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Sample records for kras mutation detection

  1. Detection of EGFR and KRAS Mutation by Pyrosequencing Analysis in Cytologic Samples of Non-Small Cell Lung Cancer.

    PubMed

    Lee, Seung Eun; Lee, So-Young; Park, Hyung-Kyu; Oh, Seo-Young; Kim, Hee-Joung; Lee, Kye-Young; Kim, Wan-Seop

    2016-08-01

    EGFR and KRAS mutations are two of the most common mutations that are present in lung cancer. Screening and detecting these mutations are of issue these days, and many different methods and tissue samples are currently used to effectively detect these two mutations. In this study, we aimed to evaluate the testing for EGFR and KRAS mutations by pyrosequencing method, and compared the yield of cytology versus histology specimens in a consecutive series of patients with lung cancer. We retrospectively reviewed EGFR and KRAS mutation results of 399 (patients with EGFR mutation test) and 323 patients (patients with KRAS mutation test) diagnosed with lung cancer in Konkuk University Medical Center from 2008 to 2014. Among them, 60 patients had received both EGFR and KRAS mutation studies. We compared the detection rate of EGFR and KRAS tests in cytology, biopsy, and resection specimens. EGFR and KRAS mutations were detected in 29.8% and 8.7% of total patients, and the positive mutation results of EGFR and KRAS were mutually exclusive. The detection rate of EGFR mutation in cytology was higher than non-cytology (biopsy or resection) materials (cytology: 48.5%, non-cytology: 26.1%), and the detection rate of KRAS mutation in cytology specimens was comparable to non-cytology specimens (cytology: 8.3%, non-cytology: 8.7%). We suggest that cytology specimens are good alternatives that can readily substitute tissue samples for testing both EGFR and KRAS mutations. Moreover, pyrosequencing method is highly sensitive in detecting EGFR and KRAS mutations in lung cancer patients.

  2. A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients

    PubMed Central

    MATSUNAGA, MOTOTSUGU; KANETA, TOSHIKADO; MIWA, KEISUKE; ICHIKAWA, WATARU; FUJITA, KEN-ICHI; NAGASHIMA, FUMIO; FURUSE, JUNJI; KAGE, MASAYOSHI; AKAGI, YOSHITO; SASAKI, YASUTSUNA

    2016-01-01

    There is currently no standard method for the detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status in colorectal tumors. In the present study, we compared the KRAS mutation detection ability of four methods: direct sequencing, Scorpion-ARMS assaying, pyrosequencing and multi-analyte profiling (Luminex xMAP). We evaluated 73 cases of metastatic colorectal cancer (mCRC) resistant to irinotecan, oxaliplatin and fluoropyrimidine that were enrolled in an all-case study of cetuximab. The KRAS mutation detection capacity of the four analytical methods was compared using DNA samples extracted from tumor tissue, and the detection success rate and concordance of the detection results were evaluated. KRAS mutations were detected by direct sequencing, Scorpion-ARMS assays, pyrosequencing and Luminex xMAP at success rates of 93.2%, 97.3%, 95.9% and 94.5%, respectively. The concordance rates of the detection results by Scorpion-ARMS, pyrosequencing and Luminex xMAP with those of direct sequencing were 0.897, 0.923 and 0.900 (κ statistics), respectively. The direct sequencing method could not determine KRAS mutation status in five DNA samples. Of these, Scorpion-ARMS, pyrosequencing and Luminex xMAP successfully detected three, two and one KRAS mutation statuses, respectively. Three cases demonstrated inconsistent results, whereby Luminex xMAP detected mutated KRAS in two samples while wild-type KRAS was detected by the other methods. In the remaining case, direct sequencing detected wild-type KRAS, which was identified as mutated KRAS by the other methods. In conclusion, we confirmed that Scorpion-ARMS, pyrosequencing and Luminex xMAP were equally reliable in detecting KRAS mutation status in mCRC. However, in rare cases, the KRAS status was differentially diagnosed using these methods. PMID:27347117

  3. A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients.

    PubMed

    Matsunaga, Mototsugu; Kaneta, Toshikado; Miwa, Keisuke; Ichikawa, Wataru; Fujita, Ken-Ichi; Nagashima, Fumio; Furuse, Junji; Kage, Masayoshi; Akagi, Yoshito; Sasaki, Yasutsuna

    2016-07-01

    There is currently no standard method for the detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status in colorectal tumors. In the present study, we compared the KRAS mutation detection ability of four methods: direct sequencing, Scorpion-ARMS assaying, pyrosequencing and multi-analyte profiling (Luminex xMAP). We evaluated 73 cases of metastatic colorectal cancer (mCRC) resistant to irinotecan, oxaliplatin and fluoropyrimidine that were enrolled in an all-case study of cetuximab. The KRAS mutation detection capacity of the four analytical methods was compared using DNA samples extracted from tumor tissue, and the detection success rate and concordance of the detection results were evaluated. KRAS mutations were detected by direct sequencing, Scorpion-ARMS assays, pyrosequencing and Luminex xMAP at success rates of 93.2%, 97.3%, 95.9% and 94.5%, respectively. The concordance rates of the detection results by Scorpion-ARMS, pyrosequencing and Luminex xMAP with those of direct sequencing were 0.897, 0.923 and 0.900 (κ statistics), respectively. The direct sequencing method could not determine KRAS mutation status in five DNA samples. Of these, Scorpion-ARMS, pyrosequencing and Luminex xMAP successfully detected three, two and one KRAS mutation statuses, respectively. Three cases demonstrated inconsistent results, whereby Luminex xMAP detected mutated KRAS in two samples while wild-type KRAS was detected by the other methods. In the remaining case, direct sequencing detected wild-type KRAS, which was identified as mutated KRAS by the other methods. In conclusion, we confirmed that Scorpion-ARMS, pyrosequencing and Luminex xMAP were equally reliable in detecting KRAS mutation status in mCRC. However, in rare cases, the KRAS status was differentially diagnosed using these methods.

  4. Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR

    PubMed Central

    Orue, Andrea; Rieber, Manuel

    2016-01-01

    Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies. Conclusion. Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting. PMID:27632281

  5. Rapid KRAS Mutation Detection via Hybridization-Induced Aggregation of Microbeads.

    PubMed

    Sloane, Hillary S; Kelly, Kimberly A; Landers, James P

    2015-10-20

    Using hybridization-induced aggregation (HIA), a unique bead-based DNA detection technology scalable for a microchip platform, we describe a simplistic, low-cost method for rapid mutation testing. HIA utilizes a pair of sequence-specific oligonucleotide probes bound to magnetic microbeads. Hybridization to a target DNA strand tethers the beads together, inducing bead aggregation. By simply using the extent of bead aggregation as a measure of the hybridization efficiency, we avoid the need for additional labels and sophisticated analytical equipment. Through strategic manipulation of the assay design and experimental parameters, we use HIA to facilitate, for the first time, the detection of single base mutations in a gene segment and, specifically, the detection of activating KRAS mutations. Following the development and optimization of the assay, we apply it for KRAS mutation analysis of four human cancer cell lines. Ultimately, we present a proof-of-principle method for detecting any of the common KRAS mutations in a single-step, 2 min assay, using only one set of oligonucleotide probes, for a total analysis time of less than 10 min post-PCR. The assay is performed at room temperature and uses simple, inexpensive instrumentation that permits multiplexed analysis.

  6. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    PubMed Central

    Olmedillas López, Susana; García-Olmo, Dolores C.; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  7. Detection of KRAS mutations using double-stranded toehold-exchange probes.

    PubMed

    Wu, Zhenhua; Ma, Tianle; Coll, Jean-Luc; Liu, Fangming; Zhang, Honglian; Ma, Yunfei; Wang, Zhishuo; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2016-06-15

    Detection of KRAS mutations in cancer tissues is immensely valuable for the identification of personalized genotype-based therapy. Here, we employed a double-stranded toehold-exchange probe, which is labeled with fluorescent molecules (FAM) and quenchers (Dabcyl), to detect KRAS mutations in cancer tissues. This probe was able to differentiate the intended mutation in a sample containing as little as 5% mutant alleles in a background of wild-type DNA. This probe also performed robustly at a wide range of conditions, for examples, from 4 °C to 37 °C, from 200 mM Na(+) to 1M Na(+), and from 200 mM K(+) to 500 mM K(+). Furthermore, we validated the practicality of this probe in a clinical setting using 8 pairs of cancer tissue samples and their NT (corresponding adjacent nontumorous tissue) samples. All the results generated from the probe detection agreed with those from direct sequencing. Combining features of extreme high specificity and robustness, this probe is a valuable tool for reliable diagnosis of cancer-related mutations.

  8. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

    PubMed Central

    Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation. PMID:26999437

  9. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    PubMed

    Itonaga, Masahiro; Matsuzaki, Ibu; Warigaya, Kenji; Tamura, Takaaki; Shimizu, Yuki; Fujimoto, Masakazu; Kojima, Fumiyoshi; Ichinose, Masao; Murata, Shin-Ichi

    2016-01-01

    Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

  10. KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test

    PubMed Central

    De Luca, Caterina; Vigliar, Elena; d’Anna, Melania; Pisapia, Pasquale; Bellevicine, Claudio; Malapelle, Umberto; Troncone, Giancarlo

    2017-01-01

    Background: Molecular techniques are relevant to modern cytopathology, but their implementation is difficult without molecular expertise and infrastructure. The assessment of KRAS mutational status on cytological preparations may be useful either to refine uncertain diagnoses on pancreatic aspirates or to yield predictive information to plan targeted treatment of metastatic colorectal cancer (mCRC). The novel test Idylla™ enables fully automated KRAS genotyping in approximately 2 h, even in less experienced hands. Materials and Methods: This study aims to validate this methodology to detect KRAS mutations on archival cytological preparations of pancreatic cancer (n = 9) and mCRC (n = 9) by comparing the Idylla™ performance to that of standard real-time polymerase chain reaction. Results: The same 11 mutations (n = 4: p.G12D; n = 2: p.G12V; n = 2: p.A59E/G/T; n = 1: p.G12R; n = 1: p.G13D; n = 1: p.Q61H) were detected by both techniques. Conclusion: Even in less experienced laboratories, a cytopathologist may easily integrate morphological diagnostic report with accurate KRAS mutation detection, which is relevant for diagnostic and treatment decisions. PMID:28331530

  11. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    EPA Science Inventory

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

    Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
    <...

  12. K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

    PubMed

    Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

    2016-07-01

    K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

  13. Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations.

    PubMed

    Botezatu, Irina V; Nechaeva, Irina O; Stroganova, Аnna М; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2015-12-15

    The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5-3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity-all of the qualities required for clinical genotyping.

  14. Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

    PubMed Central

    Duan, Guang-Jie; Shi, Yan; Deng, Guo-Hong; Xia, Han; Xu, Han-Qing; Zhao, Na; Fu, Wei-Ling; Huang, Qing

    2015-01-01

    The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔCq method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene. PMID:26701781

  15. Effectiveness of circulating tumor DNA for detection of KRAS gene mutations in colorectal cancer patients: a meta-analysis

    PubMed Central

    Hao, Yi-Xin; Fu, Qiang; Guo, Yan-Yan; Ye, Ming; Zhao, Hui-Xia; Wang, Qi; Peng, Xiu-Mei; Li, Qiu-Wen; Wang, Ru-Liang; Xiao, Wen-Hua

    2017-01-01

    Circulating tumor DNA (ctDNA) can be identified in the peripheral blood of patients and harbors the genomic alterations found in tumor tissues, which provides a noninvasive approach for detection of gene mutations. We conducted this meta-analysis to investigate whether ctDNA can be used for monitoring KRAS gene mutations in colorectal cancer (CRC) patients. Medline, Embase, Cochrane Library and Web of Science were searched for the included eligible studies in English, and data were extracted for statistical analysis according to the numbers of true-positive (TP), true-negative (TN), false-positive (FP) and false-negative (FN) cases. Sensitivity, specificity and diagnostic odds ratio (DOR) were calculated, and the area under the receiver operating characteristic curve (AUROC) was used to evaluate the diagnostic performance. After independent searching and reviewing, 21 studies involving 1,812 cancer patients were analyzed. The overall sensitivity, specificity and DOR were 0.67 (95% confidence interval [CI] =0.55–0.78), 0.96 (95% CI =0.93–0.98) and 53.95 (95% CI =26.24–110.92), respectively. The AUROC was 0.95 (95% CI =0.92–0.96), which indicated the high diagnostic accuracy of ctDNA. After stratified analysis, we found the higher diagnostic accuracy in subgroup of patients detected in blood sample of plasma. The ctDNA may be an ideal source for detection of KRAS gene mutations in CRC patients with high specificity and diagnostic value. PMID:28243130

  16. Comparison of COBAS 4800 KRAS, TaqMan PCR and high resolution melting PCR assays for the detection of KRAS somatic mutations in formalin-fixed paraffin embedded colorectal carcinomas.

    PubMed

    Harlé, Alexandre; Busser, Benoit; Rouyer, Marie; Harter, Valentin; Genin, Pascal; Leroux, Agnès; Merlin, Jean-Louis

    2013-03-01

    Many studies documented the influence of KRAS mutation status on the response of patients with metastatic colorectal cancer (mCRC) to anti-EGFR monoclonal antibodies. The COBAS 4800 KRAS is an assay using real time PCR and TaqMelt technology, CE-IVD validated, for the detection of 19 KRAS somatic mutations in exons 2 and 3. We compared COBAS with previously validated PCR TaqMan and High Resolution Melting (HRM) assays on 156 formalin-fixed paraffin embedded (FFPE) specimens of colorectal carcinoma. DNA extraction procedures, using the Qiagen QiAMP kit and the Roche COBAS DNA kit, were also compared. Of the 156 samples, 132 were interpretable using COBAS and TaqMan and 92 using COBAS and HRM. No statistically significant difference was found between COBAS/TaqMan and COBAS/HRM (k = 0.937; p < 0.001 - four discordant cases were found, mostly concerning codon 61 mutations and k = 0.891; p < 0.001 - five discordant cases were found, three regarding codon 61 and two on codon 12/13, respectively). No difference was found between the two DNA extraction methods (t = 1.7185; dol = 39; α = 5 %). The three assays were found suitable to detect accurately KRAS mutations in colon FFPE specimens. COBAS and TaqMan were found to be more robust than HRM, as they yielded fewer non-interpretable results. DNA extraction kits were found to provide equivalent results. The present study shows that pre-screening using COBAS with further TaqMan mutation characterization constitutes an easy and reliable approach for routine diagnostic purposes.

  17. Coexistence of MSI with KRAS mutation is associated with worse prognosis in colorectal cancer

    PubMed Central

    Hu, Jing; Yan, Wen-Yue; Xie, Li; Cheng, Lei; Yang, Mi; Li, Li; Shi, Jiong; Liu, Bao-Rui; Qian, Xiao-Ping

    2016-01-01

    Abstract Kristen rat sarcoma viral oncogene homolog (KRAS) and microsatellite instability (MSI) are prognostic markers of colorectal cancer (CRC). However, the clinical value is still not fully understood, when giving the consideration to both the molecular makers. Five hundred fifty-one patients with CRC were retrospectively assessed by determining their clinicopathological features. KRAS mutations were detected by polymerase chain reaction. MSI, a defect in the mismatch repair (MMR) system, was detected by immunohistochemistry. The prognostic value of KRAS in combination with MSI was studied. Among 551 CRC patients, mutations in KRAS codon 12 and KRAS codon 13 were detected in 34.5% and 10.5% of patients, respectively. Four hundred one tumors were randomly selected to detect for MMR proteins expression. In this analysis, 30 (7.5%) tumors that had at least 1 MMR protein loss were defined as MMR protein-deficient (MMR-D), and the remaining tumors were classed as MMR protein-intact (MMR-I). According to KRAS mutation and MSI status, CRC was classified into 4 groups: Group 1, KRAS-mutated and MMR-I; Group 2, KRAS-mutated and MMR-D; Group 3, KRAS wild and MMR-I; and Group 4, KRAS wild and MMR-D. We found that patients in Group4 had the best prognosis. In conclusion, combination status of KRAS and MSI status may be used as a prognostic biomarker for CRC patient, if validated by larger studies. PMID:27977612

  18. Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips.

    PubMed

    Albrecht, Jennifer Coyne; Kotani, Akira; Lin, Jennifer S; Soper, Steven A; Barron, Annelise E

    2013-02-01

    We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated "sample-in/answer-out" devices with amplification, LDR, and detection all on one platform.

  19. Amplification-free In Situ KRAS Point Mutation Detection at 60 copies/mL in Urine in a Background of 1000-fold Wild Type

    PubMed Central

    KirimLi, Ceyhun E.; Shih, Wei-Heng; Shih, Wan Y.

    2016-01-01

    We have examined in situ detection of single-nucleotide KRAS mutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35 (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to the KRAS mutation. To enhance in situ mutant (MT) DNA detection specificity against the wild type (WT), the detection was carried out in a flow with a flow rate of 4 mL/min and at 63°C with the PEPS vertically situated at the center of the flow in which both the temperature and the flow impingement force discriminated the wild type. Under such conditions, PEPS was shown to specifically detect KRAS MT in situ with 60 copies/mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, the detection was followed with detection in a mixture of blue MT fluorescent reporter microspheres (FRMs) (MT FRMs) that bound to only the captured MT and orange WT FRMs that bound to only the captured WT. Microscopic examinations showed that the captured blue MT FRMs still outnumbered the orange WT FRMs by a factor of 4 to 1 even though WT was 1000-fold of MT in urine. Finally, multiplexed specific mutation detection was demonstrated using a 6-PEPS array each with a probe DNA targeting one of the 6 codon-12 KRAS mutations. PMID:26783561

  20. K-ras oncogene mutation in pterygium.

    PubMed

    Ozturk, B T; Yıldırım, M S; Zamani, A; Bozkurt, B

    2017-03-01

    PurposePterygium is claimed to be a benign proliferation triggered by prolonged exposure to ultraviolet radiation. The frequency of K-ras oncogene mutation, which is among the initial mutations in tumorigenesis, is evaluated in this study.Patients and methodsIn this prospective randomized clinical, trial pterygium tissues and normal conjunctiva tissue specimens are obtained from the superotemporal quadrant of patients who underwent primary pterygium excision with autograft transplantation. DNA extraction from tissues was performed using the QIAamp DNA FFPE tissue kit. A PCR reaction was performed to amplify sequences containing codons 12, 13, and 61 of the K-ras gene in DNA. These PCR products then underwent the 'pyrosequencing' procedure for mutations at these codons.ResultsPterygium and normal conjunctival tissue samples of 25 patients (10 females, 15 males) were evaluated in the study. The mean age of the patients was 54.54±13.13 years. Genetic analysis revealed no K-ras mutations in normal conjunctival tissues, whereas pterygium tissues of the same cases demonstrated mutation at codon 12 in one case and mutations at codon 61 in seven cases, which was statistically significant (P<0.05). The point missense mutations at codon 61 were glutamine to arginine (Glu61Arg CAA>CGA) in four cases and glutamine to leucine (Glu61Leu CAA>CTA) in three cases.ConclusionThe significantly higher frequency of codon 61 mutation of the ras oncogene in primary and bilateral pterygium specimens compared with normal conjunctiva supports the tumoral origin of pterygium, and thus set the stage for research into a targeted therapy for pterygium with better outcomes than surgical excision.

  1. A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

    PubMed Central

    Han, Kyudong; Lee, Tae Yoon; Nikitopoulos, Dimitris E.; Soper, Steven A.; Murphy, Michael C.

    2011-01-01

    Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well-trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user-base for these types of molecular assays, a vertically-stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employed a primary PCR coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers and ExoSAP-IT® purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top instrumentation. Purifying the post-PCR products with the ExoSAP-IT® enzyme led to optimized LDR performance minimizing false positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25 μL sample, equivalent to DNA from 42 mutant cells. PMID:21771577

  2. The S492R EGFR ectodomain mutation is never detected in KRAS wild-type colorectal carcinoma before exposure to EGFR monoclonal antibodies.

    PubMed

    Esposito, Claudia; Rachiglio, Anna Maria; La Porta, Maria Libera; Sacco, Alessandra; Roma, Cristin; Iannaccone, Alessia; Tatangelo, Fabiana; Forgione, Laura; Pasquale, Raffaella; Barbaro, Americo; Botti, Gerardo; Ciardiello, Fortunato; Normanno, Nicola

    2013-12-01

    The activity of the epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab in metastatic colorectal carcinoma (mCRC) is significantly limited by molecular mechanisms leading to intrinsic or acquired resistance. The S492R mutation of the EGFR, which is caused by either the 1476C>A or the 1474A>C substitution, interferes with binding to cetuximab but not to panitumumab, and has been detected in mCRC with acquired resistance to cetuximab. Since mechanisms of acquired and intrinsic resistance to EGFR monoclonal antibodies in CRC significantly overlap, we evaluated the frequency of the S492R mutation in a series of KRAS-exon 2 wild-type CRC patients. Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues that were obtained from 505 systemic therapy-naïve CRC patients. A PCR/sequencing method for the detection of the S492R mutation was developed, by using as positive control a plasmid in which the 1474A>C mutation was generated by site directed mutagenesis. The lowest level of detection of this assay was approximately 10% mutant DNA in a background of wild-type DNA. PCR sequencing analysis revealed no S492R mutations in any of the analyzed 505 CRC specimens. Our findings suggest that the S492R mutation is not involved in primary resistance to cetuximab in CRC. Therefore, patients with mCRC should not be routinely screened for this mutation prior therapy with cetuximab.

  3. Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients

    PubMed Central

    Bacchi, Carlos E.; Ciol, Heloísa; Queiroga, Eduardo M.; Benine, Lucimara C.; Silva, Luciana H.; Ojopi, Elida B.

