Sample records for absolute tumor dna

  1. GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

    PubMed Central

    Lyng, Heidi; Lando, Malin; Brøvig, Runar S; Svendsrud, Debbie H; Johansen, Morten; Galteland, Eivind; Brustugun, Odd T; Meza-Zepeda, Leonardo A; Myklebost, Ola; Kristensen, Gunnar B; Hovig, Eivind; Stokke, Trond

    2008-01-01

    Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers. PMID:18500990

  2. Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors.

    PubMed Central

    Groner, B; Hynes, N E

    1980-01-01

    The Southern DNA filter transfer technique was used to characterize the genomic location of the mouse mammary tumor proviral DNA in different inbred strains of mice. Two of the strains (C3H and CBA) arose from a cross of a Bagg albino (BALB/c) mouse and a DBA mouse. The mouse mammary tumor virus-containing restriction enzyme DNA fragments of these strains had similar patterns, suggesting that the proviruses of these mice are in similar genomic locations. Conversely, the pattern arising from the DNA of the GR mouse, a strain genetically unrelated to the others, appeared different, suggesting that its mouse mammary tumor proviruses are located in different genomic sites. The structure of another gene, that coding for beta-globin, was also compared. The mice strains which we studied can be categorized into two classes, expressing either one or two beta-globin proteins. The macroenvironment of the beta-globin gene appeared similar among the mice strains belonging to one genetic class. Female mice of the C3H strain exogenously transmit mouse mammary tumor virus via the milk, and their offspring have a high incidence of mammary tumor occurrence. DNA isolated from individual mammary tumors taken from C3H mice or from BALB/c mice foster nursed on C3H mothers was analyzed by the DNA filter transfer technique. Additional mouse mammary tumor virus-containing fragments were found in the DNA isolated from each mammary tumor. These proviral sequences were integrated into different genomic sites in each tumor. Images PMID:6245257

  3. DNA Clutch Probes for Circulating Tumor DNA Analysis.

    PubMed

    Das, Jagotamoy; Ivanov, Ivaylo; Sargent, Edward H; Kelley, Shana O

    2016-08-31

    Progress toward the development of minimally invasive liquid biopsies of disease is being bolstered by breakthroughs in the analysis of circulating tumor DNA (ctDNA): DNA released from cancer cells into the bloodstream. However, robust, sensitive, and specific methods of detecting this emerging analyte are lacking. ctDNA analysis has unique challenges, since it is imperative to distinguish circulating DNA from normal cells vs mutation-bearing sequences originating from tumors. Here we report the electrochemical detection of mutated ctDNA in samples collected from cancer patients. By developing a strategy relying on the use of DNA clutch probes (DCPs) that render specific sequences of ctDNA accessible, we were able to readout the presence of mutated ctDNA. DCPs prevent reassociation of denatured DNA strands: they make one of the two strands of a dsDNA accessible for hybridization to a probe, and they also deactivate other closely related sequences in solution. DCPs ensure thereby that only mutated sequences associate with chip-based sensors detecting hybridization events. The assay exhibits excellent sensitivity and specificity in the detection of mutated ctDNA: it detects 1 fg/μL of a target mutation in the presence of 100 pg/μL of wild-type DNA, corresponding to detecting mutations at a level of 0.01% relative to wild type. This approach allows accurate analysis of samples collected from lung cancer and melanoma patients. This work represents the first detection of ctDNA without enzymatic amplification.

  4. Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.

    PubMed

    Bondurant, Amy E; Huang, Zhiqing; Whitaker, Regina S; Simel, Lauren R; Berchuck, Andrew; Murphy, Susan K

    2011-12-01

    Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Absolute cross-sections for DNA strand breaks and crosslinks induced by low energy electrons

    PubMed Central

    Chen, Wenzhuang; Chen, Shiliang; Dong, Yanfang; Cloutier, Pierre; Sanche, Léon

    2016-01-01

    Absolute cross sections (CSs) for the interaction of low energy electrons with condensed macromolecules are essential parameters to accurately model ionizing radiation induced reactions. To determine CSs for various conformational DNA damage induced by 2–20 eV electrons, we investigated the influence of the attenuation length (AL) and penetration factor (f) using a mathematical model. Solid films of super-coiled plasmid DNA with thicknesses of 10, 15 and 20 nm were irradiated with 4.6, 5.6, 9.6 and 14.6 eV electrons. DNA conformational changes were quantified by gel electrophoresis, and the respective yields were extrapolated from exposure–response curves. The absolute CS, AL and f values were generated by applying the model developed by Rezaee et al. The values of AL were found to lie between 11 and 16 nm with the maximum at 14.6 eV. The absolute CSs for the loss of the supercoiled (LS) configuration and production of crosslinks (CL), single strand breaks (SSB) and double strand breaks (DSB) induced by 4.6, 5.6, 9.6 and 14.6 eV electrons are obtained. The CSs for SSB are smaller, but similar to those for LS, indicating that SSB are the main conformational damage. The CSs for DSB and CL are about one order of magnitude smaller than those of LS and SSB. The value of f is found to be independent of electron energy, which allows extending the absolute CSs for these types of damage within the range 2–20 eV, from previous measurements of effective CSs. When comparison is possible, the absolute CSs are found to be in good agreement with those obtained from previous similar studies with double-stranded DNA. The high values of the absolute CSs of 4.6 and 9.6 eV provide quantitative evidence for the high efficiency of low energy electrons to induce DNA damage via the formation of transient anions. PMID:27878170

  6. Absolute cross-sections for DNA strand breaks and crosslinks induced by low energy electrons.

    PubMed

    Chen, Wenzhuang; Chen, Shiliang; Dong, Yanfang; Cloutier, Pierre; Zheng, Yi; Sanche, Léon

    2016-12-07

    Absolute cross sections (CSs) for the interaction of low energy electrons with condensed macromolecules are essential parameters to accurately model ionizing radiation induced reactions. To determine CSs for various conformational DNA damage induced by 2-20 eV electrons, we investigated the influence of the attenuation length (AL) and penetration factor (f) using a mathematical model. Solid films of supercoiled plasmid DNA with thicknesses of 10, 15 and 20 nm were irradiated with 4.6, 5.6, 9.6 and 14.6 eV electrons. DNA conformational changes were quantified by gel electrophoresis, and the respective yields were extrapolated from exposure-response curves. The absolute CS, AL and f values were generated by applying the model developed by Rezaee et al. The values of AL were found to lie between 11 and 16 nm with the maximum at 14.6 eV. The absolute CSs for the loss of the supercoiled (LS) configuration and production of crosslinks (CL), single strand breaks (SSB) and double strand breaks (DSB) induced by 4.6, 5.6, 9.6 and 14.6 eV electrons are obtained. The CSs for SSB are smaller, but similar to those for LS, indicating that SSB are the main conformational damage. The CSs for DSB and CL are about one order of magnitude smaller than those of LS and SSB. The value of f is found to be independent of electron energy, which allows extending the absolute CSs for these types of damage within the range 2-20 eV, from previous measurements of effective CSs. When comparison is possible, the absolute CSs are found to be in good agreement with those obtained from previous similar studies with double-stranded DNA. The high values of the absolute CSs of 4.6 and 9.6 eV provide quantitative evidence for the high efficiency of low energy electrons to induce DNA damage via the formation of transient anions.

  7. Fragment Length of Circulating Tumor DNA

    PubMed Central

    Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

    2016-01-01

    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

  8. Fragment Length of Circulating Tumor DNA.

    PubMed

    Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

    2016-07-01

    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

  9. Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

    PubMed

    Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

    2015-11-19

    We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

  10. DNA methylation profiles distinguish different subtypes of gastroenteropancreatic neuroendocrine tumors.

    PubMed

    How-Kit, Alexandre; Dejeux, Emelyne; Dousset, Bertrand; Renault, Victor; Baudry, Marion; Terris, Benoit; Tost, Jörg

    2015-01-01

    Most studies have considered gastroenteropancreatic neuroendocrine tumors (GEP-NETs) as a homogenous group of samples or distinguish only gastrointestinal from pancreatic endocrine tumors. This article investigates if DNA methylation patterns could distinguish subtypes of GEP-NETs. The DNA methylation level of 807 cancer-related genes was investigated in insulinomas, gastrinomas, non-functioning pancreatic endocrine tumors and small intestine endocrine tumors. DNA methylation patterns were found to be tumor type specific for each of the pancreatic tumor subtypes and identified two distinct methylation-based groups in small intestine endocrine tumors. Differences of DNA methylation levels were validated by pyrosequencing for 20 candidate genes and correlated with differences at the transcriptional level for four candidate genes. The heterogeneity of DNA methylation patterns in the different subtypes of gastroenteropancreatic neuroendocrine tumors suggests different underlying pathways and, therefore, these tumors should be considered as distinct entities in molecular and clinical studies.

  11. Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons.

    PubMed

    Tan, Carlyn Rose C; Zhou, Lanlan; El-Deiry, Wafik S

    2016-06-01

    Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are emerging noninvasive multifunctional biomarkers in liquid biopsy allowing for early diagnosis, accurate prognosis, therapeutic target selection, spatiotemporal monitoring of metastasis, as well as monitoring response and resistance to treatment. CTCs and ctDNA are released from different tumor types at different stages and contribute complementary information for clinical decision. Although big strides have been taken in technology development for detection, isolation and characterization of CTCs and sensitive and specific detection of ctDNA, CTC-, and ctDNA-based liquid biopsies may not be widely adopted for routine cancer patient care until the suitability, accuracy, and reliability of these tests are validated and more standardized protocols are corroborated in large, independent, prospectively designed trials. This review covers CTC- and ctDNA-related technologies and their application in colorectal cancer. The promise of CTC-and ctDNA-based liquid biopsies is envisioned.

  12. A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

    EPA Science Inventory

    In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

  13. Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

    PubMed

    van Ginkel, Joost H; Huibers, Manon M H; van Es, Robert J J; de Bree, Remco; Willems, Stefan M

    2017-06-19

    During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data. In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%. Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

  14. DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy.

    PubMed

    Yata, Tomoya; Takahashi, Yuki; Tan, Mengmeng; Nakatsuji, Hirotaka; Ohtsuki, Shozo; Murakami, Tatsuya; Imahori, Hiroshi; Umeki, Yuka; Shiomi, Tomoki; Takakura, Yoshinobu; Nishikawa, Makiya

    2017-11-01

    Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons

    PubMed Central

    Tan, Carlyn Rose C.; Zhou, Lanlan

    2016-01-01

    Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are emerging noninvasive multifunctional biomarkers in liquid biopsy allowing for early diagnosis, accurate prognosis, therapeutic target selection, spatiotemporal monitoring of metastasis, as well as monitoring response and resistance to treatment. CTCs and ctDNA are released from different tumor types at different stages and contribute complementary information for clinical decision. Although big strides have been taken in technology development for detection, isolation and characterization of CTCs and sensitive and specific detection of ctDNA, CTC-, and ctDNA-based liquid biopsies may not be widely adopted for routine cancer patient care until the suitability, accuracy, and reliability of these tests are validated and more standardized protocols are corroborated in large, independent, prospectively designed trials. This review covers CTC- and ctDNA-related technologies and their application in colorectal cancer. The promise of CTC-and ctDNA-based liquid biopsies is envisioned. PMID:27516729

  16. Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

    PubMed

    Wu, Zhenhua; Bai, Yanan; Cheng, Zule; Liu, Fangming; Wang, Ping; Yang, Dawei; Li, Gang; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2017-10-15

    Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Capturing tumor heterogeneity and clonal evolution in solid cancers using circulating tumor DNA analysis.

    PubMed

    Perdigones, Nieves; Murtaza, Muhammed

    2017-06-01

    Circulating tumor DNA analysis has emerged as a potential noninvasive alternative to tissue biopsies for tumor genotyping in patients with metastatic cancer. This is particularly attractive in cases where tissue biopsies are contraindicated or repeat genotyping after progression on treatment is required. However, tissue and plasma analysis results are not always concordant and clinical interpretation of discordant results is not completely understood. Discordant results could arise due to analytical limits of assays used for tumor and plasma DNA analysis or due to low overall contribution of tumor-specific DNA in plasma. Once these factors are ruled out, tissue-plasma concordance and quantitative levels of somatic mutations in plasma can capture tumor heterogeneity. During longitudinal follow-up of patients, this feature can be leveraged to track subclonal evolution and to guide combination or sequential adaptive treatment. Here, we summarize recent results evaluating the opportunities and limitations of circulating tumor DNA analysis in the context of tumor heterogeneity and subclonal evolution in patients with advanced cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Circulating tumor DNA functions as an alternative for tissue to overcome tumor heterogeneity in advanced gastric cancer.

    PubMed

    Gao, Jing; Wang, Haixing; Zang, Wanchun; Li, Beifang; Rao, Guanhua; Li, Lei; Yu, Yang; Li, Zhongwu; Dong, Bin; Lu, Zhihao; Jiang, Zhi; Shen, Lin

    2017-09-01

    Overcoming tumor heterogeneity is a major challenge for personalized treatment of gastric cancer, especially for human epidermal growth factor receptor-2 targeted therapy. Analysis of circulating tumor DNA allows a more comprehensive analysis of tumor heterogeneity than traditional biopsies in lung cancer and breast cancer, but little is known in gastric cancer. We assessed mutation profiles of ctDNA and primary tumors from 30 patients with advanced gastric cancer, then performed a comprehensive analysis of tumor mutations by multiple biopsies from five patients, and finally analyzed the concordance of HER2 amplification in ctDNA and paired tumor tissues in 70 patients. By comparing with a single tumor sample, ctDNA displayed a low concordance of mutation profile, only approximately 50% (138/275) somatic mutations were found in paired tissue samples, however, when compared with multiple biopsies, most DNA mutations in ctDNA were also shown in paired tumor tissues. ctDNA had a high concordance (91.4%, Kappa index = 0.784, P < 0.001) of HER2 amplification with tumor tissues, suggesting it might be an alternative for tissue. It implied that ctDNA-based assessment could partially overcome the tumor heterogeneity, and might serve as a potential surrogate for HER2 analysis in gastric cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  19. Alcohol consumption and breast tumor mitochondrial DNA mutations.

    PubMed

    Platek, Mary E; Shields, Peter G; Tan, Duanjun; Marian, Catalin; Bonner, Matthew R; McCann, Susan E; Nie, Jing; Wilding, Gregory E; Ambrosone, Christine; Millen, Amy E; Trevisan, Maurizio; Russell, Marcia; Nochajski, Thomas H; Edge, Stephen B; Winston, Janet; Freudenheim, Jo L

    2010-06-01

    Mitochondrial DNA (mtDNA) mutations are frequent in breast tumors, but the etiology of these mutations is unknown. We hypothesized that these mutations are associated with exposures that affect oxidative stress such as alcohol metabolism. Using archived tumor blocks from incident breast cancer cases in a case control study, the Western New York Exposures and Breast Cancer (WEB) study, analysis of mtDNA mutations was conducted on 128 breast cancer cases selected based on extremes of alcohol intake. Temporal temperature gradient gel electrophoresis (TTGE) was used to screen the entire mtDNA genome and sequencing was completed for all TTGE positive samples. Case-case comparisons were completed using unconditional logistic regression to determine the relative prevalence of the mutations by exposures including alcohol consumption, manganese superoxide dismutase (MnSOD) genotype, nutrient intake related to oxidative stress and established breast cancer risk factors. Somatic mtDNA mutations were found in 60 of the 128 tumors examined. There were no differences in the prevalence of mtDNA mutations by alcohol consumption, MnSOD genotype or dietary intake. The likelihood of mtDNA mutations was reduced among those with a positive family history for breast cancer (OR = 0.33, CI = 0.12-0.92), among postmenopausal women who used hormone replacement therapy (OR = 0.46, CI = 0.19-1.08, P = 0.08) and was increased for ER negative tumors (OR = 2.05, CI = 0.95-4.43, P = 0.07). Consistent with previous studies, we found that mtDNA mutations are a frequent occurrence in breast tumors. An understanding of the etiology of mtDNA mutations may provide insight into breast carcinogenesis.

  20. Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel.

    PubMed

    Sato, Kei A; Hachiya, Tsuyoshi; Iwaya, Takeshi; Kume, Kohei; Matsuo, Teppei; Kawasaki, Keisuke; Abiko, Yukito; Akasaka, Risaburo; Matsumoto, Takayuki; Otsuka, Koki; Nishizuka, Satoshi S

    2016-01-01

    Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile. DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor. A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease. This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

  1. Circulating tumor DNA as a liquid biopsy for cancer.

    PubMed

    Heitzer, Ellen; Ulz, Peter; Geigl, Jochen B

    2015-01-01

    Targeted therapies have markedly changed the treatment of cancer over the past 10 years. However, almost all tumors acquire resistance to systemic treatment as a result of tumor heterogeneity, clonal evolution, and selection. Although genotyping is the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot, or are often difficult to obtain. To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. Because cell-free circulating tumor DNA (ctDNA) is a potential surrogate for the entire tumor genome, the use of ctDNA as a liquid biopsy may help to obtain the genetic follow-up data that are urgently needed. This review includes recent studies exploring the diagnostic, prognostic, and predictive potential of ctDNA as a liquid biopsy in cancer. In addition, it covers biological and technical aspects, including recent advances in the analytical sensitivity and accuracy of DNA analysis as well as hurdles that have to be overcome before implementation into clinical routine. Although the analysis of ctDNA is a promising area, and despite all efforts to develop suitable tools for a comprehensive analysis of tumor genomes from plasma DNA, the liquid biopsy is not yet routinely used as a clinical application. Harmonization of preanalytical and analytical procedures is needed to provide clinical standards to validate the liquid biopsy as a clinical biomarker in well-designed and sufficiently powered multicenter studies. © 2014 American Association for Clinical Chemistry.

  2. DNA Tumor Viruses and Cell Metabolism

    PubMed Central

    Mushtaq, Muhammad; Darekar, Suhas

    2016-01-01

    Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells. PMID:27034740

  3. Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer.

    PubMed

    Cheng, Feifei; Su, Li; Qian, Cheng

    2016-07-26

    Tissue biopsy is the standard diagnostic procedure for cancers and also provides a material for genotyping, which can assist in the targeted therapies of cancers. However, tissue biopsy-based cancer diagnostic procedures have limitations in their assessment of cancer development, prognosis and genotyping, due to tumor heterogeneity and evolution. Circulating tumor DNA (ctDNA) is single- or double-stranded DNA released by the tumor cells into the blood and it thus harbors the mutations of the original tumor. In recent years, liquid biopsy based on ctDNA analysis has shed a new light on the molecular diagnosis and monitoring of cancer. Studies found that the screening of genetic mutations using ctDNA is highly sensitive and specific, suggesting that ctDNA analysis may significantly improve current systems of tumor diagnosis, even facilitating early-stage detection. Moreover, ctDNA analysis is capable of accurately determining the tumor progression, prognosis and assisting in targeted therapy. Therefore, using ctDNA as a liquid biopsy may herald a revolution for tumor management. Herein, we review the biology of ctDNA, its detection methods and potential applications in tumor diagnosis, treatment and prognosis.

  4. Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer

    PubMed Central

    Cheng, Feifei; Su, Li; Qian, Cheng

    2016-01-01

    Tissue biopsy is the standard diagnostic procedure for cancers and also provides a material for genotyping, which can assist in the targeted therapies of cancers. However, tissue biopsy-based cancer diagnostic procedures have limitations in their assessment of cancer development, prognosis and genotyping, due to tumor heterogeneity and evolution. Circulating tumor DNA (ctDNA) is single- or double-stranded DNA released by the tumor cells into the blood and it thus harbors the mutations of the original tumor. In recent years, liquid biopsy based on ctDNA analysis has shed a new light on the molecular diagnosis and monitoring of cancer. Studies found that the screening of genetic mutations using ctDNA is highly sensitive and specific, suggesting that ctDNA analysis may significantly improve current systems of tumor diagnosis, even facilitating early-stage detection. Moreover, ctDNA analysis is capable of accurately determining the tumor progression, prognosis and assisting in targeted therapy. Therefore, using ctDNA as a liquid biopsy may herald a revolution for tumor management. Herein, we review the biology of ctDNA, its detection methods and potential applications in tumor diagnosis, treatment and prognosis. PMID:27223063

  5. Liposomal lipid and plasmid DNA delivery to B16/BL6 tumors after intraperitoneal administration of cationic liposome DNA aggregates.

    PubMed

    Reimer, D L; Kong, S; Monck, M; Wyles, J; Tam, P; Wasan, E K; Bally, M B

    1999-05-01

    The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.

  6. Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy.

    PubMed

    Alix-Panabières, Catherine; Pantel, Klaus

    2016-05-01

    "Liquid biopsy" focusing on the analysis of circulating tumor cells (CTC) and circulating cell-free tumor DNA (ctDNA) in the blood of patients with cancer has received enormous attention because of its obvious clinical implications for personalized medicine. Analyses of CTCs and ctDNA have paved new diagnostic avenues and are, to date, the cornerstones of liquid biopsy diagnostics. The present review focuses on key areas of clinical applications of CTCs and ctDNA, including detection of cancer, prediction of prognosis in patients with curable disease, monitoring systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms. The application of CTCs and ctDNA for the early detection of cancer is of high public interest, but it faces serious challenges regarding specificity and sensitivity of the current assays. Prediction of prognosis in patients with curable disease can already be achieved in several tumor entities, particularly in breast cancer. Monitoring the success or failure of systemic therapies (i.e., chemotherapy, hormonal therapy, or other targeted therapies) by sequential measurements of CTCs or ctDNA is also feasible. Interventional studies on treatment stratification based on the analysis of CTCs and ctDNA are needed to implement liquid biopsy into personalized medicine. Cancer Discov; 6(5); 479-91. ©2016 AACR. ©2016 American Association for Cancer Research.

  7. Targeting DNA Methyltranferases in Urological Tumors

    PubMed Central

    Marques-Magalhães, Ângela; Graça, Inês; Henrique, Rui; Jerónimo, Carmen

    2018-01-01

    Urological cancers are a heterogeneous group of malignancies accounting for a considerable proportion of cancer-related morbidity and mortality worldwide. Aberrant epigenetic traits, especially altered DNA methylation patterns constitute a hallmark of these tumors. Nonetheless, these alterations are reversible, and several efforts have been carried out to design and test several epigenetic compounds that might reprogram tumor cell phenotype back to a normal state. Indeed, several DNMT inhibitors are currently under evaluation for therapeutic efficacy in clinical trials. This review highlights the critical role of DNA methylation in urological cancers and summarizes the available data on pre-clinical assays and clinical trials with DNMT inhibitors in bladder, kidney, prostate, and testicular germ cell cancers. PMID:29706891

  8. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

    PubMed

    Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

    2017-11-06

    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

  9. Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

    PubMed Central

    Xian, Zhi-Hong; Cong, Wen-Ming; Zhang, Shu-Hui; Wu, Meng-Chao

    2005-01-01

    AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data. RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene. CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcin-ogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. PMID:15996039

  10. Association between absolute tumor burden and serum bone-specific alkaline phosphatase in canine appendicular osteosarcoma.

    PubMed

    Sternberg, R A; Pondenis, H C; Yang, X; Mitchell, M A; O'Brien, R T; Garrett, L D; Helferich, W G; Hoffmann, W E; Fan, T M

    2013-01-01

    In dogs with appendicular osteosarcoma (OSA), increased pretreatment serum bone-specific alkaline phosphatase (BALP) activity is a negative prognostic factor, associated with shorter disease-free intervals and survival times, but a biologic basis for observed differential serum BALP activities in canine OSA patients remains incompletely defined. Serum BALP activity will correlate with absolute tumor burden in dogs with OSA. This study included 96 client-owned dogs with appendicular OSA. In canine OSA cell lines, the expression and membranous release of BALP was evaluated in vitro. The correlation between serum BALP activity and radiographic primary tumor size was evaluated in OSA-bearing dogs. In dogs developing visceral OSA metastases, serial changes in serum BALP activities were evaluated in relation to progression of macroscopic metastases, and visceral metastatic OSA cells were evaluated for BALP expression. In vitro, BALP expression was not associated with either tumorigenic or metastatic phenotype, rather the quantity of membranous BALP released was proportional with cell density. In dogs devoid of macroscopic metastases, there was a positive correlation between serum BALP activity and absolute primary tumor size. In dogs with progressive OSA metastases, serum BALP activity increased and coincided with the development of macroscopic metastases. OSA cells derived from visceral metastatic lesions retained BALP expression. Tumor burden is a determinant of serum BALP activity in dogs with appendicular OSA. The association between increased pretreatment BALP activity and negative clinical prognosis may simply be attributed to greater initial tumor burden, and consequently more advanced tumor stage. Copyright © 2013 by the American College of Veterinary Internal Medicine.

  11. Biodegradable brain-penetrating DNA nanocomplexes and their use to treat malignant brain tumors

    PubMed Central

    Mastorakos, Panagiotis; Zhang, Clark; Song, Eric; Kim, Young Eun; Park, Hee Won; Berry, Sneha; Choi, Won Kyu; Hanes, Justin; Suk, Jung Soo

    2018-01-01

    The discovery of powerful genetic targets has spurred clinical development of gene therapy approaches to treat patients with malignant brain tumors. However, lack of success in the clinic has been attributed to the inability of conventional gene vectors to achieve gene transfer throughout highly disseminated primary brain tumors. Here, we demonstrate ex vivo that small nanocomplexes composed of DNA condensed by a blend of biodegradable polymer, poly(β-amino ester) (PBAE), with PBAE conjugated with 5 kDa polyethylene glycol (PEG) molecules (PBAE-PEG) rapidly penetrate healthy brain parenchyma and orthotopic brain tumor tissues in rats. Rapid diffusion of these DNA-loaded nanocomplexes observed in fresh tissues ex vivo demonstrated that they avoided adhesive trapping in the brain owing to their dense PEG coating, which was critical to achieving widespread transgene expression throughout orthotopic rat brain tumors in vivo following administration by convection enhanced delivery. Transgene expression with the PBAE/PBAE-PEG blended nanocomplexes (DNA-loaded brain-penetrating nanocomplexes, or DNA-BPN) was uniform throughout the tumor core compared to nanocomplexes composed of DNA with PBAE only (DNA-loaded conventional nanocomplexes, or DNA-CN), and transgene expression reached beyond the tumor edge, where infiltrative cancer cells are found, only for the DNA-BPN formulation. Finally, DNA-BPN loaded with anti-cancer plasmid DNA provided significantly enhanced survival compared to the same plasmid DNA loaded in DNA-CN in two aggressive orthotopic brain tumor models in rats. These findings underscore the importance of achieving widespread delivery of therapeutic nucleic acids within brain tumors and provide a promising new delivery platform for localized gene therapy in the brain. PMID:28694032

  12. Detection of tumor DNA at the margins of colorectal cancer liver metastasis

    PubMed Central

    Holdhoff, Matthias; Schmidt, Kerstin; Diehl, Frank; Aggrawal, Nishant; Angenendt, Philipp; Romans, Katharine; Edelstein, Daniel L.; Torbenson, Michael; Kinzler, Kenneth W.; Vogelstein, Bert; Choti, Michael A.; Diaz, Luis A.

    2012-01-01

    Purpose Defining an adequate resection margin of colorectal cancer liver metastases is essential for optimizing surgical technique. We have attempted to evaluate the resection margin through a combination of histopathologic and genetic analyses. Experimental Design We evaluated 88 samples of tumor margins from 12 patients with metastatic colon cancer who each underwent partial hepatectomy of one to six liver metastases. Punch biopsies of surrounding liver tissue were obtained at 4, 8, 12 and 16 mm from the tumor border. DNA from these biopsies was analyzed by a sensitive PCR-based technique, called BEAMing, for mutations of KRAS, PIK3CA, APC, or TP53 identified in the corresponding tumor. Results Mutations were identified in each patient’s resected tumor and used to analyze the 88 samples circumscribing the tumor-normal border. Tumor-specific mutant DNA was detectable in surrounding liver tissue in five of these 88 samples, all within 4 mm of the tumor border. Biopsies that were 8, 12, and 16 mm from the macroscopic visible margin were devoid of detectable mutant tumor DNA as well as of microscopically visible cancer cells. Tumors with a significant radiologic response to chemotherapy were not associated with any increase in mutant tumor DNA in beyond 4 mm of the main tumor. Conclusions Mutant tumor-specific DNA can be detected beyond the visible tumor margin, but never beyond 4 mm, even in patients whose tumors were larger prior to chemotherapy. These data provide a rational basis for determining the extent of surgical excision required in patients undergoing resection of liver metastases. PMID:21531819

  13. Importance of DNA repair in tumor suppression

    NASA Astrophysics Data System (ADS)

    Brumer, Yisroel; Shakhnovich, Eugene I.

    2004-12-01

    The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

  14. Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors.

    PubMed

    Yan, Weixin; Zhang, Aiguo; Powell, Michael J

    2016-07-21

    Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived "driver" and "drug-resistant" alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called "liquid biopsy" allows for the dynamic monitoring of the patients' tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

  15. Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice

    PubMed Central

    Alyamkina, Ekaterina A; Dolgova, Evgenia V; Likhacheva, Anastasia S; Rogachev, Vladimir A; Sebeleva, Tamara E; Nikolin, Valeriy P; Popova, Nelly A; Orishchenko, Konstantin E; Strunkin, Dmitriy N; Chernykh, Elena R; Zagrebelniy, Stanislav N; Bogachev, Sergei S; Shurdov, Mikhail A

    2009-01-01

    Background When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment. Methods Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls. Results An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection. Conclusion Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are

  16. Feasibility of cell-free circulating tumor DNA testing for lung cancer.

    PubMed

    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.

  17. LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors.

    PubMed

    Aparicio, Ana; North, Brittany; Barske, Lindsey; Wang, Xuemei; Bollati, Valentina; Weisenberger, Daniel; Yoo, Christine; Tannir, Nizar; Horne, Erin; Groshen, Susan; Jones, Peter; Yang, Allen; Issa, Jean-Pierre

    2009-04-01

    Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.

  18. Blood-Based Analyses of Cancer: Circulating Tumor Cells and Circulating Tumor DNA

    PubMed Central

    Haber, Daniel A.; Velculescu, Victor E.

    2015-01-01

    The ability to study nonhematologic cancers through noninvasive sampling of blood is one of the most exciting and rapidly advancing fields in cancer diagnostics. This has been driven both by major technologic advances, including the isolation of intact cancer cells and the analysis of cancer cell–derived DNA from blood samples, and by the increasing application of molecularly driven therapeutics, which rely on such accurate and timely measurements of critical biomarkers. Moreover, the dramatic efficacy of these potent cancer therapies drives the selection for additional genetic changes as tumors acquire drug resistance, necessitating repeated sampling of cancer cells to adjust therapy in response to tumor evolution. Together, these advanced noninvasive diagnostic capabilities and their applications in guiding precision cancer therapies are poised to change the ways in which we select and monitor cancer treatments. Significance Recent advances in technologies to analyze circulating tumor cells and circulating tumor DNA are setting the stage for real-time, noninvasive monitoring of cancer and providing novel insights into cancer evolution, invasion, and metastasis. PMID:24801577

  19. Circulating Tumor Cell and Cell-free Circulating Tumor DNA in Lung Cancer.

    PubMed

    Nurwidya, Fariz; Zaini, Jamal; Putra, Andika Chandra; Andarini, Sita; Hudoyo, Achmad; Syahruddin, Elisna; Yunus, Faisal

    2016-09-01

    Circulating tumor cells (CTCs) are tumor cells that are separated from the primary site or metastatic lesion and disseminate in blood circulation. CTCs are considered to be part of the long process of cancer metastasis. As a 'liquid biopsy', CTC molecular examination and investigation of single cancer cells create an important opportunity for providing an understanding of cancer biology and the process of metastasis. In the last decade, we have seen dramatic development in defining the role of CTCs in lung cancer in terms of diagnosis, genomic alteration determination, treatment response and, finally, prognosis prediction. The aims of this review are to understand the basic biology and to review methods of detection of CTCs that apply to the various types of solid tumor. Furthermore, we explored clinical applications, including treatment monitoring to anticipate therapy resistance as well as biomarker analysis, in the context of lung cancer. We also explored the potential use of cell-free circulating tumor DNA (ctDNA) in the genomic alteration analysis of lung cancer.

  20. DNA methylation profiles of ovarian epithelial carcinoma tumors and cell lines.

    PubMed

    Houshdaran, Sahar; Hawley, Sarah; Palmer, Chana; Campan, Mihaela; Olsen, Mari N; Ventura, Aviva P; Knudsen, Beatrice S; Drescher, Charles W; Urban, Nicole D; Brown, Patrick O; Laird, Peter W

    2010-02-22

    Epithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies. We obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited 'subtype-specific' DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene. The significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential

  1. Exosome-based tumor antigens-adjuvant co-delivery utilizing genetically engineered tumor cell-derived exosomes with immunostimulatory CpG DNA.

    PubMed

    Morishita, Masaki; Takahashi, Yuki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2016-12-01

    For cancer immunotherapy via tumor antigen vaccination in combination with an adjuvant, major challenges include the identification of a particular tumor antigen and efficient delivery of the antigen as well as adjuvant to antigen-presenting cells. In this study, we proposed an efficient exosome-based tumor antigens-adjuvant co-delivery system using genetically engineered tumor cell-derived exosomes containing endogenous tumor antigens and immunostimulatory CpG DNA. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a fusion streptavidin (SAV; a protein that binds to biotin with high affinity)-lactadherin (LA; an exosome-tropic protein) protein, yielding genetically engineered SAV-LA-expressing exosomes (SAV-exo). SAV-exo were combined with biotinylated CpG DNA to prepare CpG DNA-modified exosomes (CpG-SAV-exo). Fluorescent microscopic observation revealed the successful modification of exosomes with CpG DNA by SAV-biotin interaction. CpG-SAV-exo showed efficient and simultaneous delivery of exosomes with CpG DNA to murine dendritic DC2.4 cells in culture. Treatment with CpG-SAV-exo effectively activated DC2.4 cells and enhanced tumor antigen presentation capacity. Immunization with CpG-SAV-exo exhibited stronger in vivo antitumor effects in B16BL6 tumor-bearing mice than simple co-administration of exosomes and CpG DNA. Thus, genetically engineered CpG-SAV-exo is an effective exosome-based tumor antigens-adjuvant co-delivery system that will be useful for cancer immunotherapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

    PubMed

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-04-02

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

  3. Monitoring Daily Dynamics of Early Tumor Response to Targeted Therapy by Detecting Circulating Tumor DNA in Urine

    PubMed Central

    Husain, Hatim; Melnikova, Vladislava O.; Kosco, Karena; Woodward, Brian; More, Soham; Pingle, Sandeep C.; Weihe, Elizabeth; Park, Ben Ho; Tewari, Muneesh; Erlander, Mark G.; Cohen, Ezra; Lippman, Scott M.; Kurzrock, Razelle

    2017-01-01

    Purpose Non-invasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design We tested the hypothesis that dynamic changes in epidermal growth factor receptor (EGFR) activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second line third generation anti-EGFR tyrosine kinase inhibitor. Results Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pre-treatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1 suggesting apoptosis with an overall decrease in fragment numbers between baselines to day 7 preceding radiographic response assessed at 6-12 weeks. Conclusions These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. PMID:28420725

  4. Highly personalized detection of minimal Ewing sarcoma disease burden from plasma tumor DNA.

    PubMed

    Hayashi, Masanori; Chu, David; Meyer, Christian F; Llosa, Nicolas J; McCarty, Gregory; Morris, Carol D; Levin, Adam S; Wolinsky, Jean-Paul; Albert, Catherine M; Steppan, Diana A; Park, Ben Ho; Loeb, David M

    2016-10-01

    Even though virtually all patients with Ewing sarcoma achieve a radiographic complete response, up to 30% of patients who present with localized disease and up to 90% of those who present with metastases experience a metastatic disease recurrence, highlighting the inability to identify patients with residual disease at the end of therapy. Up to 95% of Ewing sarcomas carry a driving EWS-ETS translocation that has an intronic breakpoint that is specific to each tumor, and the authors developed a system to quantitatively detect the specific breakpoint DNA fragment in patient plasma. The authors used a long-range multiplex polymerase chain reaction (PCR) technique to identify tumor-specific EWS-ETS breakpoints in Ewing sarcoma cell lines, patient-derived xenografts, and patient tumors, and this sequence was used to design tumor-specific primer sets to detect plasma tumor DNA (ptDNA) by droplet digital PCR in xenograft-bearing mice and patients. Tumor-specific breakpoint DNA fragments were detected in the plasma of xenograft-bearing mice, and the signal correlated with tumor burden during primary tumor growth, after surgical resection, and at the time of metastatic disease recurrence. Furthermore, the authors were able to detect the specific breakpoint in plasma DNA obtained from 3 patients with Ewing sarcoma and in 2 patients the authors were able to detect ptDNA when there was radiographically undetectable disease present. The use of droplet digital PCR to detect tumor-specific EWS-ETS fusion gene breakpoint ptDNA fragments can be developed into a highly personalized biomarker of disease recurrence that can be optimized in animal studies for ultimate use in patients. Cancer 2016;122:3015-3023. © 2016 American Cancer Society. © 2016 American Cancer Society.

  5. Genome-wide analysis of DNA methylation changes induced by gestational arsenic exposure in liver tumors.

    PubMed

    Suzuki, Takehiro; Yamashita, Satoshi; Ushijima, Toshikazu; Takumi, Shota; Sano, Tomoharu; Michikawa, Takehiro; Nohara, Keiko

    2013-12-01

    Inorganic arsenic is known to be a human carcinogen. Previous studies have reported that DNA methylation changes are involved in arsenic-induced carcinogenesis, therefore, DNA methylation changes that are specific to arsenic-induced tumors would be useful to distinguish tumors induced by arsenic from tumors caused by other factors and to dissect arsenic carcinogenesis. Previous studies have shown that gestational arsenic exposure of C3H mice, which tend to spontaneously develop liver tumors, increases the incidence of tumors in male offspring. In this study we used the same experimental protocol as in those previous studies and searched for DNA regions where methylation status was specifically altered in the liver tumors of arsenic-exposed offspring by using methylated DNA immunoprecipitation-CpG island microarrays. The methylation levels of the DNA regions selected were measured by quantitative methylation-specific PCR and bisulfite sequencing. The results of this study clarified a number of regions where DNA methylation status was altered in the liver tumors in the C3H mice compared to normal liver tissues. Among such regions, we showed that a gene body region of the oncogene Fosb underwent alteration in DNA methylation by gestational arsenic exposure. We also showed that Fosb expression significantly increased corresponding to the DNA methylation level of the gene body in the arsenic-exposed group. These findings suggest that the DNA methylation status can be used to identify tumors increased by gestational arsenic exposure. © 2013 Japanese Cancer Association.

  6. Exposure-dependent incorporation of trifluridine into DNA of tumors and white blood cells in tumor-bearing mouse.

    PubMed

    Yamashita, Fumiaki; Komoto, Ikumi; Oka, Hiroaki; Kuwata, Keizo; Takeuchi, Mayuko; Nakagawa, Fumio; Yoshisue, Kunihiro; Chiba, Masato

    2015-08-01

    Trifluridine (TFT) is an antitumor component of a novel nucleoside antitumor agent, TAS-102, which consists of TFT and tipiracil hydrochloride (thymidine phosphorylase inhibitor). Incorporation of TFT into DNA is a probable mechanism of antitumor activity and hematological toxicity. The objective of this study was to examine the TFT incorporation into tumor- and white blood cell-DNA, and to elucidate the mechanism of TFT-related effect and toxicity. TFT effect on the colony formation of mouse bone marrow cells was also investigated. Pharmacokinetics of TFT was determined in nude mice after single oral administration of TAS-102, while the antitumor activity and body weight change were evaluated in the tumor-bearing nude mice after multiple oral administrations for 2 weeks. TFT concentrations in the blood- and tumor-DNA were determined by LC/MS/MS. The colony formation was evaluated by CFU-GM assay. TFT systemic exposure in plasma increased dose-dependently. The tumor growth rate and body weight gain decreased dose-dependently, but TFT concentrations in the DNA of tumor tissues and white blood cells increased dose-dependently. TFT inhibited colony formation of bone marrow cells in a concentration-dependent manner. A significant relationship between systemic exposure of TFT and pharmacological effects including the antitumor activity and body weight change was well explained by the TFT incorporation into DNA. TFT inhibited proliferations of mouse bone marrow cells and human colorectal carcinoma cells implanted to nude mice dose-dependently. The highest tolerable TFT exposure provides the highest antitumor activity, and the hematological toxicity may serve as a potential surrogate indicator of TAS-102 efficacy.

  7. DNA Tumor Virus Regulation of Host DNA Methylation and Its Implications for Immune Evasion and Oncogenesis

    PubMed Central

    Kuss-Duerkop, Sharon K.; Westrich, Joseph A.

    2018-01-01

    Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus–host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers. PMID:29438328

  8. Circulating tumor cells and circulating tumor DNA: What surgical oncologists need to know?

    PubMed

    Cabel, L; Proudhon, C; Mariani, P; Tzanis, D; Beinse, G; Bieche, I; Pierga, J-Y; Bidard, F-C

    2017-05-01

    As a result of recent progress in detection techniques, circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) can now be accurately detected in the blood of most cancer patients. While these new biomarkers can provide a better understanding of key biological mechanisms underlying cancer growth and dissemination, they also open up a wide range of possible clinical applications in medical oncology, radiation oncology and surgical oncology. In this review, we summarize the results obtained with ctDNA and CTC together with their potential future clinical applications in the field of surgical oncology, with particular focus on the perioperative setting of various types of cancer. These applications include, but are not limited to, cancer screening, early diagnosis, prognostic assessment, evaluation and management of preoperative systemic or local therapies, post-surgical detection of minimal residual disease and early detection of cancer relapse. Copyright © 2017 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

  9. DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

    PubMed

    Matsuoka, Taeko; Kawai, Koji; Ando, Satoshi; Sugita, Shintaro; Kandori, Shuya; Kojima, Takahiro; Miyazaki, Jun; Nishiyama, Hiroyuki

    2016-05-01

    DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Pilot study of p62 DNA vaccine in dogs with mammary tumors.

    PubMed

    Gabai, Vladimir; Venanzi, Franco M; Bagashova, Elena; Rud, Oksana; Mariotti, Francesca; Vullo, Cecilia; Catone, Giuseppe; Sherman, Michael Y; Concetti, Antonio; Chursov, Andrey; Latanova, Anastasia; Shcherbinina, Vita; Shifrin, Victor; Shneider, Alexander

    2014-12-30

    Our previous data demonstrated profound anti-tumor and anti-metastatic effects of p62 (sqstm1) DNA vaccine in rodents with various types of transplantable tumors. Testing anti-cancer medicine in dogs as an intermediary step of translational research program provides two major benefits. First, clinical data collected in target animals is required for FDA/USDA approval as a veterinary anti-cancer drug or vaccine. It is noteworthy that the veterinary community is in need of novel medicine for the prevention and treatment of canine and feline cancers. The second more important benefit of testing anti-cancer vaccines in dogs is that spontaneous tumors in dogs may provide invaluable information for human trials. Here, we evaluated the effect(s) of p62 DNA vaccine on mammary tumors of dogs. We found that p62 DNA vaccine administered i.m. decreased or stabilized growth of locally advanced lesions in absence of its overall toxic effects. The observed antitumor activity was associated with lymphocyte infiltration and tumor encapsulation via fibrotic reaction. This data justifies both human clinical trials and veterinary application of p62 DNA vaccine.

  11. Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

    PubMed

    Riva, Francesca; Bidard, Francois-Clement; Houy, Alexandre; Saliou, Adrien; Madic, Jordan; Rampanou, Aurore; Hego, Caroline; Milder, Maud; Cottu, Paul; Sablin, Marie-Paule; Vincent-Salomon, Anne; Lantz, Olivier; Stern, Marc-Henri; Proudhon, Charlotte; Pierga, Jean-Yves

    2017-03-01

    In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival. Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival. © 2016 American Association for Clinical Chemistry.

  12. Polyplex-microbubble hybrids for ultrasound-guided plasmid DNA delivery to solid tumors.

    PubMed

    Sirsi, Shashank R; Hernandez, Sonia L; Zielinski, Lukasz; Blomback, Henning; Koubaa, Adel; Synder, Milo; Homma, Shunichi; Kandel, Jessica J; Yamashiro, Darrell J; Borden, Mark A

    2012-01-30

    Microbubble ultrasound contrast agents are being developed as image-guided gene carriers for targeted delivery in vivo. In this study, novel polyplex-microbubbles were synthesized, characterized and evaluated for systemic circulation and tumor transfection. Branched polyethylenimine (PEI; 25 kDa) was modified with polyethylene glycol (PEG; 5 kDa), thiolated and covalently attached to maleimide groups on lipid-coated microbubbles. The PEI-microbubbles demonstrated increasingly positive surface charge and DNA loading capacity with increasing maleimide content. The in vivo ultrasound contrast persistence of PEI-microbubbles was measured in the healthy mouse kidney, and a two-compartment pharmacokinetic model accounting for free and adherent microbubbles was developed to describe the anomalous time-intensity curves. The model suggested that PEI loading dramatically reduced free circulation and increased nonspecific adhesion to the vasculature. However, DNA loading to form polyplex-microbubbles increased circulation in the bloodstream and decreased nonspecific adhesion. PEI-microbubbles coupled to a luciferase bioluminescence reporter plasmid DNA were shown to transfect tumors implanted in the mouse kidney. Site-specific delivery was achieved using ultrasound applied over the tumor area following bolus injection of the DNA/PEI-microbubbles. In vivo imaging showed over 10-fold higher bioluminescence from the tumor region compared to untreated tissue. Ex vivo analysis of excised tumors showed greater than 40-fold higher expression in tumor tissue than non-sonicated control (heart) tissue. These results suggest that the polyplex-microbubble platform offers improved control of DNA loading and packaging suitable for ultrasound-guided tissue transfection. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts

    PubMed Central

    Tseng, Chih◻Wen; Trimble, Cornelia; Zeng, Qi; Monie, Archana; Alvarez, Ronald D.; Huh, Warner K.; Hoory, Talia; Wang, Mei-Cheng; Hung, Chien-Fu; Wu, T.-C.

    2008-01-01

    Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic anti-tumor effects and the highest frequency of E7-specific CD8+ T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8+ T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic anti-tumor effects. The clinical implications of the study are discussed. PMID:18815785

  14. Self-assembled mirror DNA nanostructures for tumor-specific delivery of anticancer drugs.

    PubMed

    Kim, Kyoung-Ran; Kim, Hyo Young; Lee, Yong-Deok; Ha, Jong Seong; Kang, Ji Hee; Jeong, Hansaem; Bang, Duhee; Ko, Young Tag; Kim, Sehoon; Lee, Hyukjin; Ahn, Dae-Ro

    2016-12-10

    Nanoparticle delivery systems have been extensively investigated for targeted delivery of anticancer drugs over the past decades. However, it is still a great challenge to overcome the drawbacks of conventional nanoparticle systems such as liposomes and micelles. Various novel nanomaterials consist of natural polymers are proposed to enhance the therapeutic efficacy of anticancer drugs. Among them, deoxyribonucleic acid (DNA) has received much attention as an emerging material for preparation of self-assembled nanostructures with precise control of size and shape for tailored uses. In this study, self-assembled mirror DNA tetrahedron nanostructures is developed for tumor-specific delivery of anticancer drugs. l-DNA, a mirror form of natural d-DNA, is utilized for resolving a poor serum stability of natural d-DNA. The mirror DNA nanostructures show identical thermodynamic properties to that of natural d-DNA, while possessing far enhanced serum stability. This unique characteristic results in a significant effect on the pharmacokinetics and biodistribution of DNA nanostructures. It is demonstrated that the mirror DNA nanostructures can deliver anticancer drugs selectively to tumors with enhanced cellular and tissue penetration. Furthermore, the mirror DNA nanostructures show greater anticancer effects as compared to that of conventional PEGylated liposomes. Our new approach provides an alternative strategy for tumor-specific delivery of anticancer drugs and highlights the promising potential of the mirror DNA nanostructures as a novel drug delivery platform. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. DNA in Uninfected and Virus-Infected Cells Complementary to Avian Tumor Virus RNA

    PubMed Central

    Rosenthal, Peter N.; Robinson, Harriet L.; Robinson, William S.; Hanafusa, Teruko; Hanafusa, Hidesaburo

    1971-01-01

    The 70S RNA component of several avian tumor viruses was hybridized with DNA extracted from avian tumor virus-infected and uninfected chicken and Japanese quail cells. Tritium-labeled 70S RNAs from Rous sarcoma virus (RSV), Rous associated virus-1 (RAV-1), RAV-60, and Schmidt-Ruppin-RSV (SR-RSV) hybridize from 3 to 10 times more with DNA from uninfected chicken cells than with DNA from Escherichia coli, calfthymus, or baby hamster kidney cells. After infection of chicken cells with RSV(RAV-1), SR-RSV, or RAV-2, the amount of 70S avian tumor virus [3H]RNA hybridized increases by 1.6 times. The specificity of the hybridization reaction was shown by the specific competition of 70S SR-RSV [3H]RNA with 70S RNA from RSV(RAV-1), and not with RNA from Sendai virus or chicken cells. There was no difference in the hybridization of 70S RNA from RSV (RAV-1), RAV-1, or RAV-60 with DNA either from chicken cells that contain RAV-60 in a nonreplicating form or from chicken cells that do not appear to contain RAV-60. These results indicate that both types of uninfected chicken cells contain DNA that is complementary to RNA from several avian tumor viruses and that the amount of complementary DNA increases in such cells after infection with an avian tumor virus. The RNAs of genetically different avian tumor viruses appear to have indistinguishable base sequences by this technique. PMID:4332808

  16. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors.

    PubMed

    Cai, Jinquan; Chen, Jing; Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

    2015-07-20

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma.

  17. Circulating tumor DNA evaluated by Next-Generation Sequencing is predictive of tumor response and prolonged clinical benefit with nivolumab in advanced non-small cell lung cancer.

    PubMed

    Giroux Leprieur, Etienne; Herbretau, Guillaume; Dumenil, Coraline; Julie, Catherine; Giraud, Violaine; Labrune, Sylvie; Dumoulin, Jennifer; Tisserand, Julie; Emile, Jean-François; Blons, Hélène; Chinet, Thierry

    2018-01-01

    Nivolumab is an anti-PD1 antibody, given in second-line or later treatment in advanced non-small cell lung cancer (NSCLC). The objective of this study was to describe the predictive value of circulating tumor DNA (ctDNA) on the efficacy of nivolumab in advanced NSCLC. We prospectively included all consecutive patients with advanced NSCLC treated with nivolumab in our Department between June 2015 and October 2016. Plasma samples were obtained before the first injection of nivolumab and at the first tumor evaluation with nivolumab. ctDNA was analyzed by Next-Generation Sequencing (NGS), and the predominant somatic mutation was followed for each patient and correlated with tumor response, clinical benefit (administration of nivolumab for more than 6 months), and progression-free survival (PFS). Of 23 patients, 15 had evaluable NGS results at both times of analysis. ctDNA concentration at the first tumor evaluation and ctDNA change correlated with tumor response, clinical benefit and PFS. ROC curve analyses showed good diagnostic performances for tumor response and clinical benefit, both for ctDNA concentration at the first tumor evaluation (tumor response: positive predictive value (PPV) at 100.0% and negative predictive value (NPV) at 71.0%; clinical benefit: PPV at 83.3% and NPV 77.8%) and the ctDNA change (tumor response: PPV 100.0% and NPV 62.5%; clinical benefit: PPV 100.0% and NPV 80.0%). Patients without ctDNA concentration increase >9% at 2 months had a long-term benefit of nivolumab. In conclusion, NGS analysis of ctDNA allows the early detection of tumor response and long-term clinical benefit with nivolumab in NSCLC.

  18. Quantitative DNA methylation analyses reveal stage dependent DNA methylation and association to clinico-pathological factors in breast tumors.

    PubMed

    Klajic, Jovana; Fleischer, Thomas; Dejeux, Emelyne; Edvardsen, Hege; Warnberg, Fredrik; Bukholm, Ida; Lønning, Per Eystein; Solvang, Hiroko; Børresen-Dale, Anne-Lise; Tost, Jörg; Kristensen, Vessela N

    2013-10-05

    Aberrant DNA methylation of regulatory genes has frequently been found in human breast cancers and correlated to clinical outcome. In the present study we investigate stage specific changes in the DNA methylation patterns in order to identify valuable markers to understand how these changes affect breast cancer progression. Quantitative DNA methylation analyses of 12 candidate genes ABCB1, BRCCA1, CDKN2A, ESR1, GSTP1, IGF2, MGMT, HMLH1, PPP2R2B, PTEN, RASSF1A and FOXC1 was performed by pyrosequencing a series of 238 breast cancer tissue samples from DCIS to invasive tumors stage I to IV. Significant differences in methylation levels between the DCIS and invasive stage II tumors were observed for six genes RASSF1A, CDKN2A, MGMT, ABCB1, GSTP1 and FOXC1. RASSF1A, ABCB1 and GSTP1 showed significantly higher methylation levels in late stage compared to the early stage breast carcinoma. Z-score analysis revealed significantly lower methylation levels in DCIS and stage I tumors compared with stage II, III and IV tumors. Methylation levels of PTEN, PPP2R2B, FOXC1, ABCB1 and BRCA1 were lower in tumors harboring TP53 mutations then in tumors with wild type TP53. Z-score analysis showed that TP53 mutated tumors had significantly lower overall methylation levels compared to tumors with wild type TP53. Methylation levels of RASSF1A, PPP2R2B, GSTP1 and FOXC1 were higher in ER positive vs. ER negative tumors and methylation levels of PTEN and CDKN2A were higher in HER2 positive vs. HER2 negative tumors. Z-score analysis also showed that HER2 positive tumors had significantly higher z-scores of methylation compared to the HER2 negative tumors. Univariate survival analysis identifies methylation status of PPP2R2B as significant predictor of overall survival and breast cancer specific survival. In the present study we report that the level of aberrant DNA methylation is higher in late stage compared with early stage of invasive breast cancers and DCIS for genes mentioned above.

  19. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer.

    PubMed

    Giannopoulou, Lydia; Mastoraki, Sophia; Buderath, Paul; Strati, Areti; Pavlakis, Kitty; Kasimir-Bauer, Sabine; Lianidou, Evi S

    2018-05-25

    Estrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). ESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). ESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). We report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Loss of ATRX, associated with DNA methylation pattern of chromosome end, impacted biological behaviors of astrocytic tumors

    PubMed Central

    Zhang, Wei; Yang, Pei; Zhang, Chuanbao; Li, Mingyang; Yao, Kun; Wang, Hongjun; Li, Qingbin; Jiang, Chuanlu; Jiang, Tao

    2015-01-01

    Loss of ATRX leads to epigenetic alterations, including abnormal levels of DNA methylation at repetitive elements such as telomeres in murine cells. We conducted an extensive DNA methylation and mRNA expression profile study on a cohort of 82 patients with astrocytic tumors to study whether ATRX expression was associated with DNA methylation level in astrocytic tumors and in which cellular functions it participated. We observed that astrocytic tumors with lower ATRX expression harbored higher DNA methylation level at chromatin end and astrocytic tumors with ATRX-low had distinct gene expression profile and DNA methylation profile compared with ATRX-high tumors. Then, we uncovered that several ATRX associated biological functions in the DNA methylation and mRNA expression profile (GEP), including apoptotic process, DNA-dependent positive regulation of transcription, chromatin modification, and observed that ATRX expression was companied by MGMT methylation and expression. We also found that loss of ATRX caused by siRNA induced apoptotic cells increasing, reduced tumor cell proliferation and repressed the cell migration in glioma cells. Our results showed ATRX-related regulatory functions of the combined profiles from DNA methylation and mRNA expression in astrocytic tumors, and delineated that loss of ATRX impacted biological behaviors of astrocytic tumor cells, providing important resources for future dissection of ATRX role in glioma. PMID:25971279

  1. KRAS detection in colonic tumors by DNA extraction from FTA paper: the molecular touch-prep.

    PubMed

    Petras, Melissa L; Lefferts, Joel A; Ward, Brian P; Suriawinata, Arief A; Tsongalis, Gregory J

    2011-12-01

    DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is usually more degraded and contains more polymerase chain reaction (PCR) inhibitors than DNA isolated from nonfixed tissue. In addition, the tumor size and cellular heterogeneity found in tissue sections can often impact testing for molecular biomarkers. As a potential remedy to this situation, we evaluated the use of Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA for use in a real-time PCR-based KRAS mutation assay. Eleven colon tumor samples were collected by making a cut into the fresh tumor and applying the Whatman FTA paper to the cut surface. Matched FFPE tissue blocks from these tumors were also collected for comparison. KRAS mutation analysis was carried out using the Applied Biosystems 7500 Fast Real-time PCR System using 7 independent custom TaqMan PCR assays. Of the 11 colon tumors sampled, 6 were positive for KRAS mutations in both the Whatman FTA paper preparations and corresponding FFPE samples. Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA have many advantages including ease of use, intrinsic antimicrobial properties, long storage potential (stability of DNA over time), and a faster turnaround time for results. Extracted DNA should be suitable for most molecular diagnostic assays that use PCR techniques. This novel means of DNA preservation from surgical specimens would benefit from additional study and validation as a dependable and practical technique to preserve specimens for molecular testing.

  2. Warthin tumors do not have microsatellite instability and express normal DNA mismatch repair proteins.

    PubMed

    Hunt, Jennifer L

    2006-01-01

    Warthin tumors are controversial entities with a poorly understood etiology. Although some investigators have suggested a neoplastic origin, others have supported a developmental anomaly. A recent study described the absence of staining for hMLH1 and hMSH2 proteins in the epithelial component of Warthin tumors, suggesting that they arise secondary to defects in the DNA mismatch repair system. To determine if Warthin tumors exhibit evidence of DNA mismatch repair defects. Immunostains for hMLH1 and hMSH2 were performed using a standard approach. Microdissection of the epithelial component was followed by DNA extraction from the tissue fragments. Polymerase chain reaction and capillary electrophoresis analyses were performed for the following 5 National Cancer Institute-recommended microsatellites: D2s123, D5s346, D17s250, BAT25, and BAT26. Twelve patients with Warthin tumors were included. The immunostains for hMLH1 and hMSH2 showed preserved expression in the nuclei of the epithelial component of all Warthin tumors. No microsatellite instability was detected, and no loss of heterozygosity was seen. These results are not concordant with previously reported results showing loss of expression of the hMLH1 and hMSH2 DNA mismatch repair enzymes in the epithelial component of Warthin tumors. Furthermore, no microsatellite instability was detected in the 5 loci tested for each tumor in this series. These data demonstrate that Warthin tumors do not have evidence of DNA mismatch repair defects at the genomic or protein expression level.

  3. Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts.

