Denis, Marc G.; Thress, Kenneth S.; Ratcliffe, Marianne; Reck, Martin
2017-01-01
Cancer treatment is evolving towards therapies targeted at specific molecular abnormalities that drive tumor growth. Consequently, to determine which patients are eligible, accurate assessment of molecular aberrations within tumors is required. Obtaining sufficient tumor tissue for molecular testing can present challenges; therefore, circulating free tumor-derived DNA (ctDNA) found in blood plasma has been proposed as an alternative source of tumor DNA. The diagnostic utility of ctDNA for the detection of epidermal growth factor receptor (EGFR) mutations harbored in tumors of patients with advanced non-small-cell lung cancer (NSCLC) is supported by the results of several large studies/meta-analyses. However, recent real-world studies suggest that the performance of ctDNA testing varies between geographic regions/laboratories, demonstrating the need for standardized guidance. In this review, we outline recommendations for obtaining an accurate result using ctDNA, relating to pre-analytical plasma processing, ctDNA extraction, and appropriate EGFR mutation detection methods, based on clinical trial results. We conclude that there are several advantages associated with ctDNA, including the potential for repeated sampling particularly following progression after first-line tyrosine kinase inhibitor (TKI) therapy, as TKIs targeting resistance mutations (eg T790M) are now approved for use in the USA/EU/Japan (at time of writing). However, evidence suggests that ctDNA does not allow detection of EGFR mutations in all patients with known mutation-positive NSCLC. Therefore, although tumor tissue should be the first sample choice for EGFR testing at diagnosis, ctDNA is a promising alternative diagnostic approach. PMID:27980215
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Sambuughin, N; Nelson, T E; Jankovic, J; Xin, C; Meissner, G; Mullakandov, M; Ji, J; Rosenberg, H; Sivakumar, K; Goldfarb, L G
2001-09-01
Malignant hyperthermia is a pharmacogenetic disorder associated with mutations in Ca(2+) regulatory proteins. It manifests as a hypermetabolic crisis triggered by commonly used anesthetics. Malignant hyperthermia susceptibility is a dominantly inherited predisposition to malignant hyperthermia that can be diagnosed by using caffeine/halothane contracture tests. In a multigenerational North American family with a severe form of malignant hyperthermia that has caused four deaths, a novel RYR1 A2350T missense mutation was identified in all individuals testing positive for malignant hyperthermia susceptibility. The same A2350T mutation was identified in an Argentinean family with two known fatal MH reactions. Functional analysis in HEK-293 cells revealed an altered Ca(2+) dependence and increased caffeine sensitivity of the expressed mutant protein thus confirming the pathogenic potential of the RYR1 A2350T mutation.
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Malzone, Maria Gabriella; Campanile, Anna Cipolletta; Losito, Nunzia Simona; Longo, Francesco; Perri, Francesco; Caponigro, Francesco; Schiavone, Concetta; Ionna, Franco; Maiello, Francesco; Martinuzzi, Claudia; Nasti, Sabina; Botti, Gerardo; Fulciniti, Franco
2015-08-01
Multiple dermal cylindromas and membranous basal cell adenoma of parotid gland in a 67-year-old woman with Brooke-Spiegler syndrome (BSS) were examined by fine-needle cytology. Histology, immunochemistry, and CYLD germline mutation testing were also performed. Cytomorphology and immunochemistry of the two lesions showed basaloid neoplasms, remarkably similar, composed by proliferating epithelial cells of basal type accompanied by a smaller proportion of myoepithelial cells. CYLD gene showed a novel germline splice acceptor site mutation (c.2042-1G>C) with skipping of the entire exon 15. The occurrence of analogous tumors, dermal cylindromas, and membranous basal cell adenoma of the parotid gland, in the same patient may result from the action of a single gene on ontogenetically similar stem cells. Therefore, patients with BSS should be offered a genetic counselling for an early and correct diagnosis. © 2015 Wiley Periodicals, Inc.
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INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia.
Jin, Hongjun; Yang, Liyuan; Wang, Lu; Yang, Zailin; Zhan, Qian; Tao, Yao; Zou, Qin; Tang, Yuting; Xian, Jingrong; Zhang, Shuaishuai; Jing, Yipei; Zhang, Ling
2018-01-17
Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P 2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan-Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells. High levels of INPP4B were at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling. Finally, high expression of INPP4B showed a trend towards lower overall survival and event-free survival in NPM1-mutated AML patients. Our results indicate that INPP4B promotes leukemia cell survival via SGK3 activation, and INPP4B might be a potential target in the treatment of NPM1-mutated AML.
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Albitar, Maher; Sudarsanam, Sucha; Ma, Wanlong; Jiang, Shiping; Chen, Wayne; Funari, Vincent; Blocker, Forrest; Agersborg, Sally
2018-01-01
Background The role of MET amplification in lung cancer, particularly in relation to checkpoint inhibition and EGFR WT, has not been fully explored. In this study, we correlated PD-L1 expression with MET amplification and EGFR, KRAS, or TP53 mutation in primary lung cancer. Methods In this retrospective study, tissue collected from 471 various tumors, including 397 lung cancers, was tested for MET amplification by FISH with a MET/centromere probe. PD-L1 expression was evaluated using clone SP142 and standard immunohistochemistry, and TP53, KRAS, and EGFR mutations were tested using next generation sequencing. Results Our results revealed that PD-L1 expression in non-small cell lung cancer is inversely correlated with EGFR mutation (P=0.0003), and positively correlated with TP53 mutation (P=0.0001) and MET amplification (P=0.004). Patients with TP53 mutations had significantly higher MET amplification (P=0.007), and were more likely (P=0.0002) to be EGFR wild type. There was no correlation between KRAS mutation and overall PD-L1 expression, but significant positive correlation between PD-L1 expression and KRAS with TP53 co-mutation (P=0.0002). A cut-off for the ratio of MET: centromere signal was determined as 1.5%, and 4% of lung cancer patients were identified as MET amplified. Conclusions This data suggests that in lung cancer both MET and TP53 play direct roles in regulating PD-L1 opposing EGFR. Moreover, KRAS and TP53 co-mutation may cooperate to drive PD-L1 expression in lung cancer. Adding MET or TP53 inhibitors to checkpoint inhibitors may be an attractive combination therapy in patients with lung cancer and MET amplification. PMID:29568386
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Goriely, Anne; McGrath, John J.; Hultman, Christina M.; Wilkie, Andrew O.M.; Malaspina, Dolores
2014-01-01
Objectives There is robust evidence from epidemiological studies that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism. Here we present a novel mechanism that may contribute to this association. Methods Narrative review. Results Because the male germ cell undergoes many more cell divisions across the reproductive age range, copy-errors taking place in the paternal germline are associated with de novo mutations in the offspring of older men. Recently it has been recognized that somatic mutations in male germ cells that modify proliferation via dysregulation of the RAS pathway can lead to within-testis expansion of mutant clonal lines. First identified in association with rare paternal age-effect disorders (e.g. Apert syndrome, achondroplasia), this process is known as ‘selfish spermatogonial selection’. This mechanism will (a) favor propagation of germ cells carrying pathogenic mutations, (b) increasingly skew the mutational profile of sperm as men age, and (c) result in an enrichment of de novo mutations in the offspring of older fathers that preferentially impact on specific cellular signaling pathways. This mechanism offers a parsimonious explanation not only for the association between advanced paternal age and various neurodevelopmental disorders, but also provides insights into the genetic architecture (role of de novo mutations), neurobiological correlates (altered cell cycle) and some epidemiological features of these disorders. We outline hypotheses to test this model. Conclusions In light of our current understanding of the genetic networks involved in neurocognitive disorders and the principles of selfish spermatogonial selection, we speculate that some pathogenic mutations associated with these disorders are the consequence of a selfish mechanism originating in the aging testis. Given the secular changes for delayed parenthood in most societies, this hypothesis has important public health implications. PMID:23639989
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The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment.
Dobrovolsky, Vasily N; Miura, Daishiro; Heflich, Robert H; Dertinger, Stephen D
2010-01-01
The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.
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Duijkers, Floor A M; Gaal, José; Meijerink, Jules P P; Admiraal, Pieter; Pieters, Rob; de Krijger, Ronald R; van Noesel, Max M
2012-03-01
Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yamshchikov, Vladimir, E-mail: yaximik@gmail.com; Manuvakhova, Marina; Rodriguez, Efrain
Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzedmore » effects of E{sub 138}K and K{sub 279}M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use. - Highlights: • Further attenuation of a WN vaccine precursor is outlined. • Effect of SA14-14-2 attenuating mutations is tested. • Mechanism of attenuation is proposed and illustrated. • The need for additional attenuating mutations is justified.« less
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Shields, Kaitlyn M; Tooley, John G; Petkowski, Janusz J; Wilkey, Daniel W; Garbett, Nichola C; Merchant, Michael L; Cheng, Alan; Schaner Tooley, Christine E
2017-08-01
A subset of B-cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N-terminal trimethylase NRMT1 and the N-terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide-binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N-terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N-terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ-irradiation. © 2017 The Protein Society.
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Caumanns, Joseph J; Berns, Katrien; Wisman, G Bea A; Fehrmann, Rudolf S N; Tomar, Tushar; Klip, Harry; Meersma, Gert Jan; Hijmans, E Marielle; Gennissen, Annemiek; Duiker, Evelien W; Weening, Desiree; Itamochi, Hiroaki; Kluin, Roelof Jc; Reyners, An K L; Birrer, Michael J; Salvesen, Helga B; Vergote, Ignace; Van Nieuwenhuysen, Els; Brenton, James D; Braicu, Elena I; Kupryjanczyk, Jolanta; Spiewankiewicz, Beata; Mittempergher, Lorenza; Bernards, Rene; van der Zee, Ate G J; de Jong, Steven
2018-04-23
Advanced stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in OCCC patients and test druggability of downstream affected pathways in OCCC models. In a large set of OCCC patients (n=124), the human kinome (518 kinases) and additional cancer related genes were sequenced and copy number alterations were determined. Genetically characterized OCCC cell lines (n=17) and OCCC patient-derived xenografts (n=3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib and the mTORC1/2 inhibitor AZD8055. We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway or the ERBB family of receptor tyrosine kinases and 82% in the DNA repair pathway. Strong p-S6 staining in OCCC patients suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not towards drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models. These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Copyright ©2018, American Association for Cancer Research.
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2018-05-31
ATM Gene Mutation; ATR Gene Mutation; BARD1 Gene Mutation; BRCA1 Gene Mutation; BRCA2 Gene Mutation; BRIP1 Gene Mutation; CHEK1 Gene Mutation; CHEK2 Gene Mutation; FANCA Gene Mutation; FANCC Gene Mutation; FANCD2 Gene Mutation; FANCF Gene Mutation; FANCM Gene Mutation; NBN Gene Mutation; PALB2 Gene Mutation; RAD51 Gene Mutation; RAD51B Gene Mutation; RAD54L Gene Mutation; Recurrent Squamous Cell Lung Carcinoma; RPA1 Gene Mutation; Stage IV Squamous Cell Lung Carcinoma AJCC v7
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Walus, Mariusz; Kida, Elizabeth; Golabek, Adam A
2010-06-01
There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes tripeptidyl peptidase I (TPPI), a lysosomal aminopeptidase associated with classic late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease-associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half-life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins.
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Genotype-Phenotype Correlation in Primary Carnitine Deficiency
Rose, Emily Cornforth; di San Filippo, Cristina Amat; Ndukwe Erlingsson, Uzochi C.; Ardon, Orly; Pasquali, Marzia; Longo, Nicola
2011-01-01
Primary carnitine deficiency is caused by defective OCTN2 carnitine transporters encoded by the SLC22A5 gene. Lack of carnitine impairs fatty acid oxidation resulting in hypoketotic hypoglycemia, hepatic encephalopathy, skeletal and cardiac myopathy. Recently, asymptomatic mothers with primary carnitine deficiency were identified by low carnitine levels in their infant by newborn screening. Here we evaluate mutations in the SLC22A5 gene and carnitine transport in fibroblasts from symptomatic patients and asymptomatic women. Carnitine transport was significantly reduced in fibroblasts obtained from all patients with primary carnitine deficiency, but was significantly higher in the asymptomatic women’s than in the symptomatic patients’ fibroblasts (p<0.01). By contrast, ergothioneine transport (a selective substrate of the OCTN1 transporter, tested here as a control) was similar in cells from controls and patients with carnitine deficiency. DNA sequencing indicated an increased frequency of nonsense mutations in symptomatic patients (p<0.001). Expression of the missense mutations in CHO cells indicated that many mutations retained residual carnitine transport activity, with no difference in the average activity of missense mutations identified in symptomatic versus asymptomatic patients. These results indicate that cells from asymptomatic women have on average higher levels of residual carnitine transport activity as compared to that of symptomatic patients due to the presence of at least one missense mutation. PMID:21922592
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Andrulis, Mindaugas; Capper, David; Luft, Thomas; Hartmann, Christian; Zentgraf, Hanswalter; von Deimling, Andreas
2010-08-01
Sequencing of the acute myeloid leukemia genome revealed somatic mutations in isocitrate dehydrogenase-1. Acute myeloid leukemia frequently develops from myelodysplastic syndrome. In order to test whether myelodysplastic syndrome also carries isocitrate dehydrogenase-1 mutations, we stained a series of bone marrow samples from patients with myelodysplastic syndrome using an antibody specific for the R132H mutation. Three out of 71 patients exhibited antibody binding to myeloid precursor cells. The presence of the R132H mutation was confirmed by DNA sequencing. We demonstrated that isocitrate dehydrogenase-1 mutations occur in myelodysplasia preceding acute myeloid leukemia and that the R132H alteration can be detected by immunohistochemistry. Copyright (c) 2010 Elsevier Ltd. All rights reserved.
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Madic, Jordan; Remon, Jordi; Honoré, Aurélie; Girard, Romain; Rouleau, Etienne; André, Barbara; Besse, Benjamin; Droniou, Magali; Lacroix, Ludovic
2017-01-01
Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results. PMID:28829811
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Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.
2018-01-01
Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098
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Evidence-based best practices for EGFR T790M testing in lung cancer in Canada.
Stockley, T; Souza, C A; Cheema, P K; Melosky, B; Kamel-Reid, S; Tsao, M S; Spatz, A; Karsan, A
2018-04-01
Epidermal growth factor receptor (egfr) tyrosine kinase inhibitors (tkis) are recommended as first-line systemic therapy for patients with non-small-cell lung cancer (nsclc) having mutations in the EGFR gene. Resistance to tkis eventually occurs in all nsclc patients treated with such drugs. In patients with resistance to tkis caused by the EGFR T790M mutation, the third-generation tki osimertinib is now the standard of care. For optimal patient management, accurate EGFR T790M testing is required. A multidisciplinary working group of pathologists, laboratory medicine specialists, medical oncologists, a respirologist, and a thoracic radiologist from across Canada was convened to discuss best practices for EGFR T790M mutation testing in Canada. The group made recommendations in the areas of the testing algorithm and the pre-analytic, analytic, and post-analytic aspects of clinical testing for both tissue testing and liquid biopsy circulating tumour dna testing. The recommendations aim to improve EGFR T790M testing in Canada and to thereby improve patient care.
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Zhukova, Nataliya; Ramaswamy, Vijay; Remke, Marc; Martin, Dianna C; Castelo-Branco, Pedro; Zhang, Cindy H; Fraser, Michael; Tse, Ken; Poon, Raymond; Shih, David J H; Baskin, Berivan; Ray, Peter N; Bouffet, Eric; Dirks, Peter; von Bueren, Andre O; Pfaff, Elke; Korshunov, Andrey; Jones, David T W; Northcott, Paul A; Kool, Marcel; Pugh, Trevor J; Pomeroy, Scott L; Cho, Yoon-Jae; Pietsch, Torsten; Gessi, Marco; Rutkowski, Stefan; Bognár, Laszlo; Cho, Byung-Kyu; Eberhart, Charles G; Conter, Cecile Faure; Fouladi, Maryam; French, Pim J; Grajkowska, Wieslawa A; Gupta, Nalin; Hauser, Peter; Jabado, Nada; Vasiljevic, Alexandre; Jung, Shin; Kim, Seung-Ki; Klekner, Almos; Kumabe, Toshihiro; Lach, Boleslaw; Leonard, Jeffrey R; Liau, Linda M; Massimi, Luca; Pollack, Ian F; Ra, Young Shin; Rubin, Joshua B; Van Meir, Erwin G; Wang, Kyu-Chang; Weiss, William A; Zitterbart, Karel; Bristow, Robert G; Alman, Benjamin; Hawkins, Cynthia E; Malkin, David; Clifford, Steven C; Pfister, Stefan M; Taylor, Michael D; Tabori, Uri
2014-12-24
TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%±8.7%, respectively (p<0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%±2% vs. 57.4%±1.8% (p<0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p<0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%±1.5% in lithium treated cells vs. 56.6±3% (p<0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%±8% for lithium treated cells vs. 27%±3% for untreated controls (p=0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.
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Stanley, Pamela; Sundaram, Subha
2014-06-03
Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. Copyright © 2014 John Wiley & Sons, Inc.
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Giant cell lesion of the jaw as a presenting feature of Noonan syndrome.
Sinnott, Bridget P; Patel, Maya
2018-05-30
This is a case of a 20-year-old woman who presented with a left jaw mass which was resected and found to be a giant cell granuloma of the mandible. Her history and physical examination were suggestive for Noonan syndrome which was confirmed with genetic testing and the finding of a PTPN11 gene mutation which has rarely been associated with giant cell lesions of the jaw. Given her particular genetic mutation and the presence of a giant cell lesion, we present a case of Noonan-like/multiple giant cell lesion syndrome. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.
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Li, Yuping; Xu, Hanyan; Su, Shanshan; Ye, Junru; Chen, Junjie; Jin, Xuru; Lin, Quan; Zhang, Dongqing; Ye, Caier; Chen, Chengshui
2017-01-01
Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive epidermal growth factor receptor (EGFR) mutations detection in lung cancer patients, but the existing methods have limitations in sensitivity or in availability. In this study, we evaluated the performance of a novel assay called ADx-SuperARMS in detecting EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma. A total of 109 patients with metastatic advanced adenocarcinoma were recruited who provided both blood samples and matched tumor tissue samples. EGFR mutation status in plasma samples were tested with ADx-SuperARMS EGFR assay and tumor tissue samples were tested with ADx-ARMS EGFR assay. The clinical sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) of ADx-SuperARMS EGFR assay were calculated by using EGFR mutation status in tumor tissue as standard reference. A receiver operating characteristic (ROC) analysis was implemented and an area under the curve (AUC) was calculated to evaluate sensitivity and specificity of exon 19 deletion (E19Del) and L858R mutation detection. The objective response rate (ORR) were calculated according to the EGFR mutation status determined by ADx-superARMS as well. 0.2% analytical sensitivity and 100% specificity of the ADx-SuperARMS EGFR assays for EGFR E19Del, L858R, and T790M mutants were confirmed by using a series of diluted cell line DNA. In the clinical study, EGFR mutations were detected in 45.9% (50/109) of the plasma samples and in 56.9% (62/109) of the matched tumor tissue samples. The sensitivity, specificity, PPV and NPV of the ADx-SuperARMS EGFR assay for plasma EGFR mutation detection were 82.0% (50/61), 100% (48/48), 100% (50/50), and 81.4% (48/59), respectively. In ROC analysis, ADx-SuperARMS achieved sensitivity and specificity of 88% and 99% in E19Dels as well as sensitivity and specificity of 89% and 100% in L858R, respectively. Among the 35 patients who were plasma EGFR mutation positive and treated with first generation of EGFR-tyrosine kinase inhibitors (TKIs), 23 (65.7%) achieved partial response, 11 (31.4%) sustained disease, and 1 (2.9%) progressive disease. The ORR and disease control rate (DCR) were 65.7% and 97.1%, respectively. ADx-SuperARMS EGFR assay is likely to be a highly sensitive and specific method to noninvasively detect plasma EGFR mutations of patients with advanced lung adenocarcinoma. The EGFR mutations detected by ADx-SuperARMS EGFR assay could predict the efficacy of the treatment with first generation of EGFR-TKIs. Hence, EGFR blood testing with ADx-SuperARMS could address the unmet clinical needs.
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Multiple giant cell lesions in patients with Noonan syndrome and cardio-facio-cutaneous syndrome.
Neumann, Thomas E; Allanson, Judith; Kavamura, Ines; Kerr, Bronwyn; Neri, Giovanni; Noonan, Jacqueline; Cordeddu, Viviana; Gibson, Kate; Tzschach, Andreas; Krüger, Gabriele; Hoeltzenbein, Maria; Goecke, Timm O; Kehl, Hans Gerd; Albrecht, Beate; Luczak, Klaudiusz; Sasiadek, Maria M; Musante, Luciana; Laurie, Rohan; Peters, Hartmut; Tartaglia, Marco; Zenker, Martin; Kalscheuer, Vera
2009-04-01
Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS) are related developmental disorders caused by mutations in genes encoding various components of the RAS-MAPK signaling cascade. NS is associated with mutations in the genes PTPN11, SOS1, RAF1, or KRAS, whereas CFCS can be caused by mutations in BRAF, MEK1, MEK2, or KRAS. The NS phenotype is rarely accompanied by multiple giant cell lesions (MGCL) of the jaw (Noonan-like/MGCL syndrome (NL/MGCLS)). PTPN11 mutations are the only genetic abnormalities reported so far in some patients with NL/MGCLS and in one individual with LEOPARD syndrome and MGCL. In a cohort of 75 NS patients previously tested negative for mutations in PTPN11 and KRAS, we detected SOS1 mutations in 11 individuals, four of whom had MGCL. To explore further the relevance of aberrant RAS-MAPK signaling in syndromic MGCL, we analyzed the established genes causing CFCS in three subjects with MGCL associated with a phenotype fitting CFCS. Mutations in BRAF or MEK1 were identified in these patients. All mutations detected in these seven patients with syndromic MGCL had previously been described in NS or CFCS without apparent MGCL. This study demonstrates that MGCL may occur in NS and CFCS with various underlying genetic alterations and no obvious genotype-phenotype correlation. This suggests that dysregulation of the RAS-MAPK pathway represents the common and basic molecular event predisposing to giant cell lesion formation in patients with NS and CFCS rather than specific mutation effects.
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Multiple giant cell lesions in patients with Noonan syndrome and cardio-facio-cutaneous syndrome
Neumann, Thomas E; Allanson, Judith; Kavamura, Ines; Kerr, Bronwyn; Neri, Giovanni; Noonan, Jacqueline; Cordeddu, Viviana; Gibson, Kate; Tzschach, Andreas; Krüger, Gabriele; Hoeltzenbein, Maria; Goecke, Timm O; Kehl, Hans Gerd; Albrecht, Beate; Luczak, Klaudiusz; Sasiadek, Maria M; Musante, Luciana; Laurie, Rohan; Peters, Hartmut; Tartaglia, Marco; Zenker, Martin; Kalscheuer, Vera
2009-01-01
Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS) are related developmental disorders caused by mutations in genes encoding various components of the RAS-MAPK signaling cascade. NS is associated with mutations in the genes PTPN11, SOS1, RAF1, or KRAS, whereas CFCS can be caused by mutations in BRAF, MEK1, MEK2, or KRAS. The NS phenotype is rarely accompanied by multiple giant cell lesions (MGCL) of the jaw (Noonan-like/MGCL syndrome (NL/MGCLS)). PTPN11 mutations are the only genetic abnormalities reported so far in some patients with NL/MGCLS and in one individual with LEOPARD syndrome and MGCL. In a cohort of 75 NS patients previously tested negative for mutations in PTPN11 and KRAS, we detected SOS1 mutations in 11 individuals, four of whom had MGCL. To explore further the relevance of aberrant RAS-MAPK signaling in syndromic MGCL, we analyzed the established genes causing CFCS in three subjects with MGCL associated with a phenotype fitting CFCS. Mutations in BRAF or MEK1 were identified in these patients. All mutations detected in these seven patients with syndromic MGCL had previously been described in NS or CFCS without apparent MGCL. This study demonstrates that MGCL may occur in NS and CFCS with various underlying genetic alterations and no obvious genotype–phenotype correlation. This suggests that dysregulation of the RAS-MAPK pathway represents the common and basic molecular event predisposing to giant cell lesion formation in patients with NS and CFCS rather than specific mutation effects. PMID:18854871
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Crizotinib Synergizes with Chemotherapy in Preclinical Models of Neuroblastoma
Krytska, Kateryna; Ryles, Hannah T.; Sano, Renata; Raman, Pichai; Infarinato, Nicole R.; Hansel, Theodore D.; Makena, Monish R.; Song, Michael M.; Reynolds, C. Patrick; Mossé, Yael P.
2015-01-01
Purpose The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. Experimental Design The sensitivity of human neuroblastoma-derived cell lines, cell line-derived and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. Results In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free-survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). Conclusions Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multi-agent therapy for ALK-aberrant neuroblastoma patients. PMID:26438783
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Trametinib with or without Vemurafenib in BRAF Mutated Non-Small Cell Lung Cancer
Joshi, Monika; Rice, Shawn J.; Liu, Xin; Miller, Bruce; Belani, Chandra P.
2015-01-01
V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) mutated lung cancer is relatively aggressive and is resistant to currently available therapies. In a recent phase II study for patients with BRAF-V600E non-small cell lung cancer (NSCLC), BRAF V600E inhibitor demonstrated evidence of activity, but 30% of this selected group progressed while on treatment, suggesting a need for developing alternative strategies. We tested two different options to enhance the efficacy of vemurafenib (BRAF V600E inhibitor) in BRAF mutated NSCLC. The first option was the addition of erlotinib to vemurafenib to see whether the combination provided synergy. The second was to induce MEK inhibition (downstream of RAF) with trametinib (MEK inhibitor). We found that the combination of vemurafenib and erlotinib was not synergistic to the inhibition of p-ERK signaling in BRAF-V600E cells. Vemurafenib caused significant apoptosis, G1 arrest and upregulation of BIM in BRAF-V600 cells. Trametinib was effective as a single agent in BRAF mutated cells, either V600E or non-V600E. Finally, the combination of vemurafenib and trametinib caused a small but significant increase in apoptosis as well as a significant upregulation of BIM when compared to either single agent. Thus, hinting at the possibility of utilizing a combinational approach for the management of this group of patients. Importantly, trametinib alone caused upregulation of p-AKT in BRAF non-V600 mutated cells, while this effect was nullified with the combination. This finding suggests that, the combination of a MEK inhibitor with a BRAF inhibitor will be more efficacious in the clinical setting for patients with BRAF mutated NSCLC. PMID:25706985
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Unveiling adaptation using high-resolution lineage tracking
NASA Astrophysics Data System (ADS)
Blundell, Jamie; Levy, Sasha; Fisher, Daniel; Petrov, Dmitri; Sherlock, Gavin
2013-03-01
Human diseases such as cancer and microbial infections are adaptive processes inside the human body with enormous population sizes: between 106 -1012 cells. In spite of this our understanding of adaptation in large populations is limited. The key problem is the difficulty in identifying anything more than a handful of rare, large-effect beneficial mutations. The development and use of molecular barcodes allows us to uniquely tag hundreds of thousands of cells and enable us to track tens of thousands of adaptive mutations in large yeast populations. We use this system to test some of the key theories on which our understanding of adaptation in large populations is based. We (i) measure the fitness distribution in an evolving population at different times, (ii) identify when an appreciable fraction of clones in the population have at most a single adaptive mutation and isolate a large number of clones with independent single adaptive mutations, and (iii) use this clone collection to determine the distribution of fitness effects of single beneficial mutations.
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Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G
2001-06-15
Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.
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Lin, Ke; Sherrington, Paul D; Dennis, Michael; Matrai, Zoltan; Cawley, John C; Pettitt, Andrew R
2002-08-15
Established adverse prognostic factors in chronic lymphocytic leukemia (CLL) include CD38 expression, relative lack of IgV(H) mutation, and defects of the TP53 gene. However, disruption of the p53 pathway can occur through mechanisms other than TP53 mutation, and we have recently developed a simple screening test that detects p53 dysfunction due to mutation of the genes encoding either p53 or ATM, a kinase that regulates p53. The present study was conducted to examine the predictive value of this test and to establish the relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation. CLL cells from 71 patients were examined for IgV(H) mutation, CD38 expression, and p53 dysfunction (detected as an impaired p53/p21 response to ionizing radiation). Survival data obtained from 69 patients were analyzed according to each of these parameters. Relative lack of IgV(H) mutation (less than 5%; n = 45), CD38 positivity (antigen expressed on more than 20% of malignant cells; n = 19), and p53 dysfunction (n = 19) were independently confirmed as adverse prognostic factors. Intriguingly, all p53-dysfunctional patients and all but one of the CD38(+) patients had less [corrected] than 5% IgV(H) mutation. Moreover, patients with p53 dysfunction and/or CD38 positivity (n = 31) accounted for the short survival of the less mutated group. These findings indicate that the poor outcome associated with having less than 5% IgV(H) mutation may be due to the overrepresentation of high-risk patients with p53 dysfunction and/or CD38 positivity within this group, and that CD38(-) patients with functionally intact p53 may have a prolonged survival regardless of the extent of IgV(H) mutation.
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Mon, Sann Y; Riedlinger, Gregory; Abbott, Collette E; Seethala, Raja; Ohori, N Paul; Nikiforova, Marina N; Nikiforov, Yuri E; Hodak, Steven P
2018-05-01
Thyroid-stimulating hormone receptor (TSHR) gene mutations play a critical role in thyroid cell proliferation and function. They are found in 20%-82% of hyperfunctioning nodules, hyperfunctioning follicular thyroid cancers (FTC), and papillary thyroid cancers (PTC). The diagnostic importance of TSHR mutation testing in fine needle aspiration (FNA) specimens remains unstudied. To examine the association of TSHR mutations with the functional status and surgical outcomes of thyroid nodules, we evaluated 703 consecutive thyroid FNA samples with indeterminate cytology for TSHR mutations using next-generation sequencing. Testing for EZH1 mutations was performed in selected cases. The molecular diagnostic testing was done as part of standard of care treatment, and did not require informed consent. TSHR mutations were detected in 31 (4.4%) nodules and were located in exons 281-640, with codon 486 being the most common. Allelic frequency ranged from 3% to 45%. Of 16 cases (12 benign, 3 FTC, 1 PTC) with surgical correlation, 15 had solitary TSHR mutations and 1 PTC had comutation with BRAF V600E. Hyperthyroidism was confirmed in all 3 FTC (2 overt, 1 subclinical). Of 5 nodules with solitary TSHR mutations detected at high allelic frequency, 3 (60%) were FTC. Those at low allelic frequency (3%-22%) were benign. EZH1 mutations were detected in 2 of 4 TSHR-mutant malignant nodules and neither of 2 benign nodules. We report that TSHR mutations occur in ∼5% thyroid nodules in a large consecutive series with indeterminate cytology. TSHR mutations may be associated with an increased cancer risk when present at high allelic frequency, even when the nodule is hyperfunctioning. Benign nodules were however most strongly correlated with TSHR mutations at low allelic frequency. © 2018 Wiley Periodicals, Inc.
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NASA Astrophysics Data System (ADS)
Husimi, Yuzuru; Nishigaki, Koichi; Kinoshita, Yasunori; Tanaka, Toyosuke
1982-04-01
A bacteriophage is continuously cultured in the flow of the host bacterial cell under the control of a minicomputer. In the culture, the population of the noninfected cell is kept constant by the endogeneous regulation mechanism, so it is called the ''cellstat'' culture. Due to the high dilution rate of the host cell, the mutant cell cannot be selected in the cellstat. Therefore, the cellstat is suitable for the study of the mutation rate and the selection process of a bacteriophage under well-defined environmental conditions (including physiological condition of the host cell) without being interfered by host-cell mutations. Applications to coliphage fd, a secretion type phage, are shown as a measurement example. A chimera between fd and a plasmid pBR322 is cultured more than 100 h. The process of population changeovers by deletion mutants indicates that the deletion hot spots exist in this cloning vector and that this apparatus can be used also for testing instability of a recombinant DNA.
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CT Radiogenomic Characterization of EGFR, K-RAS, and ALK Mutations in Non-Small Cell Lung Cancer.
Rizzo, Stefania; Petrella, Francesco; Buscarino, Valentina; De Maria, Federica; Raimondi, Sara; Barberis, Massimo; Fumagalli, Caterina; Spitaleri, Gianluca; Rampinelli, Cristiano; De Marinis, Filippo; Spaggiari, Lorenzo; Bellomi, Massimo
2016-01-01
To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer. Patients undergoing chest CT and testing for the above gene mutations were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated. P-values <0.05 were considered significant. Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking. This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes). Air bronchogram, pleural retraction, small size relate to EGFR mutation in NSCLC. Pleural effusion and younger age relate to ALK mutation. Round lesion shape, nodules in non-tumour lobes relate to KRAS mutation.
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Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian
2015-01-01
Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.
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Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian
2015-01-01
Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866
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Mostert, Bianca; Jiang, Yuqiu; Sieuwerts, Anieta M; Wang, Haiying; Bolt-de Vries, Joan; Biermann, Katharina; Kraan, Jaco; Lalmahomed, Zarina; van Galen, Anne; de Weerd, Vanja; van der Spoel, Petra; Ramírez-Moreno, Raquel; Verhoef, Cornelis; Ijzermans, Jan N M; Wang, Yixin; Gratama, Jan-Willem; Foekens, John A; Sleijfer, Stefan; Martens, John W M
2013-07-01
Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior. Copyright © 2012 UICC.
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Common Variable Immunodeficiency Caused by FANC Mutations.
Sekinaka, Yujin; Mitsuiki, Noriko; Imai, Kohsuke; Yabe, Miharu; Yabe, Hiromasa; Mitsui-Sekinaka, Kanako; Honma, Kenichi; Takagi, Masatoshi; Arai, Ayako; Yoshida, Kenichi; Okuno, Yusuke; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Muramatsu, Hideki; Kojima, Seiji; Hira, Asuka; Takata, Minoru; Ohara, Osamu; Ogawa, Seishi; Morio, Tomohiro; Nonoyama, Shigeaki
2017-07-01
Common variable immunodeficiency (CVID) is the most common adult-onset primary antibody deficiency disease due to various causative genes. Several genes, which are known to be the cause of different diseases, have recently been reported as the cause of CVID in patients by performing whole exome sequencing (WES) analysis. Here, we found FANC gene mutations as a cause of adult-onset CVID in two patients. B cells were absent and CD4 + T cells were skewed toward CD45RO + memory T cells. T-cell receptor excision circles (TRECs) and signal joint kappa-deleting recombination excision circles (sjKRECs) were undetectable in both patients. Both patients had no anemia, neutropenia, or thrombocytopenia. Using WES, we identified compound heterozygous mutations of FANCE in one patient and homozygous mutation of FANCA in another patient. The impaired function of FANC protein complex was confirmed by a monoubiquitination assay and by chromosome fragility test. We then performed several immunological evaluations including quantitative lymphocyte analysis and TRECs/sjKRECs analysis for 32 individuals with Fanconi anemia (FA). In total, 22 FA patients (68.8%) were found to have immunological abnormalities, suggesting that such immunological findings may be common in FA patients. These data indicate that FANC mutations are involved in impaired lymphogenesis probably by the accumulation of DNA replication stress, leading to CVID. It is important to diagnose FA because it drastically changes clinical management. We propose that FANC mutations can cause isolated immunodeficiency in addition to bone marrow failure and malignancy.
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Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.
2014-01-01
ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity. PMID:25142579
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Pedraza-Sánchez, Sigifredo; Lezana-Fernández, Jose Luis; Gonzalez, Yolanda; Martínez-Robles, Luis; Ventura-Ayala, María Laura; Sadowinski-Pine, Stanislaw; Nava-Frías, Margarita; Moreno-Espinosa, Sarbelio; Casanova, Jean-Laurent; Puel, Anne; Boisson-Dupuis, Stephanie; Torres, Martha
2017-01-01
In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF) mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC). We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ) circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1) was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs); phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF mutation. Therefore, GOF mutations in STAT1 can induce susceptibility not only to fungal but also to mycobacterial infections by mechanisms to be determined.
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Pedraza-Sánchez, Sigifredo; Lezana-Fernández, Jose Luis; Gonzalez, Yolanda; Martínez-Robles, Luis; Ventura-Ayala, María Laura; Sadowinski-Pine, Stanislaw; Nava-Frías, Margarita; Moreno-Espinosa, Sarbelio; Casanova, Jean-Laurent; Puel, Anne; Boisson-Dupuis, Stephanie; Torres, Martha
2017-01-01
In humans, recessive loss-of-function mutations in STAT1 are associated with mycobacterial and viral infections, whereas gain-of-function (GOF) mutations in STAT1 are associated with a type of primary immunodeficiency related mainly, but not exclusively, to chronic mucocutaneous candidiasis (CMC). We studied and established a molecular diagnosis in a pediatric patient with mycobacterial infections, associated with CMC. The patient, daughter of a non-consanguineous mestizo Mexican family, had axillary adenitis secondary to BCG vaccination and was cured with resection of the abscess at 1-year old. At the age of 4 years, she had a supraclavicular abscess with acid-fast-staining bacilli identified in the soft tissue and bone, with clinical signs of disseminated infection and a positive Gene-X-pert test, which responded to anti-mycobacterial drugs. Laboratory tests of the IL-12/interferon gamma (IFN-γ) circuit showed a higher production of IL-12p70 in the whole blood from the patient compared to healthy controls, when stimulated with BCG and BCG + IFN-γ. The whole blood of the patient produced 35% less IFN-γ compared to controls assessed by ELISA and flow cytometry, but IL-17 producing T cells from patient were almost absent in PBMC stimulated with PMA plus ionomycin. Signal transduction and activator of transcription 1 (STAT1) was hyperphosphorylated at tyrosine 701 in response to IFN-γ and -α, as demonstrated by flow cytometry and Western blotting in fresh blood mononuclear cells and in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs); phosphorylation of STAT1 in EBV-LCLs from the patient was resistant to inhibition by staurosporine but sensitive to ruxolitinib, a Jak phosphorylation inhibitor. Genomic DNA sequencing showed a de novo mutation in STAT1 in cells from the patient, absent in her parents and brother; a known T385M missense mutation in the DNA-binding domain of the transcription factor was identified, and it is a GOF mutation. Therefore, GOF mutations in STAT1 can induce susceptibility not only to fungal but also to mycobacterial infections by mechanisms to be determined. PMID:29270166
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Genotoxicity assessment of nanomaterials: recommendations on best practices, assays and methods.