    2012-01-01

    OBJECTIVE: Epidermal growth factor receptor is involved in the pathogenesis of non-small cell lung cancer and has recently emerged as an important target for molecular therapeutics. The KRAS oncogene also plays an important role in the development of lung cancer. The aim of this study was to evaluate the frequency of epidermal growth factor receptor and KRAS mutations in a population of Brazilian patients with non-small cell lung cancer. METHODS: A total of 207 specimens from Brazilian patients with non-small cell lung cancer were analyzed for activating epidermal growth factor receptor and KRAS somatic mutations, and their associations with clinicopathological characteristics (including age, gender, ethnicity, smoking habits, and histological subtype) were examined. RESULTS: We identified 63 cases (30.4%) with epidermal growth factor receptor mutations and 30 cases (14.6%) with KRAS mutations. The most frequent epidermal growth factor receptor mutation we detected was a deletion in exon 19 (60.3%, 38 patients), followed by an L858R amino acid substitution in exon 21 (27%, 17 patients). The most common types of KRAS mutations were found in codon 12. There were no significant differences in epidermal growth factor receptor or KRAS mutations by gender or primary versus metastatic lung cancer. There was a higher prevalence of KRAS mutations in the non-Asian patients. Epidermal growth factor receptor mutations were more prevalent in adenocarcinomas than in non-adenocarcinoma histological types. Being a non-smoker was significantly associated with the prevalence of epidermal growth factor receptor mutations, but the prevalence of KRAS mutations was significantly associated with smoking. CONCLUSIONS: This study is the first to examine the prevalence of epidermal growth factor receptor and KRAS mutations in a Brazilian population sample with non-small cell lung cancer. PMID:22666783

  4. Prognostic role of KRAS mutations in Sardinian patients with colorectal carcinoma

    PubMed Central

    Palomba, Grazia; Cossu, Antonio; Paliogiannis, Panagiotis; Pazzola, Antonio; Baldino, Giovanni; Scartozzi, Mario; Ionta, Maria Teresa; Ortu, Salvatore; Capelli, Francesca; Lanzillo, Annamaria; Sedda, Tito; Sanna, Giovanni; Barca, Michela; Virdis, Luciano; Budroni, Mario; Palmieri, Giuseppe

    2016-01-01

    The presence of mutations in the KRAS gene is a predictor of a poor clinical response to EGFR-targeted agents in patients affected by colorectal cancer (CRC), but its significance as a global prognostic factor remains unclear. The aim of the present study was to evaluate the impact of the KRAS mutational status on time to first metastasis (TTM) and overall survival (OS) in a cohort of Sardinian CRC patients. A total of 551 patients with metastatic CRC at the time of enrolment were included. Clinical and pathological features of the disease, including follow-up information, were obtained from medical records and cancer registry data. For mutational analysis formalin-fixed paraffin-embedded tissue samples were processed using a standard protocol. The coding sequence and splice junctions of exons 2 and 3 of the KRAS gene were screened for mutations by direct automated sequencing. Overall, 186 KRAS mutations were detected in 183/551 (33%) patients: 125 (67%) were located in codon 12, 36 (19%) in codon 13, and 18 (10%) in codon 61. The remaining mutations (7; 4%) were detected in uncommonly-affected codons. No significant correlation between KRAS mutations and gender, age, anatomical location and stage of the disease at the time of diagnosis was identified. Furthermore, no prognostic value of KRAS mutations was found considering either TTM or OS. When patients were stratified by KRAS mutational status and gender, males were significantly associated with a longer TTM. The results of the present study indicate that KRAS mutation correlated with a slower metastatic progression in males with CRC from Sardinia, irrespective of the age at diagnosis and the codon of the mutation. PMID:27446446

  5. KRAS Mutation as a Potential Prognostic Biomarker of Biliary Tract Cancers

    PubMed Central

    Yokoyama, Masaaki; Ohnishi, Hiroaki; Ohtsuka, Kouki; Matsushima, Satsuki; Ohkura, Yasuo; Furuse, Junji; Watanabe, Takashi; Mori, Toshiyuki; Sugiyama, Masanori

    2016-01-01

    BACKGROUND The aim of this study was to identify the unique molecular characteristics of biliary tract cancer (BTC) for the development of novel molecular-targeted therapies. MATERIALS AND METHODS We performed mutational analysis of KRAS, BRAF, PIK3CA, and FBXW7 and immunohistochemical analysis of EGFR and TP53 in 63 Japanese patients with BTC and retrospectively evaluated the association between the molecular characteristics and clinicopathological features of BTC. RESULTS KRAS mutations were identified in 9 (14%) of the 63 BTC patients; no mutations were detected within the analyzed regions of BRAF, PIK3CA, and FBXW7. EGFR overexpression was observed in 5 (8%) of the 63 tumors, while TP53 overexpression was observed in 48% (30/63) of the patients. Overall survival of patients with KRAS mutation was significantly shorter than that of patients with the wild-type KRAS gene (P = 0.005). By multivariate analysis incorporating molecular and clinicopathological features, KRAS mutations and lymph node metastasis were identified to be independently associated with shorter overall survival (KRAS, P = 0.004; lymph node metastasis, P = 0.015). CONCLUSIONS Our data suggest that KRAS mutation is a poor prognosis predictive biomarker for the survival in BTC patients. PMID:28008299

  6. KRAS and PIK3CA mutations in colorectal adenocarcinomas correlate with aggressive histological features and behavior.

    PubMed

    Jang, Sejin; Hong, Mineui; Shin, Mi Kyung; Kim, Byung Chun; Shin, Hyung-Sik; Yu, Eunsil; Hong, Seung-Mo; Kim, Jihun; Chun, Sung Min; Kim, Tae-Im; Choi, Kyung-Chan; Ko, Young Woong; Kim, Jeong Won

    2017-02-08

    Tumor budding (TB) in colorectal carcinoma (CRC) is related to epithelial-mesenchymal transition (EMT) and has been recently characterized as an indicator of poor prognosis along with lymphovascular tumor emboli (LVE), perineural invasion (PNI), and an infiltrative growth pattern. Mutations in the genes of the Ras-MAPK and PI3K pathways are associated with EMT and an aggressive CRC phenotype and have been used in patient stratification for anti-EGF receptor therapies; however, the impact of these mutations on CRC morphology and behavior remains unclear. In this study, using a multi-gene panel, we detected KRAS, NRAS, BRAF, PIK3CA, TP53, and POLE mutations in 90 CRCs and investigated their associations with clinicopathological parameters, including TB. Our results showed that 21 of 34 tumors with high-grade TB had KRAS mutations (P=.001) and KRAS G12D and PIK3CA exon 9 variants were significantly associated with high-grade TB (P=.002 and .006, respectively); furthermore, tumors with KRAS mutations in exons 3 and 4 tended to have LVE and PNI (P=.044 and .049, respectively). PIK3CA exon 9 mutations indicated a tendency for shorter disease-free survival (P=.030), whereas BRAF mutations were associated with extracellular mucin deposition (P=.016). Our study revealed a correlation of KRAS mutations with high-grade TB, an association of certain KRAS and PIK3CA variants with aggressive clinicopathological features, as well as a possible relationship between BRAF mutations and mucin production in CRC.

  7. [Method validation according to ISO 15189 and SH GTA 04: application for the detection of KRAS mutations using PCR TaqMan assay].

    PubMed

    Harlé, Alexandre; Dubois, Cindy; Rouyer, Marie; Merlin, Jean-Louis

    2013-01-01

    Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. Thus, all medical laboratories in France must be accredited for at least part of their biological tests before the end of October 2013. Molecular biology tests are also concerned by the accreditation. Validation of molecular biology methods is made difficult, for reasons related to the methods, but also by the type of analytes that are basically rare. This article describes the validation of the qualitative detection of KRAS mutations in metastatic colorectal cancer using TaqMan PCR according to ISO 15189 and to the technical guide for accreditation in Human Health, SH-GTA-04, edited by the COFRAC.

  8. The value of KRAS mutation testing with CEA for the diagnosis of pancreatic mucinous cysts

    PubMed Central

    Kadayifci, Abdurrahman; Al-Haddad, Mohammad; Atar, Mustafa; Dewitt, John M.; Forcione, David G.; Sherman, Stuart; Casey, Brenna W.; Fernandez-del Castillo, Carlos; Schmidt, C. Max; Pitman, Martha B.; Brugge, William R.

    2016-01-01

    Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P < 0.0001 for both). KRAS mutation was significantly more frequent in malignant CMNs compared to histologically confirmed non-malignant CMNs (73 % vs. 37 %, P = 0.001). The negative predictive value of KRAS mutation was 77.6 % in differentiating non-malignant cysts. Conclusions: The detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. It outperforms CEA for sensitivity in mucinous cyst diagnosis, but the data does not support its routine use. PMID:27092317

  9. Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

    PubMed Central

    Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno; Haas, Christian; Bellosillo, Beatriz; Montagut, Clara; Smith, Matthew; O’Sullivan, Brendan; D’Haene, Nicky; Le Mercier, Marie; Grauslund, Morten; Melchior, Linea Cecilie; Burt, Emma; Cotter, Finbarr; Stieber, Daniel; Schmitt, Fernando de Lander; Motta, Valentina; Lauricella, Calogero; Colling, Richard; Soilleux, Elizabeth; Fassan, Matteo; Mescoli, Claudia; Collin, Christine; Pagès, Jean-Christophe; Sillekens, Peter

    2016-01-01

    Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients. PMID:27685259

  10. Mutations of KRAS/NRAS/BRAF predict cetuximab resistance in metastatic colorectal cancer patients

    PubMed Central

    Hsu, Hung-Chih; Thiam, Tan Kien; Lu, Yen-Jung; Yeh, Chien Yuh; Tsai, Wen-Sy; You, Jeng Fu; Hung, Hsin Yuan; Tsai, Chi-Neu; Hsu, An; Chen, Hua-Chien; Chen, Shu-Jen; Yang, Tsai-Sheng

    2016-01-01

    Approximately 45% of metastatic colorectal cancer (mCRC) patients with wild-type KRAS exon 2 are resistant to cetuximab treatment. We set out to identify additional genetic markers that might predict the response to cetuximab treatment. Fifty-three wild-type KRAS exon 2 mCRC patients were treated with cetuximab/irinotecan-based chemotherapy as a first- or third-line therapy. The mutational statuses of 10 EGFR pathway genes were analyzed in primary tumors using next-generation sequencing. BRAF, PIK3CA, KRAS (exons 3 and 4), NRAS, PTEN, and AKT1 mutations were detected in 6, 6, 5, 4, 1, and 1 patient, respectively. Four of the BRAF mutations were non-V600 variants. Four tumors harbored multiple co-existing (complex) mutations. All patients with BRAF mutations or complex mutation patterns were cetuximab non-responders. All patients but one harboring KRAS, NRAS, or BRAF mutations were non-responders. Mutations in any one of these three genes were associated with a poor response rate (7.1%) and reduced survival (PFS = 8.0 months) compared to wild-type patients (74.4% and 11.6 months). Our data suggest that KRAS, NRAS, and BRAF mutations predict response to cetuximab treatment in mCRC patients. PMID:26989027

  11. Multiple KRAS mutations in pancreatic adenocarcinoma: molecular features of neoplastic clones indicate the selection of divergent populations of tumor cells.

    PubMed

    Visani, Michela; de Biase, Dario; Baccarini, Paola; Fabbri, Carlo; Polifemo, Anna Maria; Zanini, Nicola; Pession, Annalisa; Tallini, Giovanni

    2013-12-01

    KRAS is one of the most common genes mutated in pancreatic adenocarcinoma. Multiple KRAS mutations may be detected within the same pancreatic adenocarcinoma, but it is usually unclear whether the different mutations represent biologically irrelevant molecular events or whether they indicate the coexistence of distinct sizable neoplastic clones within a given tumor. We identified a case of pancreatic adenocarcinoma with 5 different mutations in the KRAS gene and have been able to characterize the allelic distribution of the KRAS mutations and the size of the neoplastic clones using allele-specific locked nucleic acid polymerase chain reaction and next-generation sequencing (454 GS-Junior). The results indicate that the tumor is composed of 5 distinct cell populations: one is KRAS G12V mutated (~38% of neoplastic cells), the second is KRAS G12V in one allele and KRAS G12D in the other (~32%), the third is KRAS G12V in one allele and KRAS G12R in the other (~24%), and the fourth is KRAS G12V in one allele and KRAS G12C in the other (~6%). The fifth clone, representing a minority of neoplastic cells, has a KRAS Q61H mutation in addition to one of the above alterations. Microsatellite analysis identified mutation of the NR21 marker out of the 13 tested, indicating that the tumor has a defect in maintaining DNA integrity different from loss of conventional DNA mismatch repair. These results are consistent with the successive selection of divergent populations of tumor cells and underscore the relevance of nucleotide instability in pancreatic adenocarcinoma.

  12. Diagnostic application of KRAS mutation testing in uterine microglandular proliferations.

    PubMed

    Hong, Wei; Abi-Raad, Rita; Alomari, Ahmed K; Hui, Pei; Buza, Natalia

    2015-07-01

    Microglandular proliferations often pose a diagnostic challenge in small endocervical and endometrial biopsies. Microglandular hyperplasia (MGH) is one of the most common pseudoneoplastic glandular proliferations of uterine cervix, which can closely mimic endometrial adenocarcinomas (EAC) with a microglandular pattern (microglandular EAC). Although MGH is typically characterized by relatively uniform nuclei and rare to absent mitoses, atypical forms with architectural and/or cytologic deviation from the usual morphology have been previously described. Recently, a series of MGH with high mitotic activity has also been documented. Although careful morphological assessment and immunohistochemical workup can resolve the diagnostic dilemma in some cases, additional differential diagnostic tools are needed to separate both the common and atypical variants of MGH from EAC with microglandular pattern. Frequent KRAS mutation has been previously reported in endometrial complex mucinous lesions and endometrial mucinous carcinomas. However, the diagnostic utility of KRAS mutation analysis has not yet been explored in the context of cervical and endometrial microglandular lesions. Twelve mitotically active MGH cases and 15 cases of EAC with microglandular growth pattern were selected for the study. KRAS mutation analysis was performed in all cases by highly sensitive single-strand conformation polymorphism analysis. Clinical history and follow-up data were retrieved from electronic medical records. KRAS mutation was absent in all MGH cases, whereas 9 (60%) of 15 microglandular EAC cases tested positive for KRAS mutation. Our data indicate that KRAS mutation analysis may offer additional discriminatory power in separating benign MGH from EAC with microglandular pattern.

  13. KRAS mutations in blood circulating cell-free DNA: a pancreatic cancer case-control

    PubMed Central

    Le Calvez-Kelm, Florence; Foll, Matthieu; Wozniak, Magdalena B.; Delhomme, Tiffany M.; Durand, Geoffroy; Chopard, Priscilia; Pertesi, Maroulio; Fabianova, Eleonora; Adamcakova, Zora; Holcatova, Ivana; Foretova, Lenka; Janout, Vladimir; Vallee, Maxime P.; Rinaldi, Sabina; Brennan, Paul; McKay, James D.; Byrnes, Graham B.; Scelo, Ghislaine

    2016-01-01

    The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series. PMID:27705932

  14. Analysis of KRAS and BRAF genes mutation in the central nervous system metastases of non-small cell lung cancer.

    PubMed

    Nicoś, Marcin; Krawczyk, Paweł; Jarosz, Bożena; Sawicki, Marek; Szumiłło, Justyna; Trojanowski, Tomasz; Milanowski, Janusz

    2016-05-01

    KRAS mutations are associated with tumor resistance to EGFR TKIs (erlotinib, gefitinib) and to monoclonal antibody against EGFR (cetuximab). Targeted treatment of mutated RAS patients is still considered as a challenge. Inhibitors of c-Met (onartuzumab or tiwantinib) and MEK (selumetinib-a dual inhibitor of MEK1 and MEK2) signaling pathways showed activity in patients with mutations in KRAS that can became an effective approach in carriers of such disorders. BRAF mutation is very rare in patients with NSCLC, and its presence is associated with sensitivity of tumor cells to BRAF inhibitors (vemurafenib, dabrafenib). In the present study, the frequency and type of KRAS and BRAF mutation were assessed in 145 FFPE tissue samples from CNS metastases of NSCLC. In 30 patients, material from the primary tumor was simultaneously available. Real-time PCR technique with allele-specific molecular probe (KRAS/BRAF Mutation Analysis Kit, Entrogen, USA) was used for molecular tests. KRAS mutations were detected in 21.4 % of CNS metastatic lesions and in 23.3 % of corresponding primary tumors. Five mutations were identified both in primary and in metastatic lesions, while one mutation only in primary tumor and one mutation only in the metastatic tumor. Most of mutations were observed in codon 12 of KRAS; however, an individual patient had diagnosed a rare G13D and Q61R substitutions. KRAS mutations were significantly more frequent in adenocarcinoma patients and smokers. Additional analysis indicated one patient with rare coexistence of KRAS and DDR2 mutations. BRAF mutation was not detected in the examined materials. KRAS frequency appears to be similar in primary and CNS.

  15. Kras mutations increase telomerase activity and targeting telomerase is a promising therapeutic strategy for Kras-mutant NSCLC

    PubMed Central

    Shi, Bowen; Zhang, Lianmin; Qian, Dong; Li, Chenguang; Zhang, Hua; Wang, Shengguang; Zhu, Jinfang; Gao, Liuwei; Zhang, Qiang; Jia, Bin; Hao, Ligang; Wang, Changli; Zhang, Bin

    2017-01-01

    As shortened telomeres inhibit tumor formation and prolong life span in a KrasG12D mouse lung cancer model, we investigated the implications of telomerase in Kras-mutant NSCLC. We found that Kras mutations increased TERT (telomerase reverse transcriptase) mRNA expression and telomerase activity and telomere length in both immortalized bronchial epithelial cells (BEAS-2B) and lung adenocarcinoma cells (Calu-3). MEK inhibition led to reduced TERT expression and telomerase activity. Furthermore, telomerase inhibitor BIBR1532 shortened telomere length and inhibited mutant Kras-induced long-term proliferation, colony formation and migration capabilities of BEAS-2B and Calu-3 cells. Importantly, BIBR1532 sensitized oncogenic Kras expressing Calu-3 cells to chemotherapeutic agents. The Calu-3-KrasG12D xenograft mouse model confirmed that BIBR1532 enhanced the antitumor efficacy of paclitaxel in vivo. In addition, higher TERT expression was seen in Kras-mutant NSCLC than that with wild-type Kras. Our data suggest that Kras mutations increase telomerase activity and telomere length by activating the RAS/MEK pathway, which contributes to an aggressive phenotype of NSCLC. Kras mutations-induced lung tumorigenesis and chemoresistance are attenuated by telomerase inhibition. Targeting telomerase/telomere may be a promising therapeutic strategy for patients with Kras-mutant NSCLC. PMID:27329725

  16. Prevalence of K-Ras mutations in hepatocellular carcinoma: A Turkish Oncology Group pilot study

    PubMed Central

    TURHAL, NAZIM SERDAR; SAVAŞ, BERNA; ÇOŞKUN, ÖZNUR; BAŞ, EMINE; KARABULUT, BÜLENT; NART, DENIZ; KORKMAZ, TANER; YAVUZER, DILEK; DEMIR, GÖKHAN; DOĞUSOY, GÜLEN; ARTAÇ, MEHMET

    2015-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common male-predominant type of cancer worldwide. There is no effective treatment regimen available for advanced-stage disease and chemotherapy is generally ineffective in these patients. The number of studies on the prevalence of K-Ras mutations in HCC patients is currently limited. A total of 58 patients from 6 comprehensive cancer centers in 4 metropolitan cities of Turkey were enrolled in this study. Each center committed to enroll approximately 10 random patients whose formalin-fixed paraffin-embedded tumor tissues were available for K-Ras, exon 2 genotyping. Two methods were applied based on the availability of adequate amounts of tumor DNA. In the first method, the samples were processed using TheraScreen. The genomic DNA was further used to detect the 7 most frequent somatic mutations (35G>A; 35G>C; 35G>T; 34G>A; 34G>C; 34G>T and 38G>A) in codons 12 and 13 in exon 2 of the K-Ras oncogene by quantitative polymerase chain reaction (PCR). In the second method, the genomic DNA was amplified by PCR using primers specific for K-Ras exon 2 with the GML SeqFinder Sequencing System's KRAS kit. The identified DNA sequence alterations were confirmed by sequencing both DNA strands in two independent experiments with forward and reverse primers. A total of 40 samples had adequate tumor tissue for the mutation analysis. A total of 33 (82.5%) of the investigated samples harbored no mutations in exon 2. All the mutations were identified via a direct sequencing technique, whereas none were identified by TheraScreen. In conclusion, in our patients, HCC exhibited a remarkably low (<20%) K-Ras mutation rate. Patients harboring K-Ras wild-type tumors may be good candidates for treatment with epidermal growth factor inhibitors, such as cetuximab. PMID:26807232

  17. KRAS-mutation incidence and prognostic value are metastatic site-specific in lung adenocarcinoma: poor prognosis in patients with KRAS mutation and bone metastasis

    PubMed Central

    Lohinai, Zoltan; Klikovits, Thomas; Moldvay, Judit; Ostoros, Gyula; Raso, Erzsebet; Timar, Jozsef; Fabian, Katalin; Kovalszky, Ilona; Kenessey, István; Aigner, Clemens; Renyi-Vamos, Ferenc; Klepetko, Walter; Dome, Balazs; Hegedus, Balazs

    2017-01-01

    Current guidelines lack comprehensive information on the metastatic site-specific role of KRAS mutation in lung adenocarcinoma (LADC). We investigated the effect of KRAS mutation on overall survival (OS) in this setting. In our retrospective study, 500 consecutive Caucasian metastatic LADC patients with known KRAS mutational status were analyzed after excluding 32 patients with EGFR mutations. KRAS mutation incidence was 28.6%. The most frequent metastatic sites were lung (45.6%), bone (26.2%), adrenal gland (17.4%), brain (16.8%), pleura (15.6%) and liver (11%). Patients with intrapulmonary metastasis had significantly increased KRAS mutation frequency compared to those with extrapulmonary metastases (35% vs 26.5%, p = 0.0125). In contrast, pleural dissemination and liver involvement were associated with significantly decreased KRAS mutation incidence (vs all other metastatic sites; 17% (p < 0.001) and 16% (p = 0.02) vs 33%, respectively). Strikingly, we found a significant prognostic effect of KRAS status only in the bone metastatic subcohort (KRAS-wild-type vs KRAS-mutant; median OS 9.7 v 3.7 months; HR, 0.49; 95% CI, 0.31 to 0.79; p  = 0.003). Our study suggests that KRAS mutation frequency in LADC patients shows a metastatic site dependent variation and, moreover, that the presence of KRAS mutation is associated with significantly worse outcome in bone metastatic cases. PMID:28051122

  18. K-ras mutations, rectal crypt cells proliferation, and meat consumption in patients with left-sided colorectal carcinoma.

    PubMed

    O'Brien, H; Matthew, J A; Gee, J M; Watson, M; Rhodes, M; Speakman, C T; Stebbings, W S; Kennedy, H J; Johnson, I T

    2000-02-01

    It has been suggested that N-nitroso compounds derived from meat may increase the risk of K-ras mutations in the human colon. We sought evidence of associations between red meat consumption, frequency and type of K-ras mutations in resected tumours, and the rate of crypt cell proliferation (CCP) in the normal mucosa of patients with left-sided colorectal carcinoma. Meat consumption was assessed by food frequency questionnaire, and CCP was determined in rectal biopsies obtained prior to surgery. K-ras mutations in the resected tumours were determined using a PCR-based oligonucleotide hybridization assay. Fifteen K-ras mutations were detected in tumours from 43 patients; 13/15 in codon 12, 3/15 in codon 13, and 1/15 in both codons 12 and 13. All mutations were G-->A or G-->T transitions. There was no statistically significant difference between intakes of red meat in patients with a K-ras mutation (92.4 +/- 9.7 g/day) and those without (82.3 +/- 7.7 g/day). Rectal CCP was significantly higher in patients than in healthy controls, but there was no correlation with meat consumption or K-ras mutation. These data do not support the hypothesis that meat consumption is a risk factor for acquisition of K-ras mutations during colorectal carcinogenesis.

  19. KRAS insertion mutations are oncogenic and exhibit distinct functional properties

    PubMed Central

    White, Yasmine; Bagchi, Aditi; Van Ziffle, Jessica; Inguva, Anagha; Bollag, Gideon; Zhang, Chao; Carias, Heidi; Dickens, David; Loh, Mignon; Shannon, Kevin; Firestone, Ari J.