    PubMed

    Tseng, Chih-Wen; Trimble, Cornelia; Zeng, Qi; Monie, Archana; Alvarez, Ronald D; Huh, Warner K; Hoory, Talia; Wang, Mei-Cheng; Hung, Chien-Fu; Wu, T-C

    2009-05-01

    Current therapeutic approaches to treatment of patients with bulky cervical cancer are based on conventional in situ ablative modalities including cisplatin-based chemotherapy and radiation therapy. The 5-year survival of patients with nonresectable disease is dismal. Because over 99% of squamous cervical cancer is caused by persistent infection with an oncogenic strain of human papillomavirus (HPV), particularly type 16 and viral oncoproteins E6 and E7 are functionally required for disease initiation and persistence, HPV-targeted immune strategies present a compelling opportunity in which to demonstrate proof of principle. Sublethal doses of radiation and chemotherapeutic agents have been shown to have synergistic effect in combination with either vaccination against cancer-specific antigens, or with passive transfer of tumor-specific cytotoxic T lymphocytes (CTLs). Here, we explored the combination of low-dose radiation therapy with DNA vaccination with calreticulin (CRT) linked to the mutated form of HPV-16 E7 antigen (E7(detox)), CRT/E7(detox) in the treatment of E7-expressing TC-1 tumors. We observed that TC-1 tumor-bearing mice treated with radiotherapy combined with CRT/E7(detox) DNA vaccination generated significant therapeutic antitumor effects and the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of treated mice. Furthermore, treatment with radiotherapy was shown to render the TC-1 tumor cells more susceptible to lysis by E7-specific CTLs. In addition, we observed that treatment with radiotherapy during the second DNA vaccination generated the highest frequency of E7-specific CD8(+) T cells in the tumors and spleens of TC-1 tumor-bearing mice. Finally, TC-1 tumor-bearing mice treated with the chemotherapy in combination with radiation and CRT/E7(detox) DNA vaccination generate significantly enhanced therapeutic antitumor effects. The clinical implications of the study are discussed.

  4. A Method to Convert MRI Images of Temperature Change Into Images of Absolute Temperature in Solid Tumors

    PubMed Central

    Davis, Ryan M.; Viglianti, Benjamin L.; Yarmolenko, Pavel; Park, Ji-Young; Stauffer, Paul; Needham, David; Dewhirst, Mark W.

    2013-01-01

    Purpose During hyperthermia (HT), the therapeutic response of tumors varies substantially within the target temperature range (39–43°C). Current thermometry methods are either invasive or measure only temperature change, which limits the ability to study tissue responses to HT. This study combines manganese-containing low-temperature sensitive liposomes (Mn-LTSL) with proton resonance frequency shift (PRFS) thermometry to measure absolute temperature in tumors with high spatial and temporal resolution using MRI. Methods Liposomes were loaded with 300mM MnSO4. The phase transition temperature (Tm) of Mn-LTSL samples was measured by differential scanning calorimetry (DSC). The release of manganese from Mn-LTSL in saline was characterized with inductively-coupled plasma atomic emission spectroscopy. A 2T GE small animal scanner was used to acquire dynamic T1-weighted images and temperature change images of Mn-LTSL in saline phantoms and fibrosarcoma-bearing Fisher 344 rats receiving hyperthermia after Mn-LTSL injection. Results The Tm of Mn-LTSL in rat blood was 42.9 ± 0.2 °C (DSC). For Mn-LTSL samples (0.06mM – 0.5mM Mn2+ in saline) heated monotonically from 30°C to 50°C, a peak in the rate of MRI signal enhancement occurred at 43.1 ± 0.3 °C. The same peak in signal enhancement rate was observed during heating of fibrosarcoma tumors (N=3) after injection of Mn-LTSL, and the peak was used to convert temperature change images into absolute temperature. Accuracies of calibrated temperature measurements were in the range 0.9 – 1.8°C. Conclusion The release of Mn2+ from Mn-LTSL affects the rate of MR signal enhancement which enables conversion of MRI-based temperature change images to absolute temperature. PMID:23957326

  5. DNA methylation profile distinguishes clear cell sarcoma of the kidney from other pediatric renal tumors.

    PubMed

    Ueno, Hitomi; Okita, Hajime; Akimoto, Shingo; Kobayashi, Kenichiro; Nakabayashi, Kazuhiko; Hata, Kenichiro; Fujimoto, Junichiro; Hata, Jun-Ichi; Fukuzawa, Masahiro; Kiyokawa, Nobutaka

    2013-01-01

    A number of specific, distinct neoplastic entities occur in the pediatric kidney, including Wilms' tumor, clear cell sarcoma of the kidney (CCSK), congenital mesoblastic nephroma (CMN), rhabdoid tumor of the kidney (RTK), and the Ewing's sarcoma family of tumors (ESFT). By employing DNA methylation profiling using Illumina Infinium HumanMethylation27, we analyzed the epigenetic characteristics of the sarcomas including CCSK, RTK, and ESFT in comparison with those of the non-neoplastic kidney (NK), and these tumors exhibited distinct DNA methylation profiles in a tumor-type-specific manner. CCSK is the most frequently hypermethylated, but least frequently hypomethylated, at CpG sites among these sarcomas, and exhibited 490 hypermethylated and 46 hypomethylated CpG sites in compared with NK. We further validated the results by MassARRAY, and revealed that a combination of four genes was sufficient for the DNA methylation profile-based differentiation of these tumors by clustering analysis. Furthermore, THBS1 CpG sites were found to be specifically hypermethylated in CCSK and, thus, the DNA methylation status of these THBS1 sites alone was sufficient for the distinction of CCSK from other pediatric renal tumors, including Wilms' tumor and CMN. Moreover, combined bisulfite restriction analysis could be applied for the detection of hypermethylation of a THBS1 CpG site. Besides the biological significance in the pathogenesis, the DNA methylation profile should be useful for the differential diagnosis of pediatric renal tumors.

  6. Clinical applications of circulating tumor DNA and circulating tumor cells in pancreatic cancer.

    PubMed

    Riva, Francesca; Dronov, Oleksii I; Khomenko, Dmytro I; Huguet, Florence; Louvet, Christophe; Mariani, Pascale; Stern, Marc-Henri; Lantz, Olivier; Proudhon, Charlotte; Pierga, Jean-Yves; Bidard, Francois-Clement

    2016-03-01

    Pancreatic ductal adenocarcinoma (PDAC) is the most frequent pancreatic cancer type and is characterized by a dismal prognosis due to late diagnosis, local tumor invasion, frequent distant metastases and poor sensitivity to current therapy. In this context, circulating tumor cells and circulating tumor DNA constitute easily accessible blood-borne tumor biomarkers that may prove their clinical interest for screening, early diagnosis and metastatic risk assessment of PDAC. Moreover these markers represent a tool to assess PDAC mutational landscape. In this review, together with key biological findings, we summarize the clinical results obtained using "liquid biopsies" at the different stages of the disease, for early and metastatic diagnosis as well as monitoring during therapy. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

    PubMed

    Shimizu, Akinori; Tani, Haruna; Takibuchi, Gaku; Ishikawa, Kaori; Sakurazawa, Ryota; Inoue, Takafumi; Hashimoto, Tetsuo; Nakada, Kazuto; Takenaga, Keizo; Hayashi, Jun-Ichi

    2017-11-04

    In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29 H (sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29 H (sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Nanoplasmonic biosensor: detection and amplification of dual bio-signatures of circulating tumor DNA.

    PubMed

    Nguyen, Anh H; Sim, Sang Jun

    2015-05-15

    Circulating tumor DNA (ctDNA) bearing tumor-specific mutation and methylation are promising biomarkers for noninvasive cancer assessment. However, existing methods for ctDNA detection are restricted to genetic mutations. Recently, nanoplasmonics has emerged as a platform for one-step dual detection with high sensitivity and specificity. Here we present a strategy for ultrasensitive detection of tumor-specific mutations (E542K and E545K) and methylation of ctDNA of PIK3CA gene based on localized surface plasmon resonance (LSPR) and the coupling plasmon mode of gold nanoparticles (AuNPs). Peptide nucleic acids (PNA) is used as a probe to capture and enrich the 69-bp PIK3CA ctDNA. The exposure of PNA-probed AuNPs to 200 fM ctDNA generates LSPR-peak shift of 4.3 nm, corresponding to the primary response. Immunogold colloids are exploited as methylation detectors and plasmon coupling based enhancement for secondary response. LSPR-peak shifted from 4.3 nm to 11.4 nm upon the immunogold colloids binding to two methylcytosines (mCpG), which is an approximately 107% increase, compared to that of the primary response. This enhancement leads to four times (~50 fM) improvement of sensitivity and because of two mCpG sites, ctDNA was detected. These results demonstrate that the sensor can simultaneously detect the hot-spot mutation and epigenetic changes on the ctDNA. Promisingly, other specific-tumor mutants and epigenetic changes can be detected at low concentration with this platform. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Ultrasound enhances in vivo tumor expression of plasmid DNA by PEG-introduced cationized dextran.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2005-11-28

    This study is an investigation to experimentally confirm whether or not ultrasound (US) irradiation is effective in enhancing the in vivo gene expression of plasmid DNA in tumor. Dextran was cationized by introducing spermine to the hydroxyl groups to allow to polyionically complex with a plasmid DNA. The cationized dextran prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have an active ester and methoxy groups at each terminal, to obtain cationized dextran with different percentages of PEG introduced. Various cationized dextrans with or without PEG introduction were mixed with a plasmid DNA of LacZ to form cationized dextran-plasmid DNA complexes. Electrophoretical examination revealed that the plasmid DNA was complexed both with the cationized dextran and PEG-introduced cationized dextran, irrespective of the PEG introduction percentage, although the higher N/P ratio was needed for plasmid DNA complexation with the latter. By complexation with the cationized dextran, the zeta potential of plasmid DNA was changed to be positive. The charge of PEG-introduced cationized dextran-plasmid DNA complexes became close to 0 mV as their percentage of PEG introduced increased, although the molecular size was about 250 nm, irrespective of the PEG introduction. When cationized dextran-plasmid DNA complexes with or without PEG introduction were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass and the subsequent US irradiation to the tumor mass percutaneously, the PEG-introduced cationized dextran-plasmid DNA complex plus US irradiation enhanced the tumor level of gene expression to a significantly high extent compared with the cationized dextran-plasmid DNA complex and free plasmid DNA with or without US irradiation. The enhanced level depended on the time period and timing of US irradiation. Fluorescent microscopic studies revealed that the localization of plasmid DNA and the gene expression were observed in

  10. Mutation-based detection and monitoring of cell-free tumor DNA in peripheral blood of cancer patients.

    PubMed

    Benesova, L; Belsanova, B; Suchanek, S; Kopeckova, M; Minarikova, P; Lipska, L; Levy, M; Visokai, V; Zavoral, M; Minarik, M

    2013-02-15

    Prognosis of solid cancers is generally more favorable if the disease is treated early and efficiently. A key to long cancer survival is in radical surgical therapy directed at the primary tumor followed by early detection of possible progression, with swift application of subsequent therapeutic intervention reducing the risk of disease generalization. The conventional follow-up care is based on regular observation of tumor markers in combination with computed tomography/endoscopic ultrasound/magnetic resonance/positron emission tomography imaging to monitor potential tumor progression. A recent development in methodologies allowing screening for a presence of cell-free DNA (cfDNA) brings a new viable tool in early detection and management of major cancers. It is believed that cfDNA is released from tumors primarily due to necrotization, whereas the origin of nontumorous cfDNA is mostly apoptotic. The process of cfDNA detection starts with proper collection and treatment of blood and isolation and storage of blood plasma. The next important steps include cfDNA extraction from plasma and its detection and/or quantification. To distinguish tumor cfDNA from nontumorous cfDNA, specific somatic DNA mutations, previously localized in the primary tumor tissue, are identified in the extracted cfDNA. Apart from conventional mutation detection approaches, several dedicated techniques have been presented to detect low levels of cfDNA in an excess of nontumorous (nonmutated) DNA, including real-time polymerase chain reaction (PCR), "BEAMing" (beads, emulsion, amplification, and magnetics), and denaturing capillary electrophoresis. Techniques to facilitate the mutant detection, such as mutant-enriched PCR and COLD-PCR (coamplification at lower denaturation temperature PCR), are also applicable. Finally, a number of newly developed miniaturized approaches, such as single-molecule sequencing, are promising for the future. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Circulating Cell Free Tumor DNA Detection as a Routine Tool for Lung Cancer Patient Management

    PubMed Central

    Vendrell, Julie A.; Mau-Them, Frédéric Tran; Béganton, Benoît; Godreuil, Sylvain; Coopman, Peter; Solassol, Jérôme

    2017-01-01

    Circulating tumoral DNA (ctDNA), commonly named “liquid biopsy”, has emerged as a new promising noninvasive tool to detect biomarker in several cancers including lung cancer. Applications involving molecular analysis of ctDNA in lung cancer have increased and encompass diagnosis, response to treatment, acquired resistance and prognosis prediction, while bypassing the problem of tumor heterogeneity. ctDNA may then help perform dynamic genetic surveillance in the era of precision medicine through indirect tumoral genomic information determination. The aims of this review were to examine the recent technical developments that allowed the detection of genetic alterations of ctDNA in lung cancer. Furthermore, we explored clinical applications in patients with lung cancer including treatment efficiency monitoring, acquired therapy resistance mechanisms and prognosis value. PMID:28146051

  12. Real-time quantitative PCR detection of circulating tumor cells using tag DNA mediated signal amplification strategy.

    PubMed

    Mei, Ting; Lu, Xuewen; Sun, Ning; Li, Xiaomei; Chen, Jitao; Liang, Min; Zhou, Xinke; Fang, Zhiyuan

    2018-06-05

    The level of circulating tumor cell (CTCs) is a reliable marker for tumor burden and malignant progression. Quantification of CTCs remains technically challenging due to the rarity of these cells in peripheral blood. In the present study, we established a real-time quantitative PCR (Q-PCR) based method for sensitive detection of CTCs without DNA extraction. Blood sample was first turned to erythrocyte lyses and then incubated with two antibodies, tag-DNA modified CK-19 antibody and magnetic beads conjugated EpCAM antibody. Tumor cells were further enriched by magnetic separation. Tag-DNA that immobilized on tumor cells through CK-19 antibodies were also retrieved, which was further quantified by Q-PCR. This assay was able to detect single tumor cell in a 5 mL blood sample. The detection rate of clinical tumor blood sample was 92.3%. Furthermore, CTC count in patient was correlated with tumor stage and tumor status. The signal amplification was based on tag DNA rather than tumor gene, which was independent of nucleic acid extraction. With high sensitivity and convenience, this method can be a good alternative for the determination of cancer progress. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

    PubMed Central

    Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

    2011-01-01

    Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

  14. Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

    PubMed

    Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

    2011-01-01

    Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

  15. Circulating tumor DNA profiling reveals clonal evolution and real-time disease progression in advanced hepatocellular carcinoma.

    PubMed

    Cai, Zhi-Xiong; Chen, Geng; Zeng, Yong-Yi; Dong, Xiu-Qing; Lin, Min-Jie; Huang, Xin-Hui; Zhang, Da; Liu, Xiao-Long; Liu, Jing-Feng

    2017-09-01

    Circulating tumor DNA (ctDNA) provides a potential non-invasive biomarker for cancer diagnosis and prognosis, but whether it could reflect tumor heterogeneity and monitor therapeutic responses in hepatocellular carcinoma (HCC) is unclear. Focusing on 574 cancer genes known to harbor actionable mutations, we identified the mutation repertoire of HCC tissues, and monitored the corresponding ctDNA features in blood samples to evaluate its clinical significance. Analysis of 3 HCC patients' mutation profiles revealed that ctDNA could overcome tumor heterogeneity and provide information of tumor burden and prognosis. Further analysis was conducted on the 4th HCC case with multiple lesion samples and sequential plasma samples. We identified 160 subclonal SNVs in tumor tissues as well as matched peritumor tissues with PBMC as control. 96.9% of this patient's tissue mutations could be also detected in plasma samples. These subclonal SNVs were grouped into 9 clusters according to their trends of cellular prevalence shift in tumor tissues. Two clusters constituted of tumor stem somatic mutations showed circulating levels relating with cancer progression. Analysis of tumor somatic mutations revealed that circulating level of such tumor stem somatic mutations could reflect tumor burden and even predict prognosis earlier than traditional strategies. Furthermore, HCK (p.V174M), identified as a recurrent/metastatic related mutation site, could promote migration and invasion of HCC cells. Taken together, study of mutation profiles in biopsy and plasma samples in HCC patients showed that ctDNA could overcome tumor heterogeneity and real-time track the therapeutic responses in the longitudinal monitoring. © 2017 UICC.

  16. Review of the clinical applications and technological advances of circulating tumor DNA in cancer monitoring.

    PubMed

    Chang, Yi; Tolani, Bhairavi; Nie, Xiuhong; Zhi, Xiuyi; Hu, Mu; He, Biao

    2017-01-01

    Circulating cell-free DNA (cfDNA) released by tumor cells, termed ctDNA, closely reflects the heterogeneity of primary cancers and their metastases. As a noninvasive, real-time monitoring biomarker, ctDNA is a promising tool for detecting driver gene mutations, assessing tumor burden and acquired resistance, and early diagnosis. However, isolation and enrichment of cfDNA is a big challenge due to the high degree of DNA fragmentation and its relatively low abundance in the bloodstream. This review aims to provide insights into the recent technological advances in acquisition of optimal quality cfDNA, the use of preservatives, isolation methods, processing timelines, and detection techniques. It also describes clinical applications of ctDNA in cancer patient management.

  17. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases

    PubMed Central

    Li, Yingmei; Pan, Wenying; Connolly, Ian D.; Reddy, Sunil; Nagpal, Seema

    2017-01-01

    Cerebral spinal fluid (CSF) from brain tumor patients contains tumor cellular and cell-free DNA (cfDNA), which provides a less-invasive and routinely accessible method to obtain tumor genomic information. In this report, we used droplet digital PCR to test mutant tumor DNA in CSF of a patient to monitor the treatment response of metastatic melanoma leptomeningeal disease (LMD). The primary melanoma was known to have a BRAFV600E mutation, and the patient was treated with whole brain radiotherapy and BRAF inhibitors. We collected 9 CSF samples over 6 months. The mutant cfDNA fraction gradually decreased from 53 % (time of diagnosis) to 0 (time of symptom alleviation) over the first 6 time points. Three months after clinical improvement, the patient returned with severe symptoms and the mutant cfDNA was again detected in CSF at high levels. The mutant DNA fraction corresponded well with the patient’s clinical response. We used whole exome sequencing to examine the mutation profiles of the LMD tumor DNA in CSF before therapeutic response and after disease relapse, and discovered a canonical cancer mutation PTENR130* at both time points. The cellular and cfDNA revealed similar mutation profiles, suggesting cfDNA is representative of LMD cells. This study demonstrates the potential of using cellular or cfDNA in CSF to monitor treatment response for LMD. PMID:26961773

  19. Corrupting the DNA damage response: a critical role for Rad52 in tumor cell survival.

    PubMed

    Lieberman, Rachel; You, Ming

    2017-07-15

    The DNA damage response enables cells to survive, maintain genome integrity, and to safeguard the transmission of high-fidelity genetic information. Upon sensing DNA damage, cells respond by activating this multi-faceted DNA damage response leading to restoration of the cell, senescence, programmed cell death, or genomic instability if the cell survives without proper repair. However, unlike normal cells, cancer cells maintain a marked level of genomic instability. Because of this enhanced propensity to accumulate DNA damage, tumor cells rely on homologous recombination repair as a means of protection from the lethal effect of both spontaneous and therapy-induced double-strand breaks (DSBs) in DNA. Thus, modulation of DNA repair pathways have important consequences for genomic instability within tumor cell biology and viability maintenance under high genotoxic stress. Efforts are underway to manipulate specific components of the DNA damage response in order to selectively induce tumor cell death by augmenting genomic instability past a viable threshold. New evidence suggests that RAD52, a component of the homologous recombination pathway, is important for the maintenance of tumor genome integrity. This review highlights recent reports indicating that reducing homologous recombination through inhibition of RAD52 may represent an important focus for cancer therapy and the specific efforts that are already demonstrating potential.

  20. Comparative clinical utility of tumor genomic testing and cell-free DNA in metastatic breast cancer.

    PubMed

    Maxwell, Kara N; Soucier-Ernst, Danielle; Tahirovic, Emin; Troxel, Andrea B; Clark, Candace; Feldman, Michael; Colameco, Christopher; Kakrecha, Bijal; Langer, Melissa; Lieberman, David; Morrissette, Jennifer J D; Paul, Matt R; Pan, Tien-Chi; Yee, Stephanie; Shih, Natalie; Carpenter, Erica; Chodosh, Lewis A; DeMichele, Angela

    2017-08-01

    Breast cancer metastases differ biologically from primary disease; therefore, metastatic biopsies may assist in treatment decision making. Commercial genomic testing of both tumor and circulating tumor DNA have become available clinically, but utility of these tests in breast cancer management remains unclear. Patients undergoing a clinically indicated metastatic tumor biopsy were consented to the ongoing METAMORPH registry. Tumor and blood were collected at the time of disease progression before subsequent therapy, and patients were followed for response on subsequent treatment. Tumor testing (n = 53) and concurrent cell-free DNA (n = 32) in a subset of patients was performed using CLIA-approved assays. The proportion of patients with a genomic alteration was lower in tumor than in blood (69 vs. 91%; p = 0.06). After restricting analysis to alterations covered on both platforms, 83% of tumor alterations were detected in blood, while 90% of blood alterations were detected in tumor. Mutational load specific for the panel genes was calculated for both tumor and blood. Time to progression on subsequent treatment was significantly shorter for patients whose tumors had high panel-specific mutational load (HR 0.31, 95% CI 0.12-0.78) or a TP53 mutation (HR 0.35, 95% CI 0.20-0.79), after adjusting for stage at presentation, hormone receptor status, prior treatment type, and number of lines of metastatic treatment. Treating oncologists must distinguish platform differences from true biological heterogeneity when comparing tumor and cfDNA genomic testing results. Tumor and concurrent cfDNA contribute unique genomic information in metastatic breast cancer patients, providing potentially useful biomarkers for aggressive metastatic disease.

  1. A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth

    NASA Astrophysics Data System (ADS)

    Holmgren, Lars; Ambrosino, Elena; Birot, Olivier; Tullus, Carl; Veitonmäki, Niina; Levchenko, Tetyana; Carlson, Lena-Maria; Musiani, Piero; Iezzi, Manuela; Curcio, Claudia; Forni, Guido; Cavallo, Federica; Kiessling, Rolf

    2006-06-01

    Endogenous angiogenesis inhibitors have shown promise in preclinical trials, but clinical use has been hindered by low half-life in circulation and high production costs. Here, we describe a strategy that targets the angiostatin receptor angiomotin (Amot) by DNA vaccination. The vaccination procedure generated antibodies that detected Amot on the endothelial cell surface. Purified Ig bound to the endothelial cell membrane and inhibited endothelial cell migration. In vivo, DNA vaccination blocked angiogenesis in the matrigel plug assay and prevented growth of transplanted tumors for up to 150 days. We further demonstrate that a combination of DNA vaccines encoding Amot and the extracellular and transmembrane domains of the human EGF receptor 2 (Her-2)/neu oncogene inhibited breast cancer progression and impaired tumor vascularization in Her-2/neu transgenic mice. No toxicity or impairment of normal blood vessels could be detected. This work shows that DNA vaccination targeting Amot may be used to mimic the effect of angiostatin. cancer vaccines | neoplasia | neovascularization | breast cancer | angiostatin

  2. Tumor Cell-Free DNA Copy Number Instability Predicts Therapeutic Response to Immunotherapy.

    PubMed

    Weiss, Glen J; Beck, Julia; Braun, Donald P; Bornemann-Kolatzki, Kristen; Barilla, Heather; Cubello, Rhiannon; Quan, Walter; Sangal, Ashish; Khemka, Vivek; Waypa, Jordan; Mitchell, William M; Urnovitz, Howard; Schütz, Ekkehard

    2017-09-01

    Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum-derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers. Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed. Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs. Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074-81. ©2017 AACR . ©2017 American Association for Cancer Research.

  3. DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

    PubMed

    Furst, Ariel L; Barton, Jacqueline K

    2015-07-23

    DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. The landscape of actionable genomic alterations in cell-free circulating tumor DNA from 21,807 advanced cancer patients.

    PubMed

    Zill, Oliver A; Banks, Kimberly C; Fairclough, Stephen R; Mortimer, Stefanie; Vowles, James V; Mokhtari, Reza; Gandara, David R; Mack, Philip C; Odegaard, Justin I; Nagy, Rebecca J; Baca, Arthur M; Eltoukhy, Helmy; Chudova, Darya I; Lanman, Richard B; Talasaz, AmirAli

    2018-05-18

    Cell-free DNA (cfDNA) sequencing provides a non-invasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants. Experimental Design: We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality. Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (TCGA and COSMIC; r=0.90-0.99), with the principle differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR=5x10 -7 for EGFR and ERBB2 ), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients). Together these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Copyright ©2018, American Association for Cancer Research.

  5. Digital PCR analysis of circulating tumor DNA: a biomarker for chondrosarcoma diagnosis, prognostication, and residual disease detection.

    PubMed

    Gutteridge, Alice; Rathbone, Victoria M; Gibbons, Rebecca; Bi, Mark; Archard, Nicholas; Davies, Kate E J; Brown, Jake; Plagnol, Vincent; Pillay, Nischalan; Amary, Fernanda; O'Donnell, Paul; Gupta, Manu; Tirabosco, Roberto; Flanagan, Adrienne M; Forshew, Tim

    2017-10-01

    Conventional chondrosarcoma is the most common primary bone tumor in adults. Prognosis corresponds with tumor grade but remains variable, especially for individuals with grade (G) II disease. There are currently no biomarkers available for monitoring or prognostication of chondrosarcoma. Circulating tumor DNA (ctDNA) has recently emerged as a promising biomarker for a broad range of tumor types. To date, little has been done to study the presence of ctDNA and its potential utility in the management of sarcomas, including chondrosarcoma. In this study, we have assessed ctDNA levels in a cohort of 71 patients, 32 with sarcoma, including 29 individuals with central chondrosarcoma (CS) and 39 with locally aggressive and benign bone and soft tissue tumors, using digital PCR. In patients with CS, ctDNA was detected in pretreatment samples in 14/29 patients, which showed clear correlation with tumor grade as demonstrated by the detection of ctDNA in all patients with GIII and dedifferentiated disease (n = 6) and in 8/17 patients with GII disease, but never associated with GI CS. Notably detection of ctDNA preoperatively in GII disease was associated with a poor outcome. A total of 14 patients with CS had ctDNA levels assessed at multiple time points and in most patients there was a clear reduction following surgical removal. This research lays the foundation for larger studies to assess the utility of ctDNA for chondrosarcoma diagnosis, prognostication, early detection of residual disease and monitoring disease progression. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  6. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    PubMed Central

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

  7. RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA.

    PubMed

    Giannopoulou, Lydia; Chebouti, Issam; Pavlakis, Kitty; Kasimir-Bauer, Sabine; Lianidou, Evi S

    2017-03-28

    The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.

  8. RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA

    PubMed Central

    Giannopoulou, Lydia; Chebouti, Issam; Pavlakis, Kitty; Kasimir-Bauer, Sabine; Lianidou, Evi S.

    2017-01-01

    The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients. PMID:28206954

  9. BACOM2.0 facilitates absolute normalization and quantification of somatic copy number alterations in heterogeneous tumor

    NASA Astrophysics Data System (ADS)

    Fu, Yi; Yu, Guoqiang; Levine, Douglas A.; Wang, Niya; Shih, Ie-Ming; Zhang, Zhen; Clarke, Robert; Wang, Yue

    2015-09-01

    Most published copy number datasets on solid tumors were obtained from specimens comprised of mixed cell populations, for which the varying tumor-stroma proportions are unknown or unreported. The inability to correct for signal mixing represents a major limitation on the use of these datasets for subsequent analyses, such as discerning deletion types or detecting driver aberrations. We describe the BACOM2.0 method with enhanced accuracy and functionality to normalize copy number signals, detect deletion types, estimate tumor purity, quantify true copy numbers, and calculate average-ploidy value. While BACOM has been validated and used with promising results, subsequent BACOM analysis of the TCGA ovarian cancer dataset found that the estimated average tumor purity was lower than expected. In this report, we first show that this lowered estimate of tumor purity is the combined result of imprecise signal normalization and parameter estimation. Then, we describe effective allele-specific absolute normalization and quantification methods that can enhance BACOM applications in many biological contexts while in the presence of various confounders. Finally, we discuss the advantages of BACOM in relation to alternative approaches. Here we detail this revised computational approach, BACOM2.0, and validate its performance in real and simulated datasets.

  10. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors.

    PubMed

    Amatruda, James F; Ross, Julie A; Christensen, Brock; Fustino, Nicholas J; Chen, Kenneth S; Hooten, Anthony J; Nelson, Heather; Kuriger, Jacquelyn K; Rakheja, Dinesh; Frazier, A Lindsay; Poynter, Jenny N

    2013-06-27

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy.

  11. Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA.

    PubMed

    Scherer, Florian; Kurtz, David M; Newman, Aaron M; Stehr, Henning; Craig, Alexander F M; Esfahani, Mohammad Shahrokh; Lovejoy, Alexander F; Chabon, Jacob J; Klass, Daniel M; Liu, Chih Long; Zhou, Li; Glover, Cynthia; Visser, Brendan C; Poultsides, George A; Advani, Ranjana H; Maeda, Lauren S; Gupta, Neel K; Levy, Ronald; Ohgami, Robert S; Kunder, Christian A; Diehn, Maximilian; Alizadeh, Ash A

    2016-11-09

    Patients with diffuse large B cell lymphoma (DLBCL) exhibit marked diversity in tumor behavior and outcomes, yet the identification of poor-risk groups remains challenging. In addition, the biology underlying these differences is incompletely understood. We hypothesized that characterization of mutational heterogeneity and genomic evolution using circulating tumor DNA (ctDNA) profiling could reveal molecular determinants of adverse outcomes. To address this hypothesis, we applied cancer personalized profiling by deep sequencing (CAPP-Seq) analysis to tumor biopsies and cell-free DNA samples from 92 lymphoma patients and 24 healthy subjects. At diagnosis, the amount of ctDNA was found to strongly correlate with clinical indices and was independently predictive of patient outcomes. We demonstrate that ctDNA genotyping can classify transcriptionally defined tumor subtypes, including DLBCL cell of origin, directly from plasma. By simultaneously tracking multiple somatic mutations in ctDNA, our approach outperformed immunoglobulin sequencing and radiographic imaging for the detection of minimal residual disease and facilitated noninvasive identification of emergent resistance mutations to targeted therapies. In addition, we identified distinct patterns of clonal evolution distinguishing indolent follicular lymphomas from those that transformed into DLBCL, allowing for potential noninvasive prediction of histological transformation. Collectively, our results demonstrate that ctDNA analysis reveals biological factors that underlie lymphoma clinical outcomes and could facilitate individualized therapy. Copyright © 2016, American Association for the Advancement of Science.

  12. New insights in non-small-cell lung cancer: circulating tumor cells and cell-free DNA

    PubMed Central

    Duréndez-Sáez, Elena; Azkárate, Aitor; Meri, Marina; Calabuig-Fariñas, Silvia; Aguilar-Gallardo, Cristóbal; Blasco, Ana

    2017-01-01

    Lung cancer is the second most frequent tumor and the leading cause of death by cancer in both men and women. Increasing knowledge about the cancer genome and tumor environment has led to a new setting in which morphological and molecular characterization is needed to treat patients in the most personalized way in order to achieve better outcomes. Since tumor products can be detected in body fluids, the liquid biopsy, particularly, peripheral blood, has emerged as a new source for lung cancer biomarker’s analysis. A variety of tumor components can be used for this purpose. Among them, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) should be especially considered. Different detection methods for both CTCs and ctDNA have been and are being developed to improve the sensitivity and specificity of these tests. This would lead to better characterization and would solve some clinical doubts at different disease evolution times, e.g., intratumoral or temporal heterogeneity, difficulty in the obtaining a tumor sample, etc., and would also avoid the side effects of very expensive and complicated tumor obtaining interventions. CTCs and ctDNA are useful in different lung cancer settings. Their value has been shown for the early diagnosis, prognosis, prediction of treatment efficacy, monitoring responses and early detection of lung cancer relapse. CTCs have still not been validated for use in clinical settings in non-small-cell lung cancer (NSCLC), while ctDNA has been approved by the Food and Drug Administration (FDA) and European Medical Association (EMA), and the main clinical guidelines used for detect different epidermal growth factor receptor (EGFR) mutations and the monitoring and treatment choice of mutated patients with tyrosine kinase inhibitors (TKIs). This review, describes how ctDNA seem to be winning the race against CTCs from the laboratory bench to clinical practice due to easier obtaining methods, manipulation and its implementation into

  13. Tumor transfection after systemic injection of DNA lipid nanocapsules.

    PubMed

    Morille, Marie; Passirani, Catherine; Dufort, Sandrine; Bastiat, Guillaume; Pitard, Bruno; Coll, Jean-Luc; Benoit, Jean-Pierre

    2011-03-01

    With the goal of generating an efficient vector for systemic gene delivery, a new kind of nanocarrier consisting of lipid nanocapsules encapsulating DOTAP/DOPE lipoplexes (DNA LNCs) was pegylated by the post-insertion of amphiphilic and flexible polymers. The aim of this surface modification was to create a long-circulating vector, able to circulate in the blood stream and efficient in transfecting tumoral cells after passive targeting by enhanced permeability and retention effect (EPR effect). PEG conformation, electrostatic features, and hydrophylicity are known to be important factors able to influence the pharmacokinetic behaviour of vectors. In this context, the surface structure characteristics of the newly pegylated DNA LNCs were studied by measuring electrophoretic mobility as a function of ionic strength in order to establish a correlation between surface properties and in vivo performance of the vector. Finally, thanks to this PEGylation, gene expression was measured up to 84-fold higher in tumor compared to other tested organs after intravenous injection. The present results indicate that PEGylated DNA LNCs are promising carriers for an efficient cancer gene therapy. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Novel methylation panel for the early detection of colorectal tumors in stool DNA.

    PubMed

    Azuara, Daniel; Rodriguez-Moranta, Francisco; de Oca, Javier; Soriano-Izquierdo, Antonio; Mora, Josefina; Guardiola, Jordi; Biondo, Sebastiano; Blanco, Ignacio; Peinado, Miguel Angel; Moreno, Victor; Esteller, Manel; Capellá, Gabriel

    2010-07-01

    Previous studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter. The promoter methylation status of 4 tumor-related genes (RARB2, p16INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses. In the initial set, >or= 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD. Analysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.

  15. Circulating tumor DNA for triple-negative breast cancer diagnosis and treatment decisions.

    PubMed

    Saliou, Adrien; Bidard, François-Clément; Lantz, Olivier; Stern, Marc-Henri; Vincent-Salomon, Anne; Proudhon, Charlotte; Pierga, Jean-Yves

    2016-01-01

    Triple-negative breast cancer (TNBC) is a highly aggressive disease characterized by a high number of relapses and poor overall survival. The heterogeneity of the disease and the limited treatment options compared to other breast cancer subtypes mainly explain these clinical outcomes. New biomarkers are urgently needed to improve the management of TNBC. Circulating tumor DNA, identified by tumor-related molecular alterations, could be used in the context of non-invasive "liquid biopsy" and help in TNBC diagnosis and treatment decisions. In this review, we discuss the key issues related to the potential of circulating tumor DNA to improve the management of this disease and the future steps to overcome before its implementation into clinical routine within the next 5 years.

  16. Plasma cell-free DNA quantification is highly correlated to tumor burden in children with neuroblastoma.

    PubMed

    Wang, Xisi; Wang, Lijun; Su, Yan; Yue, Zhixia; Xing, Tianyu; Zhao, Wen; Zhao, Qian; Duan, Chao; Huang, Cheng; Zhang, Dawei; Jin, Mei; Cheng, Xianfeng; Chen, Shenglan; Liu, Yi; Ma, Xiaoli

    2018-06-14

    To evaluate plasma cell-free DNA (cfDNA) as a promising biomarker for neuroblastoma (NB) tumor burden. Seventy-nine eligible patients with newly diagnosed NB were recruited from Beijing Children's Hospital between April 2016 and April 2017. Additionally, from September 2011 to June 2017, 79 patients with stable NB were evaluated with a median follow-up time of 21 months. Approximately 2 mL of peripheral blood was drawn upon enrollment, and plasma cfDNA levels were measured via quantitative polymerase chain reaction (qPCR). Total cfDNA analysis was performed using the long interspersed nuclear element 1 (LINE-1) 79 bp fragment, and DNA integrity was calculated by the ratio of the LINE-1 300 bp fragment to the LINE-1 79 bp fragment. A total of 79 NB patients with a median age of 36 months comprised the group of newly diagnosed NB patients. The main primary tumor site was the retroperitoneal and adrenal region (81%). Three or more metastatic sites were found in 17.7% of patients. Stable NB patients older than 18 months comprised 98.7% of the stable NB patients. Neuron-specific enolase (NSE), lactate dehydrogenase (LDH), and cfDNA levels were dramatically increased in the newly diagnosed NB patients and significantly different from those in the stable NB patients. Moreover, the concentration of cfDNA was much higher in patients with larger tumors. By analyzing the area under the receiver operator characteristic (ROC) curve (AUC), the areas of total cfDNA, NSE, and LDH levels were 0.953, 0.929, and 0.906, respectively. The sensitivity and specificity data clarified that the level of circulating cfDNA in plasma can be considered as a reliable biomarker for describing tumor load in NB. The plasma cfDNA concentration was as good as the levels of LDH and NSE to discriminate the tumor burden in children with NB. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  17. Early circulating tumor DNA dynamics and clonal selection with palbociclib and fulvestrant for breast cancer.

    PubMed

    O'Leary, Ben; Hrebien, Sarah; Morden, James P; Beaney, Matthew; Fribbens, Charlotte; Huang, Xin; Liu, Yuan; Bartlett, Cynthia Huang; Koehler, Maria; Cristofanilli, Massimo; Garcia-Murillas, Isaac; Bliss, Judith M; Turner, Nicholas C

    2018-03-01

    CDK4/6 inhibition substantially improves progression-free survival (PFS) for women with advanced estrogen receptor-positive breast cancer, although there are no predictive biomarkers. Early changes in circulating tumor DNA (ctDNA) level may provide early response prediction, but the impact of tumor heterogeneity is unknown. Here we use plasma samples from patients in the randomized phase III PALOMA-3 study of CDK4/6 inhibitor palbociclib and fulvestrant for women with advanced breast cancer and show that relative change in PIK3CA ctDNA level after 15 days treatment strongly predicts PFS on palbociclib and fulvestrant (hazard ratio 3.94, log-rank p = 0.0013). ESR1 mutations selected by prior hormone therapy are shown to be frequently sub clonal, with ESR1 ctDNA dynamics offering limited prediction of clinical outcome. These results suggest that early ctDNA dynamics may provide a robust biomarker for CDK4/6 inhibitors, with early ctDNA dynamics demonstrating divergent response of tumor sub clones to treatment.

  18. Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system.

    PubMed

    Buoninfante, Olimpia Alessandra; Pilzecker, Bas; Aslam, Muhammad Assad; Zavrakidis, Ioannis; van der Wiel, Rianne; van de Ven, Marieke; van den Berk, Paul C M; Jacobs, Heinz

    2018-04-10

    DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18). A survey of The Cancer Genome Atlas (TCGA) revealed a high frequency of tumors presents RAD6/RAD18 bi-allelic inactivating deletions. For instance, 11% of renal cell carcinoma and 5% of pancreatic tumors have inactivating RAD18 -deletions and 7% of malignant peripheral nerve sheath tumors lack RAD6B . To determine the potential benefit for tumor-specific DDT defects, we followed a genetic approach by establishing unique sets of DDT-proficient Pcna K164 and -defective Pcna K164R lymphoma and breast cancer cell lines. In the absence of exogenous DNA damage, Pcna K164R tumors grew comparably to their Pcna K164 controls in vitro and in vivo . However, DDT-defective lymphomas and breast cancers were compared to their DDT-proficient controls hypersensitive to the chemotherapeutic drug cisplatin (CsPt), both in vitro and in vivo. CsPt strongly inhibited tumor growth and the overall survival of tumor bearing mice greatly improved in the DDT-defective condition. These insights open new therapeutic possibilities for precision cancer medicine with DNA damaging chemotherapeutics and optimize Next-Generation-Sequencing (NGS)-based cancer-diagnostics, -therapeutics, and -prognosis.

  19. Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system

    PubMed Central

    Buoninfante, Olimpia Alessandra; Pilzecker, Bas; Aslam, Muhammad Assad; Zavrakidis, Ioannis; van der Wiel, Rianne; van de Ven, Marieke; van den Berk, Paul C.M.; Jacobs, Heinz

    2018-01-01

    DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18). A survey of The Cancer Genome Atlas (TCGA) revealed a high frequency of tumors presents RAD6/RAD18 bi-allelic inactivating deletions. For instance, 11% of renal cell carcinoma and 5% of pancreatic tumors have inactivating RAD18-deletions and 7% of malignant peripheral nerve sheath tumors lack RAD6B. To determine the potential benefit for tumor-specific DDT defects, we followed a genetic approach by establishing unique sets of DDT-proficient PcnaK164 and -defective PcnaK164R lymphoma and breast cancer cell lines. In the absence of exogenous DNA damage, PcnaK164R tumors grew comparably to their PcnaK164 controls in vitro and in vivo. However, DDT-defective lymphomas and breast cancers were compared to their DDT-proficient controls hypersensitive to the chemotherapeutic drug cisplatin (CsPt), both in vitro and in vivo. CsPt strongly inhibited tumor growth and the overall survival of tumor bearing mice greatly improved in the DDT-defective condition. These insights open new therapeutic possibilities for precision cancer medicine with DNA damaging chemotherapeutics and optimize Next-Generation-Sequencing (NGS)-based cancer-diagnostics, -therapeutics, and -prognosis. PMID:29721165

  20. The Emergent Landscape of Detecting EGFR Mutations Using Circulating Tumor DNA in Lung Cancer

    PubMed Central

    Wei, Fang; Wong, David T.; Su, Wu-Chou

    2015-01-01

    The advances in targeted therapies for lung cancer are based on the evaluation of specific gene mutations especially the epidermal growth factor receptor (EGFR). The assays largely depend on the acquisition of tumor tissue via biopsy before the initiation of therapy or after the onset of acquired resistance. However, the limitations of tissue biopsy including tumor heterogeneity and insufficient tissues for molecular testing are impotent clinical obstacles for mutation analysis and lung cancer treatment. Due to the invasive procedure of tissue biopsy and the progressive development of drug-resistant EGFR mutations, the effective initial detection and continuous monitoring of EGFR mutations are still unmet requirements. Circulating tumor DNA (ctDNA) detection is a promising biomarker for noninvasive assessment of cancer burden. Recent advancement of sensitive techniques in detecting EGFR mutations using ctDNA enables a broad range of clinical applications, including early detection of disease, prediction of treatment responses, and disease progression. This review not only introduces the biology and clinical implementations of ctDNA but also includes the updating information of recent advancement of techniques for detecting EGFR mutation using ctDNA in lung cancer. PMID:26448936

  1. Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors.

    PubMed

    Gasparre, Giuseppe; Porcelli, Anna Maria; Bonora, Elena; Pennisi, Lucia Fiammetta; Toller, Matteo; Iommarini, Luisa; Ghelli, Anna; Moretti, Massimo; Betts, Christine M; Martinelli, Giuseppe Nicola; Ceroni, Alberto Rinaldi; Curcio, Francesco; Carelli, Valerio; Rugolo, Michela; Tallini, Giovanni; Romeo, Giovanni

    2007-05-22

    Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

  2. Electric field-mediated transport of plasmid DNA in tumor interstitium in vivo.

    PubMed

    Henshaw, Joshua W; Zaharoff, David A; Mossop, Brian J; Yuan, Fan

    2007-11-01

    Local pulsed electric field application is a method for improving non-viral gene delivery. Mechanisms of the improvement include electroporation and electrophoresis. To understand how electrophoresis affects pDNA delivery in vivo, we quantified the magnitude of electric field-induced interstitial transport of pDNA in 4T1 and B16.F10 tumors implanted in mouse dorsal skin-fold chambers. Four different electric pulse sequences were used in this study, each consisted of 10 identical pulses that were 100 or 400 V/cm in strength and 20 or 50 ms in duration. The interval between consecutive pulses was 1 s. The largest distance of transport was obtained with the 400 V/cm and 50 ms pulse, and was 0.23 and 0.22 microm/pulse in 4T1 and B16.F10 tumors, respectively. There were no significant differences in transport distances between 4T1 and B16.F10 tumors. Results from in vivo mapping and numerical simulations revealed an approximately uniform intratumoral electric field that was predominantly in the direction of the applied field. The data in the study suggested that interstitial transport of pDNA induced by a sequence of ten electric pulses was ineffective for macroscopic delivery of genes in tumors. However, the induced transport was more efficient than passive diffusion.

  3. Novel Methylated Biomarkers and a Robust Assay to Detect Circulating Tumor DNA in Metastatic Breast Cancer

    PubMed Central

    Fackler, Mary Jo; Bujanda, Zoila Lopez; Umbricht, Christopher; Teo, Wei Wen; Cho, Soonweng; Zhang, Zhe; Visvanathan, Kala; Jeter, Stacie; Argani, Pedram; Wang, Chenguang; Lyman, Jaclyn P.; de Brot, Marina; Ingle, James N.; Boughey, Judy; McGuire, Kandace; King, Tari A.; Carey, Lisa A.; Cope, Leslie; Wolff, Antonio C.; Sukumar, Saraswati

    2015-01-01

    The ability to consistently detect cell-free tumor-specific DNA in peripheral blood of patients with metastatic breast cancer provides the opportunity to detect changes in tumor burden and to monitor response to treatment. We developed cMethDNA, a quantitative multiplexed methylation-specific PCR assay for a panel of ten genes, consisting of novel and known breast cancer hypermethylated markers identified by mining our previously reported study of DNA methylation patterns in breast tissue (103 cancer, 21 normal on the Illumina HumanMethylation27 Beadchip) and then validating the 10-gene panel in a TCGA breast cancer methylome database. For cMethDNA, a fixed physiological level (50 copies) of artificially constructed, standard non-human reference DNA specific for each gene is introduced into in a constant volume of serum (300 μl) prior to purification of the DNA, facilitating a sensitive, specific, robust and quantitative assay of tumor DNA, with broad dynamic range. Cancer-specific methylated DNA was detected in Training (28 normal, 24 cancer) and Test (27 normal, 33 cancer) sets of recurrent Stage 4 patient sera with a sensitivity of 91% and a specificity of 96% in the test set. In a pilot study, cMethDNA assay faithfully reflected patient response to chemotherapy (N = 29). A core methylation signature present in the primary breast cancer was retained in serum and metastatic tissues collected at autopsy 2–11 years after diagnosis of the disease. Together, our data suggest that the cMethDNA assay can detect advanced breast cancer, and monitor tumor burden and treatment response in women with metastatic breast cancer. PMID:24737128

  4. A pre-protective strategy for precise tumor targeting and efficient photodynamic therapy with a switchable DNA/upconversion nanocomposite.

    PubMed

    Yu, Zhengze; Ge, Yegang; Sun, Qiaoqiao; Pan, Wei; Wan, Xiuyan; Li, Na; Tang, Bo

    2018-04-14

    Tumor-specific targeting based on folic acid (FA) is one of the most common and significant approaches in cancer therapy. However, the expression of folate receptors (FRs) in normal tissues will lead to unexpected targeting and unsatisfactory therapeutic effect. To address this issue, we develop a pre-protective strategy for precise tumor targeting and efficient photodynamic therapy (PDT) using a switchable DNA/upconversion nanocomposite, which can be triggered in the acidic tumor microenvironment. The DNA/upconversion nanocomposite is composed of polyacrylic acid (PAA) coated upconversion nanoparticles (UCNPs), the surface of which is modified using FA and chlorin e6 (Ce6) functionalized DNA sequences with different lengths. Initially, FA on the shorter DNA was protected by a longer DNA to prevent the bonding to FRs on normal cells. Once reaching the acidic tumor microenvironment, C base-rich longer DNA forms a C-quadruplex, resulting in the exposure of the FA groups and the bonding of FA and FRs on cancer cell membranes to achieve precise targeting. Simultaneously, the photosensitizer chlorin e6 (Ce6) gets close to the surface of UCNPs, enabling the excitation of Ce6 to generate singlet oxygen ( 1 O 2 ) under near infrared light via Förster resonance energy transfer (FRET). In vivo experiments indicated that higher tumor targeting efficiency was achieved and the tumor growth was greatly inhibited through the pre-protective strategy.

  5. Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes.

    PubMed

    Kim, Kyoung-Ran; Hwang, Dohyeon; Kim, Juhyeon; Lee, Chang-Yong; Lee, Wonseok; Yoon, Dae Sung; Shin, Dongyun; Min, Sun-Joon; Kwon, Ick Chan; Chung, Hak Suk; Ahn, Dae-Ro

    2018-06-28

    Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

    PubMed

    Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

    2018-01-01

    The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

  7. Different expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in mouse tumor cells.

    PubMed

    Okabe, Kyoko; Hayashi, Mai; Wakabayashi, Naoko; Yamawaki, Yasuna; Teranishi, Miki; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2010-01-01

    Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention. Copyright © 2011 S. Karger AG, Basel.

  8. Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer

    PubMed Central

    Hrebien, Sarah; O’Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Turner, Nicholas

    2016-01-01

    Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48–72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77–0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research. PMID:27760227

  9. Reproducibility of Digital PCR Assays for Circulating Tumor DNA Analysis in Advanced Breast Cancer.

    PubMed

    Hrebien, Sarah; O'Leary, Ben; Beaney, Matthew; Schiavon, Gaia; Fribbens, Charlotte; Bhambra, Amarjit; Johnson, Richard; Garcia-Murillas, Isaac; Turner, Nicholas

    2016-01-01

    Circulating tumor DNA (ctDNA) analysis has the potential to allow non-invasive analysis of tumor mutations in advanced cancer. In this study we assessed the reproducibility of digital PCR (dPCR) assays of circulating tumor DNA in a cohort of patients with advanced breast cancer and assessed delayed plasma processing using cell free DNA preservative tubes. We recruited a cohort of 96 paired samples from 71 women with advanced breast cancer who had paired blood samples processed either immediately or delayed in preservative tubes with processing 48-72 hours after collection. Plasma DNA was analysed with multiplex digital PCR (mdPCR) assays for hotspot mutations in PIK3CA, ESR1 and ERBB2, and for AKT1 E17K. There was 94.8% (91/96) agreement in mutation calling between immediate and delayed processed tubes, kappa 0.88 95% CI 0.77-0.98). Discordance in mutation calling resulted from low allele frequency and likely stochastic effects. In concordant samples there was high correlation in mutant copies per ml plasma (r2 = 0.98; p<0.0001). There was elevation of total cell free plasma DNA concentrations in 10.3% of delayed processed tubes, although overall quantification of total cell free plasma DNA had similar prognostic effects in immediate (HR 3.6) and delayed (HR 3.0) tubes. There was moderate agreement in changes in allele fraction between sequential samples in quantitative mutation tracking (r = 0.84, p = 0.0002). Delayed processing of samples using preservative tubes allows for centralized ctDNA digital PCR mutation screening in advanced breast cancer. The potential of preservative tubes in quantitative mutation tracking requires further research.

  10. Combination of PDT and a DNA demethylating agent produces anti-tumor immune response in a mouse tumor model

    NASA Astrophysics Data System (ADS)

    Mroz, Pawel; Hamblin, Michael R.

    2009-06-01

    Epigenetic mechanisms, which involve DNA methylation and histone modifications, result in the heritable silencing of genes without a change in their coding sequence. However, these changes must be actively maintained after each cell division rendering them a promising target for pharmacologic inhibition. DNA methyltransferase inhibitors like 5-aza-deoxycytidine (5-aza-dC) induce and/or up-regulate the expression of MAGE-type antigens in human and mice cancer cells. Photodynamic therapy (PDT) has been shown to be an effective locally ablative anti-cancer treatment that has the additional advantage of stimulating tumor-directed immune response. We studied the effects of a new therapy that combined the demethylating agent 5-aza-dC with PDT in the breast cancer model 4T1 syngenic to immunocompetent BALB/c mice. PDT was used as a locally ablating tumor treatment that is capable of eliciting strong and tumor directed immune response while 5-aza-dC pretreatment was used promote de novo induction of the expression of P1A.protein. This is the mouse homolog of human MAGE family antigens and is reported to function as a tumor rejection antigen in certain mouse tumors. This strategy led to an increase in PDT-mediated immune response and better treatment outcome. These results strongly suggest that the MAGE family antigens are important target for PDT mediated immune response but that their expression can be silenced by epigenetic mechanisms. Therefore the possibility that PDT can be combined with epigenetic strategies to elicit anti-tumor immunity in MAGE-positive tumor models is highly clinically significant and should be studied in detail.

  11. Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970

    PubMed Central

    Boucher, Diane M.; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A.; Milton, Sean; Murphy, Cheryl E.; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M.; Pollard, John R.

    2014-01-01

    Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients. PMID:25010037

  12. Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

    PubMed

    Hall, Amy B; Newsome, Dave; Wang, Yuxin; Boucher, Diane M; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A; Milton, Sean; Murphy, Cheryl E; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M; Pollard, John R

    2014-07-30

    Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

  13. Association of Cell-Free DNA Tumor Fraction and Somatic Copy Number Alterations With Survival in Metastatic Triple-Negative Breast Cancer

    PubMed Central

    Stover, Daniel G.; Parsons, Heather A.; Ha, Gavin; Freeman, Samuel S.; Barry, William T.; Guo, Hao; Choudhury, Atish D.; Gydush, Gregory; Reed, Sarah C.; Rhoades, Justin; Rotem, Denisse; Hughes, Melissa E.; Dillon, Deborah A.; Partridge, Ann H.; Wagle, Nikhil; Krop, Ian E.; Getz, Gad; Golub, Todd R.; Love, J. Christopher; Winer, Eric P.; Tolaney, Sara M.; Lin, Nancy U.

    2018-01-01

    Purpose Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with patient prognosis. Triple-negative breast cancer (TNBC) is characterized by few mutations but extensive somatic copy number alterations (SCNAs), yet little is known regarding SCNAs in metastatic TNBC. We sought to evaluate SCNAs in metastatic TNBC exclusively via cfDNA and determine if cfDNA tumor fraction is associated with overall survival in metastatic TNBC. Patients and Methods In this retrospective cohort study, we identified 164 patients with biopsy-proven metastatic TNBC at a single tertiary care institution who received prior chemotherapy in the (neo)adjuvant or metastatic setting. We performed low-coverage genome-wide sequencing of cfDNA from plasma. Results Without prior knowledge of tumor mutations, we determined tumor fraction of cfDNA for 96.3% of patients and SCNAs for 63.9% of patients. Copy number profiles and percent genome altered were remarkably similar between metastatic and primary TNBCs. Certain SCNAs were more frequent in metastatic TNBCs relative to paired primary tumors and primary TNBCs in publicly available data sets The Cancer Genome Atlas and METABRIC, including chromosomal gains in drivers NOTCH2, AKT2, and AKT3. Prespecified cfDNA tumor fraction threshold of ≥ 10% was associated with significantly worse metastatic survival (median, 6.4 v 15.9 months) and remained significant independent of clinicopathologic factors (hazard ratio, 2.14; 95% CI, 1.4 to 3.8; P < .001). Conclusion We present the largest genomic characterization of metastatic TNBC to our knowledge, exclusively from cfDNA. Evaluation of cfDNA tumor fraction was feasible for nearly all patients, and tumor fraction ≥ 10% is associated with significantly worse survival in this large metastatic TNBC cohort. Specific SCNAs are enriched and prognostic in metastatic TNBC, with implications for metastasis

  14. Association of Cell-Free DNA Tumor Fraction and Somatic Copy Number Alterations With Survival in Metastatic Triple-Negative Breast Cancer.

    PubMed

    Stover, Daniel G; Parsons, Heather A; Ha, Gavin; Freeman, Samuel S; Barry, William T; Guo, Hao; Choudhury, Atish D; Gydush, Gregory; Reed, Sarah C; Rhoades, Justin; Rotem, Denisse; Hughes, Melissa E; Dillon, Deborah A; Partridge, Ann H; Wagle, Nikhil; Krop, Ian E; Getz, Gad; Golub, Todd R; Love, J Christopher; Winer, Eric P; Tolaney, Sara M; Lin, Nancy U; Adalsteinsson, Viktor A

    2018-02-20

    Purpose Cell-free DNA (cfDNA) offers the potential for minimally invasive genome-wide profiling of tumor alterations without tumor biopsy and may be associated with patient prognosis. Triple-negative breast cancer (TNBC) is characterized by few mutations but extensive somatic copy number alterations (SCNAs), yet little is known regarding SCNAs in metastatic TNBC. We sought to evaluate SCNAs in metastatic TNBC exclusively via cfDNA and determine if cfDNA tumor fraction is associated with overall survival in metastatic TNBC. Patients and Methods In this retrospective cohort study, we identified 164 patients with biopsy-proven metastatic TNBC at a single tertiary care institution who received prior chemotherapy in the (neo)adjuvant or metastatic setting. We performed low-coverage genome-wide sequencing of cfDNA from plasma. Results Without prior knowledge of tumor mutations, we determined tumor fraction of cfDNA for 96.3% of patients and SCNAs for 63.9% of patients. Copy number profiles and percent genome altered were remarkably similar between metastatic and primary TNBCs. Certain SCNAs were more frequent in metastatic TNBCs relative to paired primary tumors and primary TNBCs in publicly available data sets The Cancer Genome Atlas and METABRIC, including chromosomal gains in drivers NOTCH2, AKT2, and AKT3. Prespecified cfDNA tumor fraction threshold of ≥ 10% was associated with significantly worse metastatic survival (median, 6.4 v 15.9 months) and remained significant independent of clinicopathologic factors (hazard ratio, 2.14; 95% CI, 1.4 to 3.8; P < .001). Conclusion We present the largest genomic characterization of metastatic TNBC to our knowledge, exclusively from cfDNA. Evaluation of cfDNA tumor fraction was feasible for nearly all patients, and tumor fraction ≥ 10% is associated with significantly worse survival in this large metastatic TNBC cohort. Specific SCNAs are enriched and prognostic in metastatic TNBC, with implications for metastasis

  15. Detection of Histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma.

    PubMed

    Huang, Tina Y; Piunti, Andrea; Lulla, Rishi R; Qi, Jin; Horbinski, Craig M; Tomita, Tadanori; James, C David; Shilatifard, Ali; Saratsis, Amanda M

    2017-04-17

    Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of "liquid biopsy" in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response.