Elespuru, Rosalie; Pfuhler, Stefan; Aardema, Marilyn; Chen, Tao; Doak, Shareen H; Doherty, Ann; Farabaugh, Christopher S; Kenny, Julia; Manjanatha, Mugimane; Mahadevan, Brinda; Moore, Martha M; Ouédraogo, Gladys; Stankowski, Leon F; Tanir, Jennifer Y
2018-04-26
Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.
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Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.
Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A
2015-05-20
Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.
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Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype
Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A.
2015-01-01
Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors. PMID:25980580
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Seo, Seongjin; Solivan-Timpe, Frances; Roos, Ben R; Robin, Alan L; Stone, Edwin M; Kwon, Young H; Alward, Wallace L M; Fingert, John H
2013-02-01
Copy number variations (duplications) of TANK binding kinase 1 (TBK1) have been associated with normal tension glaucoma (NTG), a common cause of blindness worldwide. Mutations in other genes involved in autophagy (TLR4 and OPTN) have been associated with NTG. Here we report searching for additional proteins involved in autophagy that may also have roles in NTG. HEK-293T cells were transfected to produce synthetic TBK1 protein with FLAG and S tags. Proteins that associate with TBK1 were isolated from HEK-293T lysates using tandem affinity purification (TAP) and polyacrylamide gel electrophoresis (PAGE). Isolated proteins were identified with mass spectrometry. A cohort of 148 NTG patients and 77 controls from Iowa were tested for glaucoma-causing mutations in genes that encode identified proteins that interact with TBK1 using high resolution melt (HRM) analysis and DNA sequencing. TAP studies show that three proteins expressed in HEK-293T cells (NAP1, TANK and TBKBP1) interact with TBK1. Testing cohorts of NTG and normal controls for disease-causing mutations in TANK, identified a total of nine unique variants including three non-synonymous changes, one synonymous changes and five intronic changes. When analyzed alone or as a group, the non-synonymous TBK1 coding sequence changes were not associated with either NTG or primary open angle glaucoma. TAP showed that NAP1, TANK and TBKBP1 interact with TBK1 and are good candidates for contributing to NTG. A mutation screen of TANK detected three non-synonymous variants. Although, it remains possible that one or more of these TANK mutations may have a role in NTG, the data in this report do not provide statistical support for an association between TANK variants and NTG.
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Thress, Kenneth S; Brant, Roz; Carr, T Hedley; Dearden, Simon; Jenkins, Suzanne; Brown, Helen; Hammett, Tracey; Cantarini, Mireille; Barrett, J Carl
2015-12-01
To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients. A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas(®) EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR]). Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78-100%) and specificity (93-100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas(®) EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82-87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas(®) EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma. The cobas(®) EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may explain the reduced specificity observed with plasma-based detection of T790M mutations versus tissue. These data support the use of both platforms in the AZD9291 clinical development program. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
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Development of an in vitro PIG-A gene mutation assay in human cells
Rees, Benjamin J; Tate, Matthew; Lynch, Anthony M; Thornton, Catherine A; Jenkins, Gareth J; Walmsley, Richard M; Johnson, George E
2017-01-01
Abstract Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella ‘Ames’ reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell’s extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing. PMID:28057708
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Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth.
Bloch, Jeannine; Holzmann, Carsten; Koczan, Dirk; Helmke, Burkhard Maria; Bullerdiek, Jörn
2017-05-23
Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell cultures from nine UL from 40-50 year old Caucasian patients with at least one UL. Cultured UL cells were investigated for loss of MED12-mutated cells. Genetic characterization of native tumor samples and adjacent myometrium was done by array analysis. "Aged" primary cultures without passaging were compared to cells of three subsequent passages. Comparative analyses of the mutated/non-mutated ratios between native tissue, primary cells, and cultured tumor cells revealed a clear decrease of MED12-mutated cells. None of the tumors showed gross alterations of the array profiles, excluding the presence of gross genomic imbalances besides the MED12 mutations as a reason for the intertumoral variation in the loss of MED12-mutated cells. Albeit at a lesser rate, loss of MED12-mutated cells from cell cultures of UL occurs even without passaging thus indicating the requirement of soluble factors or matrix components lacking in vitro. Identification of these factors can help to understand the mechanisms of the growth of the most frequent type of uterine leiomyomas and to decipher novel drug targets.
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Shin, Ha Kyung; Grahame, George; McCandless, Shawn E; Kerr, Douglas S; Bedoyan, Jirair K
2017-11-01
Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia in children. Prompt and correct diagnosis of PDC deficiency and differentiating between specific vs generalized, or secondary deficiencies has important implications for clinical management and therapeutic interventions. Both genetic and enzymatic testing approaches are being used in the diagnosis of PDC deficiency. However, the diagnostic efficacy of such testing approaches for individuals affected with PDC deficiency has not been systematically investigated in this disorder. We sought to evaluate the diagnostic sensitivity and variability of the various PDC enzyme assays in females and males at the Center for Inherited Disorders of Energy Metabolism (CIDEM). CIDEM data were filtered by lactic acidosis and functional PDC deficiency in at least one cell/tissue type (blood lymphocytes, cultured fibroblasts or skeletal muscle) identifying 186 subjects (51% male and 49% female), about half were genetically resolved with 78% of those determined to have a pathogenic PDHA1 mutation. Assaying PDC in cultured fibroblasts in cases where the underlying genetic etiology is PDHA1, was highly sensitive irrespective of gender; 97% (95% confidence interval [CI]: 90%-100%) and 91% (95% CI: 82%-100%) in females and males, respectively. In contrast to the fibroblast-based testing, the lymphocyte- and muscle-based testing were not sensitive (36% [95% CI: 11%-61%, p=0.0003] and 58% [95% CI: 30%-86%, p=0.014], respectively) for identifying known PDC deficient females with pathogenic PDHA1 mutations. In males with a known PDHA1 mutation, the sensitivity of the various cell/tissue assays (75% lymphocyte, 91% fibroblast and 88% muscle) were not statistically different, and the discordance frequency due to the specific cell/tissue used for assaying PDC was 0.15±0.11. Based on this data, a practical diagnostic algorithm is proposed accounting for current molecular approaches, enzyme testing sensitivity, and variability due to gender, cell/tissue type used for testing, and successive repeat testing. Copyright © 2017 Elsevier Inc. All rights reserved.
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Andersson, Elin; Dahmcke, Christina M; Steven, Kenneth; Larsen, Louise K; Guldberg, Per
2015-01-01
Molecular analysis of cells from urine provides a convenient approach to non-invasive detection of bladder cancer. The practical use of urinary cell-based tests is often hampered by difficulties in handling and analyzing large sample volumes, the need for rapid sample processing to avoid degradation of cellular content, and low sensitivity due to a high background of normal cells. We present a filtration device, designed for home or point-of-care use, which enables collection, storage and shipment of urinary cells. A special feature of this device is a removable cartridge housing a membrane filter, which after filtration of urine can be transferred to a storage unit containing an appropriate preserving solution. In spiking experiments, the use of this device provided efficient recovery of bladder cancer cells with elimination of >99% of excess smaller-sized cells. The performance of the device was further evaluated by DNA-based analysis of urinary cells collected from 57 patients subjected to transurethral resection following flexible cystoscopy indicating the presence of a tumor. All samples were tested for FGFR3 mutations and seven DNA methylation markers (BCL2, CCNA1, EOMES, HOXA9, POU4F2, SALL3 and VIM). In the group of patients where a transitional cell tumor was confirmed at histopathological evaluation, urine DNA was positive for one or more markers in 29 out of 31 cases (94%), including 19 with FGFR3 mutation (61%). In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%). Only one patient in this group was positive for a FGFR3 mutation. This patient had a stage Ta tumor resected 6 months later. The ability to easily collect, store and ship diagnostic cells from urine using the presented device may facilitate non-invasive testing for bladder cancer.
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Andersson, Elin; Dahmcke, Christina M.; Steven, Kenneth; Larsen, Louise K.; Guldberg, Per
2015-01-01
Molecular analysis of cells from urine provides a convenient approach to non-invasive detection of bladder cancer. The practical use of urinary cell-based tests is often hampered by difficulties in handling and analyzing large sample volumes, the need for rapid sample processing to avoid degradation of cellular content, and low sensitivity due to a high background of normal cells. We present a filtration device, designed for home or point-of-care use, which enables collection, storage and shipment of urinary cells. A special feature of this device is a removable cartridge housing a membrane filter, which after filtration of urine can be transferred to a storage unit containing an appropriate preserving solution. In spiking experiments, the use of this device provided efficient recovery of bladder cancer cells with elimination of >99% of excess smaller-sized cells. The performance of the device was further evaluated by DNA-based analysis of urinary cells collected from 57 patients subjected to transurethral resection following flexible cystoscopy indicating the presence of a tumor. All samples were tested for FGFR3 mutations and seven DNA methylation markers (BCL2, CCNA1, EOMES, HOXA9, POU4F2, SALL3 and VIM). In the group of patients where a transitional cell tumor was confirmed at histopathological evaluation, urine DNA was positive for one or more markers in 29 out of 31 cases (94%), including 19 with FGFR3 mutation (61%). In the group of patients with benign histopathology, urine DNA was positive for methylation markers in 13 out of 26 cases (50%). Only one patient in this group was positive for a FGFR3 mutation. This patient had a stage Ta tumor resected 6 months later. The ability to easily collect, store and ship diagnostic cells from urine using the presented device may facilitate non-invasive testing for bladder cancer. PMID:26151138
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Esteban, E; Majem, M; Martinez Aguillo, M; Martinez Banaclocha, N; Dómine, M; Gómez Aldaravi, L; Juan, O; Cajal, R; Gonzalez Arenas, M C; Provencio, M
2015-06-01
The aim of the REASON study is to determine the frequency of EGFR mutation in advanced non-small cell lung cancer (aNSCLC) patients in Spain (all histologies), and to better understand the clinical factors (gender, smoking habits and histological subtypes) that may be associated with EGFR mutations, in an unselected sample of aNSCLC patients. All newly diagnosed aNSCLC patients from 40 selected centers in Spain were prospectively included for a 6-month period. Patient characteristics were obtained from clinical records. Mutation testing was performed on available tumor samples. Exploratory analyses were performed to characterize the clinico-pathological factors associated with presence of EGFR mutations. From March 2010 to March 2011, 1113 patients were included in the study, of which 1009 patients provided sample for EGFR mutation analysis (90.7%). Mutation analysis was not feasible in 146/1113 patients (13.1%) due to either sample unavailability (79/1113; 7.1%) or sample inadequacy (67/1113; 6.0%). Twenty-five out of 1113 patients (2.3%) were excluded due to unavailable information. Most patients (99.5%) were Caucasian, 74.5% were male, and predominantly were current (38.1%) or former smokers (44.0%). Median age was 66 years (range 25-90) and 70.7% of patients had non-squamous histology (57.8% adenocarcinoma, 1.8% bronchoalveolar, 11.1% large-cell carcinoma). Exon 19 deletions and the exon 21 L858R point mutation were analyzed in 942/1009 (93.4%) samples. Mutation rate was 11.6% (82.6% exon 19 dels and 17.4% L858R). To be never smoker (38.1%), female (25.4%), with bronchioloalveolar carcinoma (22.2%) or adenocarcinoma (15.4%) histology was associated with a higher prevalence of EGFR mutations. Exons 18, 20 and 21 (excluding L858R) were analyzed in 505/942 samples, and EGFR mutations were found in 22/505 samples (4.4%). The estimated prevalence of sensitizing EGFR mutations (exon 19 del, exon 21 L858R) in an unselected samples of newly diagnosed aNSCLC patients in Spain (all histologies) is consistent with previous published data in Caucasian patients. When a sample is available, EGFR mutation testing is feasible in over 90% of cases, and may therefore be suitable for routine clinical practice. CLINICALTRIALS. NCT01081496. Copyright © 2015 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yip, S; Coroller, T; Rios Velazquez, E
Purpose: Although PET-based radiomic features have been proposed to quantify tumor heterogeneity and shown promise in outcome prediction, little is known about their relationship with tumor genetics. This study assessed the association of [{sup 18}F]fluorodeoxyglucose (FDG)-PET-based radiomic features with non-small cell lung cancer (NSCLC) mutations. Methods: 348 NSCLC patients underwent FDG-PET/CT scans before treatment and were tested for genetic mutations. 13% (44/348) and 28% (96/348) patients were found to harbor EGFR (EGFR+) and KRAS (KRAS+) mutations, respectively. We evaluated nineteen PET-based radiomic features quantifying phenotypic traits, and compared them with conventional PET features (metabolic tumor volume (MTV) and maximum-SUV). Themore » association between the feature values and mutation status was evaluated using the Wilcoxcon-rank-sum-test. The ability of each measure to predict mutations was assessed by the area under the receiver operating curve (AUC). Noether’s test was used to determine if the AUCs were significantly from random (AUC=0.50). All p-values were corrected for multiple testing by controlling the false discovery rate (FDR{sub Wilcoxon} and FDR{sub Noether}) of 10%. Results: Eight radiomic features, MTV, and maximum-SUV, were significantly associated with the EGFR mutation (FDR{sub Wilcoxon}=0.01–0.10). However, KRAS+ demonstrated no significantly distinctive imaging features compared to KRAS− (FDR{sub Wilcoxon}≥0.92). EGFR+ and EGFR− were significantly discriminated by conventional PET features (AUC=0.61, FDR{sub Noether}=0.04 for MTV and AUC=0.64, FDR{sub Noether}=0.01 for maximum-SUV). Eight radiomic features were significantly predictive for EGFR+ compared to EGFR− (AUC=0.59–0.67, FDR{sub Noether}=0.0032–0.09). Normalized-inverse-difference-moment outperformed all features in predicting EGFR mutation (AUC=0.67, FDR{sub Noether}=0.0032). Moreover, only the radiomic feature normalized-inverse-difference-moment could significantly predict KRAS+ from EGFR+ (AUC=0.65, FDR{sub Noether}=0.05). All measures failed to predict KRAS+ from KRAS− (AUC=0.50–0.54, FDR{sub Noether}≥0.92). Conclusion: PET imaging features were strongly associated with EGFR mutations in NSCLC. Radiomic features have great potential in predicting EGFR mutations. Our study may help develop a non-invasive imaging biomarker for EGFR mutation. R.M. has consulting interests with Amgen.« less
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Protein and genome evolution in Mammalian cells for biotechnology applications.
Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J
2009-06-01
Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.
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Banaszak, Lauren G; Giudice, Valentina; Zhao, Xin; Wu, Zhijie; Gao, Shouguo; Hosokawa, Kohei; Keyvanfar, Keyvan; Townsley, Danielle M; Gutierrez-Rodrigues, Fernanda; Fernandez Ibanez, Maria Del Pilar; Kajigaya, Sachiko; Young, Neal S
2018-03-01
DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy. Published by Elsevier Inc.
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NASA Astrophysics Data System (ADS)
Nair, D. R.
2017-12-01
The purpose of this project was to determine the effect of two mutated oncogenes on the survival rate from invasive breast carcinoma when in comparison to the mutation of a single oncogene on the survival rate. An oncogene is defined as a gene, that when mutated, can lead to cancer. The two oncogenes used in this project were human epidermal growth factor receptor 2 (HER2) and c-myc (MYC). HER2 and MYC are both oncogenes that contribute to the formation of cancer. HER2 proteins are receptors on breast cells, and when the HER2 gene is mutated, there is an overexpression of HER2 protein on the breast cell. This makes the breast cells proliferate uncontrollably. MYC is a gene that codes for a transcription factor that plays a role in cell cycle progression. The overexpression of MYC also leads to the proliferation of cells. I hypothesized that if there is a mutation in both the MYC and HER2 genes, then the survival rate of invasive breast carcinoma patients will be lower compared to patients with the mutations of only MYC or HER2. To test this hypothesis, we conducted individual gene searches in CBioPortal for HER2 in the datasets from the studies titled TCGA Nature 2012, TCGA Cell 2015, and TCGA Provisional. We conducted individual gene searches in CBioPortal for MYC in the same datasets. The survival rate data was then exported and analyzed for patients with mutations of either HER2 or MYC and with mutations of both genes. To determine the cases that had both HER2 and MYC mutations, we found the overlapping cases in both HER2 and MYC groups for all three datasets. We calculated the median of the survival data for cases where either HER2 or MYC was mutated and cases where both MYC and HER2 were mutated. From the first dataset, the median of MYC data was 95.53, HER2 data was 95.83, and both HER2 and MYC data was 91.24. In the second dataset, the median of MYC data was 92.17 , HER2 data was 93.5, and both HER2 and MYC data was 87.95 . In the third dataset, the median of MYC data was 92.18, HER2 data was 94.22, and both HER2 and MYC data was 89.45. The median survival rates all showed that cases with mutations in both genes had a lower survival rate than those with single mutations. My hypothesis was supported. Some sources of error are the fewer number of cases in the TCGA Nature 2012 dataset, making this data statistically insignificant.
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Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith
2018-06-13
During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.
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Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andy; Fernandez, Soledad; Tsukahara, Tomonori; Ratner, Lee; Lairmore, Michael D.
2005-01-01
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo. PMID:16046523
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KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells
Lin, Yu-Lin; Ou, Da-Liang; Lin, Liang-In; Tseng, Li-Hui; Chang, Yih-Leong; Yeh, Kun-Huei; Cheng, Ann-Lii
2012-01-01
Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation. PMID:23209813
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Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung
2017-01-01
Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388
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A case report of Fanconi anemia diagnosed by genetic testing followed by prenatal diagnosis.
Lee, Hwa Jeen; Park, Seungman; Kang, Hyoung Jin; Jun, Jong Kwan; Lee, Jung Ae; Lee, Dong Soon; Park, Sung Sup; Seong, Moon-Woo
2012-09-01
Fanconi anemia (FA) is a rare genetic disorder affecting multiple body systems. Genetic testing, including prenatal testing, is a prerequisite for the diagnosis of many clinical conditions. However, genetic testing is complicated for FA because there are often many genes that are associated with its development, and large deletions, duplications, or sequence variations are frequently found in some of these genes. This study describes successful genetic testing for molecular diagnosis, and subsequent prenatal diagnosis, of FA in a patient and his family in Korea. We analyzed all exons and flanking regions of the FANCA, FANCC, and FANCG genes for mutation identification and subsequent prenatal diagnosis. Multiplex ligation-dependent probe amplification analysis was performed to detect large deletions or duplications in the FANCA gene. Molecular analysis revealed two mutations in the FANCA gene: a frameshift mutation c.2546delC and a novel splice-site mutation c.3627-1G>A. The FANCA mutations were separately inherited from each parent, c.2546delC was derived from the father, whereas c.3627-1G>A originated from the mother. The amniotic fluid cells were c.3627-1G>A heterozygotes, suggesting that the fetus was unaffected. This is the first report of genetic testing that was successfully applied to molecular diagnosis of a patient and subsequent prenatal diagnosis of FA in a family in Korea.
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A thermodynamic assay to test pharmacological chaperones for Fabry disease.
Andreotti, Giuseppina; Citro, Valentina; Correra, Antonella; Cubellis, Maria Vittoria
2014-03-01
The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Leung, F.; Cataldo, D.A.; Fellows, R.J.
1995-09-01
Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screenedmore » and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.« less
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Bellone, Stefania; Eliana, Bignotti; Lonardi, Silvia; Ferrari, Francesca; Centritto, Floriana; Masserdotti, Alice; Pettinella, Francesca; Black, Jonathan; Menderes, Gulden; Altwerger, Gary; Hui, Pei; Lopez, Salvatore; de Haydu, Christopher; Bonazzoli, Elena; Predolini, Federica; Zammataro, Luca; Cocco, Emiliano; Ferrari, Federico; Ravaggi, Antonella; Romani, Chiara; Facchettie, Fabio; Sartori, Enrico; Odicino, Franco E.; Silasi, Dan-Arin; Litkouhi, Babak; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E.; Santin, Alessandro D.
2016-01-01
Objective Up to 12 % of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. Methods Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9–14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. Results Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P = .001) and CD8+ (32.8 vs 13.5; P < .001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P < .001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P < 0.004). Conclusions POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity. PMID:27894751
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Beck, Raphaël; Pedrosa, Rozangela Curi; Dejeans, Nicolas; Glorieux, Christophe; Levêque, Philippe; Gallez, Bernard; Taper, Henryk; Eeckhoudt, Stéphane; Knoops, Laurent; Calderon, Pedro Buc; Verrax, Julien
2011-10-01
Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.
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Siena, S; Sartore-Bianchi, A; Garcia-Carbonero, R; Karthaus, M; Smith, D; Tabernero, J; Van Cutsem, E; Guan, X; Boedigheimer, M; Ang, A; Twomey, B; Bach, B A; Jung, A S; Bardelli, A
2018-01-01
Mutations in rat sarcoma (RAS) genes may be a mechanism of secondary resistance in epidermal growth factor receptor inhibitor-treated patients. Tumor-tissue biopsy testing has been the standard for evaluating mutational status; however, plasma testing of cell-free DNA has been shown to be a more sensitive method for detecting clonal evolution. Archival pre- and post-treatment tumor biopsy samples from a phase II study of panitumumab in combination with irinotecan in patients with metastatic colorectal cancer (mCRC) that also collected plasma samples before, during, and after treatment were analyzed for emergence of mutations during/post-treatment by next-generation sequencing and BEAMing. The rate of emergence of tumor tissue RAS mutations was 9.5% by next-generation sequencing (n = 21) and 6.3% by BEAMing (n = 16). Plasma testing of cell-free DNA by BEAMing revealed a mutant RAS emergence rate of 36.7% (n = 39). Exploratory outcomes analysis of plasma samples indicated that patients who had emergent RAS mutations at progression had similar median progression-free survival to those patients who remained wild-type at progression. Serial analysis of plasma samples showed that the first detected emergence of RAS mutations preceded progression by a median of 3.6 months (range, -0.3 to 7.5 months) and that there did not appear to be a mutant RAS allele frequency threshold that could predict near-term outcomes. This first prospective analysis in mCRC showed that serial plasma biopsies are more inclusive than tissue biopsies for evaluating global tumor heterogeneity; however, the clinical utility of plasma testing in mCRC remains to be further explored. NCT00891930. © The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
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Avrahami-Moyal, Liat; Engelberg, David; Wenger, Jared. W.; Sherlock, Gavin; Braun, Sergei
2012-01-01
We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 h-1 to 0.32 h-1. Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within six days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within two days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of 6 clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 out of 19 tolerant clones the stop-codon in ssd1-d was replaced with an aminoacid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol. PMID:22443114
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Giovannini, Marco; Robanus-Maandag, Els; Niwa-Kawakita, Michiko; van der Valk, Martin; Woodruff, James M.; Goutebroze, Laurence; Mérel, Philippe; Berns, Anton; Thomas, Gilles
1999-01-01
Specific mutations in some tumor suppressor genes such as p53 can act in a dominant fashion. We tested whether this mechanism may also apply for the neurofibromatosis type-2 gene (NF2) which, when mutated, leads to schwannoma development. Transgenic mice were generated that express, in Schwann cells, mutant NF2 proteins prototypic of natural mutants observed in humans. Mice expressing a NF2 protein with an interstitial deletion in the amino-terminal domain showed high prevalence of Schwann cell-derived tumors and Schwann cell hyperplasia, whereas those expressing a carboxy-terminally truncated protein were normal. Our results indicate that a subset of mutant NF2 alleles observed in patients may encode products with dominant properties when overexpressed in specific cell lineages. PMID:10215625
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Martin, Daniel; Abba, Martin C.; Molinolo, Alfredo A.; Vitale-Cross, Lynn; Wang, Zhiyong; Zaida, Moraima; Delic, Naomi C.; Samuels, Yardena; Lyons, J. Guy; Gutkind, J. Silvio
2014-01-01
The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration. PMID:25275298
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Legault, Jean; Larouche, Pierre-Luc; Côté, Isabelle; Bouchard, Line; Pichette, André; Robinson, Brian H; Morin, Charles
2011-01-01
Leigh syndrome French Canadian (LSFC) is a recessive disease caused by mutations in the LRPPRC gene (leucine-rich pentatricopeptide repeat containing protein). These mutations induce a cytochrome c oxidase (COX) deficiency resulting in episodes of acute acidotic crisis that will often lead to death. There is no effective treatment. Methylene blue (MB) is a redox dye that increases COX content and activity in vitro and in vivo suggesting that MB could prevent and treat LSFC. In this study, the protective effect of low-concentration MB was tested on two LSFC cell lines, including LSFC-F1, homozygous for the mutation A354V, and LSFC-F2 a compound heterozygous for the mutations A354V and C12775STOP. MB effect on metabolic activity was assessed on both LSFC cells in stable and acidotic conditions. For LSFC-F1, results showed that metabolic activity drastically decline after 96 hours in both conditions but not LSFC-F2 and normal cells. MB completely prevents the decrease of metabolic activity in LSFC-F1. Intracellular ATP content was also measured in both culture media. After 96 hours in acidotic medium, ATP content was almost completely depleted for both LSFC cells. Interestingly, MB completely restores ATP content in LSFC-F1 and LSFC-F2 cells. Finally, MB strongly improves the survival of both LSFC cells.
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Narita, Yusuke; Matsushima, Yukiko; Shiroiwa, Takeru; Chiba, Koji; Nakanishi, Yoichi; Kurokawa, Tatsuo; Urushihara, Hisashi
2015-10-01
The combination use of gefitinib and epidermal growth factor receptor (EGFR) testing is a standard first-line therapy for patients with non-small cell lung cancer (NSCLC). Here, we examined the cost-effectiveness of this approach in Japan. Our analysis compared the 'EGFR testing strategy', in which EGFR mutation testing was performed before treatment and patients with EGFR mutations received gefitinib while those without mutations received standard chemotherapy, to the 'no-testing strategy,' in which genetic testing was not conducted and all patients were treated with standard chemotherapy. A three-state Markov model was constructed to predict expected costs and outcomes for each strategy. We included only direct medical costs from the healthcare payer's perspective. Outcomes in the model were based on those reported in the Iressa Pan-Asia Study (IPASS). The incremental cost-effectiveness ratio (ICER) was calculated using quality-adjusted life-years (QALYs) gained. Sensitivity and scenario analyses were conducted. The incremental cost and effectiveness per patient of the 'EGFR testing strategy' compared to the 'no-testing strategy' was estimated to be approximately JP¥122,000 (US$1180; US$1=JP¥104 as of February 2014) and 0.036 QALYs. The ICER was then calculated to be around JP¥3.38 million (US$32,500) per QALY gained. These results suggest that the 'EGFR testing strategy' is cost-effective compared with the 'no-testing strategy' when JP¥5.0 million to 6.0 million per QALY gained is considered an acceptable threshold. These results were supported by the sensitivity and scenario analyses. The combination use of gefitinib and EGFR testing can be considered a cost-effective first-line therapy compared to chemotherapy such as carboplatin-paclitaxel for the treatment for NSCLC in Japan. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
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Nicoś, Marcin; Krawczyk, Paweł; Jarosz, Bożena; Sawicki, Marek; Szumiłło, Justyna; Trojanowski, Tomasz; Milanowski, Janusz
2016-05-01
KRAS mutations are associated with tumor resistance to EGFR TKIs (erlotinib, gefitinib) and to monoclonal antibody against EGFR (cetuximab). Targeted treatment of mutated RAS patients is still considered as a challenge. Inhibitors of c-Met (onartuzumab or tiwantinib) and MEK (selumetinib-a dual inhibitor of MEK1 and MEK2) signaling pathways showed activity in patients with mutations in KRAS that can became an effective approach in carriers of such disorders. BRAF mutation is very rare in patients with NSCLC, and its presence is associated with sensitivity of tumor cells to BRAF inhibitors (vemurafenib, dabrafenib). In the present study, the frequency and type of KRAS and BRAF mutation were assessed in 145 FFPE tissue samples from CNS metastases of NSCLC. In 30 patients, material from the primary tumor was simultaneously available. Real-time PCR technique with allele-specific molecular probe (KRAS/BRAF Mutation Analysis Kit, Entrogen, USA) was used for molecular tests. KRAS mutations were detected in 21.4 % of CNS metastatic lesions and in 23.3 % of corresponding primary tumors. Five mutations were identified both in primary and in metastatic lesions, while one mutation only in primary tumor and one mutation only in the metastatic tumor. Most of mutations were observed in codon 12 of KRAS; however, an individual patient had diagnosed a rare G13D and Q61R substitutions. KRAS mutations were significantly more frequent in adenocarcinoma patients and smokers. Additional analysis indicated one patient with rare coexistence of KRAS and DDR2 mutations. BRAF mutation was not detected in the examined materials. KRAS frequency appears to be similar in primary and CNS.
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Nocturnal frontal lobe epilepsy with paroxysmal arousals due to CHRNA2 loss of function.
Conti, Valerio; Aracri, Patrizia; Chiti, Laura; Brusco, Simone; Mari, Francesco; Marini, Carla; Albanese, Maria; Marchi, Angela; Liguori, Claudio; Placidi, Fabio; Romigi, Andrea; Becchetti, Andrea; Guerrini, Renzo
2015-04-14
We assessed the mutation frequency in nicotinic acetylcholine receptor (nAChR) subunits CHRNA4, CHRNB2, and CHRNA2 in a cohort including autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and sporadic nocturnal frontal lobe epilepsy (NFLE). Upon finding a novel mutation in CHRNA2 in a large family, we tested in vitro its functional effects. We sequenced all the coding exons and their flanking intronic regions in 150 probands (73 NFLE, 77 ADNFLE), in most of whom diagnosis had been validated by EEG recording of seizures. Upon finding a missense mutation in CHRNA2, we measured whole-cell currents in human embryonic kidney cells in both wild-type and mutant α2β4 and α2β2 nAChR subtypes stimulated with nicotine. We found a c.889A>T (p.Ile297Phe) mutation in the proband (≈0.6% of the whole cohort) of a large ADNFLE family (1.2% of familial cases) and confirmed its segregation in all 6 living affected individuals. Video-EEG studies demonstrated sleep-related paroxysmal epileptic arousals in all mutation carriers. Oxcarbazepine treatment was effective in all. Whole-cell current density was reduced to about 40% in heterozygosity and to 0% in homozygosity, with minor effects on channel permeability and sensitivity to nicotine. ADNFLE had previously been associated with CHRNA2 dysfunction in one family, in which a gain of function mutation was demonstrated. We confirm the causative role of CHRNA2 mutations in ADNFLE and demonstrate that also loss of function of α2 nAChRs may have pathogenic effects. CHRNA2 mutations are a rare cause of ADNFLE but this gene should be included in mutation screening. © 2015 American Academy of Neurology.
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New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy
Marchant, D; Yu, K; Bigot, K; Roche, O; Germain, A; Bonneau, D; Drouin‐Garraud, V; Schorderet, D F; Munier, F; Schmidt, D; Neindre, P Le; Marsac, C; Menasche, M; Dufier, J L; Fischmeister, R; Hartzell, C; Abitbol, M
2007-01-01
Purpose The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca2+‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl− channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. Methods Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl− current was examined using whole‐cell patch clamp analysis. Results Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner. Conclusions This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane. PMID:17287362
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Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib.
Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L; Khan, Tahsin M; Gainor, Justin F; Iafrate, A John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A; Shaw, Alice T
2014-11-15
The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non-small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. ©2014 American Association for Cancer Research.
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Hashimoto, Masami; Bacman, Sandra R; Peralta, Susana; Falk, Marni J; Chomyn, Anne; Chan, David C; Williams, Sion L; Moraes, Carlos T
2015-01-01
We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNALys gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials. PMID:26159306
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de Andrade, H H; Marques, E K; Schenberg, A C; Henriques, J A
1989-06-01
The induction of mitotic gene conversion and crossing-over in Saccharomyces cerevisiae diploid cells homozygous for the pso4-1 mutation was examined in comparison to the corresponding wild-type strain. The pso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for the pso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of the PSO4 gene. On the other hand, the pso4-1 mutant is mutationally defective for all agents used. Therefore, the pso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that the PSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between the pso4-1 mutation and the RecA or LexA mutation of Escherichia coli.
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Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi
2014-02-01
Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.
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Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4
Harris, Rebecca M.; Weiss, Jeffrey
2011-01-01
The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561
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Ding, Zhiyong; German, Peter; Bai, Shanshan; Feng, Zhehui; Gao, Meng; Si, Wendy; Sobieski, Mary M.; Stephan, Clifford C.; Mills, Gordon B.; Jonasch, Eric
2014-01-01
Background von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL. Methods The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds. Conclusion 786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL. PMID:22357874
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Oki, Masahide; Yatabe, Yasushi; Saka, Hideo; Kitagawa, Chiyoe; Kogure, Yoshihito; Ichihara, Shu; Moritani, Suzuko
2015-01-01
During bronchoscopy, small biopsy forceps are increasingly used for the diagnosis of peripheral pulmonary lesions. However, it is unclear whether the formalin-fixed paraffin-embedded specimens sampled with the small biopsy forceps are suitable for the determination of genotypes which become indispensable for the management decision regarding patients with non-small cell lung cancer. The aim of this study was to evaluate the feasibility and accuracy of molecular testing in the specimens obtained with 1.5-mm small biopsy forceps. We examined specimens in 91 patients, who were enrolled in our previous 3 studies on the usefulness of thin bronchoscopes and given a diagnosis of non-small cell lung cancer by bronchoscopy with the 1.5-mm biopsy forceps, and then underwent surgical resection. An experienced pathologist examined paraffin-embedded specimens obtained by bronchoscopic biopsy or surgical resection in a blind fashion on epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) rearrangements and KRAS mutations. Twenty-five (27%), 2 (2%) and 5 (5%) patients had an EGFR mutation, ALK rearrangement and KRAS mutation, respectively, based on the results in surgical specimens. EGFR, ALK and KRAS testing with bronchoscopic specimens was feasible in 82 (90%), 86 (95%) and 83 (91%) patients, respectively. If molecular testing was feasible, the accuracy of EGFR, ALK and KRAS testing with bronchoscopic specimens for the results with surgical specimens was 98, 100 and 98%, respectively. The results of molecular testing in the formalin-fixed paraffin-embedded specimens obtained with the small forceps, in which the genotype could be evaluated, correlated well with those in surgically resected specimens.
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CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation
Wu, Zhi-Jie; Zhao, Xin; Banaszak, Lauren G.; Gutierrez-Rodrigues, Fernanda; Keyvanfar, Keyvan; Gao, Shou-Guo; Raffo, Diego Quinones; Kajigaya, Sachiko; Young, Neal S.
2018-01-01
Additional sex combs-like 1 (ASXL1) is a well-known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis. PMID:29532865
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Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V
2018-03-20
Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.
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French, Deborah; Smith, Andrew; Powers, Martin P; Wu, Alan H B
2011-08-17
Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist. Copyright © 2011 Elsevier B.V. All rights reserved.
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Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo
2018-01-01
We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.
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Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).
Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R
2015-01-01
Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134). © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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A novel genotoxic aspect of thiabendazole as a photomutagen in bacteria and cultured human cells.
Watanabe-Akanuma, Mie; Ohta, Toshihiro; Sasaki, Yu F
2005-09-15
Thiabendazole (TBZ) is a post-harvest fungicide commonly used on imported citrus fruits. We recently found that TBZ showed photomutagenicity with UVA-irradiation in the Ames test using plate incorporation method. In the present study, potential of DNA-damaging activity, mutagenicity, and clastogenicity were investigated by short pulse treatment for 10 min with TBZ (50-400 microg/ml) and UVA-irradiation (320-400 nm, 250 microW/cm2) in bacterial and human cells. UVA-irradiated TBZ caused DNA damage in Escherichia coli and human lymphoblastoid WTK1 cells assayed, respectively, by the umu-test and the single cell gel electrophoresis (comet) assay. In a modified Ames test using Salmonella typhimurium and E. coli, strong induction of -1 frameshift mutations as well as base-substitution mutations were detected. TBZ at 50-100 microg/ml with UVA-irradiation significantly induced micronuclei in WTK1 cells in the in vitro cytochalasin-B micronucleus assay. Pulse treatment for 10 min with TBZ alone did not show any genotoxicity. Although TBZ is a spindle poison that induces aneuploidy, we hypothesize that the photogenotoxicity of TBZ in the present study was produced by a different mechanism, probably by DNA adduct formation. We concluded that UVA-activated TBZ is genotoxic in bacterial and human cells in vitro.
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Sewell, Andrew; Brown, Brandee; Biktasova, Asel; Mills, Gordon B; Lu, Yiling; Tyson, Darren R; Issaeva, Natalia; Yarbrough, Wendell G
2014-05-01
Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers. ©2014 AACR.