    2016-01-01

    Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-RasG60_A66dup and K-RasE62_A66dup proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications. PMID:26854029

  20. KRAS Mutations in Primary Colorectal Cancer Tumors and Related Metastases: A Potential Role in Prediction of Lung Metastasis

    PubMed Central

    Cejas, Paloma; López-Gómez, Miriam; Aguayo, Cristina; Madero, Rosario; de Castro Carpeño, Javier; Belda-Iniesta, Cristóbal; Barriuso, Jorge; Moreno García, Víctor; Larrauri, Javier; López, Rocío; Casado, Enrique; Gonzalez-Barón, Manuel; Feliu, Jaime

    2009-01-01

    Background KRAS mutations in colorectal cancer primary tumors predict resistance to anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody therapy in patients with metastatic colorectal cancer, and thus represent a true indicator of EGFR pathway activation status. Methodology/Principal Findings KRAS mutations were retrospectively studied using polymerase chain reactions and subsequent sequencing of codons 12 and 13 (exon 2) in 110 patients with metastatic colorectal tumors. These studies were performed using tissue samples from both the primary tumor and their related metastases (93 liver, 84%; 17 lung, 16%). All patients received adjuvant 5-Fluorouracil-based polychemotherapy after resection of metastases. None received anti-EGFR therapy. Mutations in KRAS were observed in 37 (34%) of primary tumors and in 40 (36%) of related metastases, yielding a 94% level of concordance (kappa index 0.86). Patients with primary tumors possessing KRAS mutations had a shorter disease-free survival period after metastasis resection (12.0 vs 18.0 months; P = 0.035) than those who did not. A higher percentage of KRAS mutations was detected in primary tumors of patiens with lung metastases than in patients with liver metastases (59% vs 32%; p = 0.054). To further evaluate this finding we analyzed 120 additional patients with unresectable metastatic colorectal cancer who previously had their primary tumors evaluated for KRAS mutational status for clinical purposes. Separately, the analysis of these 120 patients showed a tendency towards a higher degree of KRAS mutations in primary tumors of patients with lung metastases, although it did not reach statistical significance. Taken together the group of 230 patients showed that KRAS was mutated significantly more often in the primary tumors of patients with lung metastases (57% vs 35%; P = 0.006). Conclusions/Significance Our results suggest a role for KRAS mutations in the propensity of primary colorectal tumors to

  1. KRAS and TP53 mutations in bronchoscopy samples from former lung cancer patients.

    PubMed

    Gao, Weimin; Jin, Jide; Yin, Jinling; Land, Stephanie; Gaither-Davis, Autumn; Christie, Neil; Luketich, James D; Siegfried, Jill M; Keohavong, Phouthone

    2017-02-01

    Mutations in the KRAS and TP53 genes have been found frequently in lung tumors and specimens from individuals at high risk for lung cancer and have been suggested as predictive markers for lung cancer. In order to assess the prognostic value of these two genes' mutations in lung cancer recurrence, we analyzed mutations in codon 12 of the KRAS gene and in hotspot codons of the TP53 gene in 176 bronchial biopsies obtained from 77 former lung cancer patients. Forty-seven patients (61.0%) showed mutations, including 35/77 (45.5%) in the KRAS gene and 25/77 (32.5%) in the TP53 gene, among them 13/77 (16.9%) had mutations in both genes. When grouped according to past or current smoking status, a higher proportion of current smokers showed mutations, in particular those in the TP53 gene (P = 0.07), compared with ex-smokers. These mutations were found in both abnormal lesions (8/20 or 40%) and histologically normal tissues (70/156 or 44.9%) (P = 0.812). They consisted primarily of G to A transition and G to T transversion in both the KRAS (41/56 or 73.2%) and TP53 (24/34 or 70.6%) genes, consistent with mutations found in lung tumors of smoking lung cancer patients. Overall, recurrence-free survival (RFS) among all subjects could be explained by age at diagnosis, tumor stage, tumor subtype, and smoking (P < 0.05, Cox proportional hazard). Therefore, KRAS and TP53 mutations were frequently detected in bronchial tissues of former lung cancer patients. However, the presence of mutation of bronchial biopsies was not significantly associated with a shorter RFS time. © 2016 Wiley Periodicals, Inc.

  2. Picoliter droplet-based digital peptide nucleic acid clamp PCR and dielectric sorting for low abundant K-ras mutations

    NASA Astrophysics Data System (ADS)

    Zhang, Huidan; Sperling, Ralph; Rotem, Assaf; Shan, Lianfeng; Heyman, John; Zhang, Yizhe; Weitz, David

    2012-02-01

    Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the US, and the 5-year survival of metastatic CRC (mCRC) is less than 10%. Although monoclonal antibodies against epidermal growth factor receptor (EGFR) provide incremental improvements in survival, approximately 40% of mCRC patients with activating KRAS mutations won't benefit from this therapy. Peptide nucleic acid (PNA), a synthetic non-extendable oligonucleotides, can bind strongly to completely complementary wild-type KRAS by Watson-Crick base pairing and suppress its amplification during PCR, while any mutant allele will show unhindered amplification. The method is particularly suitable for the simultaneously detection of several adjoining mutant sites, just as mutations of codons 12 and 13 of KRAS gene where there are totally 12 possible mutation types. In this work, we describe the development and validation of this method, based on the droplet-based digital PCR. Using a microfluidic system, single target DNA molecule is compartmentalized in microdroplets together with PNA specific for wild-type KRAS, thermocycled and the fluorescence of each droplet was detected, followed by sorting and sequencing. It enables the precise determination of all possible mutant KRAS simultaneously, and the precise quantification of a single mutated KRAS in excess background unmutated KRAS.

  3. Cytomorphologic features of advanced lung adenocarcinomas tested for EGFR and KRAS mutations: a retrospective review of 50 cases.

    PubMed

    Marotti, Jonathan D; Schwab, Mary C; McNulty, Nancy J; Rigas, James R; DeLong, Peter A; Memoli, Vincent A; Tsongalis, Gregory J; Padmanabhan, Vijayalakshmi

    2013-01-01

    Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin-embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real-time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group.

  4. K-ras genetic mutation and influencing factor analysis for Han and Uygur nationality colorectal cancer patients

    PubMed Central

    Eli, Mayinur; Mollayup, Ablikim; Muattar; Liu, Chao; Zheng, Chao; Bao, Yong-Xing

    2015-01-01

    To investigate the K-ras genetic mutation status in colorectal cancer patients, compare the difference of K-ras genetic mutation rate in Han and Uygur nationality and analyze the influencing factor. 91 cases (52 cases of Han nationality and 39 cases of Uygur nationality) of colorectal biopsy or surgical ablation pathology specimen from the first affiliated hospital of Xinjiang Medical University during January, 2010 to March, 2013 were collected to detect the 12th and 13th code mutation status of K-ras gene exon 2 with pyrosequencing method and compare the difference of K-ras gene mutation rate between Han and Uygur nationality patients. Single factor analysis and multiple factor logistic regression analysis were utilized to analyze the influencing factor for K-ras genetic mutation. 33 cases of patients with K-ras genetic mutation were found from the 91 cases colorectal cancer patients and the total mutation rate was 36.3%. Among them, 24 cases (72.7%) were found with mutation only in the 12th code, 9 cases (27.3%) were found with mutation only in the 13th code and no one case was found with mutation in both the two codes. Mutation rate of the 12th code in the Uygur nationality was significantly higher than that in the Han nationality (P<0.05), but there were no significant difference in the comparison of the total mutation rate and the 13th code mutation rate between the two groups (P>0.05). There were no associativity (P>0.05) between the K-ras genetic mutation and sex, age, smoking history, drinking history, tumor location, macropathology type, differentiation level, staging, invasive depth, lymph nodes transferring and metastasis in colorectal cancer patients (P>0.05). K-ras genetic mutation rate is high in colorectal cancer patients. The mutation rate of 12th code in Uygur nationality is higher than that in Han nationality. There is no significant associativity between K-ras genetic mutation rate and patients’ clinical pathology characteristic. PMID:26309716

  5. K-ras genetic mutation and influencing factor analysis for Han and Uygur nationality colorectal cancer patients.

    PubMed

    Eli, Mayinur; Mollayup, Ablikim; Muattar; Liu, Chao; Zheng, Chao; Bao, Yong-Xing

    2015-01-01

    To investigate the K-ras genetic mutation status in colorectal cancer patients, compare the difference of K-ras genetic mutation rate in Han and Uygur nationality and analyze the influencing factor. 91 cases (52 cases of Han nationality and 39 cases of Uygur nationality) of colorectal biopsy or surgical ablation pathology specimen from the first affiliated hospital of Xinjiang Medical University during January, 2010 to March, 2013 were collected to detect the 12th and 13th code mutation status of K-ras gene exon 2 with pyrosequencing method and compare the difference of K-ras gene mutation rate between Han and Uygur nationality patients. Single factor analysis and multiple factor logistic regression analysis were utilized to analyze the influencing factor for K-ras genetic mutation. 33 cases of patients with K-ras genetic mutation were found from the 91 cases colorectal cancer patients and the total mutation rate was 36.3%. Among them, 24 cases (72.7%) were found with mutation only in the 12th code, 9 cases (27.3%) were found with mutation only in the 13th code and no one case was found with mutation in both the two codes. Mutation rate of the 12th code in the Uygur nationality was significantly higher than that in the Han nationality (P<0.05), but there were no significant difference in the comparison of the total mutation rate and the 13th code mutation rate between the two groups (P>0.05). There were no associativity (P>0.05) between the K-ras genetic mutation and sex, age, smoking history, drinking history, tumor location, macropathology type, differentiation level, staging, invasive depth, lymph nodes transferring and metastasis in colorectal cancer patients (P>0.05). K-ras genetic mutation rate is high in colorectal cancer patients. The mutation rate of 12th code in Uygur nationality is higher than that in Han nationality. There is no significant associativity between K-ras genetic mutation rate and patients' clinical pathology characteristic.

  6. Low somatic K-ras mutation frequency in colorectal cancer diagnosed under the age of 45 years.

    PubMed

    Alsop, Kathryn; Mead, Leeanne; Smith, Letitia D; Royce, Simon G; Tesoriero, Andrea A; Young, Joanne P; Haydon, Andrew; Grubb, Garry; Giles, Graham G; Jenkins, Mark A; Hopper, John L; Southey, Melissa C

    2006-07-01

    Somatic mutation of K-ras is known to be a common event in colorectal cancer tumourigenesis however its association with age at onset has not been widely explored. In this study, we have analyzed tumours from a population-based study of colorectal cancer diagnosed before the age of 45 years, in which cases had been previously screened for germ-line mismatch repair gene mutations and for microsatellite instability. We used a micro-dissection and sequencing approach to search for somatic K-ras mutations in codons 12, 13 and 61 in 101 early-onset colorectal cancers. Six (6%) somatic K-ras mutations were detected; five in codon 12 (4 G>T transitions and 1 G>A) and one in codon 13 (G>A transition). All codon 12 mutations were identified in microsatellite stable tumours and the codon 13 mutation was identified in a MSI-high tumour. Four cases with K-ras mutations had no reported family history of colorectal cancer and two had some family history of colorectal cancer. None were known to carry a germ-line mutation in hMSH2, hMLH1, hMSH6 or hPMS2. The role of somatic K-ras mutations in early-onset colorectal cancer carcinogenesis appears to be minor, in contrast to its significant role in colorectal cancer of later age of onset.

  7. Low frequency KRAS mutations in colorectal cancer patients and the presence of multiple mutations in oncogenic drivers in non-small cell lung cancer patients.

    PubMed

    Jiang, Liyan; Huang, Jiaqi; Morehouse, Chris; Zhu, Wei; Korolevich, Susana; Sui, Dan; Ge, Xiaoxiao; Lehmann, Kim; Liu, Zheng; Kiefer, Christine; Czapiga, Meggan; Su, Xinying; Brohawn, Philip; Gu, Yi; Higgs, Brandon W; Yao, Yihong

    2013-01-01

    Intratumor heterogeneity can confound the results of mutation analyses in oncodriver genes using traditional methods thereby challenging the application of targeted cancer therapy strategies for patients Ultradeep sequencing can detect low frequency and expanded clonal mutations in primary tumors to better inform treatment decisions. KRAS coding exons in 61 treatment-naive colorectal cancer (CRC) tumors and KRAS, EGFR, ALK, and MET in lung tumors from three Chinese non-small cell lung cancer (NSCLC) patients were sequenced using ultradeep sequencing methods. Forty-one percent of CRC patients (25/61) harbored mutations in the KRAS active domain, eight of which (13%) were not detected by Sanger sequencing. Three (of eight) had frequencies less than 10% and one patient harbored more than one mutation. Low frequency KRAS active (G12R) and EGFR kinase domain mutations (G719A) were identified in one NSCLC patient. A second NSCLC patient showed an EML4-ALK fusion with ALK, EGFR, and MET mutations. A third NSCLC patient harbored multiple low frequency mutations in KRAS, EGFR, and MET as well as ALK gene copy number increases. Within the same patient, multiple low frequency mutations occurred within a gene. A complex pattern of intrinsic low frequency driver mutations in well-known tumor oncogenes may exist prior to treatment, resulting in resistance to targeted therapies. Ultradeep sequencing can characterize intratumor heterogeneity and identify such mutations to ultimately affect treatment decisions.

  8. Autoimmune lymphoproliferative syndrome-like disease with somatic KRAS mutation.

    PubMed

    Takagi, Masatoshi; Shinoda, Kunihiro; Piao, Jinhua; Mitsuiki, Noriko; Takagi, Mari; Matsuda, Kazuyuki; Muramatsu, Hideki; Doisaki, Sayoko; Nagasawa, Masayuki; Morio, Tomohiro; Kasahara, Yoshihito; Koike, Kenichi; Kojima, Seiji; Takao, Akira; Mizutani, Shuki

    2011-03-10

    Autoimmune lymphoproliferative syndrome (ALPS) is classically defined as a disease with defective FAS-mediated apoptosis (type I-III). Germline NRAS mutation was recently identified in type IV ALPS. We report 2 cases with ALPS-like disease with somatic KRAS mutation. Both cases were characterized by prominent autoimmune cytopenia and lymphoadenopathy/splenomegaly. These patients did not satisfy the diagnostic criteria for ALPS or juvenile myelomonocytic leukemia and are probably defined as a new disease entity of RAS-associated ALPS-like disease (RALD).

  9. A novel KRAS gene mutation report in sporadic colorectal cancer, from Northwest of Iran.

    PubMed

    Dolatkhah, Roya; Somi, Mohammad Hossein; Kermani, Iraj Asvadi; Farassati, Faris; Dastgiri, Saeed

    2017-03-01

    While the role of KRAS gene mutations has been widely accepted for predicting responses to anti-EGFR therapy in patients with colorectal cancer, although this study was based on observation of a single case it gives hope that some KRAS gene mutation may have favorable prognosis. More studies are required on patients with similar mutation to validate this finding.

  10. KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay

    PubMed Central

    Lee, Sung Hak; Chung, Arthur Minwoo; Lee, Ahwon; Oh, Woo Jin; Choi, Yeong Jin; Lee, Youn-Soo; Jung, Eun Sun

    2017-01-01

    Background Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. Methods We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. Results KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. Conclusions The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC. PMID:28013534

  11. A mutation spectrum that includes GNAS, KRAS and TP53 may be shared by mucinous neoplasms of the appendix.

    PubMed

    Hara, Kieko; Saito, Tsuyoshi; Hayashi, Takuo; Yimit, Alkam; Takahashi, Michiko; Mitani, Keiko; Takahashi, Makoto; Yao, Takashi

    2015-09-01

    Appendiceal mucinous tumors (AMTs) are classified as low-grade appendiceal mucinous neoplasms (LAMNs) or mucinous adenocarcinomas (MACs), although their carcinogenesis is not well understood. As somatic activating mutations of GNAS are considered to be characteristic of LAMNs while TP53 mutations have been shown to be specific to MACs, MACs are unlikely to result from transformation of LAMNs. However, emerging evidence also shows the presence of GNAS mutations in MACs. We examined 16 AMTs (11 LAMNs and 5 MACs) for genetic alterations of GNAS, KRAS, BRAF, TP53, CTNNB1, and TERT promoter in order to elucidate the possibility of a shared genetic background in the two tumor types. Extensive histological examination revealed the presence of a low-grade component in all cases of MAC. GNAS mutations were detected in two LAMNs and in one MAC, although the GNAS mutation in this MAC was a nonsense mutation (Q227X) expected not to be activating mutation. TP53 mutations were detected in three LAMNs; they were frequently detected in MACs. KRAS mutations were detected in three LAMNs and three MACs, and CTNNB1 mutations were detected in two LAMNs. KRAS mutation and activating mutation of GNAS occurred exclusively in AMTs. BRAF and TERT mutations were not detected. Overexpression of p53 was observed in only two MACs, and p53 immunostaining clearly discriminated the high-grade lesion from a low-grade component in one. These findings suggest that p53 overexpression plays an important role in the carcinogenesis of AMTs and that, in addition to mutations of GNAS, KRAS and TP53 alterations might be shared by AMTs, thus providing evidence for the possible progression of LAMNs to MAC.

  12. The status of Her2 amplification and Kras mutations in mucinous ovarian carcinoma.

    PubMed

    Chang, Kuang-Leei; Lee, Ming-Yung; Chao, Wan-Ru; Han, Chih-Ping

    2016-12-28

    Jayson GC et al. remarked in Lancet that nearly 100% of mucinous ovarian cancer cases have Kras mutation as well as a high frequency of Her2 amplification. Using the Abbott PathVysion Her2 DNA Probe Kit and Kras mutant-enriched PCR Kits (FemtoPath®), 21 samples of primary ovarian mucinous cystadenocarcinomas from Taiwanese patients were examined to determine the status of Her2 amplification and Kras mutations. Our results showed the Her2 amplification rates were 33.33%, while the Kras mutation rates were 61.90%. We present here our results in order to enlighten the readership that the ~100% Kras mutant frequency and the high Her2 amplification rate reported by Jayson et al. may be too exaggerated to be applicable into all populations. Additionally, we report another 2 novel Kras mutations (A11V, V14I).

  13. Single Synonymous Mutations in KRAS Cause Transformed Phenotypes in NIH3T3 Cells

    PubMed Central

    Waters, Andrew M.; Bagni, Rachel; Portugal, Franklin; Hartley, James L.

    2016-01-01

    Synonymous mutations in the KRAS gene are clustered at G12, G13, and G60 in human cancers. We constructed 9 stable NIH3T3 cell lines expressing KRAS, each with one of these synonymous mutations. Compared to the negative control cell line expressing the wild type human KRAS gene, all the synonymous mutant lines expressed more KRAS protein, grew more rapidly and to higher densities, and were more invasive in multiple assays. Three of the cell lines showed dramatic loss of contact inhibition, were more refractile under phase contrast, and their refractility was greatly reduced by treatment with trametinib. Codon usage at these glycines is highly conserved in KRAS compared to HRAS, indicating selective pressure. These transformed phenotypes suggest that synonymous mutations found in driver genes such as KRAS may play a role in human cancers. PMID:27684555

  14. Loopback rolling circle amplification for ultrasensitive detection of Kras gene.

    PubMed

    Xu, Huo; Wu, Dong; Jiang, Yifan; Zhang, Rongbo; Wu, Qingzheng; Liu, Yiyun; Li, Feng; Wu, Zai-Sheng

    2017-03-01

    Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100fM to 20nM), and the detection limit is down to 16fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics.

  15. Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

    PubMed

    Tuononen, Katja; Mäki-Nevala, Satu; Sarhadi, Virinder Kaur; Wirtanen, Aino; Rönty, Mikko; Salmenkivi, Kaisa; Andrews, Jenny M; Telaranta-Keerie, Aino I; Hannula, Sari; Lagström, Sonja; Ellonen, Pekka; Knuuttila, Aija; Knuutila, Sakari

    2013-05-01

    The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

  16. Comparative Roles of Overexpressed and Mutated H- and K-ras in Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-08-01

    initiated tumors ) using the mismatch amplification mutation assay ( MAMA ) developed by Cha et al (5). Our initial studies indicated that there was...fold more potent at inducing mammary tumors than the activated K-ras gene. Yet, the K-ras oncogene was still effective at mammary carcinoma induction...transgenic rats harboring a H-ras gene (HrHr transgenics) or K-ras gene (HrKr transgenics) controlled by H-ras gene regulatory elements. Mammary tumor

  17. Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma

    PubMed Central

    Stefanius, Karoliina; Ylitalo, Laura; Tuomisto, Anne; Kuivila, Rami; Kantola, Tiina; Sirniö, Päivi; Karttunen, Tuomo J; Mäkinen, Markus J

    2011-01-01

    Aims To define the occurrence of KRAS and BRAF mutations, microsatellite instability (MSI), and MGMT and hMLH1 methylation and expression in colorectal serrated adenocarcinoma. Methods and results KRAS codon 12/13 and 59/61 and BRAF V600E mutations, MSI, and MGMT and hMLH1 methylation and expression in 42 serrated adenocarcinomas and 17 serrated adenomas were compared with those in 59 non-serrated colorectal carcinomas (CRCs) and nine adenomas. KRAS and BRAF mutations were observed in 45% and 33% of serrated adenocarcinomas and in 27% and 0% of non-serrated CRCs (P < 0.001). The KRAS c12G→A transition was the predominant type of mutation in serrated adenocarcinomas. Forty-two per cent of BRAF-mutated serrated adenocarcinomas showed high-level MSI (MSI-H) (P = 0.075), 100% showed hMLH1 methylation (P = 0.001) and 90.9% showed MGMT methylation (P = 0.019). Fifty-six per cent of serrated adenocarcinomas with microsatellite stability/low-level microsatellite instability harboured KRAS mutations. In non-serrated cancers, KRAS mutations were not associated with MSI status. Conclusions A high combined mutation rate (79–82%) of KRAS and BRAF in serrated adenomas and adenocarcinomas indicates that mitogen-activated protein kinase activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. A high frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors, thus challenging the traditional model of Vogelstein. PMID:21457162

  18. Assessment of DDR2, BRAF, EGFR and KRAS mutations as therapeutic targets in non-adenocarcinoma lung cancer patients.

    PubMed

    Yashima, Hideaki; Shimizu, Kimihiro; Araki, Takuya; Aomori, Tohru; Ohtaki, Yoichi; Nagashima, Toshiteru; Enokida, Yasuaki; Atsumi, Jun; Nakamura, Tomonori; Takeyoshi, Izumi; Yamamoto, Koujirou

    2014-09-01

    Molecular-targeted therapy has not been established in non-adenocarcinoma lung cancer (non-AdLC), as no targets that affect the clinical efficacy of molecular-targeted drugs have yet been identified. In this study, we investigated the frequency of genetic variations in discoidin domain receptor 2 (DDR2), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-AdLC patients, in order to evaluate the possibility of genetic mutations in these genes being used as therapeutic targets for the treatment of patients with non-AdLC. For this purpose, we enrolled 150 non-AdLC patients who had undergone surgery at the Gunma University Hospital between December, 2003 and December, 2012. Genetic mutations in the EGFR, KRAS, DDR2 and BRAF genes were detected by a sequencing method or probe assay using DNA derived from cancer tissues. No somatic mutations in DDR2 or BRAF were detected in non-AdLC patients. Conversely, genetic mutations in EGFR exon 19 were found in 3 squamous cell carcinoma (SCC) and 3 adenosquamous carcinoma patients, whereas KRAS codon 12 mutations were also found in 3 SCC patients and 1 large-cell neuroendocrine carcinoma patient. EGFR and KRAS mutations were mutually exclusive. This study indicated that, although DDR2 and BRAF mutations may only rarely be used as therapeutic targets, EGFR and KRAS mutations may represent candidate therapeutic targets, at least in the non-AdLC patients investigated.