  16. Personalized Circulating Tumor DNA Biomarkers Dynamically Predict Treatment Response and Survival In Gynecologic Cancers.

    PubMed

    Pereira, Elena; Camacho-Vanegas, Olga; Anand, Sanya; Sebra, Robert; Catalina Camacho, Sandra; Garnar-Wortzel, Leopold; Nair, Navya; Moshier, Erin; Wooten, Melissa; Uzilov, Andrew; Chen, Rong; Prasad-Hayes, Monica; Zakashansky, Konstantin; Beddoe, Ann Marie; Schadt, Eric; Dottino, Peter; Martignetti, John A

    2015-01-01

    High-grade serous ovarian and endometrial cancers are the most lethal female reproductive tract malignancies worldwide. In part, failure to treat these two aggressive cancers successfully centers on the fact that while the majority of patients are diagnosed based on current surveillance strategies as having a complete clinical response to their primary therapy, nearly half will develop disease recurrence within 18 months and the majority will die from disease recurrence within 5 years. Moreover, no currently used biomarkers or imaging studies can predict outcome following initial treatment. Circulating tumor DNA (ctDNA) represents a theoretically powerful biomarker for detecting otherwise occult disease. We therefore explored the use of personalized ctDNA markers as both a surveillance and prognostic biomarker in gynecologic cancers and compared this to current FDA-approved surveillance tools. Tumor and serum samples were collected at time of surgery and then throughout treatment course for 44 patients with gynecologic cancers, representing 22 ovarian cancer cases, 17 uterine cancer cases, one peritoneal, three fallopian tube, and one patient with synchronous fallopian tube and uterine cancer. Patient/tumor-specific mutations were identified using whole-exome and targeted gene sequencing and ctDNA levels quantified using droplet digital PCR. CtDNA was detected in 93.8% of patients for whom probes were designed and levels were highly correlated with CA-125 serum and computed tomography (CT) scanning results. In six patients, ctDNA detected the presence of cancer even when CT scanning was negative and, on average, had a predictive lead time of seven months over CT imaging. Most notably, undetectable levels of ctDNA at six months following initial treatment was associated with markedly improved progression free and overall survival. Detection of residual disease in gynecologic, and indeed all cancers, represents a diagnostic dilemma and a potential critical inflection

  17. [Exploration of Epigenetic Changes and DNA Methylation Markers Associated with Liver Tumors Induced by Inorganic Arsenite Exposure in Mice].

    PubMed

    Suzuki, Takehiro; Nohara, Keiko

    2015-01-01

    Naturally occurring inorganic arsenic is known to increase the risk of cancers of the skin and several other organs, including the urinary bladder, lung, and liver. Epidemiological studies have also indicated that gestational arsenic exposure is associated with increased incidences of cancers in several organs, including the bladder and liver, in adulthood. Previous studies have shown that epigenetic changes are involved in arsenic-induced carcinogenesis. Among epigenetic changes, DNA methylation changes that are specific to arsenic-induced tumors would be useful for distinguishing such tumors from tumors induced by other factors and for clarifying arsenic carcinogenesis. It has been reported that gestational arsenic exposure of C3H mice, whose males tend to spontaneously develop liver tumors, increases the incidence of tumors in the male offspring. Using the same experimental protocol, we found a number of regions where the DNA methylation status was altered in the liver tumors compared with the normal liver tissues by the methylated DNA immunoprecipitation (MeDIP)-CpG island microarray method. Among such regions, we demonstrated using real-time methylation-specific PCR and bisulfite sequencing that a gene body region of the oncogene Fosb underwent alteration in DNA methylation following gestational arsenic exposure. We also showed that the Fosb expression level significantly increased following gestational arsenic exposure. These findings suggest that the DNA methylation status of the Fosb region is implicated in tumor augmentation and can also be utilized for characterizing tumors induced by gestational arsenic exposure.

  18. Pretreatment dietary intake is associated with tumor suppressor DNA methylation in head and neck squamous cell carcinomas

    PubMed Central

    Colacino, Justin A.; Arthur, Anna E.; Dolinoy, Dana C.; Sartor, Maureen A.; Duffy, Sonia A.; Chepeha, Douglas B.; Bradford, Carol R.; Walline, Heather M.; McHugh, Jonathan B.; D'Silva, Nisha; Carey, Thomas E.; Wolf, Gregory T.; Taylor, Jeremy M.G.; Peterson, Karen E.; Rozek, Laura S.

    2012-01-01

    Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression. PMID:22722388

  19. Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy.

    PubMed

    Chen, Yu-Hsiang; Hancock, Bradley A; Solzak, Jeffrey P; Brinza, Dumitru; Scafe, Charles; Miller, Kathy D; Radovich, Milan

    2017-01-01

    Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival ( p  < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.

  20. DNA methylation mediated control of gene expression is critical for development of crown gall tumors.

    PubMed

    Gohlke, Jochen; Scholz, Claus-Juergen; Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

    2013-01-01

    Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA-encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA-mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene

  1. Integrated digital error suppression for improved detection of circulating tumor DNA

    PubMed Central

    Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.

    2016-01-01

    High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

  2. SOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer.

    PubMed

    Balgkouranidou, Ioanna; Chimonidou, Maria; Milaki, Georgia; Tsaroucha, Emily; Kakolyris, Stylianos; Georgoulias, Vasilis; Lianidou, Evi

    2016-08-01

    SOX17 belongs to the high-mobility group-box transcription factor superfamily and down-regulates the Wnt pathway. The aim of our study was to evaluate the prognostic significance of SOX17 promoter methylation in circulating tumor DNA (ctDNA) in plasma of non-small cell lung cancer (NSCLC) patients. We examined the methylation status of SOX17 promoter in 57 operable NSCLC primary tumors and paired adjacent non-cancerous tissues and in ctDNA isolated from 48 corresponding plasma samples as well as in plasma from 74 patients with advanced NSCLC and 49 healthy individuals. SOX17 promoter methylation was examined by Methylation Specific PCR (MSP). In operable NSCLC, SOX17 promoter was fully methylated in primary tumors (57/57, 100%), and in corresponding ctDNA (27/48, 56.2%) while it was detected in only 1/49 (2.0%) healthy individuals. In advanced NSCLC, SOX17 promoter was methylated in ctDNA in 27/74 (36.4%) patients and OS was significantly different in favor of patients with non-methylated SOX17 promoter (p=0.012). Multivariate analysis revealed that SOX17 promoter methylation in ctDNA was an independent prognostic factor associated with OS in patients with advanced but not operable NSCLC. Our results show that SOX17 promoter is highly methylated in primary tumors and in corresponding plasma samples both in operable and advanced NSCLC. In the advanced setting, SOX17 promoter methylation in plasma ctDNA has a statistical significant influence on NSCLC patient's survival time. Detection of SOX17 promoter methylation in plasma provides prognostic information and merits to be further evaluated as a circulating tumor biomarker in patients with operable and advanced NSCLC.

  3. Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data.

    PubMed

    Favero, F; Joshi, T; Marquard, A M; Birkbak, N J; Krzystanek, M; Li, Q; Szallasi, Z; Eklund, A C

    2015-01-01

    Exome or whole-genome deep sequencing of tumor DNA along with paired normal DNA can potentially provide a detailed picture of the somatic mutations that characterize the tumor. However, analysis of such sequence data can be complicated by the presence of normal cells in the tumor specimen, by intratumor heterogeneity, and by the sheer size of the raw data. In particular, determination of copy number variations from exome sequencing data alone has proven difficult; thus, single nucleotide polymorphism (SNP) arrays have often been used for this task. Recently, algorithms to estimate absolute, but not allele-specific, copy number profiles from tumor sequencing data have been described. We developed Sequenza, a software package that uses paired tumor-normal DNA sequencing data to estimate tumor cellularity and ploidy, and to calculate allele-specific copy number profiles and mutation profiles. We applied Sequenza, as well as two previously published algorithms, to exome sequence data from 30 tumors from The Cancer Genome Atlas. We assessed the performance of these algorithms by comparing their results with those generated using matched SNP arrays and processed by the allele-specific copy number analysis of tumors (ASCAT) algorithm. Comparison between Sequenza/exome and SNP/ASCAT revealed strong correlation in cellularity (Pearson's r = 0.90) and ploidy estimates (r = 0.42, or r = 0.94 after manual inspecting alternative solutions). This performance was noticeably superior to previously published algorithms. In addition, in artificial data simulating normal-tumor admixtures, Sequenza detected the correct ploidy in samples with tumor content as low as 30%. The agreement between Sequenza and SNP array-based copy number profiles suggests that exome sequencing alone is sufficient not only for identifying small scale mutations but also for estimating cellularity and inferring DNA copy number aberrations. © The Author 2014. Published by Oxford University Press on behalf of

  4. Role of specific DNA mutations in the peripheral blood of colorectal cancer patients for the assessment of tumor stage and residual disease following tumor resection

    PubMed Central

    Norcic, Gregor; Jelenc, Franc; Cerkovnik, Petra; Stegel, Vida; Novakovic, Srdjan

    2016-01-01

    In the present study, the detection of tumor-specific KRAS proto-oncogene, GTPase (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations in the peripheral blood of colorectal cancer (CRC) patients at all stages and adenomas was used for the estimation of disease stage prior to surgery and for residual disease following surgery. A total of 65 CRC patients were enrolled. The primary tumor tested positive for the specific mutations (KRAS mutations in codons 12, 13, 61, 117 or 146 and BRAF mutations in codon 600) in 35 patients. In all these patients, the specimen of normal bowel resected with the tumor was also tested for the presence of the same mutations in order to exclude the germ-line mutations. Only patients who tested positive for the specific mutation in the primary tumor were included in further analysis for the presence of tumor-specific mutation in the peripheral blood. No statistically significant differences were found between the detection rates of tumor mutations in the blood and different tumor stages (P=0.491). However, statistically significant differences in the proportions of patients with detected tumor-specific DNA mutations in the peripheral blood were found when comparing the groups of patients with R0 and R2 resections (P=0.038). Tumor-specific DNA mutations in the peripheral blood were more frequently detected in the patients with an incomplete surgical clearance of the tumor due to macroscopic residual disease (R2 resections). Therefore, the study concludes that the follow-up of somatic KRAS- and BRAF-mutated DNA in the peripheral blood of CRC patients may be useful in assessing the surgical clearance of the disease. PMID:27900004

  5. Recent advances in circulating tumor cells and cell-free DNA in metastatic prostate cancer: a review.

    PubMed

    Parimi, Sunil; Ko, Jenny J

    2017-10-01

    The treatment landscape of metastatic prostate cancer has changed dramatically over the past five years. As new discoveries are made and further novel therapies become available, there is a heightened urgency to develop biomarkers that can guide prognoses and predict therapy responses. Circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) in the blood have emerged as potential promising tumor avatars. Areas covered: In this review, we describe technological breakthroughs and clinical implementation of the CTCs and ctDNA. We also discuss the key challenges that must be overcome before circulating blood-based biomarkers can be universally adopted into the management of patients with metastatic prostate cancer. Expert commentary: Both CTCs and ctDNA have the potential to be incorporated into routine patient care, with increasing numbers of prospective trials incorporating them into clinical designs. CTCs and ctDNA will thus have an increasingly valuable role in augmenting our understanding of prostate cancer at a molecular level, aiding in prognostication of prostate cancer patients, acting as a surrogate for OS in clinical trials, and helping us prioritize our treatment selections by elucidating resistance mechanisms.

  6. Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo.

    PubMed

    Chumakova, Olga V; Liopo, Anton V; Andreev, Valery G; Cicenaite, Inga; Evers, B Mark; Chakrabarty, Shilla; Pappas, Todd C; Esenaliev, Rinat O

    2008-03-18

    The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

  7. DNA Methylation Status of the Estrogen Receptor α Gene in Canine Mammary Tumors.

    PubMed

    Brandão, Yara de Oliveira; Toledo, Mariana Busato; Chequin, Andressa; Cristo, Thierry Grima; Sousa, Renato Silva; Ramos, Edneia Amancio Souza; Klassen, Giseli

    2018-01-01

    Estrogen receptor α (ERα) has an important role in mammary carcinogenesis, prognosis, and treatment. In human and canine mammary cancer, the most aggressive tumors show loss of ERα expression, which in human breast cancer has been attributed to methylation of the cytosine followed by guanine (CpG) island within the estrogen receptor α gene ( ESR1) promoter. This study aimed to investigate the role of ESR1 CpG island (CGI) methylation in ERα expression in canine mammary tumors. Twenty-one canine mammary samples were sorted into three groups: malignant tumor (n = 9), benign tumor (n = 8), and normal gland (n = 4). Immunohistochemical analysis and reverse-transcription quantitative real-time PCR were performed to assess ERα expression and ESR1 mRNA levels. The methylation status was determined using sodium-bisulfite-treated DNA sequencing. All normal mammary glands and benign tumors showed high ERα expression (score range, 5-8). Six of the nine malignant tumors did not show ERα expression (score 0), two had score 2, and one had score 4. Lower ERα ( P < .005) and ESR1 mRNA levels ( P < .005) were found in malignant mammary tumors than in the other two groups. Canine ESR1 has an intragenic and non-promoter-associated CGI, different from humans. No significant variation in methylation percentage was observed among the groups, suggesting that ESR1 is not regulated by DNA methylation, unlike that in humans. This difference should be considered in further research using ERα as a biomarker for mammary tumors in canine studies on ERα-targeting therapy.

  8. Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

    PubMed

    Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Kato, Mamoru; Shibata, Tatsuhiro; Yachida, Shinichi

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.

  9. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    PubMed

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  10. [Hot topics of circulating tumor DNA testing in breast cancer].

    PubMed

    Liu, Y H; Zhou, B; Xu, L; Xin, L

    2017-02-01

    The progress of gene detection technologies represented by next generation sequencing (NGS) and digital PCR laid a foundation for studies of circulating tumor DNA (ctDNA) in breast cancer. In 2014, the NGS workgroup organized by the College of American Pathologists (CAP) published the College of American Pathologists ' Laboratory Standards for Next - Generation Sequencing Clinical Tests, which provides a blueprint for the standardization of gene testing. In 2015, the Guidelines for Diagnostic Next - generation Sequencing published by the European Society of Human Genetics claimed that NGS is unacceptable in clinical practice before studies guided by guidelines are approved. Although existing studies show the benefits of ctDNA testing in disease monitoring and prognosis analyzing, we have a ways to go to normalize the procedure and build strict detection criteria.

  11. Detection of somatic mutations in the mitochondrial DNA control region D-loop in brain tumors: The first report in Malaysian patients.

    PubMed

    Mohamed Yusoff, Abdul Aziz; Mohd Nasir, Khairol Naaim; Haris, Khalilah; Mohd Khair, Siti Zulaikha Nashwa; Abdul Ghani, Abdul Rahman Izaini; Idris, Zamzuri; Abdullah, Jafri Malin

    2017-11-01

    Although the role of nuclear-encoded gene alterations has been well documented in brain tumor development, the involvement of the mitochondrial genome in brain tumorigenesis has not yet been fully elucidated and remains controversial. The present study aimed to identify mutations in the mitochondrial DNA (mtDNA) control region D-loop in patients with brain tumors in Malaysia. A mutation analysis was performed in which DNA was extracted from paired tumor tissue and blood samples obtained from 49 patients with brain tumors. The D-loop region DNA was amplified using the PCR technique, and genetic data from DNA sequencing analyses were compared with the published revised Cambridge sequence to identify somatic mutations. Among the 49 brain tumor tissue samples evaluated, 25 cases (51%) had somatic mutations of the mtDNA D-loop, with a total of 48 mutations. Novel mutations that had not previously been identified in the D-loop region (176 A-deletion, 476 C>A, 566 C>A and 16405 A-deletion) were also classified. No significant associations between the D-loop mutation status and the clinicopathological parameters were observed. To the best of our knowledge, the current study presents the first evidence of alterations in the mtDNA D-loop regions in the brain tumors of Malaysian patients. These results may provide an overview and data regarding the incidence of mitochondrial genome alterations in Malaysian patients with brain tumors. In addition to nuclear genome aberrations, these specific mitochondrial genome alterations may also be considered as potential cancer biomarkers for the diagnosis and staging of brain cancers.

  12. PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.

    PubMed

    Hosseinkhani, Hossein; Tabata, Yasuhiko

    2004-05-31

    The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the

  13. An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply

    DTIC Science & Technology

    2006-05-01

    W81XWH-04-1-0489 TITLE: An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply PRINCIPAL...Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply 5b. GRANT NUMBER W81XWH-04-1-0489 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...blocking renewal of blood vessel growth in the tumor bed, have been proposed as suitable antitumor strategies. Endoglin (CD105) is a suitable

  14. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    PubMed

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  15. Properties of internalization factors contributing to the uptake of extracellular DNA into tumor-initiating stem cells of mouse Krebs-2 cell line.

    PubMed

    Dolgova, Evgeniya V; Potter, Ekaterina A; Proskurina, Anastasiya S; Minkevich, Alexandra M; Chernych, Elena R; Ostanin, Alexandr A; Efremov, Yaroslav R; Bayborodin, Sergey I; Nikolin, Valeriy P; Popova, Nelly A; Kolchanov, Nikolay A; Bogachev, Sergey S

    2016-05-25

    Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA. The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA). We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 μg DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR. The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of

  16. ATRX Loss Promotes Tumor Growth and Impairs Non-Homologous End Joining DNA Repair in Glioma

    PubMed Central

    Koschmann, Carl; Calinescu, Anda-Alexandra; Nunez, Felipe J.; Mackay, Alan; Fazal-Salom, Janet; Thomas, Daniel; Mendez, Flor; Kamran, Neha; Dzaman, Marta; Mulpuri, Lakshman; Krasinkiewicz, Johnathon; Doherty, Robert; Lemons, Rosemary; Brosnan-Cashman, Jackie A.; Li, Youping; Roh, Soyeon; Zhao, Lili; Appelman, Henry; Ferguson, David; Gorbunova, Vera; Meeker, Alan; Jones, Chris; Lowenstein, Pedro R.; Castro, Maria G.

    2017-01-01

    Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and show that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs non-homologous end joining (NHEJ) and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated, but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival. PMID:26936505

  17. Monitoring of treatment responses and clonal evolution of tumor cells by circulating tumor DNA of heterogeneous mutant EGFR genes in lung cancer.

    PubMed

    Imamura, Fumio; Uchida, Junji; Kukita, Yoji; Kumagai, Toru; Nishino, Kazumi; Inoue, Takako; Kimura, Madoka; Oba, Shigeyuki; Kato, Kikuya

    2016-04-01

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have dramatic effects on EGFR-mutant non-small-cell lung cancer (NSCLC). However, most patients experience disease recurrences, approximately half of which are T790M-mediated. Monitoring EGFR status with re-biopsy has spatiotemporal limitations. EGFR circulating tumor DNA (ctDNA) in serial plasma samples was amplified and 10(5) of them were sequenced with a next-generation sequencer. Plasma mutation (PM) score was defined as the number of reads containing deletions/substitutions in 10(5)EGFR cell free DNA (cfDNA). PM scores of various EGFR mutations showed dynamic, case-specific changes during EGFR-TKI treatments in 52 patients. The effects of the treatment on EGFR ctDNA were evaluated in 38 patients with elevated pre-treatment PM scores. The ctDNA responses correlated well with radiologic responses in radiologic good responders, whereas correlation was poor in non-responders. In addition to the peaks for the most prevalent ctDNA, small peaks of ctDNA with different types of activating EGFR mutations or the T790M mutation (early T790M ctDNA) appeared transiently in 10.5% and 26.3%, respectively. Early T790M ctDNA disappeared in all patients, including 7 who eventually developed acquired resistance accompanied by elevated levels of T790M ctDNA. Monitoring ctDNA is useful in evaluating treatment responses and monitoring driver oncogene status in NSCLC. ctDNA revealed clonal heterogeneity and genetic processes of cancer evolution in individual patients. The simple presence of the T790M mutation may be insufficient to confer EGFR-TKI resistance to tumor cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. DNA-PKcs deficiency leads to persistence of oxidatively-induced clustered DNA lesions in human tumor cells

    PubMed Central

    Peddi, Prakash; Loftin, Charles W.; Dickey, Jennifer S.; Hair, Jessica M.; Burns, Kara J.; Aziz, Khaled; Francisco, Dave C.; Panayiotidis, Mihalis I.; Sedelnikova, Olga A.; Bonner, William M.; Winters, Thomas A.; Georgakilas, Alexandros G.

    2010-01-01

    DNA-dependent protein kinase (DNA-PK) is a key non-homologous end joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. In order to examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three different models of DNA-PK deficiency i.e. chemical inactivation of its kinase activity by novel inhibitors IC86621 and NU7026, knock-down and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). Compromised DNA-PK repair pathway has lead to accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively-induced non-DSB clustered DNA lesions measured using a modified version of pulsed field gel electrophoresis or single cell gel electrophoresis (Comet assay). In all cases, DNA-PK inactivation lead to a higher level of lesion persistence even after 24–72 hrs of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters by first compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions due to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important not only for understating cancer etiology in the presence of a NHEJ deficiency but also lead to a better understanding of cancer treatments based on the induction of oxidative stress and inhibition of cluster repair. PMID:20193758

  19. DNA Methylation Mediated Control of Gene Expression Is Critical for Development of Crown Gall Tumors

    PubMed Central

    Kneitz, Susanne; Weber, Dana; Fuchs, Joerg; Hedrich, Rainer; Deeken, Rosalia

    2013-01-01

    Crown gall tumors develop after integration of the T-DNA of virulent Agrobacterium tumefaciens strains into the plant genome. Expression of the T-DNA–encoded oncogenes triggers proliferation and differentiation of transformed plant cells. Crown gall development is known to be accompanied by global changes in transcription, metabolite levels, and physiological processes. High levels of abscisic acid (ABA) in crown galls regulate expression of drought stress responsive genes and mediate drought stress acclimation, which is essential for wild-type-like tumor growth. An impact of epigenetic processes such as DNA methylation on crown gall development has been suggested; however, it has not yet been investigated comprehensively. In this study, the methylation pattern of Arabidopsis thaliana crown galls was analyzed on a genome-wide scale as well as at the single gene level. Bisulfite sequencing analysis revealed that the oncogenes Ipt, IaaH, and IaaM were unmethylated in crown galls. Nevertheless, the oncogenes were susceptible to siRNA–mediated methylation, which inhibited their expression and subsequently crown gall growth. Genome arrays, hybridized with methylated DNA obtained by immunoprecipitation, revealed a globally hypermethylated crown gall genome, while promoters were rather hypomethylated. Mutants with reduced non-CG methylation developed larger tumors than the wild-type controls, indicating that hypermethylation inhibits plant tumor growth. The differential methylation pattern of crown galls and the stem tissue from which they originate correlated with transcriptional changes. Genes known to be transcriptionally inhibited by ABA and methylated in crown galls became promoter methylated upon treatment of A. thaliana with ABA. This suggests that the high ABA levels in crown galls may mediate DNA methylation and regulate expression of genes involved in drought stress protection. In summary, our studies provide evidence that epigenetic processes regulate gene

  20. PMS2 gene mutation results in DNA mismatch repair system failure in a case of adult granulosa cell tumor.

    PubMed

    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.

  1. Absolute cross section for low-energy-electron damage to condensed macromolecules: A case study of DNA

    PubMed Central

    Rezaee, Mohammad; Cloutier, Pierre; Bass, Andrew D.; Michaud, Marc; Hunting, Darel J.; Sanche, Léon

    2013-01-01

    Cross sections (CSs) for the interaction of low-energy electrons (LEE) with condensed macromolecules are essential parameters for accurate modeling of radiation-induced molecular decomposition and chemical synthesis. Electron irradiation of dry nanometer-scale macromolecular solid films has often been employed to measure CSs and other quantitative parameters for LEE interactions. Since such films have thicknesses comparable with electron thermalization distances, energy deposition varies throughout the film. Moreover, charge accumulation occurring inside the films shields a proportion of the macromolecules from electron irradiation. Such effects complicate the quantitative comparison of the CSs obtained in films of different thicknesses and limit the applicability of such measurements. Here, we develop a simple mathematical model, termed the molecular survival model, that employs a CS for a particular damage process together with an attenuation length related to the total CS, to investigate how a measured CS might be expected to vary with experimental conditions. As a case study, we measure the absolute CS for the formation of DNA strand breaks (SBs) by electron irradiation at 10 and 100 eV of lyophilized plasmid DNA films with thicknesses between 10 and 30 nm. The measurements are shown to depend strongly on the thickness and charging condition of the nanometer-scale films. Such behaviors are in accord with the model and support its validity. Via this analysis, the CS obtained for SB damage is nearly independent of film thickness and charging effects. In principle, this model can be adapted to provide absolute CSs for electron-induced damage or reactions occurring in other molecular solids across a wider range of experimental conditions. PMID:23030950

  2. [Effect of Mn(II) on the error-prone DNA polymerase iota activity in extracts from human normal and tumor cells].

    PubMed

    Lakhin, A V; Efremova, A S; Makarova, I V; Grishina, E E; Shram, S I; Tarantul, V Z; Gening, L V

    2013-01-01

    The DNA polymerase iota (Pol iota), which has some peculiar features and is characterized by an extremely error-prone DNA synthesis, belongs to the group of enzymes preferentially activated by Mn2+ instead of Mg2+. In this work, the effect of Mn2+ on DNA synthesis in cell extracts from a) normal human and murine tissues, b) human tumor (uveal melanoma), and c) cultured human tumor cell lines SKOV-3 and HL-60 was tested. Each group displayed characteristic features of Mn-dependent DNA synthesis. The changes in the Mn-dependent DNA synthesis caused by malignant transformation of normal tissues are described. It was also shown that the error-prone DNA synthesis catalyzed by Pol iota in extracts of all cell types was efficiently suppressed by an RNA aptamer (IKL5) against Pol iota obtained in our work earlier. The obtained results suggest that IKL5 might be used to suppress the enhanced activity of Pol iota in tumor cells.

  3. Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

    PubMed

    Perets, Ruth; Greenberg, Orli; Shentzer, Talia; Semenisty, Valeria; Epelbaum, Ron; Bick, Tova; Sarji, Shada; Ben-Izhak, Ofer; Sabo, Edmond; Hershkovitz, Dov

    2018-05-01

    Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers

  4. TLR9 agonists oppositely modulate DNA repair genes in tumor versus immune cells and enhance chemotherapy effects.

    PubMed

    Sommariva, Michele; De Cecco, Loris; De Cesare, Michelandrea; Sfondrini, Lucia; Ménard, Sylvie; Melani, Cecilia; Delia, Domenico; Zaffaroni, Nadia; Pratesi, Graziella; Uva, Valentina; Tagliabue, Elda; Balsari, Andrea

    2011-10-15

    Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.

  5. Liquid Biopsy for Cancer: Circulating Tumor Cells, Circulating Free DNA or Exosomes?

    PubMed

    Zhang, Wei; Xia, Wenjie; Lv, Zhengye; Ni, Chao; Xin, Yin; Yang, Liu

    2017-01-01

    Precision medicine and personalized medicine are based on the development of biomarkers, and liquid biopsy has been reported to be able to detect biomarkers that carry information on tumor development and progression. Compared with traditional 'solid biopsy', which cannot always be performed to determine tumor dynamics, liquid biopsy has notable advantages in that it is a noninvasive modality that can provide diagnostic and prognostic information prior to treatment, during treatment and during progression. In this review, we describe the source, characteristics, technology for detection and current situation of circulating tumor cells, circulating free DNA and exosomes used for diagnosis, recurrence monitoring, prognosis assessment and medication planning. © 2017 The Author(s)Published by S. Karger AG, Basel.

  6. Circulating tumor DNA changes for early monitoring of anti-PD1 immunotherapy: a proof-of-concept study.

    PubMed

    Cabel, L; Riva, F; Servois, V; Livartowski, A; Daniel, C; Rampanou, A; Lantz, O; Romano, E; Milder, M; Buecher, B; Piperno-Neumann, S; Bernard, V; Baulande, S; Bieche, I; Pierga, J Y; Proudhon, C; Bidard, F-C

    2017-08-01

    Recent clinical results support the use of new immune checkpoint blockers (ICB), such as anti-PD-1 (e.g. nivolumab and pembrolizumab) and anti-PD-L1 antibodies. Radiological evaluation of ICB efficacy during therapy is challenging due to tumor immune infiltration. Changes of circulating tumor DNA (ctDNA) levels during therapy could be a promising tool for very accurate monitoring of treatment efficacy, but data are lacking with ICB. This prospective pilot study was conducted in patients with nonsmall cell lung cancer, uveal melanoma, or microsatellite-instable colorectal cancer treated by nivolumab or pembrolizumab monotherapy at Institut Curie. ctDNA levels were assessed at baseline and after 8 weeks (w8) by bidirectional pyrophosphorolysis-activated polymerization, droplet digital PCR or next-generation sequencing depending on the mutation type. Radiological evaluation of efficacy of treatment was carried out by using immune-related response criteria. ctDNA was detected at baseline in 10 out of 15 patients. At w8, a significant correlation (r = 0.86; P = 0.002) was observed between synchronous changes in ctDNA levels and tumor size. Patients in whom ctDNA levels became undetectable at w8 presented a marked and lasting response to therapy. ctDNA detection at w8 was also a significant prognostic factor in terms of progression-free survival (hazard ratio = 10.2; 95% confidence interval 2.5-41, P < 0.001) and overall survival (hazard ratio = 15; 95% confidence interval 2.5-94.9, P = 0.004). This proof-of-principle study is the first to demonstrate that quantitative ctDNA monitoring is a valuable tool to assess tumor response in patients treated with anti-PD-1 drugs. © The Author 2017. 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.

  7. Relationship between pretreatment level of plasma Epstein-Barr virus DNA, tumor burden, and metabolic activity in advanced nasopharyngeal carcinoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ma, Brigette; King, Ann; Lo, Y.M. Dennis

    Purpose: Plasma Epstein-Barr virus DNA (pEBV DNA) is an important prognostic marker in nasopharyngeal carcinoma (NPC). This study tested the hypotheses that pEBV DNA reflects tumor burden and metabolic activity by evaluating its relationship with tumor volume and {sup 18}F-fluorodeoxyglucose ({sup 18}F-FDG) uptake in NPC. Methods and Materials: Pre-treatment pEBV DNA analysis, {sup 18}F-FDG positron emission tomography-computed tomography scan (PET-CT) and magnetic resonance imaging (MRI) of the head and neck were performed in 57 patients. Net volume (cm{sup 3}) of the primary tumor (T{sub vol}) and regional nodes (N{sub vol}) were quantified on MRI. {sup 18}F-FDG uptake was expressed asmore » the maximum standardized uptake value (SUV{sub max}) at the primary tumor (T{sub suv}) and regional nodes (N{sub suv}). Lesions with SUV{sub max} {>=} 2.5 were considered malignant. Relationship between SUV{sub max}, natural logarithm (log) of pEBV DNA, and square root (sq) of MRI volumes was analyzed using the Wilcoxon test. A linear regression model was constructed to test for any interaction between variables and disease stage. Results: Log-pEBV DNA showed significant correlation with sq-T{sub vol} (r = 0.393), sq-N{sub vol} (r = 0.452), total tumor volume (sq-Total{sub vol} = T{sub vol} + N{sub vol}, r = 0.554), T{sub suv} (r = 0.276), N{sub suv} (r = 0.434), and total SUV{sub max} (Total{sub suv} = T{sub suv} + N{sub suv}, r = 0.457). Likewise, sq-T{sub vol} was correlated to T{sub suv} (r 0.426), and sq-N{sub vol} with N{sub suv} (r = 0.651). Regression analysis showed that only log-pEBV DNA was significantly associated with sq-Total{sub vol} (p < 0.001; parameter estimate = 8.844; 95% confidence interval = 3.986-13.703), whereas Sq-T{sub vol} was significantly associated with T{sub suv} (p = 0.002; parameter estimate = 3.923; 95% confidence interval = 1.498-6.348). Conclusion: This study supports the hypothesis that cell-free plasma EBV DNA is a marker of tumor

  8. Rapid electrochemical assessment of tumor suppressor gene methylations in raw human serum, and tumor cells and tissues using immuno-magnetic beads and selective DNA hybridization.

    PubMed

    Povedano, Eloy; Valverde, Alejandro; Ruiz-Valdepeñas Montiel, Víctor; Pedrero, María; Yáñez-Sedeño, Paloma; Barderas, Rodrigo; San Segundo-Acosta, Pablo; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Campuzano, Susana; Pingarron, José Manuel

    2018-05-09

    We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody specific for 5-methylcytosines (5-mC) are employed for the selective capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by selective hybridization with a synthetic biotinylated DNA sequence, further labeled with an HRP streptavidin conjugate. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor exhibits a dynamic range from 3.9 to 500 pM and a detection limit of 1.2 pM for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The applicability of this strategy is demonstrated through the 45 min-analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait

    PubMed Central

    Decock, Anneleen; Ongenaert, Maté; De Wilde, Bram; Brichard, Bénédicte; Noguera, Rosa; Speleman, Frank; Vandesompele, Jo

    2016-01-01

    ABSTRACT Stage 4S neuroblastoma (NB) is a special type of NB found in infants with metastases at diagnosis and is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. As genomics have not been able to explain this intriguing clinical presentation, we here aimed at profiling the DNA methylome of stage 4S NB to better understand this phenomenon. To this purpose, differential methylation analyses between International Neuroblastoma Staging System (INSS) stage 4S, stage 4 and stage 1/2 were performed, using methyl-CpG-binding domain (MBD) sequencing data of 14 stage 4S, 14 stage 4, and 13 stage 1/2 primary NB tumors (all MYCN non-amplified in order not to confound results). Stage 4S-specific hyper- and hypomethylated promoters were determined and further characterized for genomic localization and function by cytogenetic band enrichment, gene set enrichment, transcription factor target enrichment and differential RNA expression analyses. We show that specific chromosomal locations are enriched for stage 4S differentially methylated promoters and that stage 4S tumors show characteristic hypermethylation of specific subtelomeric promoters. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S tumors. Based on these findings, we describe new biological mechanisms possibly contributing to the stage 4S-specific tumor biology and spontaneous regression. In conclusion, this study is the first to describe the highly characteristic stage 4S DNA methylome. These findings will open new avenues to further unravel the NB pathology in general and stage 4S disease specifically. PMID:27599161

  10. Stage 4S neuroblastoma tumors show a characteristic DNA methylation portrait.

    PubMed

    Decock, Anneleen; Ongenaert, Maté; De Wilde, Bram; Brichard, Bénédicte; Noguera, Rosa; Speleman, Frank; Vandesompele, Jo

    2016-09-06

    Stage 4S neuroblastoma (NB) is a special type of NB found in infants with metastases at diagnosis and is associated with an excellent outcome due to its remarkable capacity to undergo spontaneous regression. As genomics have not been able to explain this intriguing clinical presentation, we here aimed at profiling the DNA methylome of stage 4S NB to better understand this phenomenon. To this purpose, differential methylation analyses between International Neuroblastoma Staging System (INSS) stage 4S, stage 4 and stage 1/2 were performed, using methyl-CpG-binding domain (MBD) sequencing data of 14 stage 4S, 14 stage 4, and 13 stage 1/2 primary NB tumors (all MYCN non-amplified in order not to confound results). Stage 4S-specific hyper- and hypo-methylated promoters were determined and further characterized for genomic localization and function by cytogenetic band enrichment, gene set enrichment, transcription factor target enrichment and differential RNA expression analyses. We show that specific chromosomal locations are enriched for stage 4S differentially methylated promoters and that stage 4S tumors show characteristic hypermethylation of subtelomeres. Furthermore, genes involved in important oncogenic pathways, in neural crest development and differentiation, and in epigenetic processes are differentially methylated and expressed in stage 4S tumors. Based on these findings, we describe new biological mechanisms possibly contributing to the stage 4S-specific tumor biology and spontaneous regression. In conclusion, this study is the first to describe the highly characteristic stage 4S DNA methylome. These findings will open new avenues to further unravel the NB pathology in general and stage 4S disease specifically.

  11. Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review.

    PubMed

    Merker, Jason D; Oxnard, Geoffrey R; Compton, Carolyn; Diehn, Maximilian; Hurley, Patricia; Lazar, Alexander J; Lindeman, Neal; Lockwood, Christina M; Rai, Alex J; Schilsky, Richard L; Tsimberidou, Apostolia M; Vasalos, Patricia; Billman, Brooke L; Oliver, Thomas K; Bruinooge, Suanna S; Hayes, Daniel F; Turner, Nicholas C

    2018-06-01

    Purpose Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from ASCO and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research. Methods An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including pre-analytical variables, analytical validity, interpretation and reporting, and clinical validity and utility. Results The literature search identified 1,338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion. Conclusion The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity and clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, re-evaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

  12. Endogenous DNA damage and testicular germ cell tumors

    PubMed Central

    Cook, Michael B.; Sigurdson, Alice J.; Jones, Irene M.; Thomas, Cynthia B.; Graubard, Barry I.; Korde, Larissa; Greene, Mark H.; McGlynn, Katherine A.

    2008-01-01

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable levels of net endogenous DNA damage. To test our hypothesis, we conducted a case-case analysis of 51 seminoma and 61 nonseminoma patients using data and specimens from the Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort. A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR50th percentile=3.31, 95%CI: 1.00, 10.98; OR75th percentile=3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR50th percentile=2.27, 95%CI: 0.75, 6.87; OR75th percentile=2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that net endogenous levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma. PMID:18657195

  13. Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

    PubMed Central

    Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

    2012-01-01

    Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

  14. Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

    PubMed

    Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

    2015-12-16

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.

  15. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

    PubMed

    Rothé, F; Laes, J-F; Lambrechts, D; Smeets, D; Vincent, D; Maetens, M; Fumagalli, D; Michiels, S; Drisis, S; Moerman, C; Detiffe, J-P; Larsimont, D; Awada, A; Piccart, M; Sotiriou, C; Ignatiadis, M

    2014-10-01

    Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology

  16. Neratinib Efficacy and Circulating Tumor DNA Detection of HER2 Mutations in HER2 Nonamplified Metastatic Breast Cancer.

    PubMed

    Ma, Cynthia X; Bose, Ron; Gao, Feng; Freedman, Rachel A; Telli, Melinda L; Kimmick, Gretchen; Winer, Eric; Naughton, Michael; Goetz, Matthew P; Russell, Christy; Tripathy, Debu; Cobleigh, Melody; Forero, Andres; Pluard, Timothy J; Anders, Carey; Niravath, Polly Ann; Thomas, Shana; Anderson, Jill; Bumb, Caroline; Banks, Kimberly C; Lanman, Richard B; Bryce, Richard; Lalani, Alshad S; Pfeifer, John; Hayes, Daniel F; Pegram, Mark; Blackwell, Kimberly; Bedard, Philippe L; Al-Kateb, Hussam; Ellis, Matthew J C

    2017-10-01

    Purpose: Based on promising preclinical data, we conducted a single-arm phase II trial to assess the clinical benefit rate (CBR) of neratinib, defined as complete/partial response (CR/PR) or stable disease (SD) ≥24 weeks, in HER2 mut nonamplified metastatic breast cancer (MBC). Secondary endpoints included progression-free survival (PFS), toxicity, and circulating tumor DNA (ctDNA) HER2 mut detection. Experimental Design: Tumor tissue positive for HER2 mut was required for eligibility. Neratinib was administered 240 mg daily with prophylactic loperamide. ctDNA sequencing was performed retrospectively for 54 patients (14 positive and 40 negative for tumor HER2 mut ). Results: Nine of 381 tumors (2.4%) sequenced centrally harbored HER2 mut (lobular 7.8% vs. ductal 1.6%; P = 0.026). Thirteen additional HER2 mut cases were identified locally. Twenty-one of these 22 HER2 mut cases were estrogen receptor positive. Sixteen patients [median age 58 (31-74) years and three (2-10) prior metastatic regimens] received neratinib. The CBR was 31% [90% confidence interval (CI), 13%-55%], including one CR, one PR, and three SD ≥24 weeks. Median PFS was 16 (90% CI, 8-31) weeks. Diarrhea (grade 2, 44%; grade 3, 25%) was the most common adverse event. Baseline ctDNA sequencing identified the same HER2 mut in 11 of 14 tumor-positive cases (sensitivity, 79%; 90% CI, 53%-94%) and correctly assigned 32 of 32 informative negative cases (specificity, 100%; 90% CI, 91%-100%). In addition, ctDNA HER2 mut variant allele frequency decreased in nine of 11 paired samples at week 4, followed by an increase upon progression. Conclusions: Neratinib is active in HER2 mut , nonamplified MBC. ctDNA sequencing offers a noninvasive strategy to identify patients with HER2 mut cancers for clinical trial participation. Clin Cancer Res; 23(19); 5687-95. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies

    PubMed Central

    2013-01-01

    The formalin-fixed, paraffin-embedded (FFPE) biopsy is a challenging sample for molecular assays such as targeted next-generation sequencing (NGS). We compared three methods for FFPE DNA quantification, including a novel PCR assay (‘QFI-PCR’) that measures the absolute copy number of amplifiable DNA, across 165 residual clinical specimens. The results reveal the limitations of commonly used approaches, and demonstrate the value of an integrated workflow using QFI-PCR to improve the accuracy of NGS mutation detection and guide changes in input that can rescue low quality FFPE DNA. These findings address a growing need for improved quality measures in NGS-based patient testing. PMID:24001039

  18. O6-Methylguanine DNA Methyltransferase Status Does Not Predict Response or Resistance to Alkylating Agents in Well-Differentiated Pancreatic Neuroendocrine Tumors.

    PubMed

    Raj, Nitya; Klimstra, David S; Horvat, Natally; Zhang, Liying; Chou, Joanne F; Capanu, Marinela; Basturk, Olca; Do, Richard Kinh Gian; Allen, Peter J; Reidy-Lagunes, Diane

    2017-07-01

    Alkylating agents have activity in well-differentiated pancreatic neuroendocrine tumors (WD panNETs). In glioblastoma multiforme, decreased activity of O-methylguanine DNA methyltransferase (MGMT) predicts response; in panNETs, MGMT relevance is unknown. We identified patients with WD panNETs treated with alkylating agents, determined best overall response by Response Evaluation Criteria In Solid Tumors (RECIST) 1.1, and performed MGMT activity testing. Fifty-six patients were identified; 26 (46%) of the 56 patients experienced partial response, 24 (43%) of 56 experienced stable disease, and 6 (11%) of 56 experienced progression of disease. O-methylguanine DNA methyltransferase status was available for 36 tumors. For tumors with partial response, 10 (67%) of 15 were MGMT deficient, and 5 (33%) of 15 were MGMT intact. For tumors with stable disease, 7 (47%) of 15 were MGMT deficient, and 8 (53%) of 15 were MGMT intact. For tumors with progression of disease, 3 (50%) of 6 were MGMT deficient, and 3 (50%) of 6 were MGMT intact. We observed response and resistance to alkylating agents in MGMT-deficient and MGMT-intact tumors. O-methylguanine DNA methyltransferase status should not guide alkylating agent therapy in WD panNETs.

  19. Integrated genomic classification of melanocytic tumors of the central nervous system using mutation analysis, copy number alterations and DNA methylation profiling.

    PubMed

    Griewank, Klaus; Koelsche, Christian; van de Nes, Johannes A P; Schrimpf, Daniel; Gessi, Marco; Möller, Inga; Sucker, Antje; Scolyer, Richard A; Buckland, Michael E; Murali, Rajmohan; Pietsch, Torsten; von Deimling, Andreas; Schadendorf, Dirk

    2018-06-11

    In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria can be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis. Targeted next-generation-sequencing, array-based genome-wide methylation analysis and BAP1 immunohistochemistry was performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, incl. 47 primary tumors of the central nervous system, 16 uveal melanomas. 13 cutaneous melanoma metastasis and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation and copy-number profiles were correlated with clinicopathological features. Combining mutation, copy-number and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group. Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations markers of poor prognosis. Copyright ©2018, American Association for Cancer Research.

  20. New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

    PubMed

    Saini, Karan Singh; Hamidullah; Ashraf, Raghib; Mandalapu, Dhanaraju; Das, Sharmistha; Siddiqui, Mohd Quadir; Dwivedi, Sonam; Sarkar, Jayanta; Sharma, Vishnu Lal; Konwar, Rituraj

    2017-04-01

    Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Smurf2 Regulates DNA Repair and Packaging to Prevent Tumors | Center for Cancer Research

    Cancer.gov

    The blueprint for all of a cell’s functions is written in the genetic code of DNA sequences as well as in the landscape of DNA and histone modifications. DNA is wrapped around histones to package it into chromatin, which is stored in the nucleus. It is important to maintain the integrity of the chromatin structure to ensure that the cell continues to behave appropriately. Recently, Ying Zhang, Ph.D., Senior Investigator in CCR’s Laboratory of Cellular and Molecular Biology, and her colleagues showed that alterations in the organization of the DNA can lead to tumor growth in a variety of tissues. This study appeared in the February 2012 issue of Nature Medicine and was featured as a cover story of that issue.

  2. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification

    EPA Science Inventory

    Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, t...

  3. Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kiss, R.; Camby, I.; Salmon, I.

    1995-06-01

    Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen-stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA ploidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic).more » In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high-grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low-grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high-risk clinical subgroups and medium-risk subgroups in clearly circumscribed histopathological groups. 38 refs., 5 figs., 2 tabs.« less

  4. Efficient systemic DNA delivery to the tumor by self-assembled nanoparticle

    NASA Astrophysics Data System (ADS)

    Tang, Hailin; Xie, Xinhua; Guo, Jiaoli; Wei, Weidong; Wu, Minqing; Liu, Peng; Kong, Yanan; Yang, Lu; Hung, Mien-Chie; Xie, Xiaoming

    2014-01-01

    There are few delivery agents that could deliver gene with high efficiency and low toxicity, especially for animal experiments. Therefore, creating vectors with good delivery efficiency and safety profile is a meaningful work. We have developed a self-assembled gene delivery system (XM001), which can more efficiently deliver DNA to multiple cell lines and breast tumor, as compared to commercial delivery agents. In addition, systemically administrated XM001-BikDD (BikDD is a mutant form of proapoptotic gene Bik) significantly inhibited the growth of human breast cancer cells and prolonged the life span in implanted nude mice. This study demonstrates that XM001 is an efficient and widespread transfection agent, which could be a promising tumor delivery vector for cancer targeted therapy.

  5. Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

    PubMed Central

    Krug, A K; Enderle, D; Karlovich, C; Priewasser, T; Bentink, S; Spiel, A; Brinkmann, K; Emenegger, J; Grimm, D G; Castellanos-Rizaldos, E; Goldman, J W; Sequist, L V; Soria, J -C; Camidge, D R; Gadgeel, S M; Wakelee, H A; Raponi, M; Noerholm, M; Skog, J

    2018-01-01

    Abstract Background A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials NCT01526928 PMID:29216356

  6. Identification of I1171N resistance mutation in ALK-positive non-small-cell lung cancer tumor sample and circulating tumor DNA.

    PubMed

    Johnson, Alison C; Dô, Pascal; Richard, Nicolas; Dubos, Catherine; Michels, Jean Jacques; Bonneau, Jessica; Gervais, Radj

    2016-09-01

    Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is sensitive to ALK inhibitor therapy, but resistance invariably develops and can be mediated by certain secondary mutations. The detection of these mutations is useful to guide treatment decisions, but tumors are not always easily accessible to re-biopsy. We report the case of a patient with ALK-rearranged NSCLC who presented acquired resistance to crizotinib and then alectinib. Sequencing analyses of DNA from a liver metastasis biopsy sample and circulating tumor DNA both found the same I1171N ALK kinase domain mutation, known to confer resistance to certain ALK inhibitors. However, the patient then received ceritinib, a 2nd generation ALK inhibitor, and achieved another partial response. This case underlines how ALK resistance mutation detection in peripheral blood could be a reliable, safer, and less invasive alternative to tissue-based samples in NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. DNA methylation profiling identifies global methylation differences and markers of adrenocortical tumors.

    PubMed

    Rechache, Nesrin S; Wang, Yonghong; Stevenson, Holly S; Killian, J Keith; Edelman, Daniel C; Merino, Maria; Zhang, Lisa; Nilubol, Naris; Stratakis, Constantine A; Meltzer, Paul S; Kebebew, Electron

    2012-06-01

    It is not known whether there are any DNA methylation alterations in adrenocortical tumors. The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors.

  8. Prostate tumor DNA methylation is associated with cigarette smoking and adverse prostate cancer outcomes.

    PubMed

    Shui, Irene M; Wong, Chao-Jen; Zhao, Shanshan; Kolb, Suzanne; Ebot, Ericka M; Geybels, Milan S; Rubicz, Rohina; Wright, Jonathan L; Lin, Daniel W; Klotzle, Brandy; Bibikova, Marina; Fan, Jian-Bing; Ostrander, Elaine A; Feng, Ziding; Stanford, Janet L

    2016-07-15

    DNA methylation has been hypothesized as a mechanism for explaining the association between smoking and adverse prostate cancer (PCa) outcomes. This study was aimed at assessing whether smoking is associated with prostate tumor DNA methylation and whether these alterations may explain in part the association of smoking with PCa recurrence and mortality. A total of 523 men had radical prostatectomy as their primary treatment, detailed smoking history data, long-term follow-up for PCa outcomes, and tumor tissue profiled for DNA methylation. Ninety percent of the men also had matched tumor gene expression data. A methylome-wide analysis was conducted to identify differentially methylated regions (DMRs) by smoking status. To select potential functionally relevant DMRs, their correlation with the messenger RNA (mRNA) expression of corresponding genes was evaluated. Finally, a smoking-related methylation score based on the top-ranked DMRs was created to assess its association with PCa outcomes. Forty DMRs were associated with smoking status, and 10 of these were strongly correlated with mRNA expression (aldehyde oxidase 1 [AOX1], claudin 5 [CLDN5], early B-cell factor 1 [EBF1], homeobox A7 [HOXA7], lectin galactoside-binding soluble 3 [LGALS3], microtubule-associated protein τ [MAPT], protocadherin γ A [PCDHGA]/protocadherin γ B [PCDHGB], paraoxonase 3 [PON3], synaptonemal complex protein 2 like [SYCP2L], and zinc finger and SCAN domain containing 12 [ZSCAN12]). Men who were in the highest tertile for the smoking-methylation score derived from these DMRs had a higher risk of recurrence (odds ratio [OR], 2.29; 95% confidence interval [CI], 1.42-3.72) and lethal disease (OR, 4.21; 95% CI, 1.65-11.78) in comparison with men in the lower 2 tertiles. This integrative molecular epidemiology study supports the hypothesis that smoking-associated tumor DNA methylation changes may explain at least part of the association between smoking and adverse PCa outcomes. Future studies

  9. Circulating tumor DNA as a liquid biopsy target for detection of pancreatic cancer.

    PubMed

    Takai, Erina; Yachida, Shinichi

    2016-10-14

    Most pancreatic cancer patients present with advanced metastatic disease, resulting in extremely poor 5-year survival, mainly because of the lack of a reliable modality for early detection and limited therapeutic options for advanced disease. Therefore, there is a need for minimally-invasive diagnostic tools for detecting pancreatic cancer at an early stage, when curative surgery and also novel therapeutic approaches including precision medicine may be feasible. The "liquid biopsy" addresses these unmet clinical needs based on the concept that simple peripheral blood sampling and detection of circulating tumor DNA (ctDNA) could provide diagnostic information. In this review, we provide an overview of the current status of blood-based tests for diagnosis of pancreatic cancer and the potential utility of ctDNA for precision medicine. We also discuss challenges that remain to be addressed in developing practical ctDNA-based liquid biopsy approaches for early diagnosis of pancreatic cancer.

  10. EG-07CELL CYCLE SIGNATURE AND TUMOR PHYLOGENY ARE ENCODED IN THE EVOLUTIONARY DYNAMICS OF DNA METHYLATION IN GLIOMA

    PubMed Central

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E.; Hong, Chibo; Bell, Robert J.A.; Smirnov, Ivan V.; Reis, Gerald F.; Phillips, Joanna J.; Barnes, Michael; Bollen, Andrew W.; Taylor, Barry S.; Molinaro, Annette M.; Olshen, Adam B.; Song, Jun S.; Berger, Mitchel S.; Chang, Susan M.; Costello, Joseph F.

    2014-01-01

    The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states, including DNA methylation, are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover tumor phylogeny. Here we examined the spatial and temporal dynamics of DNA methylation in a clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. WHO grade II gliomas are diffuse, infiltrative tumors that frequently recur and may undergo malignant progression to a higher grade with a worse prognosis. The extent to which epigenetic alterations contribute to the evolution of low-grade gliomas, including malignant progression, is unknown. While all gliomas in the cohort exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent malignant progression to high-grade glioblastoma (GBM) had a unique signature of DNA hypomethylation enriched for active enhancers, as well as sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic trees robustly recapitulated phylogenetic trees derived from somatic mutations in the same patients. These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent glioma.

  11. STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors.

    PubMed

    Deng, Liufu; Liang, Hua; Xu, Meng; Yang, Xuanming; Burnette, Byron; Arina, Ainhoa; Li, Xiao-Dong; Mauceri, Helena; Beckett, Michael; Darga, Thomas; Huang, Xiaona; Gajewski, Thomas F; Chen, Zhijian J; Fu, Yang-Xin; Weichselbaum, Ralph R

    2014-11-20

    Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-? induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-? treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer

    PubMed Central

    Soave, Armin; Rink, Michael

    2017-01-01

    Urothelial carcinoma of the bladder (UCB) is a complex disease, which is associated with highly aggressive tumor biologic behavior, especially in patients with muscle-invasive and advanced tumors. Despite multimodal therapy options including surgery, radiotherapy and chemotherapy, UCB patients frequently suffer from poor clinical outcome. Indeed, the potential of diverse opportunities for modern targeted therapies is not sufficiently elucidated in UCB yet. To improve the suboptimal treatment situation in UCB, biomarkers are urgently needed that help detecting minimal residual disease (MRD), predicting therapy response and subsequently prognosis as well as enabling patient stratification for further therapies and therapy monitoring, respectively. To date, decision making regarding treatment planning is mainly based on histopathologic evaluation of biopsies predominantly derived from the primary tumors and on clinical staging. However, both methods are imperfect for sufficient outcome prediction. During disease progression, individual disseminated tumor cells and consecutively metastases can acquire characteristics that do not match those of the corresponding primary tumors, and often are only hardly assessable for further evaluation. Therefore, during recent years, strong efforts were directed to establish non-invasive biomarkers from liquid biopsies. Urine cytology and serum tumor markers have been established for diagnostic purposes, but are still insufficient as universal biomarkers for decision-making and treatment of UCB patients. To date, the clinical relevance of various newly established blood-based biomarkers comprising circulating tumor cells (CTCs), circulating cell-free nucleic acids or tumor-educated platelets is being tested in cancer patients. In this review we summarize the current state and clinical application of CTCs and circulating cell-free tumor DNA originating from blood as biomarkers in patients with different UCB stages. PMID:29354496

  13. Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

    PubMed Central

    Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

    2013-01-01

    SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

  14. Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

    PubMed Central

    2017-01-01

    Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy. PMID:28880866

  15. An integrated genomics analysis of epigenetic subtypes in human breast tumors links DNA methylation patterns to chromatin states in normal mammary cells.

    PubMed

    Holm, Karolina; Staaf, Johan; Lauss, Martin; Aine, Mattias; Lindgren, David; Bendahl, Pär-Ola; Vallon-Christersson, Johan; Barkardottir, Rosa Bjork; Höglund, Mattias; Borg, Åke; Jönsson, Göran; Ringnér, Markus

    2016-02-29

    Aberrant DNA methylation is frequently observed in breast cancer. However, the relationship between methylation patterns and the heterogeneity of breast cancer has not been comprehensively characterized. Whole-genome DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChip arrays was performed on 188 human breast tumors. Unsupervised bootstrap consensus clustering was performed to identify DNA methylation epigenetic subgroups (epitypes). The Cancer Genome Atlas data, including methylation profiles of 669 human breast tumors, was used for validation. The identified epitypes were characterized by integration with publicly available genome-wide data, including gene expression levels, DNA copy numbers, whole-exome sequencing data, and chromatin states. We identified seven breast cancer epitypes. One epitype was distinctly associated with basal-like tumors and with BRCA1 mutations, one epitype contained a subset of ERBB2-amplified tumors characterized by multiple additional amplifications and the most complex genomes, and one epitype displayed a methylation profile similar to normal epithelial cells. Luminal tumors were stratified into the remaining four epitypes, with differences in promoter hypermethylation, global hypomethylation, proliferative rates, and genomic instability. Specific hyper- and hypomethylation across the basal-like epitype was rare. However, we observed that the candidate genomic instability drivers BRCA1 and HORMAD1 displayed aberrant methylation linked to gene expression levels in some basal-like tumors. Hypomethylation in luminal tumors was associated with DNA repeats and subtelomeric regions. We observed two dominant patterns of aberrant methylation in breast cancer. One pattern, constitutively methylated in both basal-like and luminal breast cancer, was linked to genes with promoters in a Polycomb-repressed state in normal epithelial cells and displayed no correlation with gene expression levels. The second pattern

  16. Alteration of the Tumor Stroma Using a Consensus DNA Vaccine Targeting Fibroblast Activation Protein (FAP) Synergizes with Antitumor Vaccine Therapy in Mice.

    PubMed

    Duperret, Elizabeth K; Trautz, Aspen; Ammons, Dylan; Perales-Puchalt, Alfredo; Wise, Megan C; Yan, Jian; Reed, Charles; Weiner, David B

    2018-03-01

    Purpose: Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts and is an interesting target for cancer immune therapy, with prior studies indicating a potential to affect the tumor stroma. Our aim was to extend this earlier work through the development of a novel FAP immunogen with improved capacity to break tolerance for use in combination with tumor antigen vaccines. Experimental Design: We used a synthetic consensus (SynCon) sequence approach to provide MHC class II help to support breaking of tolerance. We evaluated immune responses and antitumor activity of this novel FAP vaccine in preclinical studies, and correlated these findings to patient data. Results: This SynCon FAP DNA vaccine was capable of breaking tolerance and inducing both CD8 + and CD4 + immune responses. In genetically diverse, outbred mice, the SynCon FAP DNA vaccine was superior at breaking tolerance compared with a native mouse FAP immunogen. In several tumor models, the SynCon FAP DNA vaccine synergized with other tumor antigen-specific DNA vaccines to enhance antitumor immunity. Evaluation of the tumor microenvironment showed increased CD8 + T-cell infiltration and a decreased macrophage infiltration driven by FAP immunization. We extended this to patient data from The Cancer Genome Atlas, where we find high FAP expression correlates with high macrophage and low CD8 + T-cell infiltration. Conclusions: These results suggest that immune therapy targeting tumor antigens in combination with a microconsensus FAP vaccine provides two-fisted punch-inducing responses that target both the tumor microenvironment and tumor cells directly. Clin Cancer Res; 24(5); 1190-201. ©2018 AACR . ©2018 American Association for Cancer Research.