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Li, S; Li, L; Zhu, Y; Huang, C; Qin, Y; Liu, H; Ren-Heidenreich, L; Shi, B; Ren, H; Chu, X; Kang, J; Wang, W; Xu, J; Tang, K; Yang, H; Zheng, Y; He, J; Yu, G; Liang, N
2014-01-01
Background: Determining the somatic mutations of epidermal growth factor receptor (EGFR)-pathway networks is the key to effective treatment for non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs).The somatic mutation frequencies and their association with gender, smoking history and histology was analysed and reported in this study. Methods: Five thousand one hundred and twenty-five NSCLC patients' pathology samples were collected, and EGFR, KRAS, BRAF and PIK3CA mutations were detected by multiplex testing. The mutation status of EGFR, KRAS, BRAF and PIK3CA and their association with gender, age, smoking history and histological type were evaluated by appropriate statistical analysis. Results: EGFR, KRAS, BRAF and PIK3CA mutation rates revealed 36.2%, 8.4%, 0.5% and 3.3%, respectively, across the 5125 pathology samples. For the first time, evidence of KRAS mutations were detected in two female, non-smoking patients, age 5 and 14, with NSCLC. Furthermore, we identified 153 double and coexisting mutations and 7 triple mutations. Interestingly, the second drug-resistant mutations, T790M or E545K, were found in 44 samples from patients who had never received TKI treatments. Conclusions: EGFR exons 19, 20 and 21, and BRAF mutations tend to happen in females and non-smokers, whereas KRAS mutations were more inclined to males and smokers. Activating and resistant mutations to EGFR-TKI drugs can coexist and ‘second drug-resistant mutations', T790M or E545K, may be primary mutations in some patients. These results will help oncologists to decide candidates for mutation testing and EGFR-TKI treatment. PMID:24743704
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Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.
Mikami, Masafumi; Toki, Seiichi; Endo, Masaki
2015-10-01
Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.
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Wen, Miaomiao; Wang, Xuejiao; Sun, Ying; Xia, Jinghua; Fan, Liangbo; Xing, Hao; Zhang, Zhipei; Li, Xiaofei
2016-01-01
Echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ (2) test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent.
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Wen, Miaomiao; Wang, Xuejiao; Sun, Ying; Xia, Jinghua; Fan, Liangbo; Xing, Hao; Zhang, Zhipei; Li, Xiaofei
2016-01-01
Purpose Echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR) define specific molecular subsets of lung cancer with distinct clinical features. We aimed at revealing the clinical features of EML4-ALK fusion gene and EGFR mutation in non-small-cell lung cancer (NSCLC). Methods We enrolled 694 Chinese patients with NSCLC for analysis. EML4-ALK fusion gene was analyzed by real-time polymerase chain reaction, and EGFR mutations were analyzed by amplified refractory mutation system. Results Among the 694 patients, 60 (8.65%) patients had EML4-ALK fusions. In continuity correction χ2 test analysis, EML4-ALK fusion gene was correlated with sex, age, smoking status, and histology, but no significant association was observed between EML4-ALK fusion gene and clinical stage. A total of 147 (21.18%) patients had EGFR mutations. In concordance with previous reports, EGFR mutation was correlated with age, smoking status, histology, and clinical stage, whereas patient age was not significantly associated with EGFR mutation. Meanwhile, to our surprise, six (0.86%) patients had coexisting EML4-ALK fusions and EGFR mutations. Conclusion EML4-ALK fusion gene defines a new molecular subset in patients with NSCLC. Six patients who harbored both EML4-ALK fusion genes and EGFR mutations were identified in our study. The EGFR mutations and the EML4-ALK fusion genes are coexistent. PMID:27103824
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Wu, Yi-Long; Sequist, Lecia V; Hu, Cheng-Ping; Feng, Jifeng; Lu, Shun; Huang, Yunchao; Li, Wei; Hou, Mei; Schuler, Martin; Mok, Tony; Yamamoto, Nobuyuki; O'Byrne, Kenneth; Hirsh, Vera; Gibson, Neil; Massey, Dan; Kim, Miyoung; Yang, James Chih-Hsin
2017-01-01
Background: In the Phase III LUX-Lung 3/6 (LL3/LL6) trials in epidermal growth factor receptor (EGFR) mutation-positive lung adenocarcinoma patients, we evaluated feasibility of EGFR mutation detection using circulating cell-free DNA (cfDNA) and prognostic and predictive utility of cfDNA positivity (cfDNA+). Methods: Paired tumour and blood samples were prospectively collected from randomised patients. Mutations were detected using cfDNA from serum (LL3) or plasma (LL6) by a validated allele-specific quantitative real-time PCR kit. Results: EGFR mutation detection rates in cfDNA were 28.6% (serum) and 60.5% (plasma). Mutation detection in blood was associated with advanced disease characteristics, including higher performance score, number of metastatic sites and bone/liver metastases, and poorer prognosis. In patients with common EGFR mutations, afatinib improved progression-free survival vs chemotherapy in cfDNA+ (LL3: HR, 0.35; P=0.0009; LL6: HR, 0.25; P<0.0001) and cfDNA− (LL3: HR, 0.46; P<0.0001; LL6: HR, 0.12; P<0.0001) cohorts. A trend towards overall survival benefit with afatinib was observed in cfDNA+ patients. Conclusions: Plasma cfDNA is a promising alternative to biopsy for EGFR testing. Detectable mutation in blood was associated with more advanced disease and poorer prognosis. Afatinib improved outcomes in EGFR mutation-positive patients regardless of blood mutation status. PMID:28006816
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Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells.
Hollenbach, Jessica P; Resch, Alissa M; Palakodeti, Dasaradhi; Graveley, Brenton R; Heinen, Christopher D
2011-01-01
Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.
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Loss of DNA Mismatch Repair Imparts a Selective Advantage in Planarian Adult Stem Cells
Hollenbach, Jessica P.; Resch, Alissa M.; Palakodeti, Dasaradhi; Graveley, Brenton R.; Heinen, Christopher D.
2011-01-01
Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis. PMID:21747960
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Choi, M; Kadara, H; Zhang, J; Parra, E R; Rodriguez-Canales, J; Gaffney, S G; Zhao, Z; Behrens, C; Fujimoto, J; Chow, C; Kim, K; Kalhor, N; Moran, C; Rimm, D; Swisher, S; Gibbons, D L; Heymach, J; Kaftan, E; Townsend, J P; Lynch, T J; Schlessinger, J; Lee, J; Lifton, R P; Herbst, R S; Wistuba, I I
2017-01-01
Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.
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Lee, H; Li, D; Prior, T; Casto, B C; Weghorst, C M; Shuler, C F; Milo, G E
1997-10-01
Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells' ability to exhibit a malignant potential in nude mice.
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Hereditary diffuse gastric cancer: surgery, surveillance and unanswered questions.
Cisco, Robin M; Norton, Jeffrey A
2008-08-01
Hereditary diffuse gastric cancer (HDGC) is an inherited cancer-susceptibility syndrome characterized by autosomal dominance and high penetrance. In 30-50% of cases, a causative germline mutation in CDH1, the E-cadherin gene, may be identified. Female carriers of CDH1 mutations also have an increased (20-40%) risk of lobular breast cancer. Endoscopic surveillance of patients with CDH1 mutations is ineffective because early foci of HDGC are typically small and underlie normal mucosa. CDH1 mutation carriers are therefore offered the option of prophylactic gastrectomy, which commonly reveals early foci of invasive signet-ring cell cancer. We review recommendations for genetic testing, surveillance and prophylactic surgery in HDGC. Areas for future research are discussed, including development of new screening modalities, optimal timing of prophylactic gastrectomy, identification of additional causative mutations in HDGC, management of patients with CDH1 missense mutations and prevention/early detection of lobular breast cancer in CDH1 mutation carriers.
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Akin, C; Kirshenbaum, A S; Semere, T; Worobec, A S; Scott, L M; Metcalfe, D D
2000-02-01
The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.
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Tsui, Nancy B Y; Kadir, Rezan A; Chan, K C Allen; Chi, Claudia; Mellars, Gillian; Tuddenham, Edward G; Leung, Tak Y; Lau, Tze K; Chiu, Rossa W K; Lo, Y M Dennis
2011-03-31
Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.
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Somatic mutations reveal asymmetric cellular dynamics in the early human embryo
Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...
2017-03-22
Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less
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Somatic mutations reveal asymmetric cellular dynamics in the early human embryo
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz
Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less
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HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib.
Yang, Guang; Buhrlage, Sara J; Tan, Li; Liu, Xia; Chen, Jie; Xu, Lian; Tsakmaklis, Nicholas; Chen, Jiaji G; Patterson, Christopher J; Brown, Jennifer R; Castillo, Jorge J; Zhang, Wei; Zhang, Xiaofeng; Liu, Shuai; Cohen, Philip; Hunter, Zachary R; Gray, Nathanael; Treon, Steven P
2016-06-23
Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. © 2016 by The American Society of Hematology.
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A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.
O'Connell, K F; Leys, C M; White, J G
1998-01-01
A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522
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ROS1 fusions rarely overlap with other oncogenic drivers in non-small cell lung cancer
Lin, Jessica J.; Ritterhouse, Lauren L.; Ali, Siraj M.; Bailey, Mark; Schrock, Alexa B.; Gainor, Justin F.; Ferris, Lorin A.; Mino-Kenudson, Mari; Miller, Vincent A.; Iafrate, Anthony J.; Lennerz, Jochen K.; Shaw, Alice T.
2017-01-01
Introduction Chromosomal rearrangements involving the ROS proto-oncogene 1 receptor tyrosine kinase gene (ROS1) define a distinct molecular subset of non-small cell lung cancer (NSCLC) with sensitivity to ROS1 inhibitors. Recent reports have suggested a significant overlap between ROS1 fusions and other oncogenic driver alterations, including mutations in epidermal growth factor receptor (EGFR) and KRAS proto-oncogene (KRAS). Methods We identified patients at our institution with ROS1-rearranged NSCLC who had undergone testing for genetic alterations in additional oncogenes, including EGFR, KRAS, and anaplastic lymphoma kinase (ALK). Clinicopathologic features and genetic testing results were reviewed. We also examined a separate database of ROS1-rearranged NSCLCs identified through a commercial FoundationOne assay. Results Among 62 patients with ROS1-rearranged NSCLC evaluated at our institution, none harbored concurrent ALK fusions (0%) or EGFR activating mutations (0%). KRAS mutations were detected in two cases (3.2%), one of which harbored a concurrent non-canonical KRAS I24N mutation of unknown biological significance. In a separate ROS1 FISH-positive case, targeted sequencing failed to confirm a ROS1 fusion, but instead identified a KRAS G13D mutation. No concurrent mutations in BRAF, ERBB2, PIK3CA, AKT1, or MAP2K1 were detected. Analysis of an independent dataset of 166 ROS1-rearranged NSCLCs identified by FoundationOne demonstrated rare cases with co-occurring driver mutations in EGFR (1/166) and KRAS (3/166), and no cases with co-occurring ROS1 and ALK rearrangements. Conclusions ROS1 rearrangements rarely overlap with alterations in EGFR, KRAS, ALK, or other targetable oncogenes in NSCLC. PMID:28088512
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Pathogenesis and treatment of adult-type granulosa cell tumor of the ovary.
Färkkilä, Anniina; Haltia, Ulla-Maija; Tapper, Johanna; McConechy, Melissa K; Huntsman, David G; Heikinheimo, Markku
2017-08-01
Adult-type granulosa cell tumor is a clinically and molecularly unique subtype of ovarian cancer. These tumors originate from the sex cord stromal cells of the ovary and represent 3-5% of all ovarian cancers. The majority of adult-type granulosa cell tumors are diagnosed at an early stage with an indolent prognosis. Surgery is the cornerstone for the treatment of both primary and relapsed tumor, while chemotherapy is applied only for advanced or non-resectable cases. Tumor stage is the only factor consistently associated with prognosis. However, every third of the patients relapse, typically in 4-7 years from diagnosis, leading to death in 50% of these patients. Anti-Müllerian Hormone and inhibin B are currently the most accurate circulating biomarkers. Adult-type granulosa cell tumors are molecularly characterized by a pathognomonic somatic missense point mutation 402C->G (C134W) in the transcription factor FOXL2. The FOXL2 402C->G mutation leads to increased proliferation and survival of granulosa cells, and promotes hormonal changes. Histological diagnosis of adult-type granulosa cell tumor is challenging, therefore testing for the FOXL2 mutation is crucial for differential diagnosis. Large international collaborations utilizing molecularly defined cohorts are essential to improve and validate new treatment strategies for patients with high-risk or relapsed adult-type granulosa cell tumor. Key Messages: Adult-type granulosa cell tumor is a unique ovarian cancer with an indolent, albeit unpredictable disease course. Adult-type granulosa cell tumors harbor a pathognomonic somatic missense mutation in transcription factor FOXL2. The key challenges in the treatment of patients with adult-type granulosa cell tumor lie in the identification and management of patients with high-risk or relapsed disease.
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Ehrentraut, Stefan; Schneider, Björn; Nagel, Stefan; Pommerenke, Claudia; Quentmeier, Hilmar; Geffers, Robert; Feist, Maren; Kaufmann, Maren; Meyer, Corinna; Kadin, Marshall E; Drexler, Hans G; MacLeod, Roderick A F
2016-06-07
We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 - STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.
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ALK-rearrangements and testing methods in non-small cell lung cancer: a review
Shackelford, Rodney E.; Vora, Moiz; Mayhall, Kim; Cotelingam, James
2014-01-01
The anaplastic lymphoma tyrosine kinase (ALK) gene was first described as a driver mutation in anaplastic non-Hodgkin's lymphoma. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies, including 4-9% of non-small cell lung cancers (NSCLC). The tyrosine kinase inhibitor crizotinib is more effective than standard chemotherapeutic agents in treating ALK positive NSCLC, making molecular diagnostic testing for dysregulated ALK expression a necessary step in identifying optimal treatment modalities. Here we review ALKmediated signal transduction pathways and compare the molecular protocols used to identify dysregulated ALK expression in NSCLC. We also discuss the use of crizotinib and second generation ALK tyrosine kinase inhibitors in the treatment of ALK positive NSCLC, and the known mechanisms of crizotinib resistance in NSCLC. PMID:24955213
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Huang, Hsien-Neng; Chiang, Ying-Cheng; Cheng, Wen-Fang; Chen, Chi-An; Lin, Ming-Chieh; Kuo, Kuan-Ting
2015-02-01
Recently, mutations of telomerase reverse transcriptase (TERT) promoter were found in several types of cancer. A few reports demonstrate TERT promoter mutations in ovarian clear cell carcinomas but endometrial clear cell carcinoma has not been studied. The aims of this study were to compare differences of molecular alterations and clinical factors, and identify their prognostic impact in endometrial and ovarian clear cell carcinomas. We evaluated mutations of the TERT promoter and PIK3CA, expression of ARID1A, and other clinicopathological factors in 56 ovarian and 14 endometrial clear cell carcinomas. We found that TERT promoter mutations were present in 21% (3/14) of endometrial clear cell carcinomas and 16% (9/56) of ovarian clear cell carcinomas. Compared with ovarian clear cell carcinomas, endometrial clear cell carcinomas showed older mean patient age (P<0.001), preserved ARID1A immunoreactivity (P=0.017) and infrequent PIK3CA mutation (P=0.025). In ovarian clear cell carcinomas, TERT promoter mutations were correlated with patient age >45 (P=0.045) and preserved ARID1A expression (P=0.003). In cases of endometrial clear cell carcinoma, TERT promoter mutations were not statistically associated with any other clinicopathological factors. In ovarian clear cell carcinoma patients with early FIGO stage (stages I and II), TERT promoter mutation was an independent prognostic factor and correlated with a shorter disease-free survival and overall survival (P=0.015 and 0.009, respectively). In recurrent ovarian clear cell carcinoma patients with early FIGO stage, TERT promoter mutations were associated with early relapse within 6 months (P=0.018). We concluded that TERT promoter mutations were present in endometrial and ovarian clear cell carcinomas. Distinct molecular alteration patterns in endometrial and ovarian clear cell carcinomas implied different processes of tumorigenesis in these morphologically similar tumors. In ovarian clear cell carcinoma of early FIGO stage, patients with TERT promoter mutation require close follow-up during the initial 6 months following chemotherapy.
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Go, Derek E.; Talati, Ish; Ying, Yong; Rao, Jianyu; Kulkarni, Rajan P.; Di Carlo, Dino
2013-01-01
Evaluation of pleural fluids for metastatic cells is a key component of diagnostic cytopathology. However, a large background of smaller leukocytes and/or erythrocytes can make accurate diagnosis difficult and reduce specificity in identification of mutations of interest for targeted anti-cancer therapies. Here, we describe an automated microfluidic system (Centrifuge Chip) which employs microscale vortices for the size-based isolation and concentration of cancer cells and mesothelial cells from a background of blood cells. We are able to process non-diluted pleural fluids at 6 mL/min and enrich target cells significantly over the background; we achieved improved purity in all patient samples analyzed. The resulting isolated and viable cells are readily available for immunostaining, cytological analysis, and detection of gene mutations. To demonstrate the utility towards aiding companion diagnostics, we also show improved detection accuracy of KRAS gene mutations in lung cancer cells processed using the Centrifuge Chip, leading to an increase in the area under the curve (AUC) of the receiver operating characteristic from 0.90 to 0.99. The Centrifuge Chip allows for rapid concentration and processing of large volumes of bodily fluid samples for improved cytological diagnosis and purification of cells of interest for genetic testing, which will be helpful for enhancing diagnostic accuracy. PMID:24205153
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Proof-of-principle rapid noninvasive prenatal diagnosis of autosomal recessive founder mutations
Zeevi, David A.; Altarescu, Gheona; Weinberg-Shukron, Ariella; Zahdeh, Fouad; Dinur, Tama; Chicco, Gaya; Herskovitz, Yair; Renbaum, Paul; Elstein, Deborah; Levy-Lahad, Ephrat; Rolfs, Arndt; Zimran, Ari
2015-01-01
BACKGROUND. Noninvasive prenatal testing can be used to accurately detect chromosomal aneuploidies in circulating fetal DNA; however, the necessity of parental haplotype construction is a primary drawback to noninvasive prenatal diagnosis (NIPD) of monogenic disease. Family-specific haplotype assembly is essential for accurate diagnosis of minuscule amounts of circulating cell-free fetal DNA; however, current haplotyping techniques are too time-consuming and laborious to be carried out within the limited time constraints of prenatal testing, hampering practical application of NIPD in the clinic. Here, we have addressed this pitfall and devised a universal strategy for rapid NIPD of a prevalent mutation in the Ashkenazi Jewish (AJ) population. METHODS. Pregnant AJ couples, carrying mutation(s) in GBA, which encodes acid β-glucosidase, were recruited at the SZMC Gaucher Clinic. Targeted next-generation sequencing of GBA-flanking SNPs was performed on peripheral blood samples from each couple, relevant mutation carrier family members, and unrelated individuals who are homozygotes for an AJ founder mutation. Allele-specific haplotypes were constructed based on linkage, and a consensus Gaucher disease–associated founder mutation–flanking haplotype was fine mapped. Together, these haplotypes were used for NIPD. All test results were validated by conventional prenatal or postnatal diagnostic methods. RESULTS. Ten parental alleles in eight unrelated fetuses were diagnosed successfully based on the noninvasive method developed in this study. The consensus mutation–flanking haplotype aided diagnosis for 6 of 9 founder mutation alleles. CONCLUSIONS. The founder NIPD method developed and described here is rapid, economical, and readily adaptable for prenatal testing of prevalent autosomal recessive disease-causing mutations in an assortment of worldwide populations. FUNDING. SZMC, Protalix Biotherapeutics Inc., and Centogene AG. PMID:26426075
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Chiu, Yi-Jen; Nam, Mun-Kit; Tsai, Yueh-Ting; Huang, Chun-Chi; Tsai, Cheng-Chih
2013-01-01
Genotoxicity assessment is carried out on freeze dried powder of cultured probiotics containing Lactobacillus rhamnosus LCR177, Bifidobacterium adolescentis BA286, and Pediococcus acidilactici PA318. Ames tests, in vitro mammalian chromosome aberration assay, and micronucleus tests in mouse peripheral blood are performed. For 5 strains of Salmonella Typhimurium, the Ames tests show no increased reverse mutation upon exposure to the test substance. In CHO cells, the frequency of chromosome aberration does not increase in responding to the treatment of probiotics. Likewise, the frequency of micronucleated reticulocytes in probiotics-fed mice is indistinguishable from that in the negative control group. Taken together, the toxicity assessment studies suggest that the multispecies probiotic mixture does not have mutagenic effects on various organisms.
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Novel POC1A mutation in primordial dwarfism reveals new insights for centriole biogenesis.
Koparir, Asuman; Karatas, Omer F; Yuceturk, Betul; Yuksel, Bayram; Bayrak, Ali O; Gerdan, Omer F; Sagiroglu, Mahmut S; Gezdirici, Alper; Kirimtay, Koray; Selcuk, Ece; Karabay, Arzu; Creighton, Chad J; Yuksel, Adnan; Ozen, Mustafa
2015-10-01
POC1A encodes a WD repeat protein localizing to centrioles and spindle poles and is associated with short stature, onychodysplasia, facial dysmorphism and hypotrichosis (SOFT) syndrome. These main features are related to the defect in cell proliferation of chondrocytes in growth plate. In the current study, we aimed at identifying the molecular basis of two patients with primordial dwarfism (PD) in a single family through utilization of whole-exome sequencing. A novel homozygous p.T120A missense mutation was detected in POC1A in both patients, a known causative gene of SOFT syndrome, and confirmed using Sanger sequencing. To test the pathogenicity of the detected mutation, primary fibroblast cultures obtained from the patients and a control individual were used. For evaluating the global gene expression profile of cells carrying p.T120A mutation in POC1A, we performed the gene expression array and compared their expression profiles to those of control fibroblast cells. The gene expression array analysis showed that 4800 transcript probes were significantly deregulated in cells with p.T120A mutation in comparison to the control. GO term association results showed that deregulated genes are mostly involved in the extracellular matrix and cytoskeleton. Furthermore, the p.T120A missense mutation in POC1A caused the formation of abnormal mitotic spindle structure, including supernumerary centrosomes, and changes in POC1A were accompanied by alterations in another centrosome-associated WD repeat protein p80-katanin. As a result, we identified a novel mutation in POC1A of patients with PD and showed that this mutation causes the formation of multiple numbers of centrioles and multipolar spindles with abnormal chromosome arrangement. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Yoshida, Noriaki; Miyoshi, Hiroaki; Kato, Takeharu; Sakata-Yanagimoto, Mamiko; Niino, Daisuke; Taniguchi, Hiroaki; Moriuchi, Yukiyoshi; Miyahara, Masaharu; Kurita, Daisuke; Sasaki, Yuya; Shimono, Joji; Kawamoto, Keisuke; Utsunomiya, Atae; Imaizumi, Yoshitaka; Seto, Masao; Ohshima, Koichi
2016-04-01
Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Jin, Hyun-Seok; Kim, Jeonhyun; Kwak, Woori; Jeong, Hyeonsoo; Lim, Gyu-Bin
2017-01-01
Congenital cataracts can occur as a non-syndromic isolated ocular disease or as a part of genetic syndromes accompanied by a multi-systemic disease. Approximately 50% of all congenital cataract cases have a heterogeneous genetic basis. Here, we describe three generations of a family with an autosomal dominant inheritance pattern and common complex phenotypes, including bilateral congenital cataracts, short stature, macrocephaly, and minor skeletal anomalies. We did not find any chromosomal aberrations or gene copy number abnormalities using conventional genetic tests; accordingly, we conducted whole-exome sequencing (WES) to identify disease-causing genetic alterations in this family. Based on family WES data, we identified a novel BRD4 missense mutation as a candidate causal variant and performed cell-based experiments by ablation of endogenous BRD4 expression in human lens epithelial cells. The protein expression levels of connexin 43, p62, LC3BII, and p53 differed significantly between control cells and cells in which endogenous BRD4 expression was inhibited. We inferred that a BRD4 missense mutation was the likely disease-causing mutation in this family. Our findings may improve the molecular diagnosis of congenital cataracts and support the use of WES to clarify the genetic basis of complex diseases. PMID:28076398
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Bauer, Thomas R; Adler, Rima L; Hickstein, Dennis D
2009-01-01
Genetic mutations involving the cellular components of the hematopoietic system--red blood cells, white blood cells, and platelets--manifest clinically as anemia, infection, and bleeding. Although gene targeting has recapitulated many of these diseases in mice, these murine homologues are limited as translational models by their small size and brief life span as well as the fact that mutations induced by gene targeting do not always faithfully reflect the clinical manifestations of such mutations in humans. Many of these limitations can be overcome by identifying large animals with genetic diseases of the hematopoietic system corresponding to their human disease counterparts. In this article, we describe human diseases of the cellular components of the hematopoietic system that have counterparts in large animal species, in most cases carrying mutations in the same gene (CD18 in leukocyte adhesion deficiency) or genes in interacting proteins (DNA cross-link repair 1C protein and protein kinase, DNA-activated catalytic polypeptide in radiation-sensitive severe combined immunodeficiency). Furthermore, we describe the potential of these animal models to serve as disease-specific preclinical models for testing the efficacy and safety of clinical interventions such as hematopoietic stem cell transplantation or gene therapy before their use in humans with the corresponding disease.
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Mutation dynamics and fitness effects followed in single cells.
Robert, Lydia; Ollion, Jean; Robert, Jerome; Song, Xiaohu; Matic, Ivan; Elez, Marina
2018-03-16
Mutations have been investigated for more than a century but remain difficult to observe directly in single cells, which limits the characterization of their dynamics and fitness effects. By combining microfluidics, time-lapse imaging, and a fluorescent tag of the mismatch repair system in Escherichia coli , we visualized the emergence of mutations in single cells, revealing Poissonian dynamics. Concomitantly, we tracked the growth and life span of single cells, accumulating ~20,000 mutations genome-wide over hundreds of generations. This analysis revealed that 1% of mutations were lethal; nonlethal mutations displayed a heavy-tailed distribution of fitness effects and were dominated by quasi-neutral mutations with an average cost of 0.3%. Our approach has enabled the investigation of single-cell individuality in mutation rate, mutation fitness costs, and mutation interactions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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TERT Polymorphism rs2736100-C Is Associated with EGFR Mutation-Positive Non-Small Cell Lung Cancer
Zheng, Yonglan; Niu, Xiaomin; Weng, Xiaoling; Zhang, Hong; Favus, Murray; Zhang, Lanjun; Jia, Weihua; Zeng, Yixin; Amos, Christopher I; Lu, Shun; Wang, Hui-Yun; Liu, Yun; Liu, Wanqing
2015-01-01
Purpose Epidermal growth factor receptor (EGFR) mutation-positive (EGFRmut+) non-small cell lung cancer (NSCLC) may be a unique orphan disease. Previous studies suggested that the telomerase reverse transcriptase (TERT) gene polymorphism is associated with demographic and clinical features strongly associated with EGFR mutations, e.g. adenocarcinoma histology, never-smoking history and female gender. We aim to test the association between TERT polymorphism and EGFRmut+ NSCLC. Experimental Design We conducted a genetic association study in Chinese NSCLC patients (n=714) and healthy controls (n=2,520), between the rs2736100 polymorphism and EGFRmut+ NSCLC. We further tested the association between the EGFR mutation status and mean leukocyte telomere length (LTL). The potential function of rs2736100 in lung epithelial cells was also explored. Results The rs2736100-C allele was significantly associated with EGFRmut+ NSCLC (OR=1.52, 95%CI=1.28–1.80, p=1.6×10−6) but not EGFRmut− NSCLC (OR=1.07, 95%CI=0.92–1.24, p=0.4). While NSCLC patients as a whole have significantly longer LTL compared to healthy controls (p≤10−13), the EGFRmut+ patients have even longer LTL compared to EGFRmut-patients (p=0.008). Meanwhile, rs2736100 was significantly associated with TERT mRNA expression in both normal and tumor lung tissues. All results remained significant after controlling for age, gender, smoking status and histology (p<0.05 for all tests). Moreover, the rs2736100 DNA sequence has an allele-specific affinity to nuclear proteins extracted from lung epithelial cells, which led to an altered enhancer activity of the sequence in vitro. Conclusion Our study suggests that telomerase and telomere function may be essential for carcinogenesis of EGFRmut+ NSCLC. Further investigation for the underlying mechanism is warranted. PMID:26149460
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Correa, Bruna R.; Bettoni, Fabiana; Koyama, Fernanda C.; Navarro, Fabio C.P.; Perez, Rodrigo O.; Mariadason, John; Sieber, Oliver M.; Strausberg, Robert L.; Simpson, Andrew J.G.; Jardim, Denis L.F.; Reis, Luiz Fernando L.; Parmigiani, Raphael B.; Galante, Pedro A.F.; Camargo, Anamaria A.
2014-01-01
We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of “general” immune checkpoint drugs in this subset of patients. PMID:25193853
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Mitochondrial DNA mutations in single human blood cells.
Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S
2015-09-01
Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. Copyright © 2015 Elsevier B.V. All rights reserved.
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Bone Marrow Transplantation in Mice as a Tool to Generate Genetically Modified Animals
NASA Astrophysics Data System (ADS)
Rőszer, Tamás; Pintye, Éva; Benkő, Ilona
2008-12-01
Transgenic mice can be used either as models of known inherited human diseases or can be applied to perform phenotypic tests of genes with unknown function. In some special applications of gene modification we have to create a tissue specific mutation of a given gene. In some cases however the gene modification can be lethal in the intrauterine life, therefore we should engraft the mutated cells in the postnatal life period. After total body irradiation transplantation of bone marrow cells can be a solution to introduce mutant hematopoietic stem cells into a mature animal. Bone marrow transplantation is a useful and novel tool to study the role of hematopoietic cells in the pathogenesis of inflammation, autoimmune syndromes and many metabolic alterations coupled recently to leukocyte functions.
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Sato, Keisaku; Pollock, Neil; Stowell, Kathryn M
2010-06-01
Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.
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Turner, Gareth D. H.; Dudka-Ruszkowska, Wioleta; Taylor, Stephen; Meyts, Ewa Rajpert-De; Goriely, Anne; Wilkie, Andrew O. M.
2012-01-01
The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia–caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively–represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection. PMID:22879958
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Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y
2014-05-22
Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.
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Datta, Sandipan; Tomilov, Alexey; Cortopassi, Gino
2016-01-01
Inherited mitochondrial complex I mutations cause blinding Leber's hereditary Optic Neuropathy (LHON), for which no curative therapy exists. A specific biochemical consequence of LHON mutations in the presence of trace rotenone was observed: deficient complex I-dependent ATP synthesis (CIDAS) and mitochondrial O2 consumption, proportional to the clinical severity of the three primary LHON mutations. We optimized a high-throughput assay of CIDAS to screen 1600 drugs to 2, papaverine and zolpidem, which protected CIDAS in LHON cells concentration-dependently. TSPO and cAMP were investigated as protective mechanisms, but a conclusive mechanism remains to be elucidated; next steps include testing in animal models. PMID:27497748
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Liu, B; Zhou, P; Miao, Q
1997-05-01
Mutations of suppressor gene p53 was studied in 36 cases of silica related lung cancer and 6 cases of welding fume related lung cancer with immunohistochemical and PCR-SSCP methods. Cancer tissues were embedded in paraffin and stored for 13.4 years in average. Results revealed that there was abnormal mobility shift of electrophoresis in 18 cases with 20 point mutations of 42 specimens tested, accounted for 42.9%, and 50% (10/20) of the mutations were clustered in exon 8. This finding differed from mutational spectrum of gene in non-occupational lung cancer, in which mutation frequency of exon 8 ranged from 17.5% to 23.5%. Gene mutation frequency in varied pathological categories of pneumoconiosis related lung cancer also differed from that in common lung cancer. In the latter, the highest one was in small cell lung cancer (70%) and the lowest in adenocarcinoma (33%), but in the former, the highest in adenocarcinoma (53.9%) and the lowest in small cell lung cancer (30.8%). Immunohistochemical observations also showed a very high prevalence of p53 gene mutation expression (46.9%). Sequencing, which was determined in two cases of this study, revealed that two point mutations all occurred in non-hotspot codon 144 of p53 gene. Difference in gene mutation spectrum suggests that there exist specific carcinogens and carcinogenesis in silica and welding fume related lung cancer.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing
VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less
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Almejun, Maria B.; Cols, Montserrat; Zelazko, Marta; Oleastro, Matias; Cerutti, Andrea; Oppezzo, Pablo; Cunningham-Rundles, Charlotte; Danielian, Silvia
2013-01-01
Mutations in the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B-cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R-naive B cells. Likewise, these stimuli induced less expression of activation-induced cytidine deaminase, I(γ)1-C(μ), and I(γ)1-C(μ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL-mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R-mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88-binding site in TACI to trigger CSR. PMID:23225259
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Feretti, Donatella; Pedrazzani, Roberta; Ceretti, Elisabetta; Zerbini, Ilaria; Gozio, Eleonora; Belotti, Caterina; Alias, Carlotta; Donato, Francesco; Gelatti, Umberto
2009-01-01
The aim of this study was to evaluate the mutagenicity of several traditional detergents and that of newer more biodegradable detergents, by using a bacterial test (Ames test), a plant cell test (Allium cepa micronuclei test) and a human leucocyte test (Comet test). All tests were conducted using a wide range of doses (1-2000 mg/l). None of the examined detergents induced mutations in S.typhimurium. One traditional detergent showed a genotoxic effect with the A. cepa test, while all newer detergents and one traditional detergent were shown by the Comet test to be capable of inducing DNA damage.
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Hall, Allison R; Anderson, Corey L; Smith, Jennifer L; Mirshahi, Tooraj; Elayi, Claude S; January, Craig T; Delisle, Brian P
2018-01-01
KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K + current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention.
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Analysis of mutational spectra by denaturant capillary electrophoresis
Ekstrøm, Per O.; Khrapko, Konstantin; Li-Sucholeiki, Xiao-Cheng; Hunter, Ian W.; Thilly, William G.
2009-01-01
Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75–250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA “clamp” is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human gene-common disease associations and the discovery of origins of point mutations in human development and carcinogenesis. PMID:18600220
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An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.
Di Lullo, A M; Scorza, M; Amato, F; Comegna, M; Raia, V; Maiuri, L; Ilardi, G; Cantone, E; Castaldo, G; Iengo, M
2017-06-01
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%. © Copyright by Società Italiana di Otorinolaringologia e Chirurgia Cervico-Facciale, Rome, Italy.
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Yang, Guangdie; Yao, Yinan; Zhou, Jianya; Zhao, Qiong
2012-06-01
Epidermal growth factor receptor (EGFR) is one of the most promising targets for non-small cell lung cancer (NSCLC). Our study demonstrated the antitumor effects of icotinib hydrochloride, a highly selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), in two EGFR-mutated lung cancer cell lines compared to A549, a cell line without EGFR mutations. We incubated PC-9 and HCC827 human lung cancer cell lines both with (E746-A750) mutations with various concentrations of icotinib and gefitinib for 48 h. Cell proliferation and migration were determined using a real-time cell invasion and migration assay and cytotoxicity assay. Apoptosis was assessed by measuring Annexin V staining using flow cytometry. The antitumor effects of icotinib compared to gefitinib were similar and were most effective in reducing the proliferation of EGFR-mutated cells compared to non-mutated controls. Our results suggest the possibility of icotinib as a new therapeutic agent of EGFR-mutated cancer cells, which has the potential to be used in the first-line treatment of EGFR-mutated NSCLC.
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Recombinant EphB4-HSA Fusion Protein and Pembrolizumab, MK-3475
2018-03-30
ALK Gene Mutation; BRAF Gene Mutation; EGFR Gene Mutation; Head and Neck Squamous Cell Carcinoma; Metastatic Head and Neck Carcinoma; Recurrent Head and Neck Carcinoma; Recurrent Non-Small Cell Lung Carcinoma; ROS1 Gene Mutation; Stage III Non-Small Cell Lung Cancer; Stage IIIA Non-Small Cell Lung Cancer; Stage IIIB Non-Small Cell Lung Cancer; Stage IV Non-Small Cell Lung Cancer
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Li, Xin; Young, Nathan M.; Tropp, Stephen; Hu, Diane; Xu, Yanhua; Hallgrímsson, Benedikt; Marcucio, Ralph S.
2013-01-01
Fibroblast growth factor (FGF) signaling mutations are a frequent contributor to craniofacial malformations including midfacial anomalies and craniosynostosis. FGF signaling has been shown to control cellular mechanisms that contribute to facial morphogenesis and growth such as proliferation, survival, migration and differentiation. We hypothesized that FGF signaling not only controls the magnitude of growth during facial morphogenesis but also regulates the direction of growth via cell polarity. To test this idea, we infected migrating neural crest cells of chicken embryos with replication-competent avian sarcoma virus expressing either FgfR2C278F, a receptor mutation found in Crouzon syndrome or the ligand Fgf8. Treated embryos exhibited craniofacial malformations resembling facial dysmorphologies in craniosynostosis syndrome. Consistent with our hypothesis, ectopic activation of FGF signaling resulted in decreased cell proliferation, increased expression of the Sprouty class of FGF signaling inhibitors, and repressed phosphorylation of ERK/MAPK. Furthermore, quantification of cell polarity in facial mesenchymal cells showed that while orientation of the Golgi body matches the direction of facial prominence outgrowth in normal cells, in FGF-treated embryos this direction is randomized, consistent with aberrant growth that we observed. Together, these data demonstrate that FGF signaling regulates cell proliferation and cell polarity and that these cell processes contribute to facial morphogenesis. PMID:23906837
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Landsiedel, Robert; Ma-Hock, Lan; Van Ravenzwaay, Ben; Schulz, Markus; Wiench, Karin; Champ, Samantha; Schulte, Stefan; Wohlleben, Wendel; Oesch, Franz
2010-12-01
Titanium dioxide and zinc oxide nanomaterials, used as UV protecting agents in sunscreens, were investigated for their potential genotoxicity in in vitro and in vivo test systems. Since standard OECD test methods are designed for soluble materials and genotoxicity testing for nanomaterials is still under revision, a battery of standard tests was used, covering different endpoints. Additionally, a procedure to disperse the nanomaterials in the test media and careful characterization of the dispersed test item was added to the testing methods. No genotoxicity was observed in vitro (Ames' Salmonella gene mutation test and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micronucleus test and Comet DNA damage assay in lung cells from rats exposed by inhalation). These results add to the still limited data base on genotoxicity test results with nanomaterials and provide congruent results of a battery of standard OECD test methods applied to nanomaterials.