  19. EGFR and KRAS mutations in Turkish non-small cell lung cancer patients: a pilot study.

    PubMed

    Bircan, Sema; Baloglu, Huseyin; Kucukodaci, Zafer; Bircan, Ahmet

    2014-08-01

    EGFR and KRAS mutation profile in non-small cell lung cancers (NSCLCs) shows wide variations due to geographic and ethnic background. We aimed to determine the frequency and types of EGFR and KRAS mutations in a sample group of Turkish NSCLC cases. The study included 14 adenocarcinomas (ACs), 11 squamous cell carcinoma (SCC) patients selected from archival material including small biopsy or surgical specimens. Their formalin fixed paraffin-embedded tumor tissues were used for genomic DNA extraction for EGFR exon 19 and 21, and KRAS exon 2 mutations. Eleven NSCLCs (44 %) had EGFR mutations. Exon 19 and 21 mutations were found in 8 (32 %) and 5 (20 %) cases. Two cases showed double EGFR mutations. In ACs, 5 (35.7 %) patients had EGFR gene mutation, 3 in exon 19 and 3 in exon 21. In SCCs, 6 (54.5 %) cases had EGFR mutation, 5 in exon 19 and 2 in exon 21. All exon 19 mutations were deletion-type mutations. For exon 21, 3 cases had L858R point mutation (CTG>CGG) and two cases showed deletion-type mutations. Six (24 %) NSCLCs showed KRAS mutations (three ACC, three SCC), 5 codon 12 mutations (G>T, T>C, G>A) and one codon 13 mutation (G>T). Three NSCLC cases showed both EGFR and KRAS mutations together. The profile of KRAS mutation in our AC cases was quite similar to those seen in the Western countries; however, frequency and clustering of EGFR mutations were similar to those seen in the Eastern countries.

  20. KRAS mutational status as a predictor of epidermal growth factor receptor inhibitor efficacy in colorectal cancer.

    PubMed

    Baynes, Roy D; Gansert, Jennifer

    2009-01-01

    Inhibitors of the epidermal growth factor receptor (EGFR) have demonstrated promising potential in the treatment of advanced colorectal cancer. However, a proportion of patients do not respond to therapy with EGFR inhibitors, and therefore, there has been interest in identifying those patients most likely to benefit from therapy with these agents. KRAS, a member of the RAS family of signaling proteins, plays an important role in EGFR-mediated regulation of cellular proliferation and survival. Although there is still some debate regarding the prognostic importance of KRAS mutations in patients with metastatic colorectal cancer, several recent phase 2 and 3 studies have identified the presence of mutations at codons 12 and 13 of KRAS as predictors of poor response to the anti-EGFR monoclonal antibodies panitumumab and cetuximab. Patients with wild-type KRAS were found to have significantly better progression-free survival, overall survival, and/or objective response rate compared with patients harboring KRAS mutations. As a result, there has been growing interest in the development of KRAS mutational status as a biomarker for predicting patient response to EGFR-targeted therapy. Screening colorectal tumors for the absence of KRAS mutations may help identify patients most likely to benefit from anti-EGFR therapies.

  1. Association between coffee drinking and K-ras mutations in exocrine pancreatic cancer. PANKRAS II Study Group

    PubMed Central

    Porta, M.; Malats, N.; Guarner, L.; Carrato, A.; Rifa, J.; Salas, A.; Corominas, J. M.; Andreu, M.; Real, F. X.

    1999-01-01

    STUDY OBJECTIVE: To analyse the relation between coffee consumption and mutations in the K-ras gene in exocrine pancreatic cancer. DESIGN: Case- case study. Consumption of coffee among cases with the activating mutation in the K-ras gene was compared with that of cases without the mutation. SETTING AND PATIENTS: All cases of pancreatic cancer newly diagnosed at five hospitals in Spain during three years were included in the PANKRAS II Study (n = 185, of whom 121 whose tissue was available for molecular analysis are the object of the present report). Over 88% were personally interviewed in hospital. DNA was amplified from paraffin wax embedded tissues, and mutations in codon 12 of K-ras were detected by the artificial RFLP technique. MAIN RESULTS: Mutations were found in tumours from 94 of 121 patients (77.7%). Mutations were more common among regular coffee drinkers than among non-regular coffee drinkers (82.0% v 55.6%, p = 0.018, n = 107). The odds ratio adjusted by age, sex, smoking and alcohol drinking was 5.41 (95% CI 1.64, 17.78). The weekly intake of coffee was significantly higher among patients with a mutated tumour (mean of 14.5 cups/week v 8.8 among patients with a wild type tumour, p < 0.05). With respect to non- regular coffee drinkers, the odds ratio of a mutated tumour adjusted by age, sex, smoking and alcohol drinking was 3.26 for drinkers of 2-7 cups/week, 5.77 for drinkers of 8-14 cups/week and 9.99 for drinkers of > or = 15 cups/week (p < 0.01, test for trend). CONCLUSIONS: Pancreatic cancer cases without activating mutations in the K-ras gene had drank significantly less coffee than cases with a mutation, with a significant dose response relation: the less they drank, the less likely their tumours were to harbour a mutation. In exocrine pancreatic cancer the K-ras gene may be activated less often among non-regular coffee drinkers than among regular drinkers. Caffeine, other coffee compounds or other factors with which coffee drinking is

  2. K-ras cancer gene mutations in lung tumors from female Swiss (CD-1) mice exposed transplacentally to 3'-azido-3'-deoxythymidine.

    PubMed

    Koujitani, Takatoshi; Ton, Tai-Vu T; Lahousse, Stephanie A; Hong, Hue-Hua L; Wakamatsu, Nobuko; Sills, Robert C

    2008-12-01

    A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg 3'-azido-3'-deoxythymidine (AZT)/kg body weight (BW) daily for the duration of gestation (18-19 days) [National Toxicology Program,2006]. The incidence of alveolar/bronchiolar adenomas and carcinomas in the 200 and 300 mg/kg groups was significantly higher (P = 0.027 and 0.007, respectively) in male offspring, but not in females (P = 0.338 and 0.315, respectively). The purpose of the present study was to evaluate K-ras mutation status in lung tumors from the female offspring in AZT exposed groups and to determine whether at the molecular level there were signature K-ras mutations in lung tumors that were different from spontaneous tumors. K-ras mutation was detected by cycle sequencing of polymerase chain reaction (PCR)-amplified DNA, isolated from formalin-fixed, paraffin-embedded lung tumors. K-ras mutations were detected in 17 of 28 (61%) lung tumors from the female offspring in AZT exposed groups. No K-ras mutations were detected in the 8 tumors examined from the female control group. The predominant mutations were Codon 12 G-->T transversions in the 50, 100, and 300 mg/kg groups, and Codon 12 G-->C transversions in the 200 and 300 mg/kg groups. K-ras Codon 12 G-->T transversions (TGT mutations) may be induced by oxidative DNA damage and 8-oxoguanine (8-oxoG), while K-ras Codon 12 G-->C transversions (CGT mutations) may be due to further oxidative lesions of guanine and 8-oxoG.

  3. KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment

    PubMed Central

    Normanno, Nicola; Pinto, Carmine; Castiglione, Francesca; Bardelli, Alberto; Gambacorta, Marcello; Botti, Gerardo; Nappi, Oscar; Siena, Salvatore; Ciardiello, Fortunato; Taddei, GianLuigi; Marchetti, Antonio

    2011-01-01

    Background Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. Methodology/Principal Findings In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. Conclusions The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers

  4. KRAS mutations: variable incidences in a Brazilian cohort of 8,234 metastatic colorectal cancer patients

    PubMed Central

    2014-01-01

    Background KRAS mutations are frequently found in colorectal cancer (CRC) indicating the importance of its genotyping in the study of the molecular mechanisms behind this disease. Although major advances have occurred over the past decade, there are still important gaps in our understanding of CRC carcinogenesis, particularly whether sex-linked factors play any role. Methods The profile of KRAS mutations in the Brazilian population was analyzed by conducting direct sequencing of KRAS codons 12 and 13 belonging to 8,234 metastatic CRC patient samples. DNA was extracted from paraffin-embedded tissue, exon 1 was amplified by PCR and submitted to direct sequencing. The data obtained was analysed comparing different geographical regions, gender and age. Results The median age was 59 years and the overall percentage of wild-type and mutated KRAS was 62.8% and 31.9%, respectively. Interestingly, different percentages of mutated KRAS patients were observed between male and female patients (32.5% versus 34.8%, respectively; p = 0.03). KRAS Gly12Asp mutation was the most prevalent for both genders and for most regions, with the exception of the North where Gly12Val was the most frequent mutation found. Conclusions To the best of our knowledge this is one of the largest cohorts of KRAS genotyping in CRC patients and the largest to indicate a higher incidence of KRAS mutation in females compared to males in Brazil. Nevertheless, further research is required to better address the impact of gender differences in colorectal cancer. PMID:24720724

  5. Driver mutations among never smoking female lung cancer tissues in China identify unique EGFR and KRAS mutation pattern associated with household coal burning.

    PubMed

    Hosgood, H Dean; Pao, William; Rothman, Nathaniel; Hu, Wei; Pan, Yumei Helen; Kuchinsky, Kyle; Jones, Kirk D; Xu, Jun; Vermeulen, Roel; Simko, Jeff; Lan, Qing

    2013-11-01

    Lung cancer in never smokers, which has been partially attributed to household solid fuel use (i.e., coal), is etiologically and clinically different from lung cancer attributed to tobacco smoking. To explore the spectrum of driver mutations among lung cancer tissues from never smokers, specifically in a population where high lung cancer rates have been attributed to indoor air pollution from domestic coal use, multiplexed assays were used to detect >40 point mutations, insertions, and deletions (EGFR, KRAS, BRAF, HER2, NRAS, PIK3CA, MEK1, AKT1, and PTEN) among the lung tumors of confirmed never smoking females from Xuanwei, China [32 adenocarcinomas (ADCs), 7 squamous cell carcinomas (SCCs), 1 adenosquamous carcinoma (ADSC)]. EGFR mutations were detected in 35% of tumors. 46% of these involved EGFR exon 18 G719X, while 14% were exon 21 L858R mutations. KRAS mutations, all of which were G12C_34G>T, were observed in 15% of tumors. EGFR and KRAS mutations were mutually exclusive, and no mutations were observed in the other tested genes. Most point mutations were transversions and were also found in tumors from patients who used coal in their homes. Our high mutation frequencies in EGFR exon 18 and KRAS and low mutation frequency in EGFR exon 21 are strikingly divergent from those in other smoking and never smoking populations from Asia. Given that our subjects live in a region where coal is typically burned indoors, our findings provide new insights into the pathogenesis of lung cancer among never smoking females exposed to indoor air pollution from coal.

  6. Mutational analysis of HRAS and KRAS genes in oral carcinoma cell lines.

    PubMed

    Maemoto, Sachiko; Yumoto, Megumi; Ibata, Masato; Torizuka, Sho; Ozawa, Naohumi; Tatsumi, Shunsuke; Hashido, Moeko; Morikawa, Masako; Maeda, Genta; Imai, Kazushi

    2012-07-01

    RAS overexpression and its active mutations are involved in malignant tumorigenesis. However, the mutation rates in oral carcinoma cells differ between populations. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) or normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets, spanning HRAS and KRAS exons. Nucleotide substitutions were analyzed by single strand conformation polymorphism. In contrast to no substitutions in KRAS, nine different substitutions were detected in HRAS. Of the nine, six substitutions were located at intron 1 (HSC2 and HSC3 cells) or intron 2 (HSC3, SCCKN and Ca9.22 cells), and one each of exon 1 (all cells), exon 2 (HOC313, TSU, HSC2 and HSC3 cells) and the 5' upstream region (all cells). Substitutions at exons 1 and 2 did not affect the amino acid sequence; the exon 1 substitution was positioned at the 5' untranslated region, which may be a single nucleotide polymorphism (SNP) sequence because all the cells were isolated from a Japanese population, and the mutations at exon 2 was a silent mutation. A substitution at the 5' upstream region was an SNP. These data demonstrate that SNPs and point mutations observed in HRAS do not change the amino acid sequence, and suggest that the mutations affecting the amino acid sequence may be a rare event in oral carcinomas of the Japanese population.

  7. Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

    PubMed Central

    McFadden, David G.; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K.; Song, Xiaoling; Pirun, Mono; Santiago, Philip M.; Kim-Kiselak, Caroline; Platt, James T.; Lee, Emily; Hodges, Emily; Rosebrock, Adam P.; Bronson, Roderick T.; Socci, Nicholas D.; Hannon, Gregory J.; Jacks, Tyler; Varmus, Harold

    2016-01-01

    Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. PMID:27702896

  8. Across the universe of K-RAS mutations in non-small-cell-lung cancer.

    PubMed

    Piva, Sheila; Ganzinelli, Monica; Garassino, Marina Chiara; Caiola, Elisa; Farina, Gabriella; Broggini, Massimo; Marabese, Mirko

    2014-01-01

    RAS family proteins are important signaling molecules that regulate cell growth, survival and differentiation by coupling receptor activation to downstream effector pathways. Three distinct genes encode for the three different proteins H-, K-, and N- RAS. These proteins share high sequence homology, particularly at the N-Terminal domain. Among them, K-RAS is one of the most frequently mutated in human cancer. The majority of the mutations present in K-RAS are at codon 12 (from 80 to 100%) followed by codon 13 and 61. In all cases, aminoacid change leads to a constitutively activated protein. K-RAS mutations have a role in tumor development as well as in tumor progression and resistance. Despite the various studies which have been published, the prognostic and predictive role of K-RAS mutations is still under debate. Keeping in mind that the glycine present at position 12 can be substituted by valine, aspartic acid or cysteine, it could be well understood that each different substitution plays a different role in K-RAS-dependent processes. The present article focuses on the molecular and biological characteristics of K-RAS protein, its role in NSCLC tumor development and progression. We also present an overview of the preclinical models both in vitro and in vivo available to determine the role of K-RAS in tumor progression and response to treatment and on the recent results obtained in this field. Finally, we have considered the impact of KRAS mutations in clinical practice, analyzing the different recent trials that have taken into consideration K-RAS.

  9. Evaluation of EGFR, KRAS and BRAF gene mutations in renal cell carcinoma

    PubMed Central

    Sen, Haluk; Bulut, Ersan; Cengiz, Beyhan; Karakok, Metin; Erturhan, Sakip; Seckiner, Ilker

    2014-01-01

    A subset of renal cell carcinoma (RCC) patients has been shown to respond to anti-EGFR therapy. As KRAS and BRAF mutations are associated with poor response to anti-EGFR therapy in some cancers, it has been suggested that screening for KRAS and BRAF mutations in RCC may be a promising strategy to identify patients who might respond to EGFR-targeted therapy. The aim of this study was to investigate the mutation status of EGFR, KRAS and BRAF in RCC patients. Renal tumors and normal renal samples from forty-eight patients who underwent radical or partial nephrectomy for kidney cancer were used in this study. Histological classification of the tumors was performed according to International Union against Cancer (UICC) / American Joint Committee on Cancer (AJCC) classification. Seventeen patients (48%) had clear-cell RCC, 7 (20%) had chromophobe RCC, and 11 patients (32%) had papillary RCC. DNA isolated from the samples was subjected to melting curve mutation analysis for EGFR, BRAF and KRAS using ABI-3130 DNA sequencer. DNA sequencing analysis of RCC samples, when compared with morphologically normal matched regions, did not show any exon mutations. Our results do not support the notion that EGFR, KRAS and BRAF might be mutated in RCC.

  10. Characterization of a novel oncogenic K-ras mutation in colon cancer

    SciTech Connect

    Akagi, Kiwamu . E-mail: akagi@cancer-c.pref.saitama.jp; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

    2007-01-19

    Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.

  11. Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

    PubMed Central

    Vincenzi, Bruno; Cremolini, Chiara; Sartore-Bianchi, Andrea; Russo, Antonio; Mannavola, Francesco; Perrone, Giuseppe; Pantano, Francesco; Loupakis, Fotios; Rossini, Daniele; Ongaro, Elena; Bonazzina, Erica; Dell'Aquila, Emanuela; Imperatori, Marco; Zoccoli, Alice; Bronte, Giuseppe; De Maglio, Giovanna; Fontanini, Gabriella; Natoli, Clara; Falcone, Alfredo; Santini, Daniele; Onetti-Muda, Andrea; Siena, Salvatore; Tonini, Giuseppe; Aprile, Giuseppe

    2015-01-01

    Introduction: Activating mutations of K-Ras gene have a well-established role as predictors of resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) patients. Their prognostic value is controversial, and no data regarding the prognostic value of mutation rate, defined as the percentage of mutated alleles/tumor sample, are available. We aimed to evaluate the prognostic value of K-Rasmutation rate in a homogenous cohort of mCRC patients receiving first-line doublet plus bevacizumab. Patients and Methods: This retrospective study enrolled 397 K-Ras mutant mCRC patients from 6 Italian centers, and 263 patients were fully evaluable for our analysis. K-Ras mutation rate was assessed by pyrosequencing. Patients with less than 60% of cancer cells in tumor tissue were excluded. No patients received anti-EGFR containing anticancer therapy, at any time. Median mutation rate was 40% and was adopted as cut-off. The primary and secondary endpoints were PFS and OS respectively. Results: At univariate analysis, K-Ras mutation rate higher than 40% was significantly associated with lower PFS (7.3 vs 9.1 months; P < 0.0001) and OS (21 vs 31 months; P = 0.004). A multivariate model adjusted for age at diagnosis, site of origin of tumor tissue (primary vs metastases), referral center, number of metastatic sites, and first-line chemotherapy backbone, showed that K-Ras mutation rate remained a significant predictor of PFS and OS in the whole population. Discussion: Our data demonstrate an association between K-Ras mutation rate and prognosis in mCRC patients treated with bevacizumab-containing first-line therapy. These data deserve to be verified in an independent validation set. PMID:26384309

  12. KRAS driven expression signature has prognostic power superior to mutation status in non‐small cell lung cancer

    PubMed Central

    Nagy, Ádám; Pongor, Lőrinc Sándor; Szabó, András; Santarpia, Mariacarmela

    2016-01-01

    KRAS is the most frequently mutated oncogene in non‐small cell lung cancer (NSCLC). However, the prognostic role of KRAS mutation status in NSCLC still remains controversial. We hypothesize that the expression changes of genes affected by KRAS mutation status will have the most prominent effect and could be used as a prognostic signature in lung cancer. We divided NSCLC patients with mutation and RNA‐seq data into KRAS mutated and wild type groups. Mann‐Whitney test was used to identify genes showing altered expression between these cohorts. Mean expression of the top five genes was designated as a “transcriptomic fingerprint” of the mutation. We evaluated the effect of this signature on clinical outcome in 2,437 NSCLC patients using univariate and multivariate Cox regression analysis. Mutation of KRAS was most common in adenocarcinoma. Mutation status and KRAS expression were not correlated to prognosis. The transcriptomic fingerprint of KRAS include FOXRED2, KRAS, TOP1, PEX3 and ABL2. The KRAS signature had a high prognostic power. Similar results were achieved when using the second and third set of strongest genes. Moreover, all cutoff values delivered significant prognostic power (p < 0.01). The KRAS signature also remained significant (p < 0.01) in a multivariate analysis including age, gender, smoking history and tumor stage. We generated a “surrogate signature” of KRAS mutation status in NSCLC patients by computationally linking genotype and gene expression. We show that secondary effects of a mutation can have a higher prognostic relevance than the primary genetic alteration itself. PMID:27859136

  13. APC, K-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance.

    PubMed

    Hsieh, Jan-Sing; Lin, Shiu-Ru; Chang, Mei-Yin; Chen, Fang-Ming; Lu, Chien-Yu; Huang, Tsung-Jen; Huang, Yu-Sheng; Huang, Che-Jen; Wang, Jaw-Yuan

    2005-04-01

    Current researches have proposed a genetic model for colorectal cancer (CRC), in which the sequential accumulation of mutations in specific cancer-related genes, including adenomatous polyposis coli (APC), K-ras, and p53, drives the transition from normal epithelium through increasing adenomatous dysplasia to colorectal cancer. To identify patients with an increased risk of tumor recurrence or metastasis and evaluate the prognostic values of APC, K-ras, and p53 gene mutations, we investigated the frequency of these three mutated genes in tumors and sera of CRC patients. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired preoperative serum samples of 118 CRC patients were detected by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, followed by direct DNA sequencing of the PCR-amplified genomic DNA. Subsequently, serum molecular markers were analyzed for their correlation with patients' clinicopathologic features and presence of postoperative recurrence/metastasis. We did not observe any significant difference in the association of APC or K-ras or p53 gene mutations in primary tumors with patients' demographic data (all were P > 0.05). In contrast, both serum APC and p53 molecular markers were closely correlated with lymph node metastasis and TNM stage (both P < 0.05). Moreover, the serum overall molecular markers (at least one of the three markers) were prominently associated with depth of tumor invasion (P = 0.033), lymph node metastasis (P < 0.001), and TNM stage (P < 0.001). In addition, a significantly higher postoperative metastasis/recurrence rate in patients positive for overall molecular markers compared to those negative for these molecular markers were also demonstrated (P < 0.001). APC and K-ras molecular markers were more frequently observed in patients with locoregional metastasis (both P < 0.05), while p53 molecular marker was usually detected in the cases of peritoneal metastasis (P

  14. The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

    PubMed

    Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

    2017-03-01

    RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.

  15. Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.

    PubMed

    Lichtenstein, A V; Serdjuk, O I; Sukhova, T I; Melkonyan, H S; Umansky, S R

    2001-09-01

    The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.

  16. Analysis of EGFR, KRAS and P53 mutations in lung cancer using cells in the curette lavage fluid obtained by bronchoscopy.