  17. Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

    PubMed Central

    Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq; Povirk, Lawrence F.; Hendrickson, Eric A.; Gewirtz, David A.

    2016-01-01

    Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative

  18. Fractionation of a tumor-initiating UV dose introduces DNA damage-retaining cells in hairless mouse skin and renders subsequent TPA-promoted tumors non-regressing

    PubMed Central

    van de Glind, Gerline; Rebel, Heggert; van Kempen, Marika; Tensen, Kees; de Gruijl, Frank

    2016-01-01

    Sunburns and especially sub-sunburn chronic UV exposure are associated with increased risk of squamous cell carcinomas (SCCs). Here we focus on a possible difference in tumor initiation from a single severe-sunburn dose (on day 1, 21 hairless mice) and from an equal dose fractionated into very low sub-sunburn doses not causing any (growth-promoting) epidermal hyperplasia (40 days daily exposure, n=20). From day 47 all mice received 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications (2x/wk) for 20 weeks to promote tumor development within the lifetime of the animals. After the sub-sunburn regimen sparse DNA damage-retaining basal cells (quiescent stem cells, QSCs) remained in the non-hyperplastic epidermis. These cells were forced to divide by TPA. After discontinuation of TPA tumors regressed and disappeared in the ‘sunburn group’ but persisted and grew in the ‘sub-sunburn group’ (0.06 vs 2.50 SCCs and precursors ≥4mm/mouse after 280 days, p=0.03). As the tumors carried no mutations in p53, H/K/N-Ras and Notch1/2, these ‘usual suspects' were not involved in the UV-driven tumor initiation. Although we could not selectively eliminate QSCs (unknown phenotype) to establish causality, our data suggest that forcing specifically DNA damage-retaining QSCs to divide – with high mutagenic risk - gives rise to persisting (mainly ‘in situ’) skin carcinomas. PMID:26797757

  19. Circulating tumor DNA as a liquid biopsy target for detection of pancreatic cancer

    PubMed Central

    Takai, Erina; Yachida, Shinichi

    2016-01-01

    Most pancreatic cancer patients present with advanced metastatic disease, resulting in extremely poor 5-year survival, mainly because of the lack of a reliable modality for early detection and limited therapeutic options for advanced disease. Therefore, there is a need for minimally-invasive diagnostic tools for detecting pancreatic cancer at an early stage, when curative surgery and also novel therapeutic approaches including precision medicine may be feasible. The “liquid biopsy” addresses these unmet clinical needs based on the concept that simple peripheral blood sampling and detection of circulating tumor DNA (ctDNA) could provide diagnostic information. In this review, we provide an overview of the current status of blood-based tests for diagnosis of pancreatic cancer and the potential utility of ctDNA for precision medicine. We also discuss challenges that remain to be addressed in developing practical ctDNA-based liquid biopsy approaches for early diagnosis of pancreatic cancer. PMID:27784960

  20. Light at night and melatonin have opposite effects on breast cancer tumors in mice assessed by growth rates and global DNA methylation.

    PubMed

    Schwimmer, Hagit; Metzer, Avishag; Pilosof, Yonit; Szyf, Moshe; Machnes, Ziv M; Fares, Fuad; Harel, Orna; Haim, Abraham

    2014-02-01

    Light-at-night (LAN) is a worldwide problem co-distributed with breast cancer prevalence. We hypothesized that exposure to LAN is coincided with a decreased melatonin (MLT) secretion level, followed by epigenetic modifications and resulted in higher breast cancer tumors growth-rate. Accordingly, we studied the effect of LAN exposure and exogenous MLT on breast cancer tumors growth-rate. 4T1 cells were inoculated into BALB/c short day-acclimated mice, resulting in tumors growth. Growth rates were followed under various light exposures and global DNA methylations were measured. Results demonstrated the positive effect of LAN on tumors growth-rate, reversed by MLT through global DNA methylation.

  1. The Prognostic Value of Plasma Epstein-Barr Viral DNA and Tumor Response to Neoadjuvant Chemotherapy in Advanced-Stage Nasopharyngeal Carcinoma

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Li-Ting; Tang, Lin-Quan; Chen, Qiu-Yan

    Purpose: To explore the prognostic value of the plasma load of Epstein-Barr viral (EBV) DNA and the tumor response to neoadjuvant chemotherapy (NACT) in advanced-stage nasopharyngeal carcinoma (NPC). Patients and Methods: In all, 185 consecutive patients with stage III to IVb NPC treated with NACT followed by concurrent chemoradiation therapy (CCRT) were prospectively enrolled. The primary endpoint was progression-free survival (PFS), and the secondary endpoints included locoregional relapse–free survival (LRFS) and distant metastasis–free survival (DMFS). Results: EBV DNA was detected in 165 (89%) patients before treatment but was undetectable in 127 (69%) patients after NACT. Detectable EBV DNA levels aftermore » NACT were correlated with poor prognosis (3-year PFS 71.8% vs 85.2%, P=.008 and 3-year DMFS 82.5% vs 92.3%, P=.013). An unsatisfactory tumor response (stable disease or disease progression) after NACT was also correlated with poor clinical outcome (3-year PFS 71.1% vs 85.9%, P=.005 and 3-year LRFS 82.7% vs 93.5%, P=.012). Multivariate analysis showed that the EBV DNA level after NACT (hazard ratio [HR] 2.31, 95% CI 1.18-4.54, P=.015) and the tumor response to NACT (HR 2.84, 95% CI 1.42-5.67, P=.003) were both significant prognostic factors for PFS. Multivariate analysis also showed that EBV DNA after NACT was the only significant predictor of DMFS (HR 2.99, 95% CI 1.25-7.15, P=.014) and that tumor response to NACT was the only significant predictor of LRFS (HR 3.31, 95% CI 1.21-9.07, P=.020). Conclusion: Detectable EBV DNA levels and an unsatisfactory tumor response (stable disease or disease progression) after NACT serve as predictors of poor prognosis for patients with advanced-stage NPC. These findings will facilitate further risk stratification, early treatment modification, or both before CCRT.« less

  2. Complex engagement of DNA damage response pathways in human cancer and in lung tumor progression.

    PubMed

    Nuciforo, Paolo Giovanni; Luise, Chiara; Capra, Maria; Pelosi, Giuseppe; d'Adda di Fagagna, Fabrizio

    2007-10-01

    Tumor initiation and progression provide a multitude of occasions for the generation of DNA damage and the consequent activation of the DNA damage response (DDR) pathway. DDR signaling involves the engagement of key factors such as ATM, CHK2, 53BP1 and the phosphorylation of histone H2AX (gamma-H2AX). The systematic study of DDR in human tumors and normal tissues by high-throughput tissue microarrays revealed that ATM and gamma-H2AX were engaged in cancer but the extent of their activation was strongly affected by the organ and cell type involved, whereas 53BP1 loss was the most consistent feature among the tumor studied. Unexpectedly, we also observed activated DDR markers in morphologically normal tissues, also in association with inflammation. Analysis of the dynamic engagement of DDR along the different stages of lung tumorigenesis showed that 53BP1 loss occurs early at the transition from normal to dysplastic change whereas the activated forms of ATM and CHK2, but not gamma-H2AX, initially accumulate in pre-invasive lesions and are then lost during tumor progression. In individual lung tumors, the activation of ATM, CHK2 and the presence of 53BP1 were consistently correlated, whereas gamma-H2AX did not correlate with activated ATM. Finally, the study of associations between critical clinicopathological parameters and activated DDR factors highlighted a statistically meaningful correlation between reduced local tumor extension and the phosphorylation of ATM, CHK2 and the presence of 53BP1, whereas no significant correlations with parameters such as survival or relapse of early-stage lung carcinomas were found.

  3. Absolute cross sections for electronic excitation of condensed tetrahydrofuran (THF) by 11-16 eV electrons.

    PubMed

    Lemelin, V; Bass, A D; Cloutier, P; Sanche, L

    2016-11-07

    Absolute cross section (CS) data on the interaction of low energy electrons with DNA and its molecular constituents are required as input parameters in Monte-Carlo type simulations, for several radiobiological applications. Previously [V. Lemelin et al., J. Chem. Phys. 144, 074701 (2016)], we measured absolute vibrational CSs for low-energy electron scattering from condensed tetrahydrofuran, a convenient surrogate for the deoxyribose. Here we report absolute electronic CSs for energy losses of between 6 and 11.5 eV, by electrons with energies between 11 and 16 eV. The variation of these CSs with incident electron energy shows no evidence of transient anion states, consistent with theoretical and other experimental results, indicating that initial electron capture leading to DNA strand breaks occurs primarily on DNA bases or the phosphate group.

  4. Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

    PubMed

    Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

    2005-11-01

    Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

  5. Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors.

    PubMed

    Olkhov-Mitsel, Ekaterina; Siadat, Farshid; Kron, Ken; Liu, Liyang; Savio, Andrea J; Trachtenberg, John; Fleshner, Neil; van der Kwast, Theodorus; Bapat, Bharati

    2017-07-01

    The aim of the present study was to explore DNA methylation aberrations in association with cribriform architecture and intraductal carcinoma (IDC) of the prostate, as there is robust evidence that these morphological features are associated with aggressive disease and have significant clinical implications. Herein, the associations of a panel of seven known prognostic DNA methylation biomarkers with cribriform and IDC features were examined in a series of 91 Gleason pattern (GP) 4 tumors derived from Gleason score 7 radical prostatectomies. Gene specific DNA methylation was compared between cribriform and/or IDC positive vs. negative cases, and in association with clinicopathological features, using Chi square and Mann-Whitney U tests. DNA methylation of the adenomatous polyposis coli, Ras association domain family member 1 and T-box 15 genes was significantly elevated in GP4 tumors with cribriform and/or IDC features compared with negative cases (P=0.045, P=0.007 and P=0.013, respectively). To the best of our knowledge, this provides the first evidence for an association between cribriform and/or IDC and methylation biomarkers, and warrants further investigation of additional DNA methylation events in association with various architectural patterns in prostate cancer.

  6. Do Circulating Tumor Cells, Exosomes, and Circulating Tumor Nucleic Acids Have Clinical Utility?

    PubMed Central

    Gold, Bert; Cankovic, Milena; Furtado, Larissa V.; Meier, Frederick; Gocke, Christopher D.

    2016-01-01

    Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided. PMID:25908243

  7. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    NASA Astrophysics Data System (ADS)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-06-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  8. Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

    PubMed Central

    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

  9. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    PubMed Central

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie; Liévin, Jacques; Körzdörfer, Thomas; Rotaru, Alexandru; Gothelf, Kurt V.; Besenbacher, Flemming; Bald, Ilko

    2014-01-01

    The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5′-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections between 2.66 · 10−14 cm2 and 7.06 · 10−14 cm2. The highest cross section was found for 5′-TT(ATA)3TT and 5′-TT(ABrUA)3TT, respectively. BrU is a radiosensitizer, which was discussed to be used in cancer radiation therapy. The replacement of T by BrU into the investigated DNA sequences leads to a slight increase of the absolute strand break cross sections resulting in sequence-dependent enhancement factors between 1.14 and 1.66. Nevertheless, the variation of strand break cross sections due to the specific nucleotide sequence is considerably higher. Thus, the present results suggest the development of targeted radiosensitizers for cancer radiation therapy. PMID:25487346

  10. Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

    PubMed Central

    Oike, Takahiro; Niimi, Atsuko; Okonogi, Noriyuki; Murata, Kazutoshi; Matsumura, Akihiko; Noda, Shin-Ei; Kobayashi, Daijiro; Iwanaga, Mototaro; Tsuchida, Keisuke; Kanai, Tatsuaki; Ohno, Tatsuya; Shibata, Atsushi; Nakano, Takashi

    2016-01-01

    Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy. PMID:26925533

  11. Long-circulating DNA lipid nanocapsules as new vector for passive tumor targeting.

    PubMed

    Morille, Marie; Montier, Tristan; Legras, Pierre; Carmoy, Nathalie; Brodin, Priscille; Pitard, Bruno; Benoît, Jean-Pierre; Passirani, Catherine

    2010-01-01

    Systemic gene delivery systems are needed for therapeutic application to organs that are inaccessible by percutaneous injection. Currently, the main objective is the development of a stable and non-toxic vector that can encapsulate and deliver foreign genetic material to target cells. To this end, DNA, complexed with cationic lipids i.e. DOTAP/DOPE, was encapsulated into lipid nanocapsules (LNCs) leading to the formation of stable nanocarriers (DNA LNCs) with a size inferior to 130 nm. Amphiphilic and flexible poly (ethylene glycol) (PEG) polymer coatings [PEG lipid derivative (DSPE-mPEG(2000)) or F108 poloxamer] at different concentrations were selected to make DNA LNCs stealthy. Some of these coated lipid nanocapsules were able to inhibit complement activation and were not phagocytized in vitro by macrophagic THP-1 cells whereas uncoated DNA LNCs accumulated in the vacuolar compartment of THP-1 cells. These results correlated with a significant increase of in vivo circulation time in mice especially for DSPE-mPEG(2000) 10 mm and an early half-life time (t(1/2) of distribution) 5-fold greater than for non-coated DNA LNCs (7.1 h vs 1.4 h). Finally, a tumor accumulation assessed by in vivo fluorescence imaging system was evidenced for these coated LNCs as a passive targeting without causing any hepatic damage.

  12. Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

    PubMed Central

    Sherman, Michael Y.; Gabai, Vladimir; Kiselev, Oleg; Komissarov, Andrey; Grudinin, Mikhail; Shartukova, Maria; Romanovskaya-Romanko, Ekaterina A.; Kudryavets, Yuri; Bezdenezhnykh, Natalya; Lykhova, Oleksandra; Semesyuk, Nadiia; Concetti, Antonio; Tsyb, Anatoly; Filimonova, Marina; Makarchuk, Victoria; Yakubovsky, Raisa; Chursov, Andrey; Shcherbinina, Vita; Shneider, Alexander

    2013-01-01

    Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials. PMID:24121124

  13. LINE1 and Alu repetitive element DNA methylation in tumors and white blood cells from epithelial ovarian cancer patients.

    PubMed

    Akers, Stacey N; Moysich, Kirsten; Zhang, Wa; Collamat Lai, Golda; Miller, Austin; Lele, Shashikant; Odunsi, Kunle; Karpf, Adam R

    2014-02-01

    We determined whether DNA methylation of repetitive elements (RE) is altered in epithelial ovarian cancer (EOC) patient tumors and white blood cells (WBC), compared to normal tissue controls. Two different quantitative measures of RE methylation (LINE1 and Alu bisulfite pyrosequencing) were used in normal and tumor tissues from EOC cases and controls. Tissues analyzed included: i) EOC, ii) normal ovarian surface epithelia (OSE), iii) normal fallopian tube surface epithelia (FTE), iv) WBC from EOC patients, obtained before and after treatment, and v) WBC from demographically-matched controls. REs were significantly hypomethylated in EOC compared to OSE and FTE, and LINE1 and Alu methylation showed a significant direct association in these tissues. In contrast, WBC RE methylation was significantly higher in EOC cases compared to controls. RE methylation in patient-matched EOC tumors and pre-treatment WBC did not correlate. EOC shows robust RE hypomethylation compared to normal tissues from which the disease arises. In contrast, RE are generally hypermethylated in EOC patient WBC compared to controls. EOC tumor and WBC methylation did not correlate in matched patients, suggesting that RE methylation is independently controlled in tumor and normal tissues. Despite the significant differences observed over the population, the range of RE methylation in patient and control WBC overlapped, limiting their specific utility as an EOC biomarker. However, our data demonstrate that DNA methylation is deranged in normal tissues from EOC patients, supporting further investigation of WBC DNA methylation biomarkers suitable for EOC risk assessment. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

    PubMed Central

    Srivenugopal, Kalkunte S.

    2014-01-01

    The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O6-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2–DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O6-benzylguanine; nevertheless, the 24–36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF. PMID:24193513

  15. Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers.

    PubMed

    Huang, Rui-Lan; Gu, Fei; Kirma, Nameer B; Ruan, Jianhua; Chen, Chun-Liang; Wang, Hui-Chen; Liao, Yu-Ping; Chang, Cheng-Chang; Yu, Mu-Hsien; Pilrose, Jay M; Thompson, Ian M; Huang, Hsuan-Cheng; Huang, Tim Hui-Ming; Lai, Hung-Cheng; Nephew, Kenneth P

    2013-06-01

    Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating "hit" during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan-Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.

  16. Mitochondrial DNA copy number threshold in mtDNA depletion myopathy.

    PubMed

    Durham, S E; Bonilla, E; Samuels, D C; DiMauro, S; Chinnery, P F

    2005-08-09

    The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.

  17. Nucleoprotein Changes in Plant Tumor Growth

    PubMed Central

    Rasch, Ellen; Swift, Hewson; Klein, Richard M.

    1959-01-01

    Tumor cell transformation and growth were studied in a plant neoplasm, crown gall of bean, induced by Agrobacterium rubi. Ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA), histone, and total protein were estimated by microphotometry of nuclei, nucleoli, and cytoplasm in stained tissue sections. Transformation of normal cells to tumor cells was accompanied by marked increases in ribonucleoprotein content of affected tissues, reaching a maximum 2 to 3 days after inoculation with virulent bacteria. Increased DNA levels were in part associated with increased mitotic frequency, but also with progressive accumulation of nuclei in the higher DNA classes, formed by repeated DNA doubling without intervening reduction by mitosis. Some normal nuclei of the higher DNA classes (with 2, 4, or 8 times the DNA content of diploid nuclei) were reduced to diploid levels by successive cell divisions without intervening DNA synthesis. The normal relation between DNA synthesis and mitosis was thus disrupted in tumor tissue. Nevertheless, clearly defined DNA classes, as found in homologous normal tissues, were maintained in the tumor at all times. PMID:13673042

  18. Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

    PubMed

    Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

    2016-07-26

    Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

  19. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    NASA Technical Reports Server (NTRS)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  20. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    PubMed

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

    2012-10-09

    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

  1. Next-Generation Sequencing of Circulating Tumor DNA Reveals Frequent Alterations in Advanced Hepatocellular Carcinoma.

    PubMed

    Ikeda, Sadakatsu; Tsigelny, Igor F; Skjevik, Åge A; Kono, Yuko; Mendler, Michel; Kuo, Alexander; Sicklick, Jason K; Heestand, Gregory; Banks, Kimberly C; Talasaz, AmirAli; Lanman, Richard B; Lippman, Scott; Kurzrock, Razelle

    2018-05-01

    Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood-derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC. We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild-type allele fraction was calculated. All patients (100%) had somatic alterations (median = 3 alterations/patient [range, 1-8]); median mutant allele fraction, 0.29% (range, 0.1%-37.77%). Mutations were identified in several genes: TP53 (57% of patients), CTNNB1 (29%), PTEN (7%), CDKN2A (7%), ARID1A (7%), and MET (7%); amplifications, in CDK6 (14%), EGFR (14%), MYC (14%), BRAF (7%), RAF1 (7%), FGFR1 (7%), CCNE1 (7%), PIK3CA (7%), and ERBB2/HER2 (7%). Eleven patients (79%) had ≥1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a CDKN2A -inactivating and a CTNNB1 -activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX-2/Wnt inhibitor); des-gamma-carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ≤7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a PTEN -inactivating and a MET -activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0-15 ng/mL]). ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC. This study reports that blood-derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy. © AlphaMed Press 2018.

  2. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer

    PubMed Central

    Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C.

    2016-01-01

    Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AI). We developed ultra-high sensitivity multiplexed digital PCR assays for ESR1 mutations in circulating tumor DNA (ctDNA) and used these to investigate the clinical relevance and origin of ESR1 mutations in a cohort of 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies, and could be assessed in samples shipped at room temperature in preservative tubes without loss of accuracy. ESR1 mutations were found exclusively in patients with estrogen receptor positive breast cancer previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy (HR 3.1, 95%CI 1.9-23.1, log rank p=0.0041). ESR1 mutation prevalence differed markedly between patients that were first exposed to AI during the adjuvant and metastatic settings (5.8% (3/52) vs 36.4% (16/44) respectively, p=0.0002). In an independent cohort, ESR1 mutations were identified in 0% (0/32, 95%CI 0-10.9%) tumor biopsies taken after progression on adjuvant AI. In a patient with serial samples taken during metastatic treatment, ESR1 mutation was selected during metastatic AI therapy, to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI therapy, but are commonly selected by therapy for metastatic disease, providing evidence that the mechanisms of resistance to targeted therapy may be substantially different between the treatment of micro-metastatic and overt metastatic cancer. PMID:26560360

  3. Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

    PubMed

    Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C

    2015-11-11

    Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer. Copyright © 2015, American Association for the Advancement of Science.

  4. FSensitive detection of mono- and polyclonal ESR1 mutations in primary tumors, metastatic lesions and cell free DNA of breast cancer patients

    PubMed Central

    Wang, Peilu; Bahreini, Amir; Gyanchandani, Rekha; Lucas, Peter C.; Hartmaier, Ryan J.; Watters, Rebecca J.; Jonnalagadda, Amruth R.; Trejo Bittar, Humberto E.; Berg, Aaron; Hamilton, Ronald L.; Kurland, Brenda F.; Weiss, Kurt R.; Mathew, Aju; Leone, Jose Pablo; Davidson, Nancy E; Nikiforova, Marina N.; Brufsky, Adam M.; Ambros, Tadeu F.; Stern, Andrew M.; Puhalla, Shannon L.; Lee, Adrian V.; Oesterreich, Steffi

    2015-01-01

    Purpose Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell free DNA (cfDNA). Patients and Methods Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n=43), bone (n=12) and brain metastases (n=38), and cfDNA (n=29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results ESR1 mutations were detected for D538G (n=13), Y537S (n=3) and Y537C (n=1), and not for K303R, S463P or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3 to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n=5), mutations were detected in both (n=3) or in cfDNA only (n=2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1 mutant clones are enriched by endocrine therapy. Further studies should address if sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. PMID:26500237

  5. DNA Vaccines for Prostate Cancer

    PubMed Central

    Zahm, Christopher D.; Colluru, Viswa Teja; McNeel, Douglas G.

    2017-01-01

    DNA vaccines offer many advantages over other anti-tumor vaccine approaches due to their simplicity, ease of manufacturing, and safety. Results from several clinical trials in patients with cancer have demonstrated that DNA vaccines are safe and can elicit immune responses. However, to date few DNA vaccines have progressed beyond phase I clinical trial evaluation. Studies into the mechanism of action of DNA vaccines in terms of antigen-presenting cell types able to directly present or cross-present DNA-encoded antigens, and the activation of innate immune responses due to DNA itself, have suggested opportunities to increase the immunogenicity of these vaccines. In addition, studies into the mechanisms of tumor resistance to anti-tumor vaccination have suggested combination approaches that can increase the antitumor effect of DNA vaccines. This review focuses on these mechanisms of action and mechanisms of resistance using DNA vaccines, and how this information is being used to improve the anti-tumor effect of DNA vaccines. These approaches are then specifically discussed in the context of human prostate cancer, a disease for which DNA vaccines have been and continue to be explored as treatments. PMID:28185916

  6. Short report: Monitoring ESR1 mutations by circulating tumor DNA in aromatase inhibitor resistant metastatic breast cancer.

    PubMed

    Sefrioui, David; Perdrix, Anne; Sarafan-Vasseur, Nasrin; Dolfus, Claire; Dujon, Antoine; Picquenot, Jean-Michel; Delacour, Julien; Cornic, Marie; Bohers, Elodie; Leheurteur, Marianne; Rigal, Olivier; Tennevet, Isabelle; Thery, Jean-Christophe; Alexandru, Cristina; Guillemet, Cécile; Moldovan, Cristian; Veyret, Corinne; Frebourg, Thierry; Di Fiore, Frédéric; Clatot, Florian

    2015-11-15

    Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring. © 2015 UICC.

  7. Treating kaposiform hemangioendothelioma with Kasabach-Merritt phenomenon by intralesional injection of absolute ethanol.

    PubMed

    Shen, Weimin; Cui, Jie; Chen, Jianbin; Zou, Jijun; Xiaoying, Zhang

    2014-11-01

    Kasabach-Merritt phenomenon (KMP) is characterized by thrombocytopenia, microangiopathic hemolytic anemia, consumptive coagulopathy, and an enlarging vascular lesion. It is a rare and life-threatening disease of vascular tumor. The purpose of this study was to assess the effectiveness of absolute ethanol in the treatment of KMP. We treated 8 pediatric patients with KMP by using absolute ethanol injections, whose cases failed previously when applied steroid therapy. We reviewed the clinical and laboratory data of these 8 cases at Nanjing Children's Hospital in China. Eight pediatric patients (5 female and 3 male) showed tumor regression after treatments. Core needle biopsy had been performed on the 8 patients, and the results included 7 kaposiform hemangioendotheliomas and one tufted angioma. All patients had an increase in platelet count. All coagulopathies were corrected. Complications included one patient with flush on face, one patient with needle scars, and one patient with a small area of local tissue necrosis. Absolute ethanol therapy presents a safe option in the treatment of KMP. Treatment-associated complications seemed to be reversible and acceptable by severity levels. Direct intralesional injection of absolute ethanol provides a simple and reliable alternative treatment for KMP among infants and may be used as the second-line therapy.

  8. A feasibility study of colorectal cancer diagnosis via circulating tumor DNA derived CNV detection.

    PubMed

    Molparia, Bhuvan; Oliveira, Glenn; Wagner, Jennifer L; Spencer, Emily G; Torkamani, Ali

    2018-01-01

    Circulating tumor DNA (ctDNA) has shown great promise as a biomarker for early detection of cancer. However, due to the low abundance of ctDNA, especially at early stages, it is hard to detect at high accuracies while keeping sequencing costs low. Here we present a pilot stage study to detect large scale somatic copy numbers variations (CNVs), which contribute more molecules to ctDNA signal compared to point mutations, via cell free DNA sequencing. We show that it is possible to detect somatic CNVs in early stage colorectal cancer (CRC) patients and subsequently discriminate them from normal patients. With 25 normal and 24 CRC samples, we achieve 100% specificity (lower bound confidence interval: 86%) and ~79% sensitivity (95% confidence interval: 63% - 95%,), though the performance should be considered with caution given the limited sample size. We report a lack of concordance between the CNVs detected via cfDNA sequencing and CNVs identified in parent tissue samples. However, recent findings suggest that a lack of concordance is expected for CNVs in CRC because of their sub-clonal nature. Finally, the CNVs we detect very likely contribute to cancer progression as they lie in functionally important regions, and have been shown to be associated with CRC specifically. This study paves the path for a larger scale exploration of the potential of CNV detection for both diagnoses and prognoses of cancer.

  9. Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

    PubMed Central

    Gallerani, Giulia; Cocchi, Claudia; Bocchini, Martine; Piccinini, Filippo; Fabbri, Francesco

    2017-01-01

    Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments. PMID:29286485

  10. Tumors overexpressing RNF168 show altered DNA repair and responses to genotoxic treatments, genomic instability and resistance to proteotoxic stress.

    PubMed

    Chroma, K; Mistrik, M; Moudry, P; Gursky, J; Liptay, M; Strauss, R; Skrott, Z; Vrtel, R; Bartkova, J; Kramara, J; Bartek, J

    2017-04-27

    Chromatin DNA damage response (DDR) is orchestrated by the E3 ubiquitin ligase ring finger protein 168 (RNF168), resulting in ubiquitin-dependent recruitment of DDR factors and tumor suppressors breast cancer 1 (BRCA1) and p53 binding protein 1 (53BP1). This ubiquitin signaling regulates pathway choice for repair of DNA double-strand breaks (DSB), toxic lesions whose frequency increases during tumorigenesis. Recruitment of 53BP1 curbs DNA end resection, thereby limiting homologous recombination (HR) and directing DSB repair toward error-prone non-homologous end joining (NHEJ). Under cancer-associated ubiquitin starvation conditions reflecting endogenous or treatment-evoked proteotoxic stress, the ubiquitin-dependent accrual of 53BP1 and BRCA1 at the DNA damage sites is attenuated or lost. Challenging this current paradigm, here we identified diverse human cancer cell lines that display 53BP1 recruitment to DSB sites even under proteasome inhibitor-induced proteotoxic stress, that is, under substantial depletion of free ubiquitin. We show that central to this unexpected phenotype is overabundance of RNF168 that enables more efficient exploitation of the residual-free ubiquitin. Cells with elevated RNF168 are more resistant to combined treatment by ionizing radiation and proteasome inhibition, suggesting that such aberrant RNF168-mediated signaling might reflect adaptation to chronic proteotoxic and genotoxic stresses experienced by tumor cells. Moreover, the overabundant RNF168 and the ensuing unorthodox recruitment patterns of 53BP1, RIF1 and REV7 (monitored on laser micro-irradiation-induced DNA damage) shift the DSB repair balance from HR toward NHEJ, a scenario accompanied by enhanced chromosomal instability/micronuclei formation and sensitivity under replication stress-inducing treatments with camptothecin or poly(ADP-ribose) polymerase (PARP) inhibitor. Overall, our data suggest that the deregulated RNF168/53BP1 pathway could promote tumorigenesis by selecting

  11. Mutations in Mitochondrial DNA From Pancreatic Ductal Adenocarcinomas Associate With Survival Times of Patients and Accumulate as Tumors Progress.

    PubMed

    Hopkins, Julia F; Denroche, Robert E; Aguiar, Jennifer A; Notta, Faiyaz; Connor, Ashton A; Wilson, Julie M; Stein, Lincoln D; Gallinger, Steven; Boutros, Paul C

    2018-05-01

    Somatic mutations have been found in the mitochondria in different types of cancer cells, but it is not clear whether these affect tumorigenesis or tumor progression. We analyzed mitochondrial genomes of 268 early-stage, resected pancreatic ductal adenocarcinoma tissues and paired non-tumor tissues. We defined a mitochondrial somatic mutation (mtSNV) as a position where the difference in heteroplasmy fraction between tumor and normal sample was ≥0.2. Our analysis identified 304 mtSNVs, with at least 1 mtSNV in 61% (164 of 268) of tumor samples. The noncoding control region had the greatest proportion of mtSNVs (60 of 304 mutations); this region contains sites that regulate mitochondrial DNA transcription and replication. Frequently mutated genes included ND5, RNR2, and CO1, plus 29 mutations in transfer RNA genes. mtSNVs in 2 separate mitochondrial genes (ND4 and ND6) were associated with shorter overall survival time. This association appeared to depend on the level of mtSNV heteroplasmy. Non-random co-occurrence between mtSNVs and mutations in nuclear genes indicates interactions between nuclear and mitochondrial DNA. In an analysis of primary tumors and metastases from 6 patients, we found tumors to accumulate mitochondrial mutational mutations as they progress. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

  12. Clinical prognostic value of DNA methylation in hepatoblastoma: Four novel tumor suppressor candidates.

    PubMed

    Honda, Shohei; Minato, Masashi; Suzuki, Hiromu; Fujiyoshi, Masato; Miyagi, Hisayuki; Haruta, Masayuki; Kaneko, Yasuhiko; Hatanaka, Kanako C; Hiyama, Eiso; Kamijo, Takehiko; Okada, Tadao; Taketomi, Akinobu

    2016-06-01

    Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  13. Breast tumor DNA methylation patterns associated with smoking in the Carolina Breast Cancer Study.

    PubMed

    Conway, Kathleen; Edmiston, Sharon N; Parrish, Eloise; Bryant, Christopher; Tse, Chiu-Kit; Swift-Scanlan, Theresa; McCullough, Lauren E; Kuan, Pei Fen

    2017-06-01

    Tobacco smoking is a risk factor in several cancers, yet its roles as a putative etiologic exposure or poor prognostic factor in breast cancer are less clear. Altered DNA methylation contributes to breast cancer development and may provide a mechanistic link between smoking and gene expression changes leading to cancer development or progression. Using a cancer-focused array, we examined methylation at 933 CpGs in 517 invasive breast tumors in the Carolina Breast Cancer Study to determine whether methylation patterns differ by exposure to tobacco smoke. Multivariable generalized linear regression models were used to compare tumor methylation profiles between smokers and never smokers, overall, or stratified on hormone receptor (HR) status. Modest differences in CpG methylation were detected at p < 0.05 in breast tumors from current or ever smokers compared with never smokers. In stratified analyses, HR- tumors from smokers exhibited primarily hypomethylation compared with tumors from never smokers; hypomethylation was similarly detected within the more homogeneous basal-like subtype. Most current smoking-associated CpG loci exhibited methylation levels in former smokers that were intermediate between those in current and never smokers and exhibited progressive changes in methylation with increasing duration of smoking. Among former smokers, restoration of methylation toward baseline (never smoking) levels was observed with increasing time since quitting. Moreover, smoking-related hypermethylation was stronger in HR+ breast tumors from blacks than in whites. Our results suggest that breast tumor methylation patterns differ with tobacco smoke exposure; however, additional studies are needed to confirm these findings.

  14. Acrolein- and 4-Aminobiphenyl-DNA adducts in human bladder mucosa and tumor tissue and their mutagenicity in human urothelial cells

    PubMed Central

    Weng, Mao-wen; Hu, Yu; Chen, Wei-sheng; Chou, David; Liu, Yan; Donin, Nicholas; Huang, William C.; Lepor, Herbert; Wu, Xue-Ru; Wang, Hailin; Beland, Frederick A.; Tang, Moon-shong

    2014-01-01

    Tobacco smoke (TS) is a major cause of human bladder cancer (BC). Two components in TS, 4-aminobiphenyl (4-ABP) and acrolein, which also are environmental contaminants, can cause bladder tumor in rat models. Their role in TS related BC has not been forthcoming. To establish the relationship between acrolein and 4-ABP exposure and BC, we analyzed acrolein-deoxyguanosine (dG) and 4-ABP-DNA adducts in normal human urothelial mucosa (NHUM) and bladder tumor tissues (BTT), and measured their mutagenicity in human urothelial cells. We found that the acrolein-dG levels in NHUM and BTT are 10-30 fold higher than 4-ABP-DNA adduct levels and that the acrolein-dG levels in BTT are 2 fold higher than in NHUM. Both acrolein-dG and 4-ABP-DNA adducts are mutagenic; however, the former are 5 fold more mutagenic than the latter. These two types of DNA adducts induce different mutational signatures and spectra. We found that acrolein inhibits nucleotide excision and base excision repair and induces repair protein degradation in urothelial cells. Since acrolein is abundant in TS, inhaled acrolein is excreted into urine and accumulates in the bladder and because acrolein inhibits DNA repair and acrolein-dG DNA adducts are mutagenic, we propose that acrolein is a major bladder carcinogen in TS. PMID:24939871

  15. Liquid biopsy - Performance of the PAXgene® Blood ccfDNA Tubes for the isolation and characterization of cell-free plasma DNA from tumor patients.

    PubMed

    Schmidt, Bernd; Reinicke, Dana; Reindl, Iris; Bork, Ines; Wollschläger, Bettina; Lambrecht, Nina; Fleischhacker, Michael

    2017-06-01

    In most research laboratories the use of EDTA tubes for the isolation of plasma DNA from tumor patients is standard. Unfortunately these tubes do not allow for an extended storage of samples before processing and prevent EDTA tubes from being shipped at ambient temperature. The aim of our study was to compare the quantity and quality of plasma DNA isolated from EDTA and PAXgene® Blood ccfDNA Tubes in different downstream applications. We enrolled 29 patients in our study. Blood samples were drawn into EDTA and PAXgene® Blood ccfDNA Tubes and were processed on day 0 and day 7 after storage at ambient temperature. The plasma DNA from 10 patients was isolated manually. For the DNA isolation from the plasma of 19 additional patients we used the automated QIAsymphony system. The total amount DNA from all samples was measured with a quantitative real-time PCR assay. In addition the amount of methylated mSHOX2 plasma DNA was determined. While the 7day storage lead to an increased amount of total DNA in almost all EDTA tubes, this effect was only seen in very few PAXgene® Blood ccfDNA Tubes. The stabilization solution which prevents the lysis of blood cells had no effect on the method for quantification of methylated sequences in these samples. The quantity and quality of plasma DNA from both types of blood draw tubes are comparable. DNA from PAXgene® Blood ccfDNA was successfully used for PCR-based quantification of total amount of cell-free DNA and for methylation analysis as well. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The DNA mismatch repair genes Msh3 and Msh6 cooperate in intestinal tumor suppression.

    PubMed

    Edelmann, W; Umar, A; Yang, K; Heyer, J; Kucherlapati, M; Lia, M; Kneitz, B; Avdievich, E; Fan, K; Wong, E; Crouse, G; Kunkel, T; Lipkin, M; Kolodner, R D; Kucherlapati, R

    2000-02-15

    Repair of mismatches in DNA in mammalian cells is mediated by a complex of proteins that are members of two highly conserved families of genes referred to as MutS and MutL homologues. Germline mutations in several members of these families, MSH2, MSH6, MLH1, and PMS2, but not MSH3, are responsible for hereditary non-polyposis colorectal cancer. To examine the role of MSH3, we generated a mouse with a null mutation in this gene. Cells from Msh3-/- mice are defective in repair of insertion/ deletion mismatches but can repair base-base mismatches. Msh3-/- mice develop tumors at a late age. When the Msh3-/- and Msh6-/- mutations are combined, the tumor predisposition phenotype is indistinguishable from Msh2-/- or Mlh1-/- mice. These results suggest that MSH3 cooperates with MSH6 in tumor suppression.

  17. Quantitative DNA Methylation Profiling in Cancer.

    PubMed

    Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

    2016-01-01

    Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

  18. The DNA methylation profile of liver tumors in C3H mice and identification of differentially methylated regions involved in the regulation of tumorigenic genes.

    PubMed

    Matsushita, Junya; Okamura, Kazuyuki; Nakabayashi, Kazuhiko; Suzuki, Takehiro; Horibe, Yu; Kawai, Tomoko; Sakurai, Toshihiro; Yamashita, Satoshi; Higami, Yoshikazu; Ichihara, Gaku; Hata, Kenichiro; Nohara, Keiko

    2018-03-22

    C3H mice have been frequently used in cancer studies as animal models of spontaneous liver tumors and chemically induced hepatocellular carcinoma (HCC). Epigenetic modifications, including DNA methylation, are among pivotal control mechanisms of gene expression leading to carcinogenesis. Although information on somatic mutations in liver tumors of C3H mice is available, epigenetic aspects are yet to be clarified. We performed next generation sequencing-based analysis of DNA methylation and microarray analysis of gene expression to explore genes regulated by DNA methylation in spontaneous liver tumors of C3H mice. Overlaying these data, we selected cancer-related genes whose expressions are inversely correlated with DNA methylation levels in the associated differentially methylated regions (DMRs) located around transcription start sites (TSSs) (promoter DMRs). We further assessed mutuality of the selected genes for expression and DNA methylation in human HCC using the Cancer Genome Atlas (TCGA) database. We obtained data on genome-wide DNA methylation profiles in the normal and tumor livers of C3H mice. We identified promoter DMRs of genes which are reported to be related to cancer and whose expressions are inversely correlated with the DNA methylation, including Mst1r, Slpi and Extl1. The association between DNA methylation and gene expression was confirmed using a DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in Hepa1c1c7 cells and Hepa1-6 cells. Overexpression of Mst1r in Hepa1c1c7 cells illuminated a novel downstream pathway via IL-33 upregulation. Database search indicated that gene expressions of Mst1r and Slpi are upregulated and the TSS upstream regions are hypomethylated also in human HCC. These results suggest that DMRs, including those of Mst1r and Slpi, are involved in liver tumorigenesis in C3H mice, and also possibly in human HCC. Our study clarified genome wide DNA methylation landscape of C3H mice. The data provide useful information

  19. Genome-wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma.

    PubMed

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J; Almutairi, Bader; Etchevers, Heather C; McConville, Carmel; Malik, Karim T A; Brown, Keith W

    2017-04-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

  20. Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

    PubMed Central

    Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R.; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J.; Almutairi, Bader; Etchevers, Heather C.; McConville, Carmel; Malik, Karim T. A.

    2016-01-01

    Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27862318

  1. Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients.

    PubMed

    Wang, Peilu; Bahreini, Amir; Gyanchandani, Rekha; Lucas, Peter C; Hartmaier, Ryan J; Watters, Rebecca J; Jonnalagadda, Amruth R; Trejo Bittar, Humberto E; Berg, Aaron; Hamilton, Ronald L; Kurland, Brenda F; Weiss, Kurt R; Mathew, Aju; Leone, Jose Pablo; Davidson, Nancy E; Nikiforova, Marina N; Brufsky, Adam M; Ambros, Tadeu F; Stern, Andrew M; Puhalla, Shannon L; Lee, Adrian V; Oesterreich, Steffi

    2016-03-01

    Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. See related commentary by Gu and Fuqua, p. 1034. ©2015 American Association for Cancer Research.

  2. Circulating Tumor DNA Measurement by Picoliter Droplet-Based Digital PCR and Vemurafenib Plasma Concentrations in Patients with Advanced BRAF-Mutated Melanoma.

    PubMed

    Garlan, Fanny; Blanchet, Benoit; Kramkimel, Nora; Puszkiel, Alicja; Golmard, Jean-Louis; Noe, Gaelle; Dupin, Nicolas; Laurent-Puig, Pierre; Vidal, Michel; Taly, Valerie; Thomas-Schoemann, Audrey

    2017-06-01

    Circulating tumor DNA (ctDNA) has been reported as a prognostic marker in melanoma. In BRAF V600-mutant melanoma, a plasma under-exposure to vemurafenib could favor emerging resistance but no biological data are available to support this hypothesis. We aimed to investigate the relationship between vemurafenib plasma concentrations and the ctDNA plasma concentration during follow-up of BRAF-mutated melanoma patients. Eleven patients treated with single-agent vemurafenib for advanced BRAF V600-mutant melanoma were analyzed in an exploratory monocentric study. The vemurafenib plasma concentration was measured by liquid chromatography. ctDNA was extracted from plasma samples and the ctDNA concentration was evaluated using picoliter droplet-based digital PCR with Taqman ® detection probes targeting the BRAF p.V600E/K mutation and wild-type BRAF sequences. At baseline, plasma ctDNA was detectable in 72% (n = 8/11) of patients and the ctDNA concentration decreased in 88% of these patients (n = 7/8) from day (D) 0 to D15 after vemurafenib initiation. During follow-up, an increased ctDNA concentration was detected in nine patients: in five patients, the first increase in ctDNA concentrations followed a decrease in vemurafenib concentrations. More interestingly, an inverse correlation between vemurafenib concentration and ctDNA concentrations was demonstrated (p = 0.026). The ctDNA concentration at baseline was associated with overall survival (hazard ratio = 2.61, 95% CI 1.04-6.56; p = 0.04). This study demonstrates the relevance of vemurafenib plasma monitoring during the follow-up of metastatic melanoma patients. Plasma drug monitoring and ctDNA concentrations could be combined to monitor tumor evolution in melanoma patients treated with anti-BRAF therapies.

  3. Absolute cross section for loss of supercoiled topology induced by 10 eV electrons in highly uniform /DNA/1,3-diaminopropane films deposited on highly ordered pyrolitic graphite

    PubMed Central

    Boulanouar, Omar; Fromm, Michel; Bass, Andrew D.; Cloutier, Pierre; Sanche, Léon

    2013-01-01

    It was recently shown that the affinity of doubly charged, 1–3 diaminopropane (Dap2+) for DNA permits the growth on highly ordered pyrolitic graphite (HOPG) substrates, of plasmid DNA films, of known uniform thickness [O. Boulanouar, A. Khatyr, G. Herlem, F. Palmino, L. Sanche, and M. Fromm, J. Phys. Chem. C 115, 21291–21298 (2011)]. Post-irradiation analysis by electrophoresis of such targets confirms that electron impact at 10 eV produces a maximum in the yield of single strand breaks that can be associated with the formation of a DNA− transient anion. Using a well-adapted deterministic survival model for the variation of electron damage with fluence and film thickness, we have determined an absolute cross section for strand-break damage by 10 eV electrons and inelastic scattering attenuation length in DNA-Dap complex films. PMID:23927289

  4. Radiation induction of drug resistance in RIF-1 tumors and tumor cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hopwood, L.E.; Moulder, J.E.

    1989-11-01

    The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not formore » ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.« less

  5. Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

    PubMed

    Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P

    2017-09-01

    We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  6. ESR1 Mutations in Circulating Plasma Tumor DNA from Metastatic Breast Cancer Patients.

    PubMed

    Chu, David; Paoletti, Costanza; Gersch, Christina; VanDenBerg, Dustin A; Zabransky, Daniel J; Cochran, Rory L; Wong, Hong Yuen; Toro, Patricia Valda; Cidado, Justin; Croessmann, Sarah; Erlanger, Bracha; Cravero, Karen; Kyker-Snowman, Kelly; Button, Berry; Parsons, Heather A; Dalton, W Brian; Gillani, Riaz; Medford, Arielle; Aung, Kimberly; Tokudome, Nahomi; Chinnaiyan, Arul M; Schott, Anne; Robinson, Dan; Jacks, Karen S; Lauring, Josh; Hurley, Paula J; Hayes, Daniel F; Rae, James M; Park, Ben Ho

    2016-02-15

    Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. ©2015 American Association for Cancer Research.

  7. Phenothiazine Inhibitors of TLKs Affect Double-Strand Break Repair and DNA Damage Response Recovery and Potentiate Tumor Killing with Radiomimetic Therapy

    PubMed Central

    Ronald, Sharon; Awate, Sanket; Rath, Abhijit; Carroll, Jennifer; Galiano, Floyd; Dwyer, Donard; Kleiner-Hancock, Heather; Mathis, J. Michael; Vigod, Simone

    2013-01-01

    The Tousled-like kinases (TLKs) are involved in chromatin assembly, DNA repair, and transcription. Two TLK genes exist in humans, and their expression is often dysregulated in cancer. TLKs phosphorylate Asf1 and Rad9, regulating double-strand break (DSB) repair and the DNA damage response (DDR). TLKs maintain genomic stability and are important therapeutic intervention targets. We identified specific inhibitors of TLKs from several compound libraries, some of which belong to the family of phenothiazine antipsychotics. The inhibitors prevented the TLK-mediated phosphorylation of Rad9(S328) and impaired checkpoint recovery and DSB repair. The inhibitor thioridazine (THD) potentiated tumor killing with chemotherapy and also had activity alone. Staining for γ-H2AX revealed few positive cells in untreated tumors, but large numbers in mice treated with low doxorubicin or THD alone, possibly the result of the accumulation of DSBs that are not promptly repaired as they may occur in the harsh tumor growth environment. PMID:23946870

  8. Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors.

    PubMed

    Chen, Xiaofei; Liu, Liang; Mims, Jade; Punska, Elizabeth C; Williams, Kristin E; Zhao, Weiling; Arcaro, Kathleen F; Tsang, Allen W; Zhou, Xiaobo; Furdui, Cristina M

    2015-01-01

    Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2'deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.

  9. Mannosylated protamine as a novel DNA vaccine carrier for effective induction of anti-tumor immune responses.

    PubMed

    Zeng, Zhaoyan; Dai, Shuang; Jiao, Yan; Jiang, Lei; Zhao, Yuekui; Wang, Bo; Zong, Li

    2016-06-15

    Gene immunotherapy has been developed as a promising strategy for inhibition of tumor growth. In the study, mannosylated protamine sulphate (MPS) was used as a novel DNA vaccine carrier to enhance transfection efficiency and anti-tumor immune responses. Anti-GRP DNA vaccine (pGRP) was selected as a model gene and condensed by MPS to form MPS/pGRP nanoparticles. The cellular uptake and transfection efficiency of MPS/pGRP nanoparticles in macrophages were evaluated. The effect of the nanoparticles in enhancing GRP-specific humoral immune response was then evaluated by nasal vaccination of nanoparticles in mice. The results demonstrated that both the cellular uptake and transfection efficiency of MPS nanoparticles in macrophages were higher than those of protamine nanoparticles. MPS/pGRP nanoparticles stimulated the production of higher titers (3.9×10(3)) of specific antibodies against GRP than those of protamine/pGRP nanoparticles (6.4×10(2), p<0.01) and intramuscular injection pGRP solution (2.5×10(3), p<0.05). Furthermore, the inhibitory rate in MPS/pGRP nanoparticles group (65.80%) was significantly higher than that in protamine/pGRP nanoparticles group (35.13%) and pGRP solution group (43.39%). Hence, it is evident that MPS is an efficient targeting gene delivery carrier which could improve in vitro transfection efficiency as well as anti-tumor immunotherapy in mice. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Roles of polysaccharide from Branchiostoma belcheri in anti-DNA oxidation and anti-tumor activity in S180 mice

    NASA Astrophysics Data System (ADS)

    Liang, Hui; Zhang, Shicui

    2009-11-01

    In this study, we isolated a polysaccharide from Branchiostoma belcheri (PBB) by enzymatic protein hydrolysis and alcohol precipitation. We investigated the effects of PBB supplementation on DNA oxidation and growth of the transplanted tumor cells Sarcoma (S180) in mice. Sixty healthy Kunming mice weighing between 18 and 25 g were randomly assigned to 6 groups, each consisting of 10 animals. All the mice, except for the blank control group, were inoculated with S180 sarcoma cells into the axilla of the left foreleg. PBB was given to mice by gavage at doses of 0 (model control), 25, 50, or 100 mg/kg b.w. in 0.2 ml saline for 30 days. The fifth group of S180-mice was given cytoxan (50 mg/kg) by peritoneal injection as a positive control group. The animals had free access to food and water. The mice were sacrificed after the final treatment and blood was quickly collected. Spontaneous and oxidized DNA damage of peripheral lymphocytes induced by H2O2 were analyzed by SCGE. O6-methyl-guanine (O6-MeG) was measured by high-performance capillary zone electrophoresis. The average tumor weights (0.856-1.118 g) of the three PBB groups were significantly lower than that of the model control group (1.836 g) ( p<0.05). The tumor inhibition ratios of the PBB groups were 39.1%-53.4% and similar to the cytoxan positive group (57.5%). There were no significant differences in spontaneous DNA damage in peripheral lymphocytes among the groups. The oxidative DNA damage induced by 10 µmol/L H2O2 in the 50 and 100 mg/kg b.w. groups were 246.1 AU and 221.7 AU, respectively, both of which were significantly lower than that in the model group (289.0 AU; p<0.05). The plasma concentrations of O6-MeG in the 25, 50, and 100 mg/kg supplemented groups were 2.09 µmol/L, 1.86 µmol/L, and 1.63 µmol/L, respectively, all of which were significantly lower than that of the model group (2.67 µmol/L; p<0.05). These results indicated that PBB may have antioxidative activity and thus reduce oxidation

  11. Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

    PubMed

    Maohuai, C; Chang, A R; Lo, D

    2001-06-01

    To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.

  12. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    PubMed Central

    Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

  13. A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors

    PubMed Central

    Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D.; Ambrosone, Christine B.; Higgins, Michael J.; Sucheston-Campbell, Lara E.

    2017-01-01

    Background DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. Results For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER– tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Materials and Methods Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER−) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. Conclusions FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues. PMID:28118602

  14. A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

    PubMed

    Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

    2017-02-28

    DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

  15. Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

    PubMed

    Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

    2018-06-13

    The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

  16. INDUCTION OF DNA ADDUCTS, TUMORS, AND KI-RAS ONCOGENE MUTATIONS IN STRAIN A/J MOUSE LUNG BY IP. ADMINISTRATION OF DIBENZ[A,H]ANTHRACENE

    EPA Science Inventory

    Induction of DNA adducts, tumors, and Ki-ras oncogene mutations in strain AlJ mouse lung by ip. administration of dibenz[a,h]anthracene

    Previous studies of polycyclic aromatic hydrocarbon (P AH) induced lung tumors in the strain NJ mouse model system have demonstrated qua...

  17. Absolute Quantitation of DNA Methylation of 28 Candidate Genes in Prostate Cancer Using Pyrosequencing

    PubMed Central

    Vasiljeviš, Nataڑa; Wu, Keqiang; Brentnall, Adam R.; Kim, Dae Cheol; Thorat, Mangesh A.; Kudahetti, Sakunthala C.; Mao, Xueying; Xue, Liyan; Yu, Yongwei; Shaw, Greg L.; Beltran, Luis; Lu, Yong-Jie; Berney, Daniel M.; Cuzick, Jack; Lorincz, Attila T.

    2011-01-01

    Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa from BPH. Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001). Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential. PMID:21694441

  18. Chemo brain or tumor brain - that is the question: the presence of extracranial tumors profoundly affects molecular processes in the prefrontal cortex of TumorGraft mice

    PubMed Central

    Kovalchuk, Anna; Ilnytskyy, Yaroslav; Rodriguez-Juarez, Rocio; Shpyleva, Svitlana; Melnyk, Stepan; Pogribny, Igor; Katz, Amanda; Sidransky, David; Kovalchuk, Olga; Kolb, Bryan

    2017-01-01

    Cancer chemotherapy causes numerous persistent central nervous system complications. This condition is known as chemo brain. Cognitive impairments occur even before treatment, and hence are referred to as cancer associated cognitive changes, or tumor brain. There is much yet to be learned about the mechanisms of both chemo brain and tumor brain. The frequency and timing of chemo brain and tumor brain occurrence and persistence strongly suggest they may be epigenetic in nature and associated with altered gene expression. Here we used TumorGraftTM models wherein part of a patient's tumor is removed and grafted into immune-deficient mice and conducted global gene expression and DNA methylation analysis. We show that malignant non-central nervous system tumor growth causes profound molecular alterations in the brain. Mice harbouring triple negative or progesterone positive breast cancer TumorGrafts exhibited altered gene expression, decreased levels of DNA methylation, increased levels of DNA hydroxymethylation, and oxidative stress in the prefrontal cortex. Interestingly, chemotherapy did not have any additional synergistic effects on the analyzed processes. The molecular changes observed in this study are known signs of neurodegeneration and brain aging. This study provides an important roadmap for future large-scale analysis of the molecular and cellular mechanisms of tumor brain. PMID:28758896

  19. Small-molecule inhibitors of DNA damage-repair pathways: an approach to overcome tumor resistance to alkylating anticancer drugs

    PubMed Central

    Srinivasan, Ajay; Gold, Barry

    2013-01-01

    A major challenge in the future development of cancer therapeutics is the identification of biological targets and pathways, and the subsequent design of molecules to combat the drug-resistant cells hiding in virtually all cancers. This therapeutic approach is justified based upon the limited advances in cancer cures over the past 30 years, despite the development of many novel chemotherapies and earlier detection, which often fail due to drug resistance. Among the various targets to overcome tumor resistance are the DNA repair systems that can reverse the cytotoxicity of many clinically used DNA-damaging agents. Some progress has already been made but much remains to be done. We explore some components of the DNA-repair process, which are involved in repair of alkylation damage of DNA, as targets for the development of novel and effective molecules designed to improve the efficacy of existing anticancer drugs. PMID:22709253

  20. DNA methylation screening of primary prostate tumors identifies SRD5A2 and CYP11A1 as candidate markers for assessing risk of biochemical recurrence.

    PubMed

    Horning, Aaron M; Awe, Julius A; Wang, Chiou-Miin; Liu, Joseph; Lai, Zhao; Wang, Vickie Yao; Jadhav, Rohit R; Louie, Anna D; Lin, Chun-Lin; Kroczak, Tad; Chen, Yidong; Jin, Victor X; Abboud-Werner, Sherry L; Leach, Robin J; Hernandez, Javior; Thompson, Ian M; Saranchuk, Jeff; Drachenberg, Darrel; Chen, Chun-Liang; Mai, Sabine; Huang, Tim Hui-Ming

    2015-11-01

    Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. . © 2015 Wiley Periodicals, Inc.

  1. Thymine DNA Glycosylase (TDG) is involved in the pathogenesis of intestinal tumors with reduced APC expression.

    PubMed

    Xu, Jinfei; Cortellino, Salvatore; Tricarico, Rossella; Chang, Wen-Chi; Scher, Gabrielle; Devarajan, Karthik; Slifker, Michael; Moore, Robert; Bassi, Maria Rosaria; Caretti, Elena; Clapper, Margie; Cooper, Harry; Bellacosa, Alfonso

    2017-10-27

    Thymine DNA Glycosylase (TDG) is a base excision repair enzyme that acts as a thymine and uracil DNA N-glycosylase on G:T and G:U mismatches, thus protecting CpG sites in the genome from mutagenesis by deamination. In addition, TDG has an epigenomic function by removing the novel cytosine derivatives 5-formylcytosine and 5-carboxylcytosine (5caC) generated by Ten-Eleven Translocation (TET) enzymes during active DNA demethylation. We and others previously reported that TDG is essential for mammalian development. However, its involvement in tumor formation is unknown. To study the role of TDG in tumorigenesis, we analyzed the effects of its inactivation in a well-characterized model of tumor predisposition, the Apc Min mouse strain. Mice bearing a conditional Tdg flox allele were crossed with Fabpl ::Cre transgenic mice, in the context of the Apc Min mutation, in order to inactivate Tdg in the small intestinal and colonic epithelium. We observed an approximately 2-fold increase in the number of small intestinal adenomas in the test Tdg -mutant Apc Min mice in comparison to control genotypes (p=0.0001). This increase occurred in female mice, and is similar to the known increase in intestinal adenoma formation due to oophorectomy. In the human colorectal cancer (CRC) TCGA database, the subset of patients with TDG and APC expression in the lowest quartile exhibits an excess of female cases. We conclude that TDG inactivation plays a role in intestinal tumorigenesis initiated by mutation/underexpression of APC . Our results also indicate that TDG may be involved in sex-specific protection from CRC.

  2. The construction of cDNA library and the screening of related antigen of ascitic tumor cells of ovarian cancer.

    PubMed

    Hou, Q; Chen, K; Shan, Z

    2015-01-01

    To construct the cDNA library of the ascites tumor cells of ovarian cancer, which can be used to screen the related antigen for the early diagnosis of ovarian cancer and therapeutic targets of immune treatment. Four cases of ovarian serous cystadenocarcinoma, two cases of ovarian mucinous cystadenocarcinoma, and two cases of ovarian endometrial carcinoma in patients with ascitic tumor cells which were used to construct the cDNA library. To screen the ovarian cancer antigen gene, evaluate the enzyme, and analyze nucleotide sequence, serological analysis of recombinant tumor cDNA expression libraries (SEREX) and suppression subtractive hybridization technique (SSH) techniques were utilized. The detection method of recombinant expression-based serological mini-arrays (SMARTA) was used to detect the ovarian cancer antigen and the positive reaction of 105 cases of ovarian cancer patients and 105 normal women's autoantibodies correspondingly in serum. After two rounds of serologic screening and glycosides sequencing analysis, 59 candidates of ovarian cancer antigen gene fragments were finally identified, which corresponded to 50 genes. They were then divided into six categories: (1) the homologous genes which related to the known ovarian cancer genes, such as BARD 1 gene, etc; (2) the homologous genes which were associated with other tumors, such as TM4SFI gene, etc; (3) the genes which were expressed in a special organization, such as ILF3, FXR1 gene, etc; (4) the genes which were the same with some protein genes of special function, such as TIZ, ClD gene; (5) the homologous genes which possessed the same source with embryonic genes, such as PKHD1 gene, etc; (6) the remaining genes were the unknown genes without the homologous sequence in the gene pool, such as OV-189 genes. SEREX technology combined with SSH method is an effective research strategy which can filter tumor antigen with high specific character; the corresponding autoantibodies of TM4SFl, ClD, TIZ, BARDI

  3. Relative expression of rRNA transcripts and 45S rDNA promoter methylation status are dysregulated in tumors in comparison with matched-normal tissues in breast cancer.