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Cole, Christopher B.; Russler-Germain, David A.; Ketkar, Shamika; Verdoni, Angela M.; Smith, Amanda M.; Bangert, Celia V.; Helton, Nichole M.; Guo, Mindy; O’Laughlin, Shelly; Fronick, Catrina; Fulton, Robert; Chang, Gue Su; Petti, Allegra A.; Miller, Christopher A.; Ley, Timothy J.
2017-01-01
The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/– mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/– mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/– mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear. PMID:28872462
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TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.
Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf
2017-06-01
Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Role of the Transcription Factor Sox4 in Insulin Secretion and Impaired Glucose Tolerance
Goldsworthy, Michelle; Hugill, Alison; Freeman, Helen; Horner, Emma; Shimomura, Kenju; Bogani, Debora; Pieles, Guido; Mijat, Vesna; Arkell, Ruth; Bhattacharya, Shoumo; Ashcroft, Frances M.; Cox, Roger D.
2008-01-01
OBJECTIVES— To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion. RESEARCH DESIGN AND METHODS— Haploinsufficiency of the insulin receptor and associated mild insulin resistance has been used to sensitize an N-ethyl-N-nitrosourea (ENU) screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. The new impaired glucose tolerance 4 (IGT4) model was selected using an intraperitoneal glucose tolerance test and inheritance of the phenotype confirmed by generation of backcross progeny. Segregation of the phenotype was correlated with genotype information to map the location of the gene and candidates sequenced for mutations. The function of the SRY-related high mobility group (HMG)-box 4 (Sox4) gene in insulin secretion was tested using another ENU allele and by small interfering RNA silencing in insulinoma cells. RESULTS— We describe two allelic autosomal dominant mutations in the highly conserved HMG box of the transcription factor Sox4. Previously associated with pancreas development, Sox4 mutations in the adult mouse result in an insulin secretory defect, which exhibits impaired glucose tolerance in association with insulin receptor+/−–induced insulin resistance. Elimination of the Sox4 transcript in INS1 and Min6 cells resulted in the abolition of glucose-stimulated insulin release similar to that observed for silencing of the key metabolic enzyme glucokinase. Intracellular calcium measurements in treated cells indicate that this defect lies downstream of the ATP-sensitive K+ channel (KATP channel) and calcium influx. CONCLUSIONS— IGT4 represents a novel digenic model of insulin resistance coupled with an insulin secretory defect. The Sox4 gene has a role in insulin secretion in the adult β-cell downstream of the KATP channel. PMID:18477811
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2018-01-31
Anemia; ASXL1 Gene Mutation; EZH2 Gene Mutation; IDH1 Gene Mutation; IDH2 Gene Mutation; Plasma Cell Myeloma; Primary Myelofibrosis; Recurrent Plasma Cell Myeloma; Secondary Myelofibrosis; Thrombocytopenia
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RADIATION INDUCED VIABILITY MUTATIONS IN THE HONEY BEE
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, W.R.
The frequency of recessive detrimental mutations expressed in the haploid drone honey bee was investigated and compared with recessive and dominant lethal mutations detected in the haploid drone and diploid worker. A single queen was inseminated by a drone homozygous for three genetic markers. Viability of progeny was determined, and hybrid daughters bearing the genetic markers were stored in colonies. The spermatheca of the queen was then irradiated with 2600 r kvp x rays. Morphological defects and viability were studied in progeny and grand-progeny. A total of 92 pairs was tested during one season. Results showed that 60.8% of themore » sperm cells receiving radiation contained at least one or more dominant lethals. Correcting for the saturation effect on the assumption of independence of each dominant lethal, an average proportion of 0.94 dominant lethals were found per cell. The average reduction in embryonic viability was 28%. Forty per cent of the queens tested contained one or more recessive lethals. Corrections in procedure and plans for future work, as well as work in progress, are described. (H.M.G.)« less
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van der Post, Rachel S; Vogelaar, Ingrid P; Carneiro, Fátima; Guilford, Parry; Huntsman, David; Hoogerbrugge, Nicoline; Caldas, Carlos; Schreiber, Karen E Chelcun; Hardwick, Richard H; Ausems, Margreet G E M; Bardram, Linda; Benusiglio, Patrick R; Bisseling, Tanya M; Blair, Vanessa; Bleiker, Eveline; Boussioutas, Alex; Cats, Annemieke; Coit, Daniel; DeGregorio, Lynn; Figueiredo, Joana; Ford, James M; Heijkoop, Esther; Hermens, Rosella; Humar, Bostjan; Kaurah, Pardeep; Keller, Gisella; Lai, Jennifer; Ligtenberg, Marjolijn J L; O'Donovan, Maria; Oliveira, Carla; Pinheiro, Hugo; Ragunath, Krish; Rasenberg, Esther; Richardson, Susan; Roviello, Franco; Schackert, Hans; Seruca, Raquel; Taylor, Amy; Ter Huurne, Anouk; Tischkowitz, Marc; Joe, Sheena Tjon A; van Dijck, Benjamin; van Grieken, Nicole C T; van Hillegersberg, Richard; van Sandick, Johanna W; Vehof, Rianne; van Krieken, J Han; Fitzgerald, Rebecca C
2015-06-01
Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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van der Post, Rachel S; Vogelaar, Ingrid P; Carneiro, Fátima; Guilford, Parry; Huntsman, David; Hoogerbrugge, Nicoline; Caldas, Carlos; Schreiber, Karen E Chelcun; Hardwick, Richard H; Ausems, Margreet G E M; Bardram, Linda; Benusiglio, Patrick R; Bisseling, Tanya M; Blair, Vanessa; Bleiker, Eveline; Boussioutas, Alex; Cats, Annemieke; Coit, Daniel; DeGregorio, Lynn; Figueiredo, Joana; Ford, James M; Heijkoop, Esther; Hermens, Rosella; Humar, Bostjan; Kaurah, Pardeep; Keller, Gisella; Lai, Jennifer; Ligtenberg, Marjolijn J L; O'Donovan, Maria; Oliveira, Carla; Ragunath, Krish; Rasenberg, Esther; Richardson, Susan; Roviello, Franco; Schackert, Hans; Seruca, Raquel; Taylor, Amy; ter Huurne, Anouk; Tischkowitz, Marc; Joe, Sheena Tjon A; van Dijck, Benjamin; van Grieken, Nicole C T; van Hillegersberg, Richard; van Sandick, Johanna W; Vehof, Rianne; van Krieken, J Han; Fitzgerald, Rebecca C
2015-01-01
Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored. PMID:25979631
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Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns; Denning, Chris
2013-10-15
With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.
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Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns
2013-01-01
With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870
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Myeloproliferative Neoplasms: Molecular Drivers and Therapeutics.
Reuther, G W
2016-01-01
Activating mutations in genes that drive neoplastic cell growth are numerous and widespread in cancer, and specific genetic alterations are associated with certain types of cancer. For example, classic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that affect cells of the myeloid lineage, including erythrocytes, platelets, and granulocytes. An activating mutation in the JAK2 tyrosine kinase is prevalent in these diseases. In MPN patients that lack such a mutation, other genetic changes that lead to activation of the JAK2 signaling pathway are present, indicating deregulation of JAK2 signaling plays an etiological driving role in MPNs, a concept supported by significant evidence from in vivo experimental MPN systems. Thus, small molecules that inhibit JAK2 activity are ideal drugs to impede the progression of disease in MPN patients. However, even though JAK inhibitors provide significant symptomatic relief, they have failed as a remission-inducing therapy. Nonetheless, the progress made understanding the molecular etiology of MPNs since 2005 is significant and has provided insight for the development and testing of novel molecular targeted therapeutic approaches. The current understanding of driver mutations in MPNs and an overview of current and potential therapeutic strategies for MPN patients will be discussed. © 2016 Elsevier Inc. All rights reserved.
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Transcriptional specificity in various p53-mutant cells.
Okaichi, Kumio; Izumi, Nanaka; Takamura, Yuma; Fukui, Shoichi; Kudo, Takashi
2013-03-01
Mutation of the tumor suppressor gene p53 is the most common genetic alteration observed in human tumors. However, the relationship between the mutation point of p53 and the transcriptional specificity is not so obvious. We prepared Saos-2 cells with various mutations of p53 that are found in human tumors, and examined the resulting transcriptional alterations in the cells. Loss of function and gain of function were observed in all p53 mutants. Hot-spot mutations of p53 are frequently found in tumor cells. We compared hot-spot mutations and other mutations of p53 and found that a more than 2-fold transcription of CADPS2, PIWIL4 and TRIM9 was induced by hot spot mutations, but not by other mutations. As PIWIL4 suppresses the p16(INK4A) and ARF pathway, restraining cell growth and genomic instability, induction of PIWIL4 expression may be one reason why hot-spot mutations are frequently found in tumor cells.
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Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.
2013-01-01
Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593
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Mutagenic effect of accelerated heavy ions on bacterial cells
NASA Astrophysics Data System (ADS)
Boreyko, A. V.; Krasavin, E. A.
2011-11-01
The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific features of energy transfer of the radiations that affect the character of induced DNA damage, and the efficiency inducible and constitutive cell repair systems. The growth of relative biological efficiency of heavy charged particles is determined by the growth of the damage yield of the DNA participating in the formation of radiation-induced effects, and higher efficiency of inducible repair systems. It was established that the LET value ( L max) for which the maximum (according to the applied irradiation criteria) coefficients of relative biological efficiency are observed varies depending on the character of the registered radiation induced effect. It was demonstrated that for gene mutations and induction of precision excision of mobile elements the values of L max are realized in a LET range of ≈20 keV/μm. For lethal effects of irradiation and induction of deletion mutations the value of L max is ≈ 100 and 50 keV/μm, respectively. The differences in the L max for the studied radiation gene effectis are determined by the different type of DNA damage participating in the mutation process. A molecular model of the formation of gene mutations in Escherichia coli cells under the action of ionizing radiation was proposed. Basic DNA radiation damage and main repair ways were considered in the framework of this model. The basis is the idea of the decisive role of mutagenic, error-prone, branch of SOS repair in fixing premutation DNA damage into point mutations. It was demonstrated that the central mechanism in this process is the formation of an inducible multi-enzymatic complex including the DNA polymerase V (Umu C), RecA-protease, SSB proteins, subunits of DNA polymerase III, performing erroneous DNA synthesis on the damaged matrix. A mathematical model of induction of gene mutations under ultraviolet cell irradiation was developed based on the molecular model.
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Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael
2018-04-06
Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.
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Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael
2018-01-01
Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection. PMID:29719623
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Ben Jehuda, Ronen; Eisen, Binyamin; Shemer, Yuval; Mekies, Lucy N; Szantai, Agnes; Reiter, Irina; Cui, Huanhuan; Guan, Kaomei; Haron-Khun, Shiraz; Freimark, Dov; Sperling, Silke R; Gherghiceanu, Mihaela; Arad, Michael; Binah, Ofer
2018-02-01
Mutations in the PRKAG2 gene encoding the γ-subunit of adenosine monophosphate kinase (AMPK) cause hypertrophic cardiomyopathy (HCM) and familial Wolff-Parkinson-White (WPW) syndrome. Patients carrying the R302Q mutation in PRKAG2 present with sinus bradycardia, escape rhythms, ventricular preexcitation, supraventricular tachycardia, and atrioventricular block. This mutation affects AMPK activity and increases glycogen storage in cardiomyocytes. The link between glycogen storage, WPW syndrome, HCM, and arrhythmias remains unknown. The purpose of this study was to investigate the pathological changes caused by the PRKAG2 mutation. We tested the hypothesis that patient's induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) display clinical aspects of the disease. Using clustered regularly interspaced short palindromic repeats (CRISPR) technology, we corrected the mutation and then generated isogenic iPSC-CMs. Action potentials were recorded from spontaneously firing and paced cardiomyocytes using the patch clamp technique. Using a microelectrode array setup, we recorded electrograms from iPSC-CMs clusters. Transmission electron microscopy was used to detect ultrastructural abnormalities in the mutated iPSC-CMs. PRKAG2-mutated iPSC-CMs exhibited abnormal firing patterns, delayed afterdepolarizations, triggered arrhythmias, and augmented beat rate variability. Importantly, CRISPR correction eliminated the electrophysiological abnormalities, the augmented glycogen, storage, and cardiomyocyte hypertrophy. PRKAG2-mutated iPSC-CMs displayed functional and structural abnormalities, which were abolished by correcting the mutation in the patient's iPSCs using CRISPR technology. Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.
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Roberts, Patrick J; Stinchcombe, Thomas E; Der, Channing J; Socinski, Mark A
2010-11-01
In patients with metastatic colorectal cancer, the predictive value of KRAS mutational status in the selection of patients for treatment with anti-epidermal growth factor (EGFR) monoclonal antibodies is established. In patients with non-small-cell lung cancer (NSCLC), the utility of determining KRAS mutational status to predict clinical benefit to anti-EGFR therapies remains unclear. This review will provide a brief description of Ras biology, provide an overview of aberrant Ras signaling in NSCLC, and summarize the clinical data for using KRAS mutational status as a negative predictive biomarker to anti-EGFR therapies. Retrospective investigations of KRAS mutational status as a negative predictor of clinical benefit from anti-EGFR therapies in NSCLC have been performed; however, small samples sizes as a result of low prevalence of KRAS mutations and the low rate of tumor sample collection have limited the strength of these analyses. Although an association between the presence of KRAS mutation and lack of response to EGFR tyrosine kinase inhibitors (TKIs) has been observed, it remains unclear whether there is an association between KRAS mutation and EGFR TKI progression-free and overall survival. Unlike colorectal cancer, KRAS mutations do not seem to identify patients who do not benefit from anti-EGFR monoclonal antibodies in NSCLC. The future value of testing for KRAS mutational status may be to exclude the possibility of an EGFR mutation or anaplastic lymphoma kinase translocation or to identify a molecular subset of patients with NSCLC in whom to pursue a drug development strategy that targets the KRAS pathway.
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Gu, Jincui; Xu, Siqi; Huang, Lixia; Li, Shaoli; Wu, Jian; Xu, Junwen; Feng, Jinlun; Liu, Baomo; Zhou, Yanbin
2018-02-01
We sought to investigate the associations between pretreatment serum Carcinoembryonic antigen (CEA) level, 18 F-Fluoro-2-deoxyglucose ( 18 F-FDG) uptake value of primary tumor and epidermal growth factor receptor ( EGFR ) mutation status in non-small cell lung cancer (NSCLC). We retrospectively reviewed medical records of 210 NSCLC patients who underwent EGFR mutation test and 18 F-FDG positron emission tomography/computed tomography (PET/CT) scan before anti-tumor therapy. The associations between EGFR mutations and patients' characteristics, serum CEA, PET/CT imaging characteristics maximal standard uptake value (SUVmax) of the primary tumor were analyzed. Receiver-operating characteristic (ROC) curve was used to assess the predictive value of these factors. EGFR mutations were found in 70 patients (33.3%). EGFR mutations were more common in high CEA group (CEA ≥7.0 ng/mL) than in low CEA group (CEA <7.0 ng/mL) (40.4% vs . 27.6%; P=0.05). Females (P<0.001), non-smokers (P<0.001), patients with adenocarcinoma (P<0.001) and SUVmax <9.0 (P=0.001) were more likely to be EGFR mutation-positive. Multivariate analysis revealed that gender, tumor histology, pretreatment serum CEA level, and SUVmax were the most significant predictors for EGFR mutations. The ROC curve revealed that combining these four factors yielded a higher calculated AUC (0.80). Gender, histology, pretreatment serum CEA level and SUVmax are significant predictors for EGFR mutations in NSCLC. Combining these factors in predicting EGFR mutations has a moderate diagnostic accuracy, and is helpful in guiding anti-tumor treatment.
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Cell lineage analysis in human brain using endogenous retroelements
Evrony, Gilad D.; Lee, Eunjung; Mehta, Bhaven K.; Benjamini, Yuval; Johnson, Robert M.; Cai, Xuyu; Yang, Lixing; Haseley, Psalm; Lehmann, Hillel S.; Park, Peter J.; Walsh, Christopher A.
2015-01-01
Summary Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sub-lineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development, and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain. PMID:25569347
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Broncy, Lucile; Njima, Basma Ben; Méjean, Arnaud; Béroud, Christophe; Romdhane, Khaled Ben; Ilie, Marius; Hofman, Veronique; Muret, Jane; Hofman, Paul; Bouhamed, Habiba Chaabouni; Paterlini-Bréchot, And Patrizia
2018-04-13
Circulating Rare Cells (CRC) are non-haematological cells circulating in blood. They include Circulating Cancer Cells (CCC) and cells with uncertain malignant features (CRC-UMF) according to cytomorphology. Clear cell renal cell carcinomas frequently bear a mutated Von Hippel-Lindau (VHL) gene. To match blind genetic analysis of CRC and tumor samples with CRC cytopathological diagnosis. 29/30 patients harboured CRC (20 harboured CCC, 29 CRC-UMF) and 25/29 patients carried VHL mutations in their tumour. 205 single CRC (64 CCC, 141 CRC-UMF) provided genetic data. 57/57 CCC and 104/125 CRC-UMF from the 25 patients with VHL-mutated tumor carried the same VHL mutation detected in the tumor. Seven CCC and 16 CRC-UMF did not carry VHL mutations but were found in patients with wild-type VHL tumor tissue. All the CCC and 83,2% (104/125) of the CRC-UMF were found to carry the same VHL mutation identified in the corresponding tumorous tissue, validating cytopathological identification of CCC in patients with clear cell renal cell carcinoma. The blood of 30 patients with clear cell renal cell carcinoma was treated by ISET ® for CRC isolation, cytopathology and single-cell VHL mutations analysis, performed blindly and compared to VHL mutations of corresponding tumor tissues and leukocytes.
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CVID-associated TACI mutations affect autoreactive B cell selection and activation
Romberg, Neil; Chamberlain, Nicolas; Saadoun, David; Gentile, Maurizio; Kinnunen, Tuure; Ng, Yen Shing; Virdee, Manmeet; Menard, Laurence; Cantaert, Tineke; Morbach, Henner; Rachid, Rima; Martinez-Pomar, Natalia; Matamoros, Nuria; Geha, Raif; Grimbacher, Bodo; Cerutti, Andrea; Cunningham-Rundles, Charlotte; Meffre, Eric
2013-01-01
Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications. PMID:24051380
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Kim, H S; Jung, M; Kang, H N; Kim, H; Park, C-W; Kim, S-M; Shin, S J; Kim, S H; Kim, S G; Kim, E K; Yun, M R; Zheng, Z; Chung, K Y; Greenbowe, J; Ali, S M; Kim, T-M; Cho, B C
2017-06-08
Despite remarkable progress in cutaneous melanoma genomic profiling, the mutational landscape of primary mucosal melanomas (PMM) remains unclear. Forty-six PMMs underwent targeted exome sequencing of 111 cancer-associated genes. Seventy-six somatic nonsynonymous mutations in 42 genes were observed, and recurrent mutations were noted on eight genes, including TP53 (13%), NRAS (13%), SNX31 (9%), NF1 (9%), KIT (7%) and APC (7%). Mitogen-activated protein kinase (MAPK; 37%), cell cycle (20%) and phosphatidylinositol 3-kinase (PI3K)-mTOR (15%) pathways were frequently mutated. We biologically characterized a novel ZNF767-BRAF fusion found in a vemurafenib-refractory respiratory tract PMM, from which cell line harboring ZNF767-BRAF fusion were established for further molecular analyses. In an independent data set, NFIC-BRAF fusion was identified in an oral PMM case and TMEM178B-BRAF fusion and DGKI-BRAF fusion were identified in two malignant melanomas with a low mutational burden (number of mutation per megabase, 0.8 and 4, respectively). Subsequent analyses revealed that the ZNF767-BRAF fusion protein promotes RAF dimerization and activation of the MAPK pathway. We next tested the in vitro and in vivo efficacy of vemurafenib, trametinib, BKM120 or LEE011 alone and in combination. Trametinib effectively inhibited tumor cell growth in vitro, but the combination of trametinib and BKM120 or LEE011 yielded more than additive anti-tumor effects both in vitro and in vivo in a melanoma cells harboring the BRAF fusion. In conclusion, BRAF fusions define a new molecular subset of PMM that can be targeted therapeutically by the combination of a MEK inhibitor with PI3K or cyclin-dependent kinase 4/6 inhibitors.
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Lin, Chia-Chi; Shih, Jin-Yuan; Yu, Chong-Jen; Ho, Chao-Chi; Liao, Wei-Yu; Lee, Jih-Hsing; Tsai, Tzu-Hsiu; Su, Kang-Yi; Hsieh, Min-Shu; Chang, Yih-Leong; Bai, Ya-Ying; Huang, Derek De-Rui; Thress, Kenneth S; Yang, James Chih-Hsin
2018-02-01
Osimertinib is approved for the treatment of non-small-cell lung cancer in patients who develop the EGFR Thr790Met mutation after treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKIs). We assessed outcomes in patients with non-small-cell lung cancer and the EGFR Thr790Met mutation who were treated with osimertinib, a third-generation EGFR TKI, after previous treatment failure with one or more other EGFR TKIs. Eligible patients had been enrolled at one centre in the AURA study, had shown resistance to a previous EGFR TKI, and had EGFR-activating mutations and acquired Thr790Met mutation detectable in tumour tissue or plasma. Patients took 20-240 mg osimertinib per day until disease progression or development of intolerable side-effects. Plasma samples were collected every 6 weeks and tumour tissue biopsy was done at study entry and was optional after disease progression. We tested samples for resistance mechanisms, including EGFR-activating, Thr790Met, and Cys797Ser mutations, and assessed associations with overall survival, progression-free survival, and survival after disease progression. Of 71 patients enrolled in AURA, 53 were eligible for this analysis. Median progression-free survival was 11·1 months (95% CI 8·4-13·9) and overall survival was 16·9 months (11·7-29·1). 47 patients had disease progression. Median overall survival after osimertinib progression was 5·4 months (95% CI 4·1-10·0). Plasma samples were available for 40 patients after disease progression. 12 (30%) of these had the Thr790Met mutation (four of whom also had Cys797Ser mutations). Patients without detectable EGFR-activating mutations in plasma before treatment had the best overall and post-progression survival (22·4 months, 95% CI 15·6-not reached, and 10·8 months, 7·2-not reached, respectively). Loss of the Thr790Met mutation but presence of EGFR-activating mutations in plasma were associated with the shortest progression-free survival (median 2·6 months, 95% CI 1·3-not reached). In 22 post-progression tumour samples, we found one squamous cell and two small-cell transformations. We detected Thr790Met in nine (50%) of 18 samples, Cys797Ser in two (17%) of 12, cMET amplification in five (50%) of ten, BRAF mutation in one (8%) of 13, and KRAS mutation in one (8%) of 13. Heterogeneous resistance mechanisms developed in patients receiving osimertinib. Differences in resistance mechanisms might dictate future development strategies for osimertinib in clinical trials. AstraZeneca, Taiwan Ministry of Science and Technology. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Yang, Zhenli; Li, Zhanwen; Feng, Hailiang; Bian, Xiaocui; Liu, Yanyan; Liu, Yuqin
2014-09-01
To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms. A549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot. Both GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax. For single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.
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HPV-negative penile squamous cell carcinoma: disruptive mutations in the TP53 gene are common.
Kashofer, Karl; Winter, Elke; Halbwedl, Iris; Thueringer, Andrea; Kreiner, Marisa; Sauer, Stefan; Regauer, Sigrid
2017-07-01
The majority of penile squamous cell carcinomas is caused by transforming human papilloma virus (HPV) infection. The etiology of HPV-negative cancers is unclear, but TP53 mutations have been implicated. Archival tissues of 108 invasive squamous cell carcinoma from a single pathology institution in a low-incidence area were analyzed for HPV-DNA and p16 ink4a overexpression and for TP53 mutations by ion torrent next-generation sequencing. Library preparation failed in 32/108 squamous cell carcinomas. Institutional review board approval was obtained. Thirty of 76 squamous cell carcinomas (43%; average 63 years) were HPV-negative with 8/33 squamous cell carcinomas being TP53 wild-type (24%; average 63 years). Twenty-five of 33 squamous cell carcinomas (76%; average 65 years) showed 32 different somatic TP53 mutations (23 missense mutations in exons 5-8, 6 nonsense, 1 frameshift and 2 splice-site mutations). Several hotspot mutations were detected multiple times (R175H, R248, R282, and R273). Eighteen of 19 squamous cell carcinomas with TP53 expression in immunohistochemistry had TP53 mutations. Fifty percent of TP53-negative squamous cell carcinomas showed mostly truncating loss-of-function TP53 mutations. Patients without mutations had longer survival (5 years: 86% vs 61%; 10 years: 60% vs 22%), but valid clinically relevant conclusions cannot be drawn due to different tumor stages and heterogeneous treatment of the cases presented in this study. Somatic TP53 mutations are a common feature in HPV-negative penile squamous cell carcinomas and offer an explanation for HPV-independent penile carcinogenesis. About half of HPV-negative penile cancers are driven by oncogenic activation of TP53, while a quarter is induced by loss of TP53 tumor suppressor function. Detection of TP53 mutations should be carried out by sequencing, as immunohistochemical TP53 staining could not identify all squamous cell carcinomas with TP53 mutations.
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NASA Astrophysics Data System (ADS)
Fei, Rong
Purpose: Lung cancer is one of the most common cancers and non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancers. 70% of individuals with NSCLC harboring somatic mutations in exons of the epidermal growth factor receptor (EGFR) gene that encode tyrosine kinase domain. EGFR tyrosine kinase inhibitors (TKIs) are promising molecular targeted therapy for NSCLC with sensitizing EGFR mutations. However, secondary mutation of EGFR after treatment of TKIs develops resistance. Vandetanib is introduced to overcome erlotinib resistance as a multi-targeted TKI. However, its anticancer effect is still compromised by EGFR T790M mutation. Therefore, new molecular anticancer strategies are necessarily needed. In this study, vandetanib is incorporated with Pt-based anticancer agents as hybrid compounds, aiming to circumvent TKI resistance. Furthermore, hybrid compounds are investigated in cisplatin resistant problem to expect to overcome resistance by introduction of vandetanib. Methods: Three novel Pt-vandetanib hybrid compounds were synthesized and its physicochemical properties were characterized. Anticancer activity and cytotoxicity were evaluated by sulforhodamine B assay and lactate dehydrogenase release. Docking simulation was performed to investigate the interaction of compounds with EGFR harboring different mutations. Inhibition efficacy of hybrids to kinases was evaluated by kinase inhibition profiling service and cell-free kinase inhibition assay. Mechanistic studies on cytotoxicity activity of the hybrid compounds were carried out. DNA damage response of hybrid compounds was further investigated in KB cells. The cytotoxicity of hybrids was tested in cisplatin resistant KB CP20 cells. Mechanistic of anticancer activity was studied to test inhibition on oncoprotein CIP2Aand DNA damage. Results: Platinum-vandetanib hybrid compounds were synthesized and test to be stable under extracellular condition. Hybrids reacted with 5'-GMP2- and glutathione, and both of them formed mono-dentate adducts. Moreover, hybrid compounds exhibited low toxicity in human normal kidney cells. Compounds maintained the inhibition selectivity towards EGFR from the results of kinase inhibition profiling and cell-free kinase inhibition assay. Hybrids formed strong H-bond at D800 on EGFR. Pt-vandetanib hybrids were highly effective against HCC827 cells harboring sensitizing EGFR mutation. Importantly, relative resistant rate of hybrids were much smaller than vandetanib in H1975 cells. Western blot analysis results revealed that the hybrid compounds could efficiently inhibit EGFR phosphorylation in a dose dependent manner in HCC827. While, inhibition of p-EGFR was not as good as the original TKI in H1975 cells. However, the hybrid compounds induced DNA damage and caused apoptosis of the NSCLC cells. Both of the two pathways were contributed to cancer cell death and overcome vandetanib resistance. Pt-vandetanib hybrids showed little resistance in cisplatin resistant cell line KB-CP20. Drug accumulation evaluation revealed that cisplatin accumulation in CP20 cells decreased to one eighth of that in the parental KB3.1 cells. While hybrids maintained similar drug accumulation extent in both cells lines. Mechanistic study showed that hybrid compounds could induce DNA damage and cause apoptosis, whereas cisplatin failed to cause DNA damage in KB-CP20 cells. Oncoprotein CIP2A was overexpressed in CP20 cell and was ascribed to CDDP resistance. The hybrids inhibited CIP2A expression and downstream AKT phosphorylation. It was hypothesized that downregulation of CIP2A contributed to circumvention platinum resistance. Conclusion: Novel Pt-vandetanib hybrid compounds were able to overcome vandetanib resistance in H1975 cells by maintaining inhibition to the EGFR and inducing DNA damage and apoptosis. Moreover, Pt-vandetanib hybrid compounds behaved low toxicity and overcome cisplatin resistance by being "non-substrate" to efflux transporter and successfully causing DNA damage. Hybrids were found to downregulate oncogene CIP2A expression level. The novel Pt-vandetanib hybrid compounds are potent for further development.
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Mok, Tony; Ladrera, Guia; Srimuninnimit, Vichien; Sriuranpong, Virote; Yu, Chong-Jen; Thongprasert, Sumitra; Sandoval-Tan, Jennifer; Lee, Jin Soo; Fuerte, Fatima; Shames, David S; Klughammer, Barbara; Truman, Matt; Perez-Moreno, Pablo; Wu, Yi-Long
2016-08-01
The FASTACT-2 study of intercalated erlotinib with chemotherapy in Asian patients found that EGFR mutations were the main driver behind the significant progression-free survival (PFS) benefit noted in the overall population. Further exploratory biomarker analyses were conducted to provide additional insight. This multicenter, randomized, placebo-controlled, double-blind, phase III study investigated intercalated first-line erlotinib or placebo with gemcitabine/platinum, followed by maintenance erlotinib or placebo, for patients with stage IIIB/IV non-small cell lung cancer (NSCLC). Provision of samples for biomarker analysis was encouraged but not mandatory. The following biomarkers were analyzed (in order of priority): EGFR mutation by cobas(®) test, KRAS mutation by cobas(®)KRAS test, HER2 by immunohistochemistry (IHC), HER3 by IHC, ERCC1 by IHC, EGFR gene copy number by fluorescence in-situ hybridization (FISH) and EGFR by IHC. All subgroups were assessed for PFS (primary endpoint), overall survival (OS), non-progression rate and objective response rate. Overall, 256 patients provided samples for analysis. Considerable overlap was noted among biomarkers, except for EGFR and KRAS mutations, which are mutually exclusive. Other than EGFR mutations (p<0.0001), no other biomarkers were significantly predictive of outcomes in a treatment-by-biomarker interaction test, although ERCC1 IHC-positive status was predictive of improved OS for the erlotinib arm versus placebo in EGFR wild-type patients (median 18.4 vs 9.5 months; hazard ratio [HR] HR=0.32, 95% confidence intervals [CI]: 0.14-0.69, p=0.0024). Activating EGFR mutations were predictive for improved treatment outcomes with a first-line intercalated regimen of chemotherapy and erlotinib in NSCLC. ERCC1 status may have some predictive value in EGFR wild-type disease, but requires further investigation. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
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Harms, Paul W; Collie, Angela M B; Hovelson, Daniel H; Cani, Andi K; Verhaegen, Monique E; Patel, Rajiv M; Fullen, Douglas R; Omata, Kei; Dlugosz, Andrzej A; Tomlins, Scott A; Billings, Steven D
2016-03-01
Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes with CK20-positive Merkel cell carcinoma, including RB1 mutations restricted to Merkel cell polyomavirus-negative tumors. However, some CK20-negative Merkel cell carcinomas harbor mutations not previously described in Merkel cell carcinoma. Hence, CK20-negative Merkel cell carcinomas harbor diverse oncogenic drivers which may represent therapeutic targets in individual tumors.
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Harms, Paul W.; Collie, Angela M. B.; Hovelson, Daniel H.; Cani, Andi K.; Verhaegen, Monique E.; Patel, Rajiv M.; Fullen, Douglas R.; Omata, Kei; Dlugosz, Andrzej A.; Tomlins, Scott A.; Billings, Steven D.
2016-01-01
Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin-20 (CK20) is expressed in approximately 95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (ten Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%)) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes with CK20-positive Merkel cell carcinoma, including RB1 mutations restricted to Merkel cell polyomavirus-negative tumors. However, some CK20-negative Merkel cell carcinomas harbor mutations not previously described in Merkel cell carcinoma. Hence, CK20-negative Merkel cell carcinomas harbor diverse oncogenic drivers which may represent therapeutic targets in individual tumors. PMID:26743471
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Multiple mutant clones in blood rarely coexist
NASA Astrophysics Data System (ADS)
Dingli, David; Pacheco, Jorge M.; Traulsen, Arne
2008-02-01
Leukemias arise due to mutations in the genome of hematopoietic (blood) cells. Hematopoiesis has a multicompartment architecture, with cells exhibiting different rates of replication and differentiation. At the root of this process, one finds a small number of stem cells, and hence the description of the mutation-selection dynamics of blood cells calls for a stochastic approach. We use stochastic dynamics to investigate to which extent acquired hematopoietic disorders are associated with mutations of single or multiple genes within developing blood cells. Our analysis considers the appearance of mutations both in the stem cell compartment as well as in more committed compartments. We conclude that in the absence of genomic instability, acquired hematopoietic disorders due to mutations in multiple genes are most likely very rare events, as multiple mutations typically require much longer development times compared to those associated with a single mutation.
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Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.
Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S
2016-09-01
To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Feasibility of cell-free circulating tumor DNA testing for lung cancer.
Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael
2016-01-01
Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications.
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Hyperforin Exhibits Antigenotoxic Activity on Human and Bacterial Cells.
Imreova, Petronela; Feruszova, Jana; Kyzek, Stanislav; Bodnarova, Kristina; Zduriencikova, Martina; Kozics, Katarina; Mucaji, Pavel; Galova, Eliska; Sevcovicova, Andrea; Miadokova, Eva; Chalupa, Ivan
2017-01-21
Hyperforin (HF), a substance that accumulates in the leaves and flowers of Hypericum perforatum L. (St. John's wort), consists of a phloroglucinol skeleton with lipophilic isoprene chains. HF exhibits several medicinal properties and is mainly used as an antidepressant. So far, the antigenotoxicity of HF has not been investigated at the level of primary genetic damage, gene mutations, and chromosome aberrations, simultaneously. The present work is designed to investigate the potential antigenotoxic effects of HF using three different experimental test systems. The antigenotoxic effect of HF leading to the decrease of primary/transient promutagenic genetic changes was detected by the alkaline comet assay on human lymphocytes. The HF antimutagenic effect leading to the reduction of gene mutations was assessed using the Ames test on the standard Salmonella typhimurium (TA97, TA98, and TA100) bacterial strains, and the anticlastogenic effect of HF leading to the reduction of chromosome aberrations was evaluated by the in vitro mammalian chromosome aberration test on the human tumor cell line HepG2 and the non-carcinogenic cell line VH10. Our findings provided evidence that HF showed antigenotoxic effects towards oxidative mutagen zeocin in the comet assay and diagnostic mutagen (4-nitroquinoline-1-oxide) in the Ames test. Moreover, HF exhibited an anticlastogenic effect towards benzo(a)pyrene and cisplatin in the chromosome aberration test.
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Nagy, Ádám; Pongor, Lőrinc Sándor; Szabó, András; Santarpia, Mariacarmela; Győrffy, Balázs
2017-02-15
KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC). However, the prognostic role of KRAS mutation status in NSCLC still remains controversial. We hypothesize that the expression changes of genes affected by KRAS mutation status will have the most prominent effect and could be used as a prognostic signature in lung cancer. We divided NSCLC patients with mutation and RNA-seq data into KRAS mutated and wild type groups. Mann-Whitney test was used to identify genes showing altered expression between these cohorts. Mean expression of the top five genes was designated as a "transcriptomic fingerprint" of the mutation. We evaluated the effect of this signature on clinical outcome in 2,437 NSCLC patients using univariate and multivariate Cox regression analysis. Mutation of KRAS was most common in adenocarcinoma. Mutation status and KRAS expression were not correlated to prognosis. The transcriptomic fingerprint of KRAS include FOXRED2, KRAS, TOP1, PEX3 and ABL2. The KRAS signature had a high prognostic power. Similar results were achieved when using the second and third set of strongest genes. Moreover, all cutoff values delivered significant prognostic power (p < 0.01). The KRAS signature also remained significant (p < 0.01) in a multivariate analysis including age, gender, smoking history and tumor stage. We generated a "surrogate signature" of KRAS mutation status in NSCLC patients by computationally linking genotype and gene expression. We show that secondary effects of a mutation can have a higher prognostic relevance than the primary genetic alteration itself. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.
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Gaber, Rania; Watermann, Iris; Kugler, Christian; Vollmer, Ekkehard; Perner, Sven; Reck, Martin; Goldmann, Torsten
2017-01-01
Targeting epidermal growth factor receptor (EGFR) in patients with non-small-cell lung cancer (NSCLC) having EGFR mutations is associated with an improved overall survival. The aim of this study is to verify, if EGFR mutations detected by immunohistochemistry (IHC) is a convincing way to preselect patients for DNA-sequencing and to figure out, the statistical association between EGFR mutation, wild-type EGFR overexpression, gene copy number gain, which are the main factors inducing EGFR tumorigenic activity and the clinicopathological data. Two hundred sixteen tumor tissue samples of primarily chemotherapeutic naïve NSCLC patients were analyzed for EGFR mutations E746-A750del and L858R and correlated with DNA-sequencing. Two hundred six of which were assessed by IHC, using 6B6 and 43B2 specific antibodies followed by DNA-sequencing of positive cases and 10 already genotyped tumor tissues were also included to investigate debugging accuracy of IHC. In addition, EGFR wild-type overexpression was IHC evaluated and EGFR gene copy number determination was performed by fluorescence in situ hybridization (FISH). Forty-one÷206 (19.9%) cases were positive for mutated EGFR by IHC. Eight of them had EGFR mutations of exons 18-21 by DNA-sequencing. Hit rate of 10 already genotyped NSCLC mutated cases was 90% by IHC. Positive association was found between EGFR mutations determined by IHC and both EGFR overexpression and increased gene copy number (p=0.002 and p<0.001, respectively). Additionally, positive association was detected between EGFR mutations, high tumor grade and clinical stage (p<0.001). IHC staining with mutation specific antibodies was demonstrated as a possible useful screening test to preselect patients for DNA-sequencing.