    PubMed

    Yamaguchi, Fumihiro; Kugawa, Satoshi; Tateno, Hidetsugu; Kokubu, Fumio; Fukuchi, Kunihiko

    2012-12-01

    Histopathological samples are commonly used for molecular testing to detect both oncogenes and tumor-suppressor genes in lung cancer. The purpose of this study was to determine the efficacy of using curette lavage fluid for molecular testing to detect EGFR, KRAS and P53 mutations in lung cancer patients. Samples were obtained from 77 lung cancer patients by bronchoscopy at the time of diagnosis, collected by scraping the site of the primary tumor lesion with a curette. DNA was extracted from cells in the curette lavage fluid, and PCRs were performed to amplify mutation hot spot regions in the EGFR, KRAS and P53 genes. The PCR products were direct-sequenced to detect mutations of each gene. The reference sequence of each gene was obtained from GenBank. Overall, 27% (21 of 77) were found with EGFR mutations, 1% (1 of 77) with KRAS mutations, and 36% (28 of 77) with P53 mutations. KRAS mutations were not detected in patients harboring mutations in either EGFR or P53. P53 mutations were identified in 38% (8 of 21) of the patients with EGFR mutations, all of who had advanced lung cancer. Of these patients, a 62-year-old female current smoker was given EGFR-TKI as third-line therapy, with no improvement in clinical symptoms or results of radiographic examination. Multivariate analysis indicated that P53 mutation rates in advanced-stage lung cancer were significantly higher than those in early-stage lung cancer (P=.017). In contrast, EGFR mutation rates were not significantly associated with staging. L747S in EGFR, described as a mutation associated with secondary resistance to EGFR-TKI, was detected in three patients who had never received EGFR-TKI, including one SCLC patient. It is possible to analyze EGFR, KRAS and P53 mutations using curette lavage fluid collected from lung cancer patients. This is useful when sufficient amounts of tumor samples cannot be obtained. Data from the current study suggest that EGFR mutations in concert with P53 mutations accelerate cancer

  17. Lung adenocarcinoma: Sustained subtyping with immunohistochemistry and EGFR, HER2 and KRAS mutational status.

    PubMed

    Sousa, Vitor; Rodrigues, Carolina; Silva, Maria; Alarcão, Ana Maria; Carvalho, Lina

    2015-01-01

    Pulmonary adenocarcinomas are still in the process of achieving morphological, immunohistochemical and genetic standardization. The ATS/ERS/IASLC proposed classification for lung adenocarcinomas supports the value of the identification of histological patterns, specifically in biopsies. Thirty pulmonary adenocarcinomas were subjected to immunohistochemical study (CK7, CK5, 6, 18, CK20, TTF1, CD56, HER2, EGFR and Ki-67), FISH and PCR followed by sequencing and fragment analysis for EGFR, HER2 and KRAS. Solid pattern showed lower TTF1 and higher Ki-67 expression. TTF1 expression was higher in non-mucinous lepidic and micropapillary patterns when compared to acinar and solid and acinar, solid and mucinous respectively. Higher Ki67 expression was present in lepidic and solid patterns compared to mucinous. EGFR membranous staining had increasing expression from non-mucinous lepidic/BA pattern to solid pattern and micropapillary until acinar pattern. EGFR mutations, mainly in exon 19, were more frequent in females, together with non-smoking status, while KRAS exon 2 mutations were statistically more frequent in males, especially in solid pattern. FISH EGFR copy was correlated gross, with mutations. HER2 copy number was raised in female tumours without mutations, in all cases. Although EGFR and KRAS mutations are generally considered mutually exclusive, in rare cases they can coexist as it happened in one of this series, and was represented in acinar pattern with rates of 42.9% and 17.9%, respectively. EGFR mutations were more frequent in lepidic/BA and acinar patterns. Some cases showed different EGFR mutations. The differences identified between the adenocarcinoma patterns reinforce the need to carefully identify the patterns present, with implications in diagnosis and in pathogenic understanding. EGFR and KRAS mutational status can be determined in biopsies representing bronchial pulmonary carcinomas because when a mutation is present it is generally present in all the

  18. KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer

    PubMed Central

    Niitsu, H; Hinoi, T; Kawaguchi, Y; Sentani, K; Yuge, R; Kitadai, Y; Sotomaru, Y; Adachi, T; Saito, Y; Miguchi, M; Kochi, M; Sada, H; Shimomura, M; Oue, N; Yasui, W; Ohdan, H

    2016-01-01

    KRAS mutations occur in 30–40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apcflox/flox; LSL-KrasG12D and CDX2P-G22Cre;Apcflox/flox mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription–PCR (qRT–PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT–PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin–nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin–NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC. PMID:27526107

  19. Somatic mutation analysis of KRAS, BRAF, HER2 and PTEN in EGFR mutation-negative non-small cell lung carcinoma: determination of frequency, distribution pattern and identification of novel deletion in HER2 gene from Indian patients.

    PubMed

    Bhaumik, Sangeet; Ahmad, Firoz; Das, Bibhu Ranjan

    2016-10-01

    Somatic mutations of KRAS, BRAF, HER2, PTEN genes are the most important molecular markers after the EGFR gene mutation. The current study evaluated the frequency and distribution pattern of KRAS, BRAF, HER2, PTEN mutation in Indian non-small cell lung carcinoma patients. The frequency of KRAS, BRAF, HER2, PTEN mutations was 6.4 % (14/204), 1.5 % (3/204), 1.5 % (3/204), 0 % (0/204), respectively. KRAS, BRAF, HER2 mutations were more prevalent in males than in females. KRAS and HER2 showed a trend of a higher frequency of mutation in the age group of <60 years, whereas BRAF mutations were more frequent in the age group of ≥60 years. Sequencing analysis of KRAS gene revealed c.34G>T (G12C) (n = 8), c.35G>A (G12D) (n = 3), c.35G>T (G12 V) (n = 1) and c.34G>T (G12C)/c.41T>C (V14A) (n = 2) mutations. Three different BRAF mutations (L584P: n = 1, V600E: n = 1, K601E: n = 1) were detected. Two cases harboured c.2324_2325ins12 (ATACGTGATGGC duplication) in HER2 gene, and one case was positive for NG_007503.2 (NM_001005862.2):c.2218-4del. It is less certain, but still quite possible that this mutation will affect splicing as the deletion of one C actually brings in one additional purine into the region. In conclusion, the present study demonstrates an instance of diverse nature of KRAS, BRAF, HER2 and PTEN gene in Indian patients and confirms that the frequency of these gene mutations varies globally. To the best of our knowledge, this is the first Indian study to evaluate KRAS, BRAF, HER2 and PTEN gene mutations.

  20. KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

    SciTech Connect

    Erben, Philipp; Stroebel, Philipp; Horisberger, Karoline; Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin; Kaehler, Georg; Kienle, Peter; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas

    2011-11-15

    Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

  1. High-Throughput Mutation Profiling of Primary and Metastatic Endometrial Cancers Identifies KRAS, FGFR2 and PIK3CA to Be Frequently Mutated

    PubMed Central

    Seidel, Danila; Kusonmano, Kanthida; Petersen, Kjell; Mjøs, Siv; Hoivik, Erling A.; Wik, Elisabeth; Halle, Mari Kyllesø; Øyan, Anne M.; Kalland, Karl-Henning; Werner, Henrica Maria Johanna; Trovik, Jone; Salvesen, Helga

    2012-01-01

    Background Despite being the most common pelvic gynecologic malignancy in industrialized countries, no targeted therapies are available for patients with metastatic endometrial carcinoma. In order to improve treatment, underlying molecular characteristics of primary and metastatic disease must be explored. Methodology/Principal Findings We utilized the mass spectrometric-based mutation detection technology OncoMap to define the types and frequency of point somatic mutations in endometrial cancer. 67 primary tumors, 15 metastases corresponding to 7 of the included primary tumors and 11 endometrial cancer cell lines were screened for point mutations in 28 known oncogenes. We found that 27 (40.3%) of 67 primary tumors harbored one or more mutations with no increase in metastatic lesions. FGFR2, KRAS and PIK3CA were consistently the most frequently mutated genes in primary tumors, metastatic lesions and cell lines. Conclusions/Significance Our results emphasize the potential for targeting FGFR2, KRAS and PIK3CA mutations in endometrial cancer for development of novel therapeutic strategies. PMID:23300780

  2. Molecular pathological epidemiology of colorectal cancer in Chinese patients with KRAS and BRAF mutations

    PubMed Central

    Li, Wenbin; Qiu, Tian; Ling, Yun; Guo, Lei; Li, Lin; Ying, Jianming

    2015-01-01

    An investigation of interactive effects of exogenous and endogenous factors and tumor molecular changes can lead to a better understanding of tumor molecular signatures in colorectal cancer. We here report a molecular pathological epidemiology study in a large cohort of 945 colorectal cancer patients. Mutations of KRAS (36.6%) and BRAF (3.46%) were nearly mutually exclusive. KRAS-mutated tumors were more common in female patients (odds ratio [OR] = 1.68; P = 0.0001) and never smokers (OR = 1.60; P = 0.001). Whereas BRAF-mutated tumors demonstrated no discrepancy in aspects of gender and smoking status compared with wild-type tumors. In addition, tumors with BRAF or KRAS mutations were in correlation with elevated serum level of carbohydrate antigen (CA19-9) and carcinoma embryonic antigen (CEA) and the combination of serum biomarkers and molecular mutation status may enhance the more precise risk stratification of CRC patients. Further studies are needed to define the mechanism brought about by the aforementioned epidemiologic and clinicopathologic characteristics that may help optimize cancer prevention and precision therapy. PMID:26530529

  3. Dietary heme iron and the risk of colorectal cancer with specific mutations in KRAS and APC.

    PubMed

    Gilsing, Anne M J; Fransen, Fiona; de Kok, Theo M; Goldbohm, Alexandra R; Schouten, Leo J; de Bruïne, Adriaan P; van Engeland, Manon; van den Brandt, Piet A; de Goeij, Anton F P M; Weijenberg, Matty P

    2013-12-01

    Red meat intake has been linked to increased colorectal cancer (CRC) risk. Although the underlying mechanisms remain unclear, experimental studies suggest a role for dietary heme iron. Because heme iron was shown to promote specific mutations, it would be insightful to link heme iron data to CRC with mutations in key genes in an observational, population-based study. We investigated the association between dietary heme iron intake and risk of CRC with mutations in APC (adenomatous polyposis coli) and KRAS (Kirsten ras) and P53 overexpression in the Netherlands Cohort Study. After 7.3 years of follow-up, excluding the first 2.3 years due to incomplete coverage of the pathology registry and to avoid preclinical disease, adjusted hazard ratios (including adjustment for total meat) and 95% confidence intervals were calculated, using 4026 subcohort members (aged 55-69 years at baseline), 435 colon and 140 rectal cancer patients. When comparing the highest with the lowest tertile of intake, heme iron intake was associated with an increased risk of CRC harboring activating mutations in KRAS (hazard ratio = 1.71, 95% confidence interval: 1.15-2.57; P for trend = 0.03) and CRC without truncating mutations in APC (hazard ratio = 1.79, 95% confidence interval: 1.23-2.60; P for trend = 0.003). We observed a positive association between heme iron intake and the risk of CRC with activating G>A mutations in KRAS (P for trend = 0.01) and overall G>A mutations in APC (P for trend = 0.005). No associations were found with CRC harboring G>T mutations in KRAS/APC. Heme iron intake was positively associated with the risk of P53 overexpressed tumors but not with tumors without P53 overexpression (Pheterogeneity = 0.12). Heme iron intake was associated with an increased risk of colorectal tumors harboring G>A transitions in KRAS and APC and overexpression of P53. These novel findings suggest that alkylating rather than oxidative DNA-damaging mechanisms are involved in heme

  4. Conformational SERS Classification of K-Ras Point Mutations for Cancer Diagnostics.

    PubMed

    Morla-Folch, Judit; Gisbert-Quilis, Patricia; Masetti, Matteo; Garcia-Rico, Eduardo; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2017-02-20

    Point mutations in Ras oncogenes are routinely screened for diagnostics and treatment of tumors (especially in colorectal cancer). Here, we develop an optical approach based on direct SERS coupled with chemometrics for the study of the specific conformations that single-point mutations impose on a relatively large fragment of the K-Ras gene (141 nucleobases). Results obtained offer the unambiguous classification of different mutations providing a potentially useful insight for diagnostics and treatment of cancer in a sensitive, fast, direct and inexpensive manner.

  5. Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection

    PubMed Central

    Ostrow, Sheli L.; Simon, Einav; Prinz, Elad; Bick, Tova; Shentzer, Talia; Nagawkar, Sima S.; Sabo, Edmond; Ben-Izhak, Ofer; Hershberg, Ruth; Hershkovitz, Dov

    2016-01-01

    Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor. PMID:26902163

  6. Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection.

    PubMed

    Ostrow, Sheli L; Simon, Einav; Prinz, Elad; Bick, Tova; Shentzer, Talia; Nagawkar, Sima S; Sabo, Edmond; Ben-Izhak, Ofer; Hershberg, Ruth; Hershkovitz, Dov

    2016-02-23

    Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor.

  7. CYP1B1 polymorphisms and k-ras mutations in patients with pancreatic ductal adenocarcinoma.

    PubMed

    Crous-Bou, Marta; De Vivo, Immaculata; Porta, Miquel; Pumarega, José A; López, Tomàs; Alguacil, Joan; Morales, Eva; Malats, Núria; Rifà, Juli; Hunter, David J; Real, Francisco X

    2008-05-01

    The frequency of CYP1B1 polymorphisms in pancreatic cancer has never been reported. There is also no evidence on the relationship between CYP1B1 variants and mutations in ras genes (K-, H- or N-ras) in any human neoplasm. We analyzed the following CYP1B1 polymorphisms in 129 incident cases of pancreatic ductal adenocarcinoma (PDA): the m1 allele (Val to Leu at codon 432) and the m2 allele (Asn to Ser at codon 453). The calculated frequencies for the m1 Val and m2 Asn alleles were 0.45 and 0.68, respectively. CYP1B1 genotypes were out of Hardy-Weinberg equilibrium; this was largely due to K-ras mutated PDA cases. The Val/Val genotype was over five times more frequent in PDA cases with a K-ras mutation than in wild-type cases (OR = 5.25; P = 0.121). In PDA, polymorphisms in CYP1B1 might be related with K-ras activation pathways.

  8. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

    PubMed

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

    2016-10-04

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

  9. Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

    PubMed Central

    Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy te; Basso, Giuseppe

    2016-01-01

    To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations. PMID:27698462

  10. Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole-Imidazole Polyamide seco-CBI Conjugates.

    PubMed

    Taylor, Rhys D; Chandran, Anandhakumar; Kashiwazaki, Gengo; Hashiya, Kaori; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2015-10-12

    Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N-methylpyrrole-N-methylimidazole polyamide seco-1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one conjugate (conjugate 4) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer-minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one-base-pair mismatch sequence. Conjugate 4 was modified for next-generation sequencing. Bind-n-Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild-type sequence.

  11. Dietary acrylamide intake and the risk of colorectal cancer with specific mutations in KRAS and APC.

    PubMed

    Hogervorst, Janneke G F; de Bruijn-Geraets, Daisy; Schouten, Leo J; van Engeland, Manon; de Kok, Theo M C M; Goldbohm, R Alexandra; van den Brandt, Piet A; Weijenberg, Matty P

    2014-05-01

    Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings.

  12. Autoimmunity Including Intestinal Behçet Disease Bearing the KRAS Mutation in Lymphocytes: A Case Report.

    PubMed

    Moritake, Hiroshi; Takagi, Masatoshi; Kinoshita, Mariko; Ohara, Osamu; Yamamoto, Shojiro; Moriguchi, Sayaka; Nunoi, Hiroyuki

    2016-03-01

    We experienced the case of a 3-year-old male with a very rare combination of autoimmunity, including immune thrombocytopenia, recurrent Henoch-Schönlein purpura and intestinal Behçet disease. Exome sequencing of the patient's peripheral blood mononuclear cells identified a KRAS G13C mutation. Interestingly, the KRAS G13C mutation was observed in T and B lymphocytes, as well as natural killer cells, but not granulocytes. Our case was completely phenotypically different from RASopathies and did not meet the criteria for Ras-associated lymphoproliferative disease or juvenile myelomonocytic leukemia. This is the first reported case in which the KRAS mutation existed only in the lymphoid lineage. Based on the findings of our case and the current literature, it is clear that the RAS mutation in lymphoid cells is tightly linked with various autoimmune symptoms. The presence of the RAS mutation in lymphocytes should be reconsidered as a pathogenesis in cases of autoimmunity.

  13. Pooled Analysis of the Prognostic and Predictive Effects of KRAS Mutation Status and KRAS Mutation Subtype in Early-Stage Resected Non–Small-Cell Lung Cancer in Four Trials of Adjuvant Chemotherapy

    PubMed Central

    Shepherd, Frances A.; Domerg, Caroline; Hainaut, Pierre; Jänne, Pasi A.; Pignon, Jean-Pierre; Graziano, Stephen; Douillard, Jean-Yves; Brambilla, Elizabeth; Le Chevalier, Thierry; Seymour, Lesley; Bourredjem, Abderrahmane; Teuff, Gwénaël Le; Pirker, Robert; Filipits, Martin; Rosell, Rafael; Kratzke, Robert; Bandarchi, Bizhan; Ma, Xiaoli; Capelletti, Marzia; Soria, Jean-Charles; Tsao, Ming-Sound

    2013-01-01

    Purpose We undertook this analysis of KRAS mutation in four trials of adjuvant chemotherapy (ACT) versus observation (OBS) to clarify the prognostic/predictive roles of KRAS in non–small-cell lung cancer (NSCLC). Methods KRAS mutation was determined in blinded fashion. Exploratory analyses were performed to characterize relationships between mutation status and subtype and survival outcomes using a multivariable Cox model. Results Among 1,543 patients (763 OBS, 780 ACT), 300 had KRAS mutations (codon 12, n = 275; codon 13, n = 24; codon 14, n = 1). In OBS patients, there was no prognostic difference for overall survival for codon-12 (mutation v wild type [WT] hazard ratio [HR] = 1.04; 95% CI, 0.77 to 1.40) or codon-13 (HR = 1.01; 95% CI, 0.47 to 2.17) mutations. No significant benefit from ACT was observed for WT-KRAS (ACT v OBS HR = 0.89; 95% CI, 0.76 to 1.04; P = .15) or codon-12 mutations (HR = 0.95; 95% CI, 0.67 to 1.35; P = .77); with codon-13 mutations, ACT was deleterious (HR = 5.78; 95% CI, 2.06 to 16.2; P < .001; interaction P = .002). There was no prognostic effect for specific codon-12 amino acid substitution. The effect of ACT was variable among patients with codon-12 mutations: G12A or G12R (HR = 0.66; P = .48), G12C or G12V (HR = 0.94; P = .77) and G12D or G12S (HR = 1.39; P = .48; comparison of four HRs, including WT, interaction P = .76). OBS patients with KRAS-mutated tumors were more likely to develop second primary cancers (HR = 2.76, 95% CI, 1.34 to 5.70; P = .005) but not ACT patients (HR = 0.66; 95% CI, 0.25 to 1.75; P = .40; interaction, P = .02). Conclusion KRAS mutation status is not significantly prognostic. The potential interaction in patients with codon-13 mutations requires validation. At this time, KRAS status cannot be recommended to select patients with NSCLC for ACT. PMID:23630215

  14. KRAS and TP53 mutations in inflammatory bowel disease-associated colorectal cancer: a meta-analysis.

    PubMed

    Du, Lijun; Kim, John J; Shen, Jinhua; Chen, Binrui; Dai, Ning

    2017-01-07

    Although KRAS and TP53 mutations are common in both inflammatory bowel disease-associated colorectal cancer (IBD-CRC) and sporadic colorectal cancer (S-CRC), molecular events leading to carcinogenesis may be different. Previous studies comparing the frequency of KRAS and TP53 mutations in IBD-CRC and S-CRC were inconsistent. We performed a meta-analysis to compare the presence of KRAS and TP53 mutations among patients with IBD-CRC, S-CRC, and IBD without dysplasia. A total of 19 publications (482 patients with IBD-CRC, 4,222 with S-CRC, 281 with IBD without dysplasia) met the study inclusion criteria. KRAS mutation was less frequent (RR=0.71, 95%CI 0.56-0.90; P=0.004) while TP53 mutation was more common (RR=1.24, 95%CI 1.10-1.39; P<0.001) in patients with IBD-CRC compared to S-CRC. Both KRAS (RR=3.09, 95%CI 1.47-6.51; P=0.003) and TP53 (RR=2.15, 95%CI 1.07-4.31 P=0.03) mutations were more prevalent in patients with IBD-CRC compared to IBD without dysplasia. In conclusion, IBD-CRC and S-CRC appear to have biologically different molecular pathways. TP53 appears to be more important than KRAS in IBD-CRC compared to S-CRC. Our findings suggest possible roles of TP53 and KRAS as biomarkers for cancer and dysplasia screening among patients with IBD and may also provide targeted therapy in patients with IBD-CRC.

  15. Anti-tumor activity of ESX1 on cancer cells harboring oncogenic K-ras mutation

    SciTech Connect

    Nakajima, Junta; Ishikawa, Susumu; Hamada, Jun-Ichi; Yanagihara, Masatomo; Koike, Takao; Hatakeyama, Masanori

    2008-05-23

    Human ESX1 is a 65-kilodalton (kDa) paired-like homeoprotein that is proteolytically processed into N-terminal 45-kDa and C-terminal 20-kDa fragments. The N-terminal ESX1 fragment, which contains the homeodomain, localizes to the nucleus and represses mRNA transcription from the K-ras gene. When we inoculated human colorectal carcinoma HCT116 constitutive expressing N-terminal region of ESX1 (N-ESX1) into nude mice, transfectant cells uniformly showed decreased tumor-forming activity compared with that of the parental cells. Furthermore, pretreatment of HCT116 carcinoma cells with a fusion protein consisting of N-ESX1 and the protein-transduction domain derived from the human immunodeficiency virus type-1 TAT protein gave rise to a dramatic reduction in the tumorigenicity of HCT116 cells in nude mice. Our results provide first in vivo evidence for the molecular targeting therapeutic application of the K-ras repressor ESX1, especially TAT-mediated transduction of N-ESX1, in the treatment of human cancers having oncogenic K-ras mutations.

  16. LUNG TUMOR KRAS AND TP53 MUTATIONS IN NON-SMOKERS REFLECT EXPOSURE TO PAH-RICH COAL COMBUSTION EMISSIONS

    EPA Science Inventory


    Abstract

    We determined the TP53 and codon 12 KRAS mutations in lung tumors from 24 nonsmokers whose tumors were associated with exposure to smoky coal. Among any tumors studied previously, these showed the highest percentage of mutations that (a) were G -+ T transver...

  17. EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells.

    PubMed

    Roda, Desamparados; Castillo, Josefa; Telechea-Fernández, Marcelino; Gil, Anabel; López-Rodas, Gerardo; Franco, Luís; González-Rodríguez, Patricia; Roselló, Susana; Pérez-Fidalgo, J Alejandro; García-Trevijano, Elena R; Cervantes, Andrés; Zaragozá, Rosa

    2015-01-01

    KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRASG13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRASA146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRASA146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRASG13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.

  18. [Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

    PubMed

    Botezatu, I V; Panchuk, I O; Stroganova, A M; Senderovich, A I; Kondratova, V N; Shelepov, V P; Lichtenstein, A V

    2017-01-01

    Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

  19. Lung Adenocarcinoma: Predictive Value of KRAS Mutation Status in Assessing Local Recurrence in Patients Undergoing Image-guided Ablation.