    PubMed

    Karahan, Gurbet; Sayar, Nilufer; Gozum, Gokcen; Bozkurt, Betul; Konu, Ozlen; Yulug, Isik G

    2015-06-01

    Ribosomal RNA (rRNA) expression, one of the most important factors regulating ribosome production, is primarily controlled by a CG-rich 45 S rDNA promoter. However, the DNA methylation state of the 45 S rDNA promoter, as well as its effect on rRNA gene expression in types of human cancers is controversial. In the present study we analyzed the methylation status of the rDNA promoter (-380 to +53 bp) as well as associated rRNA expression levels in breast cancer cell lines and breast tumor-normal tissue pairs. We found that the aforementioned regulatory region was extensively methylated (74-96%) in all cell lines and in 68% (13/19 tumor-normal pairs) of the tumors. Expression levels of rRNA transcripts 18 S, 28 S, 5.8 S and 45 S external transcribed spacer (45 S ETS) greatly varied in the breast cancer cell lines regardless of their methylation status. Analyses of rRNA transcript expression levels in the breast tumor and normal matched tissues showed no significant difference when normalized with TBP. On the other hand, using the geometric mean of the rRNA expression values (GM-rRNA) as reference enabled us to identify significant changes in the relative expression of rRNAs in the tissue samples. We propose GM-rRNA normalization as a novel strategy to analyze expression differences between rRNA transcripts. Accordingly, the 18S rRNA/GM-rRNA ratio was significantly higher whereas the 5.8S rRNA/GM-rRNA ratio was significantly lower in breast tumor samples than this ratio in the matched normal samples. Moreover, the 18S rRNA/GM-rRNA ratio was negatively correlated with the 45 S rDNA promoter methylation level in the normal breast tissue samples, yet not in the breast tumors. Significant correlations observed between the expression levels of rRNA transcripts in the normal samples were lost in the tumor samples. We showed that the expression of rRNA transcripts may not be based solely on promoter methylation. Carcinogenesis may cause dysregulation of the correlation

  4. Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dumaz, N.; Drougard, C.; Sarasin, A.

    1993-11-15

    The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a goodmore » target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC [yields] TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues.« less

  5. Do circulating tumor cells, exosomes, and circulating tumor nucleic acids have clinical utility? A report of the association for molecular pathology.

    PubMed

    Gold, Bert; Cankovic, Milena; Furtado, Larissa V; Meier, Frederick; Gocke, Christopher D

    2015-05-01

    Diagnosing and screening for tumors through noninvasive means represent an important paradigm shift in precision medicine. In contrast to tissue biopsy, detection of circulating tumor cells (CTCs) and circulating tumor nucleic acids provides a minimally invasive method for predictive and prognostic marker detection. This allows early and serial assessment of metastatic disease, including follow-up during remission, characterization of treatment effects, and clonal evolution. Isolation and characterization of CTCs and circulating tumor DNA (ctDNA) are likely to improve cancer diagnosis, treatment, and minimal residual disease monitoring. However, more trials are required to validate the clinical utility of precise molecular markers for a variety of tumor types. This review focuses on the clinical utility of CTCs and ctDNA testing in patients with solid tumors, including somatic and epigenetic alterations that can be detected. A comparison of methods used to isolate and detect CTCs and some of the intricacies of the characterization of the ctDNA are also provided. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  6. A novel strategy for highly efficient isolation and analysis of circulating tumor-specific cell-free DNA from lung cancer patients using a reusable conducting polymer nanostructure.

    PubMed

    Lee, HyungJae; Jeon, SeungHyun; Seo, Jin-Suck; Goh, Sung-Ho; Han, Ji-Youn; Cho, Youngnam

    2016-09-01

    We have developed a reusable nanostructured polypyrrole nanochip and demonstrated its use in the electric field-mediated recovery of circulating cell-free DNA (cfDNA) from the plasma of lung cancer patients. Although cfDNA has been recognized and widely studied as a versatile and promising biomarker for the diagnosis and prognosis of cancers, the lack of efficient strategies to directly isolate cfDNA from the plasma has become a great hindrance to its potential clinical use. As a proof-of-concept study, we demonstrated a technique for the rapid and efficient isolation of cfDNA with high yield and purity. In particular, the synergistic effects of the electro-activity and the nanostructured features of the polypyrrole polymer enabled repeated retrieval of cfDNA using a single platform. Moreover, polypyrrole nanochip facilitated the amplification of tumor-specific DNA fragments from the plasma samples of patients with lung cancer characterized by mutations in exons 21 of the epidermal growth factor receptor gene (EGFR). Overall, the proposed polypyrrole nanochip has enormous potential for industrial and clinical applications with significantly enhanced efficiency in the recovery of tumor-associated circulating cfDNA. This may ultimately contribute to more robust and reliable evaluation of gene mutations in peripheral blood. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Adenomatous tumors of the middle ear and temporal bone: clinical, morphological and tumor biological characteristics of challenging neoplastic lesions.

    PubMed

    Duderstadt, M; Förster, Christine; Welkoborsky, H-J; Ostertag, H

    2012-03-01

    Adenomatous tumors of the middle ear and temporal bone are rare tumors. In this retrospective study, we examined nine patients who underwent surgery for an adenomatous tumor of the middle ear, mastoid cavity or eustachian tube. In seven patients, a middle ear adenoma (MEA) and in two patients an aggressive papillary tumor (APT) was diagnosed. We report the clinical, radiologic, morphologic, immunohistochemical and DNA image cytometrical characteristics that can help to correctly classify these tumors. Therapy consisted of surgical excision of the tumors in eight cases. In one elderly patient, only a large biopsy was taken, because this patient suffered from cardial and kidney disorders and was not suitable for an extended surgical approach. This patient received stereotactic radiotherapy. Seven patients underwent planned second look operation. Recurrences occurred in three patients (one with APT, two with MEA), whereas in two of these cases rather a residual tumor due to initial incomplete tumor resection occurred. By image analysis, DNA cytometry MEA were considered benign, whereas the appearance of aneuploid tumor cells in APT confirmed these tumors as low grade malignant lesions. The proliferation rates were equally low in both entities. APT and MEA are tumor entities which can only be correctly classified by a synopsis of histopathology, immunohistochemistry and DNA image cytometry. The recommended therapy is the complete tumor excision. In cases of APT, von Hippel-Lindau syndrome has to be excluded.

  8. Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study

    PubMed Central

    Zonta, Eleonora; Didelot, Audrey; Combe, Pierre; Thibault, Constance; Gibault, Laure; Lours, Camille; Taly, Valérie; Laurent-Puig, Pierre

    2016-01-01

    Background Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive “real-time” biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). Methods and Findings We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01–3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30–3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66–6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR

  9. Topical application of ochratoxin A causes DNA damage and tumor initiation in mouse skin.

    PubMed

    Kumar, Rahul; Ansari, Kausar M; Chaudhari, Bhushan P; Dhawan, Alok; Dwivedi, Premendra D; Jain, Swatantra K; Das, Mukul

    2012-01-01

    Skin cancer is one of the most common forms of cancer and 2-3 million new cases are being diagnosed globally each year. Along with UV rays, environmental pollutants/chemicals including mycotoxins, contaminants of various foods and feed stuffs, could be one of the aetiological factors of skin cancer. In the present study, we evaluated the DNA damaging potential and dermal carcinogenicity of a mycotoxin, ochratoxin A (OTA), with the rationale that dermal exposure to OTA in workers may occur during their involvement in pre and post harvest stages of agriculture. A single topical application of OTA (20-80 µg/mouse) resulted in significant DNA damage along with elevated γ-H2AX level in skin. Alteration in oxidative stress markers such as lipid peroxidation, protein carbonyl, glutathione content and antioxidant enzymes was observed in a dose (20-80 µg/mouse) and time-dependent (12-72 h) manner. The oxidative stress was further emphasized by the suppression of Nrf2 translocation to nucleus following a single topical application of OTA (80 µg/mouse) after 24 h. OTA (80 µg/mouse) application for 12-72 h caused significant enhancement in- (a) reactive oxygen species generation, (b) activation of ERK1/2, p38 and JNK MAPKs, (c) cell cycle arrest at G0/G1 phase (37-67%), (d) induction of apoptosis (2.0-11.0 fold), (e) expression of p53, p21/waf1, (f) Bax/Bcl-2 ratio, (g) cytochrome c level, (h) activities of caspase 9 (1.2-1.8 fold) and 3 (1.7-2.2 fold) as well as poly ADP ribose polymerase cleavage. In a two-stage mouse skin tumorigenesis protocol, it was observed that a single topical application of OTA (80 µg/mouse) followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate for 24 week leads to tumor formation. These results suggest that OTA has skin tumor initiating property which may be related to oxidative stress, MAPKs signaling and DNA damage.

  10. Nontoxic concentration of DNA-PK inhibitor NU7441 radio-sensitizes lung tumor cells with little effect on double strand break repair.

    PubMed

    Sunada, Shigeaki; Kanai, Hideki; Lee, Younghyun; Yasuda, Takeshi; Hirakawa, Hirokazu; Liu, Cuihua; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

    2016-09-01

    High-linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non-toxic concentration and carbon ions. NU7441-treated non-small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X-rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence-associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio-sensitization in tumor cells exposed to X-rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53-null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA-PK inhibitor contributes to various modes of cell death in a p53-dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio-sensitized by a low concentration of DNA-PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  11. Measurement of telomerase activity in dog tumors.

    PubMed

    Yazawa, M; Okuda, M; Setoguchi, A; Nishimura, R; Sasaki, N; Hasegawa, A; Watari, T; Tsujimoto, H

    1999-10-01

    Telomeres are specific structures present at the end of liner chromosomes. DNA polymerase can not synthesize the end of liner DNA and, as a result, the telomeres become progressively shortened by successive cell divisions. To overcome the end replication problem, telomerase adds new telomeric sequences to the end of chromosomal DNA. The enzyme activity is undetectable in most normal human adult somatic cells, in which shortening of the telomere is thought to limit the somatic-cell life span. In contrast to normal somatic cells, many human tumors possess telomerase activity. The present study looked at whether telomerase activity might serve as a marker for canine tumors. Telomerase activity was measured using the telomeric repeat amplification protocol assay. Normal dog somatic tissues showed little or no telomerase activity, while normal testis exhibited a high level of telomerase activity. We measured telomerase activity in tumor samples from 45 dogs; 21 mammary gland tumors, 16 tumors developed in the skin and oral cavity, 7 vascular tumors and 1 Sertoli cell tumor. Greater than 95% of the tumor samples contained telomerase activity (3-924 U/2 micrograms protein). The results obtained in this study indicated that telomerase should be a useful diagnostic marker for a variety of dog tumors, and it may serve as a target for antitumor chemotherapy.

  12. Prime-boost immunization by both DNA vaccine and oncolytic adenovirus expressing GM-CSF and shRNA of TGF-β2 induces anti-tumor immune activation

    PubMed Central

    Choi, Hye Jin; Joo, Yeonsoo; Kim, Joo-Hang; Song, Jae J.

    2017-01-01

    A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-β2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-β2 (shmTGF-β2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-β2 (AdGshT) with that of oncolytic adenovirus expressing mGM-CSF only (AdG), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received AdGshT showed delayed tumor growth than those that received AdG. Heterologous prime-boost immunization was combined with oncolytic AdGshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-β2, tumor growth inhibition by shmTGF-β2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse

  13. Prime-boost immunization by both DNA vaccine and oncolytic adenovirus expressing GM-CSF and shRNA of TGF-β2 induces anti-tumor immune activation.

    PubMed

    Kim, So Young; Kang, Dongxu; Choi, Hye Jin; Joo, Yeonsoo; Kim, Joo-Hang; Song, Jae J

    2017-02-28

    A successful DNA vaccine for the treatment of tumors should break established immune tolerance to tumor antigen. However, due to the relatively low immunogenicity of DNA vaccines, compared to other kinds of vaccines using live virus or protein, a recombinant viral vector was used to enhance humoral and cellular immunity. In the current study, we sought to develop a novel anti-cancer agent as a complex of DNA and oncolytic adenovirus for the treatment of malignant melanoma in the C57BL/6 mouse model. MART1, a human melanoma-specific tumor antigen, was used to induce an increased immune reaction, since a MART1-protective response is required to overcome immune tolerance to the melanoma antigen MelanA. Because GM-CSF is a potent inducer of anti-tumor immunity and TGF-β2 is involved in tumor survival and host immune suppression, mouse GM-CSF (mGM-CSF) and shRNA of mouse TGF-β2 (shmTGF-β2) genes were delivered together with MART1 via oncolytic adenovirus. MART1 plasmid was also used for antigen-priming. To compare the anti-tumor effect of oncolytic adenovirus expressing both mGM-CSF and shmTGF-β2 (AdGshT) with that of oncolytic adenovirus expressing mGM-CSF only (AdG), each virus was intratumorally injected into melanoma-bearing C57BL/6 mice. As a result, mice that received AdGshT showed delayed tumor growth than those that received AdG. Heterologous prime-boost immunization was combined with oncolytic AdGshT and MART1 expression to result in further delayed tumor growth. This regression is likely due to the following 4 combinations: MART1-derived mouse melanoma antigen-specific immune reaction, immune stimulation by mGM-CSF/shmTGF-β2, tumor growth inhibition by shmTGF-β2, and tumor cell-specific lysis via an oncolytic adenovirus. Immune activation was mainly induced by mature tumor-infiltrating dendritic cell (TIDC) and lowered regulatory T cells in tumor-infiltrating lymphocytes (TIL). Taken together, these findings demonstrate that human MART1 induces a mouse

  14. Polyphenols in brewed green tea inhibit prostate tumor xenograft growth by localizing to the tumor and decreasing oxidative stress and angiogenesis

    PubMed Central

    Henning, Susanne M.; Wang, Piwen; Said, Jonathan; Magyar, Clara; Castor, Brandon; Doan, Ngan; Tosity, Carmen; Moro, Aune; Gao, Kun; Li, Luyi; Heber, David

    2011-01-01

    It has been demonstrated in various animal models that the oral administration of green tea (GT) extracts in drinking water can inhibit tumor growth, but the effects of brewed GT on factors promoting tumor growth, including oxidant damage of DNA and protein, angiogenesis, and DNA methylation, have not been tested in an animal model. To explore these potential mechanisms, brewed GT was administered instead of drinking water to male severe combined immunodeficiency (SCID) mice with androgen-dependent human LAPC4 prostate cancer cell subcutaneous xenografts. Tumor volume was decreased significantly in mice consuming GT, and tumor size was significantly correlated with GT polyphenol (GTP) content in tumor tissue. There was a significant reduction in hypoxia-inducible factor 1-alpha and vascular endothelial growth factor protein expression. GT consumption significantly reduced oxidative DNA and protein damage in tumor tissue as determined by 8-hydroxydeoxyguanosine/deoxyguanosine ratio and protein carbonyl assay, respectively. Methylation is known to inhibit antioxidative enzymes such as glutathione S-transferase pi (GSTp1) to permit reactive oxygen species promotion of tumor growth. GT inhibited tumor 5-cytosine DNA methyltransferase 1 (DNMT1) mRNA and protein expression significantly, which may contribute to the inhibition of tumor growth by reactivation of antioxidative enzymes. This study advances our understanding of tumor growth inhibition by brewed GT in an animal model by demonstrating tissue localization of GTPs in correlation with inhibition of tumor growth. Our results suggest that the inhibition of tumor growth is due to GTP-mediated inhibition of oxidative stress and angiogenesis in the LAPC4 xenograft prostate tumor in SCID mice. PMID:22405694

  15. Global DNA methylation analysis reveals miR-214-3p contributes to cisplatin resistance in pediatric intracranial nongerminomatous malignant germ cell tumors.

    PubMed

    Hsieh, Tsung-Han; Liu, Yun-Ru; Chang, Ting-Yu; Liang, Muh-Lii; Chen, Hsin-Hung; Wang, Hsei-Wei; Yen, Yun; Wong, Tai-Tong

    2018-03-27

    Pediatric central nervous system germ cell tumors (CNSGCTs) are rare and heterogeneous neoplasms, which can be divided into germinomas and nongerminomatous germ cell tumors (NGGCTs). NGGCTs are further subdivided into mature teratomas and nongerminomatous malignant GCTs (NGMGCTs). Clinical outcomes suggest that NGMGCTs have poor prognosis and survival and that they require more extensive radiotherapy and adjuvant chemotherapy. However, the mechanisms underlying this difference are still unclear. DNA methylation alteration is generally acknowledged to cause therapeutic resistance in cancers. We hypothesized that the pediatric NGMGCTs exhibit a different genome-wide DNA methylation pattern, which is involved in the mechanism of its therapeutic resistance. We performed methylation and hydroxymethylation DNA immunoprecipitation sequencing, mRNA expression microarray, and small RNA sequencing (smRNA-seq) to determine methylation-regulated genes, including microRNAs (miRNAs). The expression levels of 97 genes and 8 miRNAs were correlated with promoter DNA methylation and hydroxymethylation status, such as the miR-199/-214 cluster, and treatment with DNA demethylating agent 5-aza-2'-deoxycytidine elevated its expression level. Furthermore, smRNA-seq analysis showed 27 novel miRNA candidates with differential expression between germinomas and NGMGCTs. Overexpresssion of miR-214-3p in NCCIT cells leads to reduced expression of the pro-apoptotic protein BCL2-like 11 and induces cisplatin resistance. We interrogated the differential DNA methylation patterns between germinomas and NGMGCTs and proposed a mechanism for chemoresistance in NGMGCTs. In addition, our sequencing data provide a roadmap for further pediatric CNSGCT research and potential targets for the development of new therapeutic strategies.

  16. Calling Chromosome Alterations, DNA Methylation Statuses, and Mutations in Tumors by Simple Targeted Next-Generation Sequencing: A Solution for Transferring Integrated Pangenomic Studies into Routine Practice?

    PubMed

    Garinet, Simon; Néou, Mario; de La Villéon, Bruno; Faillot, Simon; Sakat, Julien; Da Fonseca, Juliana P; Jouinot, Anne; Le Tourneau, Christophe; Kamal, Maud; Luscap-Rondof, Windy; Boeva, Valentina; Gaujoux, Sebastien; Vidaud, Michel; Pasmant, Eric; Letourneur, Franck; Bertherat, Jérôme; Assié, Guillaume

    2017-09-01

    Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  17. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

    PubMed

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

    2016-10-04

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

  18. Novel insights into the apoptosis mechanism of DNA topoisomerase I inhibitor isoliquiritigenin on HCC tumor cell

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Ze-xin; Li, Jian; Li, Yan

    The inhibitory effect of DNA topoisomerase (Top I) by isoliquiritigenin(ISO) were investigated and their interaction mechanism was evaluated using methods including UV–vis absorption, fluorescence, coupled with molecular simulation, and using the MTT method of inhibition rate of HCC tumor cell SNU475 proliferation assay, finally, the interaction of ISO with calf thymus DNA was investigated by melting measurements and molecular docking studies. It was found that isoliquiritigenin reversibly inhibited DNA Top I in a competitive manner with the concentrations of ISO resulting in 50% activity lost (IC{sub 50}) were estimated to be 0.178 ± 0.12 mM. Isoliquiritigenin exhibited a strong ability to quench themore » intrinsic fluorescence of Top I through a static quenching procedure. The positive values of enthalpy change and entropy change suggested that the binding of isoliquiritigenin to Top I was driven mainly by hydrophobic interactions. The molecular docking results revealed isoliquiritigenin actually interacted with the primary amino acid residues on the active site of Top I, and the detection results of fluorescence staining and the inhibitory effect on the growth of HCC SUN475 showed that isoliquiritigenin induced the apoptosis cells increased gradually. The interaction of ISO with DNA can cause the denaturation temperature to be increased, which indicated that the stabilization of the DNA helix was increased in the presence of ISO, which indicated that the results provide strong evidence for intercalative binding of ISO with DNA. - Highlights: • ISO reversibly inhibits TOP I activity in an A dose dependent manner. • Hydrophobic interactions play a major role in ISO–TOP I interaction. • ISO has a high affinity close to the active site pocket of TOP I. • The binding of ISO to DNA induces the stability of the structure of DNA.« less

  19. DNA replication stress and cancer chemotherapy.

    PubMed

    Kitao, Hiroyuki; Iimori, Makoto; Kataoka, Yuki; Wakasa, Takeshi; Tokunaga, Eriko; Saeki, Hiroshi; Oki, Eiji; Maehara, Yoshihiko

    2018-02-01

    DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  20. Polymerization by DNA polymerase eta is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells.

    PubMed

    Chijiwa, Shotaro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori; Kuraoka, Isao

    2010-03-01

    cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

  1. Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients.

    PubMed

    Koeppel, Florence; Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

    2017-01-01

    Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

  2. Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients

    PubMed Central

    Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

    2017-01-01

    Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation. PMID:29161279

  3. Detection of an ALK Fusion in Colorectal Carcinoma by Hybrid Capture-Based Assay of Circulating Tumor DNA.

    PubMed

    Lai, Andrea Z; Schrock, Alexa B; Erlich, Rachel L; Ross, Jeffrey S; Miller, Vincent A; Yakirevich, Evgeny; Ali, Siraj M; Braiteh, Fadi

    2017-07-01

    ALK rearrangements have been observed in 0.05%-2.5% of patients with colorectal cancers (CRCs) and are predicted to be oncogenic drivers largely mutually exclusive of KRAS, NRAS, or BRAF alterations. Here we present the case of a patient with metastatic CRC who was treatment naïve at the time of molecular testing. Initial ALK immunohistochemistry (IHC) staining was negative, but parallel genomic profiling of both circulating tumor DNA (ctDNA) and tissue using similar hybrid capture-based assays each identified an identical STRN-ALK fusion. Subsequent ALK IHC staining of the same specimens was positive, suggesting that the initial result was a false negative. This report is the first instance of an ALK fusion in CRC detected using a ctDNA assay. Current guidelines for colorectal cancer (CRC) only recommend genomic assessment of KRAS, NRAS, BRAF, and microsatellite instability (MSI) status. ALK rearrangements are rare in CRC, but patients with activating ALK fusions have responded to targeted therapies ALK rearrangements can be detected by genomic profiling of ctDNA from blood or tissue, and this methodology may be informative in cases where immunohistochemistry (IHC) or other standard testing is negative. © AlphaMed Press 2017.

  4. Teaching Absolute Value Meaningfully

    ERIC Educational Resources Information Center

    Wade, Angela

    2012-01-01

    What is the meaning of absolute value? And why do teachers teach students how to solve absolute value equations? Absolute value is a concept introduced in first-year algebra and then reinforced in later courses. Various authors have suggested instructional methods for teaching absolute value to high school students (Wei 2005; Stallings-Roberts…

  5. Inhibition of DNA damage repair by the CDK4/6 inhibitor palbociclib delays irradiated intracranial atypical teratoid rhabdoid tumor and glioblastoma xenograft regrowth.

    PubMed

    Hashizume, Rintaro; Zhang, Ali; Mueller, Sabine; Prados, Michael D; Lulla, Rishi R; Goldman, Stewart; Saratsis, Amanda M; Mazar, Andrew P; Stegh, Alexander H; Cheng, Shi-Yuan; Horbinski, Craig; Haas-Kogan, Daphne A; Sarkaria, Jann N; Waldman, Todd; James, C David

    2016-11-01

    Radiation therapy is the most commonly used postsurgical treatment for primary malignant brain tumors. Consequently, investigating the efficacy of chemotherapeutics combined with radiation for treating malignant brain tumors is of high clinical relevance. In this study, we examined the cyclin-dependent kinase 4/6 inhibitor palbociclib, when used in combination with radiation for treating human atypical teratoid rhabdoid tumor (ATRT) as well as glioblastoma (GBM). Evaluation of treatment antitumor activity in vitro was based upon results from cell proliferation assays, clonogenicity assays, flow cytometry, and immunocytochemistry for DNA double-strand break repair. Interpretation of treatment antitumor activity in vivo was based upon bioluminescence imaging, animal subject survival analysis, and staining of tumor sections for markers of proliferation and apoptosis. For each of the retinoblastoma protein (RB)-proficient tumor models examined (2 ATRTs and 2 GBMs), one or more of the combination therapy regimens significantly (P < .05) outperformed both monotherapies with respect to animal subject survival benefit. Among the combination therapy regimens, concurrent palbociclib and radiation treatment and palbociclib treatment following radiation consistently outperformed the sequence in which radiation followed palbociclib treatment. In vitro investigation revealed that the concurrent use of palbociclib with radiation, as well as palbociclib following radiation, inhibited DNA double-strand break repair and promoted increased tumor cell apoptosis. Our results support further investigation and possible clinical translation of palbociclib as an adjuvant to radiation therapy for patients with malignant brain tumors that retain RB expression. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Cytogenetic and molecular aspects of absolute teratozoospermia: comparison between polymorphic and monomorphic forms.

    PubMed

    Brahem, Sonia; Elghezal, Hatem; Ghédir, Houda; Landolsi, Hanène; Amara, Abdelbacett; Ibala, Samira; Gribaa, Moez; Saad, Ali; Mehdi, Meriem

    2011-12-01

    To compare the results of cytogenetic and molecular analysis between absolute polymorphic and monomorphic teratozoospermia. The semen samples from patients with polymorphic teratozoospermia (n = 20), globozoospermia (n = 8), or macrocephalic sperm head syndrome (n = 12), and healthy fertile men (n = 20) were analyzed according to the World Health Organization criteria. The constitutional blood karyotype of the patients was performed on cultured lymphocytes, according to standard techniques. Microdeletion analysis of the Y chromosomes used a sequence tagged site-polymerase chain reaction technique. Triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18 were used to analyze the meiotic segregation. DNA fragmentation was detected using the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Whatever the type of teratozoospermia, a normal karyotype and an absence of Y chromosome microdeletion were shown for all patients. A significant increase in the sperm aneuploidy rate and DNA fragmentation were shown, regardless of the type of teratozoospermia. Spermatozoa of the patients with globozoospermia carry an abnormal chromosomal constitution and DNA damage rate with the same frequency as that found in the sperm of patients with absolute polymorphic teratozoospermia. However, a greater sperm aneuploidy rate and DNA fragmentation were found in patients whose teratozoospermia was mainly characterized by increased rates of spermatozoa with macrocephalic head and multiple flagella. Our data have demonstrated that DNA fragmentation and sperm aneuploidy are critical tests in teratozoospermic men, because the results could negatively affect the intracytoplasmic sperm injection outcomes and might play an important role in the counseling of couples considering intracytoplasmic sperm injection. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Enzymatic cleavage of uracil-containing single-stranded DNA linkers for the efficient release of affinity-selected circulating tumor cells.

    PubMed

    Nair, Soumya V; Witek, Małgorzata A; Jackson, Joshua M; Lindell, Maria A M; Hunsucker, Sally A; Sapp, Travis; Perry, Caroline E; Hupert, Mateusz L; Bae-Jump, Victoria; Gehrig, Paola A; Wysham, Weiya Z; Armistead, Paul M; Voorhees, Peter; Soper, Steven A

    2015-02-21

    We report a novel strategy to enzymatically release affinity-selected cells, such as circulating tumor cells (CTCs), from surfaces with high efficiency (∼90%) while maintaining cell viability (>85%). The strategy utilizes single-stranded DNAs that link a capture antibody to the surfaces of a CTC selection device. The DNA linkers contain a uracil residue that can be cleaved.

  8. Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

    PubMed

    Stachler, Matthew; Jia, Yonghui; Sharaf, Nematullah; Wade, Jacqueline; Longtine, Janina; Garcia, Elizabeth; Sholl, Lynette M

    2015-01-01

    Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

  9. Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

    PubMed

    Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

    2015-10-01

    The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto

  10. DNA Damage Response Genes and the Development of Cancer Metastasis

    PubMed Central

    Broustas, Constantinos G.; Lieberman, Howard B.

    2014-01-01

    DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

  11. Genome-scale analysis of aberrant DNA methylation in colorectal cancer

    PubMed Central

    Hinoue, Toshinori; Weisenberger, Daniel J.; Lange, Christopher P.E.; Shen, Hui; Byun, Hyang-Min; Van Den Berg, David; Malik, Simeen; Pan, Fei; Noushmehr, Houtan; van Dijk, Cornelis M.; Tollenaar, Rob A.E.M.; Laird, Peter W.

    2012-01-01

    Colorectal cancer (CRC) is a heterogeneous disease in which unique subtypes are characterized by distinct genetic and epigenetic alterations. Here we performed comprehensive genome-scale DNA methylation profiling of 125 colorectal tumors and 29 adjacent normal tissues. We identified four DNA methylation–based subgroups of CRC using model-based cluster analyses. Each subtype shows characteristic genetic and clinical features, indicating that they represent biologically distinct subgroups. A CIMP-high (CIMP-H) subgroup, which exhibits an exceptionally high frequency of cancer-specific DNA hypermethylation, is strongly associated with MLH1 DNA hypermethylation and the BRAFV600E mutation. A CIMP-low (CIMP-L) subgroup is enriched for KRAS mutations and characterized by DNA hypermethylation of a subset of CIMP-H-associated markers rather than a unique group of CpG islands. Non-CIMP tumors are separated into two distinct clusters. One non-CIMP subgroup is distinguished by a significantly higher frequency of TP53 mutations and frequent occurrence in the distal colon, while the tumors that belong to the fourth group exhibit a low frequency of both cancer-specific DNA hypermethylation and gene mutations and are significantly enriched for rectal tumors. Furthermore, we identified 112 genes that were down-regulated more than twofold in CIMP-H tumors together with promoter DNA hypermethylation. These represent ∼7% of genes that acquired promoter DNA methylation in CIMP-H tumors. Intriguingly, 48/112 genes were also transcriptionally down-regulated in non-CIMP subgroups, but this was not attributable to promoter DNA hypermethylation. Together, we identified four distinct DNA methylation subgroups of CRC and provided novel insight regarding the role of CIMP-specific DNA hypermethylation in gene silencing. PMID:21659424

  12. A common deletion in two gamma ray induced rat pulmonary tumor cell lines.

    PubMed

    Van Klaveren, P; De Bruijne, J; Van der Winden, H; Kal, H B; Bentvelzen, P

    1994-01-01

    Subtraction hybridization was performed on normal WAG/Rij rat DNA with DNA from a syngeneic Ir-192 induced pulmonary tumor cell line L37. The residual DNA was amplified by means of sequence-independent PCR. This procedure yielded a sequence, of which multiple copies are present in normal rat DNA. In the tumor line L37 two restriction fragments hybridizing with this repeat sequence are lacking. In another Ir-192 induced pulmonary tumor line, L33, one of these fragments was also lacking. This indicates a common deletion in the two tumor lines.

  13. Glucosylated polyethylenimine as a tumor-targeting gene carrier.

    PubMed

    Park, In-Kyu; Cook, Seung-Eun; Kim, You-Kyoung; Kim, Hyun-Woo; Cho, Myung-Haing; Jeong, Hwan-Jeong; Kim, Eun-Mi; Nah, Jae-Woon; Bom, Hee-Seung; Cho, Chong-Su

    2005-11-01

    Glucosylated polyethylenimine (GPEI) was synthesized as a tumor-targeting gene carrier through facilitative glucose metabolism by tumor glucose transporter. Particle sizes of GPEI/DNA complex increased in proportion to glucose content of GPEI, whereas surface charge of the complex was not dependent on glucosylation, partially due to inefficient shielding of the short hydrophilic group introduced. GPEI with higher glucosylation (36 mol-%) had no cytotoxic effect on cells even at polymer concentrations higher than 200 microg/mL. Compared to unglucosylated PEI, glucosylation induced less than one-order decrease of transfection efficiency. Transfection of GPEI/DNA complex into tumor cells possibly occurred through specific interaction between glucose-related cell receptors and glucose moiety of GPEI. Gamma imaging technique revealed GPEI/DNA complex was distributed in liver, spleen, and tumors.

  14. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

    PubMed Central

    Doolittle-Hall, Janet M.; Cunningham Glasspoole, Danielle L.; Seaman, William T.; Webster-Cyriaque, Jennifer

    2015-01-01

    Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures. PMID:26569308

  15. HPV circulating tumor DNA to monitor the efficacy of anti-PD-1 therapy in metastatic squamous cell carcinoma of the anal canal: A case report.

    PubMed

    Cabel, Luc; Bidard, François-Clément; Servois, Vincent; Cacheux, Wulfran; Mariani, Pascale; Romano, Emanuela; Minsat, Mathieu; Bieche, Ivan; Farkhondeh, Fereshteh; Jeannot, Emmanuelle; Buecher, Bruno

    2017-10-15

    Squamous cell carcinoma of the anal canal (SCCA) is a rare HPV-associated cancer with limited sensitivity to standard chemotherapy. In a phase 2 study, nivolumab, an anti PD-1 immune checkpoint inhibitor, demonstrated significant efficacy as single-agent therapy in metastatic SCCA patients. Nevertheless, imaging assessment by standard RECIST criteria of the efficacy of immune therapy can be difficult in some patients due to tumor immune cell infiltration, and biomarkers of treatment efficacy are needed. We have previously developed a quantitative droplet digital PCR (ddPCR) technique to detect HPV circulating tumor DNA (HPV ctDNA), with excellent sensitivity and specificity. Here, we report, for the first time, the kinetics of HPV ctDNA during therapy in a patient with metastatic SCCA, who obtained sustained partial response to single-agent nivolumab. We observed an early and very significant decrease of HPV ctDNA during therapy from the baseline level of 3713 copies/ml plasma to 564 copies/ml plasma at 4 weeks, and 156 copies/ml at 6 weeks, followed by a plateau. This observation provides proof-of-concept that HPV ctDNA can be used as a noninvasive early dynamic biomarker to monitor the efficacy of new immunotherapy agents. © 2017 UICC.

  16. MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

    PubMed

    Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

    2017-07-01

    Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Combined DNA methylation and gene expression profiling in gastrointestinal stromal tumors reveals hypomethylation of SPP1 as an independent prognostic factor.

    PubMed

    Haller, Florian; Zhang, Jitao David; Moskalev, Evgeny A; Braun, Alexander; Otto, Claudia; Geddert, Helene; Riazalhosseini, Yasser; Ward, Aoife; Balwierz, Aleksandra; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, B Michael; Agaimy, Abbas; Fletcher, Jonathan A; Hoheisel, Jörg; Hartmann, Arndt; Werner, Martin; Wiemann, Stefan; Sahin, Ozgür

    2015-03-01

    Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category. © 2014 UICC.

  18. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    NASA Astrophysics Data System (ADS)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  19. Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dittmann, Klaus H.; Mayer, Claus; Ohneseit, Petra A.

    2008-01-01

    Purpose: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. Methods and Materials: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by {gamma}H{sub 2}AX foci assay. Results: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observedmore » radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual {gamma}H2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. Conclusions: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.« less

  20. Easy Absolute Values? Absolutely

    ERIC Educational Resources Information Center

    Taylor, Sharon E.; Mittag, Kathleen Cage

    2015-01-01

    The authors teach a problem-solving course for preservice middle-grades education majors that includes concepts dealing with absolute-value computations, equations, and inequalities. Many of these students like mathematics and plan to teach it, so they are adept at symbolic manipulations. Getting them to think differently about a concept that they…

  1. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

    PubMed Central

    Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck

    2010-01-01

    Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973

  2. Tumor targeting of gene expression through metal-coordinated conjugation with dextran.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-03-07

    Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivatives with chelate residues based on metal coordination. Diethylenetriamine pentaacetic acid (DTPA), spermidine (Sd), and spermine (Sm) were chemically introduced to the hydroxyl groups of dextran to obtain dextran-DTPA, dextran-Sd and dextran-Sm derivatives. Conjugation of the dextran derivative by Zn(2+) coordination decreased the apparent size of the plasmid DNA, depending on the derivative type. The negative zeta potential of plasmid DNA became almost 0 mV after Zn(2+)-coordinated conjugation with dextran-Sm. When the dextran derivative-plasmid DNA conjugates with Zn(2+) coordination were intravenously injected subcutaneously into mice bearing Meth-AR-1 fibrosarcoma, the dextran-Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to the conjugate of other dextran derivatives and free plasmid DNA. The enhanced gene expression produced by the Zn(2+)-coordinated dextran-Sm-plasmid DNA conjugate was specific to the tumor, whereas a simple mixture of dextran-Sm and plasmid DNA was not effective. The level of gene expression depended on the percentage of chelate residues introduced, the mixing weight ratio of the plasmid DNA/Sm residue used for conjugate preparation, and the plasmid DNA dose. A fluorescent microscopic study revealed that localization of plasmid DNA in the tumor tissue was observed only after injection of the dextran-Sm-plasmid DNA conjugate with Zn(2+) coordination. In addition, the gene expression induced by the conjugate lasted for more than 10 days after the injection. We conclude that Zn(2+)-coordinated dextran-Sm conjugation is a promising way to enable plasmid DNA to target the tumor in gene expression as well as to prolong the duration of gene expression.

  3. Absolutely relative or relatively absolute: violations of value invariance in human decision making.

    PubMed

    Teodorescu, Andrei R; Moran, Rani; Usher, Marius

    2016-02-01

    Making decisions based on relative rather than absolute information processing is tied to choice optimality via the accumulation of evidence differences and to canonical neural processing via accumulation of evidence ratios. These theoretical frameworks predict invariance of decision latencies to absolute intensities that maintain differences and ratios, respectively. While information about the absolute values of the choice alternatives is not necessary for choosing the best alternative, it may nevertheless hold valuable information about the context of the decision. To test the sensitivity of human decision making to absolute values, we manipulated the intensities of brightness stimuli pairs while preserving either their differences or their ratios. Although asked to choose the brighter alternative relative to the other, participants responded faster to higher absolute values. Thus, our results provide empirical evidence for human sensitivity to task irrelevant absolute values indicating a hard-wired mechanism that precedes executive control. Computational investigations of several modelling architectures reveal two alternative accounts for this phenomenon, which combine absolute and relative processing. One account involves accumulation of differences with activation dependent processing noise and the other emerges from accumulation of absolute values subject to the temporal dynamics of lateral inhibition. The potential adaptive role of such choice mechanisms is discussed.

  4. The rationale for liquid biopsy in colorectal cancer: a focus on circulating tumor cells.

    PubMed

    Gazzaniga, Paola; Raimondi, Cristina; Nicolazzo, Chiara; Carletti, Raffaella; di Gioia, Cira; Gradilone, Angela; Cortesi, Enrico

    2015-01-01

    Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells.

  5. MUC1 and survivin combination tumor gene vaccine generates specific immune responses and anti-tumor effects in a murine melanoma model.

    PubMed

    Zhang, Haihong; Liu, Chenlu; Zhang, Fangfang; Geng, Fei; Xia, Qiu; Lu, Zhenzhen; Xu, Ping; Xie, Yu; Wu, Hui; Yu, Bin; Wu, Jiaxin; Yu, Xianghui; Kong, Wei

    2016-05-23

    MUC1 and survivin are ideal tumor antigens. Although many cancer vaccines targeting survivin or MUC1 have entered clinical trials, no vaccine combining MUC1 and survivin have been reported. Due to tumor heterogeneity, vaccines containing a combination of antigens may have improved efficacy and coverage of a broader spectrum of cancer targets. Here, cellular responses and anti-tumor activities induced by a combination of DNA vaccine targeting MUC1 and survivin (MS) were evaluated. Results showed that CTL activity and inhibition of tumor growth were obviously enhanced in mice immunized with the combined vaccine in a protection assay. However, in order to enhance the therapeutic effect in the treatment assay, a recombinant adenovirus (rAd) vaccine expressing MUC1 and survivin (Ad-MS) was used as a booster following the DNA vaccine prime. Meanwhile, IL-2 promoting T cell proliferation was used as an immunoadjuvant for the DNA vaccine. Results showed that the CTL activity response to the DNA vaccine was enhanced nearly 200% when boosted by the rAd vaccine and was further enhanced by nearly 60% when combined with the IL-2 adjuvant. Therefore, DNA prime combined with rAd boost and IL-2 (MS/IL2/Ad-MS) adjuvant was considered as the best strategy and further evaluated. Multiple cytokines promoting cellular immune responses were shown to be greatly enhanced in mice immunized with MS/IL2/Ad-MS. Moreover, in the treatment assay, the tumor inhibition rate of MS/IL2/Ad-MS reached up to 50.1%, which may be attributed to the enhancement of immune responses and reduction of immunosuppressive factors in tumor-bearing mice. These results suggested that immunization with the combination vaccine targeting MUC1 and survivin using a DNA prime-rAd boost strategy along with IL-2 adjuvant may be an effective method for breaking through immune tolerance to tumors expressing these antigens with potential therapeutic benefits in melanoma cancer. Copyright © 2016. Published by Elsevier Ltd.

  6. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.

    PubMed

    de Smith, Adam J; Walsh, Kyle M; Hansen, Helen M; Endicott, Alyson A; Wiencke, John K; Metayer, Catherine; Wiemels, Joseph L

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  7. Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: evidence for increased tumor cell apoptosis in vivo.

    PubMed

    Hans, M A; Müller, M; Meyer-Ficca, M; Bürkle, A; Küpper, J H

    1999-11-25

    Poly(ADP-ribose) polymerase (PARP4) catalyzes the formation of ADP-ribose polymers covalently attached to proteins by using NAD+ as substrate. PARP is strongly activated by DNA single- or double-strand breaks and is thought to be involved in cellular responses to DNA damage. We characterized a dominant negative PARP mutant, i.e. the DNA-binding domain of this enzyme, whose overexpression in cells leads to increased genetic instability following DNA damage. In order to study whether PARP activity is also implicated in the process of tumorigenesis, we generated stably transfected HeLa cell clones with constitutive overexpression of dominant negative PARP and investigated tumor formation of these clones in nude mice. We found that inhibition of PARP activity dramatically reduces tumor forming ability of HeLa cells. Moreover, we provide strong evidence that the observed reduction in tumor forming ability is due to increased tumor cell apoptosis in vivo. Viewed together, our data and those from other groups show that inhibition of PARP enzyme activity interferes with DNA base excision repair and leads to increased genetic instability and recombination but, on the other hand, can sensitize cells to apoptotic stimuli and by this mechanism may prevent tumor formation.

  8. DNA origami as an in vivo drug delivery vehicle for cancer therapy.

    PubMed

    Zhang, Qian; Jiang, Qiao; Li, Na; Dai, Luru; Liu, Qing; Song, Linlin; Wang, Jinye; Li, Yaqian; Tian, Jie; Ding, Baoquan; Du, Yang

    2014-07-22

    Many chemotherapeutics used for cancer treatments encounter issues during delivery to tumors in vivo and may have high levels of systemic toxicity due to their nonspecific distribution. Various materials have been explored to fabricate nanoparticles as drug carriers to improve delivery efficiency. However, most of these materials suffer from multiple drawbacks, such as limited biocompatibility and inability to engineer spatially addressable surfaces that can be utilized for multifunctional activity. Here, we demonstrate that DNA origami possessed enhanced tumor passive targeting and long-lasting properties at the tumor region. Particularly, the triangle-shaped DNA origami exhibits optimal tumor passive targeting accumulation. The delivery of the known anticancer drug doxorubicin into tumors by self-assembled DNA origami nanostructures was performed, and this approach showed prominent therapeutic efficacy in vivo. The DNA origami carriers were prepared through the self-assembly of M13mp18 phage DNA and hundreds of complementary DNA helper strands; the doxorubicin was subsequently noncovalently intercalated into these nanostructures. After conducting fluorescence imaging and safety evaluation, the doxorubicin-containing DNA origami exhibited remarkable antitumor efficacy without observable systemic toxicity in nude mice bearing orthotopic breast tumors labeled with green fluorescent protein. Our results demonstrated the potential of DNA origami nanostructures as innovative platforms for the efficient and safe drug delivery of cancer therapeutics in vivo.

  9. MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia.

    PubMed

    Tagde, Ashujit; Rajabi, Hasan; Stroopinsky, Dina; Gali, Reddy; Alam, Maroof; Bouillez, Audrey; Kharbanda, Surender; Stone, Richard; Avigan, David; Kufe, Donald

    2016-06-28

    Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38- population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML.

  10. Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

    PubMed

    Kishikawa, Takahiro; Otsuka, Motoyuki; Suzuki, Tatsunori; Seimiya, Takahiro; Sekiba, Kazuma; Ishibashi, Rei; Tanaka, Eri; Ohno, Motoko; Yamagami, Mari; Koike, Kazuhiko

    2018-05-10

    Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis in vivo , we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase. Implications: Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. Mol Cancer Res; 1-8. ©2018 AACR. ©2018 American Association for Cancer Research.

  11. DNA Vaccination Against Metastatic Breast Cancer

    DTIC Science & Technology

    2002-07-01

    Although DNA vaccines have shown effectiveness in clinical trials , it is essential to demonstrate pre- clinical effectiveness for anti-tumor DNA vaccines...been shown to induce strong anti-tumor immunity in mice (3). Although gene vaccines have shown effectiveness in clinical trials for infectious...stronger justification for a clinical trial . REFERENCES: 1. Fornier, M., P. Munster, and A. D. Seidman. 1999. Update on the management of advanced breast

  12. Aneuploidy in benign tumors and nonneoplastic lesions of musculoskeletal tissues.

    PubMed

    Alho, A; Skjeldal, S; Pettersen, E O; Melvik, J E; Larsen, T E

    1994-02-15

    Aneuploidy in DNA flow cytometry (FCM) of musculoskeletal tumors is generally considered to be a sign of malignancy. Previously, giant cell tumor of the bone has been reported to contain aneuploid (near-diploid) DNA stemlines. Otherwise, only spordic cases have been reported. The authors wanted to study the relationships among DNA FCM, histology, and clinical course of nonmalignant musculoskeletal lesions. Twenty-eight histologically benign tumors and seven nonneoplastic lesions were subjected to DNA FCM: After tissue preparation mechanically and with ribonuclease and trypsin, the isolated nuclei were stained with propidium iodine using chicken and rainbow trout erythrocytes as controls. In the DNA FCM histograms, ploidy and cell cycle fractions were determined using a computerized mathematical model. The histologic diagnoses were made without knowledge of the DNA FCM results. Aneuploidy was found in eight lesions. A shoulder in the diploid peak, suggesting a diploid and a near-diploid population, was found in DNA histograms of a condensing osteitis of the clavicle (a benign inflammatory process) and of a giant cell tumor of bone. The latter lesion also had a tetraploid population. Six benign tumors--two enchondromas, one osteochondroma, one subcutaneous and one intramuscular lipoma, and a calcifying aponeurotic fibroma--showed clear aneuploidy with separate peaks. The S-phase fraction was less than 10% in all cases. The highest aneuploid population, DNA index = 1.70, in a subcutaneous lipoma, was small, with an undetectable S phase. Despite nonradical operations in seven lesions, no recurrences were observed during a median follow-up of 49 months (range, 28-73 months). Small aneuploid populations with low DNA synthetic activity may be compatible with a benign histologic picture and uneventful clinical course of the musculoskeletal lesion.

  13. Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.

    PubMed

    Jackson, Jennifer B; Choi, Daniel S; Luketich, James D; Pennathur, Arjun; Ståhlberg, Anders; Godfrey, Tony E

    2016-03-01

    Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. Absolute biological needs.

    PubMed

    McLeod, Stephen

    2014-07-01

    Absolute needs (as against instrumental needs) are independent of the ends, goals and purposes of personal agents. Against the view that the only needs are instrumental needs, David Wiggins and Garrett Thomson have defended absolute needs on the grounds that the verb 'need' has instrumental and absolute senses. While remaining neutral about it, this article does not adopt that approach. Instead, it suggests that there are absolute biological needs. The absolute nature of these needs is defended by appeal to: their objectivity (as against mind-dependence); the universality of the phenomenon of needing across the plant and animal kingdoms; the impossibility that biological needs depend wholly upon the exercise of the abilities characteristic of personal agency; the contention that the possession of biological needs is prior to the possession of the abilities characteristic of personal agency. Finally, three philosophical usages of 'normative' are distinguished. On two of these, to describe a phenomenon or claim as 'normative' is to describe it as value-dependent. A description of a phenomenon or claim as 'normative' in the third sense does not entail such value-dependency, though it leaves open the possibility that value depends upon the phenomenon or upon the truth of the claim. It is argued that while survival needs (or claims about them) may well be normative in this third sense, they are normative in neither of the first two. Thus, the idea of absolute need is not inherently normative in either of the first two senses. © 2013 John Wiley & Sons Ltd.

  15. Doxycycline down-regulates DNA-PK and radiosensitizes tumor initiating cells: Implications for more effective radiation therapy

    PubMed Central

    Lamb, Rebecca; Fiorillo, Marco; Chadwick, Amy; Ozsvari, Bela; Reeves, Kimberly J.; Smith, Duncan L.; Clarke, Robert B.; Howell, Sacha J.; Cappello, Anna Rita; Martinez-Outschoorn, Ubaldo E.; Peiris-Pagès, Maria; Sotgia, Federica; Lisanti, Michael P.

    2015-01-01

    DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by “chemical proteomics”. In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-derived mammospheres. Interestingly, genetic or pharmacological inhibition of DNA-PK in MCF7 cells is sufficient to functionally block mammosphere formation. Thus, it appears that active DNA-repair is required for the clonal expansion of CSCs. Mechanistically, doxycycline treatment dramatically reduced the oxidative mitochondrial capacity and the glycolytic activity of cancer cells, consistent with previous studies linking DNA-PK expression to the proper maintenance of mitochondrial DNA integrity and copy number. Using a luciferase-based assay, we observed that doxycycline treatment quantitatively reduces the anti-oxidant response (NRF1/2) and effectively blocks signaling along multiple independent pathways normally associated with stem cells, including STAT1/3, Sonic Hedgehog (Shh), Notch, WNT and TGF-beta signaling. In conclusion, we propose that the efficacy of doxycycline as a DNA-PK inhibitor should be tested in Phase-II clinical trials, in combination with radio-therapy. Doxycycline has

  16. Molecular Analysis of Medaka Tumors: New Models for Carcinogenicity Tests

    DTIC Science & Technology

    1993-07-07

    analysis, 5 were reported to have either a 8 cholangiocarcinoma or a hepatocellular carcinoma . Only DNA from the hepatocellular carcinomas were able to... hepatocellular carcinoma were able to induce tumors in nude mice in 3-4 weeks (Table II). In summary, DNA isolated from these tumors had relatively low...DNAs from tumor-bearing livers (cholangiocarcinomas, hepatocellular carcinomas and one mixed type) were able to induce significant numbers of foci

  17. Refractory testicular germ cell tumors are highly sensitive to the second generation DNA methylation inhibitor guadecitabine.

    PubMed

    Albany, Costantine; Hever-Jardine, Mary P; von Herrmann, Katherine M; Yim, Christina Y; Tam, Janice; Warzecha, Joshua M; Shin, Leah; Bock, Sarah E; Curran, Brian S; Chaudhry, Aneeq S; Kim, Fred; Sandusky, George E; Taverna, Pietro; Freemantle, Sarah J; Christensen, Brock C; Einhorn, Lawrence H; Spinella, Michael J

    2017-01-10

    Testicular germ cell tumors (TGCTs) are the most common cancers of young males. A substantial portion of TGCT patients are refractory to cisplatin. There are no effective therapies for these patients, many of whom die from progressive disease. Embryonal carcinoma (EC) are the stem cells of TGCTs. In prior in vitro studies we found that EC cells were highly sensitive to the DNA methyltransferase inhibitor, 5-aza deoxycytidine (5-aza). Here, as an initial step in bringing demethylation therapy to the clinic for TGCT patients, we evaluated the effects of the clinically optimized, second generation demethylating agent guadecitabine (SGI-110) on EC cells in an animal model of cisplatin refractory testicular cancer. EC cells were exquisitely sensitive to guadecitabine and the hypersensitivity was dependent on high levels of DNA methyltransferase 3B. Guadecitabine mediated transcriptional reprogramming of EC cells included induction of p53 targets and repression of pluripotency genes. As a single agent, guadecitabine completely abolished progression and induced complete regression of cisplatin resistant EC xenografts even at doses well below those required to impact somatic solid tumors. Low dose guadecitabine also sensitized refractory EC cells to cisplatin in vivo. Genome-wide analysis indicated that in vivo antitumor activity was associated with activation of p53 and immune-related pathways and the antitumor effects of guadecitabine were dependent on p53, a gene rarely mutated in TGCTs. These preclinical findings suggest that guadecitabine alone or in combination with cisplatin is a promising strategy to treat refractory TGCT patients.

  18. The DNA methylation landscape of human melanoma.

    PubMed

    Jin, Seung-Gi; Xiong, Wenying; Wu, Xiwei; Yang, Lu; Pfeifer, Gerd P

    2015-12-01

    Using MIRA-seq, we have characterized the DNA methylome of metastatic melanoma and normal melanocytes. Individual tumors contained several thousand hypermethylated regions. We discovered 179 tumor-specific methylation peaks present in all (27/27) melanomas that may be effective disease biomarkers, and 3113 methylation peaks were seen in >40% of the tumors. We found that 150 of the approximately 1200 tumor-associated methylation peaks near transcription start sites (TSSs) were marked by H3K27me3 in melanocytes. DNA methylation in melanoma was specific for distinct H3K27me3 peaks rather than for broadly covered regions. However, numerous H3K27me3 peak-associated TSS regions remained devoid of DNA methylation in tumors. There was no relationship between BRAF mutations and the number of methylation peaks. Gene expression analysis showed upregulated immune response genes in melanomas presumably as a result of lymphocyte infiltration. Down-regulated genes were enriched for melanocyte differentiation factors; e.g., KIT, PAX3 and SOX10 became methylated and downregulated in melanoma. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest

    PubMed Central

    Lundholm, L; Hååg, P; Zong, D; Juntti, T; Mörk, B; Lewensohn, R; Viktorsson, K

    2013-01-01

    Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC

  20. Transcatheter Arterial Embolization with a Mixture of Absolute Ethanol and Iodized Oil for Poorly Visualized Endophytic Renal Masses Prior to CT-Guided Percutaneous Cryoablation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Michimoto, Kenkichi, E-mail: michikoo@jikei.ac.jp; Shimizu, Kanichiro; Kameoka, Yoshihiko

    PurposeTo retrospectively evaluate the feasibility of transcatheter arterial embolization (TAE) using a mixture of absolute ethanol and iodized oil to improve localization of endophytic renal masses on unenhanced computed tomography (CT) prior to CT-guided percutaneous cryoablation (PCA).Materials and MethodsOur institutional review board approved this retrospective study. From September 2011 to June 2015, 17 patients (mean age, 66.8 years) with stage T1a endophytic renal masses (mean diameter, 26.5 mm) underwent TAE using a mixture of absolute ethanol and iodized oil to improve visualization of small and endophytic renal masses on unenhanced CT prior to CT-guided PCA. TAE was considered successful that accumulated iodizedmore » oil depicted whole of the tumor edge on CT. PCA was considered successful when the iceball covered the entire tumor with over a 5 mm margin. Oncological and renal functional outcomes and complications were also evaluated.ResultsTAE was successfully performed in 16 of 17 endophytic tumors. The 16 tumors were performed under CT-guided PCA with their distinct visualization of localization and safe ablated margin. During the mean follow-up period of 15.4 ± 5.1 months, one patient developed local recurrence. Estimated glomerular filtration rate declined by 8 % with statistical significance (P = 0.01). There was no procedure-related significant complication.ConclusionTAE using a mixture of absolute ethanol and iodized oil to improve visualization of endophytic renal masses facilitated tumor localization on unenhanced CT, permitting depiction of the tumor edge as well as a safe margin for ablation during CT-guided PCA, with an acceptable decline in renal function.« less

  1. Tumor microenvironment dual-responsive core-shell nanoparticles with hyaluronic acid-shield for efficient co-delivery of doxorubicin and plasmid DNA.

    PubMed

    Wang, Tianqi; Yu, Xiaoyue; Han, Leiqiang; Liu, Tingxian; Liu, Yongjun; Zhang, Na

    2017-01-01

    As the tumor microenvironment (TME) develops, it is critical to take the alterations of pH value, reduction and various enzymes of the TME into consideration when constructing the desirable co-delivery systems. Herein, TME pH and enzyme dual-responsive core-shell nanoparticles were prepared for the efficient co-delivery of chemotherapy drug and plasmid DNA (pDNA). A novel pH-responsive, positively charged drug loading material, doxorubicin (DOX)-4-hydrazinobenzoic acid (HBA)-polyethyleneimine (PEI) conjugate (DOX-HBA-PEI, DHP), was synthesized to fabricate positively charged polyion complex inner core DHP/DNA nanoparticles (DDN). Hyaluronic acid (HA) was an enzyme-responsive shell which could protect the core and enhance the co-delivery efficiency through CD44-mediated endocytosis. The HA-shielded pH and enzyme dual-responsive nanoparticles (HDDN) were spherical with narrow distribution. The particle size of HDDN was 148.3±3.88 nm and the zeta potential was changed to negative (-18.1±2.03 mV), which led to decreased cytotoxicity. The cumulative release of DOX from DHP at pH 5.0 (66.4%) was higher than that at pH 7.4 (30.1%), which indicated the pH sensitivity of DHP. The transfection efficiency of HDDN in 10% serum was equal to that in the absence of serum, while the transfection of DDN was significantly decreased in the presence of 10% serum. Furthermore, cellular uptake studies and co-localization assay showed that HDDN were internalized effectively through CD44-mediated endocytosis in the tumor cells. The efficient co-delivery of DOX and pEGFP was confirmed by fluorescent image taken by laser confocal microscope. It can be concluded that TME dual-responsive HA-shielded core-shell nanoparticles could be considered as a promising platform for the co-delivery of chemotherapy drug and pDNA.