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Autosomal dominant polycystic kidney disease caused by somatic and germline mosaicism.
Tan, A Y; Blumenfeld, J; Michaeel, A; Donahue, S; Bobb, W; Parker, T; Levine, D; Rennert, H
2015-04-01
Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by loss of function mutations of PKD1 or PKD2 genes. Although PKD1 is highly polymorphic and the new mutation rate is relatively high, the role of mosaicism is incompletely defined. Herein, we describe the molecular analysis of ADPKD in a 19-year-old female proband and her father. The proband had a PKD1 truncation mutation c.10745dupC (p.Val3584ArgfsX43), which was absent in paternal peripheral blood lymphocytes (PBL). However, very low quantities of this mutation were detected in the father's sperm DNA, but not in DNA from his buccal cells or urine sediment. Next generation sequencing (NGS) analysis determined the level of this mutation in the father's PBL, buccal cells and sperm to be ∼3%, 4.5% and 10%, respectively, consistent with somatic and germline mosaicism. The PKD1 mutation in ∼10% of her father's sperm indicates that it probably occurred early in embryogenesis. In ADPKD cases where a de novo mutation is suspected because of negative PKD gene testing of PBL, additional evaluation with more sensitive methods (e.g. NGS) of the proband PBL and paternal sperm can enhance detection of mosaicism and facilitate genetic counseling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.
Wasielewski, Marijke; Hanifi-Moghaddam, Pejman; Hollestelle, Antoinette; Merajver, Sofia D; van den Ouweland, Ans; Klijn, Jan G M; Ethier, Stephen P; Schutte, Mieke
2009-01-01
The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.
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Therapeutic targeting of RNA splicing in myelodysplasia.
Kim, Young Joon; Abdel-Wahab, Omar
2017-07-01
Genomic analysis of patients with myelodysplastic syndromes (MDS) has identified that mutations within genes encoding RNA splicing factors represent the most common class of genetic alterations in MDS. These mutations primarily affect SF3B1, SRSF2, U2AF1, and ZRSR2. Current data suggest that these mutations perturb RNA splicing catalysis in a manner distinct from loss of function but how exactly the global changes in RNA splicing imparted by these mutations result in MDS is not well delineated. At the same time, cells bearing mutations in RNA splicing factors are exquisitely dependent on the presence of the remaining wild-type (WT) allele to maintain residual normal splicing for cell survival. The high frequency of these mutations in MDS, combined with their mutual exclusivity and noteworthy dependence on the WT allele, make targeting RNA splicing attractive in MDS. To this end, two promising therapeutic approaches targeting RNA splicing are being tested clinically currently. These include molecules targeting core RNA splicing catalysis by interfering with the ability of the SF3b complex to interact with RNA, as well as molecules degrading the auxiliary RNA splicing factor RBM39. The preclinical and clinical evaluation of these compounds are discussed here in addition to their potential as therapies for spliceosomal mutant MDS. Copyright © 2017 Elsevier Inc. All rights reserved.
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Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Pozzi, Gianni
2016-01-01
Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10−9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth. PMID:27959952
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Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Gennaro, Maria Laura; Pozzi, Gianni
2016-01-01
Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.
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Wang, Rui; Zhang, Yang; Pan, Yunjian; Li, Yuan; Hu, Haichuan; Cai, Deng; Li, Hang; Ye, Ting; Luo, Xiaoyang; Zhang, Yiliang; Li, Bin; Shen, Lei; Sun, Yihua; Chen, Haiquan
2015-10-27
To determine the frequency of driver mutations in Chinese non-small cell lung cancer (NSCLC) patients. Comprehensive mutational analysis was performed in 1356 lung adenocarcinoma, 503 squamous cell carcinoma, 57 adenosquamous lung carcinoma, 19 large cell carcinoma and 8 sarcomatoid carcinoma. The effect of EGFR tyrosine kinase inhibitors (TKIs) on EGFR-mutated lung adenocarcinoma patients after disease recurrence was investigated. Mutations in EGFR kinase domain, HER2 kinase domain, KRAS, BRAF, ALK, ROS1 and RET were mutually exclusive. In lung adenocarcinoma cases "pan-negative" for the seven above-mentioned driver mutations, we also detected two oncogenic EGFR extracellular domain mutations (A289D and R324L), two HER2 extracellular and transmembrane domain mutations (S310Y and V659E), one ARAF S214C mutation and two CD74-NRG1 fusions. Six (1.2%) FGFR3 activating mutations were identified in lung squamous cell carcinoma (five S249C and one R248C). There were three (15.8%) EGFR mutations and four (21.1%) KRAS mutations in large cell carcinoma. Three (37.5%) KRAS mutations were detected in sarcomatoid carcinoma. In EGFR-mutated lung adenocarcinoma patients who experienced disease recurrence, treatment with EGFR TKIs was an independent predictor of better overall survival (HR = 0.299, 95% CI: 0.172-0.519, P < 0.001). We determined the frequency of driver mutations in a large series of Chinese NSCLC patients. EGFR TKIs might improve the survival outcomes of EGFR-mutated lung adenocarcinoma patients who experienced disease recurrence.
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Burdon, Kathryn P.; Dave, Alpana; Jamieson, Robyn V.; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E.
2008-01-01
Purpose Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. Methods All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Results Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. Conclusions This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions. PMID:18949062
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Sharma, Shiwani; Burdon, Kathryn P; Dave, Alpana; Jamieson, Robyn V; Yaron, Yuval; Billson, Frank; Van Maldergem, Lionel; Lorenz, Birgit; Gécz, Jozef; Craig, Jamie E
2008-01-01
Nance-Horan syndrome is typically characterized by severe bilateral congenital cataracts and dental abnormalities. Truncating mutations in the Nance-Horan syndrome (NHS) gene cause this X-linked genetic disorder. NHS encodes two isoforms, NHS-A and NHS-1A. The ocular lens expresses NHS-A, the epithelial and neuronal cell specific isoform. The NHS-A protein localizes in the lens epithelium at the cellular periphery. The data to date suggest a role for this isoform at cell-cell junctions in epithelial cells. This study aimed to identify the causative mutations in new patients diagnosed with Nance-Horan syndrome and to investigate the effect of mutations on subcellular localization of the NHS-A protein. All coding exons of NHS were screened for mutations by polymerase chain reaction (PCR) and sequencing. PCR-based mutagenesis was performed to introduce three independent mutations in the NHS-A cDNA. Expression and localization of the mutant proteins was determined in mammalian epithelial cells. Truncating mutations were found in 6 out of 10 unrelated patients from four countries. Each of four patients carried a novel mutation (R248X, P264fs, K1198fs, and I1302fs), and each of the two other patients carried two previously reported mutations (R373X and R879X). No mutation was found in the gene in four patients. Two disease-causing mutations (R134fs and R901X) and an artificial mutation (T1357fs) resulted in premature truncation of the NHS-A protein. All three mutant proteins failed to localize to the cellular periphery in epithelial cells and instead were found in the cytoplasm. This study brings the total number of mutations identified in NHS to 18. The mislocalization of the mutant NHS-A protein, revealed by mutation analysis, is expected to adversely affect cell-cell junctions in epithelial cells such as the lens epithelium, which may explain cataractogenesis in Nance-Horan syndrome patients. Mutation analysis also shed light on the significance of NHS-A regions for its localization and, hence, its function at epithelial cell junctions.
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Waide, Emily H; Dekkers, Jack CM; Ross, Jason W; Rowland, Raymond RR; Wyatt, Carol R; Ewen, Catherine L; Evans, Alyssa B; Thekkoot, Dinesh M; Boddicker, Nicholas J; Serão, Nick VL; Ellinwood, N Matthew; Tuggle, Christopher K
2017-01-01
Mutations in over 30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as severe combined immunodeficiency (SCID). SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T− B− NK+ SCID, representing the first example of naturally occurring SCID in pigs. Here, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed two mutations were present in the Artemis gene, which in homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented here reveals two mutations in the Artemis gene that cause T− B− NK+ SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues. PMID:26320255
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Heymann, Jonas J.; Bulman, William A.; Maxfield, Roger A.; Powell, Charles A.; Halmos, Balazs; Sonett, Joshua; Beaubier, Nike T.; Crapanzano, John P.; Mansukhani, Mahesh M.; Saqi, Anjali
2014-01-01
Background: Lung cancer is a leading cause of mortality, and patients often present at a late stage. More recently, advances in screening, diagnosing, and treating lung cancer have been made. For instance, greater numbers of minimally invasive procedures are being performed, and identification of lung adenocarcinoma driver mutations has led to the implementation of targeted therapies. Advances in molecular techniques enable use of scant tissue, including cytology specimens. In addition, per recently published consensus guidelines, cytology-derived cell blocks (CBs) are preferred over direct smears. Yet, limited comparison of molecular testing of fine-needle aspiration (FNA) CBs and corresponding histology specimens has been performed. This study aimed to establish concordance of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma (KRAS) virus homolog testing between FNA CBs and histology samples from the same patients. Materials and Methods: Patients for whom molecular testing for EGFR or KRAS was performed on both FNA CBs and histology samples containing lung adenocarcinoma were identified retrospectively. Following microdissection, when necessary, concordance of EGFR and KRAS molecular testing results between FNA CBs and histology samples was evaluated. Results: EGFR and/or KRAS testing was performed on samples obtained from 26 patients. Concordant results were obtained for all EGFR (22/22) and KRAS (17/17) mutation analyses performed. Conclusions: Identification of mutations in lung adenocarcinomas affects clinical decision-making, and it is important that results from small samples be accurate. This study demonstrates that molecular testing on cytology CBs is as sensitive and specific as that on histology. PMID:24987443
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Riccio, Gennaro; Maisto, Maria; Bottone, Sara; Badolati, Nadia; Rossi, Giovanni Battista; Tenore, Gian Carlo; Stornaiuolo, Mariano; Novellino, Ettore
2017-11-18
Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. 'Annurca' and Malus domestica cv 'Limoncella'; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract.
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Maisto, Maria; Bottone, Sara; Badolati, Nadia; Rossi, Giovanni Battista; Novellino, Ettore
2017-01-01
Inhibitors of the Wingless-related Integration site (WNT)/β-catenin pathway have recently been under consideration as potential chemopreventive agents against Familial Adenomatous Polyposis (FAP). This autosomal-dominant syndrome is caused by germline mutations in the gene coding for the protein APC and leads to hyperactivation of the WNT/β-catenin signaling pathway, uncontrolled intestinal cell proliferation and formation of adenocarcinomas. The aim of the present work was to: (i) test, on in vitro cultures of cells carrying FAP mutations and on ex vivo biopsies of FAP patients, the WNT inhibitory activity of extracts from two common southern Italian apples, Malus pumila Miller cv. ‘Annurca’ and Malus domestica cv ‘Limoncella’; (ii) identify the mechanisms underpinning their activities and; (iii) evaluate their potency upon gastrointestinal digestion. We here show that both Annurca and Limoncella apple extracts act as WNT inhibitors, mostly thanks to their polyphenolic contents. They inhibit the pathway in colon cells carrying FAP mutations with active dilutions falling in ranges close to consumer-relevant concentrations. Food-grade manufacturing of apple extracts increases their WNT inhibitory activity as result of the conversion of quercetin glycosides into the aglycone quercetin, a potent WNT inhibitor absent in the fresh fruit extract. However, in vitro simulated gastrointestinal digestion severely affected WNT inhibitory activity of apple extracts, as result of a loss of polyphenols. In conclusion, our results show that apple extracts inhibit the WNT pathway in colon cells carrying FAP mutations and represent a potential nutraceutical alternative for the treatment of this pathology. Enteric coating is advisable to preserve the activity of the extracts in the colon-rectal section of the digestive tract. PMID:29156563
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Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models.
Brasil, Aline A; Magalhães, Rayne S S; De Carvalho, Mariana D C; Paiva, Isabel; Gerhardt, Ellen; Pereira, Marcos D; Outeiro, Tiago F; Eleutherio, Elis C A
2018-06-01
Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.
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Cocco, Emiliano; Lopez, Salvatore; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L; Menderes, Gulden; Zammataro, Luca; Buza, Natalia; Hui, Pei; Wong, Serena; Zhao, Siming; Bai, Yalai; Rimm, David L; Ratner, Elena; Litkouhi, Babak; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D
2016-01-01
Background: Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets. Methods: Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs. Results: We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs. Conclusions: Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours. PMID:27351214
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McKell, Allison O.; LaConte, Leslie E. W.
2017-01-01
ABSTRACT Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1. PMID:28100623
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Genotoxicity evaluation of Mequindox in different short-term tests.
Ihsan, Awais; Wang, Xu; Tu, Hong-Gong; Zhang, Wei; Dai, Meng-Hong; Peng, De-Peng; Wang, Yu-Lian; Huang, Ling-Li; Chen, Dong-Mei; Mannan, Shazia; Tao, Yan-Fei; Liu, Zhen-Li; Yuan, Zong-Hui
2013-01-01
Quinoxaline-1,4-dioxides (QdNOs) are the potent heterocyclic N-oxides with interesting biological properties such as antibacterial, anticandida, antitubercular, anticancer and antiprotozoal activities. Here, we tested and compared the mequindox (MEQ) for mutagenic abilities in a battery of different short term tests according to OECD guidelines. When compared with the controls, a strong mutagenicity of MEQ and carbadox (CBX) was observed with an approximate concentration-effect relationship in Salmonella reverse mutation test, chromosome aberration test, unscheduled DNA synthesis assay and HGPRT gene mutation test, in the absence and presence of S(9)-mix. In in vivo micronucleus test, CBX produced significant increase in the proportion of micronucleus formation than MEQ in mice bone marrow cells. From these results, we can conclude that MEQ had a strong genotoxic potential to mammalian cells in vitro as well as in vivo and its mutagenicity is slightly higher than CBX. Our results, for the 1st time, discuss the genotoxic potential of MEQ. These results not only confirm the earlier findings about CBX but also extend the knowledge and awareness about the genotoxic risk of QdNO derivatives. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Salgia, Ravi
2016-01-01
ABSTRACT Introduction: Advances in the biology of non-small-cell lung cancer, especially adenocarcinoma, reveal multiple molecular subtypes driving oncogenesis. Accordingly, individualized targeted therapeutics are based on mutational diagnostics. Areas covered: Advances in strategies and techniques for individualized treatment, particularly of adenocarcinoma, are described through literature review. Approved therapies are established for some molecular subsets, with new driver mutations emerging that represent increasing proportions of patients. Actionable mutations are de novo oncogenic drivers or acquired resistance mediators, and mutational profiling is important for directing therapy. Patients should be monitored for emerging actionable resistance mutations. Liquid biopsy and associated multiplex diagnostics will be important means to monitor patients during treatment. Expert commentary: Outcomes with targeted agents may be improved by integrating mutation screens during treatment to optimize subsequent therapy. In order for this to be translated into impactful patient benefit, appropriate platforms and strategies need to be optimized and then implemented universally. PMID:27139190
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Effect of Mutation Order on Myeloproliferative Neoplasms
Nangalia, Jyoti; Silber, Yvonne; Wedge, David C.; Grinfeld, Jacob; Baxter, E. Joanna; Massie, Charles E.; Papaemmanuil, Elli; Menon, Suraj; Godfrey, Anna L.; Dimitropoulou, Danai; Guglielmelli, Paola; Bellosillo, Beatriz; Besses, Carles; Döhner, Konstanze; Harrison, Claire N.; Vassiliou, George S.; Vannucchi, Alessandro; Campbell, Peter J.; Green, Anthony R.
2015-01-01
BACKGROUND Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as “TET2-first patients”), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first (“JAK2-first patients”) had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2–TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.) PMID:25671252
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Hall, Michael J; Innocent, Julie; Rybak, Christina; Veloski, Colleen; Scott, Walter J; Wu, Hong; Ridge, John A; Hoffman, John P; Borghaei, Hossein; Turaka, Aruna; Daly, Mary B
2015-01-01
Introduction Multiple endocrine neoplasia 1 (MEN1) is a cancer syndrome resulting from mutations of the MEN1 gene. The syndrome is characterized by neoplasia of the parathyroid and pituitary glands, and malignant tumors of the endocrine pancreas. Other manifestations include benign lipomas, angiofibromas, and carcinoid tumors commonly originating in the colon, thymus, and lung. This is the first report of MEN1 syndrome manifesting as bilateral granulosa cell ovarian tumors, and which is associated with a rare intronic mutation of the MEN1 gene. Case report A 41-year-old woman presented with abdominal pain, increasing abdominal girth, and dysmenorrhea. Ultrasound demonstrated enlarged ovaries and uterine fibroids. After an exploratory laparotomy, she subsequently underwent bilateral salpingo–oophorectomy with hysterectomy where the pathology revealed bilateral cystic granulosa cell tumors of the ovaries. Additional workup including computed tomography imaging discovered a thymic mass, which the pathology showed was malignant, along with a pancreatic mass suspicious for a neuroendocrine tumor. Hyperparathyroidism was also discovered and was found to be secondary to a parathyroid adenoma. Genetic testing revealed an exceedingly rare mutation in the MEN1 gene (c.654 + 1 G>A). Discussion Mutations of the menin gene leading to MEN1 syndrome are classically nonsense or missense mutations producing a dysfunctional protein product. Recently, researchers described a novel mutation of MEN1 (c.654 + 1 G>A) in a male proband meeting the criteria for clinical MEN1 syndrome. Functional analysis performed on the stable mutant protein showed selective disruption of the transforming growth factor beta signaling pathway, yet it maintained its wild-type ability to inhibit nuclear factor kappa B and to suppress JunD transcriptional activity. Conclusion To our knowledge, this is the first report of MEN1 syndrome associated with bilateral granulosa cell malignancy. We postulate that this presentation may be due to the novel menin gene mutation recently described. PMID:25733923
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Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi
2017-05-01
Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.
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Zhang, Hui; Zhang, Jinshan; Wei, Pengliang; Zhang, Botao; Gou, Feng; Feng, Zhengyan; Mao, Yanfei; Yang, Lan; Zhang, Heng; Xu, Nanfei; Zhu, Jian-Kang
2014-08-01
The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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Soo, Ross A; Kubo, Akihito; Ando, Masahiko; Kawaguchi, Tomoya; Ahn, Myung-Ju; Ou, Sai-Hong Ignatius
2017-09-01
Molecular studies have demonstrated actionable driver oncogene alterations are more frequent in never-smokers with non-small-cell lung cancer (NSCLC). The etiology of these driver oncogenes in patients with NSCLC remains unknown, and environmental tobacco smoke (ETS) is a potential cause in these cases. We assembled clinical and genetic information for never-smoker patients with NSCLC accrued in Japan, Korea, Singapore, and the United States. To determine an association between cumulative ETS and activating EGFR mutations or ALK rearrangements, the Mantel extension test was used. Multivariate analysis on activating EGFR and ALK gene rearrangements was performed using the generalized linear mixed model with nations as a random effect. From July 2007 to December 2012, 498 never-smokers with pathologically proven NSCLC were registered and tested for the association between ETS and EGFR and ALK status. EGFR mutations were more frequent in the ever-ETS cohort (58.4%) compared with the never-ETS cohort (39.6%), and the incidence of EGFR mutations was significantly associated with the increment of cumulative ETS (cETS) in female never-smokers (P = .033), whereas the incidence of ALK rearrangements was not significantly different between the ever-ETS and never-ETS cohorts. Odds ratio for EGFR mutations for each 10-year increment in cETS was 1.091 and 0.89 for female and male never-smokers (P = .031 and P = .263, respectively). Increased ETS exposure was closely associated with EGFR mutations in female never-smokers with NSCLC in the expanded multinational cohort. However, the association of ETS and ALK rearrangements in never-smokers with NSCLC was not significant. Copyright © 2017 Elsevier Inc. All rights reserved.
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Feasibility of re-biopsy and EGFR mutation analysis in patients with non-small cell lung cancer.
Kim, Tae-Ok; Oh, In-Jae; Kho, Bo Gun; Park, Ha Young; Chang, Jin Sun; Park, Cheol-Kyu; Shin, Hong-Joon; Lim, Jung-Hwan; Kwon, Yong-Soo; Kim, Yu-Il; Lim, Sung-Chul; Kim, Young-Chul; Choi, Yoo-Duk
2018-05-14
In cases of EGFR-tyrosine kinase inhibitor (TKI) failure, re-biopsy may be useful to understand resistance mechanisms and guide further treatment decisions. However, performing re-biopsy is challenging because of several hurdles. We assessed the feasibility of re-biopsy in advanced non-small cell lung cancer (NSCLC) patients in real-world clinical practice. We retrospectively reviewed the clinical and pathologic data of advanced NSCLC patients who experienced disease progression after previous treatment with EGFR-TKIs at a single tertiary hospital in Korea between January 2014 and December 2016. Re-biopsy specimens included small biopsy, surgical tissue, or liquid-based cytology. EGFR mutation was tested using peptide nucleic acid-mediated clamping PCR. Of the 230 NSCLC patients that experienced progression after EGFR-TKI therapy, 105 (45.7%) underwent re-biopsy. Re-biopsy was successfully performed in 94 (89.5%) patients, and 11 patients were diagnosed with no malignancy. The complication rate was 8.6%, including seven cases of pneumothorax. EGFR mutation testing was performed on 75 patients using re-biopsy specimens. Of the 57 patients who had sensitizing mutations at diagnosis, T790M mutations were found in 19 (33.3%), while 38 (66.7%) had no T790M mutation. Multivariate analysis showed that the re-biopsy group was younger (P = 0.002) and exhibited a previous response to EGFR-TKIs (P < 0.001). Re-biopsy in advanced NSCLC is feasible in real world clinical practice, particularly in younger patients and those who achieved a previous response to EGFR-TKIs. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.
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Osoegawa, Atsushi; Hashimoto, Takafumi; Takumi, Yohei; Abe, Miyuki; Yamada, Tomonori; Kobayashi, Ryoji; Miyawaki, Michiyo; Takeuchi, Hideya; Okamoto, Tatsuro; Sugio, Kenji
2018-03-28
Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.
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Sajed, Dipti P; Faquin, William C; Carey, Chris; Severson, Eric A; H Afrogheh, Amir; A Johnson, Carl; Blacklow, Stephen C; Chau, Nicole G; Lin, Derrick T; Krane, Jeffrey F; Jo, Vickie Y; Garcia, Joaquín J; Sholl, Lynette M; Aster, Jon C
2017-11-01
NOTCH1 is frequently mutated in adenoid cystic carcinoma (ACC). To test the idea that immunohistochemical (IHC) staining can identify ACCs with NOTCH1 mutations, we performed IHC for activated NOTCH1 (NICD1) in 197 cases diagnosed as ACC from 173 patients. NICD1 staining was positive in 194 cases (98%) in 2 major patterns: subset positivity, which correlated with tubular/cribriform histology; and diffuse positivity, which correlated with a solid histology. To determine the relationship between NICD1 staining and NOTCH1 mutational status, targeted exome sequencing data were obtained on 14 diffusely NICD1-positive ACC specimens from 11 patients and 15 subset NICD1-positive ACC specimens from 15 patients. This revealed NOTCH1 gain-of-function mutations in 11 of 14 diffusely NICD1-positive ACC specimens, whereas all subset-positive tumors had wild-type NOTCH1 alleles. Notably, tumors with diffuse NICD1 positivity were associated with significantly worse outcomes (P=0.003). To determine whether NOTCH1 activation is unique among tumors included in the differential diagnosis with ACC, we performed NICD1 IHC on a cohort of diverse salivary gland and head and neck tumors. High fractions of each of these tumor types were positive for NICD1 in a subset of cells, particularly in basaloid squamous cell carcinomas; however, sequencing of basaloid squamous cell carcinomas failed to identify NOTCH1 mutations. These findings indicate that diffuse NICD1 positivity in ACC correlates with solid growth pattern, the presence of NOTCH1 gain-of-function mutations, and unfavorable outcome, and suggest that staining for NICD1 can be helpful in distinguishing ACC with solid growth patterns from other salivary gland and head and neck tumors.
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Chakraborty, Jayashree B; Mahato, Sanjit K; Joshi, Kalpana; Shinde, Vaibhav; Rakshit, Srabanti; Biswas, Nabendu; Choudhury Mukherjee, Indrani; Mandal, Labanya; Ganguly, Dipyaman; Chowdhury, Avik A; Chaudhuri, Jaydeep; Paul, Kausik; Pal, Bikas C; Vinayagam, Jayaraman; Pal, Churala; Manna, Anirban; Jaisankar, Parasuraman; Chaudhuri, Utpal; Konar, Aditya; Roy, Siddhartha; Bandyopadhyay, Santu
2012-01-01
Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings. © 2011 Japanese Cancer Association.
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Identification of constrained cancer driver genes based on mutation timing.
Sakoparnig, Thomas; Fried, Patrick; Beerenwinkel, Niko
2015-01-01
Cancer drivers are genomic alterations that provide cells containing them with a selective advantage over their local competitors, whereas neutral passengers do not change the somatic fitness of cells. Cancer-driving mutations are usually discriminated from passenger mutations by their higher degree of recurrence in tumor samples. However, there is increasing evidence that many additional driver mutations may exist that occur at very low frequencies among tumors. This observation has prompted alternative methods for driver detection, including finding groups of mutually exclusive mutations and incorporating prior biological knowledge about gene function or network structure. Dependencies among drivers due to epistatic interactions can also result in low mutation frequencies, but this effect has been ignored in driver detection so far. Here, we present a new computational approach for identifying genomic alterations that occur at low frequencies because they depend on other events. Unlike passengers, these constrained mutations display punctuated patterns of occurrence in time. We test this driver-passenger discrimination approach based on mutation timing in extensive simulation studies, and we apply it to cross-sectional copy number alteration (CNA) data from ovarian cancer, CNA and single-nucleotide variant (SNV) data from breast tumors and SNV data from colorectal cancer. Among the top ranked predicted drivers, we find low-frequency genes that have already been shown to be involved in carcinogenesis, as well as many new candidate drivers. The mutation timing approach is orthogonal and complementary to existing driver prediction methods. It will help identifying from cancer genome data the alterations that drive tumor progression.
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Identification of Constrained Cancer Driver Genes Based on Mutation Timing
Sakoparnig, Thomas; Fried, Patrick; Beerenwinkel, Niko
2015-01-01
Cancer drivers are genomic alterations that provide cells containing them with a selective advantage over their local competitors, whereas neutral passengers do not change the somatic fitness of cells. Cancer-driving mutations are usually discriminated from passenger mutations by their higher degree of recurrence in tumor samples. However, there is increasing evidence that many additional driver mutations may exist that occur at very low frequencies among tumors. This observation has prompted alternative methods for driver detection, including finding groups of mutually exclusive mutations and incorporating prior biological knowledge about gene function or network structure. Dependencies among drivers due to epistatic interactions can also result in low mutation frequencies, but this effect has been ignored in driver detection so far. Here, we present a new computational approach for identifying genomic alterations that occur at low frequencies because they depend on other events. Unlike passengers, these constrained mutations display punctuated patterns of occurrence in time. We test this driver–passenger discrimination approach based on mutation timing in extensive simulation studies, and we apply it to cross-sectional copy number alteration (CNA) data from ovarian cancer, CNA and single-nucleotide variant (SNV) data from breast tumors and SNV data from colorectal cancer. Among the top ranked predicted drivers, we find low-frequency genes that have already been shown to be involved in carcinogenesis, as well as many new candidate drivers. The mutation timing approach is orthogonal and complementary to existing driver prediction methods. It will help identifying from cancer genome data the alterations that drive tumor progression. PMID:25569148
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Galán, Maria; García-Herrero, Carmen-Maria; Azriel, Sharona; Gargallo, Manuel; Durán, Maria; Gorgojo, Juan-Jose; Andía, Victor-Manuel; Navas, Maria-Angeles
2011-01-01
Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype. PMID:21170474
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Cancer-associated TERT promoter mutations abrogate telomerase silencing
Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk
2015-01-01
Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells. DOI: http://dx.doi.org/10.7554/eLife.07918.001 PMID:26194807
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Driver, Brandon R; Portier, Bryce P; Mody, Dina R; Deavers, Michael; Bernicker, Eric H; Kim, Min P; Teh, Bin S; Santacruz, Jose F; Kopas, Lisa; Munden, Reginald F; Cagle, Philip T
2016-04-01
The classification of pulmonary large cell carcinoma has undergone a major revision with the recent World Health Organization (WHO) 2015 Classification. Many large cell carcinomas are now reassigned to either adenocarcinoma with solid pattern or nonkeratinizing squamous cell carcinoma based on immunopositivity for adenocarcinoma markers or squamous cell carcinoma markers, respectively. Large cell carcinomas that are negative for adenocarcinoma and squamous cell carcinoma immunomarkers are now classified as large cell carcinoma with null immunohistochemical features (LCC-N). Although a few studies investigated the mutation profile of large cell carcinomas grouped by immunostain profile before the publication of the new WHO classification, investigation of tumors previously diagnosed as large cell carcinoma and reclassified according to the 2015 WHO classification has not, to our knowledge, been reported. To determine the mutation profiles of pulmonary large cell carcinomas reclassified by WHO 2015 criteria. Archival cases of non-small cell lung carcinoma with large cell carcinoma morphology (n = 17) were reclassified according to 2015 WHO criteria. To determine mutation profile, we employed Ion Torrent (Life Technologies, Carlsbad, California)-based next-generation sequencing (50 genes; more than 2800 mutations) in addition to real-time quantitative reverse transcription polymerase chain reaction for ALK translocation detection. Two of 17 cases (12%) were reclassified as LCC-N, and both had mutations-BRAF D594N in one case and KRAS G12C in the other case. Seven of 17 cases (41%) were reclassified in the adenocarcinoma with solid pattern group, which showed one KRAS G12C and one EGFR E709K + G719C double mutation in addition to mutations in TP53. Eight of 17 cases (47%) were reclassified in the nonkeratinizing squamous cell carcinoma group, which showed mutations in PIK3CA, CDKN2A, and TP53. No ALK translocations or amplifications were detected. The adenocarcinoma with solid pattern group showed mutations typical of adenocarcinoma, whereas the nonkeratinizing squamous cell carcinoma group showed mutations typical of squamous cell carcinoma. Both LCC-N cases had mutations associated with adenocarcinoma, supporting the hypothesis that LCC-N is related to adenocarcinoma.
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Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi
2015-01-01
Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383
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The Role of Notch Signaling Pathway in Breast Cancer Pathogenesis
2005-07-01
breast cancer cells, I tested whether ErbB2 overexpression will cooperate with Notch in HMLE cells. While overexpression of activated Notch1 failed to...tyrosine kinase upstream of Ras normally found overexpressed in many breast cancers , also failed to transform HMLE cells. These observations suggested...cooperation between Notch1IC and ErbB2 signaling in transforming HMLE cells. Breast cancers typically do not harbor oncogenic Ras mutations; nevertheless
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Ng, Isaac Ks; Lee, Joanne; Ng, Christopher; Kosmo, Bustamin; Chiu, Lily; Seah, Elaine; Mok, Michelle Meng Huang; Tan, Karen; Osato, Motomi; Chng, Wee-Joo; Yan, Benedict; Tan, Lip Kun
2018-01-01
Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations ( RUNX1 , GATA2 , DNMT3A , BCORL1 , BCOR , 2 PHF6 and CDKN2A ) were identified in the pre-induction sample, of which 5 remained ( RUNX1 , DNMT3A , BCORL1 , BCOR and 1 out of 2 PHF6 ) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1 -mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.
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Pritchard, Colin C; Smith, Christina; Salipante, Stephen J; Lee, Ming K; Thornton, Anne M; Nord, Alex S; Gulden, Cassandra; Kupfer, Sonia S; Swisher, Elizabeth M; Bennett, Robin L; Novetsky, Akiva P; Jarvik, Gail P; Olopade, Olufunmilayo I; Goodfellow, Paul J; King, Mary-Claire; Tait, Jonathan F; Walsh, Tom
2012-07-01
Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome.
Daga, Sergio; Baldassarri, Margherita; Lo Rizzo, Caterina; Fallerini, Chiara; Imperatore, Valentina; Longo, Ilaria; Frullanti, Elisa; Landucci, Elisa; Massella, Laura; Pecoraro, Carmine; Garosi, Guido; Ariani, Francesca; Mencarelli, Maria Antonietta; Mari, Francesca; Renieri, Alessandra; Pinto, Anna Maria
2018-02-01
Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end-stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes-targeted therapeutic strategies have been hampered by the difficulty of non-invasively isolating them and transcripts-based diagnostic approaches are complicated by the inaccessibility of other COL4 chains-expressing cells. We firstly isolated podocyte-lineage cells from ATS patients' urine samples, in a non-invasive way. RT-PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patient's urine derived podocyte-lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease-relevant cells. © 2017 Wiley Periodicals, Inc.
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[Clinical Tests Testing New Therapies for Stargardt Disease].
Kousal, B; Ďuďáková, Ľ; Hlavatá, L; Lišková, P
2016-02-01
To provide information on currently ongoing clinical trials for Stargardt disease. We have searched the clinical trial register (www.clinicaltrials.gov) for the keyword "Stargardt" and list active ongoing studies. There are currently eight registered clinical trials enrolling patients with Stargardt disease; all in phase I or II aiming at four mechanisms of action: inhibition of the production of vitamin A toxic dimers, gene therapy restoring wild type transcription of the ABCA4 gene, neuroprotection preventing retinal cells from oxidative damage, and replacement of the damaged retinal pigment epithelium using stem cell therapy. The basic prerequisite for enrolment in the vast majority of clinical trials is confirmation of the clinical diagnosis by mutational analysis. The wide variety of therapies that are registered as clinical trials for Stargardt disease significantly raises the possibility that effective treatments will be available in the near future for this currently incurable condition and that molecular genetic testing should be increasingly considered. Stargardt disease, clinical trial, ABCA4, mutation.
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Cytotoxicity of Zardaverine in Embryonal Rhabdomyosarcoma from a Costello Syndrome Patient
Cartledge, Donna M.; Robbins, Katherine M.; Drake, Katherine M.; Sternberg, Rachel; Stabley, Deborah L.; Gripp, Karen W.; Kolb, E. Anders; Sol-Church, Katia; Napper, Andrew D.
2017-01-01
Costello syndrome (CS) patients suffer from a very high 10% incidence of embryonal rhabdomyosarcoma (ERMS). As tools to discover targeted therapeutic leads, we used a CS patient-derived ERMS cell line (CS242 ERMS) harboring a homozygous p.G12A mutation in HRAS, and a control cell line derived from the same patient comprising non-malignant CS242 fibroblasts with a heterozygous p.G12A HRAS mutation. A library of 2,000 compounds with known pharmacological activities was screened for their effect on CS242 ERMS cell viability. Follow-up testing in a panel of cell lines revealed that various compounds originally developed for other indications were remarkably selective; notably, the phosphodiesterase (PDE) inhibitor zardaverine was at least 1,000-fold more potent in CS242 ERMS than in the patient-matched non-malignant CS242 fibroblasts, other ERMS, or normal fibroblasts. Chronic treatment with zardaverine led to the emergence of resistant cells, consistent with CS242 ERMS comprising a mixed population of cells. Many PDE inhibitors in addition to zardaverine were tested on CS242 ERMS, but almost all had no effect. Interestingly, zardaverine and analogs showed a similar cytotoxicity profile in CS242 ERMS and cervical carcinoma-derived HeLa cells, suggesting a mechanism of action common to both cell types that does not require the presence of an HRAS mutation (HeLa contains wild type HRAS). Two recent studies presented possible mechanistic explanations for the cytotoxicity of zardaverine in HeLa cells. One revealed that zardaverine inhibited a HeLa cell-based screen measuring glucocorticoid receptor (GR) activation; however, using engineered HeLa cells, we ruled out a specific effect of zardaverine on signaling through the GR. The second attributed zardaverine toxicity in HeLa cells to promotion of the interaction of phosphodiesterase 3A and the growth regulatory protein Schlafen 12. We speculate that this work may provide a possible mechanism for zardaverine action in CS242 ERMS, although we have not yet tested this hypothesis. In conclusion, we have identified zardaverine as a potent cytotoxic agent in a CS-derived ERMS cell line and in HeLa. Although we have ruled out some possibilities, the mechanism of action of zardaverine in CS242 ERMS remains to be determined. PMID:28421158
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Evaluation of the genotoxicity of extracts of Houttuynia cordata Thunb.
Kang, Chang Keun; Hah, Dae Sik; Kim, Chung Hui; Kim, Euikyung; Kim, Jong Shu
2012-01-01
The present study was conducted to evaluate the activity of methanol extracts from Houttuynia cordata Thunb. (HC) in a reverse mutation assay in Salmonella typhimurium, and a chromosome aberration assay in the Chinese hamster ovary (CHO) cell line and to evaluate its effect on the occurrence of polychromatic erythrocytes in mice. In the reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2urvA(-), methanol extracts of HC (5, 2.5, 1.25, 0.62, or 0.312 mg/plate) did not induce reverse mutations in the presence or absence of an S9 metabolic activation mixture. In the chromosome aberration test using CHO cells, methanol extracts (1.25, 2.5 or 5 μg/ml) caused a few incidences of structural and numerical aberrations, in both of absence or presence of an S9 metabolic activation mixture, but in comparison with the positive control group, these incidences were not significantly increased. In the mouse micronucleus test, no significant increases in the occurrence of micronucleated polychromatic erythrocytes were observed in male ICR mice that were orally administered methanol extracts of HC at doses of 2.0, 1.0, or 0.5 g/kg. From these results, we concluded that the methanol extracts of HC did not induce harmful effects on genes in bacteria, a mammalian cell system or in mouse bone marrow cells. Thus, HC's use for health promotion and/or a sick remedy for humans may be safe.