    PubMed

    Ziv, Etay; Erinjeri, Joseph P; Yarmohammadi, Hooman; Boas, F Edward; Petre, Elena N; Gao, Song; Shady, Waleed; Sofocleous, Constantinos T; Jones, David R; Rudin, Charles M; Solomon, Stephen B

    2017-01-01

    Purpose To establish the relationship between KRAS mutation status and local recurrence after image-guided ablation of lung adenocarcinoma. Materials and Methods This study consisted of a HIPAA-compliant institutional review board-approved retrospective review of 56 primary lung adenocarcinomas in 54 patients (24 men, 30 women; median age, 72 years; range, 54-87 years) treated with percutaneous image-guided ablation and with available genetic mutational analysis. KRAS mutation status and additional clinical and technical variables-Eastern Cooperative Oncology Group (ECOG) status, smoking history, stage at diagnosis, status (new primary or not), history of radiation, history of surgery, prior systemic treatment, modality of ablation, size of nodule, ablation margin, and presence of ground-glass appearance-were recorded and evaluated in relation to time to local recurrence, which was calculated from the time of ablation to the first radiographic evidence of recurrence. Predictors of outcome were identified by using a proportional hazards model for both univariate and multivariate analysis, with death as a competing risk. Results Technical success was 100%. Of the 56 ablated tumors, 37 (66%) were wild type for KRAS and 19 (34%) were KRAS mutants. The 1-year and 3-year cumulative incidences of recurrence were 20% and 35% for wild-type KRAS compared with 40% and 63% for KRAS mutant tumors. KRAS mutation status was a significant predictor of local recurrence at both univariate (P = .05; subdistribution hazard ratio [sHR], 2.32) and multivariate (P = .006; sHR, 3.75) analysis. At multivariate analysis, size (P = .026; sHR, 2.54) and ECOG status (P = .012; sHR, 2.23) were also independent significant predictors, whereas minimum margin (P = .066) was not. Conclusion The results of this study show that there is a relationship between KRAS mutation status and local recurrence after image-guided ablation of lung adenocarcinoma. Specifically, KRAS mutation status of the ablated

  20. Prevalence of KRAS, BRAF, PI3K and EGFR mutations among Asian patients with metastatic colorectal cancer

    PubMed Central

    PHUA, LEE CHENG; NG, HUI WEN; YEO, ANGIE HUI LING; CHEN, ELYA; LO, MICHELLE SHU MEI; CHEAH, PEH YEAN; CHAN, ERIC CHUN YONG; KOH, POH KOON; HO, HAN KIAT

    2015-01-01

    Mutations in oncogenes along the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the resistance to cetuximab in patients with metastatic colorectal cancer (mCRC). However, the relative significance of these mutations based on their frequencies of occurrence in the Singaporean population remains unclear. In the present study, the prevalence of Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B (BRAF), phosphoinositide 3-kinase (PI3K) and EGFR somatic mutations were determined among Singaporean patients with mCRC. DNA extracted from 45 pairs of surgically resected tumor and normal mucosa samples was subjected to direct sequencing or restriction fragment length polymorphism. Associations of the genetic mutations with various clinicopathological parameters were further explored. Mutations in either codon 12 or 13 of KRAS were confirmed as prominent phenomena among the included Singaporean mCRC patients, at a prevalence comparable with that of Caucasian and patients of other Asian ethnicities [33.3% (90% confidence interval, 21.8–44.9%)]. KRAS mutation was not associated with clinicopathological features, including age, gender and ethnicity of patients, or the tumor site, differentiation and mucinous status. Conversely, the prevalence of BRAF (0%), PI3K (2.2%) and EGFR (0%) mutations were low. The results of the present study indicate that KRAS mutations are prevalent among the studied population, and confirm the low prevalence of BRAF, PI3K and EGFR mutations. KRAS should be prioritized as an investigational gene for future studies of predictive biomarkers of cetuximab response among Singaporean patients with mCRC. PMID:26622882

  1. The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

    PubMed Central

    Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

    2016-01-01

    Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP–GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer. PMID:26902995

  2. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  3. Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.

    PubMed

    Yu, Shaorong; Xie, Li; Hou, Zhibo; Qian, Xiaoping; Yu, Lixia; Wei, Jia; Ding, Yitao; Liu, Baorui

    2011-09-01

    Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.

  4. Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis.

    PubMed

    Rau, Kun-Ming; Chen, Han-Ku; Shiu, Li-Yen; Chao, Tsai-Ling; Lo, Yi-Ping; Wang, Chin-Chou; Lin, Meng-Chih; Huang, Chao-Cheng

    2016-04-07

    Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

  5. MUTATION SPECTRA OF SMOKY COAL COMBUSTION EMMISSIONS IN SALMONELLA REFLECTS THE TP53 AND KRAS MUTATIONS IN LUNG TUMORS FROM SMOKY COAL EXPOSED INDIVIDUALS

    EPA Science Inventory


    Mutation Spectra of Smoky Coal Combustion Emissions in Salmonella Reflect the TP53
    and KRAS Mutations in Lung Tumors from Smoky Coal-Exposed Individuals

    Abstract
    Nonsmoking women in Xuan Wei County, Yunnan Province, China who use smoky coal for cooking and h...

  6. Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia.

    PubMed

    Urosević, N; Krtolica, K; Skaro-Milić, A; Knezević-Usaj, S; Dujić, A

    1993-05-08

    Human colorectal carcinoma tissue sampled from 37 patients, routinely graded into Dukes' stages A, B and C and histologically examined for the level of differentiation, were analyzed for the presence of point mutations in the K-ras oncogene. Seventeen cases out of the 37 analyzed were found to have a mutation in either the 12th or the 13th codon of the K-ras gene, giving an overall frequency of mutation of 46%. The incidence of mutations in Dukes' stages A, B and C was 33, 46 and 58% respectively. Although the frequency of mutation appears to be similar to that reported for the USA population, the spectrum of point mutations in codons 12 and 13 of the K-ras gene in the Yugoslav population appears to differ significantly. G-to-T transversions make up 77% of all mutations present, with the distribution as follows: 18% at the first base and 59% at the second base of codons 12 and 13. G-to-A transitions at the second base is the only other mutation identified, occurring mainly in codon 13 in colorectal tumors of all 3 stages.

  7. LUNG TUMOR KRAS AND TP53 MUTATIONS IN NONSMOKERS REFLECT EXPOSURE TO PAH-RICH COAL COMBUSTION EMISSIONS

    EPA Science Inventory

    Lung Tumor KRAS and TP53 Mutations in Nonsmokers Reflect Exposure to PAH-Rich
    Coal Combustion Emissions

    Use of smoky coal in unvented homes in Xuan Wei County, Yunnan Province, China, is associated with lung cancer among nonsmoking females. Such women have the highest...

  8. ALTERNATE PATHWAY TO LUNG CANCER INDICATED BY KRAS AND P53 MUTATIONS IN NONSMOKERS EXPOSED TO INDOOR SMOKY COAL EMISSIONS

    EPA Science Inventory

    Alternate Pathway to Lung Cancer Indicated by KRAS and P53 Mutations in Nonsmokers Exposed to Indoor Smoky Coal Emissions

    Use of smoky coal in unvented homes in Xuan Wei County, Yunnan Province, China, is
    associated with lung cancer among nonsmoking females. Such wome...

  9. Chronic Obstructive Pulmonary Disease Is Not Associated with KRAS Mutations in Non-Small Cell Lung Cancer

    PubMed Central

    Saber, Ali; van der Wekken, Anthonie J.; Kerner, Gerald S. M. A.; van den Berge, Maarten; Timens, Wim; Schuuring, Ed; ter Elst, Arja; van den Berg, Anke; Hiltermann, T. Jeroen N.; Groen, Harry J. M.

    2016-01-01

    Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56–4.39; p<0.001) and smoking (HR 4.10; 95% CI: 1.14–14.79; p = 0.03) were associated with KRAS mutational status. In contrast, only smoking (HR 0.11; 95% CI: 0.04–0.32; p<0.001) was inversely associated with EGFR mutational status. Smoking related G>T and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients. PMID:27008036

  10. EGFR and KRAS Mutations Predict the Incidence and Outcome of Brain Metastases in Non-Small Cell Lung Cancer

    PubMed Central

    Tomasini, Pascale; Serdjebi, Cindy; Khobta, Nataliya; Metellus, Philippe; Ouafik, L’Houcine; Nanni, Isabelle; Greillier, Laurent; Loundou, Anderson; Fina, Frederic; Mascaux, Celine; Barlesi, Fabrice

    2016-01-01

    Background: Lung cancer is the leading cause of brain metastases (BM). The identification of driver oncogenes and matched targeted therapies has improved outcome in non-small cell lung cancer (NSCLC) patients; however, a better understanding of BM molecular biology is needed to further drive the process in this field. Methods: In this observational study, stage IV NSCLC patients tested for EGFR and KRAS mutations were selected, and BM incidence, recurrence and patients’ outcome were assessed. Results: A total of 144 patients (142 Caucasian and two Asian) were selected, including 11.27% with EGFR-mutant and 33.10% with KRAS-mutant tumors, and 57.04% patients had developed BM. BM incidence was more frequent in patients with EGFR mutation according to multivariate analyses (MVA) (Odds ratio OR = 8.745 [1.743–43.881], p = 0.008). Among patients with treated BM, recurrence after local treatment was less frequent in patients with KRAS mutation (OR = 0.234 [0.078–0.699], p = 0.009). Among patients with untreated BM, overall survival (OS) was shorter for patients with KRAS mutation according to univariate analysis (OR = 7.130 [1.240–41.012], p = 0.028), but not MVA. Conclusions: EGFR and KRAS mutations have a predictive role on BM incidence, recurrence and outcome in Caucasian NSCLC patients. These results may impact the routine management of disease in these patients. Further studies are required to assess the influence of other biomarkers on NSCLC BM. PMID:27999344

  11. Associations of anthropometric factors with KRAS and BRAF mutation status of primary colorectal cancer in men and women: a cohort study.

    PubMed

    Brändstedt, Jenny; Wangefjord, Sakarias; Nodin, Björn; Eberhard, Jakob; Sundström, Magnus; Manjer, Jonas; Jirström, Karin

    2014-01-01

    Obesity is a well-established risk factor for colorectal cancer (CRC), and accumulating evidence suggests a differential influence of sex and anthropometric factors on the molecular carcinogenesis of the disease. The aim of the present study was to investigate the relationship between height, weight, bodyfat percentage, waist- and hip circumference, waist-hip ratio (WHR), body mass index (BMI) and CRC risk according to KRAS and BRAF mutation status of the tumours, with particular reference to potential sex differences. KRAS and BRAF mutations were analysed by pyrosequencing in tumours from 494 incident CRC cases in the Malmö Diet and Cancer Study. Hazard ratios of CRC risk according to anthropometric factors and mutation status were calculated using multivariate Cox regression models. While all anthropometric measures except height were associated with an increased risk of KRAS-mutated tumours, only BMI was associated with an increased risk of KRAS wild type tumours overall. High weight, hip, waist, WHR and BMI were associated with an increased risk of BRAF wild type tumours, but none of the anthropometric factors were associated with risk of BRAF-mutated CRC, neither in the overall nor in the sex-stratified analysis. In men, several anthropometric measures were associated with both KRAS-mutated and KRAS wild type tumours. In women, only a high WHR was significantly associated with an increased risk of KRAS-mutated CRC. A significant interaction was found between sex and BMI with respect to risk of KRAS-mutated tumours. In men, all anthropometric factors except height were associated with an increased risk of BRAF wild type tumours, whereas in women, only bodyfat percentage was associated with an increased risk of BRAF wild type tumours. The results from this prospective cohort study further support an influence of sex and lifestyle factors on different pathways of colorectal carcinogenesis, defined by KRAS and BRAF mutation status of the tumours.

  12. Generation and analysis of mouse intestinal tumors and organoids harboring APC and K-Ras mutations.

    PubMed

    van Es, Johan H; Clevers, Hans

    2015-01-01

    Genetically engineered mouse models of intestinal cancer are experimental systems in which mice are genetically manipulated to develop malignancies in the gastrointestinal tract. These models enable researchers to study the mechanisms of onset, progression, and metastasis of the disease. They also provide a valuable biological system which is suitable for testing (novel) drugs in vivo. Recently, an in vitro culture model has been established in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. This culture system has been applied to diseased epithelia, such as adenoma, adenocarcinoma, and Barrett's epithelium. These organoids can be particularly useful for studying the mechanisms of intestinal tumors and to test (novel) drugs in vitro. Here, we describe our current laboratory protocols to generate and analyze intestinal tumors and organoids harboring APC and K-Ras double mutations.

  13. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    PubMed

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy.

  14. Alcohol and the risk of colon and rectal cancer with mutations in the K-ras gene.

    PubMed

    Bongaerts, Brenda W C; de Goeij, Anton F P M; van den Brandt, Piet A; Weijenberg, Matty P

    2006-04-01

    The first metabolite of alcohol, acetaldehyde, may trigger replication errors and mutations in DNA, which may predispose to developing colorectal cancer (CRC). In a prospective study on colon and rectal cancer, we investigated the following hypotheses: alcohol consumption is associated with an increased risk of mutations in the K-ras oncogene, and beer consumption is associated with an increased risk of G-->A mutations in this gene. Therefore, we studied the associations between consumption of alcohol and alcoholic beverages and the risk of CRC without and with specific K-ras gene mutations. In 1986, 120,852 men and women, aged 55-69 years, completed a questionnaire on risk factors for cancer. The case-cohort approach was used for data processing and analyses. After 7.3 years of follow-up, excluding the first 2.3 years, complete data from 4,076 subcohort members, 428 colon and 150 rectal cancer patients, were available for data analyses. Incidence rate ratios (RRs) and corresponding 95% confidence intervals (95% CIs) were estimated using Cox proportional hazards models. Compared to abstaining, a total alcohol consumption of 30.0 g/day and more was associated with the risk of colon and rectal cancer with and without a K-ras mutation in both men and women. Independent from alcohol intake, liquor consumption when compared to nonliquor consumption was associated with an increased risk of rectal cancer with a wild type K-ras in men (RR: 2.25, 95% CI: 1.0-5.0). Beer consumption was not clearly associated with the risk of colon and rectal tumors harboring G-->A mutations in the K-ras gene in men. This association could not be assessed in women because of sparse beer consumption. In conclusion, alcohol does not seem to be involved in predisposing to CRC through mutations in the K-ras gene, and specifically beer consumption is not associated with colon and rectal tumors harboring a G-->A mutation.

  15. The Influence of BRAF and KRAS Mutation Status on the Association between Aspirin Use and Survival after Colon Cancer Diagnosis

    PubMed Central

    Reimers, Marlies S.; Swets, Marloes; Bastiaannet, Esther; Prinse, Bianca; van Eijk, Ronald; Lemmens, Valery E. P. P.; van Herk-Sukel, Myrthe P. P.; van Wezel, Tom; Kuppen, Peter J. K.; Morreau, Hans; van de Velde, Cornelis J. H.; Liefers, Gerrit-Jan

    2017-01-01

    Background Use of aspirin after diagnosis of colon cancer has been associated with improved survival. Identification of cancer subtypes that respond to aspirin treatment may help develop personalized treatment regimens. The aim of this study was to investigate the influence of BRAF and KRAS mutation status on the association between aspirin use and overall survival after colon cancer diagnosis. Methods A random selection of 599 patients with colon cancer were analyzed, selected from the Eindhoven Cancer Registry, and BRAF and KRAS mutation status was determined. Data on aspirin use (80 mg) were obtained from the PHARMO Database Network. Parametric survival models with exponential (Poisson) distribution were used. Results Aspirin use after colon cancer diagnosis was associated with improved overall survival in wild-type BRAF tumors, adjusted rate ratio (RR) of 0.60 (95% CI 0.44–0.83). In contrast, aspirin use in BRAF mutated tumors was not associated with an improved survival (RR 1.11, 95% CI 0.57–2.16). P-value for interaction was non-significant. KRAS mutational status did not differentiate in the association between aspirin use and survival. Conclusion Low-dose aspirin use after colon cancer diagnosis was associated with improved survival in BRAF wild-type tumors only. However, the large confidence interval of the rate ratio for the use of aspirin in patients with BRAF mutation does not rule out a possible benefit. These results preclude BRAF and KRAS mutation status to be used as a marker for individualized treatment with aspirin, if aspirin becomes regular adjuvant treatment for colon cancer patients in the future. PMID:28125730

  16. KRAS Mutation Status and Clinical Outcome of Preoperative Chemoradiation With Cetuximab in Locally Advanced Rectal Cancer: A Pooled Analysis of 2 Phase II Trials

    SciTech Connect

    Kim, Sun Young; Shim, Eun Kyung; Yeo, Hyun Yang; Baek, Ji Yeon; Hong, Yong Sang; Kim, Dae Yong; Kim, Tae Won; Kim, Jee Hyun; Im, Seock-Ah; Jung, Kyung Hae; Chang, Hee Jin

    2013-01-01

    Purpose: Cetuximab-containing chemotherapy is known to be effective for KRAS wild-type metastatic colorectal cancer; however, it is not clear whether cetuximab-based preoperative chemoradiation confers an additional benefit compared with chemoradiation without cetuximab in patients with locally advanced rectal cancer. Methods and Materials: We analyzed EGFR, KRAS, BRAF, and PIK3CA mutation status with direct sequencing and epidermal growth factor receptor (EGFR) and Phosphatase and tensin homolog (PTEN) expression status with immunohistochemistry in tumor samples of 82 patients with locally advanced rectal cancer who were enrolled in the IRIX trial (preoperative chemoradiation with irinotecan and capecitabine; n=44) or the ERBIRIX trial (preoperative chemoradiation with irinotecan and capecitabine plus cetuximab; n=38). Both trials were similarly designed except for the administration of cetuximab; radiation therapy was administered at a dose of 50.4 Gy/28 fractions and irinotecan and capecitabine were given at doses of 40 mg/m{sup 2} weekly and 1650 mg/m{sup 2}/day, respectively, for 5 days per week. In the ERBIRIX trial, cetuximab was additionally given with a loading dose of 400 mg/m{sup 2} on 1 week before radiation, and 250 mg/m{sup 2} weekly thereafter. Results: Baseline characteristics before chemoradiation were similar between the 2 trial cohorts. A KRAS mutation in codon 12, 13, and 61 was noted in 15 (34%) patients in the IRIX cohort and 5 (13%) in the ERBIRIX cohort (P=.028). Among 62 KRAS wild-type cancer patients, major pathologic response rate, disease-free survival and pathologic stage did not differ significantly between the 2 cohorts. No mutations were detected in BRAF exon 11 and 15, PIK3CA exon 9 and 20, or EGFR exon 18-24 in any of the 82 patients, and PTEN and EGFR expression were not predictive of clinical outcome. Conclusions: In patients with KRAS wild-type locally advanced rectal cancer, the addition of cetuximab to the chemoradiation with

  17. KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia.

    PubMed

    Malinowska-Ozdowy, K; Frech, C; Schönegger, A; Eckert, C; Cazzaniga, G; Stanulla, M; zur Stadt, U; Mecklenbräuker, A; Schuster, M; Kneidinger, D; von Stackelberg, A; Locatelli, F; Schrappe, M; Horstmann, M A; Attarbaschi, A; Bock, C; Mann, G; Haas, O A; Panzer-Grümayer, R

    2015-08-01

    High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

  18. K-Ras, H-Ras, N-Ras and B-Raf mutation and expression analysis in Wilms tumors: association with tumor growth.

    PubMed

    Dalpa, Efterpi; Gourvas, Victor; Soulitzis, Nikolaos; Spandidos, Demetrios A

    2017-01-01

    Nephroblastoma (Wilms tumor) is a kidney neoplasia, predominately occurring at very young age, resulting from the malignant transformation of renal stem cells. The Ras proto-oncogenes and B-Raf are members of an intracellular cascade pathway, which regulates cell growth and differentiation, and ultimately cancer development. Our objective was to determine the mutation rate and to measure the mRNA levels of the three Ras genes and of B-Raf in formalin-fixed paraffin-embedded tissue samples from 32 patients with nephroblastoma and 10 controls. No mutations were detected in the four studied genes among our Wilms tumors cases, while Ras and B-Raf expression was higher in malignant samples versus controls. Statistical analysis revealed a positive correlation of K-Ras (p < 0.001) and B-Raf (p = 0.006) with tumor size, a negative correlation of K-Ras (p = 0.041) and H-Ras (p = 0.033) with the percentage of tissue necrosis, and an association of N-Ras (p = 0.047) and B-Raf (p = 0.044) with tissue histology. From the above, we deduce that although Ras and B-Raf mutations are rare events in Wilms tumors, their expression pattern suggests that they play an important role in the development and progression of this malignancy.

  19. Treatment with the nitric oxide synthase inhibitor L-NAME provides a survival advantage in a mouse model of Kras mutation-positive, non-small cell lung cancer

    PubMed Central

    Xu, MengMeng; Counter, Christopher M.

    2016-01-01

    Oncogenic mutations in the gene KRAS are commonly detected in non-small cell lung cancer (NSCLC). This disease is inherently difficult to treat, and combinations involving platinum-based drugs remain the therapeutic mainstay. In terms of novel, pharmacologically actionable targets, nitric oxide synthases (NOS) have been implicated in the etiology of KRAS-driven cancers, including lung cancer, and small molecular weight NOS inhibitors have been developed for the treatment of other diseases. Thus, we evaluated the anti-neoplastic activity of the oral NOS inhibitor L-NAME in a randomized preclinical trial using a genetically engineered mouse model of Kras and p53 mutation-positive NSCLC. We report here that L-NAME decreased lung tumor growth in vivo, as assessed by sequential radiological imaging, and provided a survival advantage, perhaps the most difficult clinical parameter to improve upon. Moreover, L-NAME enhanced the therapeutic benefit afforded by carboplatin chemotherapy, provided it was administered as maintenance therapy after carboplatin. Collectively, these results support the clinical evaluation of L-NAME for the treatment of KRAS mutation-positive NSCLC. PMID:27285753

  20. Influence of anatomical subsite on the incidence of microsatellite instability, and KRAS and BRAF mutation rates in patients with colon carcinoma.

    PubMed

    Benedix, Frank; Meyer, Frank; Kube, Rainer; Kropf, Siegfried; Kuester, Doerthe; Lippert, Hans; Roessner, Albert; Krüger, Sabine

    2012-10-15

    There is a growing amount of data supporting the concept that cancers originating from the proximal and distal colon are distinct clinicopathological entities. The incidence of MSI and BRAF mutation is strongly associated with right sided tumor location, whereas there are conflicting results for KRAS mutation rates. However, to date, no data exist whether and to what extent defined colonic subsites influence MSI status, KRAS and BRAF mutation rates. We selected primary colon cancer from 171 patients operated on at our institution between 2007 and 2010. BRAF, KRAS mutation rates and microsatellite instability were determined and correlated with clinicopathological features and tumor location. MSI-h cancers were significantly associated with poor histological grade but a lower rate of distant metastases. KRAS-mutated tumors were linked to lower T-stage and better differentiation. Colon carcinomas with BRAF mutation were significantly associated with distant metastatic spread and poor histological grade. Furthermore, we found that MSI-h status, KRAS and BRAF mutation rates varied remarkably among the colonic subsites irrespective of right- and left-sided origin, respectively. The results of the current study provide further evidence that a simple classification into right- and left-sided colon carcinoma does not represent the complexity of this tumor entity.