  2. Practical calculation method to estimate the absolute boron concentration in tissues using 18F-FBPA PET.

    PubMed

    Watabe, Tadashi; Hanaoka, Kohei; Naka, Sadahiro; Kanai, Yasukazu; Ikeda, Hayato; Aoki, Masanao; Shimosegawa, Eku; Kirihata, Mitsunori; Hatazawa, Jun

    2017-07-01

    The purpose of this study was to establish a practical method to estimate the absolute boron concentrations in the tissues based on the standardized uptake values (SUVs) after administration of 4-borono-phenylalanine (BPA) using 4-borono-2- 18 F-fluoro-phenylalanine ( 18 F-FBPA) PET. Rat xenograft models of C6 glioma (n = 7, body weight 241 ± 28.0 g) were used for the study. PET was performed 60 min after intravenous injection of 18 F-FBPA (30.5 ± 0.7 MBq). After the PET scanning, BPA-fructose (167.3 ± 18.65 mg/kg) was administered by slow intravenous injection to the same subjects. The rats were killed 60 min after the BPA injection and tissue samples were collected from the major organs and tumors. The absolute boron concentrations (unit: ppm) in the samples were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). The boron concentrations in the tissues/tumors were also estimated from the 18 F-FBPA PET images using the following formula: estimated absolute boron concentration (ppm) = 0.0478 × [BPA dose (mg/kg)] × SUV. The measured absolute boron concentrations (mBC) by ICP-OES and the estimated boron concentrations (eBC) from the PET images were compared. The percent difference between the mBC and eBC calculated based on the SUV max was -5.2 ± 21.1% for the blood, -9.4 ± 22.3% for the brain, 1.6 ± 21.3% for the liver, -14.3 ± 16.8% for the spleen, -9.5 ± 27.5% for the pancreas, and 3.4 ± 43.2% for the tumor. Relatively large underestimation was observed for the lung (-48.4 ± 16.2%), small intestine (-37.8 ± 19.3%) and large intestine (-33.9 ± 11.0%), due to the partial volume effect arising from the air or feces contained in these organs. In contrast, relatively large overestimation was observed for the kidney (34.3 ± 29.3%), due to the influence of the high uptake in urine. The absolute boron concentrations in tissues/tumors can be estimated from the SUVs on 18 F-FBPA PET using a

  3. Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients.

    PubMed

    Gaykalova, Daria A; Vatapalli, Rajita; Wei, Yingying; Tsai, Hua-Ling; Wang, Hao; Zhang, Chi; Hennessey, Patrick T; Guo, Theresa; Tan, Marietta; Li, Ryan; Ahn, Julie; Khan, Zubair; Westra, William H; Bishop, Justin A; Zaboli, David; Koch, Wayne M; Khan, Tanbir; Ochs, Michael F; Califano, Joseph A

    2015-01-01

    Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.

  4. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cao Hongbin; Banh, Alice; Kwok, Shirley

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed bymore » p16{sup INK4a} staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using {sup 18}F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.« less

  5. Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

    PubMed Central

    Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M.; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

    2017-01-01

    Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma. PMID:28696319

  6. Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

    PubMed

    Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

    2017-07-25

    Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

  7. Communication: Electron ionization of DNA bases.

    PubMed

    Rahman, M A; Krishnakumar, E

    2016-04-28

    No reliable experimental data exist for the partial and total electron ionization cross sections for DNA bases, which are very crucial for modeling radiation damage in genetic material of living cell. We have measured a complete set of absolute partial electron ionization cross sections up to 500 eV for DNA bases for the first time by using the relative flow technique. These partial cross sections are summed to obtain total ion cross sections for all the four bases and are compared with the existing theoretical calculations and the only set of measured absolute cross sections. Our measurements clearly resolve the existing discrepancy between the theoretical and experimental results, thereby providing for the first time reliable numbers for partial and total ion cross sections for these molecules. The results on fragmentation analysis of adenine supports the theory of its formation in space.

  8. Measuring DNA Replication in Hypoxic Conditions.

    PubMed

    Foskolou, Iosifina P; Biasoli, Deborah; Olcina, Monica M; Hammond, Ester M

    2016-01-01

    It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)-a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.

  9. Gene Expression, DNA Methylation and Prognostic Significance of DNA Repair Genes in Human Bladder Cancer.

    PubMed

    Wojtczyk-Miaskowska, Anita; Presler, Malgorzata; Michajlowski, Jerzy; Matuszewski, Marcin; Schlichtholz, Beata

    2017-01-01

    This study investigated the gene expression and DNA methylation of selected DNA repair genes (MBD4, TDG, MLH1, MLH3) and DNMT1 in human bladder cancer in the context of pathophysiological and prognostic significance. To determine the relationship between the gene expression pattern, global methylation and promoter methylation status, we performed real-time PCR to quantify the mRNA of selected genes in 50 samples of bladder cancer and adjacent non-cancerous tissue. The methylation status was analyzed by methylation-specific polymerase chain reaction (MSP) or digestion of genomic DNA with a methylation-sensitive restriction enzyme and PCR with gene-specific primers (MSRE-PCR). The global DNA methylation level was measured using the antibody-based 5-mC detection method. The relative levels of mRNA for MBD4, MLH3, and MLH1 were decreased in 28% (14/50), 34% (17/50) and 36% (18/50) of tumor samples, respectively. The MBD4 mRNA expression was decreased in 46% of non-muscle invasive tumors (Ta/T1) compared with 11% found in muscle invasive tumors (T2-T4) (P<0.003). Analysis of mRNA expression for TDG did not show any significant differences between Ta/T1 and T2-T4 tumors. The frequency of increased DNMT1 mRNA expression was higher in T2-T4 (52%) comparing to Ta/T1 (16%). The overall methylation rates in tumor tissue were 18% for MBD4, 25% for MLH1 and there was no evidence of MLH3 promoter methylation. High grade tumors had significantly lower levels of global DNA methylation (P=0.04). There was a significant association between shorter survival and increased expression of DNMT1 mRNA (P=0.002), decreased expression of MLH1 mRNA (P=0.032) and the presence of MLH1 promoter methylation (P=0.006). This study highlights the importance of DNA repair pathways and provides the first evidence of the role of MBD4 and MLH3 in bladder cancer. In addition, our findings suggest that DNMT1 mRNA and MLH1 mRNA expression, as well as the status of MLH1 promoter methylation, are attractive

  10. Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors.

    PubMed

    Chen, Yan; Sun, Ji; Lu, Ying; Tao, Chun; Huang, Jingbin; Zhang, He; Yu, Yuan; Zou, Hao; Gao, Jing; Zhong, Yanqiang

    2013-01-01

    pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/-]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy.

  11. Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors

    PubMed Central

    Chen, Yan; Sun, Ji; Lu, Ying; Tao, Chun; Huang, Jingbin; Zhang, He; Yu, Yuan; Zou, Hao; Gao, Jing; Zhong, Yanqiang

    2013-01-01

    pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/−]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy. PMID:23637529

  12. Mouse mammary tumor virus-like RNA transcripts and DNA are found in affected cells of human breast cancer.

    PubMed

    Ford, Caroline E; Faedo, Margaret; Rawlinson, William D

    2004-11-01

    Identifiable risk factors for the development of breast cancer include age, diet, family history, and lifetime estrogen exposure. An infectious agent (mouse mammary tumor virus; MMTV) is known to cause murine breast tumors and may be involved in the pathogenesis of human disease. Multiple studies have detected MMTV-like sequences in 30 to 60% of breast cancer samples and up to 1.8% of samples from normal breast. Using in situ PCR of MMTV-like sequences of formalin-fixed, paraffin-embedded breast tissue, viral sequences have been located in cancerous epithelial cells in breast acini of male and female breast tumors, but not in adjacent nonmalignant cells. MMTV-like sequences were also located in the epithelial cells of male gynecomastia samples. Using reverse transcriptase in situ PCR, RNA transcripts from the env gene were also detected within cancerous epithelial cells of 78% of DNA-positive tumors, 80% of gynecomastia samples, and 0% of normal tissues screened. This suggests the virus may be replicating in these cells. The epidemiologic and histopathological data are consistent with the association of an MMTV-like virus with breast cancers in men and women. The association with gynecomastia, a benign, possibly premalignant condition suggests hormonal influences, rather than cancer per se, may be the dominant factor in determining viral presence and replication.

  13. Jasminum flexile flower absolute from India--a detailed comparison with three other jasmine absolutes.

    PubMed

    Braun, Norbert A; Kohlenberg, Birgit; Sim, Sherina; Meier, Manfred; Hammerschmidt, Franz-Josef

    2009-09-01

    Jasminum flexile flower absolute from the south of India and the corresponding vacuum headspace (VHS) sample of the absolute were analyzed using GC and GC-MS. Three other commercially available Indian jasmine absolutes from the species: J. sambac, J. officinale subsp. grandiflorum, and J. auriculatum and the respective VHS samples were used for comparison purposes. One hundred and twenty-one compounds were characterized in J. flexile flower absolute, with methyl linolate, benzyl salicylate, benzyl benzoate, (2E,6E)-farnesol, and benzyl acetate as the main constituents. A detailed olfactory evaluation was also performed.

  14. Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

    PubMed Central

    Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

    2011-01-01

    Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

  15. DNA-dependent protein kinase is a molecular target for the development of noncytotoxic radiation-sensitizing drugs.

    PubMed

    Shinohara, Eric T; Geng, Ling; Tan, Jiahui; Chen, Heidi; Shir, Yu; Edwards, Eric; Halbrook, James; Kesicki, Edward A; Kashishian, Adam; Hallahan, Dennis E

    2005-06-15

    DNA-dependent protein kinase (DNA-PK)-defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.

  16. Total DNA input is a crucial determinant of the sensitivity of plasma cell-free DNA EGFR mutation detection using droplet digital PCR

    PubMed Central

    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

  17. Marine natural products. XXXII. Absolute configurations of C-4 of the manoalide family, biologically active sesterterpenes from the marine sponge Hyrtios erecta.

    PubMed

    Kobayashi, M; Okamoto, T; Hayashi, K; Yokoyama, N; Sasaki, T; Kitagawa, I

    1994-02-01

    Cytotoxic sesterterpenes, manoalide 25-acetals (1a, 1b), seco-manoalide (2), (E)-neomanoalide (3), (Z)-neomanoalide (4), and heteronemin (6), were isolated from the marine sponge Hyrtios erecta (collected at Amami Island, Kagoshima Prefecture, Japan) by bioassay-guided separation and the absolute configurations of these manoalide family members have been determined. Manoalide 25-acetals (1a, 1b) were shown to exhibit in vivo antitumor activity and to inhibit the DNA-relaxing activity of mouse DNA topoisomerase I and the DNA-unknotting activity of calf thymus DNA topoisomerase II.

  18. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  19. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  20. DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

    PubMed Central

    Sheffield, Nathan C; Pierron, Gaelle; Klughammer, Johanna; Datlinger, Paul; Schönegger, Andreas; Schuster, Michael; Hadler, Johanna; Surdez, Didier; Guillemot, Delphine; Lapouble, Eve; Freneaux, Paul; Champigneulle, Jacqueline; Bouvier, Raymonde; Walder, Diana; Ambros, Ingeborg M; Hutter, Caroline; Sorz, Eva; Amaral, Ana T; de Álava, Enrique; Schallmoser, Katharina; Strunk, Dirk; Rinner, Beate; Liegl-Atzwanger, Bernadette; Huppertz, Berthold; Leithner, Andreas; de Pinieux, Gonzague; Terrier, Philippe; Laurence, Valérie; Michon, Jean; Ladenstein, Ruth; Holter, Wolfgang; Windhager, Reinhard; Dirksen, Uta; Ambros, Peter F; Delattre, Olivier; Kovar, Heinrich; Bock, Christoph; Tomazou, Eleni M

    2018-01-01

    Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine. PMID:28134926

  1. A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma.

    PubMed

    Connolly, Ian David; Li, Yingmei; Pan, Wenying; Johnson, Eli; You, Linya; Vogel, Hannes; Ratliff, John; Hayden Gephart, Melanie

    2017-10-01

    Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.

  2. A single digital droplet PCR assay to detect multiple KIT exon 11 mutations in tumor and plasma from patients with gastrointestinal stromal tumors.

    PubMed

    Boonstra, Pieter A; Ter Elst, Arja; Tibbesma, Marco; Bosman, Lisette J; Mathijssen, Ron; Atrafi, Florence; van Coevorden, Frits; Steeghs, Neeltje; Farag, Sheima; Gelderblom, Hans; van der Graaf, Winette T A; Desar, Ingrid M E; Maier, Jacqueline; Overbosch, Jelle; Suurmeijer, Albert J H; Gietema, Jourik; Schuuring, Ed; Reyners, Anna K L

    2018-03-02

    Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients' plasma. A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

  3. The Value of DNA Sequencing - TCGA

    Cancer.gov

    DNA sequencing: what it tells us about DNA changes in cancer, how looking across many tumors will help to identify meaningful changes and potential drug targets, and how genomics is changing the way we think about cancer.

  4. Communication: Electron ionization of DNA bases

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rahman, M. A.; Krishnakumar, E., E-mail: ekkumar@tifr.res.in

    2016-04-28

    No reliable experimental data exist for the partial and total electron ionization cross sections for DNA bases, which are very crucial for modeling radiation damage in genetic material of living cell. We have measured a complete set of absolute partial electron ionization cross sections up to 500 eV for DNA bases for the first time by using the relative flow technique. These partial cross sections are summed to obtain total ion cross sections for all the four bases and are compared with the existing theoretical calculations and the only set of measured absolute cross sections. Our measurements clearly resolve themore » existing discrepancy between the theoretical and experimental results, thereby providing for the first time reliable numbers for partial and total ion cross sections for these molecules. The results on fragmentation analysis of adenine supports the theory of its formation in space.« less

  5. Targeting DNA repair pathways for cancer treatment: what's new?

    PubMed Central

    Kelley, Mark R; Logsdon, Derek; Fishel, Melissa L

    2014-01-01

    Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses. PMID:24947262

  6. Selective Targeting of Brain Tumors with Gold Nanoparticle-Induced Radiosensitization

    PubMed Central

    Joh, Daniel Y.; Sun, Lova; Stangl, Melissa; Al Zaki, Ajlan; Murty, Surya; Santoiemma, Phillip P.; Davis, James J.; Baumann, Brian C.; Alonso-Basanta, Michelle; Bhang, Dongha; Kao, Gary D.; Tsourkas, Andrew; Dorsey, Jay F.

    2013-01-01

    Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature. PMID:23638079

  7. Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

    PubMed

    Kuo, Lu-Ting; Lu, Hsueh-Yi; Lee, Chien-Chang; Tsai, Jui-Chang; Lai, Hong-Shiee; Tseng, Ham-Min; Kuo, Meng-Fai; Tu, Yong-Kwang

    2016-08-01

    Aberrant methylation has been associated with transcriptional inactivation of tumor-related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan-Meier survival curve analysis demonstrated longer duration of progression-free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression-free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression-free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  8. Codon 61 mutations in the c-Harvey-ras gene in mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene plus okadaic acid class tumor promoters.

    PubMed

    Fujiki, H; Suganuma, M; Yoshizawa, S; Kanazawa, H; Sugimura, T; Manam, S; Kahn, S M; Jiang, W; Hoshina, S; Weinstein, I B

    1989-01-01

    Three okadaic acid class tumor promoters, okadaic acid, dinophysistoxin-1, and calyculin A, have potent tumor-promoting activity in two-stage carcinogenesis experiments on mouse skin. DNA isolated from tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and each of these tumor promoters revealed the same mutation at the second nucleotide of codon 61 (CAA----CTA) in the c-Ha-ras gene, determined by the polymerase chain reaction procedure and DNA sequencing. Three potent 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoters, TPA, teleocidin, and aplysiatoxin, showed the same effects. These results provide strong evidence that this mutation in the c-Ha-ras gene is due to a direct effect of DMBA rather than a selective effect of specific tumor promoters.

  9. Migration of mitochondrial DNA in the nuclear genome of colorectal adenocarcinoma.

    PubMed

    Srinivasainagendra, Vinodh; Sandel, Michael W; Singh, Bhupendra; Sundaresan, Aishwarya; Mooga, Ved P; Bajpai, Prachi; Tiwari, Hemant K; Singh, Keshav K

    2017-03-29

    Colorectal adenocarcinomas are characterized by abnormal mitochondrial DNA (mtDNA) copy number and genomic instability, but a molecular interaction between mitochondrial and nuclear genome remains unknown. Here we report the discovery of increased copies of nuclear mtDNA (NUMT) in colorectal adenocarcinomas, which supports link between mtDNA and genomic instability in the nucleus. We name this phenomenon of nuclear occurrence of mitochondrial component as numtogenesis. We provide a description of NUMT abundance and distribution in tumor versus matched blood-derived normal genomes. Whole-genome sequence data were obtained for colon adenocarcinoma and rectum adenocarcinoma patients participating in The Cancer Genome Atlas, via the Cancer Genomics Hub, using the GeneTorrent file acquisition tool. Data were analyzed to determine NUMT proportion and distribution on a genome-wide scale. A NUMT suppressor gene was identified by comparing numtogenesis in other organisms. Our study reveals that colorectal adenocarcinoma genomes, on average, contains up to 4.2-fold more somatic NUMTs than matched normal genomes. Women colorectal tumors contained more NUMT than men. NUMT abundance in tumor predicted parallel abundance in blood. NUMT abundance positively correlated with GC content and gene density. Increased numtogenesis was observed with higher mortality. We identified YME1L1, a human homolog of yeast YME1 (yeast mitochondrial DNA escape 1) to be frequently mutated in colorectal tumors. YME1L1 was also mutated in tumors derived from other tissues. We show that inactivation of YME1L1 results in increased transfer of mtDNA in the nuclear genome. Our study demonstrates increased somatic transfer of mtDNA in colorectal tumors. Our study also reveals sex-based differences in frequency of NUMT occurrence and that NUMT in blood reflects NUMT in tumors, suggesting NUMT may be used as a biomarker for tumorigenesis. We identify YME1L1 as the first NUMT suppressor gene in human and

  10. Diagnostic accuracy of droplet digital PCR for detection of EGFR T790M mutation in circulating tumor DNA

    PubMed Central

    Tong, Xiang; Wang, Ye; Wang, Chengdi; Jin, Jing; Tian, Panwen; Li, Weimin

    2018-01-01

    Objectives Although different methods have been established to detect epidermal growth factor receptor (EGFR) T790M mutation in circulating tumor DNA (ctDNA), a wide range of diagnostic accuracy values were reported in previous studies. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for droplet digital PCR (ddPCR) in the diagnosis of EGFR T790M mutation based on ctDNA. Materials and methods A systematic review and meta-analysis were carried out based on resources from Pubmed, Web of Science, Embase and Cochrane Library up to October 11, 2017. Data were extracted to assess the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio (NLR), diagnostic OR (DOR), and areas under the summary receiver-operating characteristic curve (SROC). Results Eleven of 311 studies identified have met the including criteria. The sensitivity and specificity of ddPCR for the detection of T790M mutation in ctDNA ranged from 0.0% to 100.0% and 63.2% to 100.0%, respectively. For the pooled analysis, ddPCR had a performance of 70.1% (95% CI, 62.7%–76.7%) sensitivity, 86.9 % (95% CI, 80.6%–91.7%) specificity, 3.67 (95% CI, 2.33–5.79) PLR, 0.41 (95% CI, 0.32–0.55) NLR, and 10.83 (95% CI, 5.86–20.03) DOR, with the area under the SROC curve being 0.82. Conclusion The ddPCR harbored a good performance for detection of EGFR T790M mutation in ctDNA. PMID:29844700

  11. Theranostic GO-based nanohybrid for tumor induced imaging and potential combinational tumor therapy.

    PubMed

    Qin, Si-Yong; Feng, Jun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Liu, Xiang-Ji; Luo, Guo-Feng; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2014-02-12

    Graphene oxide (GO)-based theranostic nanohybrid is designed for tumor induced imaging and potential combinational tumor therapy. The anti-tumor drug, Doxorubicin (DOX) is chemically conjugated to the poly(ethylenimine)-co-poly(ethylene glycol) (PEI-PEG) grafted GO via a MMP2-cleavable PLGLAG peptide linkage. The therapeutic efficacy of DOX is chemically locked and its intrinsic fluorescence is quenched by GO under normal physiological condition. Once stimulated by the MMP2 enzyme over-expressed in tumor tissues, the resulting peptide cleavage permits the unloading of DOX for tumor therapy and concurrent fluorescence recovery of DOX for in situ tumor cell imaging. Attractively, this PEI-bearing nanohybrid can mediate efficient DNA transfection and shows great potential for combinational drug/gene therapy. This tumor induced imaging and potential combinational therapy will open a window for tumor treatment by offering a unique theranostic approach through merging the diagnostic capability and pathology-responsive therapeutic function. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A DNA methylation map of human cancer at single base-pair resolution

    PubMed Central

    Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

    2017-01-01

    Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

  13. A DNA methylation map of human cancer at single base-pair resolution.

    PubMed

    Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

    2017-10-05

    Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

  14. Analysis of Fecal DNA Methylation to Detect Gastrointestinal Neoplasia

    PubMed Central

    Tanaka, Noriaki; Cullings, Harry M.; Sun, Dong-Sheng; Sasamoto, Hiromi; Uchida, Takuyuki; Koi, Minoru; Nishida, Naoshi; Naomoto, Yoshio; Boland, C. Richard; Matsubara, Nagahide; Goel, Ajay

    2009-01-01

    Background The development of noninvasive screening tests is important to reduce mortality from gastrointestinal neoplasia. We sought to develop such a test by analysis of DNA methylation from exfoliated cancer cells in feces. Methods We first analyzed methylation of the RASSF2 and SFRP2 gene promoters from 788 primary gastric and colorectal tissue specimens to determine whether methylation patterns could act as stage-dependent biomarkers of gastrointestinal tumorigenesis. Next, we developed a novel strategy that uses single-step modification of DNA with sodium bisulfite and fluorescence polymerase chain reaction methodology to measure aberrant methylation in fecal DNA. Methylation of the RASSF2 and SFRP2 promoters was analyzed in 296 fecal samples obtained from a variety of patients, including 21 with gastric tumors, 152 with colorectal tumors, and 10 with non-neoplastic or inflammatory lesions in the gastrointestinal lumen. Results Analysis of DNA from tissues showed presence of extensive methylation in both gene promoters exclusively in advanced gastric and colorectal tumors. The assay successfully identified one or more methylated markers in fecal DNA from 57.1% of patients with gastric cancer, 75.0% of patients with colorectal cancer, and 44.4% of patients with advanced colorectal adenomas, but only 10.6% of subjects without neoplastic or active diseases (difference, gastric cancer vs undiseased  =  46.5%, 95% confidence interval (CI)  =  24.6% to 68.4%, P < .001; difference, colorectal cancer vs undiseased = 64.4%, 95% CI = 53.5% to 75.2%, P < .001; difference, colorectal adenoma vs undiseased = 33.8%, 95% CI = 14.2% to 53.4%, P < .001). Conclusions Methylation of the RASSF2 and SFRP2 promoters in fecal DNA is associated with the presence of gastrointestinal tumors relative to non-neoplastic conditions. Our novel fecal DNA methylation assay provides a possible means to noninvasively screen not only for colorectal tumors but also for gastric tumors

  15. Reduced mitochondrial DNA content associates with poor prognosis of prostate cancer in African American men.

    PubMed

    Koochekpour, Shahriar; Marlowe, Timothy; Singh, Keshav K; Attwood, Kristopher; Chandra, Dhyan

    2013-01-01

    Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. Although imbalanced mtDNA content is known to occur in prostate cancer, differences in mtDNA content between African American (AA) and Caucasian American (CA) men are not defined. We provide the first evidence that tumors in AA men possess reduced level of mtDNA compared to CA men. The median tumor mtDNA content was reduced in AA men. mtDNA content was also reduced in normal prostate tissues of AA men compared to CA men, suggesting a possible predisposition to cancer in AA men. mtDNA content was also reduced in benign prostatic hyperplasia (BPH) tissue from AA men. Tumor and BPH tissues from patients ≥ 60 years of age possess reduced mtDNA content compared to patients <60 years of age. In addition, mtDNA content was higher in normal tissues from patients with malignant T3 stage disease compared to patients with T2 stage disease. mtDNA levels in matched normal prostate tissues were nearly doubled in Gleason grade of >7 compared to ≤ 7, whereas reduced mtDNA content was observed in tumors of Gleason grade >7 compared to ≤ 7. Together, our data suggest that AA men possess lower mtDNA levels in normal and tumor tissues compared to CA men, which could contribute to higher risk and more aggressive prostate cancer in AA men.

  16. The innate immune system in host mice targets cells with allogenic mitochondrial DNA

    PubMed Central

    Ishikawa, Kaori; Nakada, Kazuto; Morimoto, Mami; Imanishi, Hirotake; Yoshizaki, Mariko; Sasawatari, Shigemi; Niikura, Mamoru; Takenaga, Keizo; Yonekawa, Hiromichi

    2010-01-01

    Mitochondrial DNA (mtDNA) has been proposed to be involved in respiratory function, and mtDNA mutations have been associated with aging, tumors, and various disorders, but the effects of mtDNA imported into transplants from different individuals or aged subjects have been unclear. We examined this issue by generating trans-mitochondrial tumor cells and embryonic stem cells that shared the syngenic C57BL/6 (B6) strain–derived nuclear DNA background but possessed mtDNA derived from allogenic mouse strains. We demonstrate that transplants with mtDNA from the NZB/B1NJ strain were rejected from the host B6 mice, not by the acquired immune system but by the innate immune system. This rejection was caused partly by NK cells and involved a MyD88-dependent pathway. These results introduce novel roles of mtDNA and innate immunity in tumor immunology and transplantation medicine. PMID:20937705

  17. Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair

    PubMed Central

    Gelincik, Ozkan; Blecua, Pedro; Edelmann, Winfried; Kucherlapati, Raju; Zhou, Kathy; Jasin, Maria; Gümüş, Zeynep H.; Lipkin, Steven M.

    2017-01-01

    Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression. PMID:29069730

  18. Intraperitoneal Administration of a Tumor-Associated Antigen SART3, CD40L, and GM-CSF Gene-Loaded Polyplex Micelle Elicits a Vaccine Effect in Mouse Tumor Models

    PubMed Central

    Furugaki, Kouichi; Cui, Lin; Kunisawa, Yumi; Osada, Kensuke; Shinkai, Kentaro; Tanaka, Masao; Kataoka, Kazunori; Nakano, Kenji

    2014-01-01

    Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype. PMID:25013909

  19. The case for DNA methylation based molecular profiling to improve diagnostic accuracy for central nervous system embryonal tumors (not otherwise specified) in adults.

    PubMed

    Halliday, Gail C; Junckerstorff, Reimar C; Bentel, Jacqueline M; Miles, Andrew; Jones, David T W; Hovestadt, Volker; Capper, David; Endersby, Raelene; Cole, Catherine H; van Hagen, Tom; Gottardo, Nicholas G

    2018-01-01

    Central nervous system primitive neuro-ectodermal tumors (CNS-PNETs), have recently been re-classified in the most recent 2016 WHO Classification into a standby catch all category, "CNS Embryonal Tumor, not otherwise specified" (CNS embryonal tumor, NOS) based on epigenetic, biologic and histopathologic criteria. CNS embryonal tumors (NOS) are a rare, histologically and molecularly heterogeneous group of tumors that predominantly affect children, and occasionally adults. Diagnosis of this entity continues to be challenging and the ramifications of misdiagnosis of this aggressive class of brain tumors are significant. We report the case of a 45-year-old woman who was diagnosed with a central nervous system embryonal tumor (NOS) based on immunohistochemical analysis of the patient's tumor at diagnosis. However, later genome-wide methylation profiling of the diagnostic tumor undertaken to guide treatment, revealed characteristics most consistent with IDH-mutant astrocytoma. DNA sequencing and immunohistochemistry confirmed the presence of IDH1 and ATRX mutations resulting in a revised diagnosis of high-grade small cell astrocytoma, and the implementation of a less aggressive treatment regime tailored more appropriately to the patient's tumor type. This case highlights the inadequacy of histology alone for the diagnosis of brain tumours and the utility of methylation profiling and integrated genomic analysis for the diagnostic verification of adults with suspected CNS embryonal tumor (NOS), and is consistent with the increasing realization in the field that a combined diagnostic approach based on clinical, histopathological and molecular data is required to more accurately distinguish brain tumor subtypes and inform more effective therapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    PubMed Central

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  1. Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

    PubMed Central

    Ishii, Hidenobu; Azuma, Koichi; Sakai, Kazuko; Kawahara, Akihiko; Yamada, Kazuhiko; Tokito, Takaaki; Okamoto, Isamu; Nishio, Kazuto; Hoshino, Tomoaki

    2015-01-01

    As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs. PMID:26334838

  2. A DNA enzyme with N-glycosylase activity

    NASA Technical Reports Server (NTRS)

    Sheppard, T. L.; Ordoukhanian, P.; Joyce, G. F.

    2000-01-01

    In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10(6)-fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an N-glycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.

  3. Histones: Controlling Tumor Signaling Circuitry

    PubMed Central

    Martins, Manoela D.; Castilho, Rogerio M.

    2014-01-01

    Epigenetic modifications constitute the next frontier in tumor biology research. Post-translation modification of histones dynamically influences gene expression independent of alterations to the DNA sequence. These mechanisms are often mediated by histone linkers or by proteins associated with the recruitment of DNA-binding proteins, HDAC I and II interacting proteins and transcriptional activators, coactivators or corepressors. Early evidence suggested that histones and their modifiers are involved in sophisticated processes that modulate tumor behavior and cellular phenotype. In this review, we discuss how recent discoveries about chromatin modifications, particularly histone acetylation, are shaping our knowledge of cell biology and our understanding of the molecular circuitry governing tumor progression and consider whether recent insights may extend to novel therapeutic approaches. Furthermore, we discuss the latest oncogenomic findings in Head and Neck Squamous Cell Carcinoma (HNSCC) from studies using Next Generation Sequencing (NGS) technology and highlight the impact of mutations identified in histones and their modifiers. PMID:25177526

  4. Helical-Tip Needle for Transthoracic Percutaneous Image-Guided Biopsy of Lung Tumors: Results of a Pilot Prospective Comparative Study with a Standard Tru-Cut Needle

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Veltri, Andrea, E-mail: andrea.veltri@unito.it; Busso, Marco; Sardo, Diego

    PurposeTo prospectively evaluate feasibility and diagnostic performance of the 14-gauge helical-tip (Spirotome™, Cook{sup ®} Medical, Bloomington, USA) needle in transthoracic needle biopsy (TTNB) of lung lesions, compared to a conventional 18-gauge Tru-Cut needle.Materials and MethodsStudy was institutional review board approved, with informed consent obtained. Data from synchronous Spirotome and Tru-Cut image-guided TTNB of 20 consecutive patients with malignant peripheral lung tumors larger than 3 cm were enrolled for pathologic characterization and mutational analysis. Samples obtained with Spirotome and Tru-Cut needle were compared for fragmentation, length, weight, morphologic and immunohistochemistry typifying, tumor cellularity (TC) and DNA concentration.ResultsThe technical success rate for TTNBmore » with Spirotome was 100%, and no major complications occurred. Less fragmentation (mean 2 vs. 3 fragments, P = .418), greater weight (mean 13 vs. 8.5 mg, P = .027) and lower length (mean 10.2 vs. 12.6 mm, P = .174) were observed with Spirotome compared to Tru-Cut needle. Accuracy of Spirotome and Tru-Cut needle in defining cancer histotype was similar (90%). Absolute and relative TC (mean 42 vs. 38, 124 vs. 108/10HPF), and DNA concentration (mean 49.6 vs. 39.0 ng/μl) were higher with Spirotome compared to Tru-Cut needle, with no statistical significance (P = .787 and P = .140, respectively).Conclusions Percutaneous 14-gauge Spirotome TTNB of selected lesions is feasible and accurate. It provides adequate samples for diagnosis, comparable to 18-gauge Tru-Cut needle, with a higher amount of tumor tissue (weight, TC, DNA concentration) even in shorter samples.« less

  5. KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue.

    PubMed

    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.

  6. Clones from a shooty tobacco crown gall tumor I: deletions, rearrangements and amplifications resulting in irregular T-DNA structures and organizations.

    PubMed

    Peerbolte, R; Leenhouts, K; Hooykaas-van Slogteren, G M; Hoge, J H; Wullems, G J; Schilperoort, R A

    1986-07-01

    Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having a Tn1831 insertion in the auxin locus, were investigated for their T-DNA structure and expression. In addition to clones with the expected phenotype, i.e. phytohormone autonomy, regeneration of non-rooting shoots and octopine synthesis (Aut(+)Reg(+)Ocs(+) 'type I' clones), clones were obtained with an aberrant phenotype. Among these were the Aut(-)Reg(-)Ocs(+) 'type II' clones. Two shooty type I clones and three type II callus clones (all randomly chosen) as well as a rooting shoot regenerated from a type II clone via a high kinetin treatment, all had a T-DNA structure which differed significantly from 'regular' T-DNA structures. No Tn1831 DNA sequences were detected in these clones. The two type I clones were identical: they both contained the same highly truncated T-DNA segments. One TL-DNA segment of approximately 0.7 kb, originating form the left part of the TL-region, was present at one copy per diploid tobacco genome. Another segment with a maximum size of about 7 kb was derived from the right hand part of the TL-region and was present at minimally two copies. Three copies of a truncated TR-DNA segment were detected, probably starting at the right TR-DNA border repeat and ending halfway the regular TR-region. Indications have been obtained that at least some of the T-DNA segments are closely linked, sometimes via intervening plant DNA sequences. The type I clones harbored TL-DNA transcripts 4, 6a/b and 3 as well as TR-DNA transcript 0'. The type II clones harbored three to six highly truncated T-DNA segments, originating from the right part of the TL-region. In addition they had TR-DNA segments, similar to those of the type I clones. On Northern blots TR-DNA transcripts 0' and 1' were detected as well as the TL-DNA transcripts 3 and 6a/b and an 1800 bp hybrid transcript (tr.Y) containing gene 6b sequences. Possible origins of the observed

  7. ctDNA DLBCL Detection Lancet Oncology

    Cancer.gov

    Measurement of circulating tumor DNA in blood can be used to detect disease recurrence in patients with a curable form of cancer known as diffuse large B-cell lymphoma (DLBCL). In most patients, measurement of ctDNA enabled detection of microscopic diseas

  8. Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

    PubMed

    Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

    2005-09-01

    1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

  9. Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

    PubMed Central

    Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

    2016-01-01

    Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

  10. Computational Approach to Explore the B/A Junction Free Energy in DNA.

    PubMed

    Kulkarni, Mandar; Mukherjee, Arnab

    2016-01-04

    Protein-DNA interactions induce conformational changes in DNA such as B- to A-form transitions at a local level. Such transitions are associated with a junction free energy cost at the boundary of two different conformations in a DNA molecule. In this study, we performed umbrella sampling simulations to find the free energy values of the B-A transition at the dinucleotide and trinucleotide level of DNA. Using a combination of dinucleotide and trinucleotide free energy costs obtained from simulations, we calculated the B/A junction free energy. Our study shows that the B/A junction free energy is 0.52 kcal mol(-1) for the A-philic GG step and 1.59 kcal mol(-1) for the B-philic AA step. This observation is in agreement with experimentally derived values. After excluding junction effects, we obtained an absolute free energy cost for the B- to A-form conversion for all the dinucleotide steps. These absolute free energies may be used for predicting the propensity of structural transitions in DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    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

  12. Nuclear dna amounts in angiosperms.

    PubMed

    Bennett, M D; Smith, J B

    1976-05-27

    The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication of a more comprehensive list is over due. This paper lists absolute nuclear DNA amounts for 753 angiosperm species. The dats were assembled primarily for reference purposes, and so the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists whom, it is anticipated, will be among its major users. The paper also reviews aspects of the history, nomenclature, methods, accuracy and problems of nuclear DNA estimation in angiosperms. No attempt is made to reconsider those aspects of nuclear DNA estimation which have been fully revised previously, although the bibliography of such aspects is given. Instead, the paper is intended as a source of basic information regarding the terminology, practice and limitations of nuclear DNA estimation, especially by Feulgen microdensitometry, as currently practiced.

  13. Tumor prevalence and biomarkers of genotoxicity in brown bullhead (Ameiurus nebulosus) in Chesapeake Bay tributaries

    USGS Publications Warehouse

    Pinkney, Alfred E.; Harshbarger, John C.; Karouna-Renier, Natalie K.; Jenko, Kathryn; Balk, Lennart; Skarphéðinsdóttir, Halldora; Liewenborg, Birgitta; Rutter, Michael A.

    2011-01-01

    We surveyed four Chesapeake Bay tributaries for skin and liver tumors in brown bullhead (Ameiurus nebulosus). We focused on the South River, where the highest skin tumor prevalence (53%) in the Bay watershed had been reported. The objectives were to 1) compare tumor prevalence with nearby rivers (Severn and Rhode) and a more remote river (Choptank); 2) investigate associations between tumor prevalence and polynuclear aromatic hydrocarbons (PAHs) and alkylating agents; and 3) statistically analyze Chesapeake Bay bullhead tumor data from 1992 through 2008. All four South River collections exhibited high skin tumor prevalence (19% to 58%), whereas skin tumor prevalence was 2%, 10%, and 52% in the three Severn collections; 0% and 2% in the Choptank collections; and 5.6% in the Rhode collection. Liver tumor prevalence was 0% to 6% in all but one South River collection (20%) and 0% to 6% in the three other rivers. In a subset of samples, PAH-like biliary metabolites and 32P-DNA adducts were used as biomarkers of exposure and response to polycyclic aromatic compounds (PACs). Adducts from alkylating agents were detected as O6-methyl-2'-deoxyguanosine (O6Me-dG) and O6-ethyl-2'-deoxyguanosine (O6Et-dG) modified DNA. Bullheads from the contaminated Anacostia River were used as a positive control for DNA adducts. 32P-DNA adduct concentrations were significantly higher in Anacostia bullhead livers compared with the other rivers. We identified alkyl DNA adducts in bullhead livers from the South and Anacostia, but not the Choptank. Neither the PAH-like bile metabolite data, sediment PAH data, nor the DNA adduct data suggest an association between liver or skin tumor prevalence and exposure to PACs or alkylating agents in the South, Choptank, Severn, or Rhode rivers. Logistic regression analysis of the Chesapeake Bay database revealed that sex and length were significant covariates for liver tumors and length was a significant covariate for skin tumors.

  14. Oxidative DNA damage induced by a hydroperoxide derivative of cyclophosphamide.

    PubMed

    Murata, Mariko; Suzuki, Toshinari; Midorikawa, Kaoru; Oikawa, Shinji; Kawanishi, Shosuke

    2004-09-15

    Interstrand DNA cross-linking has been considered to be the primary action mechanism of cyclophosphamide (CP) and its hydroperoxide derivative, 4-hydroperoxycyclophosphamide (4-HC). To clarify the mechanism of anti-tumor effects by 4-HC, we investigated DNA damage in a human leukemia cell line, HL-60, and its H(2)O(2)-resistant clone HP100. Apoptosis DNA ladder formation was detected in HL-60 cells treated with 4-HC, whereas it was not observed in HP100 cells. 4-HC significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, a marker of oxidative DNA damage, in HL-60 cells. On the other hand, CP did not significantly induce 8-oxodG formation and apoptosis in HL-60 cells under the same conditions as did 4-HC. Using (32)P-labeled DNA fragments from the human p53 tumor suppressor gene, 4-HC was found to cause Cu(II)-mediated oxidative DNA damage, but CP did not. Catalase inhibited 4-HC-induced DNA damage, including 8-oxodG formation, suggesting the involvement of H(2)O(2). The generation of H(2)O(2) during 4-HC degradation was ascertained by procedures using scopoletin and potassium iodide. We conclude that, in addition to DNA cross-linking, oxidative DNA damage through H(2)O(2) generation may participate in the anti-tumor effects of 4-HC.

  15. Estimating the absolute wealth of households.

    PubMed

    Hruschka, Daniel J; Gerkey, Drew; Hadley, Craig

    2015-07-01

    To estimate the absolute wealth of households using data from demographic and health surveys. We developed a new metric, the absolute wealth estimate, based on the rank of each surveyed household according to its material assets and the assumed shape of the distribution of wealth among surveyed households. Using data from 156 demographic and health surveys in 66 countries, we calculated absolute wealth estimates for households. We validated the method by comparing the proportion of households defined as poor using our estimates with published World Bank poverty headcounts. We also compared the accuracy of absolute versus relative wealth estimates for the prediction of anthropometric measures. The median absolute wealth estimates of 1,403,186 households were 2056 international dollars per capita (interquartile range: 723-6103). The proportion of poor households based on absolute wealth estimates were strongly correlated with World Bank estimates of populations living on less than 2.00 United States dollars per capita per day (R(2)  = 0.84). Absolute wealth estimates were better predictors of anthropometric measures than relative wealth indexes. Absolute wealth estimates provide new opportunities for comparative research to assess the effects of economic resources on health and human capital, as well as the long-term health consequences of economic change and inequality.

  16. Structure of the Tetrameric p53 Tumor Suppressor Bound to DNA

    DTIC Science & Technology

    2002-05-01

    been unable to prepare suitable p53/DNA cocrystals for structure determination. Nonetheless, we have successfully determined the medium resolution (2.7A... cocrystallization with longer DNA targets or DNA targets assembled into nucleosome core particles. The structure of tetrameric p53 bound to DNA will provide

  17. Fanconi anemia (cross)linked to DNA repair.

    PubMed

    Niedernhofer, Laura J; Lalai, Astrid S; Hoeijmakers, Jan H J

    2005-12-29

    Fanconi anemia is characterized by hypersensitivity to DNA interstrand crosslinks (ICLs) and susceptibility to tumor formation. Despite the identification of numerous Fanconi anemia (FANC) genes, the mechanism by which proteins encoded by these genes protect a cell from DNA interstrand crosslinks remains unclear. The recent discovery of two DNA helicases that, when defective, cause Fanconi anemia tips the balance in favor of the direct involvement of the FANC proteins in DNA repair and the bypass of DNA lesions.

  18. Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors

    PubMed Central

    Kalra, Sean; Cleaver, Timothy G.; Merrick, Daniel; Wang, Xiao-Jing; Malkoski, Stephen P.

    2015-01-01

    Non-small cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer, however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC datasets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms including mutation, homozygous deletion, and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4−/− mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage while Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4 deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4 deficient tumors can be exploited by specific therapeutic strategies. PMID:25893305

  19. Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines

    PubMed Central

    Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

    2013-01-01

    The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer. PMID:24149212

  20. Recurrent patterns of DNA methylation in the ZNF154, CASP8, and VHL promoters across a wide spectrum of human solid epithelial tumors and cancer cell lines.

    PubMed

    Sánchez-Vega, Francisco; Gotea, Valer; Petrykowska, Hanna M; Margolin, Gennady; Krivak, Thomas C; DeLoia, Julie A; Bell, Daphne W; Elnitski, Laura

    2013-12-01

    The study of aberrant DNA methylation in cancer holds the key to the discovery of novel biological markers for diagnostics and can help to delineate important mechanisms of disease. We have identified 12 loci that are differentially methylated in serous ovarian cancers and endometrioid ovarian and endometrial cancers with respect to normal control samples. The strongest signal showed hypermethylation in tumors at a CpG island within the ZNF154 promoter. We show that hypermethylation of this locus is recurrent across solid human epithelial tumor samples for 15 of 16 distinct cancer types from TCGA. Furthermore, ZNF154 hypermethylation is strikingly present across a diverse panel of ENCODE cell lines, but only in those derived from tumor cells. By extending our analysis from the Illumina 27K Infinium platform to the 450K platform, to sequencing of PCR amplicons from bisulfite treated DNA, we demonstrate that hypermethylation extends across the breadth of the ZNF154 CpG island. We have also identified recurrent hypomethylation in two genomic regions associated with CASP8 and VHL. These three genes exhibit significant negative correlation between methylation and gene expression across many cancer types, as well as patterns of DNaseI hypersensitivity and histone marks that reflect different chromatin accessibility in cancer vs. normal cell lines. Our findings emphasize hypermethylation of ZNF154 as a biological marker of relevance for tumor identification. Epigenetic modifications affecting the promoters of ZNF154, CASP8, and VHL are shared across a vast array of tumor types and may therefore be important for understanding the genomic landscape of cancer.

  1. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

    USDA-ARS?s Scientific Manuscript database

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

  2. Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

    PubMed Central

    Fukai, Junya; Koizumi, Fumiaki; Nakao, Naoyuki

    2014-01-01

    Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance. PMID:25111384

  3. Intratumoral heterogeneity in colorectal carcinoma: trucut sampling for DNA ploidy analysis.

    PubMed

    Giovagnoli, M R; Giarnieri, E; Midiri, G; Tesoriere, A; Ferraro, S; Vecchione, A

    1999-01-01

    Solid tumors, such as colorectal carcinomas, consist of cell subpopulations that differ both genetically and in their clinical behavior. Many authors have examined cell kinetics and DNA content in colorectal tumors in correlation to clinical and pathological variables with different results. The interpretation of those results present some difficulties related to tumor heterogeneity that to date are unsolved. Our study is based on a new method of colon cancer sampling for DNA content determination. The aim of this work was to reduce the risk of incorrect DNA evaluation due to tumor heterogeneity. Our study was based on eleven selected cases of T3 colorectal carcinoma. Fresh surgical specimens from the primary tumor site were taken during surgery. For each case at least four samples were taken using a 23 gauge trucut from the outside of the serosa through the tumor to the lumen of the colon. The specimens were stained according to a modified Feulgen method and DNA content was measured by image analysis. Three parameters were evaluated: DNA index, ploidy and proliferation level (considered as the sum of elements corresponding to the S and G2 phases). One of the eleven (9.1%) tumors showed a diploid pattern; four out of eleven (36.4%) cases showed a tetra/polyploid pattern and six out of eleven (54.5%) cases showed an aneuploid pattern. Three tumors were monoclonal (27.3%), one diploid and two aneuploid. Eight were polyclonal (72.7%). Considering the single specimen, seven out of sixty-eight specimens (10.3%) were inadequate because of scanty material. Twenty-five out of the sixty-one adequate specimens (41%) were monoclonal and thirty-six (59%) were polyclonal. Five tumors (three monoclonal and two polyclonal) showed the same cell clones on all the examined samples. The remaining six tumors showed interregional variability. The six of the eight polyclonal cases (75%) multiple stem lines were evident, analyzing only one sample taken close to colon serosa, while in one

  4. Intratumor DNA methylation heterogeneity reflects clonal evolution in aggressive prostate cancer.

    PubMed

    Brocks, David; Assenov, Yassen; Minner, Sarah; Bogatyrova, Olga; Simon, Ronald; Koop, Christina; Oakes, Christopher; Zucknick, Manuela; Lipka, Daniel Bernhard; Weischenfeldt, Joachim; Feuerbach, Lars; Cowper-Sal Lari, Richard; Lupien, Mathieu; Brors, Benedikt; Korbel, Jan; Schlomm, Thorsten; Tanay, Amos; Sauter, Guido; Gerhäuser, Clarissa; Plass, Christoph

    2014-08-07

    Despite much evidence on epigenetic abnormalities in cancer, it is currently unclear to what extent epigenetic alterations can be associated with tumors' clonal genetic origins. Here, we show that the prostate intratumor heterogeneity in DNA methylation and copy-number patterns can be explained by a unified evolutionary process. By assaying multiple topographically distinct tumor sites, premalignant lesions, and lymph node metastases within five cases of prostate cancer, we demonstrate that both DNA methylation and copy-number heterogeneity consistently reflect the life history of the tumors. Furthermore, we show cases of genetic or epigenetic convergent evolution and highlight the diversity in the evolutionary origins and aberration spectrum between tumor and metastatic subclones. Importantly, DNA methylation can complement genetic data by serving as a proxy for activity at regulatory domains, as we show through identification of high epigenetic heterogeneity at androgen-receptor-bound enhancers. Epigenome variation thereby expands on the current genome-centric view on tumor heterogeneity. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Insights into the Pathogenesis of Anaplastic Large-Cell Lymphoma through Genome-wide DNA Methylation Profiling.

    PubMed

    Hassler, Melanie R; Pulverer, Walter; Lakshminarasimhan, Ranjani; Redl, Elisa; Hacker, Julia; Garland, Gavin D; Merkel, Olaf; Schiefer, Ana-Iris; Simonitsch-Klupp, Ingrid; Kenner, Lukas; Weisenberger, Daniel J; Weinhaeusel, Andreas; Turner, Suzanne D; Egger, Gerda

    2016-10-04

    Aberrant DNA methylation patterns in malignant cells allow insight into tumor evolution and development and can be used for disease classification. Here, we describe the genome-wide DNA methylation signatures of NPM-ALK-positive (ALK+) and NPM-ALK-negative (ALK-) anaplastic large-cell lymphoma (ALCL). We find that ALK+ and ALK- ALCL share common DNA methylation changes for genes involved in T cell differentiation and immune response, including TCR and CTLA-4, without an ALK-specific impact on tumor DNA methylation in gene promoters. Furthermore, we uncover a close relationship between global ALCL DNA methylation patterns and those in distinct thymic developmental stages and observe tumor-specific DNA hypomethylation in regulatory regions that are enriched for conserved transcription factor binding motifs such as AP1. Our results indicate similarity between ALCL tumor cells and thymic T cell subsets and a direct relationship between ALCL oncogenic signaling and DNA methylation through transcription factor induction and occupancy. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Aberrantly methylated DNA as a biomarker in breast cancer.

    PubMed

    Kristiansen, Søren; Jørgensen, Lars M; Guldberg, Per; Sölétormos, György

    2013-01-01

    Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

  7. Estimating the absolute wealth of households

    PubMed Central

    Gerkey, Drew; Hadley, Craig

    2015-01-01

    Abstract Objective To estimate the absolute wealth of households using data from demographic and health surveys. Methods We developed a new metric, the absolute wealth estimate, based on the rank of each surveyed household according to its material assets and the assumed shape of the distribution of wealth among surveyed households. Using data from 156 demographic and health surveys in 66 countries, we calculated absolute wealth estimates for households. We validated the method by comparing the proportion of households defined as poor using our estimates with published World Bank poverty headcounts. We also compared the accuracy of absolute versus relative wealth estimates for the prediction of anthropometric measures. Findings The median absolute wealth estimates of 1 403 186 households were 2056 international dollars per capita (interquartile range: 723–6103). The proportion of poor households based on absolute wealth estimates were strongly correlated with World Bank estimates of populations living on less than 2.00 United States dollars per capita per day (R2 = 0.84). Absolute wealth estimates were better predictors of anthropometric measures than relative wealth indexes. Conclusion Absolute wealth estimates provide new opportunities for comparative research to assess the effects of economic resources on health and human capital, as well as the long-term health consequences of economic change and inequality. PMID:26170506

  8. A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non-Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial.

    PubMed

    Yanagita, Masahiko; Redig, Amanda J; Paweletz, Cloud P; Dahlberg, Suzanne E; O'Connell, Allison; Feeney, Nora; Taibi, Myriam; Boucher, David; Oxnard, Geoffrey R; Johnson, Bruce E; Costa, Daniel B; Jackman, David M; Jänne, Pasi A

    2016-12-15

    Genotype-directed therapy is the standard of care for advanced non-small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010-20. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. Factors influencing the specific interaction of Neisseria gonorrhoeae with transforming DNA.

    PubMed Central

    Goodman, S D; Scocca, J J

    1991-01-01

    The specific interaction of transformable Neisseria gonorrhoeae with DNA depends on the recognition of specific 10-residue target sequences. The relative affinity for DNA between 3 and 17 kb in size appears to be linearly related to the frequency of targets on the segment and is unaffected by absolute size. The average frequency of targets in chromosomal DNA of N. gonorrhoeae appears to be approximately one per 1,000 bp. PMID:1909325

  10. Role of Escherichia coli dnaA gene and its integrative suppression in M13 Coliphage DNA synthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mitra, S.; Stallions, D.R.

    An F/sup +/ derivative of Escherichia coli E508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508(ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also,more » but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dnaA function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dnaA function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.« less

  11. Phylogenetic ctDNA analysis depicts early stage lung cancer evolution

    PubMed Central

    Abbosh, Christopher; Birkbak, Nicolai J.; Wilson, Gareth A.; Jamal-Hanjani, Mariam; Constantin, Tudor; Salari, Raheleh; Le Quesne, John; Moore, David A; Veeriah, Selvaraju; Rosenthal, Rachel; Marafioti, Teresa; Kirkizlar, Eser; Watkins, Thomas B K; McGranahan, Nicholas; Ward, Sophia; Martinson, Luke; Riley, Joan; Fraioli, Francesco; Al Bakir, Maise; Grönroos, Eva; Zambrana, Francisco; Endozo, Raymondo; Bi, Wenya Linda; Fennessy, Fiona M.; Sponer, Nicole; Johnson, Diana; Laycock, Joanne; Shafi, Seema; Czyzewska-Khan, Justyna; Rowan, Andrew; Chambers, Tim; Matthews, Nik; Turajlic, Samra; Hiley, Crispin; Lee, Siow Ming; Forster, Martin D.; Ahmad, Tanya; Falzon, Mary; Borg, Elaine; Lawrence, David; Hayward, Martin; Kolvekar, Shyam; Panagiotopoulos, Nikolaos; Janes, Sam M; Thakrar, Ricky; Ahmed, Asia; Blackhall, Fiona; Summers, Yvonne; Hafez, Dina; Naik, Ashwini; Ganguly, Apratim; Kareht, Stephanie; Shah, Rajesh; Joseph, Leena; Quinn, Anne Marie; Crosbie, Phil; Naidu, Babu; Middleton, Gary; Langman, Gerald; Trotter, Simon; Nicolson, Marianne; Remmen, Hardy; Kerr, Keith; Chetty, Mahendran; Gomersall, Lesley; Fennell, Dean; Nakas, Apostolos; Rathinam, Sridhar; Anand, Girija; Khan, Sajid; Russell, Peter; Ezhil, Veni; Ismail, Babikir; Irvin-sellers, Melanie; Prakash, Vineet; Lester, Jason; Kornaszewska, Malgorzata; Attanoos, Richard; Adams, Haydn; Davies, Helen; Oukrif, Dahmane; Akarca, Ayse U; Hartley, John A; Lowe, Helen L; Lock, Sara; Iles, Natasha; Bell, Harriet; Ngai, Yenting; Elgar, Greg; Szallasi, Zoltan; Schwarz, Roland F; Herrero, Javier; Stewart, Aengus; Quezada, Sergio A; Peggs, Karl S.; Van Loo, Peter; Dive, Caroline; Lin, Jimmy; Rabinowitz, Matthew; Aerts, Hugo JWL; Hackshaw, Allan; Shaw, Jacqui A; Zimmermann, Bernhard G.; Swanton, Charles

    2017-01-01

    Summary The early detection of relapse following primary surgery for non-small cell lung cancer and the characterization of emerging subclones seeding metastatic sites might offer new therapeutic approaches to limit tumor recurrence. The potential to non-invasively track tumor evolutionary dynamics in ctDNA of early-stage lung cancer is not established. Here we conduct a tumour-specific phylogenetic approach to ctDNA profiling in the first 100 TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy (Rx)) study participants, including one patient co-recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and perform tumor volume limit of detection analyses. Through blinded profiling of post-operative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients destined to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastases, providing a new approach for ctDNA driven therapeutic studies PMID:28445469

  12. An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors including DNA repair proteins and Nrf2

    PubMed Central

    Cardnell, Robert J.G.; Behrens, Carmen; Diao, Lixia; Fan, YouHong; Tang, Ximing; Tong, Pan; John D., Minna; Mills, Gordon B.; Heymach, John V.; Wistuba, Ignacio I.; Wang, Jing; Byers., Lauren A.

    2015-01-01

    Purpose Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15-20%) does not express TTF1 and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Experimental Design Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and the TCGA, we interrogated proteomic profiles (by reverse-phase protein array (RPPA)), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Results Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho=0.57, p<0.001) and gene expression (NKX2-1, Rho=0.61, p<0.001). Established driver mutations (e.g. BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (p=0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3K/MAPK signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Conclusions Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD includingKEAP1/Nrf2 and DNA repair pathways. PMID:25878335

  13. CancerLocator: non-invasive cancer diagnosis and tissue-of-origin prediction using methylation profiles of cell-free DNA.

    PubMed

    Kang, Shuli; Li, Qingjiao; Chen, Quan; Zhou, Yonggang; Park, Stacy; Lee, Gina; Grimes, Brandon; Krysan, Kostyantyn; Yu, Min; Wang, Wei; Alber, Frank; Sun, Fengzhu; Dubinett, Steven M; Li, Wenyuan; Zhou, Xianghong Jasmine

    2017-03-24

    We propose a probabilistic method, CancerLocator, which exploits the diagnostic potential of cell-free DNA by determining not only the presence but also the location of tumors. CancerLocator simultaneously infers the proportions and the tissue-of-origin of tumor-derived cell-free DNA in a blood sample using genome-wide DNA methylation data. CancerLocator outperforms two established multi-class classification methods on simulations and real data, even with the low proportion of tumor-derived DNA in the cell-free DNA scenarios. CancerLocator also achieves promising results on patient plasma samples with low DNA methylation sequencing coverage.

  14. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  15. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  16. Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma.

    PubMed

    Nakaoka, Toshiaki; Saito, Yoshimasa; Saito, Hidetsugu

    2017-05-23

    Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1 , p14 , p16 , death-associated protein kinase ( DAPK ), miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for the diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors holds considerable promise for the treatment of cholangiocarcinoma through the reactivation of tumor suppressor genes and miRNAs as well as the induction of an anti-viral immune response.

  17. Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma

    PubMed Central

    Nakaoka, Toshiaki; Saito, Yoshimasa; Saito, Hidetsugu

    2017-01-01

    Cholangiocarcinoma is an epithelial malignancy arising in the region between the intrahepatic bile ducts and the ampulla of Vater at the distal end of the common bile duct. The effect of current chemotherapy regimens against cholangiocarcinoma is limited, and the prognosis of patients with cholangiocarcinoma is poor. Aberrant DNA methylation and histone modification induce silencing of tumor suppressor genes and chromosomal instability during carcinogenesis. Studies have shown that the tumor suppressor genes and microRNAs (miRNAs) including MLH1, p14, p16, death-associated protein kinase (DAPK), miR-370 and miR-376c are frequently methylated in cholangiocarcinoma. Silencing of these tumor suppressor genes and miRNAs plays critical roles in the initiation and progression of cholangiocarcinoma. In addition, recent studies have demonstrated that DNA methylation inhibitors induce expression of endogenous retroviruses and exert the anti-tumor effect of via an anti-viral immune response. Aberrant DNA methylation of tumor suppressor genes and miRNAs could be a powerful biomarker for the diagnosis and treatment of cholangiocarcinoma. Epigenetic therapy with DNA methylation inhibitors holds considerable promise for the treatment of cholangiocarcinoma through the reactivation of tumor suppressor genes and miRNAs as well as the induction of an anti-viral immune response. PMID:28545228

  18. Chemotherapeutic Drugs: DNA Damage and Repair in Glioblastoma.

    PubMed

    Annovazzi, Laura; Mellai, Marta; Schiffer, Davide

    2017-05-26

    Despite improvements in therapeutic strategies, glioblastoma (GB) remains one of the most lethal cancers. The presence of the blood-brain barrier, the infiltrative nature of the tumor and several resistance mechanisms account for the failure of current treatments. Distinct DNA repair pathways can neutralize the cytotoxicity of chemo- and radio-therapeutic agents, driving resistance and tumor relapse. It seems that a subpopulation of stem-like cells, indicated as glioma stem cells (GSCs), is responsible for tumor initiation, maintenance and recurrence and they appear to be more resistant owing to their enhanced DNA repair capacity. Recently, attention has been focused on the pivotal role of the DNA damage response (DDR) in tumorigenesis and in the modulation of therapeutic treatment effects. In this review, we try to summarize the knowledge concerning the main molecular mechanisms involved in the removal of genotoxic lesions caused by alkylating agents, emphasizing the role of GSCs. Beside their increased DNA repair capacity in comparison with non-stem tumor cells, GSCs show a constitutive checkpoint expression that enables them to survive to treatments in a quiescent, non-proliferative state. The targeted inhibition of checkpoint/repair factors of DDR can contribute to eradicate the GSC population and can have a great potential therapeutic impact aiming at sensitizing malignant gliomas to treatments, improving the overall survival of patients.