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CRISPR/Cas9 system and its applications in human hematopoietic cells.
Hu, Xiaotang
2016-11-01
Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.
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Garcia-Montero, Andres C; Jara-Acevedo, Maria; Alvarez-Twose, Ivan; Teodosio, Cristina; Sanchez-Muñoz, Laura; Muñiz, Carmen; Muñoz-Gonzalez, Javier I; Mayado, Andrea; Matito, Almudena; Caldas, Carolina; Morgado, Jose M; Escribano, Luis; Orfao, Alberto
2016-02-11
Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. © 2016 by The American Society of Hematology.
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JAM-C regulates tight junctions and integrin-mediated cell adhesion and migration.
Mandicourt, Guillaume; Iden, Sandra; Ebnet, Klaus; Aurrand-Lions, Michel; Imhof, Beat A
2007-01-19
Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.
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Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran
2018-03-01
Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.
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Gentry, Sara N; Jackson, Trachette L
2013-01-01
Hierarchical organized tissue structures, with stem cell driven cell differentiation, are critical to the homeostatic maintenance of most tissues, and this underlying cellular architecture is potentially a critical player in the development of a many cancers. Here, we develop a mathematical model of mutation acquisition to investigate how deregulation of the mechanisms preserving stem cell homeostasis contributes to tumor initiation. A novel feature of the model is the inclusion of both extrinsic and intrinsic chemical signaling and interaction with the niche to control stem cell self-renewal. We use the model to simulate the effects of a variety of types and sequences of mutations and then compare and contrast all mutation pathways in order to determine which ones generate cancer cells fastest. The model predicts that the sequence in which mutations occur significantly affects the pace of tumorigenesis. In addition, tumor composition varies for different mutation pathways, so that some sequences generate tumors that are dominated by cancerous cells with all possible mutations, while others are primarily comprised of cells that more closely resemble normal cells with only one or two mutations. We are also able to show that, under certain circumstances, healthy stem cells diminish due to the displacement by mutated cells that have a competitive advantage in the niche. Finally, in the event that all homeostatic regulation is lost, exponential growth of the cancer population occurs in addition to the depletion of normal cells. This model helps to advance our understanding of how mutation acquisition affects mechanisms that influence cell-fate decisions and leads to the initiation of cancers.
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Midha, Anita; Dearden, Simon; McCormack, Rose
2015-01-01
Mutations in the epidermal growth factor receptor (EGFR) gene are commonly observed in non-small-cell lung cancer (NSCLC), particularly in tumors of adenocarcinoma (ADC) histology (NSCLC/ADC). Robust data exist regarding the prevalence of EGFR mutations in Western and Asian patients with NSCLC/ADC, yet there is a lack of data for patients of other ethnicities. This review collated available data with the aim of creating a complete, global picture of EGFR mutation frequency in patients with NSCLC/ADC by ethnicity. Worldwide literature reporting EGFR mutation frequency in patients with NSCLC/ADC was reviewed, to create a map of the world populated with EGFR mutation frequency by country (a ‘global EGFR mutMap’). A total of 151 worldwide studies (n=33162 patients with NSCLC/ADC, of which 9749 patients had EGFR mutation-positive NSCLC/ADC) were included. There was substantial variation in EGFR mutation frequency between studies, even when grouped by geographic region or individual country. As expected, the Asia-Pacific NSCLC/ADC subgroup had the highest EGFR mutation frequency (47% [5958/12819; 87 studies; range 20%-76%]) and the lowest EGFR mutation frequency occurred in the Oceania NSCLC/ADC subgroup (12% [69/570; 4 studies; range 7%-36%]); however, comparisons between regions were limited due to the varying sizes of the patient populations studied. In all regional (geographic) subgroups where data were available, EGFR mutation frequency in NSCLC/ADC was higher in women compared with men, and in never-compared with ever-smokers. This review provides the foundation for a global map of EGFR mutation frequency in patients with NSCLC/ADC. The substantial lack of data from several large geographic regions of the world, notably Africa, the Middle East, Central Asia, and Central and South America, highlights a potential lack of routine mutation testing and the need for further investigations in these regions. PMID:26609494
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Ulivi, Paola; Chiadini, Elisa; Dazzi, Claudio; Dubini, Alessandra; Costantini, Matteo; Medri, Laura; Puccetti, Maurizio; Capelli, Laura; Calistri, Daniele; Verlicchi, Alberto; Gamboni, Alessandro; Papi, Maximilian; Mariotti, Marita; De Luigi, Nicoletta; Scarpi, Emanuela; Bravaccini, Sara; Turolla, Gian Michele; Amadori, Dino; Crinò, Lucio; Delmonte, Angelo
2016-09-01
Epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, and echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation are generally considered to be mutually exclusive. However, concomitant mutations are found in a small number of patients and the effect of these on response to targeted therapy is still unknown. We considered 380 non-small-cell lung cancer (NSCLC) patients who underwent nonsequential testing for EGFR and EML4-ALK translocation. KRAS mutation analysis was also performed on 282 patients. We found 1.6%, 1.1%, and 2.5% of patients who showed a double mutation comprising EGFR and EML4-ALK, EGFR and KRAS, and EML4-ALK and KRAS, respectively. Twenty-eight patients with EGFR mutation underwent first-line therapy with a tyrosine kinase receptor; a clinical benefit was observed in 81.8% of patients with EGFR mutations only and in 67% of those who also showed an EML4-ALK translocation. Twelve patients with an EML4-ALK translocation received crizotinib and 7 of these had disease progression within 3 months (2 had a concomitant KRAS mutation and 1 had a concomitant EGFR mutation). Two patients showed stable disease, 1 of whom also had a KRAS mutation. Two patients obtained a partial response and 1 had a complete response; all harbored an EML4-ALK translocation only. The median overall survival of patients who carried an EML4-ALK translocation alone or concomitant with a KRAS mutation was 57.1 (range, 10.7-not reached) and 10.7 (range, 4.6-not reached) months, respectively. Concomitant EGFR, EML4-ALK, or KRAS mutations can occur in NSCLC. Concomitant KRAS mutation and EML4-ALK translocation represents the most common double alteration and confers a poor prognosis. Copyright © 2015 Elsevier Inc. All rights reserved.
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Chen, Xi; Zhang, Yu; Xu, Bin; Cai, Zhongqi; Wang, Lin; Tian, Jinwen; Liu, Yuqi; Li, Yang
2016-09-01
Recent studies have shown that the mitochondrial DNA mutations are involved in the pathogenesis of hypertension. Our previous study identified mitochondrial tRNA(Ile) A4263G mutation in a large Chinese Han family with maternally-inherited hypertension. This mutation may contribute to mitochondrial Ca(2+) cycling dysfuntion, but the mechanism is unclear. Lymphoblastoid cell lines were derived from hypertensive and normotensive individuals, either with or without tRNA(Ile) A4263G mutation. The mitochondrial calcium ([Ca(2+)]m) in cells from hypertensive subjects with the tRNA(Ile) A4263G mutation, was lower than in cells from normotension or hypertension without mutation, or normotension with mutation (P<0.05). Meanwhile, cytosolic calcium ([Ca(2+)]c) in hypertensive with mutation cells was higher than another three groups. After exposure to caffeine, which could increase the [Ca(2+)]c by activating ryanodine receptor on endoplasmic reticulum, [Ca(2+)]c/[Ca(2+)]m increased higher than in hypertensive with mutation cells from another three groups. Moreover, MCU expression was decreased in hypertensive with mutation cells compared with in another three groups (P<0.05). [Ca(2+)]c increased and [Ca(2+)]m decreased after treatment with Ru360 (an inhibitor of MCU) or an siRNA against MCU. In this study we found decreased MCU expression in hypertensive with mutation cells contributed to dysregulated Ca(2+) uptake into the mitochondria, and cytoplasmic Ca(2+) overload. This abnormality might be involved in the underlying mechanisms of maternally inherited hypertension in subjects carrying the mitochondrial tRNA(Ile) A4263G mutation. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M
2015-06-01
The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.
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Michaelis, M; Rothweiler, F; Barth, S; Cinatl, J; van Rikxoort, M; Löschmann, N; Voges, Y; Breitling, R; von Deimling, A; Rödel, F; Weber, K; Fehse, B; Mack, E; Stiewe, T; Doerr, H W; Speidel, D; Cinatl, J
2011-01-01
Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells. PMID:22170099
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75 FR 51388 - 2-methyl-1,3-propanediol; Exemption from the Requirement of a Tolerance
Federal Register 2010, 2011, 2012, 2013, 2014
2010-08-20
... mutagenic in an in vitro chromosome aberration test, bacterial gene mutation test, and mammalian cell gene... Research on Cancer (IARC). Based on available studies, there is no evidence of genotoxic activity. There is... hazard endpoint, the Agency has determined that a quantitative risk assessment using safety factors...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Liu Shukun; Wu Mei; Zhang Zunzhen, E-mail: zhangzunzhen@163.co
2010-08-01
Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol {beta} +/+) and homozygous pol {beta} null cell (pol {beta} -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here,more » cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol {beta} -/- cells were more sensitive to ADM compared with pol {beta} +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol {beta} -/- cells. ROS level in pol {beta} -/- cells increased along with decreased activity of SOD. These results demonstrated that pol {beta} deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol {beta} -/- cells to ADM. These findings suggested that pol {beta} is vital for repairing oxidative damage induced by ADM.« less
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Sebastiano, Vittorio; Maeder, Morgan L; Angstman, James F; Haddad, Bahareh; Khayter, Cyd; Yeo, Dana T; Goodwin, Mathew J; Hawkins, John S; Ramirez, Cherie L; Batista, Luis F Z; Artandi, Steven E; Wernig, Marius; Joung, J Keith
2011-11-01
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications. Copyright © 2011 AlphaMed Press.
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Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke
2015-10-20
Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.
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NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells.
Di Ianni, Mauro; Baldoni, Stefano; Del Papa, Beatrice; Aureli, Patrizia; Dorillo, Erica; De Falco, Filomena; Albi, Elisa; Varasano, Emanuela; Di Tommaso, Ambra; Giancola, Raffaella; Accorsi, Patrizia; Rotta, Gianluca; Rompietti, Chiara; Silva Barcelos, Estevão Carlos; Campese, Antonio Francesco; Di Bartolomeo, Paolo; Screpanti, Isabella; Rosati, Emanuela; Falzetti, Franca; Sportoletti, Paolo
2018-01-01
To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed NOTCH1 mutation/expression in hematopoietic stem cells (HSCs). In NOTCH1- mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. NOTCH1 mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless NOTCH1 mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.
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Russell, Prudence A; Rogers, Toni-Maree; Solomon, Benjamin; Alam, Naveed; Barnett, Stephen A; Rathi, Vivek; Williams, Richard A; Wright, Gavin M; Conron, Matthew
2017-10-01
We investigated correlations between diagnosis according to the 2015 World Health Organization (WHO) classification of unresected lung tumours, molecular analysis and TTF1 expression in small biopsy and cytology specimens from 344 non-small cell lung carcinoma (NSCLC) patients. One case failed testing for EGFR, KRAS and ALK abnormalities and six had insufficient tumour for ALK testing. Overall mutation rate in 343 cases was 48% for the genes tested, with 19% EGFR, 33% KRAS and 4% BRAF mutations, and 5% ALK rearrangements detected. More EGFR-mutant (78%) and ALK-rearranged (75%) tumours had morphologic adenocarcinoma than KRAS-mutant (56%) tumours. Despite no significant difference in the overall rate of any molecular abnormality between morphologic adenocarcinoma (52%) and NSCLC, favour adenocarcinoma (47%) (p = 0.18), KRAS mutations were detected more frequently in the latter group. No significant difference in the overall rate of any molecular abnormality between TTF1 positive (49%) and TTF1 negative tumours (44%) (p = 0.92) was detected, but more EGFR-mutant (97%) and ALK-rearranged tumours (92%) were TTF1 positive than KRAS-mutant tumours (68%). Rates of EGFR, KRAS and BRAF mutations and ALK rearrangements in this Australian NSCLC patient population are consistent with the published international literature. Our findings suggest that 2015 WHO classification of unresected tumours may assist in identifying molecular subsets of advanced NSCLC. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.
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Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L; Bown, Nick; Tweddle, Deborah A
2016-12-27
Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ~85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression.
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Acquired initiating mutations in early hematopoietic cells of CLL patients.
Damm, Frederik; Mylonas, Elena; Cosson, Adrien; Yoshida, Kenichi; Della Valle, Véronique; Mouly, Enguerran; Diop, M'boyba; Scourzic, Laurianne; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Kikushige, Yoshikane; Davi, Frederick; Lambert, Jérôme; Gautheret, Daniel; Merle-Béral, Hélène; Sutton, Laurent; Dessen, Philippe; Solary, Eric; Akashi, Koichi; Vainchenker, William; Mercher, Thomas; Droin, Nathalie; Ogawa, Seishi; Nguyen-Khac, Florence; Bernard, Olivier A
2014-09-01
Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL. ©2014 American Association for Cancer Research.
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Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek
2017-01-01
Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
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Devine, Helen; Patani, Rickie
2017-04-01
The induced pluripotent state represents a decade-old Nobel prize-winning discovery. Human-induced pluripotent stem cells (hiPSCs) are generated by the nuclear reprogramming of any somatic cell using a variety of established but evolving methods. This approach offers medical science unparalleled experimental opportunity to model an individual patient's disease "in a dish." HiPSCs permit developmentally rationalized directed differentiation into any cell type, which express donor cell mutation(s) at pathophysiological levels and thus hold considerable potential for disease modeling, drug discovery, and potentially cell-based therapies. This review will focus on the translational potential of hiPSCs in clinical neurology and the importance of integrating this approach with complementary model systems to increase the translational yield of preclinical testing for the benefit of patients. This strategy is particularly important given the expected increase in prevalence of neurodegenerative disease, which poses a major burden to global health over the coming decades.
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Osimertinib for the treatment of non-small cell lung cancer.
Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Park, Keunchil; Ahn, Myung-Ju
2017-02-01
The T790 M mutation of the epidermal growth factor receptor (EGFR) gene is the most common mechanism underlying resistance to first- or second-generation EGFR tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). Osimertinib, a third-generation EGFR TKI, shows robust clinical efficacy in patients with T790 M-mutated lung cancer. Areas covered: We analyzed and reviewed clinical data for which patients who experienced acquired resistance to first- or second-generation EGFR TKIs. In addition, we briefly reviewed the potential role of osimertinib as a first-line therapy. Expert opinion: Osimertinib was recently licensed for use in NSCLC patients with acquired resistance to other EGFR TKIs due to a T790 M mutation. However, unresolved issues surrounding the optimal application of osimertinib remain, specifically the development of a plasma-based mutation test to overcome the difficulty of repeat biopsy, the efficacy of osimertinib for brain or leptomeningeal metastases, the development of resistance to osimertinib, and the use of osimertinib therapy as a first-line treatment. Many ongoing studies are currently exploring these issues.
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Melchardt, Thomas; Hufnagl, Clemens; Weinstock, David M; Kopp, Nadja; Neureiter, Daniel; Tränkenschuh, Wolfgang; Hackl, Hubert; Weiss, Lukas; Rinnerthaler, Gabriel; Hartmann, Tanja N; Greil, Richard; Weigert, Oliver; Egle, Alexander
2016-08-09
Little information is available about the role of certain mutations for clonal evolution and the clinical outcome during relapse in diffuse large B-cell lymphoma (DLBCL). Therefore, we analyzed formalin-fixed-paraffin-embedded tumor samples from first diagnosis, relapsed or refractory disease from 28 patients using next-generation sequencing of the exons of 104 coding genes. Non-synonymous mutations were present in 74 of the 104 genes tested. Primary tumor samples showed a median of 8 non-synonymous mutations (range: 0-24) with the used gene set. Lower numbers of non-synonymous mutations in the primary tumor were associated with a better median OS compared with higher numbers (28 versus 15 months, p=0.031). We observed three patterns of clonal evolution during relapse of disease: large global change, subclonal selection and no or minimal change possibly suggesting preprogrammed resistance. We conclude that targeted re-sequencing is a feasible and informative approach to characterize the molecular pattern of relapse and it creates novel insights into the role of dynamics of individual genes.
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Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme
Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.; ...
2014-12-05
The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less
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Molecular crowding overcomes the destabilizing effects of mutations in a bacterial ribozyme
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, Hui-Ting; Kilburn, D.; Behrouzi, R.
The native structure of the Azoarcus group I ribozyme is stabilized by the cooperative formation of tertiary interactions between double helical domains. Thus, even single mutations that break this network of tertiary interactions reduce ribozyme activity in physiological Mg2+ concentrations. Here, we report that molecular crowding comparable to that in the cell compensates for destabilizing mutations in the Azoarcus ribozyme. Small angle X-ray scattering, native polyacrylamide gel electrophoresis and activity assays were used to compare folding free energies in dilute and crowded solutions containing 18% PEG1000. Crowder molecules allowed the wild-type and mutant ribozymes to fold at similarly low Mg2+more » concentrations and stabilized the active structure of the mutant ribozymes under physiological conditions. This compensation helps explains why ribozyme mutations are often less deleterious in the cell than in the test tube. Nevertheless, crowding did not rescue the high fraction of folded but less active structures formed by double and triple mutants. We conclude that crowding broadens the fitness landscape by stabilizing compact RNA structures without improving the specificity of self-assembly.« less
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Wiehe, Kevin; Bradley, Todd; Meyerhoff, R Ryan; Hart, Connor; Williams, Wilton B; Easterhoff, David; Faison, William J; Kepler, Thomas B; Saunders, Kevin O; Alam, S Munir; Bonsignori, Mattia; Haynes, Barton F
2018-06-13
HIV-1 broadly neutralizing antibodies (bnAbs) require high levels of activation-induced cytidine deaminase (AID)-catalyzed somatic mutations for optimal neutralization potency. Probable mutations occur at sites of frequent AID activity, while improbable mutations occur where AID activity is infrequent. One bottleneck for induction of bnAbs is the evolution of viral envelopes (Envs) that can select bnAb B cell receptors (BCR) with improbable mutations. Here we define the probability of bnAb mutations and demonstrate the functional significance of key improbable mutations in three bnAb B cell lineages. We show that bnAbs are enriched for improbable mutations, which implies that their elicitation will be critical for successful vaccine induction of potent bnAb B cell lineages. We discuss a mutation-guided vaccine strategy for identification of Envs that can select B cells with BCRs that have key improbable mutations required for bnAb development. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
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Heterozygous ABCC8 mutations are a cause of MODY.
Bowman, P; Flanagan, S E; Edghill, E L; Damhuis, A; Shepherd, M H; Paisey, R; Hattersley, A T; Ellard, S
2012-01-01
The ABCC8 gene encodes the sulfonylurea receptor 1 (SUR1) subunit of the pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel. Inactivating mutations cause congenital hyperinsulinism (CHI) and activating mutations cause transient neonatal diabetes (TNDM) or permanent neonatal diabetes (PNDM) that can usually be treated with sulfonylureas. Sulfonylurea sensitivity is also a feature of HNF1A and HNF4A MODY, but patients referred for genetic testing with clinical features of these types of diabetes do not always have mutations in the HNF1A/4A genes. Our aim was to establish whether mutations in the ABCC8 gene cause MODY that is responsive to sulfonylurea therapy. We sequenced the ABCC8 gene in 85 patients with a BMI <30 kg/m², no family history of neonatal diabetes and who were deemed sensitive to sulfonylureas by the referring clinician or were sulfonylurea-treated. All had tested negative for mutations in the HNF1A and HNF4A genes. ABCC8 mutations were found in seven of the 85 (8%) probands. Four patients were heterozygous for previously reported mutations and four novel mutations, E100K, G214R, Q485R and N1245D, were identified. Only four probands fulfilled MODY criteria, with two diagnosed after 25 years and one patient, who had no family history of diabetes, as a result of a proven de novo mutation. ABCC8 mutations can cause MODY in patients whose clinical features are similar to those with HNF1A/4A MODY. Therefore, sequencing of ABCC8 in addition to the known MODY genes should be considered if such features are present, to facilitate optimal clinical management of these patients.
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[Diagnostic molecular pathology of lymphatic and myeloid neoplasms].
Klapper, W; Kreipe, H
2015-03-01
Molecular pathology has been an integral part of the diagnostics of tumors of the hematopoietic system substantially longer than for solid neoplasms. In contrast to solid tumors, the primary objective of molecular pathology in hematopoietic neoplasms is not the prediction of drug efficacy but the diagnosis itself by excluding reactive proliferation and by using molecular features for tumor classification. In the case of malignant lymphomas, the most commonly applied molecular tests are those for gene rearrangements for immunoglobulin heavy chains and T-cell receptors. However, this article puts the focus on new and diagnostically relevant assays in hematopathology. Among these are mutations of MYD88 codon 265 in lymphoplasmacytic lymphomas, B-raf V600E in hairy cell leukemia and Stat3 exon 21 in indolent T-cell lymphomas. In myeloproliferative neoplasms, MPL W515, calreticulin exon 9 and the BCR-ABL and JAK2 V617F junctions are the most frequently analyzed differentiation series. In myelodysplastic and myeloproliferative neoplasms, SRSF2, SETBP1 and CSF3R mutations provide important differential diagnostic information. Genes mutated in myelodysplastic syndromes (MDS) are particularly diverse but their analysis significantly improves the differential diagnostics between reactive conditions and MDS. The most frequent changes in MDS include mutations of TET2 and various genes encoding splicing factors.
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A quantitative assay measuring the function of lipase maturation factor 1
Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós
2009-01-01
Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043
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JAK2 mutation in a patient with CLL with coexistent myeloproliferative neoplasm (MPN).
Kodali, Srinivas; Chen, Chi; Rathnasabapathy, Chenthilmurugan; Wang, Jen Chin
2009-12-01
JAK2 mutation has not been described in patients with chronic lymphocytic leukemia (CLL). We found JAK2 mutation in a patient with CLL and coexisting myeloproliferative neoplasm (MPN). In this patient, we demonstrated the presence of the JAK2 mutation in CD34(+) progenitor cells, myeloid lineage cells, megakaryocytes, B lymphocytes but not in T lymphocytes. This case represents the first case report of JAK2 mutation in CLL and may also suggest that, JAK2 mutation most likely represents a secondary event from primary gene mutations involving the primitive stem cells which give rise to MPN and CLL. Furthermore, in this case, we believe that we are the first to demonstrate that JAK2 mutation in myeloid and B lymphoid cells but not T lymphocytes in a case of coexisting CLL and MPN.
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Baradat, Maryse; Jouanin, Isabelle; Dalleau, Sabine; Taché, Sylviane; Gieules, Mathilde; Debrauwer, Laurent; Canlet, Cécile; Huc, Laurence; Dupuy, Jacques; Pierre, Fabrice H F; Guéraud, Françoise
2011-11-21
Animal and epidemiological studies suggest that dietary heme iron would promote colorectal cancer. Oxidative properties of heme could lead to the formation of cytotoxic and genotoxic secondary lipid oxidation products, such as 4-hydroxy-2(E)-nonenal (HNE). This compound is more cytotoxic to mouse wild-type colon cells than to isogenic cells with a mutation on the adenomatous polyposis coli (APC) gene. The latter thus have a selective advantage, possibly leading to cancer promotion. This mutation is an early and frequent event in human colorectal cancer. To explain this difference, the HNE biotransformation capacities of the two cell types have been studied using radiolabeled and stable isotope-labeled HNE. Apc-mutated cells showed better biotransformation capacities than nonmutated cells did. Thiol compound conjugation capacities were higher for mutated cells, with an important advantage for the extracellular conjugation to cysteine. Both cells types were able to reduce HNE to 4-hydroxynonanal, a biotransformation pathway that has not been reported for other intestinal cells. Mutated cells showed higher capacities to oxidize 4-hydroxynonanal into 4-hydroxynonanoic acid. The mRNA expression of different enzymes involved in HNE metabolism such as aldehyde dehydrogenase 1A1, 2 and 3A1, glutathione transferase A4-4, or cystine transporter xCT was upregulated in mutated cells compared with wild-type cells. In conclusion, this study suggests that Apc-mutated cells are more efficient than wild-type cells in metabolizing HNE into thiol conjugates and 4-hydroxynonanoic acid due to the higher expression of key biotransformation enzymes. These differential biotransformation capacities would explain the differences of susceptibility between normal and Apc-mutated cells regarding secondary lipid oxidation products.
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Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A.; Verma, Amit; Boultwood, Jacqueline
2015-01-01
Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival. PMID:26623729
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Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A; Verma, Amit; Boultwood, Jacqueline
2015-12-29
Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.
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Johncilla, Melanie; Stachler, Matthew; Misdraji, Joseph; Lisovsky, Mikhail; Yozu, Masato; Lindeman, Neal; Lauwers, Gregory Y; Odze, Robert D; Srivastava, Amitabh
2018-02-08
There is limited data on the spectrum of molecular alterations in goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids of the appendix. We used next generation sequencing to determine mutations of potential pathogenetic and therapeutic significance in this rare group of tumors. Adequate DNA was successfully extracted in 34/46 cases and the final group included 18 goblet cell carcinoids and 16 adenocarcinoma ex goblet cell carcinoids. Illumina TruSeq™ was used for sequencing exons of a custom 282 gene panel using an Illumina HiSeq 2000. All cases had a minimum coverage depth of at least 50 reads. After filtering through the Exome Sequencing Project, the number of mutations per case ranged from 0-9 (mean:3). The mutational burden in adenocarcinoma ex goblet cell carcinoids was significantly higher than goblet cell carcinoids (mean 5 vs. 3; p < 0.05) but the spectrum of alterations overlapped between the two groups. The most frequent mutations included ARID1A (4/34), ARID2 (4/34), CDH1 (4/34), RHPN2 (4/34), and MLL2 (3/34). Some mutations typically seen in conventional colorectal adenocarcinomas were also identified but with much lower frequency (APC :4/34; KRAS :2/34). MLL2 and KRAS mutations were only seen in adenocarcinoma ex goblet cell carcinoids and TP53 mutations were limited to poorly differentiated adenocarcinoma ex goblet cell carcinoids (2/34). Copy number changes could be evaluated in 15/34 cases and showed low copy number gains in CDKN1B (6/15) and NFKBIA (6/15), among others. The overlapping molecular alterations suggest that goblet cell carcinoids and adenocarcinoma ex goblet cell carcinoids are best considered two grades of differentiation of the same tumor rather than two distinct histological types. Mutations in TP53, CDH1 and MLL2 mutations were predominantly present in the adenocarcinoma ex goblet cell carcinoid group consistent with transformation to a higher grade lesion. The unique mutational profile also offers an explanation for the poor chemosensitivity in these tumors and highlights the need for developing new targeted therapies.
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CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.
He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu
2016-01-01
The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.
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Analysis of the genes encoding neuroligins NLGN3 and NLGN4 in Bulgarian patients with autism.
Avdjieva-Tzavella, D M; Todorov, T P; Todorova, A P; Kirov, A V; Hadjidekova, S P; Rukova, B B; Litvinenko, I O; Hristova-Naydenova, D N; Tincheva, R S; Toncheva, D I
2012-01-01
Many studies have supported a genetic aetiology for autism. Neuroligins are postsynaptically located cell-adhesion molecules. Mutations in two X-linked neuroligin genes, NLGN3 and NLGN4, have been implicated in pathogenesis of autism. In order to confirm these causative mutations in our autistic population and to determine their frequency we screened 20 individuals affected with autism. We identified one patient with a point mutation in NLGN4 gene that substituted a Met for Thr 787 - c.2360C > T, p.(Thr787Met) and three patients with identical polymorphisms in the same gene: c.933C > T, p.(Thr311Thr) in combination with c.[1777C > T+1779C > G, p.(Leu593Leu)]. All patients tested for NLGN3 mutations were negative. These results indicate that mutations in these genes are responsible for at most a small fraction of autism cases.
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LATS2 tumour specific mutations and down-regulation of the gene in non-small cell carcinoma.
Strazisar, Mojca; Mlakar, Vid; Glavac, Damjan
2009-06-01
LATS2 is a new member of the LATS tumour suppressor family. The human LATS2 gene is located at chromosome 13q11-12, a hot spot (67%) for loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). We screened 129 non-small cell lung cancer samples and 13 lung cancer cell lines, initially for mutations in the LATS2 gene and subsequently for mutations in P53 and K-RAS genes. Either polymorphisms or mutations were identified in over 50 percent of analysed tumours. A novel missense mutation, S1073R, and a large deletion of 8 amino acids in the PAPA-repeat region were detected in 9 and 2 NSCLC tumours, respectively. Those mutations were not identified in the 13 lung cancer cell lines. Mutations were tumour specific and were absent from adjacent normal tissue and healthy controls. Down-regulation of the LATS2 gene was observed in most NSCLC tumours but was not related to any mutation or polymorphism. Tumours with a LATS2 mutation often also harbour a P53 but not K-RAS gene mutation and were mostly in an advanced stage of development, with regional lymph node involvement.
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Genetic predictors of MEK dependence in non-small cell lung cancer.
Pratilas, Christine A; Hanrahan, Aphrothiti J; Halilovic, Ensar; Persaud, Yogindra; Soh, Junichi; Chitale, Dhananjay; Shigematsu, Hisayuki; Yamamoto, Hiromasa; Sawai, Ayana; Janakiraman, Manickam; Taylor, Barry S; Pao, William; Toyooka, Shinichi; Ladanyi, Marc; Gazdar, Adi; Rosen, Neal; Solit, David B
2008-11-15
Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.
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Hu, Jiancheng; Yu, Haiyang; Kornev, Alexandr P; Zhao, Jianping; Filbert, Erin L; Taylor, Susan S; Shaw, Andrey S
2011-04-12
Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.
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Omar-Nazir, Laila; Shi, Xiaopei; Moller, Anders; Mousseau, Timothy; Byun, Soohyun; Hancock, Samuel; Seymour, Colin; Mothersill, Carmel
2018-08-01
The impact of the Chernobyl NPP accident on the environment is documented to be greater than expected, with higher mutation rates than expected at the current, chronic low dose rate. In this paper we suggest that the historic acute exposure and resulting non-targeted effects (NTE) such as delayed mutations and genomic instability could account at least in part for currently measured mutation rates and provide an initial test of this concept. Data from Møller and Mousseau on the phenotypic mutation rates of Chernobyl birds 9-11 generations post the Chernobyl accident were used and the reconstructed dose response for mutations was compared with delayed reproductive death dose responses (as a measure of genomic instability) in cell cultures exposed to a similar range of doses. The dose to birds present during the Chernobyl NPP accident was reconstructed through the external pathway due to Cs-137 with an estimate of the uncertainty associated with such reconstruction. The percentage of Chernobyl birds several generations after the accident without mutations followed the general shape of the clonogenic survival percentage of the progeny of irradiated cells, and it plateaued at low doses. This is the expected result if NTE of radiation are involved. We suggest therefore, that NTE induced by the historic dose may play a role in generating mutations in progeny many generations following the initial disaster. Copyright © 2018 Elsevier Inc. All rights reserved.
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Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi
2012-06-15
G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.
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Li, Wenbin; Zhang, Zhihui; Guo, Lei; Qiu, Tian; Ling, Yun; Cao, Jian; Guo, Huiqin; Zhao, Huan; Li, Lin; Ying, Jianming
2016-02-16
To investigate the use of molecular testing on cytological specimens in selecting advanced non-small cell lung cancer (NSCLC) patients who are adequate for targeted treatment, a total of 137 NSCLC cases were analyzed by fluorescence in situ hybridization (FISH) for anaplastic lymphoma kinase (ALK) rearrangements, and Epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were evaluated by quantitative real-time PCR (qRT-PCR) platform combining amplification refractory mutation system (ARMS) primers and TaqMan probes. Cytological specimens included 91 fine-needle aspirates, 5 fibreoptic bronchoscopic derived samples and 41 pleural effusions. Among 137 NSCLCs analyzed for ALK FISH, 16 (11.7%, of 137) were detected to harbor ALK rearrangement. FISH positive cases were all defined as adenocarcinoma (ADC) histologic subtype and the FNA samples showed the highest ALK positive rate (13.2%, 12/91). Of the 9 ALK FISH positive patients who received crizotinib treatment, 8 (88.9%) patients exhibited tumor regression. In addition, 60 (44.8%, of 134) cases were found to harbor EGFR mutations and 22 patients with EGFR sensitive mutations who received gefitinib or erlotinib treatment showed a median PFS of 16.0 months. Mutations of KRAS occurred in 8 (6.0%, of 134) cases and this was mutually exclusive from EGFR mutation. Our results demonstrated that ALK FISH and EGFR, KRAS mutational analysis on cytological specimens are sensitive methods for screening advanced stage NSCLC patients who are adequate for targeted treatment.
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Syrigos, Konstantinos N; Georgoulias, Vasilis; Zarogoulidis, Konstantinos; Makrantonakis, Paris; Charpidou, Andriani; Christodoulou, Christos
2018-06-01
Real-world evidence regarding the prevalence of epidermal growth factor receptor (EGFR) mutation-positive status (M+) and the clinicopathological characteristics associated with the presence of EGFR mutations in advanced non-small cell lung cancer (NSCLC) is scarce, especially among Caucasian populations. The present study aimed to bridge this gap, as well as to record treatment patterns and outcomes in routine-care settings. REASON (NCT01153399) was a prospective study of patients with stage IIIB/IV NSCLC and known EGFR mutation status. Clinicopathological, treatment characteristics and clinical outcomes were recorded and correlated with EGFR mutation testing results. Of 575 enrolled patients, EGFR mutations were detected in 15.7% of them. Male gender (p=0.008) and smoking (p<0.001), but not adenocarcinoma, were associated with EGFR M+ status. In the EGFR M+ subpopulation (n=88), absence of bone and/or brain metastasis and presence of exon 19 EGFR M+ status at diagnosis were independently associated with longer progression-free survival (PFS) (p=0.011 and p=0.040, respectively). In our population, males and smokers had decreased odds of harboring an EGFR mutation, while adenocarcinoma histology was not a significant predictor of EGFR M+ status. EGFR M+ patients with bone and/or brain metastases at diagnosis or mutations other than exon 19 deletions were at increased risk for earlier disease progression. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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Identification of Genetically Modified Maraba Virus as an Oncolytic Rhabdovirus
Brun, Jan; McManus, Dan; Lefebvre, Charles; Hu, Kang; Falls, Theresa; Atkins, Harold; Bell, John C; McCart, J. Andrea; Mahoney, Douglas; Stojdl, David F
2010-01-01
To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations. PMID:20551913
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Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.
Brun, Jan; McManus, Dan; Lefebvre, Charles; Hu, Kang; Falls, Theresa; Atkins, Harold; Bell, John C; McCart, J Andrea; Mahoney, Douglas; Stojdl, David F
2010-08-01
To expand our current array of safe and potent oncolytic viruses, we screened a variety of wild-type (WT) rhabdoviruses against a panel of tumor cell lines. Our screen identified a number of viruses with varying degrees of killing activity. Maraba virus was the most potent of these strains. We built a recombinant system for the Maraba virus platform, engineered a series of attenuating mutations to expand its therapeutic index, and tested their potency in vitro and in vivo. A double mutant (MG1) strain containing both G protein (Q242R) and M protein (L123W) mutations attenuated Maraba virus in normal diploid cell lines, yet appeared to be hypervirulent in cancer cells. This selective attenuation was mediated through interferon (IFN)-dependent and -independent mechanisms. Finally, the Maraba MG1 strain had a 100-fold greater maximum tolerable dose (MTD) than WT Maraba in vivo and resulted in durable cures when systemically administered in syngeneic and xenograft models. In summary, we report a potent new oncolytic rhabdovirus platform with unique tumor-selective attenuating mutations.
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Amyloid-like aggregation of provasopressin in diabetes insipidus and secretory granule sorting.
Beuret, Nicole; Hasler, Franziska; Prescianotto-Baschong, Cristina; Birk, Julia; Rutishauser, Jonas; Spiess, Martin
2017-01-26
Aggregation of peptide hormone precursors in the trans-Golgi network is an essential process in the biogenesis of secretory granules in endocrine cells. It has recently been proposed that this aggregation corresponds to the formation of functional amyloids. Our previous finding that dominant mutations in provasopressin, which cause cell degeneration and diabetes insipidus, prevent native folding and produce fibrillar aggregates in the endoplasmic reticulum (ER) might thus reflect mislocalized amyloid formation by sequences that evolved to mediate granule sorting. Here we identified two sequences responsible for fibrillar aggregation of mutant precursors in the ER: the N-terminal vasopressin nonapeptide and the C-terminal glycopeptide. To test their role in granule sorting, the glycopeptide was deleted and/or vasopressin mutated to inactivate ER aggregation while still permitting precursor folding and ER exit. These mutations strongly reduced sorting into granules and regulated secretion in endocrine AtT20 cells. The same sequences - vasopressin and the glycopeptide - mediate physiological aggregation of the wild-type hormone precursor into secretory granules and the pathological fibrillar aggregation of disease mutants in the ER. These findings support the amyloid hypothesis for secretory granule biogenesis.
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Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin
2015-05-12
The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Glass, H.B.