  1. K-Ras gene mutation status as a prognostic and predictive factor in patients with colorectal cancer undergoing irinotecan- or oxaliplatin-based chemotherapy

    PubMed Central

    Stec, Rafał; Bodnar, Lubomir; Charkiewicz, Radosław; Korniluk, Jan; Rokita, Marta; Smoter, Marta; Ciechowicz, Marzena; Chyczewski, Lech; Nikliński, Jacek; Kozłowski, Wojciech; Szczylik, Cezary

    2012-01-01

    Background: CRC caused more than 600,000 estimated deaths in 2008. Dysregulated signaling through the RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway due to mutations in K-Ras and B-Raf are common events in CRC. Methods: Incidence of mutations in codons 12 and 13 of K-Ras and exons 11 and 15 of B-Raf were analyzed in amplified PCR products from primary tumors of 273 patients with CRC, and their prognostic and predictive significance was assessed. The prognostic role of clinical and pathological factors was also examined. Results: K-Ras mutations were present in 89 patients (32.6%), of whom 76 (85.4%) had mutations in codon 12 and 10 (11.2%) had mutations in codon 13. B-Raf gene mutations were present in 17 patients (6.9%), of whom 6 (35.3%) had mutations in exon 15. Multivariate analysis revealed a predictive significance for K-Ras mutations with respect to time to progression in patients treated with irinotecan and oxaliplatin as first-line chemotherapy. There was no predictive significance for B-Raf gene mutation status in these patients. The following risk factors were found to affect overall survival (OS) rates: primary tumor location, lymph node involvement grade, carcinoembryonic antigen (CEA) level before treatment, and performance status according to WHO criteria. Conclusions: Based on the results of this study, K-Ras mutation status may be a suitable indicator of patient eligibility and a prognostic indicator for responsiveness to anti-EGFR therapy alone, or in combination with chemotherapy. Also, K-Ras mutation status may predict time to progression in patients treated with irinotecan and oxaliplatin. PMID:22909976

  2. Mutated KRAS Results in Overexpression of DUSP4, a MAP-Kinase Phosphatase, and SMYD3, a Histone Methyltransferase, in Rectal Carcinomas

    PubMed Central

    Gaedcke, Jochen; Grade, Marian; Jung, Klaus; Camps, Jordi; Jo, Peter; Emons, Georg; Gehoff, Anastasia; Sax, Ulrich; Schirmer, Markus; Becker, Heinz; Beissbarth, Tim; Ried, Thomas; Ghadimi, B. Michael

    2012-01-01

    Mutations of the KRAS oncogene are predictive for resistance to treatment with antibodies against the epithelial growth factor receptor in patients with colorectal cancer. Overcoming this therapeutic dilemma could potentially be achieved by the introduction of drugs that inhibit signaling pathways that are activated by KRAS mutations. To identify comprehensively such signaling pathways we profiled pretreatment biopsies and normal mucosa from 65 patients with locally advanced rectal cancer - 30 of which carried mutated KRAS - using global gene expression microarrays. By comparing all tumor tissues exclusively to matched normal mucosa, we could improve assay sensitivity, and identified a total of 22,297 features that were differentially expressed (adjusted P-value <0.05) between normal mucosa and cancer, including several novel potential rectal cancer genes. We then used this comprehensive description of the rectal cancer transcriptome as the baseline for identifying KRAS-dependent alterations. The presence of activating KRAS mutations is significantly correlated to an upregulation of 13 genes (adjusted P-value <0.05), among them DUSP4, a MAP-kinase phosphatase, and SMYD3, a histone methyltransferase. Inhibition of the expression of both genes has previously been shown using the MEK1-inhibitor PD98059 and the antibacterial compound Novobiocin, respectively. These findings suggest a potential approach to overcome resistance to treatment with antibodies against the epithelial growth factor receptor in patients with KRAS-mutant rectal carcinomas. PMID:20725992

  3. Cutaneous metastasis as an initial presentation of lung adenocarcinoma with KRAS mutation: a case report and literature review

    PubMed Central

    Wu, Shenhong; Karbowitz, Stephen R.; Morgenstern, Nora; Rose, David R.

    2014-01-01

    Cutaneous metastasis as an initial presentation occurs in 0.8% of patients with internal malignancies, and is poorly understood in its molecular pathogenesis. We reported a case in which a 61-year-old male patient initially presented with rapidly growing skin nodule on his left chest wall, then developed dyspnea and loss of weight. Echocardiogram showed a large pericardial effusion with right ventricular collapse. PET/CT revealed moderate pleural effusion and multiple lymphadenopathies with hypermetabolic concentration of radiotracer in the lymph nodes as well as in the chest wall skin mass. Biopsy of the skin mass and pericardial/pleural fluids revealed metastatic adenocarcinoma consistent with lung primary with KRAS mutation. Palliative chemotherapy was administered without resulting in any improvement. This is the first case report to show that KRAS-mutant lung adenocarcinoma can be associated with cutaneous metastasis. PMID:27358853

  4. Association of K-ras codon 12 transversions with short survival in non-small cell lung cancer.

    PubMed

    Vega, F; Iniesta, P; Caldes, T; Sanchez, A; Lopez, J; Dejuan, C; Diazrubio, E; Torres, A; Balibrea, J; Benito, M

    1996-12-01

    K-ras activating point mutations appear to have a role in human lung cancer, however, the prognostic significance of these abnormalities remains unclear. The aim of our work was to clarify the role of K-ras mutations as prognostic indicators in patients affected by non-small cell lung cancer (NSCLC). We studied 94 resected primary NSCLCs for K-ras mutations by the PCR-RFLP technique, followed by sequencing. K-ras activating mutations were present in 34% of tumors, a higher incidence being detected in adenocarcinomas. Comparing the impact of K-ras mutation types, we found that K-ras transversions were associated with a shorter survival in NSCLC.

  5. Prevalence and coexistence of KRAS, BRAF, PIK3CA, NRAS, TP53, and APC mutations in Indian colorectal cancer patients: Next-generation sequencing-based cohort study.

    PubMed

    Jauhri, Mayank; Bhatnagar, Akanksha; Gupta, Satish; Bp, Manasa; Minhas, Sachin; Shokeen, Yogender; Aggarwal, Shyam

    2017-02-01

    Colorectal cancer incidences are on a rise in India. In this study, we have analyzed the mutation frequencies of six potential biomarkers, their coexistence, association with clinicopathological characteristics, and tumor location in Indian colorectal cancer patients. Next-generation sequencing was performed to identify mutations in the six potential biomarker genes using formalin-fixed paraffin-embedded tissue blocks of 112 colorectal cancer patients. The mutation frequency observed in KRAS, BRAF, PIK3CA, NRAS, TP53, and APC was 35.7%, 7.1%, 16.1%, 6.3%, 39.3%, and 29.5%, respectively. The significant associations of mutations were KRAS with age less than 60 years (p = 0.041), PIK3CA with males (p = 0.032), tumor stage I-II (p = 0.013), lack of metastasis in lymph nodes (p = 0.040), NRAS with rectum (p = 0.002), and APC with T2 stage of tumor growth (p = 0.013). No single patient harbored mutations in these six genes or any five genes simultaneously. Significance was noted in coexistence of KRAS with APC (p = 0.024) and mutual exclusion of KRAS with BRAF (p = 0.029). PIK3CA exon 9 was observed to be more frequently associated with KRAS mutations than PIK3CA exon 20 (p = 0.072). NRAS mutations were mutually exclusive with BRAF and PIK3CA mutations. As per our knowledge, this is the first next-generation sequencing-based biomarker study in Indian colorectal cancer patients. Frequent coexistence of gene mutations in pairs and triplets suggests that synergistic effect of overlapping mutations might further trigger the disease. In addition, infrequent coexistence of multiple gene mutations hints toward different signaling pathways for colorectal cancer tumorigenesis.

  6. A combined oncogenic pathway signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction

    PubMed Central

    Tian, Sun; Simon, Iris; Moreno, Victor; Roepman, Paul; Tabernero, Josep; Snel, Mireille; van't Veer, Laura; Salazar, Ramon; Bernards, Rene

    2013-01-01

    Objective To develop gene expression profiles that characterise KRAS-, BRAF- or PIK3CA-activated- tumours, and to explore whether these profiles might be helpful in predicting the response to the epidermal growth factor receptor (EGFR) pathway inhibitors better than mutation status alone. Design Fresh frozen tumour samples from 381 colorectal cancer (CRC) patients were collected and mutations in KRAS, BRAF and PIK3CA were assessed. Using microarray data, three individual oncogenic and a combined model were developed and validated in an independent set of 80 CRC patients, and in a dataset from metastatic CRC patients treated with cetuximab. Results 175 tumours (45.9%) harboured oncogenic mutations in KRAS (30.2%), BRAF (11.0%) and PIK3CA (11.5%). Activating mutation signatures for KRAS (75 genes), for BRAF (58 genes,) and for PIK3CA (49 genes) were developed. The development of a combined oncogenic pathway signature-classified tumours as ‘activated oncogenic’, or as ‘wildtype-like’ with a sensitivity of 90.3% and a specificity of 61.7%. The identified signature revealed other mechanisms that can activate ERK/MAPK pathway in KRAS, BRAF and PIK3CA wildtype patients. The combined signature is associated with response to cetuximab treatment in patients with metastatic CRC (HR 2.51, p<0.0009). Conclusion A combined oncogenic pathway signature allows the identification of patients with an active EGFR-signalling pathway that could benefit from downstream pathway inhibition. PMID:22798500

  7. Assessment of MAGE-A expression in resected non-small cell lung cancer in relation to clinicopathologic features and mutational status of EGFR and KRAS.

    PubMed

    Ayyoub, Maha; Memeo, Lorenzo; Alvarez-Fernández, Emilio; Colarossi, Cristina; Costanzo, Rosario; Aiello, Eleonora; Martinetti, Daniela; Valmori, Danila

    2014-10-01

    Non-small cell lung cancer (NSCLC) is a major public health problem, accounting for more cancer-related deaths than any other cancer. Both immunotherapy, based on the expression of tumor-specific antigens, and targeted therapy, based on the presence of oncogenic mutations, are under development for NSCLC. In this study, we analyzed the expression of MAGE-A, a cancer-testis antigen, in tumors from a cohort of patients with resected NSCLC with respect to their clinicopathologic characteristics and their mutational status for the EGFR and KRAS genes. We found MAGE-A expression by IHC in 43% of the tumors. MAGE-A expression was significantly more frequent in squamous tumors than in adenocarcinomas, did not correlate with disease stage, but was correlated significantly with high tumor grade and worse survival. EGFR and KRAS mutations were present in adenocarcinomas, but not in squamous tumors. Whereas the presence of EGFR mutations did not seem to affect survival, the presence of KRAS mutations was associated with early-stage disease and better survival. MAGE-A expression was absent from adenocarcinomas with KRAS mutations, but not significantly different in tumors with or without EGFR mutations. Together, the reported results provide guidance for the design of combination therapies in patients with NSCLC.

  8. Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal.

    PubMed

    Hong, Hue-Hua L; Hoenerhoff, Mark J; Ton, Thai-Vu; Herbert, Ronald A; Kissling, Grace E; Hooth, Michelle J; Behl, Mamta; Witt, Kristine L; Smith-Roe, Stephanie L; Sills, Robert C; Pandiri, Arun R

    2015-08-01

    Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kirsten rat sarcoma oncogene homolog (Kras), epidermal growth factor receptor (Egfr), and tumor protein 53 (Tp53) mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD)-induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominant in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assay indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents.

  9. Kras, Egfr, and Tp53 Mutations in B6C3F1/N Mouse and F344/NTac Rat Alveolar/Bronchiolar Carcinomas Resulting from Chronic Inhalation Exposure to Cobalt Metal

    PubMed Central

    Hong, Hue-Hua L.; Hoenerhoff, Mark J.; Ton, Thai-Vu; Herbert, Ronald A.; Kissling, Grace E.; Hooth, Michelle J.; Behl, Mamta; Witt, Kristine L.; Smith-Roe, Stephanie L.; Sills, Robert C.; Pandiri, Arun R.

    2015-01-01

    Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kras, Egfr and Tp53 mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD) induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors, and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors, and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominantly in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assays indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents. PMID:26059825

  10. Endoscopic ultrasound-guided fine-needle aspiration plus KRAS and GNAS mutation in malignant intraductal papillary mucinous neoplasm of the pancreas

    PubMed Central

    Bournet, Barbara; Vignolle-Vidoni, Alix; Grand, David; Roques, Céline; Breibach, Florence; Cros, Jérome; Muscari, Fabrice; Carrère, Nicolas; Selves, Janick; Cordelier, Pierre; Buscail, Louis

    2016-01-01

    Background: KRAS and GNAS mutations are common in intraductal papillary mucinous neoplasia of the pancreas (IPMN). The aims of this study were to assess the role of pre-therapeutic cytopathology combined with KRAS and GNAS mutation assays within cystic fluid sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to predict malignancy of IPMN. Patients and methods: We prospectively included 37 IPMN patients with clinical and/or imaging predictors of malignancy (men: 24; mean age: 69.5 years). Cytopathology (performed on cystic fluid and/or IPMN nodules), KRAS (Exon 2, codon 12) and GNAS (Exon 8, codon 201) mutations assays (using TaqMan® allelic discrimination) were performed on EUS-FNA material. The final diagnosis was obtained from IPMN resections (n = 18); surgical biopsies, EUS-FNA analyses, and follow-up (n = 19): 10 and 27 IPMN were benign and malignant, respectively. Results: Sensitivity, specificity, positive and negative predictive values, and accuracy of cytopathology alone to diagnose IPMN malignancy were 55 %, 100 %, 100 %, 45 %, and 66 %, respectively. When KRAS-mutation analysis was combined with cytopathology these values were 92 %, 50 %, 83 %, 71 %, and 81 %, respectively. GNAS assays did not improve the performances of cytopathology alone or those of cytopathology plus a KRAS assay. Conclusions: In patients with a likelihood of malignant IPMN at pre-therapeutic investigation, testing for KRAS mutations in cystic fluid sampling by EUS-FNA improved the results of cytopathology for the diagnosis of malignancy whereas GNAS mutation assay did not. PMID:27995180

  11. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma

    PubMed Central

    Park, Jangchul; Kobayashi, Yoshihisa; Urayama, Kevin Y.; Yamaura, Hidekazu; Yatabe, Yasushi; Hida, Toyoaki

    2016-01-01

    This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB–IV) and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups’ characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159), KRAS mutations (n = 55), or ALK rearrangements (n = 51). Among the three groups, we evaluated only patients with stage IIIB–IV lung adenocarcinoma who had EGFR mutations (n = 126), KRAS mutations (n = 35), or ALK rearrangements (n = 47). We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009). Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003). Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively). Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049). Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively). Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively). In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  12. Sequence-specific DNA alkylation targeting for Kras codon 13 mutation by pyrrole-imidazole polyamide seco-CBI conjugates.

    PubMed

    Taylor, Rhys Dylan; Asamitsu, Sefan; Takenaka, Tomohiro; Yamamoto, Makoto; Hashiya, Kaori; Kawamoto, Yusuke; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2014-01-27

    Hairpin N-methylpyrrole-N-methylimidazole polyamide seco-CBI conjugates 2-6 were designed for synthesis by Fmoc solid-phase synthesis, and their DNA-alkylating activities against the Kras codon 13 mutation were compared by high-resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5'-ACGTCACCA-3' (site 2), including minor 1 bp-mismatch alkylation against wild type 5'-ACGCCACCA-3' (site 3). Conjugate 6, which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5'-ACGTCACCA-3' (site 2). The hairpin polyamide seco-CBI conjugate 5 thus alkylates according to Dervan's pairing rule with the pairing recognition which β/β pair targets T-A and A-T pairs. SPR and a computer-minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.

  13. Higher quality of molecular testing, an unfulfilled priority: Results from external quality assessment for KRAS mutation testing in colorectal cancer.

    PubMed

    Tembuyser, Lien; Ligtenberg, Marjolijn J L; Normanno, Nicola; Delen, Sofie; van Krieken, J Han; Dequeker, Elisabeth M C

    2014-05-01

    Precision medicine is now a key element in clinical oncology. RAS mutational status is a crucial predictor of responsiveness to anti-epidermal growth factor receptor agents in metastatic colorectal cancer. In an effort to guarantee high-quality testing services in molecular pathology, the European Society of Pathology has been organizing an annual KRAS external quality assessment program since 2009. In 2012, 10 formalin-fixed, paraffin-embedded samples, of which 8 from invasive metastatic colorectal cancer tissue and 2 artificial samples of cell line material, were sent to more than 100 laboratories from 26 countries with a request for routine KRAS testing. Both genotyping and clinical reports were assessed independently. Twenty-seven percent of the participants genotyped at least 1 of 10 samples incorrectly. In total, less than 5% of the distributed specimens were genotyped incorrectly. Genotyping errors consisted of false negatives, false positives, and incorrectly genotyped mutations. Twenty percent of the laboratories reported a technical error for one or more samples. A review of the written reports showed that several essential elements were missing, most notably a clinical interpretation of the test result, the method sensitivity, and the use of a reference sequence. External quality assessment serves as a valuable educational tool in assessing and improving molecular testing quality and is an important asset for monitoring quality assurance upon incorporation of new biomarkers in diagnostic services.

  14. Molecular spectrum of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC somatic gene mutations in Arab patients with colorectal cancer: determination of frequency and distribution pattern

    PubMed Central

    Al-Shamsi, Humaid O.; Jones, Jeremy; Fahmawi, Yazan; Dahbour, Ibrahim; Tabash, Aziz; Abdel-Wahab, Reham; Abousamra, Ahmed O. S.; Shaw, Kenna R.; Xiao, Lianchun; Hassan, Manal M.; Kipp, Benjamin R.; Kopetz, Scott; Soliman, Amr S.; McWilliams, Robert R.; Wolff, Robert A.

    2016-01-01

    Background The frequency rates of mutations such as KRAS, NRAS, BRAF, and PIK3CA in colorectal cancer (CRC) differ among populations. The aim of this study was to assess mutation frequencies in the Arab population and determine their correlations with certain clinicopathological features. Methods Arab patients from the Arab Gulf region and a population of age- and sex-matched Western patients with CRC whose tumors were evaluated with next-generation sequencing (NGS) were identified and retrospectively reviewed. The mutation rates of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC were recorded, along with clinicopathological features. Other somatic mutation and their rates were also identified. Fisher’s exact test was used to determine the association between mutation status and clinical features. Results A total of 198 cases were identified; 99 Arab patients and 99 Western patients. Fifty-two point seven percent of Arab patients had stage IV disease at initial presentation, 74.2% had left-sided tumors. Eighty-nine point two percent had tubular adenocarcinoma and 10.8% had mucinous adenocarcinoma. The prevalence rates of KRAS, NRAS, BRAF, PIK3CA, TP53, APC, SMAD, FBXW7 mutations in Arab population were 44.4%, 4%, 4%, 13.1%, 52.5%, 27.3%, 2% and 3% respectively. Compared to 48.4%, 4%, 4%, 12.1%, 47.5%, 24.2%, 11.1% and 0% respectively in matched Western population. Associations between these mutations and patient clinicopathological features were not statistically significant. Conclusions This is the first study to report comprehensive hotspot mutations using NGS in Arab patients with CRC. The frequency of KRAS, NRAS, BRAF, TP53, APC and PIK3CA mutations were similar to reported frequencies in Western population except SMAD4 that had a lower frequency and higher frequency of FBXW7 mutation. PMID:28078112

  15. K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine.

    PubMed

    Femia, Angelo Pietro; Tarquini, Elena; Salvadori, Maddalena; Ferri, Stefania; Giannini, Augusto; Dolara, Piero; Caderni, Giovanna

    2008-01-01

    K-ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin-depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2-dimethylhydrazine (DMH). The frequency of lesions with K-ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K-ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up-regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K-ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors.

  16. The expression of SALL4 is significantly associated with EGFR, but not KRAS or EML4-ALK mutations in lung cancer

    PubMed Central

    Jia, Xiangbo; Qian, Rulin; Zhang, Binbin

    2016-01-01

    Background Lung cancer is the leading cause of cancer-related deaths worldwide; unfortunately, its prognosis is still very poor. Therefore, developing the target molecular is very important for lung cancer diagnosis and treatment, especially in the early stage. With this in view, spalt-like transcription factor 4 (SALL4) is considered a potential biomarker for diagnosis and prognosis in cancers, including lung cancer. Methods In order to better investigate the association between the expression of SALL4 and driver genes mutation, 450 histopathologically diagnosed patients with lung cancer and 11 non-cancer patients were enrolled to test the expression of SALL4 and the status of driver genes mutation. This investigation included epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene homolog (KRAS), and a fusion gene of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK). Results The results of the study showed that females harbored more EGFR mutation in adenocarcinoma (ADC). The mutation rate of KRAS and EML4-ALK was about 5%, and the double mutations of EGFR/EML4-ALK were higher than EGFR/KRAS. In the expression analysis, the expression of SALL4 was much higher in cancer tissues than normally expected, especially in tissues that carried EGFR mutation (P<0.05), however, there were no significant differences between different mutation types. Likewise, there were no significant differences between expression of SALL4 and KRAS and EML4-ALK mutations. Conclusions SALL4 is up regulated in lung cancer specimens and harbors EGFR mutation; this finding indicates that SALL4 expression may be relevant with EGFR, which could provide a new insight to lung cancer therapy. The mechanism needs further investigation and analysis. PMID:27867542

  17. Correlation of immunohistochemical staining p63 and TTF-1 with EGFR and K-ras mutational spectrum and diagnostic reproducibility in non small cell lung carcinoma.

    PubMed

    Thunnissen, Erik; Boers, Evan; Heideman, Daniëlle A M; Grünberg, Katrien; Kuik, Dirk J; Noorduin, Arnold; van Oosterhout, Matthijs; Pronk, Divera; Seldenrijk, Cees; Sietsma, Hannie; Smit, Egbert F; van Suylen, Robertjan; von der Thusen, Jan; Vrugt, Bart; Wiersma, Anne; Witte, Birgit I; den Bakker, Michael

    2012-12-01

    For treatment purposes, distinction between squamous cell carcinoma and adenocarcinoma is important. The aim of this study is to examine the diagnostic accuracy on lung cancer small biopsies for the distinction between adenocarcinoma and squamous cell carcinoma and relate these to immunohistochemical and KRAS and EGFR mutation analysis. An interobserver study was performed on 110 prospectively collected biopsies obtained by bronchoscopy or transthoracic needle biopsy of patients with non-small cell lung cancer. The diagnosis was correlated with immunohistochemical (IHC) analysis for markers of adeno- (TTF1 and/or mucin positivity) and squamous cell differentiation (P63 and CK5/6) as well as KRAS and EGFR mutation analysis. Eleven observers independently read H&E-stained slides of 110 cases, resulting in a kappa value of 0.55 ± 0.10. The diagnosis non-small cell lung cancer not otherwise specified was given on average on 29.5 % of the biopsies. A high concordance was observed between hematoxylin-eosin-based consensus diagnosis (≥8/11 readings concordant) and IHC markers. In all cases with EGFR (n = 1) and KRAS (n = 20) mutations, adenodifferentiation as determined by IHC was present and p63 staining was absent. In 2 of 25 cases with a consensus diagnosis of squamous cell carcinoma, additional stainings favored adenodifferentation, and a KRAS mutation was present. P63 is most useful for distinction between EGFR/KRAS mutation positive and negative patients. In the diagnostic work-up of non-small cell lung carcinoma the limited reproducibility on small biopsies is optimized with immunohistochemical analysis, resulting in reliable delineation for predictive analysis.