  19. 20 CFR 404.1205 - Absolute coverage groups.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false Absolute coverage groups. 404.1205 Section... Covered § 404.1205 Absolute coverage groups. (a) General. An absolute coverage group is a permanent... are not under a retirement system. An absolute coverage group may include positions which were...

  20. DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

    PubMed

    Schuler, Nadine; Palm, Jan; Kaiser, Mareike; Betten, Dominik; Furtwängler, Rhoikos; Rübe, Christian; Graf, Norbert; Rübe, Claudia E

    2014-01-01

    In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA) suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

  1. DNA Array-Based Gene Profiling

    PubMed Central

    Mocellin, Simone; Provenzano, Maurizio; Rossi, Carlo Riccardo; Pilati, Pierluigi; Nitti, Donato; Lise, Mario

    2005-01-01

    Cancer is a heterogeneous disease in most respects, including its cellularity, different genetic alterations, and diverse clinical behaviors. Traditional molecular analyses are reductionist, assessing only 1 or a few genes at a time, thus working with a biologic model too specific and limited to confront a process whose clinical outcome is likely to be governed by the combined influence of many genes. The potential of functional genomics is enormous, because for each experiment, thousands of relevant observations can be made simultaneously. Accordingly, DNA array, like other high-throughput technologies, might catalyze and ultimately accelerate the development of knowledge in tumor cell biology. Although in its infancy, the implementation of DNA array technology in cancer research has already provided investigators with novel data and intriguing new hypotheses on the molecular cascade leading to carcinogenesis, tumor aggressiveness, and sensitivity to antiblastic agents. Given the revolutionary implications that the use of this technology might have in the clinical management of patients with cancer, principles of DNA array-based tumor gene profiling need to be clearly understood for the data to be correctly interpreted and appreciated. In the present work, we discuss the technical features characterizing this powerful laboratory tool and review the applications so far described in the field of oncology. PMID:15621987

  2. Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery.

    PubMed

    Buckel, Lisa; Savariar, Elamprakash N; Crisp, Jessica L; Jones, Karra A; Hicks, Angel M; Scanderbeg, Daniel J; Nguyen, Quyen T; Sicklick, Jason K; Lowy, Andrew M; Tsien, Roger Y; Advani, Sunil J

    2015-04-01

    Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. ©2015 American Association for Cancer Research.

  3. Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

    PubMed Central

    Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

    2015-01-01

    Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

  4. Circulating Cell-free DNA for Metastatic Cervical Cancer Detection, Genotyping, and Monitoring.

    PubMed

    Kang, Zhigang; Stevanović, Sanja; Hinrichs, Christian S; Cao, Liang

    2017-11-15

    Purpose: Circulating cell-free (ccf) human papillomavirus (HPV) DNA may serve as a unique tumor marker for HPV-associated malignancies, including cervical cancer. We developed a method to genotype and quantify circulating HPV DNA in patients with HPV16- or HPV18-positive metastatic cervical cancer for potential disease monitoring and treatment-related decision making. Experimental Design: In this retrospective study, HPV ccfDNA was measured in serum samples from 19 metastatic cervical cancer patients by duplex digital droplet PCR (ddPCR). Nine patients had received tumor-infiltrating lymphocyte (TIL) immunotherapy. ccfDNA data were aligned with the tumor HPV genotype, drug treatment, and clinical outcome. Results: In blinded tests, HPV ccfDNA was detected in 19 of 19 (100%) patients with HPV-positive metastatic cervical cancer but not in any of the 45 healthy blood donors. The HPV genotype harbored in the patients' tumors was correctly identified in 87 of 87 (100%) sequential patient serum samples from 9 patients who received TIL immunotherapy. In three patients who experienced objective cancer regression after TIL treatment, a transient HPV ccfDNA peak was detected 2-3 days after TIL infusion. Furthermore, persistent clearance of HPV ccfDNA was only observed in two patients who experienced complete response (CR) after TIL immunotherapy. Conclusions: HPV ccfDNA represents a promising tumor marker for noninvasive HPV genotyping and may be used in selecting patients for HPV type-specific T-cell-based immunotherapies. It may also have value in detecting antitumor activity of therapeutic agents and in the long-term follow-up of cervical cancer patients in remission. Clin Cancer Res; 23(22); 6856-62. ©2017 AACR . ©2017 American Association for Cancer Research.

  5. Effect of seven days of spaceflight on hindlimb muscle protein, RNA and DNA in adult rats

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1985-01-01

    Effects of seven days of spaceflight on skeletal muscle (soleus, gastrocnemius, EDL) content of protein, RNA and DNA were determined in adult rats. Whereas total protein contents were reduced in parallel with muscle weights, myofibrillar protein appeared to be more affected. There were no significant changes in absolute DNA contents, but a significant (P less than 0.05) increase in DNA concentration (microgram/milligram) in soleus muscles from flight rats. Absolute RNA contents were significantly (P less than 0.025) decreased in the soleus and gastrocnemius muscles of flight rats, with RNA concentrations reduced 15-30 percent. These results agree with previous ground-based observations on the suspended rat with unloaded hindlimbs and support continued use of this model.

  6. DNA Trojan Horses: Self-Assembled Floxuridine-Containing DNA Polyhedra for Cancer Therapy.

    PubMed

    Mou, Quanbing; Ma, Yuan; Pan, Gaifang; Xue, Bai; Yan, Deyue; Zhang, Chuan; Zhu, Xinyuan

    2017-10-02

    Based on their structural similarity to natural nucleobases, nucleoside analogue therapeutics were integrated into DNA strands through conventional solid-phase synthesis. By elaborately designing their sequences, floxuridine-integrated DNA strands were synthesized and self-assembled into well-defined DNA polyhedra with definite drug-loading ratios as well as tunable size and morphology. As a novel drug delivery system, these drug-containing DNA polyhedra could ideally mimic the Trojan Horse to deliver chemotherapeutics into tumor cells and fight against cancer. Both in vitro and in vivo results demonstrate that the DNA Trojan horse with buckyball architecture exhibits superior anticancer capability over the free drug and other formulations. With precise control over the drug-loading ratio and structure of the nanocarriers, the DNA Trojan horse may play an important role in anticancer treatment and exhibit great potential in translational nanomedicine. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. DNA repair and tumorigenesis: lessons from hereditary cancer syndromes.

    PubMed

    Heinen, Christopher D; Schmutte, Christoph; Fishel, Richard

    2002-01-01

    The discovery that alterations of the DNA mismatch repair system (MMR) were linked to the common human cancer susceptibility syndrome hereditary nonpolyposis colon cancer (HNPCC) resulted in the declaration of a third class of genes involved in tumor development. In addition to oncogenes and tumor suppressors, alterations of DNA repair genes involved in maintaining genomic stability were found to be a clear cause of tum the level of the single nucleotides or chromosomes. This observation suggested that the establishment of genomic instability, termed the Mutator Phenotype, was an important aspect of tumor development.(1,2) Since the initial identification of the human MutS homolog hMSH2 nearly a decade ago,(3,4) more links have been described between human cancers and genes involved in maintaining genomic stability. Work in recent years has revealed that DNA repair proteins may also function in signaling pathways that provoke cell cycle arrest and apoptosis. This review will focus on the genetic and biochemical functions of DNA repair genes linked to hereditary cancer predisposition characterized by genomic instability (Table 1). Interestingly, the protein products of these genes have been directly or indirectly linked to the DNA damage-induce cell cycle arrest and apoptosis. We conclude that a robust connection between DNA repair proteins and damage-induced apoptosis may be as important for tumorigenesis as their role in maintaining genome stability.

  8. Electroporation driven delivery of both an IL-12 expressing plasmid and cisplatin synergizes to inhibit B16 melanoma tumor growth through an NK cell mediated tumor killing mechanism.

    PubMed

    Kim, Ha; Sin, Jeong-Im

    2012-11-01

    Combined therapy using chemotherapeutic drugs and immunotherapeutics offers some promise for treating patients with cancer. In this study, we evaluated whether cisplatin delivered by intratumoral (IT)-electroporation (EP) might enhance antitumor activity against established B16 melanoma and whether further addition of intramuscular (IM)-EP of IL-12 cDNA to IT-EP of cisplatin might augment antitumor therapeutic activity, with a focus on the underlining antitumor mechanism(s). When tumor (7 mm)-bearing animals were treated locally with cisplatin by IT-EP, they showed tumor growth inhibition significantly more than those without IT-EP. Moreover, IL-12 cDNA delivered by IM-EP was also able to inhibit tumor growth significantly more than control vector delivery. This tumor growth inhibition was mediated by NK cells, but not CD4+ T or CD8+ T cells, as determined by immune cell subset depletion and IFN-γ induction. Moreover, concurrent therapy using IT-EP of cisplatin plus IM-EP of IL-12 cDNA displayed antitumor therapeutic synergy. This therapeutic synergy appeared to be mediated by increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. Taken together, these data support that cisplatin delivery by IT-EP plus IL-12 gene delivery by IM-EP are more effective at inducing antitumor therapeutic responses through increased sensitivity of cisplatin-treated tumors to NK cell-mediated tumor killing. This combined approach might have some implication for treating melanoma in patients.

  9. The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

    PubMed Central

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2013-01-01

    The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment. PMID:24276378

  10. Homologous recombination deficiency and host anti-tumor immunity in triple-negative breast cancer.

    PubMed

    Telli, M L; Stover, D G; Loi, S; Aparicio, S; Carey, L A; Domchek, S M; Newman, L; Sledge, G W; Winer, E P

    2018-05-07

    Triple-negative breast cancer (TNBC) is associated with worse outcomes relative to other breast cancer subtypes. Chemotherapy remains the standard-of-care systemic therapy for patients with localized or metastatic disease, with few biomarkers to guide benefit. We will discuss recent advances in our understanding of two key biological processes in TNBC, homologous recombination (HR) DNA repair deficiency and host anti-tumor immunity, and their intersection. Recent advances in our understanding of homologous recombination (HR) deficiency, including FDA approval of PARP inhibitor olaparib for BRCA1 or BRCA2 mutation carriers, and host anti-tumor immunity in TNBC offer potential for new and biomarker-driven approaches to treat TNBC. Assays interrogating HR DNA repair capacity may guide treatment with agents inducing or targeting DNA damage repair. Tumor infiltrating lymphocytes (TILs) are associated with improved prognosis in TNBC and recent efforts to characterize infiltrating immune cell subsets and activate host anti-tumor immunity offer promise, yet challenges remain particularly in tumors lacking pre-existing immune infiltrates. Advances in these fields provide potential biomarkers to stratify patients with TNBC and guide therapy: induction of DNA damage in HR-deficient tumors and activation of existing or recruitment of host anti-tumor immune cells. Importantly, these advances provide an opportunity to guide use of existing therapies and development of novel therapies for TNBC. Efforts to combine therapies that exploit HR deficiency to enhance the activity of immune-directed therapies offer promise. HR deficiency remains an important biomarker target and potentially effective adjunct to enhance immunogenicity of 'immune cold' TNBCs.

  11. Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities

    DTIC Science & Technology

    2016-10-01

    Report We will continue to recruit African American patients and bank their prostate tissue . We will continue dissecting tumor samples into tumor...in prostate tumors and adjacent normal tissue derived from both AA and EA individuals. We will determine if DNA methylation patterns in prostate... tissue (both cancerous and normal tissue ) differ between AA and EA individuals. We will also identify methylation features that differ between tumor

  12. A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma

    PubMed Central

    Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D.; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

    2016-01-01

    Background Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Results Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. Conclusions The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as ‘passengers’ and consequently have no discernible effect in this type of cancer. PMID:27351283

  13. A comprehensive characterization of rare mitochondrial DNA variants in neuroblastoma.

    PubMed

    Calabrese, Francesco Maria; Clima, Rosanna; Pignataro, Piero; Lasorsa, Vito Alessandro; Hogarty, Michael D; Castellano, Aurora; Conte, Massimo; Tonini, Gian Paolo; Iolascon, Achille; Gasparre, Giuseppe; Capasso, Mario

    2016-08-02

    Neuroblastoma, a tumor of the developing sympathetic nervous system, is a common childhood neoplasm that is often lethal. Mitochondrial DNA (mtDNA) mutations have been found in most tumors including neuroblastoma. We extracted mtDNA data from a cohort of neuroblastoma samples that had undergone Whole Exome Sequencing (WES) and also used snap-frozen samples in which mtDNA was entirely sequenced by Sanger technology. We next undertook the challenge of determining those mutations that are relevant to, or arisen during tumor development. The bioinformatics pipeline used to extract mitochondrial variants from matched tumor/blood samples was enriched by a set of filters inclusive of heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. Our in silico multistep workflow applied both on WES and Sanger-sequenced neuroblastoma samples, allowed us to identify a limited burden of somatic and germline mitochondrial mutations with a potential pathogenic impact. The few singleton germline and somatic mitochondrial mutations emerged, according to our in silico analysis, do not appear to impact on the development of neuroblastoma. Our findings are consistent with the hypothesis that most mitochondrial somatic mutations can be considered as 'passengers' and consequently have no discernible effect in this type of cancer.

  14. Calculating tumor trajectory and dose-of-the-day using cone-beam CT projections

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jones, Bernard L., E-mail: bernard.jones@ucdenver.edu; Westerly, David; Miften, Moyed

    2015-02-15

    Purpose: Cone-beam CT (CBCT) projection images provide anatomical data in real-time over several respiratory cycles, forming a comprehensive picture of tumor movement. The authors developed and validated a method which uses these projections to determine the trajectory of and dose to highly mobile tumors during each fraction of treatment. Methods: CBCT images of a respiration phantom were acquired, the trajectory of which mimicked a lung tumor with high amplitude (up to 2.5 cm) and hysteresis. A template-matching algorithm was used to identify the location of a steel BB in each CBCT projection, and a Gaussian probability density function for themore » absolute BB position was calculated which best fit the observed trajectory of the BB in the imager geometry. Two modifications of the trajectory reconstruction were investigated: first, using respiratory phase information to refine the trajectory estimation (Phase), and second, using the Monte Carlo (MC) method to sample the estimated Gaussian tumor position distribution. The accuracies of the proposed methods were evaluated by comparing the known and calculated BB trajectories in phantom-simulated clinical scenarios using abdominal tumor volumes. Results: With all methods, the mean position of the BB was determined with accuracy better than 0.1 mm, and root-mean-square trajectory errors averaged 3.8% ± 1.1% of the marker amplitude. Dosimetric calculations using Phase methods were more accurate, with mean absolute error less than 0.5%, and with error less than 1% in the highest-noise trajectory. MC-based trajectories prevent the overestimation of dose, but when viewed in an absolute sense, add a small amount of dosimetric error (<0.1%). Conclusions: Marker trajectory and target dose-of-the-day were accurately calculated using CBCT projections. This technique provides a method to evaluate highly mobile tumors using ordinary CBCT data, and could facilitate better strategies to mitigate or compensate for motion during

  15. Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

    PubMed

    Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Amuran, Gökçe Güllü; Güllüoğlu, Bahadır M; Kaya, Handan; Özer, Ayşe

    2015-11-10

    IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

  16. Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis

    PubMed Central

    Srivastava, Shikha; Somasagara, Ranganatha R.; Hegde, Mahesh; Nishana, Mayilaadumveettil; Tadi, Satish Kumar; Srivastava, Mrinal; Choudhary, Bibha; Raghavan, Sathees C.

    2016-01-01

    Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy. PMID:27068577

  17. A comprehensive characterization of mitochondrial DNA mutations in glioblastoma multiforme.

    PubMed

    Vidone, Michele; Clima, Rosanna; Santorsola, Mariangela; Calabrese, Claudia; Girolimetti, Giulia; Kurelac, Ivana; Amato, Laura Benedetta; Iommarini, Luisa; Trevisan, Elisa; Leone, Marco; Soffietti, Riccardo; Morra, Isabella; Faccani, Giuliano; Attimonelli, Marcella; Porcelli, Anna Maria; Gasparre, Giuseppe

    2015-06-01

    Glioblastoma multiforme (GBM) is the most malignant brain cancer in adults, with a poor prognosis, whose molecular stratification still represents a challenge in pathology and clinics. On the other hand, mitochondrial DNA (mtDNA) mutations have been found in most tumors as modifiers of the bioenergetics state, albeit in GBM a characterization of the mtDNA status is lacking to date. Here, a characterization of the burden of mtDNA mutations in GBM samples was performed. First, investigation of tumor-specific vs. non tumor-specific mutations was carried out with the MToolBox bioinformatics pipeline by analyzing 45 matched tumor/blood samples, from whole genome or whole exome sequencing datasets obtained from The Cancer Genome Atlas (TCGA) consortium. Additionally, the entire mtDNA sequence was obtained in a dataset of 104 fresh-frozen GBM samples. Mitochondrial mutations with potential pathogenic interest were prioritized based on heteroplasmic fraction, nucleotide variability, and in silico prediction of pathogenicity. A preliminary biochemical analysis of the activity of mitochondrial respiratory complexes was also performed on fresh-frozen GBM samples. Although a high number of mutations was detected, we report that the large majority of them does not pass the prioritization filters. Therefore, a relatively limited burden of pathogenic mutations is indeed carried by GBM, which did not appear to determine a general impairment of the respiratory chain. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer

    PubMed Central

    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

  19. Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.

    PubMed

    Hartford, Suzanne A; Luo, Yunhai; Southard, Teresa L; Min, Irene M; Lis, John T; Schimenti, John C

    2011-10-25

    Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

  20. Therapy-induced antitumor vaccination by targeting tumor necrosis factor alpha to tumor vessels in combination with melphalan.

    PubMed

    Mortara, Lorenzo; Balza, Enrica; Sassi, Francesca; Castellani, Patrizia; Carnemolla, Barbara; De Lerma Barbaro, Andrea; Fossati, Sara; Tosi, Giovanna; Accolla, Roberto S; Borsi, Laura

    2007-12-01

    Treatment of tumor-bearing mice with mouse (m)TNF-alpha, targeted to tumor vasculature by the anti-ED-B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF-alpha and melphalan resulted in long-lasting antitumor immune memory, accompanied by a mixed Th1/Th2-type response and significant in vitro tumor-specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor-draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long-lasting antitumor adaptive immune response.

  1. Measurement of inelastic cross sections for low-energy electron scattering from DNA bases.

    PubMed

    Michaud, Marc; Bazin, Marc; Sanche, Léon

    2012-01-01

    To determine experimentally the absolute cross sections (CS) to deposit various amount of energies into DNA bases by low-energy electron (LEE) impact. Electron energy loss (EEL) spectra of DNA bases were recorded for different LEE impact energies on the molecules deposited at very low coverage on an inert argon (Ar) substrate. Following their normalisation to the effective incident electron current and molecular surface number density, the EEL spectra were then fitted with multiple Gaussian functions in order to delimit the various excitation energy regions. The CS to excite a molecule into its various excitation modes were finally obtained from computing the area under the corresponding Gaussians. The EEL spectra and absolute CS for the electronic excitations of pyrimidine and the DNA bases thymine, adenine, and cytosine by electron impacts below 18 eV were reported for the molecules deposited at about monolayer coverage on a solid Ar substrate. The CS for electronic excitations of DNA bases by LEE impact were found to lie within the 10(216) to 10(218) cm(2) range. The large value of the total ionisation CS indicated that ionisation of DNA bases by LEE is an important dissipative process via which ionising radiation degrades and is absorbed in DNA.

  2. Measurement of inelastic cross sections for low-energy electron scattering from DNA bases

    PubMed Central

    Michaud, Marc; Bazin, Marc.; Sanche, Léon

    2013-01-01

    Purpose Determine experimentally the absolute cross sections (CS) to deposit various amount of energies into DNA bases by low-energy electron (LEE) impact. Materials and methods Electron energy loss (EEL) spectra of DNA bases are recorded for different LEE impact energies on the molecules deposited at very low coverage on an inert argon (Ar) substrate. Following their normalisation to the effective incident electron current and molecular surface number density, the EEL spectra are then fitted with multiple Gaussian functions in order to delimit the various excitation energy regions. The CS to excite a molecule into its various excitation modes are finally obtained from computing the area under the corresponding Gaussians. Results The EEL spectra and absolute CS for the electronic excitations of pyrimidine and the DNA bases thymine, adenine, and cytosine by electron impacts below 18 eV are reported for the molecules deposited at about monolayer coverage on a solid Ar substrate. Conclusions The CS for electronic excitations of DNA bases by LEE impact are found to lie within the 10−16 – 10−18 cm2 range. The large value of the total ionisation CS indicates that ionisation of DNA bases by LEE is an important dissipative process via which ionising radiation degrades and is absorbed in DNA. PMID:21615242

  3. Effects of Acanthus ebracteatus Vahl on tumor angiogenesis and on tumor growth in nude mice implanted with cervical cancer

    PubMed Central

    Mahasiripanth, Taksanee; Hokputsa, Sanya; Niruthisard, Somchai; Bhattarakosol, Parvapan; Patumraj, Suthiluk

    2012-01-01

    Purpose The aim of this study was to examine the effects of the crude extract of Acanthus ebracteatus Vahl (AE) on tumor growth and angiogenesis by utilizing a tumor model in which nude mice were implanted with cervical cancer cells containing human papillomavirus 16 DNA (HPV-16 DNA). Materials and methods The growth-inhibitory effect of AE was investigated in four different cell types: CaSki (HPV-16 positive), HeLa (HPV-18 positive), hepatocellular carcinoma cells (HepG2), and human dermal fibroblast cells (HDFs). The cell viabilities and IC50 values of AE were determined in cells incubated with AE for different lengths of time. To conduct studies in vivo, female BALB/c nude mice (aged 6–7 weeks, weighing 20–25 g) were used. A cervical cancer-derived cell line (CaSki) with integrated HPV-16 DNA was injected subcutaneously (1 × 107 cells/200 μL) in the middle dorsum of each animal (HPV group). One week after injection, mice were fed orally with AE crude extract at either 300 or 3000 mg/kg body weight/day for 14 or 28 days (HPV-AE groups). Tumor microvasculature and capillary vascularity were determined using laser scanning confocal microscopy. Tumor tissue was collected from each mouse to evaluate tumor histology and vascular endothelial growth factor (VEGF) immunostaining. Results The time-response curves of AE and the dose-dependent effect of AE on growth inhibition were determined. After a 48-hour incubation period, the IC50 of AE in CaSki was discovered to be significantly different from that of HDFs (P < 0.05). A microvascular network was observed around the tumor area in the HPV group on days 21 and 35. Tumor capillary vascularity in the HPV group was significantly increased compared with the control group (P < 0.001). High-dose treatment of AE extract (HPV-3000AE group) significantly attenuated the increase in VEGF expression and tumor angiogenesis in mice that received either the 14- or 28-day treatment period (P < 0.001). Conclusion Our novel

  4. The Five Immune Forces Impacting DNA-Based Cancer Immunotherapeutic Strategy

    PubMed Central

    Amara, Suneetha; Tiriveedhi, Venkataswarup

    2017-01-01

    DNA-based vaccine strategy is increasingly realized as a viable cancer treatment approach. Strategies to enhance immunogenicity utilizing tumor associated antigens have been investigated in several pre-clinical and clinical studies. The promising outcomes of these studies have suggested that DNA-based vaccines induce potent T-cell effector responses and at the same time cause only minimal side-effects to cancer patients. However, the immune evasive tumor microenvironment is still an important hindrance to a long-term vaccine success. Several options are currently under various stages of study to overcome immune inhibitory effect in tumor microenvironment. Some of these approaches include, but are not limited to, identification of neoantigens, mutanome studies, designing fusion plasmids, vaccine adjuvant modifications, and co-treatment with immune-checkpoint inhibitors. In this review, we follow a Porter’s analysis analogy, otherwise commonly used in business models, to analyze various immune-forces that determine the potential success and sustainable positive outcomes following DNA vaccination using non-viral tumor associated antigens in treatment against cancer. PMID:28304339

  5. DNA methylation aberrancies as a guide for surveillance and treatment of human cancers

    PubMed Central

    Liang, Gangning; Weisenberger, Daniel J.

    2017-01-01

    ABSTRACT DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. PMID:28358281

  6. Transgelin gene is frequently downregulated by promoter DNA hypermethylation in breast cancer.

    PubMed

    Sayar, Nilufer; Karahan, Gurbet; Konu, Ozlen; Bozkurt, Betul; Bozdogan, Onder; Yulug, Isik G

    2015-01-01

    CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation. Using microarray expression profiling of AZA- or DMSO-treated breast cancer and non-tumorigenic breast (NTB) cells, we identified for the first time TAGLN gene as a target of DNA hypermethylation in breast cancer. TAGLN expression was significantly and frequently downregulated via promoter DNA hypermethylation in breast cancer cells compared to NTB cells, and also in 13/21 (61.9 %) of breast tumors compared to matched normal tissues. Analyses of public microarray methylation data showed that TAGLN was also hypermethylated in 63.02 % of tumors compared to normal tissues; relapse-free survival of patients was worse with higher TAGLN methylation; and methylation levels could discriminate between tumors and healthy tissues with 83.14 % sensitivity and 100 % specificity. Additionally, qRT-PCR and immunohistochemistry experiments showed that TAGLN expression was significantly downregulated in two more independent sets of breast tumors compared to normal tissues and was lower in tumors with poor prognosis. Colony formation was increased in TAGLN silenced NTB cells, while decreased in overexpressing BC cells. TAGLN gene is frequently downregulated by DNA hypermethylation, and TAGLN promoter methylation profiles could serve as a future diagnostic biomarker, with possible clinical impact regarding the prognosis in breast cancer.

  7. Forecasting Error Calculation with Mean Absolute Deviation and Mean Absolute Percentage Error

    NASA Astrophysics Data System (ADS)

    Khair, Ummul; Fahmi, Hasanul; Hakim, Sarudin Al; Rahim, Robbi

    2017-12-01

    Prediction using a forecasting method is one of the most important things for an organization, the selection of appropriate forecasting methods is also important but the percentage error of a method is more important in order for decision makers to adopt the right culture, the use of the Mean Absolute Deviation and Mean Absolute Percentage Error to calculate the percentage of mistakes in the least square method resulted in a percentage of 9.77% and it was decided that the least square method be worked for time series and trend data.

  8. EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

    PubMed

    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.

  9. Cell motility and ECM proteolysis regulate tumor growth and tumor relapse by altering the fraction of cancer stem cells and their spatial scattering

    NASA Astrophysics Data System (ADS)

    Kumar, Sandeep; Kulkarni, Rahul; Sen, Shamik

    2016-06-01

    Tumors consist of multiple cell sub-populations including cancer stem cells (CSCs), transiently amplifying cells and terminally differentiated cells (TDCs), with the CSC fraction dictating the aggressiveness of the tumor and drug sensitivity. In epithelial cancers, tumor growth is influenced greatly by properties of the extracellular matrix (ECM), with cancer progression associated with an increase in ECM density. However, the extent to which increased ECM confinement induced by an increase in ECM density influences tumor growth and post treatment relapse dynamics remains incompletely understood. In this study, we use a cellular automata-based discrete modeling approach to study the collective influence of ECM density, cell motility and ECM proteolysis on tumor growth, tumor heterogeneity, and tumor relapse after drug treatment. We show that while increased confinement suppresses tumor growth and the spatial scattering of CSCs, this effect can be reversed when cells become more motile and proteolytically active. Our results further suggest that, in addition to the absolute number of CSCs, their spatial positioning also plays an important role in driving tumor growth. In a nutshell, our study suggests that, in confined environments, cell motility and ECM proteolysis are two key factors that regulate tumor growth and tumor relapse dynamics by altering the number and spatial distribution of CSCs.

  10. A Distinct DNA Methylation Shift in a Subset of Glioma CpG Island Methylator Phenotypes during Tumor Recurrence.

    PubMed

    de Souza, Camila Ferreira; Sabedot, Thais S; Malta, Tathiane M; Stetson, Lindsay; Morozova, Olena; Sokolov, Artem; Laird, Peter W; Wiznerowicz, Maciej; Iavarone, Antonio; Snyder, James; deCarvalho, Ana; Sanborn, Zachary; McDonald, Kerrie L; Friedman, William A; Tirapelli, Daniela; Poisson, Laila; Mikkelsen, Tom; Carlotti, Carlos G; Kalkanis, Steven; Zenklusen, Jean; Salama, Sofie R; Barnholtz-Sloan, Jill S; Noushmehr, Houtan

    2018-04-10

    Glioma diagnosis is based on histomorphology and grading; however, such classification does not have predictive clinical outcome after glioblastomas have developed. To date, no bona fide biomarkers that significantly translate into a survival benefit to glioblastoma patients have been identified. We previously reported that the IDH mutant G-CIMP-high subtype would be a predecessor to the G-CIMP-low subtype. Here, we performed a comprehensive DNA methylation longitudinal analysis of diffuse gliomas from 77 patients (200 tumors) to enlighten the epigenome-based malignant transformation of initially lower-grade gliomas. Intra-subtype heterogeneity among G-CIMP-high primary tumors allowed us to identify predictive biomarkers for assessing the risk of malignant recurrence at early stages of disease. G-CIMP-low recurrence appeared in 9.5% of all gliomas, and these resembled IDH-wild-type primary glioblastoma. G-CIMP-low recurrence can be characterized by distinct epigenetic changes at candidate functional tissue enhancers with AP-1/SOX binding elements, mesenchymal stem cell-like epigenomic phenotype, and genomic instability. Molecular abnormalities of longitudinal G-CIMP offer possibilities to defy glioblastoma progression. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

    PubMed

    Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

    2015-08-01

    DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Gene expression analysis using a highly sensitive DNA microarray for colorectal cancer screening.

    PubMed

    Koga, Yoshikatsu; Yamazaki, Nobuyoshi; Takizawa, Satoko; Kawauchi, Junpei; Nomura, Osamu; Yamamoto, Seiichiro; Saito, Norio; Kakugawa, Yasuo; Otake, Yosuke; Matsumoto, Minori; Matsumura, Yasuhiro

    2014-01-01

    Half of all patients with small, right-sided, non-metastatic colorectal cancer (CRC) have negative results for the fecal occult blood test (FOBT). In the present study, the usefulness of CRC screening with a highly sensitive DNA microarray was evaluated in comparison with that by FOBT using fecal samples. A total of 53 patients with CRC and 61 healthy controls were divided into "training" and "validation sets". For the gene profiling, total RNA extracted from 0.5 g of feces was hybridized to a highly sensitive DNA chip. The expressions of 43 genes were significantly higher in the patients with CRC than in healthy controls (p<0.05). In the training set, the sensitivity and specificity of the DNA chip assay using six genes were 85.4% and 85.2%, respectively. On the other hand, in the validation set, the sensitivity and specificity of the DNA chip assay were 85.2% and 85.7%, respectively. The sensitivities of the DNA chip assay were higher than those of FOBT in cases of the small, right-sided, early-CRC, tumor invading up to the muscularis propria (i.e. surface tumor) subgroups. In particular, the sensitivities of the DNA chip assay in the surface tumor and early-CRC subgroups were significantly higher than those of FOBT (p=0.023 and 0.019, respectively.). Gene profiling assay using a highly sensitive DNA chip was more effective than FOBT at detecting patients with small, right-sided, surface tumor, and early-stage CRC.

  13. Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

    PubMed Central

    Peng, Shiwen; Trimble, Cornelia; Alvarez, Ronald D.; Huh, Warner K.; Lin, Zhenhua; Monie, Archana; Hung, Chien-Fu; Wu, T.-C.

    2010-01-01

    Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD8+ T cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal timeframe. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation. PMID:18401437

  14. Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.

    PubMed

    Karanam, Narasimha Kumar; Srinivasan, Kalayarasan; Ding, Lianghao; Sishc, Brock; Saha, Debabrata; Story, Michael D

    2017-03-30

    The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (P<0.05) during TTFields treatment. DNA double-strand break (DSB) repair foci increased when cells were exposed to TTFields as did the appearance of chromatid-type aberrations, suggesting an interphase mechanism responsible for cell death involving DNA repair. Exposing cells to TTFields immediately following ionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents.

  15. Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.

    PubMed

    Amemiya, Kenji; Hirotsu, Yosuke; Goto, Taichiro; Nakagomi, Hiroshi; Mochizuki, Hitoshi; Oyama, Toshio; Omata, Masao

    2016-12-01

    Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time-consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]-seq), to detect somatic mutations in tumors. We prepared FFPE tissues and TIC specimens from tumors in nine lung cancer patients and one patient with breast cancer. We found that the quality and quantity of TIC DNA was higher than that of FFPE DNA, which requires microdissection to enrich DNA from target tissues. Targeted sequencing using a next-generation sequencer obtained sufficient sequence data using TIC DNA. Most (92%) somatic mutations in lung primary tumors were found to be consistent between TIC and FFPE DNA. We also applied TIC DNA to primary and metastatic tumor tissues to analyze tumor heterogeneity in a breast cancer patient, and showed that common and distinct mutations among primary and metastatic sites could be classified into two distinct histological subtypes. TIC-seq is an alternative and feasible method to analyze genomic alterations in tumors by simply touching the cut surface of specimens to slides. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  16. Tumor immunology.

    PubMed

    Mocellin, Simone; Lise, Mario; Nitti, Donato

    2007-01-01

    Advances in tumor immunology are supporting the clinical implementation of several immunological approaches to cancer in the clinical setting. However, the alternate success of current immunotherapeutic regimens underscores the fact that the molecular mechanisms underlying immune-mediated tumor rejection are still poorly understood. Given the complexity of the immune system network and the multidimensionality of tumor/host interactions, the comprehension of tumor immunology might greatly benefit from high-throughput microarray analysis, which can portrait the molecular kinetics of immune response on a genome-wide scale, thus accelerating the discovery pace and ultimately catalyzing the development of new hypotheses in cell biology. Although in its infancy, the implementation of microarray technology in tumor immunology studies has already provided investigators with novel data and intriguing new hypotheses on the molecular cascade leading to an effective immune response against cancer. Although the general principles of microarray-based gene profiling have rapidly spread in the scientific community, the need for mastering this technique to produce meaningful data and correctly interpret the enormous output of information generated by this technology is critical and represents a tremendous challenge for investigators, as outlined in the first section of this book. In the present Chapter, we report on some of the most significant results obtained with the application of DNA microarray in this oncology field.

  17. Clinicopathologic Risk Factor Distributions for MLH1 Promoter Region Methylation in CIMP-Positive Tumors.

    PubMed

    Levine, A Joan; Phipps, Amanda I; Baron, John A; Buchanan, Daniel D; Ahnen, Dennis J; Cohen, Stacey A; Lindor, Noralane M; Newcomb, Polly A; Rosty, Christophe; Haile, Robert W; Laird, Peter W; Weisenberger, Daniel J

    2016-01-01

    The CpG island methylator phenotype (CIMP) is a major molecular pathway in colorectal cancer. Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations, and family colorectal cancer history with MLH1 methylation status in a large population-based sample of CIMP-positive colorectal cancers defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, and more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR, 0.50; 95% confidence interval, 0.31-0.82). These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. MLH1 DNA methylation status should be taken into account in etiologic studies. ©2015 American Association for Cancer Research.

  18. Clinicopathological risk factor distributions for MLH1 promoter region methylation in CIMP positive tumors

    PubMed Central

    Levine, A. Joan; Phipps, Amanda I.; Baron, John A.; Buchanan, Daniel D.; Ahnen, Dennis J.; Cohen, Stacey A.; Lindor, Noralane M.; Newcomb, Polly A.; Rosty, Christophe; Haile, Robert W.; Laird, Peter W.; Weisenberger, Daniel J.

    2015-01-01

    Background The CpG Island Methylator Phenotype (CIMP) is a major molecular pathway in colorectal cancer (CRC). Approximately 25% to 60% of CIMP tumors are microsatellite unstable (MSI-H) due to DNA hypermethylation of the MLH1 gene promoter. Our aim was to determine if the distributions of clinicopathologic factors in CIMP-positive tumors with MLH1 DNA methylation differed from those in CIMP-positive tumors without DNA methylation of MLH1. Methods We assessed the associations between age, sex, tumor-site, MSI status BRAF and KRAS mutations and family CRC history with MLH1 methylation status in a large population-based sample of CIMP-positive CRCs defined by a 5-marker panel using unconditional logistic regression to assess the odds of MLH1 methylation by study variables. Results Subjects with CIMP-positive tumors without MLH1 methylation were significantly younger, more likely to be male, more likely to have distal colon or rectal primaries and the MSI-L phenotype. CIMP-positive MLH1-unmethylated tumors were significantly less likely than CIMP-positive MLH1-methylated tumors to harbor a BRAF V600E mutation and significantly more likely to harbor a KRAS mutation. MLH1 methylation was associated with significantly better overall survival (HR=0.50; 95% Confidence Interval (0.31, 0.82)). Conclusions These data suggest that MLH1 methylation in CIMP-positive tumors is not a completely random event and implies that there are environmental or genetic determinants that modify the probability that MLH1 will become methylated during CIMP pathogenesis. Impact MLH1 DNA methylation status should be taken into account in etiologic studies. PMID:26512054

  19. A Children's Oncology Group and TARGET initiative exploring the genetic landscape of Wilms tumor.

    PubMed

    Gadd, Samantha; Huff, Vicki; Walz, Amy L; Ooms, Ariadne H A G; Armstrong, Amy E; Gerhard, Daniela S; Smith, Malcolm A; Auvil, Jaime M Guidry; Meerzaman, Daoud; Chen, Qing-Rong; Hsu, Chih Hao; Yan, Chunhua; Nguyen, Cu; Hu, Ying; Hermida, Leandro C; Davidsen, Tanja; Gesuwan, Patee; Ma, Yussanne; Zong, Zusheng; Mungall, Andrew J; Moore, Richard A; Marra, Marco A; Dome, Jeffrey S; Mullighan, Charles G; Ma, Jing; Wheeler, David A; Hampton, Oliver A; Ross, Nicole; Gastier-Foster, Julie M; Arold, Stefan T; Perlman, Elizabeth J

    2017-10-01

    We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

  20. Heterogeneity of DNA methylation in multifocal prostate cancer.

    PubMed

    Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

    2015-01-01

    Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p < 0.001). Hierarchical cluster analysis revealed a divergent methylation profile of paired PCa foci, while the foci from separate cases with biochemical recurrence showed similar methylation profile and the highest mean levels of DNA methylation. Our findings suggest that PCa tissue is heterogeneous, as between paired foci differences in DNA methylation status were found. Common epigenetic profile of recurrent tumors can be inferred from our data.

  1. Tumor Environmental Factors Glucose Deprivation and Lactic Acidosis Induce Mitotic Chromosomal Instability – An Implication in Aneuploid Human Tumors

    PubMed Central

    Zhu, Chunpeng; Hu, Xun

    2013-01-01

    Mitotic chromosomal instability (CIN) plays important roles in tumor progression, but what causes CIN is incompletely understood. In general, tumor CIN arises from abnormal mitosis, which is caused by either intrinsic or extrinsic factors. While intrinsic factors such as mitotic checkpoint genes have been intensively studied, the impact of tumor microenvironmental factors on tumor CIN is largely unknown. We investigate if glucose deprivation and lactic acidosis – two tumor microenvironmental factors – could induce cancer cell CIN. We show that glucose deprivation with lactic acidosis significantly increases CIN in 4T1, MCF-7 and HCT116 scored by micronuclei, or aneuploidy, or abnormal mitosis, potentially via damaging DNA, up-regulating mitotic checkpoint genes, and/or amplifying centrosome. Of note, the feature of CIN induced by glucose deprivation with lactic acidosis is similar to that of aneuploid human tumors. We conclude that tumor environmental factors glucose deprivation and lactic acidosis can induce tumor CIN and propose that they are potentially responsible for human tumor aneuploidy. PMID:23675453

  2. 20 CFR 404.1205 - Absolute coverage groups.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 20 Employees' Benefits 2 2014-04-01 2014-04-01 false Absolute coverage groups. 404.1205 Section... INSURANCE (1950- ) Coverage of Employees of State and Local Governments What Groups of Employees May Be Covered § 404.1205 Absolute coverage groups. (a) General. An absolute coverage group is a permanent...

  3. 20 CFR 404.1205 - Absolute coverage groups.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 20 Employees' Benefits 2 2013-04-01 2013-04-01 false Absolute coverage groups. 404.1205 Section... INSURANCE (1950- ) Coverage of Employees of State and Local Governments What Groups of Employees May Be Covered § 404.1205 Absolute coverage groups. (a) General. An absolute coverage group is a permanent...

  4. 20 CFR 404.1205 - Absolute coverage groups.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 20 Employees' Benefits 2 2012-04-01 2012-04-01 false Absolute coverage groups. 404.1205 Section... INSURANCE (1950- ) Coverage of Employees of State and Local Governments What Groups of Employees May Be Covered § 404.1205 Absolute coverage groups. (a) General. An absolute coverage group is a permanent...

  5. Dendritic Cell-Based Immunotherapy of Breast Cancer: Modulation by CpG DNA

    DTIC Science & Technology

    2005-09-01

    tumor-associated antigens and bacterial DNA oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNA) further augment the immune priming...associated antigens by cytotoxic T lymphocytes, and bacterial DNA oligodeoxy- nucleotides containing unmethylated CpG sequences (CpG DNA) can further...further amplify their immunostimulatory capacity and bacterial DNA oligodeoxynucleotides (ODN) containing unmethylated CpG sequences (CpG DNA) provide such

  6. [The clinical study of percutaneous transhepatic radiofrequency ablation combined with tumor edge of percutaneous absolute ethanol injection on liver cancer adjacent to major blood vessels].

    PubMed

    Du, Jun-dong; Liu, Rong; Jiao, Hua-bo; Xiang, De-dong; Yin, Hui-nan; Li, Zhen-cai; Li, Tao; Zhu, Zi-man; Li, Zhan-liang

    2011-05-01

    To explore the effects of percutaneous transhepatic radiofrequency ablation (PRFA) combined with tumor edge of percutaneous absolute ethanol injection (PEI) on liver cancer adjacent to major blood vessels. Seventy five patients with liver cancer adjacent to major blood vessels were randomly divided into two groups: PRFA+PEI therapy group (38 cases) and PRFA control group (37 cases). Tumor necrosis rate, AFP levels, local recurrence rate, median for survival time and cum survival were used as the evaluation index to evaluate the efficacies of the two methods. Tumor necrosis rates of the therapy group and the control group were 84.2% and 54.1% (P < 0.01), respectively; AFP levels of therapy group and control group at 1, 3, 6 and 12 months after treatment were (105.0 ± 35.5) μg/L, (28.4 ± 4.3) μg/L, (58.6 ± 6.7) μg/L, (89.5 ± 12.5) μg/L and (137.2 ± 34.6) μg/L, (84.2 ± 18.4) μg/L, (106.6 ± 20.3) μg/L, (173.7 ± 32.0) μg/L, respectively. The rates of therapy group was significantly lower than of control group. Local recurrence rates of the therapy group and control group were 2.6%, 7.9%, 13.2% and 31.6% vs 10.8%, 21.6% , 40.5% and 62.1% (P < 0.05) at 3, 6, 12 and 24 months after treatment, respectively. Median for survival time of the therapy group and control group were 28.0 ± 2.8 months and 19.0 ± 3.6 months, respectively. Cum survival of the therapy group and control group were 84.2%, 78.9%, 60.5% and 31.6% vs 78.4%, 67.6%, 37.8% and 8.1% (P < 0.05) at 6, 12, 24 and 36 months after treatment, respectively. PEI as a supplementary treatment of PRFA can effectively improve the treatment of liver cancer adjacent to major blood vessels and significantly reduce the local recurrence rate and improve long-term survival rates.

  7. Fibrinolysis in Tumor Associated Angiogenesis

    DTIC Science & Technology

    2005-07-01

    tPA expression in mammary vessel assays and in animals with use of retroviral vectors to deliver antisense RNA . We still intend to use that strategy...pads of nude mice. RNA obtained from tumor- and mam- mary fat pad-associated endothelial cells was used to synthesize cDNA and cDNA libraries, which... RNA (mRNA) for tPA and MT1-MMP, with some upregulation of uPA expression. BODY 1. Confirm differential expression of tPA and MT1-MIP with GAPDH control

  8. Biomarkers in Cerebrospinal Fluid: Analysis of Cell-Free Circulating Mitochondrial DNA by Digital PCR.

    PubMed

    Podlesniy, Petar; Trullas, Ramon

    2018-01-01

    Cerebrospinal fluid (CSF) contains molecules directly linked with brain function because it permeates brain tissue. The analysis of protein biomarkers in CSF is currently recommended for the diagnosis of neurodegenerative disorders, but the clinical sensitivity and specificity are still being investigated. A major drawback is that most of the currently used biomarkers of neurodegenerative diseases are proteins that are found at very low concentrations in CSF and need to be measured by immunoassays that provide relative values, which sometimes are difficult to reproduce between laboratories. In contrast, the recent availability of digital PCR platforms allows the absolute quantification of nucleic acids at single-molecule resolution, but their presence in CSF has not been characterized. CSF contains cell-free mitochondrial DNA (mtDNA) and changes in the concentration of this nucleic acid are linked to neurodegeneration. Here we describe a method to measure the concentration of cell-free circulating mtDNA directly in unpurified CSF using droplet digital PCR with either hydrolysis probes or fluorescent DNA-binding dye methods. This protocol allows the detection and absolute quantification of mtDNA content in the CSF with high analytical sensitivity, specificity, and accuracy.

  9. Characterization of DNA isolated from normal and cancerous ovarian tissues by ultraviolet resonance Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Zhao, Xiaojie; Vinson, Michael A.; Malins, Donald C.; Spiro, Thomas G.

    2000-05-01

    We report significant differences in UV resonance Raman (UVRR) spectra of DNA samples from normal and cancerous tissues. The four bases of DNA, adenosine, thymine, guanosine and cytidine, can be enhanced in UVRR spectra, and their intensities are very sensitive to base stacking and DNA H-bonding. 14 DNA samples from patients at different stages of ovarian cancer, 5 from normal, 2 from primary, 3 from metastasis primary and 4 from distant metastasis tumor tissues, were characterized by 257, 238, 229, 220 and 210 nm-excited UVRR spectra. Raman spectral difference between normal and tumor DNA could be readily detected.

  10. DNA Vaccination Against Metastatic Breast Cancer

    DTIC Science & Technology

    2001-07-01

    cells. Although DNA vaccines have shown effectiveness in clinical trials , it is essential to demonstrate pre- clinical effectiveness for anti-tumor... clinical trials for infectious diseases (4), it is essential to (5-7)demonstrate pre- clinical effectiveness for anti-tumor vaccines before clinical testing...Program Clinical Translational Research (CTR) award to perform a Phase I clinical trial of ELVIS2-neu. Our preliminary application was selected for

  11. Tumor Suppression by BRCA-1: A Critical Role at DNA Replication Forks

    DTIC Science & Technology

    2006-10-01

    replication defect. We wished to test the hypothesis that BRCA1/BARD1 function during DNA replication supporting DNA transactions at replication forks. We...are using cell-free extracts derived from Xenopus laevis eggs that support: 1. Semi-conservative, cell-cycle regulated DNA replication ; 2. Many facets...complex assembles to chromatin in a DNA replication -dependent manner. Finally, we show that BRCA1/BARD1 loading to chromatin does not dramatically

  12. Regulatory Control of Breast Tumor Cell Poly (ADP-Ribose) Polymerase

    DTIC Science & Technology

    2002-08-01

    DNA replication complex (designated the DNA synthesome) from a variety of non-malignant and malignant tumor cells including breast cancer cells. We have shown that poly(ADP-ribose) polymerase PARP is among the components of the DNA synthesome. The transformation of a non-malignant human breast cell to a malignant state was accompanied by a significant alteration in the 2-D PAGE profile of specific protein components of the DNA synthesome (such as PCNA) together with a 6-8 decrease in the replication fidelity of the DNA

  13. DNA damage checkpoint recovery and cancer development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong, E-mail: lsteng@zju.edu.cn

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observedmore » to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control.

  14. A mutation screening of oncogenes, tumor suppressor gene TP53 and nuclear encoded mitochondrial complex I genes in oncocytic thyroid tumors.

    PubMed

    Evangelisti, Cecilia; de Biase, Dario; Kurelac, Ivana; Ceccarelli, Claudio; Prokisch, Holger; Meitinger, Thomas; Caria, Paola; Vanni, Roberta; Romeo, Giovanni; Tallini, Giovanni; Gasparre, Giuseppe; Bonora, Elena

    2015-03-21

    Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.

  15. Flavonoids and DNA Repair in Prostate Cancer

    DTIC Science & Technology

    2004-12-01

    AD Award Number: W81XWH-04-1-0114 TITLE: Flavonoids and DNA Repiar in Prostate Cancer PRINCIPAL INVESTIGATOR: Susanne M. Henning, Ph.D. CONTRACTING...DATES COVERED (Leave blank) December 2004 Annual (1 Dec 2003 - 30 Nov 2004) 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Flavonoids and DNA Repiar in... flavonoids are able to stimulate the repair of oxidative DNA damage. For this purpose LNCaP prostate tumor cells were exposed to FeSO4 to induce

  16. Variance computations for functional of absolute risk estimates.

    PubMed

    Pfeiffer, R M; Petracci, E

    2011-07-01

    We present a simple influence function based approach to compute the variances of estimates of absolute risk and functions of absolute risk. We apply this approach to criteria that assess the impact of changes in the risk factor distribution on absolute risk for an individual and at the population level. As an illustration we use an absolute risk prediction model for breast cancer that includes modifiable risk factors in addition to standard breast cancer risk factors. Influence function based variance estimates for absolute risk and the criteria are compared to bootstrap variance estimates.

  17. Variance computations for functional of absolute risk estimates

    PubMed Central

    Pfeiffer, R.M.; Petracci, E.

    2011-01-01

    We present a simple influence function based approach to compute the variances of estimates of absolute risk and functions of absolute risk. We apply this approach to criteria that assess the impact of changes in the risk factor distribution on absolute risk for an individual and at the population level. As an illustration we use an absolute risk prediction model for breast cancer that includes modifiable risk factors in addition to standard breast cancer risk factors. Influence function based variance estimates for absolute risk and the criteria are compared to bootstrap variance estimates. PMID:21643476

  18. Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing.

    PubMed

    Dobbs, Larry J; Madigan, Merle N; Carter, Alexis B; Earls, Lori

    2002-01-01

    Efficient methods of storing tumor specimens for molecular testing are needed in the modern surgical pathology laboratory. The FTA Gene Guard system is a novel method for the collection and room temperature storage of blood samples for DNA testing. The method uses index card-sized filter papers that provide an ideal medium on which to store tumor specimens for DNA testing. To determine whether FTA filter paper can be used in the surgical pathology laboratory to store tumor cells for DNA testing. Cell suspensions were prepared from 60 surgical specimens, and DNA was extracted either immediately or after storage on FTA paper. The DNA extracted by each method was tested by polymerase chain reaction (PCR) for the beta-globin and interferon gamma genes, and the results were compared. Fifteen lymph node specimens stored on FTA paper were then tested for immunoglobulin heavy chain (IgH) gene rearrangement by PCR, and these results were compared with those obtained for immediately extracted DNA. University medical center. The DNA extracted from cells stored on FTA paper performed as well in the PCR as the freshly extracted DNA in nearly all cases (>95%). The results of tests for IgH gene rearrangements showed 100% concordance between the 2 methods of DNA extraction.Conclusion.-Cells from surgical specimens can be stored on FTA paper for extended lengths of time, and DNA can be extracted from these cells for PCR-based testing. FTA filter paper is a reliable medium for the storage and/or transport of tumor cells for PCR-based DNA analysis.

  19. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.

  20. Systemically administered gp100 encoding DNA vaccine for melanoma using water-in-oil-in-water multiple emulsion delivery systems.

    PubMed

    Kalariya, Mayurkumar; Amiji, Mansoor M

    2013-09-10

    The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Direct DNA binding by Brca1.

    PubMed

    Paull, T T; Cortez, D; Bowers, B; Elledge, S J; Gellert, M

    2001-05-22

    The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

  2. DNA methylation-based reclassification of olfactory neuroblastoma.

    PubMed

    Capper, David; Engel, Nils W; Stichel, Damian; Lechner, Matt; Glöss, Stefanie; Schmid, Simone; Koelsche, Christian; Schrimpf, Daniel; Niesen, Judith; Wefers, Annika K; Jones, David T W; Sill, Martin; Weigert, Oliver; Ligon, Keith L; Olar, Adriana; Koch, Arend; Forster, Martin; Moran, Sebastian; Tirado, Oscar M; Sáinz-Japeado, Miguel; Mora, Jaume; Esteller, Manel; Alonso, Javier; Del Muro, Xavier Garcia; Paulus, Werner; Felsberg, Jörg; Reifenberger, Guido; Glatzel, Markus; Frank, Stephan; Monoranu, Camelia M; Lund, Valerie J; von Deimling, Andreas; Pfister, Stefan; Buslei, Rolf; Ribbat-Idel, Julika; Perner, Sven; Gudziol, Volker; Meinhardt, Matthias; Schüller, Ulrich

    2018-05-05

    Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of

  3. Prognostic value of DNA repair based stratification of hepatocellular carcinoma

    PubMed Central

    Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

    2016-01-01

    Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

  4. Absolute Summ

    NASA Astrophysics Data System (ADS)

    Phillips, Alfred, Jr.

    Summ means the entirety of the multiverse. It seems clear, from the inflation theories of A. Guth and others, that the creation of many universes is plausible. We argue that Absolute cosmological ideas, not unlike those of I. Newton, may be consistent with dynamic multiverse creations. As suggested in W. Heisenberg's uncertainty principle, and with the Anthropic Principle defended by S. Hawking, et al., human consciousness, buttressed by findings of neuroscience, may have to be considered in our models. Predictability, as A. Einstein realized with Invariants and General Relativity, may be required for new ideas to be part of physics. We present here a two postulate model geared to an Absolute Summ. The seedbed of this work is part of Akhnaton's philosophy (see S. Freud, Moses and Monotheism). Most important, however, is that the structure of human consciousness, manifest in Kenya's Rift Valley 200,000 years ago as Homo sapiens, who were the culmination of the six million year co-creation process of Hominins and Nature in Africa, allows us to do the physics that we do. .

  5. SU-D-201-04: Study On the Impact of Tumor Shape and Size On Drug Delivery to Pancreatic Tumors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Soltani, M; Bazmara, H; Sefidgar, M

    Purpose: Drug delivery to solid tumors can be expressed physically using transport phenomena such as convection and diffusion for the drug of interest within extracellular matrices. We aimed to carefully model these phenomena, and to investigate the effect of tumor shape and size on drug delivery to solid tumors in the pancreas. Methods: In this study, multiple tumor geometries as obtained from clinical PET/CT images were considered. An advanced numerical method was used to simultaneously solve fluid flow and solute transport equations. Data from n=45 pancreatic cancer patients with non-resectable locoregional disease were analyzed, and geometrical information from the tumorsmore » including size, shape, and aspect ratios were classified. To investigate effect of tumor shape, tumors with similar size but different shapes were selected and analyzed. Moreover, to investigate effect of tumor size, tumors with similar shapes but different sizes, ranging from 1 to 77 cm{sup 3}, were selected and analyzed. A hypothetical tumor similar to one of the analyzed tumors, but scaled to reduce its size below 0.2 cm{sup 3}, was also analyzed. Results: The results showed relatively similar average drug concentration profiles in tumors with different sizes. Generally, smaller tumors had higher absolute drug concentration. In the hypothetical tumor, with volume less than 0.2 cm{sup 3}, the average drug concentration was 20% higher in comparison to its counterparts. For the various real tumor geometries, however, the maximum difference between average drug concentrations was 10% for the smallest and largest tumors. Moreover, the results demonstrated that for pancreatic tumors the shape is not significant. The negligible difference of drug concentration in different tumor shapes was due to the minimum effect of convection in pancreatic tumors. Conclusion: In tumors with different sizes, smaller tumors have higher drug delivery; however, the impact of tumor shape in the case of

  6. DNA methylation intratumor heterogeneity in localized lung adenocarcinomas.

    PubMed

    Quek, Kelly; Li, Jun; Estecio, Marcos; Zhang, Jiexin; Fujimoto, Junya; Roarty, Emily; Little, Latasha; Chow, Chi-Wan; Song, Xingzhi; Behrens, Carmen; Chen, Taiping; William, William N; Swisher, Stephen; Heymach, John; Wistuba, Ignacio; Zhang, Jianhua; Futreal, Andrew; Zhang, Jianjun

    2017-03-28

    Cancers are composed of cells with distinct molecular and phenotypic features within a given tumor, a phenomenon termed intratumor heterogeneity (ITH). Previously, we have demonstrated genomic ITH in localized lung adenocarcinomas; however, the nature of methylation ITH in lung cancers has not been well investigated. In this study, we generated methylation profiles of 48 spatially separated tumor regions from 11 localized lung adenocarcinomas and their matched normal lung tissues using Illumina Infinium Human Methylation 450K BeadChip array. We observed methylation ITH within the same tumors, but to a much less extent compared to inter-individual heterogeneity. On average, 25% of all differentially methylated probes compared to matched normal lung tissues were shared by all regions from the same tumors. This is in contrast to somatic mutations, of which approximately 77% were shared events amongst all regions of individual tumors, suggesting that while the majority of somatic mutations were early clonal events, the tumor-specific DNA methylation might be associated with later branched evolution of these 11 tumors. Furthermore, our data showed that a higher extent of DNA methylation ITH was associated with larger tumor size (average Euclidean distance of 35.64 (> 3cm, median size) versus 27.24 (<= 3cm), p = 0.014), advanced age (average Euclidean distance of 34.95 (above 65) verse 28.06 (below 65), p = 0.046) and increased risk of postsurgical recurrence (average Euclidean distance of 35.65 (relapsed patients) versus 29.03 (patients without relapsed), p = 0.039).