1962-02-01
Studies of the comparative mutagenic effects of ionizing radiations on males and females of Drosophila melanogaster are described. Sex-linked recessive lethal mutations were induced in nitrogen, air, and oxygen at doses of obtained in spermatozoa were uniformly about one-third higher than the frequencies obtained for the same dose and condition of atmosphere in mature oocytes. The relative frequencies of recessive autosomal lethals in mature male and female germ cells were identical with the relative fre quencies of sex-linked recessive lethals. In studies of point mutations and deficiencies involving specific loci, the rates in the male germ cells exceeded those inmore » the female germ cells by a proportion equal to that found to apply to autosomal and sex-linked recessive lethals. Spontaneous mutation rates were determined for a number of specific loci marked by recessive genes used in the tested stocks. Fertility was lost in both males and females when they were x-rayed as 80-hr-old larvae and bred upon emerging as adults. Females recovered their fertility rapidly but the males did so at a much slower rate. The brown; scarlet'' stock was found to carry two mutants each suppressed by a particular suppressor gene. It was concluded that the two suppressors act along different metabolic pathways departing from tryplophan, but both involving an x-ray-sensitive step. A study was made of the effects on the life span of two different mating regimens: immediate and deferred. It was found that the lines previously subjected to immediate mating significantly outlived the lines previously subjected to deferred mating when the mating regimen in the test was immediate mating. Exactly the opposite happened when the mating regimen in the test was deferred mating. (M.C.G.)« less
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FLT3-ITD cooperates with inv(16) to promote progression to acute myeloid leukemia
Kim, Hyung-Gyoon; Kojima, Kyoko; Swindle, C. Scott; Cotta, Claudiu V.; Huo, Yongliang; Reddy, Vishnu
2008-01-01
The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML. PMID:17967943
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Granell, Susana; Baldini, Giovanna; Mohammad, Sameer; Nicolin, Vanessa; Narducci, Paola; Storrie, Brian
2008-01-01
A variant α1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, α1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated α1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated α1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated α1-antitrypsin in the ER. In hepatoma cells, shift of mutated α1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated α1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway. PMID:18045994
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Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.
Cheng, J; Haas, M
1990-01-01
Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611
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Zhao, Jing; Ye, Xin; Xu, Yan; Chen, Minjiang; Zhong, Wei; Sun, Yun; Yang, Zhenfan; Zhu, Guanshan; Gu, Yi; Wang, Mengzhao
2016-12-01
Central nervous system (CNS) is the prevalent site for metastases in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-relapsed NSCLC patients. To understand the EGFR mutation status in paired cerebrospinal fluid (CSF) and plasma samples after EGFR-TKI treatment failure might be useful to guide the treatment of intra- and extracranial tumors in those patients. Paired CSF and plasma samples were collected from seven NSCLC patients with CNS metastases after EGFR-TKI failure. EGFR mutations were tested by amplification refractory mutation system (ARMS) and droplet digital PCR (ddPCR) methods. Gefitinib concentrations were evaluated by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). EGFR mutations were detected in all seven CSF samples, including three of E19-Del, three of L858R and one of E19-Del&T790M by both methods. On the other hand, majority of the matched plasma samples (5/7) were negative for EGFR mutations by both methods. The other two plasma samples were positive for E19-Del&T790M by ddPCR, and one of them had undetectable T790M by ARMS. Gefitinib concentration in CSF was much lower than that in plasma (mean CSF/plasma ratio: 1.8 %). After EGFR-TKI failure, majority of the NSCLC patients with CNS metastases remained positive detection of EGFR sensitive mutations in CSF, but much less detection in the matched plasma. Significantly low exposure of gefitinib in CSF might explain the intracranial protection of the EGFR sensitive mutation positive tumor cells.
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Using single cell sequencing data to model the evolutionary history of a tumor.
Kim, Kyung In; Simon, Richard
2014-01-24
The introduction of next-generation sequencing (NGS) technology has made it possible to detect genomic alterations within tumor cells on a large scale. However, most applications of NGS show the genetic content of mixtures of cells. Recently developed single cell sequencing technology can identify variation within a single cell. Characterization of multiple samples from a tumor using single cell sequencing can potentially provide information on the evolutionary history of that tumor. This may facilitate understanding how key mutations accumulate and evolve in lineages to form a heterogeneous tumor. We provide a computational method to infer an evolutionary mutation tree based on single cell sequencing data. Our approach differs from traditional phylogenetic tree approaches in that our mutation tree directly describes temporal order relationships among mutation sites. Our method also accommodates sequencing errors. Furthermore, we provide a method for estimating the proportion of time from the earliest mutation event of the sample to the most recent common ancestor of the sample of cells. Finally, we discuss current limitations on modeling with single cell sequencing data and possible improvements under those limitations. Inferring the temporal ordering of mutational sites using current single cell sequencing data is a challenge. Our proposed method may help elucidate relationships among key mutations and their role in tumor progression.
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Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y
2014-01-01
Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR–Ras–Raf–MEK–ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [3H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras–MAPK activity could be important in its anticancer activity. PMID:24853419
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Experimental design to evaluate directed adaptive mutation in Mammalian cells.
Bordonaro, Michael; Chiaro, Christopher R; May, Tobias
2014-12-09
We describe the experimental design for a methodological approach to determine whether directed adaptive mutation occurs in mammalian cells. Identification of directed adaptive mutation would have profound practical significance for a wide variety of biomedical problems, including disease development and resistance to treatment. In adaptive mutation, the genetic or epigenetic change is not random; instead, the presence and type of selection influences the frequency and character of the mutation event. Adaptive mutation can contribute to the evolution of microbial pathogenesis, cancer, and drug resistance, and may become a focus of novel therapeutic interventions. Our experimental approach was designed to distinguish between 3 types of mutation: (1) random mutations that are independent of selective pressure, (2) undirected adaptive mutations that arise when selective pressure induces a general increase in the mutation rate, and (3) directed adaptive mutations that arise when selective pressure induces targeted mutations that specifically influence the adaptive response. The purpose of this report is to introduce an experimental design and describe limited pilot experiment data (not to describe a complete set of experiments); hence, it is an early report. An experimental design based on immortalization of mouse embryonic fibroblast cells is presented that links clonal cell growth to reversal of an inactivating polyadenylation site mutation. Thus, cells exhibit growth only in the presence of both the countermutation and an inducing agent (doxycycline). The type and frequency of mutation in the presence or absence of doxycycline will be evaluated. Additional experimental approaches would determine whether the cells exhibit a generalized increase in mutation rate and/or whether the cells show altered expression of error-prone DNA polymerases or of mismatch repair proteins. We performed the initial stages of characterizing our system and have limited preliminary data from several pilot experiments. Cell growth and DNA sequence data indicate that we have identified a cell clone that exhibits several suitable characteristics, although further study is required to identify a more optimal cell clone. The experimental approach is based on a quantum biological model of basis-dependent selection describing a novel mechanism of adaptive mutation. This project is currently inactive due to lack of funding. However, consistent with the objective of early reports, we describe a proposed study that has not produced publishable results, but is worthy of report because of the hypothesis, experimental design, and protocols. We outline the project's rationale and experimental design, with its strengths and weaknesses, to stimulate discussion and analysis, and lay the foundation for future studies in this field.
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Péterfia, Bálint; Kalmár, Alexandra; Patai, Árpád V; Csabai, István; Bodor, András; Micsik, Tamás; Wichmann, Barnabás; Egedi, Krisztina; Hollósi, Péter; Kovalszky, Ilona; Tulassay, Zsolt; Molnár, Béla
2017-01-01
Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes ( APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53 ). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations ( FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation ( APC ). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.
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Abdouh, Mohamed; Hamam, Dana; Gao, Zu-Hua; Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio
2017-08-30
Horizontal transfer of malignant traits from the primary tumor to distant organs, through blood circulating factors, has recently become a thoroughly studied metastatic pathway to explain cancer dissemination. Recently, we reported that oncosuppressor gene-mutated human cells undergo malignant transformation when exposed to cancer patients' sera. We also observed that oncosuppressor mutated cells would show an increased uptake of cancer-derived exosomes and we suggested that oncosuppressor genes might protect the integrity of the cell genome by blocking integration of cancer-derived exosomes. In the present study, we tested the hypothesis that cancer patients' sera-derived exosomes might be responsible for the malignant transformation of target cells and that oncosuppressor mutation would promote their increased uptake. We also sought to unveil the mechanisms behind the hypothesized phenomena. We used human BRCA1 knockout (BRCA1-KO) fibroblasts as target cells. Cells were treated in vitro with cancer patients' sera or cancer patients' sera-derived exosomes. Treated cells were injected into NOD-SCID mice. Immunohistochemical analyses were performed to determine the differentiation state of the xenotransplants. Mass spectrometry analyses of proteins from cancer exosomes and the BRCA1-KO fibroblasts' membrane were performed to investigate possible de novo expression of molecules involved in vesicles uptake. Blocking of the identified molecules in vitro was performed and in vivo experiments were conducted to confirm the role of these molecules in the malignant transformation carried out by cancer-derived exosomes. Cells treated with exosomes isolated from cancer patients' sera underwent malignant transformation and formed tumors when transplanted into immunodeficient mice. Histological analyses showed that the tumors were carcinomas that differentiated into the same lineage of the primary tumors of blood donors. Oncosuppressor mutation promoted the de novo expression, on the plasma membrane of target cells, of receptors, responsible for the increased uptake of cancer-derived exosomes. The selective blocking of these receptors inhibited the horizontal transfer of malignant traits. These findings strengthen the hypothesis that oncogenic factors transferred via circulating cancer exosomes, induce malignant transformation of target cells even at distance. Oncosuppressor genes might protect the integrity of the cell genome by inhibiting the uptake of cancer-derived exosomes.
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Besaratinia, Ahmad; Kim, Sang-in; Pfeifer, Gerd P.
2009-01-01
Despite the predominance of UVA relative to UVB in terrestrial sunlight, solar mutagenesis in humans and rodents is characterized by mutations specific for UVB. We have investigated the kinetics of repair of UVA- and UVB-induced DNA lesions in relation to mutagenicity in transgenic mouse fibroblasts irradiated with equilethal doses of UVA and UVB in comparison to SSL. We have also analyzed mutagenesis-derived carcinogenesis in sunlight-associated human skin cancers by compiling the published data on mutation types found in crucial genes in non-melanoma and melanoma skin cancers. Here, we demonstrate a resistance to repair of UVB-induced CPDs together with rapid removal of UVA-induced oxidized purines in the genome overall and in the cII transgene of SSL-irradiated cells. The spectra of mutation induced by both UVB- and SSL-irradiation in this experimental system are characterized by significant increases in relative frequency of C to T transitions at dipyrimidines, which are the established signature mutation of CPDs. This type of mutation is also the predominant mutation found in human non-melanoma and melanoma tumor samples in the TP53, CDKN2, PTCH, and protein kinase genes. The prevailing role of UVB over UVA in solar mutagenesis in our test system can be ascribed to different kinetics of repair for lesions induced by the respective UV-irradiation. PMID:18326785
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Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei
2016-07-01
To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.
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Metabolite toxicity determines the pace of molecular evolution within microbial populations.
Lilja, Elin E; Johnson, David R
2017-02-14
The production of toxic metabolites has shaped the spatial and temporal arrangement of metabolic processes within microbial cells. While diverse solutions to mitigate metabolite toxicity have evolved, less is known about how evolution itself is affected by metabolite toxicity. We hypothesized that the pace of molecular evolution should increase as metabolite toxicity increases. At least two mechanisms could cause this. First, metabolite toxicity could increase the mutation rate. Second, metabolite toxicity could increase the number of available mutations with large beneficial effects that selection could act upon (e.g., mutations that provide tolerance to toxicity), which consequently would increase the rate at which those mutations increase in frequency. We tested this hypothesis by experimentally evolving the bacterium Pseudomonas stutzeri under denitrifying conditions. The metabolite nitrite accumulates during denitrification and has pH-dependent toxic effects, which allowed us to evolve P. stutzeri at different magnitudes of nitrite toxicity. We demonstrate that increased nitrite toxicity results in an increased pace of molecular evolution. We further demonstrate that this increase is generally due to an increased number of available mutations with large beneficial effects and not to an increased mutation rate. Our results demonstrate that the production of toxic metabolites can have important impacts on the evolutionary processes of microbial cells. Given the ubiquity of toxic metabolites, they could also have implications for understanding the evolutionary histories of biological organisms.
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Chimeric protein identification of dystrophic, Pierson and other laminin polymerization residues
McKee, Karen K.; Aleksandrova, Maya; Yurchenco, Peter D.
2018-01-01
Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain. PMID:29408412
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Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping
2013-09-01
A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.
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Odogwu, Lauretta; Mathieu, Luckson; Goldberg, Kirsten B; Blumenthal, Gideon M; Larkins, Erin; Fiero, Mallorie H; Rodriguez, Lisa; Bijwaard, Karen; Lee, Eunice Y; Philip, Reena; Fan, Ingrid; Donoghue, Martha; Keegan, Patricia; McKee, Amy; Pazdur, Richard
2018-03-01
On March 30, 2017, the U.S. Food and Drug Administration (FDA) approved osimertinib for the treatment of patients with metastatic, epidermal growth factor receptor (EGFR) T790M mutation-positive, non-small cell lung cancer (NSCLC), as detected by an FDA-approved test, whose disease has progressed following EGFR tyrosine kinase inhibitor (TKI) therapy. Approval was based on demonstration of a statistically significant difference in the primary endpoint of progression-free survival (PFS) when comparing osimertinib with chemotherapy in an international, multicenter, open-label, randomized trial (AURA3). In this confirmatory trial, which enrolled 419 patients, the PFS hazard ratio for osimertinib compared with chemotherapy per investigator assessment was 0.30 (95% confidence interval 0.23-0.41), p < .001, with median PFS of 10.1 months in the osimertinib arm and 4.4 months in the chemotherapy arm. Supportive efficacy data included PFS per blinded independent review committee demonstrating similar PFS results and an improved confirmed objective response rate per investigator assessment of 65% and 29%, with estimated median durations of response of 11.0 months and 4.2 months, in the osimertinib and chemotherapy arms, respectively. Patients received osimertinib 80 mg once daily and had a median duration of exposure of 8 months. The toxicity profile of osimertinib compared favorably with the profile of other approved EGFR TKIs and chemotherapy. The most common adverse drug reactions (>20%) in patients treated with osimertinib were diarrhea, rash, dry skin, nail toxicity, and fatigue. Herein, we review the benefit-risk assessment of osimertinib that led to regular approval, for patients with metastatic NSCLC harboring EGFR TKI whose disease has progressed on or after EGFR TKI therapy. Osimertinib administered to metastatic non-small cell lung cancer (NSCLC) patients harboring an EGFR T790M mutation, who have progressed on or following EGFR TKI therapy, demonstrated a substantial improvement over platinum-based doublet chemotherapy as well as durable intracranial responses. The ability to test for the T790M mutation in plasma using the FDA-approved cobas EGFR Mutation Test v2 (Roche, Basel, Switzerland) identifies patients with NSCLC tumors not amenable to biopsy. Since a 40% false-negative rate has been observed with the circulating tumor DNA test, re-evaluation of the feasibility of tissue biopsy is recommended to identify patients with a false-negative plasma test result who may benefit from osimertinib. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.
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Directed evolution of cell size in Escherichia coli.
Yoshida, Mari; Tsuru, Saburo; Hirata, Naoko; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen; Yomo, Tetsuya
2014-12-17
In bacteria, cell size affects chromosome replication, the assembly of division machinery, cell wall synthesis, membrane synthesis and ultimately growth rate. In addition, cell size can also be a target for Darwinian evolution for protection from predators. This strong coupling of cell size and growth, however, could lead to the introduction of growth defects after size evolution. An important question remains: can bacterial cell size change and/or evolve without imposing a growth burden? The directed evolution of particular cell sizes, without a growth burden, was tested with a laboratory Escherichia coli strain. Cells of defined size ranges were collected by a cell sorter and were subsequently cultured. This selection-propagation cycle was repeated, and significant changes in cell size were detected within 400 generations. In addition, the width of the size distribution was altered. The changes in cell size were unaccompanied by a growth burden. Whole genome sequencing revealed that only a few mutations in genes related to membrane synthesis conferred the size evolution. In conclusion, bacterial cell size could evolve, through a few mutations, without growth reduction. The size evolution without growth reduction suggests a rapid evolutionary change to diverse cell sizes in bacterial survival strategies.
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The TCA cycle is not required for selection or survival of multidrug-resistant Salmonella
Ricci, Vito; Loman, Nick; Pallen, Mark; Ivens, Alasdair; Fookes, Maria; Langridge, Gemma C.; Wain, John; Piddock, Laura J. V.
2012-01-01
Objectives The initial aim of this study was to use a systems biology approach to analyse a ciprofloxacin-selected multidrug-resistant (MDR) Salmonella enterica serotype Typhimurium, L664. Methods The whole genome sequence and transcriptome of L664 were analysed. Site-directed mutagenesis to recreate each mutation was carried out, followed by phenotypic characterization and mutation frequency analysis. As a mutation in the TCA cycle was detected we tested the controversial hypothesis regarding the bacterial response to bactericidal antibiotics, put forward by Kohanski et al. (Cell 2007; 130: 797–810 and Mol Cell 2010; 37: 311–20), that exposure of bacteria to agents such as ciprofloxacin produces reactive oxygen species (ROS), which transiently increase the mutation rate giving rise to MDR bacteria. Results L664 contained a mutation in ramR that conferred MDR. A mutation in tctA affected the TCA cycle and conferred the inability to grow on minimal agar. The virulence of L664 was not attenuated. Ciprofloxacin exposure produced ROS in L664 and SL1344 (tctA::aph), but it was reduced and occurred later. There were no significant differences in the rates of killing or mutations per generation to antibiotic resistance between the strains. Conclusions Whilst we confirm production of ROS in response to ciprofloxacin, we have no data to support the hypothesis that this leads to selection of MDR strains. Our results indicate that the mutations in tctA and glgA were random as they did not pre-exist in the parental strain, and that the mutation in tctA did not provide a survival advantage or disadvantage in the presence of antibiotic. PMID:22186876
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Staedtler, F.; Locher, F.; Sreenan, G.
1997-10-01
In order to evaluate the in vivo genotoxic potential of three putative genotoxic mouse liver carcinogens, high doses of 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine and 2, 4-diaminotoluene were tested short term in the Big Blue{reg_sign} transgenic mouse mutation assay. Small statistically significant increases in the lacI mutant frequencies in the liver by factors 1.7 to 2.0 were found. A representative number of 347 lacI mutants isolated from liver tissue of male and female animals were analyses by DNA sequencing. The mutational spectra were examined with the Adams-Skopek algorithm. The spontaneous mutational spectra from untreated male and female animals were similar and consistent withmore » spectral Big Blue{reg_sign} control data stored in the lacI database. Most of the background mutations were located in the 5{prime} portion of the coding region of the lacI gene. Single base substitutions were most prominent. G:C to A:T transitions and G:C to T:A transversions occurred predominatly and were preferentially located at CpG sites. Despite the increases observed in the mutant frequencies of the treated animals, the corresponding mutational spectra did not differ from the controls. However, it is possible that certain classes of point mutations were substantially increased but not detected due to the limited number of sequenced mutants. In two animals treated with 2, 4- diaminotoluene unusually high mutant frequencies and the multiple occurrence of certain mutations in the liver was observed. From one of these animals six lacI mutants isolated from colon tissue were all different. Since 2, 4-diaminotoluene was shown to induce liver cell proliferation these results may reflect clonal expansion of single mutated liver cells.« less
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Two novel ALK mutations mediate acquired resistance to the next generation ALK inhibitor alectinib
Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L.; Khan, Tahsin M.; Gainor, Justin F.; Iafrate, A. John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A.; Shaw, Alice T.
2014-01-01
Purpose The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. Experimental Design We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity-relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. Results We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. Conclusions We have identified two novel ALK mutations arising after alectinib exposure which are sensitive to other next generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. PMID:25228534
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Ames Test to Detect Mutagenicity of 2-Alkylcyclobutanones: A Review.
Barbezan, Angélica B; Martins, Regiane; Bueno, Jennifer B; Villavicencio, Anna Lúcia C H
2017-07-01
Food irradiation is an effective and safe method for preservation and long-term storage, and it is approved for use in over 60 countries for various applications in a wide variety of food products. This process is performed by use of accelerated electron beams, X-rays, or gamma radiation ( 60 Co or 137 Cs). 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that have fatty acids (triglycerides) and are subjected to irradiation. Since the 1990s toxicological safety studies of 2-ACBs have been conducted extensively through synthetic compounds, then and tests to determine if the compounds have any mutagenic activity are strictly necessary. The Ames test was chosen by many researchers to assess the mutagenicity of 2-ACBs. The test uses distinct bacterial cell lines Salmonella typhimurium to detect point mutations at sites guanine-cytosine (G-C) and Escherichia coli to detect point mutations at sites adenine-thymine (A-T). This bibliographic research aims to bring together all the results obtained and a comparison and cell lines used, type of plates, and solvents. This research showed that no mutagenic activity was observed in any of the cell lines and concentrations evaluated by the works of authors, so the 2-ACBs compounds showed no mutagenic substance in concentrations detectable by the Ames test. © 2017 Institute of Food Technologists®.
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Cinti, Caterina; Leoncini, Lorenzo; Nyongo, Aggrey; Ferrari, Filomena; Lazzi, Stefano; Bellan, Cristiana; Vatti, Rosella; Zamparelli, Alessandra; Cevenini, Gabriele; osi, Gian Marco T; Claudio, Pier Paolo; Maraldi, Nadir M.; Tosi, Piero; Giordano, Antonio
2000-01-01
Alterations of cell cycle-associated genes probably contribute to the pathogenesis of Burkitt’s Lymphoma (BL), in addition to c-myc translocation. Mutations disrupting the nuclear localization signal of the retinoblastoma-related gene RB2/p130 have been documented recently in BL cell lines and primary tumors. Given the importance of the RB2/p130 gene in controlling cell growth, mutations of this gene may result in uncontrolled cell proliferation. We tested the expression and genomic organization of the RB2/p130 gene in relation to the proliferative features of a series of BL samples collected from the endemic and sporadic regions, regardless of whether the samples were acquired immune deficiency syndrome (AIDS)-related. The expression of the Rb2/p130, p107, and cell proliferation-related proteins (cyclin A and B) was determined by immunohistochemistry. The structures of exons 19 through 22 of the RB2/p130 gene, encoding for the B domain and C terminus, were analyzed by polymerase chain reaction (PCR) analysis and single-strand conformation polymorphism (SSCP) technique. The direct PCR products were sequenced to identify the actual mutations. Our results suggest that BL is composed of a mixture of molecular types with distinct genetic and phenotypic patterns, probably resulting from different pathogenetic mechanisms. In endemic BL, the RB2/p130 gene is mutated in most of the cases, and the protein is restricted to the cytoplasm. In AIDS-related BL, high levels of nuclear expression of the wild-type pRb2/p130, p107, and cell proliferation-related proteins were detected. This finding is in line with the molecular mechanisms observed in virus-linked oncogenesis. Sporadic BLs were mainly characterized by the low nuclear values of the wild-type pRb2/p130 and, conversely, the high values of p107. The increased cell proliferation due to different alterations of cell growth control by Rb-related proteins may be the first step in lymphomagenesis, during which additional genetic changes, including missense mutations of c-myc, may subsequently occur. PMID:10702389
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Elevated Cell Wall Chitin in Candida albicans Confers Echinocandin Resistance In Vivo
Lee, Keunsook K.; MacCallum, Donna M.; Jacobsen, Mette D.; Walker, Louise A.; Odds, Frank C.
2012-01-01
Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations. PMID:21986821
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Pang, Ying; Gupta, Garima; Yang, Chunzhang; Wang, Herui; Huynh, Thanh-Truc; Abdullaev, Ziedulla; Pack, Svetlana D; Percy, Melanie J; Lappin, Terence R J; Zhuang, Zhengping; Pacak, Karel
2018-03-13
The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome. A female presented with a history of JAK2 V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells. This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.
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Rubinstein, M; Japón, M A; Low, M J
1993-06-11
The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.
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Rubinstein, M; Japón, M A; Low, M J
1993-01-01
The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes. Images PMID:8392702
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Santana, A.; Salido, E.; Torres, A.; Shapiro, L. J.
2003-01-01
Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 → Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 → Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation. PMID:12777626
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Santana, A; Salido, E; Torres, A; Shapiro, L J
2003-06-10
Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 --> Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 --> Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.
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Matsuo, Miki; Cui, Longzhu; Kim, Jeeyoung; Hiramatsu, Keiichi
2013-12-01
Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) spontaneously produces VISA cells within its cell population at a frequency of 10(-6) or greater. We established a total of 45 VISA mutant strains independently obtained from hVISA Mu3 and its related strains by one-step vancomycin selection. We then performed high-throughput whole-genome sequencing of the 45 strains and their parent strains to identify the genes involved in the hVISA-to-VISA phenotypic conversion. A comparative genome study showed that all the VISA strains tested carried a unique set of mutations. All of the 45 VISA strains carried 1 to 4 mutations possibly affecting the expression of a total of 48 genes. Among them, 32 VISA strains carried only one gene affected by a single mutation. As many as 20 genes in more than eight functional categories were affected in the 32 VISA strains, which explained the extremely high rates of the hVISA-to-VISA phenotypic conversion. Five genes, rpoB, rpoC, walK, pbp4, and pp2c, were previously reported as being involved in vancomycin resistance. Fifteen remaining genes were newly identified as associated with vancomycin resistance in this study. The gene most frequently affected (6 out of 32 strains) was cmk, which encodes cytidylate kinase, followed closely by rpoB (5 out of 32), encoding the β subunit of RNA polymerase. A mutation prevalence study also revealed a sizable number of cmk mutants among clinical VISA strains (7 out of 38 [18%]). Reduced cytidylate kinase activity in cmk mutant strains is proposed to contribute to the hVISA-to-VISA phenotype conversion by thickening the cell wall and reducing the cell growth rate.
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Preusser, Matthias; Wöhrer, Adelheid; Stary, Susanne; Höftberger, Romana; Streubel, Berthold; Hainfellner, Johannes A
2011-08-01
To assess the value of anti-isocitrate dehydrogenase 1 (IDH1) immunohistochemistry for evaluating diffuse gliomas, we analyzed anti-IDH1-R132H immunohistochemistry using monoclonal antibodies DIA-H09 and IMab-1 and IDH1 gene sequencing in formalin-fixed and paraffin-embedded biopsy samples of 95 diffuse gliomas. We found concordant immunostaining results using the 2 antibodies in 94 (98.9%) of the 95 cases, but DIA-H09 generally showed a higher signal-to-background ratio than IMab-1 did. Fifty-five percent of cases showed anti-IDH1-R132H immunostaining of virtually all tumor cells and 15% of only a fraction of tumor cells. All cases with complete or partial immunostaining of the tumor tissue carried the IDH1-R132H mutation. In all cases with negative immunostaining results (approximately 30%), genetic analysis showed IDH1 wild-type or non-R132H-IDH1 mutations. In a single tiny biopsy, both anti-IDH1-R132H antibodies showed immunoreactivity, but genetic testing was inconclusive. Our data confirm anti-IDH1-R132H immunostaining as a reliable method for evaluation of IDH1 gene mutation status. They also suggest the following: (i) in some cases, nonspecific background staining or regional heterogeneity of IDH1-R132H protein expression may necessitate confirmatory genetic analysis; (ii) for individual cases, anti-IDH1-R132H immunostaining may not reliably identify infiltrating tumor cells admixed with preexisting or reactive glial cells; and (iii) in tiny biopsies, immunohistochemistry may be more sensitive for detection of IDH1-R132H mutation than genetic analysis.
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Targeted genome editing in a quail cell line using a customized CRISPR/Cas9 system.
Ahn, Jinsoo; Lee, Joonbum; Park, Ju Yeon; Oh, Keon Bong; Hwang, Seongsoo; Lee, Chang-Won; Lee, Kichoon
2017-05-01
Soon after RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease opened a new era of targeted genome editing, the CRISPR/Cas9 platform began to be extensively used to modify genes in various types of cells and organisms. However, successful CRISPR/Cas9-mediated insertion/deletion (indel) mutation remains to be demonstrated in avian cell lines. The objective of this study was to design a poultry-specific CRISPR/Cas9 system to efficiently introduce targeted deletion mutation in chromosomes of the quail muscle clone 7 (QM7) cell line using a customized quail CRISPR vector. In this study, two avian-specific promoters, quail 7SK (q7SK) promoter and CBh promoter, the hybrid form of cytomegalovirus and chicken β-actin promoters, were cloned into a CRISPR vector for the expression of guide RNA and Cas9 protein, respectively. Then, guide RNA, which was designed to target 20-base pair (bp) nucleotides in the quail melanophilin (MLPH) locus, was ligated to the modified CRISPR vector and transfected to QM7 cells. Our results showed multiple indel mutations in the quail MLPH locus in nearly half of the alleles being tested, suggesting the high efficiency of the system for targeted gene modification. The new CRISPR vector developed from this study has the potential application to generate knockout avian cell lines and knockout poultry. © 2016 Poultry Science Association Inc.
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Choi, Sung Won; Ryu, Ok Hee; Choi, Sun Jin; Song, In Sun; Bleyer, Anthony J; Hart, Thomas C
2005-10-01
As a consequence of uromodulin gene mutations, individuals develop precocious hyperuricemia, gout, and progressive renal failure. In vitro studies suggest that pathologic accumulation of uromodulin/Tamm-Horsfall glycoprotein (THP) occurs in the endoplasmic reticulum (ER), but the pathophysiology of renal damage is unclear. It was hypothesized that programmed cell death triggered by accumulation of misfolded THP in the ER causes progressive renal disease. Stably transfected human embryonic kidney 293 cells and immortalized thick ascending limb of Henle's loop cells with wild-type and mutated uromodulin cDNA were evaluated to test this hypothesis. Immunocytochemistry, ELISA, and deglycosylation studies indicated that accumulation of mutant THP occurred in the ER. FACS analyses showed a significant increase in early apoptosis signal in human embryonic kidney 293 and thick ascending limb of Henle's loop cells that were transfected with mutant uromodulin constructs. Colchicine and sodium 4-phenylbutyrate treatment increased secretion of THP from the ER to the cell membrane and into the culture media and significantly improved cell viability. These findings indicate that intracellular accumulation of THP facilitates apoptosis and that this may provide the pathologic mechanism responsible for the progressive renal damage associated with uromodulin gene mutations. Colchicine and sodium 4-phenylbutyrate reverse these processes and could potentially be beneficial in ameliorating the progressive renal damage in uromodulin-associated kidney diseases.
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Kerner, Gerald S. M. A.; Schuuring, Ed; Sietsma, Johanna; Hiltermann, Thijo J. N.; Pieterman, Remge M.; de Leede, Gerard P. J.; van Putten, John W. G.; Liesker, Jeroen; Renkema, Tineke E. J.; van Hengel, Peter; Platteel, Inge; Timens, Wim; Groen, Harry J. M.
2013-01-01
Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival. PMID:23922984
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McKell, Allison O; LaConte, Leslie E W; McDonald, Sarah M
2017-04-01
Temperature-sensitive ( ts ) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11- ts C, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11- ts C, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11- ts C genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1 L138P -GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1 L138P -GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1 L138P ) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11- ts C and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11- ts C) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11- ts C and shed new light on functional protein-protein interaction sites of VP1. Copyright © 2017 American Society for Microbiology.
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2018-04-25
EGFR Activating Mutation; EGFR Exon 19 Deletion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage III Non-Small Cell Lung Cancer AJCC v7; Stage IIIA Non-Small Cell Lung Cancer AJCC v7; Stage IIIB Non-Small Cell Lung Cancer AJCC v7; Stage IV Non-Small Cell Lung Cancer AJCC v7
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Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T
2000-02-14
The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.
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Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M
2015-01-01
Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors. PMID:25855885
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van den Oever, Jessica M E; van Minderhout, Ivonne J H M; Harteveld, Cornelis L; den Hollander, Nicolette S; Bakker, Egbert; van der Stoep, Nienke; Boon, Elles M J
2015-09-01
The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Cabanero, M.; Sangha, R.; Sheffield, B.S.; Sukhai, M.; Pakkal, M.; Kamel-Reid, S.; Karsan, A.; Ionescu, D.; Juergens, R.A.; Butts, C.; Tsao, M.S.
2017-01-01
Starting in the early 2000s, non-small-cell lung cancer (nsclc) subtypes have evolved from being histologically described to molecularly defined. Management of lung adenocarcinomas now generally requires multiple molecular tests at baseline to define the optimal treatment strategy. More recently, second biopsies performed at progression in patients treated with tyrosine kinase inhibitors (tkis) have further defined the continued use of molecularly targeted therapy. In the present article, we focus on one molecular subtype: EGFR-mutated nsclc. For that patient population, multiple lines of tki therapy are now available either clinically or in clinical trials. Each line of treatment is guided by the specific mutations (for example, L858R, T790M, C797S) identified in EGFR. We first describe the various mechanisms of acquired resistance to EGFR tki treatment. We then focus on strategies that clinicians and pathologists can both use during tissue acquisition and handling to optimize patient results. We also discuss future directions for the molecular characterization of lung cancers with driver mutations, including liquid biopsies. Finally, we provide an algorithm to guide treating physicians managing patients with EGFR-mutated nsclc. The same framework can also be applied to other molecularly defined nsclc subgroups as resistance patterns are elucidated and additional lines of treatment are developed. PMID:28490925
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Verheij, Johanna B G M; Sival, Deborah A; van der Hoeven, Johannes H; Vos, Yvonne J; Meiners, Linda C; Brouwer, Oebele F; van Essen, Anthonie J
2006-01-01
Shah-Waardenburg syndrome is a rare congenital disorder with variable clinical expression, characterised by aganglionosis of the rectosigmoïd (Hirschsprung disease), and abnormal melanocyte migration, resulting in pigmentary abnormalities and sensorineural deafness (Waardenburg syndrome). Mutations in the EDN, EDNRB and SOX10 genes can be found in patients with this syndrome. SOX10 mutations are specifically associated with a more severe phenotype called PCWH: peripheral demyelinating neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome, and Hirschsprung disease. Neuronal expression of SOX10 occurs in neural crest cells during early embryonic development and in glial cells of the peripheral and central nervous systems during late embryonic development and in adults. We present a 4-year-old girl with the PCWH phenotype associated with a de novo nonsense mutation (S384X) in SOX10. Main clinical features were mental retardation, peripheral neuropathy, deafness, Hirschsprung disease, distal arthrogryposis, white hairlock, and growth retardation. She presented with hypotonia, developmental delay, reduced peripheral nerve conduction velocities, and radiologically assessed central hypomyelination. Subsequently, the formation of abnormal myelin within the central and peripheral nervous system was functionally and radiologically assessed. Children presenting with features of Waardenburg syndrome and neurological dysfunction should be tested for mutations in the SOX10 gene to enable diagnosis and counselling.
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Qiao, Xiuli; Ai, Dan; Liang, Honglu; Mu, Dianbin; Guo, Qisen
2017-01-20
Molecular targeted therapy has gradually become an important treatment for lung cancer, the aim of this research is to analyze the clinicopathologic features associated with the gene mutation status of epidermal growth factor receptor (EGFR), echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) and Kirsten rat sarcoma viral oncogene (KRAS) in non-small cell lung cancer (NSCLC) patients and determine the most likely populations to benefit from molecular target therapy treatment. The mutation status of EGFR, EML4-ALK fusion gene, ROS1 and KARS gene were determined by Real-time PCR, the relationship between clinical pathologic features and concomitant gene were analyzed with χ2 test by SPSS software 19.0. A total of 514 specimens from Shandong tumor hospital were collected from NSCLC patients between January 2014 and May 2016. The total mutation rate of EGFR gene was 36.70%, major occurred in exon 19 (36.61%) and exon 21 (51.36%), respectively, and EGFR mutations usually occurred in female, non-smoking and adenocarcinoma patients (P<0.05). The total rearrangements rate of EML4-ALK fusion gene was 9.37%, EML4-ALK fusion gene usually occurred in younger age (≤60 yr) and non-smoking patients (P<0.05). Mutations were not related to gender and pathological type (P>0.05). ROS1 fusion gene was detected in 136 cases, the positive rate was 3.67%, all patients were 60 years old, and the difference was statistically significant (P<0.05). Only 23 samples were tested KARS gene mutations, two of them were positive and the positive rate was 8.70%. They all occurred in non-smoker and adenocarcinoma patients. No mutation was detected to coexist in EGFR, EML4-ALK and KARS gene mutation. EGFR, EML4-ALK, ROS1 and KRAS defines different molecular subset of NSCLC with distinct characteristic, which provides a new option for the clinical treatment of patients with NSCLC.
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Liu, Jessica Ai-Jia; Lai, Frank Pui-Ling; Gui, Hong-Sheng; Sham, Mai-Har; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Hui, Chi-Chung; Ngan, Elly Sau-Wai
2015-12-01
Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.
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Little, Daniel; Luft, Christin; Mosaku, Olukunbi; Lorvellec, Maëlle; Yao, Zhi; Paillusson, Sébastien; Kriston-Vizi, Janos; Gandhi, Sonia; Abramov, Andrey Y; Ketteler, Robin; Devine, Michael J; Gissen, Paul
2018-06-13
Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.
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Fontaine, Sarah N; Bauer, Scott P; Lin, Xiaoyan; Poorfarahani, Sara; Ybe, Joel A
2012-09-21
HIP1 crystal structures solved in our laboratory revealed abnormalities in the coiled-coil region, suggesting intrinsic plasticity. To test this, specific amino acids in the coiled-coil were mutated. The apparent thermal stability of HIP1 was altered when Thr528 and Glu531 were replaced by leucine, and was enhanced when Lys510 was also mutated. In cells, HIP1 mutant expression produced aggregation. MTS and flow cytometry indicate a correlation between aggregated HIP1 and enhanced cell death. These data support the idea that flexibility of the HIP1 coiled-coil domain is important for normal function and may lead to new insights into Huntington's disease. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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Li, Chunmei; Gao, Yonglin; Wang, Yunzhi; Li, Guisheng; Fan, Xiaochen; Li, Yanshen; Guo, Chenghua; Tao, Jun
2017-06-01
As part of a safety evaluation, we evaluated the potential genotoxicity of sodium formononetin-3'-sulphonate (Sul-F) using bacterial reverse mutation assay, chromosomal aberrations detection, and mouse micronucleus test. In bacterial reverse mutation assay using five strains of Salmonella typhimurium (TA97, TA98, TA100, TA102 and TA1535), Sul-F (250, 500, 1000, 2000, 4000 μg/plate) did not increase the number of revertant colonies in any tester strain with or without S9 mix. In a chromosomal assay using Chinese hamster lung fibroblast (CHL) cells, there were no increases in either kind of aberration at any dose of Sul-F (400, 800, and 1600 μg/mL) treatment groups with or without S9 metabolic activation. In an in vivo bone marrow micronucleus test in ICR mice, Sul-F at up to 2000 mg/kg (intravenous injection) showed no significant increases in the incidence of micronucleated polychromatic erythrocytes, and the proportion of immature erythrocytes to total erythrocytes. The results demonstrated that Sul-F does not show mutagenic or genotoxic potential under these test conditions. Copyright © 2017. Published by Elsevier Inc.
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Rotondi, Mario; Coperchini, Francesca; Awwad, Oriana; Di Buduo, Christian A.; Abbonante, Vittorio; Magri, Flavia; Balduini, Alessandra
2016-01-01
CXCL8 displays several tumor-promoting effects. Targeting and/or lowering CXCL8 concentrations within the tumor microenvironment would produce a therapeutic benefit. Aim of this study was to test the effect of IFNγ on the basal and TNFα-stimulated secretion of CXCL8 in TCP-1 and BCPAP thyroid cancer cell lines (harboring RET/PTC rearrangement and BRAF V600e mutation, resp.). Cells were incubated with IFNγ (1, 10, 100, and 1000 U/mL) alone or in combination with TNF-α (10 ng/mL) for 24 hours. CXCL8 and CXCL10 concentrations were measured in the cell supernatants. IFNγ inhibited in a dose-dependent and significant manner both the basal (ANOVA F: 22.759; p < 0.00001) and the TNFα-stimulated (ANOVA F: 15.309; p < 0.00001) CXCL8 secretions in BCPAP but not in TPC-1 cells (NS). On the other hand, IFNγ and IFNγ + TNF-α induced a significant secretion of CXCL10 in both BCPAP (p < 0.05) and TPC-1 (p < 0.05) cells. Transwell migration assay showed that (i) CXCL8 increased cell migration in both TPC-1 and BCPAP cells; (ii) IFNγ significantly reduced the migration only of BCPAP cells; and (iii) CXCL8 reverted the effect of IFNγ. These results constitute the first demonstration that IFNγ inhibits CXCL8 secretion and in turn the migration of a BRAF V600e mutated thyroid cell line. PMID:27555670
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Rotondi, Mario; Coperchini, Francesca; Awwad, Oriana; Pignatti, Patrizia; Di Buduo, Christian A; Abbonante, Vittorio; Magri, Flavia; Balduini, Alessandra; Chiovato, Luca
2016-01-01
CXCL8 displays several tumor-promoting effects. Targeting and/or lowering CXCL8 concentrations within the tumor microenvironment would produce a therapeutic benefit. Aim of this study was to test the effect of IFNγ on the basal and TNFα-stimulated secretion of CXCL8 in TCP-1 and BCPAP thyroid cancer cell lines (harboring RET/PTC rearrangement and BRAF V600e mutation, resp.). Cells were incubated with IFNγ (1, 10, 100, and 1000 U/mL) alone or in combination with TNF-α (10 ng/mL) for 24 hours. CXCL8 and CXCL10 concentrations were measured in the cell supernatants. IFNγ inhibited in a dose-dependent and significant manner both the basal (ANOVA F: 22.759; p < 0.00001) and the TNFα-stimulated (ANOVA F: 15.309; p < 0.00001) CXCL8 secretions in BCPAP but not in TPC-1 cells (NS). On the other hand, IFNγ and IFNγ + TNF-α induced a significant secretion of CXCL10 in both BCPAP (p < 0.05) and TPC-1 (p < 0.05) cells. Transwell migration assay showed that (i) CXCL8 increased cell migration in both TPC-1 and BCPAP cells; (ii) IFNγ significantly reduced the migration only of BCPAP cells; and (iii) CXCL8 reverted the effect of IFNγ. These results constitute the first demonstration that IFNγ inhibits CXCL8 secretion and in turn the migration of a BRAF V600e mutated thyroid cell line.
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Saini, Jasmine; Hershberg, Uri
2015-01-01
The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire towards the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased towards focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased towards using only highly skewed V genes at all stages of their response. PMID:25660968
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Saini, Jasmine; Hershberg, Uri
2015-05-01
The exceptional ability of B cells to diversify through somatic mutation and improve affinity of the repertoire toward the antigens is the cornerstone of adaptive immunity. Somatic mutation is not evenly distributed and exhibits certain micro-sequence specificities. We show here that the combination of somatic mutation targeting and the codon usage in human B cell receptor (BCR) Variable (V) genes create expected patterns of mutation and post mutation changes that are focused on their complementarity determining regions (CDR). T cell V genes are also skewed in targeting mutations but to a lesser extent and are lacking the codon usage bias observed in BCRs. This suggests that the observed skew in T cell receptors is due to their amino acid usage, which is similar to that of BCRs. The mutation targeting and the codon bias allow B cell CDRs to diversify by specifically accumulating nonconservative changes. We counted the distribution of mutations to CDR in 4 different human datasets. In all four cases we found that the number of actual mutations in the CDR correlated significantly with the V gene mutation biases to the CDR predicted by our models. Finally, it appears that the mutation bias in V genes indeed relates to their long-term survival in actual human repertoires. We observed that resting repertoires of B cells overexpressed V genes that were especially biased toward focused mutation and change in the CDR. This bias in V gene usage was somewhat relaxed at the height of the immune response to a vaccine, presumably because of the need for a wider diversity in a primary response. However, older patients did not retain this flexibility and were biased toward using only highly skewed V genes at all stages of their response. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Cellular evidence for selfish spermatogonial selection in aged human testes.
Maher, G J; Goriely, A; Wilkie, A O M
2014-05-01
Owing to a recent trend for delayed paternity, the genomic integrity of spermatozoa of older men has become a focus of increased interest. Older fathers are at higher risk for their children to be born with several monogenic conditions collectively termed paternal age effect (PAE) disorders, which include achondroplasia, Apert syndrome and Costello syndrome. These disorders are caused by specific mutations originating almost exclusively from the male germline, in genes encoding components of the tyrosine kinase receptor/RAS/MAPK signalling pathway. These particular mutations, occurring randomly during mitotic divisions of spermatogonial stem cells (SSCs), are predicted to confer a selective/growth advantage on the mutant SSC. This selective advantage leads to a clonal expansion of the mutant cells over time, which generates mutant spermatozoa at levels significantly above the background mutation rate. This phenomenon, termed selfish spermatogonial selection, is likely to occur in all men. In rare cases, probably because of additional mutational events, selfish spermatogonial selection may lead to spermatocytic seminoma. The studies that initially predicted the clonal nature of selfish spermatogonial selection were based on DNA analysis, rather than the visualization of mutant clones in intact testes. In a recent study that aimed to identify these clones directly, we stained serial sections of fixed testes for expression of melanoma antigen family A4 (MAGEA4), a marker of spermatogonia. A subset of seminiferous tubules with an appearance and distribution compatible with the predicted mutant clones were identified. In these tubules, termed 'immunopositive tubules', there is an increased density of spermatogonia positive for markers related to selfish selection (FGFR3) and SSC self-renewal (phosphorylated AKT). Here we detail the properties of the immunopositive tubules and how they relate to the predicted mutant clones, as well as discussing the utility of identifying the potential cellular source of PAE mutations. © 2013 American Society of Andrology and European Academy of Andrology.
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Biophysical Properties of 9 KCNQ1 Mutations Associated with Long QT Syndrome (LQTS)
Yang, Tao; Chung, Seo-Kyung; Zhang, Wei; Mullins, Jonathan G.L.; McCulley, Caroline H.; Crawford, Jackie; MacCormick, Judith; Eddy, Carey-Anne; Shelling, Andrew N.; French, John K.; Yang, Ping; Skinner, Jonathan R.; Roden, Dan M.; Rees, Mark I.
2009-01-01
Background Inherited long QT syndrome (LQTS) is characterized by prolonged QT interval on the EKG, syncope and sudden death due to ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modelling and phylogenetic alignments, however, biophysical testing offers a higher degree of validating evidence. Methods and Results By using in-vitro electrophysiological testing of transfected mutant and wild-type LQTS constructs into Chinese Hamster Ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand based LQTS screening programme. We demonstrate through electrophysiology and molecular modeling that seven of the missense mutations have profound pathological dominant negative loss-of-function properties confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed or fast activation kinetics. Further investigation of the A46T family has revealed an inconsistent co-segregation of the variant with the clinical phenotype. Conclusions Electrophysiological characterisation should be used to validate LQTS pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counselling and screening of such families, and alternative disease causing mechanisms should be considered. PMID:19808498
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AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.
Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter
2016-05-01
Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner. © 2016 Society for Endocrinology.
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Mutant p53 expression in fallopian tube epithelium drives cell migration.
Quartuccio, Suzanne M; Karthikeyan, Subbulakshmi; Eddie, Sharon L; Lantvit, Daniel D; Ó hAinmhire, Eoghainín; Modi, Dimple A; Wei, Jian-Jun; Burdette, Joanna E
2015-10-01
Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates. © 2015 UICC.
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Lopez-Chavez, Ariel; Thomas, Anish; Rajan, Arun; Raffeld, Mark; Morrow, Betsy; Kelly, Ronan; Carter, Corey Allan; Guha, Udayan; Killian, Keith; Lau, Christopher C.; Abdullaev, Zied; Xi, Liqiang; Pack, Svetlana; Meltzer, Paul S.; Corless, Christopher L.; Sandler, Alan; Beadling, Carol; Warrick, Andrea; Liewehr, David J.; Steinberg, Seth M.; Berman, Arlene; Doyle, Austin; Szabo, Eva; Wang, Yisong; Giaccone, Giuseppe
2015-01-01
Purpose We conducted a basket clinical trial to assess the feasibility of such a design strategy and to independently evaluate the effects of multiple targeted agents against specific molecular aberrations in multiple histologic subtypes concurrently. Patients and Methods We enrolled patients with advanced non–small-cell lung cancer (NSCLC), small-cell lung cancer, and thymic malignancies who underwent genomic characterization of oncogenic drivers. Patients were enrolled onto a not-otherwise-specified arm and treated with standard-of-care therapies or one of the following five biomarker-matched treatment groups: erlotinib for EGFR mutations; selumetinib for KRAS, NRAS, HRAS, or BRAF mutations; MK2206 for PIK3CA, AKT, or PTEN mutations; lapatinib for ERBB2 mutations or amplifications; and sunitinib for KIT or PDGFRA mutations or amplification. Results Six hundred forty-seven patients were enrolled, and 88% had their tumors tested for at least one gene. EGFR mutation frequency was 22.1% in NSCLC, and erlotinib achieved a response rate of 60% (95% CI, 32.3% to 83.7%). KRAS mutation frequency was 24.9% in NSCLC, and selumetinib failed to achieve its primary end point, with a response rate of 11% (95% CI, 0% to 48%). Completion of accrual to all other arms was not feasible. In NSCLC, patients with EGFR mutations had the longest median survival (3.51 years; 95% CI, 2.89 to 5.5 years), followed by those with ALK rearrangements (2.94 years; 95% CI, 1.66 to 4.61 years), those with KRAS mutations (2.3 years; 95% CI, 2.3 to 2.17 years), those with other genetic abnormalities (2.17 years; 95% CI, 1.3 to 2.74 years), and those without an actionable mutation (1.85 years; 95% CI, 1.61 to 2.13 years). Conclusion This basket trial design was not feasible for many of the arms with rare mutations, but it allowed the study of the genetics of less common malignancies. PMID:25667274
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Saber, Ali; van der Wekken, Anthonie J; Kerner, Gerald S M A; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T Jeroen N; Groen, Harry J M
2016-01-01
Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14-14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04-0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.
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Stubbs, Andrew P.; Vroegindeweij, Eric M.; Smits, Willem K.; van Marion, Ronald; Dinjens, Winand N. M.; Horstmann, Martin; Kuiper, Roland P.; Zaman, Guido J. R.; van der Spek, Peter J.; Pieters, Rob; Meijerink, Jules P. P.
2016-01-01
Background Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment. Methods and Findings We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor’s ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL. Conclusions Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome. PMID:27997540
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Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har
2003-10-15
SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. Copyright 2003 Wiley-Liss, Inc.
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Hb taradale [beta82(EF6)Lys-->Arg]: a novel mutation at a 2,3-diphosphoglycerate binding site.
Brennan, Stephen O; Sheen, Campbell; Chan, Tim; George, Peter M
2005-01-01
Hb Taradale [beta82(EF6)Lys-->Arg] was initially detected as a split Hb A0 peak on Hb A1c, monitoring. Red cell parameters, hemoglobin (Hb) electrophoresis and stability tests were normal. Mass spectrometry (ms) clearly identified a variant beta chain with a mass increase of 28 Da and peptide mapping located the mutation site to peptide betaT-9. DNA sequencing confirmed the presence of a novel beta82(EF6)Lys-->Arg mutation. This conservative substitution at a 2,3-diphosphoglycerate (2,3-DPG) binding site did not, however, appear to affect the P50 for oxygen binding.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, W.R.
The spectrum of viability mutations ranging from dominant lethals to detrimentals in haploids that resulted from irradiating semen from a single haploid male was studied in the honey bee. From the decrease in viability of diploid progeny following irradiation of the spermatheca of the parental queen, it was calculated that one or more dominant lethals were induced in 60.8% of the sperm cells. In a separate test using the same dosage on an unrelated queen 60.9% dominant lethals were found. Recessive mutations and mutants with incomplete dominance were detected in haploid progeny of F-1 queens. (M.C.G.)
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Su, Fei; Zheng, Ke; Fu, Yiyun; Wu, Qian; Tang, Yuan; Wang, Weiya; Jiang, Lili
2018-05-20
Epidermal growth factor receptor (EGFR) gene mutation is closely related to the EGFR-TKI target treatment and prognosis of lung adenocarcinoma patients. The mutation status of EGFR is limited by tissue detection. The purpose of this study was to investigate the difference of EGFR mutants in plasmacirculating cell-free DNA (cfDNA) obtained from patients with non-small cell lung cancer (NSCLC) in three groups: pre-therapy, after traditional chemotherapy and targeted therapy. The aim of this study was to analyze whether the plasma cfDNA could effectively determine the EGFR mutations and monitor the drug resistant gene T790M, as well as its prognostic prediction value in patients with targeted therapy. ARMS (amplification refractory mutation system)-PCR was used to detect EGFR mutations in 107 (50 of pre-therapy, 29 after traditional chemotherapy and 28 after targeted therapy) cases of paired plasma and tumor tissue specimens, followed by comparing their concordance. The sensitivity, specificity and the prognostic value of plasma cfDNA detection were also observed. The total rate of EGFR mutation was 56% (60/107) in all plasma samples and 77.6% (83/107) in corresponding tumor tissues. Completely the same mutants and wild-type EGFR were found in 68.2% cases of paired specimens. The sensitivity of plasma cfDNA detection was 72.3% and the specificity was up to 100%. Patients were sub-categorized according to therapy. The results showed that the highest consistent rate of cfDNA and tumor tissues was found in the group of pre-therapy (74%, 37/50). Whereas, the lowest consistent rate was observed in the targeted therapy group (57.1%, 16/28). It indicated that the targeted treatment could change the EGFR status in plasma cfDNA. Further analyses on inconsistent cases in this group revealed that 50% of them were compound EGFR mutations with T790M. Thereby, it suggested that targeted therapy might induce the emergence of drug resistance gene T790M. This speculation was confirmed by survival analyses. Based on plasma cfDNA results, patients with T790M mutant had significantly worse progression-free survival (PFS) and overall survival (OS). For EGFR testing, ARMS-PCR on plasma cfDNA is a promising methodology with the highest specificity and effective sensitivity. It is useful for EGFR testing in patients before treatment, especially the late-stage patients. Simultaneously, plasma cfDNA could be used to monitor the drug resistant mutation, T790M status and predict prognosis after targeted therapy.
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Molecular diagnostics of lung cancer in the clinic.
Sholl, Lynette
2017-10-01
According to current practice guidelines, all patients with advanced non-small cell lung cancer (NSCLC) should undergo predictive biomarker testing. For squamous cell carcinoma patients, PD-L1 immunohistochemistry is indicated to select patients for immunotherapy in the first line. For lung adenocarcinoma, all patients with advanced disease should undergo testing for epidermal growth factor receptor ( EGFR ) mutations, ALK and ROS1 rearrangements, and PD-L1 expression to predict response to EGFR, ALK, or ROS1 targeted inhibitors or immunotherapy, respectively. Besides these, a number of other biomarkers are under clinical investigation as predictors of response to targeted therapies, including BRAF , ERBB2 , MET splice mutations and amplification, and RET rearrangements. Successful testing for this complex array of molecular targets demands careful coordination between proceduralists, pathologists and molecular laboratories to ensure proper tumor tissue handling following biopsy as well as judicious use of diagnostic immunohistochemistry. Even so, sample failure rates due to inadequate tumor tissue are high in practice, particularly when using sequential testing methods. Use of next generation sequencing (NGS) in clinical practice can enable detection of multiple targets and multiple alteration types (mutation, gene copy change, and rearrangement) simultaneously even with small amounts of input nucleic acids, thus increasing molecular testing success rates. In patients with an established lung cancer diagnosis but with prohibitively limited amounts of tumor tissue or who are experiencing relapse, analyses of circulating tumor DNA (ctDNA) from the plasma can serve as an alternate testing substrate, however the more limited clinical sensitivity of this approach must be taken into account. This review will explore the indications for and pitfalls of routine NGS and plasma genotyping in the clinic, including the intersection of these technologies.
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Molecular diagnostics of lung cancer in the clinic
2017-01-01
According to current practice guidelines, all patients with advanced non-small cell lung cancer (NSCLC) should undergo predictive biomarker testing. For squamous cell carcinoma patients, PD-L1 immunohistochemistry is indicated to select patients for immunotherapy in the first line. For lung adenocarcinoma, all patients with advanced disease should undergo testing for epidermal growth factor receptor (EGFR) mutations, ALK and ROS1 rearrangements, and PD-L1 expression to predict response to EGFR, ALK, or ROS1 targeted inhibitors or immunotherapy, respectively. Besides these, a number of other biomarkers are under clinical investigation as predictors of response to targeted therapies, including BRAF, ERBB2, MET splice mutations and amplification, and RET rearrangements. Successful testing for this complex array of molecular targets demands careful coordination between proceduralists, pathologists and molecular laboratories to ensure proper tumor tissue handling following biopsy as well as judicious use of diagnostic immunohistochemistry. Even so, sample failure rates due to inadequate tumor tissue are high in practice, particularly when using sequential testing methods. Use of next generation sequencing (NGS) in clinical practice can enable detection of multiple targets and multiple alteration types (mutation, gene copy change, and rearrangement) simultaneously even with small amounts of input nucleic acids, thus increasing molecular testing success rates. In patients with an established lung cancer diagnosis but with prohibitively limited amounts of tumor tissue or who are experiencing relapse, analyses of circulating tumor DNA (ctDNA) from the plasma can serve as an alternate testing substrate, however the more limited clinical sensitivity of this approach must be taken into account. This review will explore the indications for and pitfalls of routine NGS and plasma genotyping in the clinic, including the intersection of these technologies. PMID:29114472
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Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J; Subbiah, Vivek; Hong, David S; Karp, Daniel D; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L; Zhao, Yue; Javle, Milind; Kopetz, E Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han-Yu; Kruglyak, Kristina M; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V; Meric-Bernstam, Funda; Mills, Gordon B; Fan, Jian-Bing; Salathia, Neeraj S
2017-09-15
Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR . ©2017 American Association for Cancer Research.
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NASA Astrophysics Data System (ADS)
Kumagai, Jun; Nakama, Mitsuo; Miyazaki, Tetsuo; Ise, Tamaki; Kodama, Seiji; Watanabe, Masami
2002-07-01
Effect of (-)-epigallocatechin-3- O-gallate (EGCg) on scavenging long-lived radicals and its biological significance were investigated using electron-spin-resonance spectroscopy and mutation assay in cultured human embryo cells. EGCg scavenged long-lived radicals in irradiated golden hamster embryo cells and albumin solution, and simultaneously reduced mutation frequency in the irradiated human embryo cells. These results indicate that long-lived radials are involved in the induction of mutation by radiation.
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2010-01-01
In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT: http://www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST CANCER: An Evidence-Based AnalysisEpidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based AnalysisK-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa(®)) or erlotinib (Tarceva(®)) in patients with advanced non-small cell lung cancer (NSCLC). TARGET POPULATION AND CONDITION With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib). Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined. EGFR MUTATION TESTING The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%. In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting? A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms: Non-Small-Cell Lung CarcinomaEpidermal Growth Factor ReceptorAn automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology. The inclusion criteria were as follows: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)PROCEDURE: EGFR mutation testing before treatment with gefitinib or erlotinibLANGUAGE: publication in EnglishPublished health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract) progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).The exclusion criteria were as follows: Studies lacking outcomes specific to those of interestStudies focused on erlotinib maintenance therapyStudies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drugGrey literature, where relevant, was also reviewed. PFSORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)OSQoL QUALITY OF EVIDENCE: The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria. Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel. In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). (ABSTRACT TRUNCATED)
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Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors
Wang, Qiong J.; Yu, Zhiya; Griffith, Kayla; Hanada, Ken-ichi; Restifo, Nicholas P.; Yang, James C.
2015-01-01
KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60–70% of pancreatic cancers and 20–30% of colorectal cancers. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCRs) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBLs) transduced with these TCRs could recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types. PMID:26701267
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Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H
2017-01-01
The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.
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Alachkar, Houda; Mutonga, Martin; Malnassy, Gregory; Park, Jae-Hyun; Fulton, Noreen; Woods, Alex; Meng, Liping; Kline, Justin; Raca, Gordana; Odenike, Olatoyosi; Takamatsu, Naofumi; Miyamoto, Takashi; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke
2015-01-01
Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML. PMID:26450903
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Zhang, Can; Browne, Andrew; Kim, Doo Yeon; Tanzi, Rudolph E
2010-02-01
Alzheimer's disease (AD) is an insidious and progressive disease with a genetically complex and heterogenous etiology. More than 200 fully penetrant mutations in the amyloid beta-protein precursor (APP), presenilin 1 (or PSEN1), and presenilin 2 (PSEN2) have been linked to early-onset familial AD (FAD). 177 PSEN1 FAD mutations have been identified so far and account for more than approximately 80% of all FAD mutations. All PSEN1 FAD mutations can increase the Abeta42:Abeta40 ratio with seemingly different and incompletely understood mechanisms. A recent study has shown that the 286 amino acid N-terminal fragment of APP (N-APP), a proteolytic product of beta-secretase-derived secreted form of APP (sAPPbeta), could bind the death receptor, DR6, and lead to neurodegeneration. Here we asked whether PSEN1 FAD mutations lead to neurodegeneration by modulating sAPPbeta levels. All four different PSEN1 FAD mutations tested (in three mammalian cell lines) did not alter sAPPbeta levels. Therefore PS1 mutations do not appear to contribute to AD pathogenesis via altered production of sAPPbeta.
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Prevalence of EGFR Mutations in Lung Cancer in Uruguayan Population
Touya, Diego; Bertoni, Bernardo; Osinaga, Eduardo; Varangot, Mario
2017-01-01
Background Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR) gene into routine clinical practice represents a milestone for personalized therapy of the non-small-cell lung cancer (NSCLC). However, the genetic testing of EGFR mutations has not yet become a routine clinical practice in developing countries. In view of different prevalence of such mutations among different ethnicities and geographic regions, as well as the limited existing data from Latin America, our aim was to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients from Uruguay. Methods We examined EGFR mutations in exons 18 through 21 in 289 NSCLC Uruguayan patients by PCR-direct sequencing. Results EGFR mutations were detected in 53 of the 289 (18.3%) patients, more frequently in women (23.4%) than in men (14.5%). The distribution by exon was similar to that generally reported in the literature. Conclusions This first epidemiological study of EGFR mutations in Uruguay reveals a wide spectrum of mutations and an overall prevalence of 18.3%. The background ethnic structure of the Uruguayan population could play an important role in explaining our findings. PMID:28744312
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GNQ-209P Mutation in Metastatic Uveal Melanoma and Treatment Outcome.
Abdel Karim, Nagla; Eldessouki, Ihab; Taftaf, Ahmad; Ayham, Deeb; Gaber, Ola; Makramalla, Abouelmagd; Correa, Zelia M
2018-01-01
Metastatic prognosis in uveal melanoma is assessed by gene expression profiling (GEP) testing of the tumor cells, usually obtained by fine needle aspiration (FNA). GEP has demonstrated high accuracy in distinguishing class I and II tumors, both having different metastatic potential. Transcriptomic studies identified distinct mutations including somatic mutations in GNAQ and GNA11 , detected in more than 80%, and contribute to the upregulation of the mitogen-activated protein kinase (MAPK) pathway and the development of uveal melanoma (UM). The role of these mutations in treatment selection and possible benefit from targeted therapy are somewhat unclear. However, until the discovery of novel agents, local versus systemic therapies remain options for treatment that can still be considered for disease control in certain cases. We report a series of patients with metastatic UM with distinct mutational profiles. One had significant liver metastases with proven GNQ-209P mutation on tissue biopsy while peripheral blood molecular profiling did not show these mutations. The other three cases had no GNQ-209P mutation. All cases received nab-paclitaxel (Abraxane) as a treatment drug, and we record their responses to treatment and their molecular-profiling results.
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Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan
2015-01-01
T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799
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Pascal, Tod A; Abrol, Ravinder; Mittal, Rahul; Wang, Ying; Prasadarao, Nemani V; Goddard, William A
2010-11-26
Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.
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Angioimmunoblastic T-cell lymphoma: more than a disease of T follicular helper cells.
Lemonnier, François; Mak, Tak W
2017-08-01
Angioimmunoblastic T-cell lymphoma (AITL) is one of the most frequent entities of peripheral T-cell lymphoma. An AITL has two components: the AITL tumour cells, which have a T follicular helper (TFH) cell phenotype, and a surrounding and extensive tumour microenvironment that is populated with various reactive cell types, including B cells. Recurrent TET2 mutations have been described in 50-80% of AITLs, possibly occurring in a haematopoietic progenitor cell. An article published recently in the Journal of Pathology describes the use of microdissection to isolate PD1 + AITL tumour cells and CD20 + B cells from the AITL microenvironment, and to show that TET2 mutations are actually more frequent in these diseases than previously thought. Whereas TET2 mutations were detected in only six of 13 AITLs, 12 of 13 samples of microdissected PD1 + AITL tumour cells possessed this mutation. Moreover, TET2 mutations were detected in CD20 + B cells from the AITL microenvironment in six of nine informative cases. These results confirm that TET2 mutation is an early event in the majority of AITL cases, and that the driving molecular anomalies are not restricted to the T lineage tumour cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Intestinal Stem Cell Dynamics: A Story of Mice and Humans.
Hodder, Michael C; Flanagan, Dustin J; Sansom, Owen J
2018-06-01
Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations. Copyright © 2018 Elsevier Inc. All rights reserved.
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Elevated germline mutation rate in teenage fathers.
Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd
2015-03-22
Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.
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Reddien, Peter W; Andersen, Erik C; Huang, Michael C; Horvitz, H Robert
2007-04-01
The genes egl-1, ced-9, ced-4, and ced-3 play major roles in programmed cell death in Caenorhabditis elegans. To identify genes that have more subtle activities, we sought mutations that confer strong cell-death defects in a genetically sensitized mutant background. Specifically, we screened for mutations that enhance the cell-death defects caused by a partial loss-of-function allele of the ced-3 caspase gene. We identified mutations in two genes not previously known to affect cell death, dpl-1 and mcd-1 (modifier of cell death). dpl-1 encodes the C. elegans homolog of DP, the human E2F-heterodimerization partner. By testing genes known to interact with dpl-1, we identified roles in cell death for four additional genes: efl-1 E2F, lin-35 Rb, lin-37 Mip40, and lin-52 dLin52. mcd-1 encodes a novel protein that contains one zinc finger and that is synthetically required with lin-35 Rb for animal viability. dpl-1 and mcd-1 act with efl-1 E2F and lin-35 Rb to promote programmed cell death and do so by regulating the killing process rather than by affecting the decision between survival and death. We propose that the DPL-1 DP, MCD-1 zinc finger, EFL-1 E2F, LIN-35 Rb, LIN-37 Mip40, and LIN-52 dLin52 proteins act together in transcriptional regulation to promote programmed cell death.
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Entezari, Maliheh; Dabaghian, Fataneh Hashem; Hashemi, Mehrdad
2014-01-01
Cancer is one of the main causes of mortality in the world which is created by the effect of enviromental physico-chemical mutagen and carcinogen agents. In the last years, many studies have been performed on the anticancer effects of flavonoids. Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The extract was evaluated in terms of antimutagenicity properties by a standard reverse mutation assay (Ames Test). This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100). Thus, it requires histidine from a foreign supply to ensure its growth. The afore mentioned strain gives rise to reverted colonies when expose to carcinogen substance (Sodium Azide). The other objective of this study was to examine the in vitro cytotoxic activity of cell death of crude methanolic extracts prepared from Echinophora platyloba on Acute Promyelocytic Leukemia cell line (NB4). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. In Ames test the extract prevented the reverted mutations and the hindrance percent was 93.4% in antimutagenicity test. Data obtained from this assay indicated that the extract significantly reduced the viability of NB4 cells and inhibited cell growth in a dose dependent manner. This study demonstrates the antimutagenicity effect of Echinophora Platyloba and suggests that it has a potential as an anticancer agent.
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2018-05-16
Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia in Remission; Hematopoietic Cell Transplantation Recipient; JAK2 Gene Mutation; Loss of Chromosome 17p; Mantle Cell Lymphoma; Minimal Residual Disease; Myelodysplastic Syndrome; Non-Hodgkin Lymphoma; Plasma Cell Myeloma; RAS Family Gene Mutation; Recurrent Diffuse Large B-Cell Lymphoma; Recurrent Hematologic Malignancy; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Therapy-Related Acute Myeloid Leukemia; Therapy-Related Myelodysplastic Syndrome; TP53 Gene Mutation
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Identification of Potential Germ-Cell Mutagens
The existence of agents that can induce germ-cell mutations in experimental systems has been recognized since 1927 with the discovery of the ability of X-rays to induce such mutations in Drosophila. Various rodent-based germ-cell mutation assays have been developed, and ~50 germ...
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Uchida, Hiroaki; Shah, Waris A; Ozuer, Ali; Frampton, Arthur R; Goins, William F; Grandi, Paola; Cohen, Justus B; Glorioso, Joseph C
2009-04-01
Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.
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Hollingsworth, Julia; Lau, Angela; Tone, Alicia; Kollara, Alexandra; Allen, Lisa; Colgan, Terence J; Dube, Valerie; Rosen, Barry; Murphy, K Joan; Greenblatt, Ellen M; Feigenberg, Tomer; Virtanen, Carl; Brown, Theodore J
2018-05-28
Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed epithelial ovarian cancer histotype. Other identified risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation, during which the FTE cells become transiently exposed to follicular fluid (FF). To test whether mtBRCA1 or mtBRCA2 nonmalignant FTE cells respond differently to periovulatory FF exposure than control patient FTE cells, gene expression profiles from primary FTE cultures derived from BRCA1 or BRCA2 mutation carriers or control patients were compared at baseline, 24 hours after FF exposure, and 24 hours after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Gene set enrichment analysis revealed increased NFκB and EGFR signaling at baseline in mtBRCA1 samples, with increased interferon target gene expression, including members of the ISGylation pathway, observed after recovery from FF exposure. Gene set enrichment analysis did not identify altered pathway signaling in mtBRCA2 samples. An inverse relationship between EGFR signaling and ISGylation with BRCA1 protein levels was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1 cDNA. Suppression of ISG15 and ISGylated protein levels by increased BRCA1 expression was found to be mediated by decreased NFκB signaling. These studies indicate that increased NFκB signaling associated with decreased BRCA1 expression results in increased ISG15 and protein ISGylation following FF exposure, which may be involved in predisposition to HGSOC. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
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Scheffler, Matthias; Bos, Marc; Gardizi, Masyar; König, Katharina; Michels, Sebastian; Fassunke, Jana; Heydt, Carina; Künstlinger, Helen; Ihle, Michaela; Ueckeroth, Frank; Albus, Kerstin; Serke, Monika; Gerigk, Ulrich; Schulte, Wolfgang; Töpelt, Karin; Nogova, Lucia; Zander, Thomas; Engel-Riedel, Walburga; Stoelben, Erich; Ko, Yon-Dschun; Randerath, Winfried; Kaminsky, Britta; Panse, Jens; Becker, Carolin; Hellmich, Martin; Merkelbach-Bruse, Sabine; Heukamp, Lukas C; Büttner, Reinhard; Wolf, Jürgen
2015-01-20
Somatic mutations of the PIK3CA gene have been described in non-small cell lung cancer (NSCLC), but limited data is available on their biological relevance. This study was performed to characterize PIK3CA-mutated NSCLC clinically and genetically. Tumor tissue collected consecutively from 1144 NSCLC patients within a molecular screening network between March 2010 and March 2012 was analyzed for PIK3CA mutations using dideoxy-sequencing and next-generation sequencing (NGS). Clinical, pathological, and genetic characteristics of PIK3CA-mutated patients are described and compared with a control group of PIK3CA-wildtype patients. Among the total cohort of 1144 patients we identified 42 (3.7%) patients with PIK3CA mutations in exon 9 and exon 20. These mutations were found with a higher frequency in sqamous cell carcinoma (8.9%) compared to adenocarcinoma (2.9%, p<0.001). The most common PIK3CA mutation was exon 9 E545K. The majority of patients (57.1%) had additional oncogenic driver aberrations. With the exception of EGFR-mutated patients, non of the genetically defined subgroups in this cohort had a significantly better median overall survival. Further, PIK3CA-mutated patients had a significantly higher incidence of malignancy prior to lung cancer (p<0.001). PIK3CA-mutated NSCLC represents a clinically and genetically heterogeneous subgroup in adenocarcinomas as well as in squamous cell carcinomas with a higher prevalence of these mutations in sqamous cell carcinoma. PIK3CA mutations have no negative impact on survival after surgery or systemic therapy. However, PIK3CA mutated lung cancer frequently develops in patients with prior malignancies.
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Visualization of tandem repeat mutagenesis in Bacillus subtilis.
Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M
2018-03-01
Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.
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Novel Mutation of the NOTCH3 Gene in a Chinese Pedigree with CADASIL.
Hou, Xiaoxia; He, Chuan; Jin, Qingwen; Niu, Qi; Ren, Guang; Cheng, Hong
2017-01-01
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) results from NOTCH3 gene mutations, which lead to the degeneration of vascular smooth muscle cells (VSMCs). The clinical presentation of CADASIL patients is dependent on the impact of other vascular risk factors and the type of NOTCH3 mutation present. Here, we report a rare pathogenic mutation on exon 14 of the NOTCH3 gene in a Chinese family affected by CADASIL with phenotypic peculiarities. We performed genetic testing, clinical and neuropsychological examination, brain magnetic resonance images (MRI), and electron microscopy (EM) in skin biopsies. NOTCH3 gene analysis revealed a c.2182CT substitution on exon 14, which is the first example of this mutation in a Chinese individual from the Han ancestry. Granular osmiophilic material (GOM) was found in the proband, and all patients had migraine, subcortical ischemic events, and mood disturbances, without progressive cognitive impairment. Cranial MRI further showed white matter hyperintensity, involving bilateral basal ganglia and multiple microbleeds (MBs), in the thalamus and brain stem. This study suggests that different missense mutations in NOTCH3 might contribute to atypical clinical features of CADASIL. This report also indicates that for individuals with a positive family history having clinical and neuroradiological findings suggestive of CADASIL, genetic testing and GOM detection should be performed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Loyer, M.; Leclerc, D.; Gravel, R.A.
1994-09-01
Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less
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Hornemann, Thorsten; Penno, Anke; Richard, Stephane; Nicholson, Garth; van Dijk, Fleur S; Rotthier, Annelies; Timmerman, Vincent; von Eckardstein, Arnold
2009-04-01
Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cells--a SPTLC1 deficient CHO cell line--could be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SNP.
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Teng, Siyong; Huang, Jian; Gao, Zhan; Hao, Jie; Yang, Yuejin; Zhang, Shu; Pu, Jielin; Hui, Rutai; Wu, Yongjian; Fan, Zheng
2017-01-01
Nonsense mutation readthrough is used as a gene-specific treatment in some genetic diseases. The response to readthrough treatment is determined by the readthrough efficiency of various nonsense mutations. In this manuscript, we aimed to explore the harmful effects of nonsense mutation suppression. HEK293 cells were transfected with two SCN5A (encode cardiac Na+ channel) nonsense mutations, p.R1623X and p.S1812X. We applied two readthrough-enhancing methods (either aminoglycosides or a siRNA-targeting eukaryotic release factor eRF3a (a GTPase that binds eRF1)) to suppress these SCN5A nonsense mutations. When either of readthrough methods was used, the sodium channel proteins were examined by western blot and immunoblotting and recorded by whole cell patch-clamp to observe the functional characterization of the restored channels. Upon readthrough treatment, the sodium currents were restored to the mutant cDNAs. These mutations reduced full-length sodium channel protein levels, and the sodium currents were reduced to 3% of wild-type. The mutant cDNA sodium currents were increased to 30% of wild-type, and the fulllength proteins also increased. However, the functional characterization of these channels from cDNAs carrying p.R1623X and p.S1812X exhibited abnormal biophysical properties, including a negative shift in steady-state sodium channel inactivation, a positive shift in sodium channel activation and robust late sodium currents. The ramp test showed prolonged QT intervals. These results demonstrated that readthrough-enhancing methods effectively suppressed nonsense mutations in SCN5A and restored the expression of full-length channels. However, the restored channels may increase the risk of arrhythmia. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.