  7. Concurrent, but not sequential, PD-1 blockade with a DNA vaccine elicits anti-tumor responses in patients with metastatic, castration-resistant prostate cancer.

    PubMed

    McNeel, Douglas G; Eickhoff, Jens C; Wargowski, Ellen; Zahm, Christopher; Staab, Mary Jane; Straus, Jane; Liu, Glenn

    2018-05-22

    T-cell checkpoint inhibitors have demonstrated dramatic clinical activity against multiple cancer types, however little activity in patients with prostate cancer. Conversely, an anti-tumor vaccine was approved for the treatment of prostate cancer, having demonstrated an improvement in overall survival, despite few objective disease responses. In murine studies, we found that PD-1 expression on CD8+ T cells increased following anti-tumor vaccination, and that PD-1/PD-L1 blockade at the time of immunization elicited greater anti-tumor responses. Based on these data we initiated a pilot trial evaluating the immunological and clinical efficacy of a DNA encoding prostatic acid phosphatase (PAP) when delivered in combination with pembrolizumab. 26 patients were treated for 12 weeks with vaccine and received pembrolizumab either during this time or during the subsequent 12 weeks. Adverse events included grade 2 and 3 fatigue, diarrhea, thyroid dysfunction, and hepatitis. Median time to radiographic progression was not different between study arms. 8/13 (62%) of patients treated concurrently, and 1/12 (8%, p=0.01) of patients treated sequentially, experienced PSA declines from baseline. Of these, two were over 50% and one was a complete PSA response. No confirmed CR or PR were observed, however 4/5 patients treated concurrently had measurable decreases in tumor volume at 12 weeks. PSA declines were associated with the development of PAP-specific Th1-biased T cell immunity and CD8+ T cell infiltration in metastatic tumor biopsy specimens. These data are the first report of a clinical trial demonstrating that the efficacy of an anti-tumor vaccine can be augmented by concurrent PD-1 blockade.

  8. Absolute optical metrology : nanometers to kilometers

    NASA Technical Reports Server (NTRS)

    Dubovitsky, Serge; Lay, O. P.; Peters, R. D.; Liebe, C. C.

    2005-01-01

    We provide and overview of the developments in the field of high-accuracy absolute optical metrology with emphasis on space-based applications. Specific work on the Modulation Sideband Technology for Absolute Ranging (MSTAR) sensor is described along with novel applications of the sensor.

  9. Effective and absolute cross sections for low-energy (1-30 eV) electron interactions with condensed biomolecules

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Sanche, Léon

    2018-06-01

    Ionizing radiation is intensively used for therapeutic [e.g., radiotherapy, brachytherapy, and targeted radionuclide therapy (TRT)], as well as for diagnostic medical imaging purposes. In these applications, the radiation dose given to the patient should be known and controlled. In conventional cancer treatments, absorbed dose calculations rely essentially on scattering cross sections (CSs) of the primary high-energy radiation. In more sophisticated treatments, such as combined radio- and chemo-therapy, a description of the details of energy deposits at the micro- and nano-scopic level is preferred to relate dose to radiobiological effectiveness or to evaluate doses at the biomolecular level, when radiopharmaceuticals emitting short-range radiation are delivered to critical molecular components of cancer cells (e.g., TRT). These highly radiotoxic compounds emit large densities of low-energy electrons (LEEs). More generally, LEE (0-30 eV) are emitted in large numbers by any type of high-energy radiation; i.e., about 30 000 per MeV of deposited primary energy. Thus, to optimize the effectiveness of several types of radiation treatments, the energy deposited by LEEs must be known at the level of the cell, nucleus, chromosome, or DNA. Such local doses can be evaluated by Monte Carlo (MC) calculations, which account event-by-event, for the slowing down of all generations of particles. In particular, these codes require as input parameters absolute LEE CSs for elastic scattering, energy losses, and direct damage to vital cellular molecules, particularly DNA, the main target of radiation therapy. In the last decade, such CSs have emerged in the literature. Furthermore, a method was developed to transform relative yields of damages into absolute CSs by measuring specific parameters in the experiments. In this review article, we first present a general description of dose calculations in biological media via MC simulation and give an overview of the CSs available from

  10. Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy.

    PubMed

    Hu, Qinglian; Wu, Min; Fang, Chun; Cheng, Changyong; Zhao, Mengmeng; Fang, Weihuan; Chu, Paul K; Ping, Yuan; Tang, Guping

    2015-04-08

    Live attenuated bacteria are of increasing importance in biotechnology and medicine in the emerging field of cancer immunotherapy. Oral DNA vaccination mediated by live attenuated bacteria often suffers from low infection efficiency due to various biological barriers during the infection process. To this end, we herein report, for the first time, a new strategy to engineer cationic nanoparticle-coated bacterial vectors that can efficiently deliver oral DNA vaccine for efficacious cancer immunotherapy. By coating live attenuated bacteria with synthetic nanoparticles self-assembled from cationic polymers and plasmid DNA, the protective nanoparticle coating layer is able to facilitate bacteria to effectively escape phagosomes, significantly enhance the acid tolerance of bacteria in stomach and intestines, and greatly promote dissemination of bacteria into blood circulation after oral administration. Most importantly, oral delivery of DNA vaccines encoding autologous vascular endothelial growth factor receptor 2 (VEGFR2) by this hybrid vector showed remarkable T cell activation and cytokine production. Successful inhibition of tumor growth was also achieved by efficient oral delivery of VEGFR2 with nanoparticle-coated bacterial vectors due to angiogenesis suppression in the tumor vasculature and tumor necrosis. This proof-of-concept work demonstrates that coating live bacterial cells with synthetic nanoparticles represents a promising strategy to engineer efficient and versatile DNA vaccines for the era of immunotherapy.

  11. Interaction of the Tumor Suppressor p53 with Replication Protein A.

    DTIC Science & Technology

    1996-08-01

    The DNA replication factor RPA physically associates with the tumor suppressor protein p53, an interaction that could be important for the function...binding single-stranded DNA, this mutant of RPA fails to support DNA replication . Therefore the region of RPA which interacts with p53 is essential for...of p53, p21/WAFl/CIPl, inhibits the cell-cycle by associating with cyclin-cdk kinases. It also inhibits DNA replication by interacting with a

  12. DNA repair pathways and mitochondrial DNA mutations in gastrointestinal carcinogenesis.

    PubMed

    Basso, Daniela; Navaglia, Filippo; Fogar, Paola; Zambon, Carlo-Federico; Greco, Eliana; Schiavon, Stefania; Fasolo, Michela; Stranges, Alessia; Falda, Alessandra; Padoan, Andrea; Fadi, Elisa; Pedrazzoli, Sergio; Plebani, Mario

    2007-05-01

    This work focuses on the main DNA repair pathways, highlighting their role in gastrointestinal carcinogenesis and the role of mitochondrial DNA (mtDNA), mutations being described in several tumor types, including those of the gastrointestinal tract. The mismatch repair (MMR) system is inherently altered in patients with hereditary non-polyposis colorectal cancer, and plays a role in carcinogenesis in a subset of sporadic colorectal, gastric and esophageal cancers. Alterations in homologous recombination (HR) and non-homologous end-joining (NHEJ) also contribute to the development of pancreatic cancer. Gene polymorphisms of some X-ray cross-complementing (XRCCs), cofactor proteins involved in the base excision repair pathway, have been investigated in relation to gastric, colorectal and pancreatic cancer. Yet only one polymorphism, XRCC1 Arg194Trp, appears to be involved in smoking-related cancers and in early onset pancreatic cancer. Although evidence in the literature indicates that mtDNA somatic mutations play a role in gastric and colorectal carcinogenesis, no sound conclusions have yet been drawn regarding this issue in pancreatic cancer, although an mtDNA variant at 16519 is believed to worsen the outcome of pancreatic cancer patients, possibly because it is involved in altering cellular metabolism.

  13. Tumor Mutational Burden Guides Therapy in a Treatment Refractory POLE-Mutant Uterine Carcinosarcoma.

    PubMed

    Bhangoo, Munveer S; Boasberg, Peter; Mehta, Pareen; Elvin, Julia A; Ali, Siraj M; Wu, Winnie; Klempner, Samuel J

    2018-05-01

    Gynecologic carcinosarcomas, previously known as malignant mixed Müllerian tumors, are uncommon malignancies that demonstrate an aggressive biology and lack a standard therapeutic approach. Molecular analyses have revealed recurrent alterations in chromatin remodeling genes, but clinical support for therapeutic significance is lacking. We prospectively identified a patient with refractory uterine carcinosarcoma whose tumor was subject to molecular profiling at diagnosis and again at radiographic progression. Initial molecular testing did not assess tumor mutational burden, DNA polymerase ɛ ( POLE ), or microsatellite status. After the failure of several lines of chemotherapy, comprehensive genomic profiling of a repeat biopsy identified two missense mutations of the exonuclease domain of POLE (P286R and T323A). Tumor mutational burden was elevated (169 mutations per DNA megabase), consistent with an ultramutator phenotype. As seen in previously reported POLE -endometrioid cases, our patient harbored alterations in PIK3CA , ARID1A , and PTEN and was microsatellite stable, with appreciable tumor-infiltrating lymphocytes. She achieved an ongoing durable response with pembrolizumab. This is the first report of programmed cell death protein 1 response in uterine carcinosarcoma. Uterine carcinosarcoma is an uncommon and aggressive histologic variant of endometrial carcinoma with a poor prognosis.Inactivating DNA polymerase ɛ ( POLE ) mutations have been associated with high tumor mutational burden (TMB) and response to immune checkpoint inhibition.To the authors' knowledge, this is the first report of response to immune checkpoint inhibitor therapy in a patient with uterine carcinosarcoma.This case further supports expanding genomic profiling to include assessment of tumor mutational burden across tumor types, given the potential for immune checkpoint inhibitor therapy in TMB-high tumors. © AlphaMed Press 2018.

  14. A global algorithm for estimating Absolute Salinity

    NASA Astrophysics Data System (ADS)

    McDougall, T. J.; Jackett, D. R.; Millero, F. J.; Pawlowicz, R.; Barker, P. M.

    2012-12-01

    The International Thermodynamic Equation of Seawater - 2010 has defined the thermodynamic properties of seawater in terms of a new salinity variable, Absolute Salinity, which takes into account the spatial variation of the composition of seawater. Absolute Salinity more accurately reflects the effects of the dissolved material in seawater on the thermodynamic properties (particularly density) than does Practical Salinity. When a seawater sample has standard composition (i.e. the ratios of the constituents of sea salt are the same as those of surface water of the North Atlantic), Practical Salinity can be used to accurately evaluate the thermodynamic properties of seawater. When seawater is not of standard composition, Practical Salinity alone is not sufficient and the Absolute Salinity Anomaly needs to be estimated; this anomaly is as large as 0.025 g kg-1 in the northernmost North Pacific. Here we provide an algorithm for estimating Absolute Salinity Anomaly for any location (x, y, p) in the world ocean. To develop this algorithm, we used the Absolute Salinity Anomaly that is found by comparing the density calculated from Practical Salinity to the density measured in the laboratory. These estimates of Absolute Salinity Anomaly however are limited to the number of available observations (namely 811). In order to provide a practical method that can be used at any location in the world ocean, we take advantage of approximate relationships between Absolute Salinity Anomaly and silicate concentrations (which are available globally).

  15. A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

    PubMed

    Pulverer, Walter; Hofner, Manuela; Preusser, Matthias; Dirnberger, Elisabeth; Hainfellner, Johannes A; Weinhaeusel, Andreas

    2014-01-01

    MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

  16. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    PubMed

    Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  17. Trial watch: Naked and vectored DNA-based anticancer vaccines.

    PubMed

    Bloy, Norma; Buqué, Aitziber; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Sautès-Fridman, Catherine; Fucikova, Jitka; Galon, Jérôme; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-05-01

    One type of anticancer vaccine relies on the administration of DNA constructs encoding one or multiple tumor-associated antigens (TAAs). The ultimate objective of these preparations, which can be naked or vectored by non-pathogenic viruses, bacteria or yeast cells, is to drive the synthesis of TAAs in the context of an immunostimulatory milieu, resulting in the (re-)elicitation of a tumor-targeting immune response. In spite of encouraging preclinical results, the clinical efficacy of DNA-based vaccines employed as standalone immunotherapeutic interventions in cancer patients appears to be limited. Thus, efforts are currently being devoted to the development of combinatorial regimens that allow DNA-based anticancer vaccines to elicit clinically relevant immune responses. Here, we discuss recent advances in the preclinical and clinical development of this therapeutic paradigm.

  18. Trial watch: Naked and vectored DNA-based anticancer vaccines

    PubMed Central

    Bloy, Norma; Buqué, Aitziber; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Sautès-Fridman, Catherine; Fucikova, Jitka; Galon, Jérôme; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    One type of anticancer vaccine relies on the administration of DNA constructs encoding one or multiple tumor-associated antigens (TAAs). The ultimate objective of these preparations, which can be naked or vectored by non-pathogenic viruses, bacteria or yeast cells, is to drive the synthesis of TAAs in the context of an immunostimulatory milieu, resulting in the (re-)elicitation of a tumor-targeting immune response. In spite of encouraging preclinical results, the clinical efficacy of DNA-based vaccines employed as standalone immunotherapeutic interventions in cancer patients appears to be limited. Thus, efforts are currently being devoted to the development of combinatorial regimens that allow DNA-based anticancer vaccines to elicit clinically relevant immune responses. Here, we discuss recent advances in the preclinical and clinical development of this therapeutic paradigm. PMID:26155408

  19. Absolute instability of the Gaussian wake profile

    NASA Technical Reports Server (NTRS)

    Hultgren, Lennart S.; Aggarwal, Arun K.

    1987-01-01

    Linear parallel-flow stability theory has been used to investigate the effect of viscosity on the local absolute instability of a family of wake profiles with a Gaussian velocity distribution. The type of local instability, i.e., convective or absolute, is determined by the location of a branch-point singularity with zero group velocity of the complex dispersion relation for the instability waves. The effects of viscosity were found to be weak for values of the wake Reynolds number, based on the center-line velocity defect and the wake half-width, larger than about 400. Absolute instability occurs only for sufficiently large values of the center-line wake defect. The critical value of this parameter increases with decreasing wake Reynolds number, thereby indicating a shrinking region of absolute instability with decreasing wake Reynolds number. If backflow is not allowed, absolute instability does not occur for wake Reynolds numbers smaller than about 38.

  20. Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

    PubMed

    Aung, Kyaw L; Donald, Emma; Ellison, Gillian; Bujac, Sarah; Fletcher, Lynn; Cantarini, Mireille; Brady, Ged; Orr, Maria; Clack, Glen; Ranson, Malcolm; Dive, Caroline; Hughes, Andrew

    2014-05-01

    BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  1. 49 CFR 236.709 - Block, absolute.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Block, absolute. 236.709 Section 236.709 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Block, absolute. A block in which no train is permitted to enter while it is occupied by another train. ...

  2. 49 CFR 236.709 - Block, absolute.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Block, absolute. 236.709 Section 236.709 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Block, absolute. A block in which no train is permitted to enter while it is occupied by another train. ...

  3. Absolute quantification of microbial taxon abundances.

    PubMed

    Props, Ruben; Kerckhof, Frederiek-Maarten; Rubbens, Peter; De Vrieze, Jo; Hernandez Sanabria, Emma; Waegeman, Willem; Monsieurs, Pieter; Hammes, Frederik; Boon, Nico

    2017-02-01

    High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

  4. Mitochondrial DNA maintenance is regulated in human hepatoma cells by glycogen synthase kinase 3β and p53 in response to tumor necrosis factor α.

    PubMed

    Vadrot, Nathalie; Ghanem, Sarita; Braut, Françoise; Gavrilescu, Laura; Pilard, Nathalie; Mansouri, Abdellah; Moreau, Richard; Reyl-Desmars, Florence

    2012-01-01

    During chronic liver inflammation, up-regulated Tumor Necrosis Factor alpha (TNF-α) targets hepatocytes and induces abnormal reactive oxygen species (ROS) production responsible for mitochondrial DNA (mtDNA) alterations. The serine/threonine Glycogen Synthase Kinase 3 beta (GSK3β) plays a pivotal role during inflammation but its involvement in the maintenance of mtDNA remains unknown. The aim of this study was to investigate its involvement in TNF-α induced mtDNA depletion and its interrelationship with p53 a protein known to maintain mtDNA copy numbers. Using quantitative polymerase chain reaction (qPCR) we found that at 30 min in human hepatoma HepG2 cells TNF-α induced 0.55±0.10 mtDNA lesions per 10 Kb and a 52.4±2.8% decrease in mtDNA content dependent on TNF-R1 receptor and ROS production. Both lesions and depletion returned to baseline from 1 to 6 h after TNF-α exposure. Luminol-amplified chemiluminescence (LAC) was used to measure the rapid (10 min) and transient TNF-α induced increase in ROS production (168±15%). A transient 8-oxo-dG level of 1.4±0.3 ng/mg DNA and repair of abasic sites were also measured by ELISA assays. Translocation of p53 to mitochondria was observed by Western Blot and co-immunoprecipitations showed that TNF-α induced p53 binding to GSK3β and mitochondrial transcription factor A (TFAM). In addition, mitochondrial D-loop immunoprecipitation (mtDIP) revealed that TNF-α induced p53 binding to the regulatory D-loop region of mtDNA. The knockdown of p53 by siRNAs, inhibition by the phosphoSer(15)p53 antibody or transfection of human mutant active GSK3βS9A pcDNA3 plasmid inhibited recovery of mtDNA content while blockade of GSK3β activity by SB216763 inhibitor or knockdown by siRNAs suppressed mtDNA depletion. This study is the first to report the involvement of GSK3β in TNF-α induced mtDNA depletion. We suggest that p53 binding to GSK3β, TFAM and D-loop could induce recovery of mtDNA content through mtDNA repair.

  5. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways

    PubMed Central

    Leitner, Wolfgang W.; Hwang, Leroy N.; Deveer, Michael J.; Zhou, Aimin; Silverman, Robert H.; Williams, Bryan R.G.; Dubensky, Thomas W.; Ying, Han; Restifo, Nicholas P.

    2006-01-01

    Cancer vaccines targeting ‘self’ antigens that are expressed at consistently high levels by tumor cells are potentially useful in immunotherapy, but immunological tolerance may block their function. Here, we describe a novel, naked DNA vaccine encoding an alphavirus replicon (self-replicating mRNA) and the self/tumor antigen tyrosinase-related protein-1. Unlike conventional DNA vaccines, this vaccine can break tolerance and provide immunity to melanoma. The vaccine mediates production of double-stranded RNA, as evidenced by the autophosphorylation of protein kinase R. Double-stranded RNA is critical to vaccine function because both the immunogenicity and the anti-tumor activity of the vaccine are blocked in mice deficient for the RNase L enzyme, a key component of the 2′,5′-linked oligoadenylate synthetase antiviral pathway involved in double-stranded RNA recognition. This study shows for the first time that alphaviral replicon-encoding DNA vaccines activate innate immune pathways known to drive antiviral immune responses, and points the way to strategies for improving the efficacy of immunization with naked DNA. PMID:12496961

  6. Single-Molecule Encoders for Tracking Motor Proteins on DNA

    NASA Astrophysics Data System (ADS)

    Lipman, Everett A.

    2012-02-01

    Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with linear or rotational motion. We have implemented this technique at the nanometer scale by labeling DNA with regularly spaced fluorescent dyes. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. These measurements do not require mechanical coupling to macroscopic instrumentation, are automatically calibrated by the underlying structure of DNA, and depend on signal periodicity rather than absolute level. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.

  7. Prognostic significance of DNA ploidy in adenocarcinoma of the pancreas. A flow cytometric study of paraffin-embedded specimens.

    PubMed

    Porschen, R; Remy, U; Bevers, G; Schauseil, S; Hengels, K J; Borchard, F

    1993-06-15

    The prognostic significance of tumor DNA ploidy in patients with cancer of the pancreas has not been defined because conflicting results have been reported. DNA content was measured in 56 ductal adenocarcinomas of the pancreas. DNA ploidy status was evaluated by flow cytometry in nuclei isolated from paraffin-embedded tumor tissues. An abnormal DNA stemline was observed in 27 (48%) patients. The percentage of aneuploid tumors was significantly increased in tumors classified as Stage III/IV (53%) compared with those classified as Stage I (22%). A borderline significant association existed between DNA ploidy and radicality of surgery (P = 0.08). The median survival of patients with diploid carcinomas was 6.9 months (standard error, +/- 0.9) in comparison to 4.5 +/- 1.2 months for patients with aneuploid tumors (P = 0.013 by generalized Wilcoxon test; P = 0.023 by generalized Savage test). Although a selection bias cannot be excluded, survival of patients with a radical resection was longer than that of patients with a nonradical resection (P = 0.0008 and P = 0.0085, respectively). In addition, presence of distant metastasis (P = 0.0006 [Wilcoxon test] and P = 0.033 [Savage test]) could be identified as a prognostic factor. In a Cox regression model, results of surgery and DNA ploidy were independent prognostic variables. Because DNA ploidy has a significant impact on prognosis in pancreatic cancer, it should be used as a variable for stratified randomization of patients in therapeutic trials.

  8. Absolute Humidity and the Seasonality of Influenza (Invited)

    NASA Astrophysics Data System (ADS)

    Shaman, J. L.; Pitzer, V.; Viboud, C.; Grenfell, B.; Goldstein, E.; Lipsitch, M.

    2010-12-01

    Much of the observed wintertime increase of mortality in temperate regions is attributed to seasonal influenza. A recent re-analysis of laboratory experiments indicates that absolute humidity strongly modulates the airborne survival and transmission of the influenza virus. Here we show that the onset of increased wintertime influenza-related mortality in the United States is associated with anomalously low absolute humidity levels during the prior weeks. We then use an epidemiological model, in which observed absolute humidity conditions temper influenza transmission rates, to successfully simulate the seasonal cycle of observed influenza-related mortality. The model results indicate that direct modulation of influenza transmissibility by absolute humidity alone is sufficient to produce this observed seasonality. These findings provide epidemiological support for the hypothesis that absolute humidity drives seasonal variations of influenza transmission in temperate regions. In addition, we show that variations of the basic and effective reproductive numbers for influenza, caused by seasonal changes in absolute humidity, are consistent with the general timing of pandemic influenza outbreaks observed for 2009 A/H1N1 in temperate regions. Indeed, absolute humidity conditions correctly identify the region of the United States vulnerable to a third, wintertime wave of pandemic influenza. These findings suggest that the timing of pandemic influenza outbreaks is controlled by a combination of absolute humidity conditions, levels of susceptibility and changes in population mixing and contact rates.

  9. Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

    PubMed

    Mandelker, Diana; Zhang, Liying; Kemel, Yelena; Stadler, Zsofia K; Joseph, Vijai; Zehir, Ahmet; Pradhan, Nisha; Arnold, Angela; Walsh, Michael F; Li, Yirong; Balakrishnan, Anoop R; Syed, Aijazuddin; Prasad, Meera; Nafa, Khedoudja; Carlo, Maria I; Cadoo, Karen A; Sheehan, Meg; Fleischut, Megan H; Salo-Mullen, Erin; Trottier, Magan; Lipkin, Steven M; Lincoln, Anne; Mukherjee, Semanti; Ravichandran, Vignesh; Cambria, Roy; Galle, Jesse; Abida, Wassim; Arcila, Marcia E; Benayed, Ryma; Shah, Ronak; Yu, Kenneth; Bajorin, Dean F; Coleman, Jonathan A; Leach, Steven D; Lowery, Maeve A; Garcia-Aguilar, Julio; Kantoff, Philip W; Sawyers, Charles L; Dickler, Maura N; Saltz, Leonard; Motzer, Robert J; O'Reilly, Eileen M; Scher, Howard I; Baselga, Jose; Klimstra, David S; Solit, David B; Hyman, David M; Berger, Michael F; Ladanyi, Marc; Robson, Mark E; Offit, Kenneth

    2017-09-05

    Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of

  10. A Photosensitizer-Loaded DNA Origami Nanosystem for Photodynamic Therapy.

    PubMed

    Zhuang, Xiaoxi; Ma, Xiaowei; Xue, Xiangdong; Jiang, Qiao; Song, Linlin; Dai, Luru; Zhang, Chunqiu; Jin, Shubin; Yang, Keni; Ding, Baoquan; Wang, Paul C; Liang, Xing-Jie

    2016-03-22

    Photodynamic therapy (PDT) offers an alternative for cancer treatment by using ultraviolet or visible light in the presence of a photosensitizer and molecular oxygen, which can produce highly reactive oxygen species that ultimately leading to the ablation of tumor cells by multifactorial mechanisms. However, this technique is limited by the penetration depth of incident light, the hypoxic environment of solid tumors, and the vulnerability of photobleaching reduces the efficiency of many imaging agents. In this work, we reported a cellular level dual-functional imaging and PDT nanosystem BMEPC-loaded DNA origami for photodynamic therapy with high efficiency and stable photoreactive property. The carbazole derivative BMEPC is a one- and two-photon imaging agent and photosensitizer with large two-photon absorption cross section, which can be fully excited by near-infrared light, and is also capable of destroying targets under anaerobic condition by generating reactive intermediates of Type I photodynamic reactions. However, the application of BMEPC was restricted by its poor solubility in aqueous environment and its aggregation caused quenching. We observed BMEPC-loaded DNA origami effectively reduced the photobleaching of BMEPC within cells. Upon binding to DNA origami, the intramolecular rotation of BMEPC became proper restricted, which intensify fluorescence emission and radicals production when being excited. After the BMEPC-loaded DNA origami are taken up by tumor cells, upon irradiation, BMEPC could generate free radicals and be released due to DNA photocleavage as well as the following partially degradation. Apoptosis was then induced by the generation of free radicals. This functional nanosystem provides an insight into the design of photosensitizer-loaded DNA origami for effective intracellular imaging and photodynamic therapy.

  11. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  12. Succinate Dehydrogenase Mutation Underlies Global Epigenomic Divergence in Gastrointestinal Stromal Tumor

    PubMed Central

    Killian, J. Keith; Kim, Su Young; Miettinen, Markku; Smith, Carly; Merino, Maria; Tsokos, Maria; Quezado, Martha; Smith, William I.; Jahromi, Mona S.; Xekouki, Paraskevi; Szarek, Eva; Walker, Robert L.; Lasota, Jerzy; Raffeld, Mark; Klotzle, Brandy; Wang, Zengfeng; Jones, Laura; Zhu, Yuelin; Wang, Yonghong; Waterfall, Joshua J.; O’Sullivan, Maureen J.; Bibikova, Marina; Pacak, Karel; Stratakis, Constantine; Janeway, Katherine A.; Schiffman, Joshua D.; Fan, Jian-Bing; Helman, Lee; Meltzer, Paul S.

    2014-01-01

    Gastrointestinal stromal tumors (GIST) harbor driver mutations of signal transduction kinases such as KIT, or, alternatively, manifest loss-of-function defects in the mitochondrial succinate dehydrogenase (SDH) complex, a component of the Krebs cycle and electron transport chain. We have uncovered a striking divergence between the DNA methylation profiles of SDH-deficient GIST (n = 24) versus KIT tyrosine kinase pathway–mutated GIST (n = 39). Infinium 450K methylation array analysis of formalin-fixed paraffin-embedded tissues disclosed an order of magnitude greater genomic hypermethylation relative to SDH-deficient GIST versus the KIT-mutant group (84.9 K vs. 8.4 K targets). Epigenomic divergence was further found among SDH-mutant paraganglioma/pheochromocytoma (n = 29), a developmentally distinct SDH-deficient tumor system. Comparison of SDH -mutant GIST with isocitrate dehydrogenase -mutant glioma, another Krebs cycle–defective tumor type, revealed comparable measures of global hypo- and hypermethylation. These data expose a vital connection between succinate metabolism and genomic DNA methylation during tumorigenesis, and generally implicate the mitochondrial Krebs cycle in nuclear epigenomic maintenance. SIGNIFICANCE This study shows that SDH deficiency underlies pervasive DNA hypermethylation in multiple tumor lineages, generally defining the Krebs cycle as mitochondrial custodian of the methylome. We propose that this phenomenon may result from a failure of maintenance CpG demethylation, secondary to inhibition of the TET 5-methylcytosine dioxgenase demethylation pathway, by inhibitory metabolites that accumulate in tumors with Krebs cycle dysfunction. PMID:23550148

  13. [Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

    PubMed

    Bartošík, M; Ondroušková, E

    Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

  14. Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors.

    PubMed

    Herstad, Kristin Marie Valand; Moen, Aina Elisabeth Fossum; Gaby, John Christian; Moe, Lars; Skancke, Ellen

    2018-01-01

    Colorectal epithelial tumors occur spontaneously in dogs, and the pathogenesis seems to parallel that of humans. The development of human colorectal tumorigenesis has been linked to alterations in the composition of the intestinal microbiota. This study characterized the fecal- and mucosa-associated microbiota in dogs with colorectal epithelial tumors (n = 10). The fecal microbiota was characterized by 16S rDNA analysis and compared with that of control dogs (n = 13). We also determined the mucosa-associated microbiota composition in colonic tumor tissue (n = 8) and in adjacent non-tumor tissue (n = 5) by 16S rDNA- and rRNA profiling. The fecal microbial community structure in dogs with tumors was different from that of control samples and was distinguished by oligotypes affiliated with Enterobacteriaceae, Bacteroides, Helicobacter, Porphyromonas, Peptostreptococcus and Streptococcus, and lower abundance of Ruminococcaceae, Slackia, Clostridium XI and Faecalibacterium. The overall community structure and populations of mucosal bacteria were not different based on either the 16S rDNA or the 16S rRNA profile in tumor tissue vs. adjacent non-tumor tissue. However, the proportion of live, potentially active bacteria appeared to be higher in non-tumor tissue compared with tumor tissue and included Slackia, Roseburia, unclass. Ruminococcaeceae, unclass. Lachnospiraceae and Oscillibacter. Colorectal tumors are rarely diagnosed in dogs, but despite this limitation, we were able to show that dogs with colorectal tumors have distinct fecal microbiota profiles. These initial results support the need for future case-control studies that are adequately powered, as well as age-matched and breed-matched, in order to evaluate the influence of bacteria on colorectal cancer etiopathogenesis and to determine whether the bacteria may have potential as biomarkers in clinical settings.

  15. Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

    PubMed

    He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

    2013-11-01

    Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

  16. Genital shedding of human immunodeficiency virus type 1 DNA during pregnancy: association with immunosuppression, abnormal cervical or vaginal discharge, and severe vitamin A deficiency.

    PubMed

    John, G C; Nduati, R W; Mbori-Ngacha, D; Overbaugh, J; Welch, M; Richardson, B A; Ndinya-Achola, J; Bwayo, J; Krieger, J; Onyango, F; Kreiss, J K

    1997-01-01

    The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1-seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1-infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count > or = 500 (95% confidence interval, 2.8-34.2). Detection of vaginal HIV- 1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1-infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge.

  17. Genital Shedding of Human Immunodeficiency Virus Type 1 DNA during Pregnancy: Association with Immunosuppression, Abnormal Cervical or Vaginal Discharge, and Severe Vitamin A Deficiency

    PubMed Central

    John, Grace C.; Nduati, Ruth W.; Mbori-Ngacha, Dorothy; Overbaugh, Julie; Welch, Mary; Richardson, Barbra A.; Ndinya-Achola, Jeckoniah; Bwayo, Job; Krieger, John; Onyango, Francis; Kreiss, Joan K.

    2012-01-01

    The presence of human immunodeficiency virus type 1 (HIV-1) in genital secretions may be a determinant of vertical HIV-1 transmission. Cervical and vaginal secretions from HIV-1–seropositive pregnant women were evaluated to determine prevalence and correlates of HIV-1–infected cells in the genital tract. HIV-1 DNA was detected by polymerase chain reaction in 32% of 212 cervical and 10% of 215 vaginal specimens. Presence of HIV-1 DNA in the cervix was associated with cervical mucopus and a significantly lower absolute CD4 cell count (354 vs. 469, P < .001). An absolute CD4 cell count <200 was associated with a 9.6-fold increased odds of cervical HIV-1 DNA detection compared with a count ≥500 (95% confidence interval, 2.8–34.2). Detection of vaginal HIV-1 DNA was associated with abnormal vaginal discharge, lower absolute CD4 cell count, and severe vitamin A deficiency. Presence of HIV-1–infected cells in genital secretions was associated with immunosuppression and abnormal cervical or vaginal discharge. PMID:8985196

  18. Flow cytometric monitoring of hormone receptor expression in human solid tumors

    NASA Astrophysics Data System (ADS)

    Krishan, Awtar

    2002-05-01

    Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

  19. Perspective on the pipeline of drugs being developed with modulation of DNA damage as a target.

    PubMed

    Plummer, Ruth

    2010-09-15

    Inhibitors of various elements of the DNA repair pathways have entered clinical development or are in late preclinical stages of drug development. It was initially considered that agents targeting DNA repair would act to overcome tumor resistance to chemotherapy and radiotherapy. More recent data have shown that targeting DNA repair pathways can be effective in selected tumors via a synthetically lethal route, with single agent activity having been shown with poly-ADP ribose polymerase (PARP) inhibitors. An increased understanding of the biology and interaction of the DNA repair pathways also means that rational combination of DNA repair inhibitors may also give great benefit in the clinic. ©2010 AACR.

  20. Noninvasive imaging of absolute PpIX concentration distribution in nonmelanoma skin tumors at pre-PDT

    NASA Astrophysics Data System (ADS)

    Sunar, Ulas; Rohrbach, Daniel; Morgan, Janet; Zeitouni, Natalie

    2013-03-01

    Photodynamic Therapy (PDT) has proven to be an effective treatment option for nonmelanoma skin cancers. The ability to quantify the concentration of drug in the treated area is crucial for effective treatment planning as well as predicting outcomes. We utilized spatial frequency domain imaging for quantifying the accurate concentration of protoporphyrin IX (PpIX) in phantoms and in vivo. We correct fluorescence against the effects of native tissue absorption and scattering parameters. First we quantified the absorption and scattering of the tissue non-invasively. Then, we corrected raw fluorescence signal by compensating for optical properties to get the absolute drug concentration. After phantom experiments, we used basal cell carcinoma (BCC) model in Gli mice to determine optical properties and drug concentration in vivo at pre-PDT.

  1. Age-specific absolute and relative organ weight distributions for B6C3F1 mice.

    PubMed

    Marino, Dale J

    2012-01-01

    The B6C3F1 mouse is the standard mouse strain used in toxicology studies conducted by the National Cancer Institute (NCI) and the National Toxicology Program (NTP). While numerous reports have been published on growth, survival, and tumor incidence, no overall compilation of organ weight data is available. Importantly, organ weight change is an endpoint used by regulatory agencies to develop toxicity reference values (TRVs) for use in human health risk assessments. Furthermore, physiologically based pharmacokinetic (PBPK) models, which utilize relative organ weights, are increasingly being used to develop TRVs. Therefore, all available absolute and relative organ weight data for untreated control B6C3F1 mice were collected from NCI/NTP studies in order to develop age-specific distributions. Results show that organ weights were collected more frequently in NCI/NTP studies at 2-wk (60 studies), 3-mo (147 studies), and 15-mo (40 studies) intervals than at other intervals, and more frequently from feeding and inhalation than drinking water studies. Liver, right kidney, lung, heart, thymus, and brain weights were most frequently collected. From the collected data, the mean and standard deviation for absolute and relative organ weights were calculated. Results show age-related increases in absolute liver, right kidney, lung, and heart weights and relatively stable brain and right testis weights. The results suggest a general variability trend in absolute organ weights of brain < right testis < right kidney < heart < liver < lung < spleen < thymus. This report describes the results of this effort.

  2. ZnO Nanoparticles Protect RNA from Degradation Better than DNA.

    PubMed

    McCall, Jayden; Smith, Joshua J; Marquardt, Kelsey N; Knight, Katelin R; Bane, Hunter; Barber, Alice; DeLong, Robert K

    2017-11-08

    Gene therapy and RNA delivery require a nanoparticle (NP) to stabilize these nucleic acids when administered in vivo. The presence of degradative hydrolytic enzymes within these environments limits the nucleic acids' pharmacologic activity. This study compared the effects of nanoscale ZnO and MgO in the protection afforded to DNA and RNA from degradation by DNase, serum or tumor homogenate. For double-stranded plasmid DNA degradation by DNase, our results suggest that the presence of MgO NP can protect DNA from DNase digestion at an elevated temperature (65 °C), a biochemical activity not present in ZnO NP-containing samples at any temperature. In this case, intact DNA was remarkably present for MgO NP after ethidium bromide staining and agarose gel electrophoresis where these same stained DNA bands were notably absent for ZnO NP. Anticancer RNA, polyinosinic-polycytidylic acid (poly I:C) is now considered an anti-metastatic RNA targeting agent and as such there is great interest in its delivery by NP. For it to function, the NP must protect it from degradation in serum and the tumor environment. Surprisingly, ZnO NP protected the RNA from degradation in either serum-containing media or melanoma tumor homogenate after gel electrophoretic analysis, whereas the band was much more diminished in the presence of MgO. For both MgO and ZnO NP, buffer-dependent rescue from degradation occurred. These data suggest a fundamental difference in the ability of MgO and ZnO NP to stabilize nucleic acids with implications for DNA and RNA delivery and therapy.

  3. Improving the yield and quality of DNA isolated from white-rot fungi.

    PubMed

    Kuhad, R C; Kapoor, R K; Lal, R

    2004-01-01

    A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.

  4. Low absolute neutrophil counts in African infants.

    PubMed

    Kourtis, Athena P; Bramson, Brian; van der Horst, Charles; Kazembe, Peter; Ahmed, Yusuf; Chasela, Charles; Hosseinipour, Mina; Knight, Rodney; Lugalia, Lebah; Tegha, Gerald; Joaki, George; Jafali, Robert; Jamieson, Denise J

    2005-07-01

    Infants of African origin have a lower normal range of absolute neutrophil counts than white infants; this fact, however, remains under appreciated by clinical researchers in the United States. During the initial stages of a clinical trial in Malawi, the authors noted an unexpectedly high number of infants with absolute neutrophil counts that would be classifiable as neutropenic using the National Institutes of Health's Division of AIDS toxicity tables. The authors argue that the relevant Division of AIDS table does not take into account the available evidence of low absolute neutrophil counts in African infants and that a systematic collection of data from many African settings might help establish the absolute neutrophil count cutpoints to be used for defining neutropenia in African populations.

  5. Absolute colorimetric characterization of a DSLR camera

    NASA Astrophysics Data System (ADS)

    Guarnera, Giuseppe Claudio; Bianco, Simone; Schettini, Raimondo

    2014-03-01

    A simple but effective technique for absolute colorimetric camera characterization is proposed. It offers a large dynamic range requiring just a single, off-the-shelf target and a commonly available controllable light source for the characterization. The characterization task is broken down in two modules, respectively devoted to absolute luminance estimation and to colorimetric characterization matrix estimation. The characterized camera can be effectively used as a tele-colorimeter, giving an absolute estimation of the XYZ data in cd=m2. The user is only required to vary the f - number of the camera lens or the exposure time t, to better exploit the sensor dynamic range. The estimated absolute tristimulus values closely match the values measured by a professional spectro-radiometer.

  6. Hormonal induction of transfected genes depends on DNA topology.

    PubMed Central

    Piña, B; Haché, R J; Arnemann, J; Chalepakis, G; Slater, E P; Beato, M

    1990-01-01

    Plasmids containing the hormone regulatory element of mouse mammary tumor virus linked to the thymidine kinase promoter of herpes simplex virus and the reporter gene chloramphenicol acetyltransferase of Escherichia coli respond to glucocorticoids and progestins when transfected into appropriate cells. In the human mammary tumor cell line T47D, the response to progestins, but not to glucocorticoids, is highly dependent on the topology of the transfected DNA. Although negatively supercoiled plasmids respond optimally to the synthetic progestin R5020, their linearized counterparts exhibit markedly reduced progestin inducibility. This is not due to changes in the efficiency of DNA transfection, since the amount of DNA incorporated into the cell nucleus is not significantly dependent on the initial topology of the plasmids. In contrast, cotransfection experiments with glucocorticoid receptor cDNA in the same cell line show no significant influence of DNA topology on induction by dexamethasone. A similar result was obtained with fibroblasts that contain endogenous glucocorticoid receptors. When the distance between receptor-binding sites or between the binding sites and the promoter was increased, the dependence of progestin induction on DNA topology was more pronounced. In contrast to the original plasmid, these constructs also revealed a similar topological dependence for induction by glucocorticoids. The differential influence of DNA topology is not due to differences in the affinity of the two hormone receptors for DNA of various topologies, but probably reflects an influence of DNA topology on the interaction between different DNA-bound receptor molecules and between receptors and other transcription factors. Images PMID:2153920

  7. Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data.

    PubMed

    Racle, Julien; de Jonge, Kaat; Baumgaertner, Petra; Speiser, Daniel E; Gfeller, David

    2017-11-13

    Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org).

  8. Simultaneous enumeration of cancer and immune cell types from bulk tumor gene expression data

    PubMed Central

    Racle, Julien; de Jonge, Kaat; Baumgaertner, Petra; Speiser, Daniel E

    2017-01-01

    Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org). PMID:29130882

  9. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells

    PubMed Central

    Dolman, M. Emmy M.; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization. PMID:26716839

  10. Mitochondrial DNA content in breast cancer: Impact on in vitro and in vivo phenotype and patient prognosis

    PubMed Central

    Weerts, Marjolein J.A.; Sieuwerts, Anieta M.; Smid, Marcel; Look, Maxime P.; Foekens, John A.; Sleijfer, Stefan; Martens, John W.M.

    2016-01-01

    Reduced mitochondrial DNA (mtDNA) content in breast cancer cell lines has been associated with transition towards a mesenchymal phenotype, but its clinical consequences concerning breast cancer dissemination remain unidentified. Here, we aimed to clarify the link between mtDNA content and a mesenchymal phenotype and its relation to prognosis of breast cancer patients. We analyzed mtDNA content in 42 breast cancer cell lines and 207 primary breast tumor specimens using a combination of quantitative PCR and array-based copy number analysis. By associating mtDNA content with expression levels of genes involved in epithelial-to-mesenchymal transition (EMT) and with the intrinsic breast cancer subtypes, we could not identify a relation between low mtDNA content and mesenchymal properties in the breast cancer cell lines or in the primary breast tumors. In addition, we explored the relation between mtDNA content and prognosis in our cohort of primary breast tumor specimens that originated from patients with lymph node-negative disease who did not receive any (neo)adjuvant systemic therapy. When patients were divided based on the tumor quartile levels of mtDNA content, those in the lowest quarter (≤ 350 mtDNA molecules per cell) showed a poorer 10-year distant metastasis-free survival than patients with > 350 mtDNA molecules per cell (HR 0.50 [95% CI 0.29–0.87], P = 0.015). The poor prognosis was independent of established clinicopathological markers (HR 0.54 [95% CI 0.30–0.97], P = 0.038). We conclude that, despite a lack of evidence between mtDNA content and EMT, low mtDNA content might provide meaningful prognostic value for distant metastasis in breast cancer. PMID:27081694

  11. Oncogenetic tree model of somatic mutations and DNA methylation in colon tumors.

    PubMed

    Sweeney, Carol; Boucher, Kenneth M; Samowitz, Wade S; Wolff, Roger K; Albertsen, Hans; Curtin, Karen; Caan, Bette J; Slattery, Martha L

    2009-01-01

    Our understanding of somatic alterations in colon cancer has evolved from a concept of a series of events taking place in a single sequence to a recognition of multiple pathways. An oncogenetic tree is a model intended to describe the pathways and sequence of somatic alterations in carcinogenesis without assuming that tumors will fall in mutually exclusive categories. We applied this model to data on colon tumor somatic alterations. An oncogenetic tree model was built using data on mutations of TP53, KRAS2, APC, and BRAF genes, methylation at CpG sites of MLH1 and TP16 genes, methylation in tumor (MINT) markers, and microsatellite instability (MSI) for 971 colon tumors from a population-based series. Oncogenetic tree analysis resulted in a reproducible tree with three branches. The model represents methylation of MINT markers as initiating a branch and predisposing to MSI, methylation of MHL1 and TP16, and BRAF mutation. APC mutation is the first alteration in an independent branch and is followed by TP53 mutation. KRAS2 mutation was placed a third independent branch, implying that it neither depends on, nor predisposes to, the other alterations. Individual tumors were observed to have alteration patterns representing every combination of one, two, or all three branches. The oncogenetic tree model assumptions are appropriate for the observed heterogeneity of colon tumors, and the model produces a useful visual schematic of the sequence of events in pathways of colon carcinogenesis.

  12. DNA Damage Repair and the Emerging Role of Poly(ADP-ribose) Polymerase Inhibition in Cancer Therapeutics.

    PubMed

    Rabenau, Karen; Hofstatter, Erin

    2016-07-01

    As a result of improved understanding of DNA repair mechanisms, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasingly recognized to play an important therapeutic role in the treatment of cancer. The aim of this article is to provide a review of PARPi function in DNA damage repair and synthetic lethality and to demonstrate how these mechanisms can be exploited to provide new PARPi-based therapies to patients with solid tumors. Literature from a range of sources, including PubMed and MEDLINE, were searched to identify recent reports regarding DNA damage repair and PARPi. DNA damage repair is central to cellular viability. The family of poly(ADP-ribose) polymerase proteins play multiple intracellular roles in DNA repair, but function primarily in the resolution of repair of single-strand DNA breaks. Insights through the discovery of germline BRCA1/2 mutations led to the understanding of synthetic lethality and the potential therapeutic role of PARPi in the treatment of cancer. Further understanding of DNA damage repair and the concept of BRCA-like tumors have catalyzed PARPi clinical investigation in multiple oncologic settings. PARPi hold great promise in the treatment of solid tumors, both as monotherapy and in combination with other cancer therapeutics. Multiple PARPi clinical trials are currently underway. Further understanding of aberrant DNA repair mechanisms in the germline and in the tumor genome will allow clinicians and researchers to apply PARPi most strategically in the era of personalized medicine. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

  13. ME-10TUMOR MICROENVIRONMENT INFILTRATING MYELOID DERIVED SUPPRESSOR CELLS INHIBIT ANTI-TUMOR T CELL RESPONSES

    PubMed Central

    Kamran, Neha; Ayala, Mariela; Li, Youping; Assi, Hikmat; Candolfi, Marianela; Dzaman, Marta; Lowenstein, Pedro; Castro, Maria

    2014-01-01

    MDSCs represent a population of immature myeloid cells at various stages of differentiation that inhibit anti-tumor T cell-mediated responses. We demonstrate the accumulation of MDSCs in GL26 induced glioma and B16 melanoma bearing mice. Absolute numbers of Ly-6G+ (Gr-1high) MDSCs showed a 200 fold increase within the tumor microenvironment (TME) 28 days post-tumor implantation. The numbers of Ly-6C+ (Gr-1low) MDSCs also showed a similar trend within the TME. While this massive influx of MDSCs was noted within intracranial tumors, MDSC levels did not increase in the dLNs, spleen or bone marrow (BM) of intracranial tumor bearing mice. MDSCs numbers were significantly elevated in the blood of GL26 intracranial tumor bearing mice at 28 days. Mice bearing B16 tumors in the flank showed a ∼5 fold increased influx of Ly-6G+ MDSCs while the Ly6C+ MDSCs increased marginally by 1.1 fold within the tumor mass. Levels of circulating MDSCs also increased by ∼10 fold, while the levels of splenic MDSCs did not change. While both Ly-6G+ and Ly6C+ MDSCs isolated from the brain TME of GL26 intracranial tumor bearing mice inhibited antigen-specific T cell proliferation, Ly6C+ MDSC were found to be more efficient. Ly6G+ or Ly6C+ MDSCs from the bone marrow of intracranial tumor bearing mice failed to suppress antigen-specific T cell proliferation. Splenic and bone marrow MDSCs from naïve mice also did not inhibit antigen-specific T cell proliferation suggesting that TME derived factors may activate MDSCs to exert their immune-suppressive properties. Microarray analysis of glioma cell lines showed elevated levels of CXCL1 mRNA and splenic MDSCs from GL26 tumor mice showed upregulation of the CXCR2 mRNA. Preliminary experiments indicate that CXCR2 signaling mediates MDSC chemotaxis. Overall, our data suggests that strategies that inhibit MDSC recruitment to the TME and/or block their activity could enhance the T cell mediated tumor clearance.

  14. Correlation of DNA content and nucleomorphometric features with World Health Organization grading of meningiomas.

    PubMed

    Grunewald, J P; Röhl, F W; Kirches, E; Dietzmann, K

    1998-02-01

    Many studies dealing with extracranial cancer showed a strong correlation of DNA ploidy to a poor clinical outcome, recurrence, or malignancy. In brain tumors, analysis of DNA content did not always provided significant diagnostic information. In this study, DNA density and karyometric parameters of 50 meningiomas (26 Grade I, 10 Grade II, 14 Grade III) were quantitatively evaluated by digital cell image analyses of Feulgen-stained nuclei. In particular, the densitometric parameter SEXT, which describes nuclear DNA content, as well as the morphometric values LENG (a computer-assisted measurement of nuclear circumference), AREA (a computer-assisted measurement of nuclear area), FCON (a parameter that describes nuclear roundness), and CONC (a describing nuclear contour), evaluated with the software IMAGE C, were correlated to World Health Organization (WHO) grading using univariate and multivariate methods. AREA and LENG values showed significant differences between tumors of Grades I and III. FCON values were unable to distinguish WHO Grade III from Grade I/II but were useful in clearly separating Grade II from Grade I tumors. CONC values detected differences between WHO Grades II and I/III tumors but not between the latter. SEXT values clearly distinguished Grade III from Grade I/II tumors. The 1c, 2c, 2.5c, and 5c exceeding rates showed no predictive values. Only the 6c exceeding rate showed a significant difference between Grades I and III. These results outline the characteristic features of the atypical (Grade II) meningiomas, which make them a recognizable tumor entity distinct from benign and anaplastic meningiomas. The combination of DNA densitometric and morphometric findings seems to be a powerful addition to the histopathologic classification of meningiomas, as suggested by the WHO.

  15. Cryogenic, Absolute, High Pressure Sensor

    NASA Technical Reports Server (NTRS)

    Chapman, John J. (Inventor); Shams. Qamar A. (Inventor); Powers, William T. (Inventor)

    2001-01-01

    A pressure sensor is provided for cryogenic, high pressure applications. A highly doped silicon piezoresistive pressure sensor is bonded to a silicon substrate in an absolute pressure sensing configuration. The absolute pressure sensor is bonded to an aluminum nitride substrate. Aluminum nitride has appropriate coefficient of thermal expansion for use with highly doped silicon at cryogenic temperatures. A group of sensors, either two sensors on two substrates or four sensors on a single substrate are packaged in a pressure vessel.

  16. Size-based separation methods of circulating tumor cells.

    PubMed

    Hao, Si-Jie; Wan, Yuan; Xia, Yi-Qiu; Zou, Xin; Zheng, Si-Yang

    2018-02-01

    Circulating tumor cells (CTCs) originate from the primary tumor mass and enter into the peripheral bloodstream. Compared to other "liquid biopsy" portfolios such as exosome, circulating tumor DNA/RNA (ctDNA/RNA), CTCs have incomparable advantages in analyses of transcriptomics, proteomics, and signal colocalization. Hence, CTCs hold the key to understanding the biology of metastasis and play a vital role in cancer diagnosis, treatment monitoring, and prognosis. Size-based enrichment features are prominent in CTC isolation. It is a label-free, simple and fast method. Enriched CTCs remain unmodified and viable for a wide range of subsequent analyses. In this review, we comprehensively summarize the differences of size and deformability between CTCs and blood cells, which would facilitate the development of technologies of size-based CTC isolation. Then we review representative size-/deformability-based technologies available for CTC isolation and highlight the recent achievements in molecular analysis of isolated CTCs. To wrap up, we discuss the substantial challenges facing the field, and elaborate on prospects. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Integrated Molecular Characterization of Testicular Germ Cell Tumors.

    PubMed

    Shen, Hui; Shih, Juliann; Hollern, Daniel P; Wang, Linghua; Bowlby, Reanne; Tickoo, Satish K; Thorsson, Vésteinn; Mungall, Andrew J; Newton, Yulia; Hegde, Apurva M; Armenia, Joshua; Sánchez-Vega, Francisco; Pluta, John; Pyle, Louise C; Mehra, Rohit; Reuter, Victor E; Godoy, Guilherme; Jones, Jeffrey; Shelley, Carl S; Feldman, Darren R; Vidal, Daniel O; Lessel, Davor; Kulis, Tomislav; Cárcano, Flavio M; Leraas, Kristen M; Lichtenberg, Tara M; Brooks, Denise; Cherniack, Andrew D; Cho, Juok; Heiman, David I; Kasaian, Katayoon; Liu, Minwei; Noble, Michael S; Xi, Liu; Zhang, Hailei; Zhou, Wanding; ZenKlusen, Jean C; Hutter, Carolyn M; Felau, Ina; Zhang, Jiashan; Schultz, Nikolaus; Getz, Gad; Meyerson, Matthew; Stuart, Joshua M; Akbani, Rehan; Wheeler, David A; Laird, Peter W; Nathanson, Katherine L; Cortessis, Victoria K; Hoadley, Katherine A

    2018-06-12

    We studied 137 primary testicular germ cell tumors (TGCTs) using high-dimensional assays of genomic, epigenomic, transcriptomic, and proteomic features. These tumors exhibited high aneuploidy and a paucity of somatic mutations. Somatic mutation of only three genes achieved significance-KIT, KRAS, and NRAS-exclusively in samples with seminoma components. Integrated analyses identified distinct molecular patterns that characterized the major recognized histologic subtypes of TGCT: seminoma, embryonal carcinoma, yolk sac tumor, and teratoma. Striking differences in global DNA methylation and microRNA expression between histology subtypes highlight a likely role of epigenomic processes in determining histologic fates in TGCTs. We also identified a subset of pure seminomas defined by KIT mutations, increased immune infiltration, globally demethylated DNA, and decreased KRAS copy number. We report potential biomarkers for risk stratification, such as miRNA specifically expressed in teratoma, and others with molecular diagnostic potential, such as CpH (CpA/CpC/CpT) methylation identifying embryonal carcinomas. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Absolute Income, Relative Income, and Happiness

    ERIC Educational Resources Information Center

    Ball, Richard; Chernova, Kateryna

    2008-01-01

    This paper uses data from the World Values Survey to investigate how an individual's self-reported happiness is related to (i) the level of her income in absolute terms, and (ii) the level of her income relative to other people in her country. The main findings are that (i) both absolute and relative income are positively and significantly…

  19. Prognostic importance of DNA ploidy in non-endometrioid, high-risk endometrial carcinomas.

    PubMed

    Sorbe, Bengt

    2016-03-01

    The present study investigated the predictive and prognostic impact of DNA ploidy together with other well-known prognostic factors in a series of non-endometrioid, high-risk endometrial carcinomas. From a complete consecutive series of 4,543 endometrial carcinomas of International Federation of Gynecology and Obstetrics (FIGO) stages I-IV, 94 serous carcinomas, 48 clear cell carcinomas and 231 carcinosarcomas were selected as a non-endometrioid, high-risk group for further studies regarding prognosis. The impact of DNA ploidy, as assessed by flow cytometry, was of particular focus. The age of the patients, FIGO stage, depth of myometrial infiltration and tumor expression of p53 were also included in the analyses (univariate and multivariate). In the complete series of cases, the recurrence rate was 37%, and the 5-year overall survival rate was 39% with no difference between the three histological subtypes. The primary cure rate (78%) was also similar for all tumor types studied. DNA ploidy was a significant predictive factor (on univariate analysis) for primary tumor cure rate, and a prognostic factor for survival rate (on univariate and multivariate analyses). The predictive and prognostic impact of DNA ploidy was higher in carcinosarcomas than in serous and clear cell carcinomas. In the majority of multivariate analyses, FIGO stage and depth of myometrial infiltration were the most important predictive (tumor recurrence) and prognostic (survival rate) factors. DNA ploidy status is a less important predictive and prognostic factor in non-endometrioid, high-risk endometrial carcinomas than in the common endometrioid carcinomas, in which FIGO and nuclear grade also are highly significant and important factors.

  20. Epigenome-Wide Tumor DNA Methylation Profiling Identifies Novel Prognostic Biomarkers of Metastatic-Lethal Progression in Men Diagnosed with Clinically Localized Prostate Cancer.

    PubMed

    Zhao, Shanshan; Geybels, Milan S; Leonardson, Amy; Rubicz, Rohina; Kolb, Suzanne; Yan, Qingxiang; Klotzle, Brandy; Bibikova, Marina; Hurtado-Coll, Antonio; Troyer, Dean; Lance, Raymond; Lin, Daniel W; Wright, Jonathan L; Ostrander, Elaine A; Fan, Jian-Bing; Feng, Ziding; Stanford, Janet L

    2017-01-01

    Aside from Gleason sum, few factors accurately identify the subset of prostate cancer patients at high risk for metastatic progression. We hypothesized that epigenetic alterations could distinguish prostate tumors with life-threatening potential. Epigenome-wide DNA methylation profiling was performed in surgically resected primary tumor tissues from a population-based (n = 430) and a replication (n = 80) cohort of prostate cancer patients followed prospectively for at least 5 years. Metastasis was confirmed by positive bone scan, MRI, CT, or biopsy, and death certificates confirmed cause of death. AUC, partial AUC (pAUC, 95% specificity), and P value criteria were used to select differentially methylated CpG sites that robustly stratify patients with metastatic-lethal from nonrecurrent tumors, and which were complementary to Gleason sum. Forty-two CpG biomarkers stratified patients with metastatic-lethal versus nonrecurrent prostate cancer in the discovery cohort, and eight of these CpGs replicated in the validation cohort based on a significant (P < 0.05) AUC (range, 0.66-0.75) or pAUC (range, 0.007-0.009). The biomarkers that improved discrimination of patients with metastatic-lethal prostate cancer include CpGs in five genes (ALKBH5, ATP11A, FHAD1, KLHL8, and PI15) and three intergenic regions. In the validation dataset, the AUC for Gleason sum alone (0.82) significantly increased with the addition of four individual CpGs (range, 0.86-0.89; all P <0.05). Eight differentially methylated CpGs that distinguish patients with metastatic-lethal from nonrecurrent tumors were validated. These novel epigenetic biomarkers warrant further investigation as they may improve prognostic classification of patients with clinically localized prostate cancer and provide new insights on tumor aggressiveness. Clin Cancer Res; 23(1); 311-9. ©2016 AACR. ©2016 American Association for Cancer Research.