  18. Roles of EGFR and KRAS Mutations in the Treatment Of Patients With Non–Small-Cell Lung Cancer

    PubMed Central

    Langer, Corey J.

    2011-01-01

    After decades of empirical treatment, molecular subtypes of non–small-cell lung cancer (NSCLC) are now emerging that may enable us to target treatment for patients and increase the likelihood of response. Of the biomarkers under evaluation, gene mutations are gaining recognition as predictive markers for anti–epidermal-growth factor receptor (EGFR) therapy. To date, unlike the situation in colorectal cancer, mutation of the v-Ki-Ras-2 Kirsten rat sarcoma viral oncogene homolog (KRAS) has an inconclusive role in NSCLC and should not be used to exclude patients from anti-EGFR therapy. For first-line NSCLC therapy, EGFR mutation status constitutes a prudent test to identify patients who are most likely to benefit from EGFR–tyrosine kinase inhibitor therapy rather than from chemotherapy. In first-line maintenance and relapsed (second-line or third-line) settings, clinical data support the use of erlotinib (Tarceva), as currently indicated, without regard to evaluation of EGFR mutation status. All patient subsets have been shown to benefit with prolonged progression-free and overall survival. PMID:21785539

  19. KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC: A meta-analysis of 41 studies.

    PubMed

    Pan, Wei; Yang, Yan; Zhu, Hongcheng; Zhang, Youcheng; Zhou, Rongping; Sun, Xinchen

    2016-02-16

    Mutation of oncogene KRAS is common in non-small cell lung cancer (NSCLC), however, its clinical significance is still controversial. Independent studies evaluating its prognostic and predictive value usually drew inconsistent conclusions. Hence, We performed a meta-analysis with 41 relative publications, retrieved from multi-databases, to reconcile these controversial results and to give an overall impression of KRAS mutation in NSCLC. According to our findings, KRAS mutation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in early stage resected NSCLC (hazard ratio or HR=1.56 and 1.57, 95% CI 1.39-1.76 and 1.17-2.09 respectively), and with inferior outcomes of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) treatment and chemotherapy (relative risk or RR=0.21 and 0.66 for objective response rate or ORR, 95% CI 0.12-0.39 and 0.54-0.81 respectively; HR=1.46 and 1.30 for progression-free survival or PFS, 95%CI 1.23-1.74 and 1.14-1.50 respectively) in advanced NSCLC. When EGFR mutant patients were excluded, KRAS mutation was still significantly associated with worse OS and PFS of EGFR-TKIs (HR=1.40 and 1.35, 95 % CI 1.21-1.61 and 1.11-1.64). Although KRAS mutant patients presented worse DFS and PFS of chemotherapy (HR=1.33 and 1.11, 95% CI 0.97-1.84 and 0.95-1.30), and lower response rate to EGFR-TKIs or chemotherapy (RR=0.55 and 0.88, 95 % CI 0.27-1.11 and 0.76-1.02), statistical differences were not met. In conclusion, KRAS mutation is a weak, but valid predictor for poor prognosis and treatment outcomes in NSCLC. There's a need for developing target therapies for KRAS mutant lung cancer and other tumors.

  20. CXCR2 signaling regulates KRAS(G12D)-induced autocrine growth of pancreatic cancer

    PubMed Central

    Purohit, Abhilasha; Varney, Michelle; Rachagani, Satyanarayana; Ouellette, Michel M.; Batra, Surinder K.; Singh, Rakesh K.

    2016-01-01

    Pharmacological inhibition of RAS, the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS(G12D)-induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL-Kras(G12D) mice. Knocking-down CXCR2 in KRAS(G12D)-bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS(G12D)-bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS(G12D)-bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS(G12D)-induced growth transformation and progression in PDAC. PMID:26771140

  1. Hyperplastic (serrated) polyps of the colorectum: relationship of CpG island methylator phenotype and K-ras mutation to location and histologic subtype.

    PubMed

    O'Brien, Michael J; Yang, Shi; Clebanoff, Jennifer L; Mulcahy, Elizabeth; Farraye, Francis A; Amorosino, Mark; Swan, Niall

    2004-04-01

    We investigated the frequency of promoter region CpG island methylation (CIM) of hMLH1, MGMT, MINT1, MINT2, and p16 and K-ras mutations in a total of 79 hyperplastic (serrated) polyps (HPs) from 75 patients and correlated the molecular profiles to polyp location in the colorectum, histologic variation, and other factors. Methylation-specific PCR (MS-PCR) was used to assay CIM status. HPs that showed CIM of one or more or two or more of the genes assayed were classified as CpG island methylator phenotype (CIMP) and CIMP-high (CIMP-H), respectively. PCR restriction fragment length polymorphism was used to assay K-ras codon 12 and 13 mutations. Logistic regression indicated a statistically significant trend for increasing odds for CIMP (P = 0.002) and CIMP-H (P < 0.001) according to proximity to the cecum or distance from the rectum. Conversely, K-ras codon 12 mutation was present in 13 of 40 (32.5%) distally located HPs compared with 2 of 39 (5.1%) proximal HPs (P = 0.006). Histologic subtype distribution varied by proximal and distal locations. Frequency of CIMP in serrated polyps with abnormal proliferation (SPAPs), differed significantly from goblet cell serrated polyps (GCSPs) (24 of 26, 92.3% vs. 6 of 13, 46.2%) (P = 0.003) and microvesicular serrated polyps (MVSPs) (26 of 38, 68.4%) (P = 0.03). Frequency of K-ras mutation in GCSPs (7 of 13, 54%) differed from that of MVSPs (6 of 38, 16%) (P = 0.01) and SPAPs (2 of 26, 8%) (P = 0.003). Location in the colorectum and histologic subtype were major determinants of the molecular profile of HPs. The molecular findings of CIMP and K-ras mutations appear to encompass most if not all HPs; CIMP profiles suggest that SPAP is the most advanced morphologic variant. We postulate that MVSP and GCSP may be precursor lesions that, if proximally located or larger, can progress to SPAP. Frequent K-ras mutations and infrequent CIMP distinguish the distal GCSP variant.

  2. K-ras oncogene DNA sequences in pink salmon in streams impacted by the Exxon Valdez oil spill: no evidence of oil-induced heritable mutations.

    PubMed

    Cronin, Matthew A; Wickliffe, Jeffrey K; Dunina, Yelena; Baker, Robert J

    2002-08-01

    It was hypothesized in previous studies that the Exxon Valdez oil spill in Prince William Sound, Alaska, induced heritable mutations and resulted in mortality of pink salmon (Oncorhynchus gorbuscha) embryos. In one of these studies, laboratory exposure of pink salmon embryos to crude oil resulted in apparent mutation-induction in exon 1 and exon 2 of the K-ras oncogene, but no fish from the area impacted by the oil spill were analyzed. We assessed K-ras exon 1 and exon 2 DNA sequences in pink salmon from five streams that were oiled and five streams that were not oiled by the Exxon Valdez oil spill in Prince William Sound, and two streams with natural oil seeps and one stream without seeps on the Alaska Peninsula. Of the 79 fish analyzed for exon 1 and the 89 fish analyzed for exon 2, none had the nucleotide substitutions representing the mutations induced in the laboratory study. Other variable nucleotides occurred in similar proportions in oiled and non-oiled streams and probably represent natural allelic variation. These data do not support the hypothesis that heritable mutations in the K-ras gene were induced by the Exxon Valdez oil spill or oil seeps.

  3. NVP-BKM120, a novel PI3K inhibitor, shows synergism with a STAT3 inhibitor in human gastric cancer cells harboring KRAS mutations

    PubMed Central

    PARK, EUNJU; PARK, JINAH; HAN, SAE-WON; IM, SEOCK-AH; KIM, TAE-YOU; OH, DO-YOUN; BANG, YUNG-JUE

    2012-01-01

    Aberrations of Phosphoinositide 3-kinase (PI3K)/AKT signaling are frequently observed in many types of cancer, promoting its emergence as a promising target for cancer treatment. PI3K can become activated by various pathways, one of which includes RAS. RAS can not only directly activate the PI3K/AKT pathway via binding to p110 of PI3K, but also regulates mTOR via ERK or RSK independently of the PI3K/AKT pathway. Thus, actively mutated RAS can constitutively activate PI3K signaling. Additionally, in RAS tumorigenic transformation, signal transducer and activator of transcription 3 (STAT3) has been known also to be required. In this study, we examined the efficacy of NVP-BKM120, a pan-class I PI3K inhibitor in human gastric cancer cells and hypothesized that the combined inhibition of PI3K and STAT3 would be synergistic in KRAS mutant gastric cancer cells. NVP-BKM120 demonstrated anti-proliferative activity in 11 human gastric cancer cell lines by decreasing mTOR downstream signaling. But NVP-BKM120 treatment increased p-AKT by subsequent abrogation of feedback inhibition by stabilizing insulin receptor substrate-1. In KRAS mutant gastric cancer cells, either p-ERK or p-STAT3 was also increased upon treatment of NVP-BKM120. The synergistic efficacy study demonstrated that dual PI3K and STAT3 blockade showed a synergism in cells harboring mutated KRAS by inducing apoptosis. The synergistic effect was not seen in KRAS wild-type cells. Together, these findings suggest for the first time that the dual inhibition of PI3K and STAT3 signaling may be an effective therapeutic strategy for KRAS mutant gastric cancer patients. PMID:22159814

  4. A RAS renaissance: emerging targeted therapies for KRAS-mutated non-small cell lung cancer.

    PubMed

    Vasan, Neil; Boyer, Julie L; Herbst, Roy S

    2014-08-01

    Of the numerous oncogenes implicated in human cancer, the most common and perhaps the most elusive to target pharmacologically is RAS. Since the discovery of RAS in the 1960s, numerous studies have elucidated the mechanism of activity, regulation, and intracellular trafficking of the RAS gene products, and of its regulatory pathways. These pathways yielded druggable targets, such as farnesyltransferase, during the 1980s to 1990s. Unfortunately, early clinical trials investigating farnesyltransferase inhibitors yielded disappointing results, and subsequent interest by pharmaceutical companies in targeting RAS waned. However, recent advances including the identification of novel regulatory enzymes (e.g., Rce1, Icmt, Pdeδ), siRNA-based synthetic lethality screens, and fragment-based small-molecule screens, have resulted in a "Ras renaissance," signified by new Ras and Ras pathway-targeted therapies that have led to new clinical trials of patients with Ras-driven cancers. This review gives an overview of KRas signaling pathways with an emphasis on novel targets and targeted therapies, using non-small cell lung cancer as a case example.

  5. A RAS Renaissance: Emerging Targeted Therapies for KRAS-Mutated Non–Small Cell Lung Cancer

    PubMed Central

    Vasan, Neil; Boyer, Julie; Herbst, Roy S.

    2017-01-01

    Of the numerous oncogenes implicated in human cancer, the most common and perhaps the most elusive to target pharmacologically is RAS. Since the discovery of RAS in the 1960s, numerous studies have elucidated the mechanism of activity, regulation, and intracellular trafficking of the RAS gene products, and of its regulatory pathways. These pathways yielded druggable targets, such as farnesyltransferase during the 1980s–1990s. Unfortunately, early clinical trials investigating farnesyltransferase inhibitors yielded disappointing results, and subsequent interest by pharmaceutical companies in targeting RAS waned. However, recent advances including the identification of novel regulatory enzymes (e.g. Rce1, Icmt, Pdeδ), siRNA-based synthetic lethality screens, and fragment-based small molecule screens have resulted in a “Ras renaissance”, signified by new Ras and Ras-pathway targeted therapies that have led to new clinical trials of patients with Ras-driven cancers. This review gives an overview of KRas signaling pathways with an emphasis on novel targets and targeted therapies, using non-small cell lung cancer (NSCLC) as a case example. PMID:24893629

  6. KRAS-mutation status dependent effect of zoledronic acid in human non-small cell cancer preclinical models

    PubMed Central

    Kenessey, István; Kói, Krisztina; Horváth, Orsolya; Cserepes, Mihály; Molnár, Dávid; Izsák, Vera; Dobos, Judit; Hegedűs, Balázs

    2016-01-01

    Background In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. Results We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. Materials and methods Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. Conclusions The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy. PMID:27780929

  7. TIMP-1 is under regulation of the EGF signaling axis and promotes an aggressive phenotype in KRAS-mutated colorectal cancer cells: A potential novel approach to the treatment of metastatic colorectal cancer

    PubMed Central

    Christensen, Ib J.; Nordgaard, Cathrine; Noer, Julie; Guren, Tormod K.; Glimelius, Bengt; Sorbye, Halfdan; Ikdahl, Tone; Kure, Elin H.; Tveit, Kjell M.; Nielsen, Hans J.

    2016-01-01

    It is now widely accepted that therapeutic antibodies targeting epidermal growth factor receptor (EGFR) can have efficacy in KRAS wild-type advanced colorectal cancer (CRC) patients. What remains to be ascertained is whether a subgroup of KRAS-mutated CRC patients might not also derive benefit from EGFR inhibitors. Metalloproteinase inhibitor 1 (TIMP-1) is a pleiotropic factor predictive of survival outcome of CRC patients. Levels of TIMP-1 were measured in pre-treatment plasma samples (n = 426) of metastatic CRC patients randomized to Nordic FLOX (5-fluorouracil and oxaliplatin) +/− cetuximab (NORDIC VII study). Multivariate analysis demonstrated a significant interaction between plasma TIMP-1 protein levels, KRAS status and treatment with patients bearing KRAS mutated tumors and high TIMP-1 plasma level (> 3rd quartile) showing a significantly longer overall survival if treated with cetuximab (HR, 0.48; 95% CI, 0.25 to 0.93). To gain mechanistic insights into this association we analyzed a set of five different CRC cell lines. We show here that EGFR signaling induces TIMP-1 expression in CRC cells, and that TIMP-1 promotes a more aggressive behavior, specifically in KRAS mutated cells. The two sets of data, clinical and in vitro, are complementary and support each other, lending strength to our contention that TIMP- 1 plasma levels can identify a subset of patients with KRAS-mutated metastatic CRC that will have benefit from EGFR-inhibition therapy. PMID:27509063

  8. Dovitinib (TKI258), a multi-target angiokinase inhibitor, is effective regardless of KRAS or BRAF mutation status in colorectal cancer

    PubMed Central

    Lee, Choong-Kun; Lee, Myung Eun; Lee, Won Suk; Kim, Jeong Min; Park, Kyu Hyun; Kim, Tae Soo; Lee, Kang Young; Ahn, Joong Bae; Chung, Hyun Cheol; Rha, Sun Young

    2015-01-01

    Introduction: We aimed to determine whether KRAS and BRAF mutant colorectal cancer (CRC) cells exhibit distinct sensitivities to the multi-target angiokinase inhibitor, TKI258 (dovitinib). Materials and methods: We screened 10 CRC cell lines by using receptor tyrosine kinase (RTK) array to identify activated RTKs. MTT assays, anchorage-independent colony-formation assays, and immunoblotting assays were performed to evaluate the in vitro anti-tumor effects of TKI258. In vivo efficacy study followed by pharmacodynamic evaluation was done. Results: Fibroblast Growth Factor Receptor 1 (FGFR1) and FGFR3 were among the most highly activated RTKs in CRC cell lines. In in vitro assays, the BRAF mutant HT-29 cells were more resistant to the TKI258 than the KRAS mutant LoVo cells. However, in xenograft assays, TKI258 equally delayed the growth of tumors induced by both cell lines. TUNEL assays showed that the apoptotic index was unchanged following TKI258 treatment, but staining for Ki-67 and CD31 was substantially reduced in both xenografts, implying an anti-angiogenic effect of the drug. TKI258 treatment was effective in delaying CRC tumor growth in vivo regardless of the KRAS and BRAF mutation status. Conclusions: Our results identify FGFRs as potential targets in CRC treatment and suggest that combined targeting of multiple RTKs with TKI258 might serve as a novel approach to improve outcome of patients with CRC. PMID:25628921

  9. Genetic Alterations in K-ras and p53 Cancer Genes in Lung Neoplasms From B6C3F1 Mice Exposed to Cumene

    PubMed Central

    Hong, Hue-Hua L.; Ton, Thai-Vu. T.; Kim, Yongbaek; Wakamatsu, Nobuko; Clayton, Natasha P.; Chan, Po-Chuen; Sills, Robert C.; Lahousse, Stephanie A.

    2009-01-01

    The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the controls. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87 % cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56 % cumene-induced neoplasms and mutations were detected in 52 % neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions and codon 133 C to T transitions. No p53 mutation and one of 7 (14 %) K-ras mutation was detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13 %) and on chromosome 6 near the K-ras gene (12 %). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene. PMID:18648094

  10. Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene.

    PubMed

    Hong, Hue-Hua L; Ton, Thai-Vu T; Kim, Yongbaek; Wakamatsu, Nobuko; Clayton, Natasha P; Chan, Po-Chuen; Sills, Robert C; Lahousse, Stephanie A

    2008-07-01

    The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

  11. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.

  12. Mutations of p53 and KRAS activate NF-κB to promote chemoresistance and tumorigenesis via dysregulation of cell cycle and suppression of apoptosis in lung cancer cells.

    PubMed

    Yang, Lina; Zhou, Yunjiao; Li, Yinghua; Zhou, Juan; Wu, Yougen; Cui, Yunqing; Yang, Gong; Hong, Yang

    2015-02-28

    Although mutations of p53 and KRAS and activation of NF-κB signaling have been highly associated with chemoresistance and tumorigenesis of lung cancer, the interactive mechanisms between two of p53, KRAS, and NF-κB are elusive. In the present study, we first observed that blocking of NF-κB function in KRAS mutant A549 cell line with an IκBα mutant (IκBαM) inhibited cell cycle progression, anti-apoptosis, chemoresistance, and tumorigenesis. Silencing of p53 or KRAS in A549 or H358 cells either enhanced or attenuated the resistance of cells to cisplatin and taxol through promotion or suppression of the NF-κB p65 nuclear translocation. Introduction of a wild type p53 into p53 null lung cancer cell lines H1299 and H358 inhibited NF-κB activity, leading to the enhanced response to chemotherapeutic drugs. Delivery of a mutant p53 or KRAS-V12 into A549/IκBαM or H1299/p53Wt cells increased cell cycle progression, anti-apoptosis, chemoresistance, and tumorigenesis due to the accumulated nuclear localization of NF-κB p65, while treatment of H1299/p53Wt/KRAS-V12 with NF-κB inhibitor PS1145 diminished these effects. Thus, we conclude that p53 deficiency and KRAS mutation activate the NF-κB signaling to control chemoresistance and tumorigenesis, and that the status of p53 and KRAS may be considered for the targeted therapy against NF-κB in lung cancer patients.

  13. Compound EGFR mutation is frequently detected with co-mutations of actionable genes and associated with poor clinical outcome in lung adenocarcinoma.

    PubMed

    Kim, Eun Young; Cho, Eun Na; Park, Heae Surng; Hong, Ji Young; Lim, Seri; Youn, Jong Pil; Hwang, Seung Yong; Chang, Yoon Soo

    2016-01-01

    Compound EGFR mutations, defined as double or multiple mutations in the EGFR tyrosine kinase domain, are frequently detected with advances in sequencing technology but its clinical significance is unclear. This study analyzed 61 cases of EGFR mutation positive lung adenocarcinoma using next-generation sequencing (NGS) based repeated deep sequencing panel of 16 genes that contain actionable mutations and investigated clinical implication of compound EGFR mutations. Compound EGFR mutation was detected in 15 (24.6%) of 61 cases of EGFR mutation-positive lung adenocarcinoma. The majority (12/15) of compound mutations are combination of the atypical mutation and typical mutations such as exon19 deletion, L858R or G719X substitutions, or exon 20 insertion whereas 3 were combinations of rare atypical mutations. The patients with compound mutation showed shorter overall survival than those with simple mutations (83.7 vs. 72.8 mo; P = 0.020, Breslow test). Among the 115 missense mutations discovered in the tested genes, a few number of actionable mutations were detected irrelevant to the subtype of EGFR mutations, including ALK rearrangement, BCL2L11 intron 2 deletion, KRAS c.35G>A, PIK3CA c.1633G>A which are possible target of crizotinib, BH3 mimetics, MEK inhibitors, and PI3K-tyrosine kinase inhibitors, respectively. 31 missense mutations were detected in the cases with simple mutations whereas 84 in those with compound mutation, showing that the cases with compound missense mutation have higher burden of missense mutations (P = 0.001, independent sample t-test). Compound EGFR mutations are detected at a high frequency using NGS-based repeated deep sequencing. Because patients with compound EGFR mutations showed poor clinical outcomes, they should be closely monitored during follow-up.

  14. Pharmacological strategies to target oncogenic KRAS signaling in pancreatic cancer.

    PubMed

    Chuang, Hsiao-Ching; Huang, Po-Hsien; Kulp, Samuel K; Chen, Ching-Shih

    2017-03-01

    The clear importance of mutated KRAS as a therapeutic target has driven the investigation of multiple approaches to inhibit oncogenic KRAS signaling at different molecular levels. However, no KRAS-targeted therapy has reached the clinic to date, which underlies the intrinsic difficulty in developing effective, direct inhibitors of KRAS. Thus, this article provides an overview of the history and recent progress in the development of pharmacological strategies to target oncogenic KRAS with small molecule agents. Mechanistically, these KRAS-targeted agents can be classified into the following four categories. (1) Small-molecule RAS-binding ligands that prevent RAS activation by binding within or outside the nucleotide-binding motif. (2) Inhibitors of KRAS membrane anchorage. (3) Inhibitors that bind to RAS-binding domains of RAS-effector proteins. (4) Inhibitors of KRAS expression. The advantage and limitation of each type of these anti-KRAS agents are discussed.

  15. The effect of forced expression of mutated K-RAS gene on gastrointestinal cancer cell lines and the IGF-1R targeting therapy.

    PubMed

    Matsunaga, Yasutaka; Adachi, Yasushi; Sasaki, Yasushi; Koide, Hideyuki; Motoya, Masayo; Nosho, Katsuhiko; Takagi, Hideyasu; Yamamoto, Hiroyuki; Sasaki, Shigeru; Arimura, Yoshiaki; Tokino, Takashi; Carbone, David P; Imai, Kohzoh; Shinomura, Yasuhisa

    2017-02-01

    Mutation in K-RAS (K-RAS-MT) plays important roles in both cancer progression and resistance to anti-epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin-like growth factor-1 receptor (IGF-1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K-RAS mutation using an anti-IGF-1R monoclonal antibody. In this study, we sought to evaluate effects of forced K-RAS-MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti-EGFR therapy and IGF-1R-targeted therapy for these transfectants. We made stable transfectants of K-RAS-MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC-3. We assessed the effect of forced expression of K-RAS-MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti-tumor effects of dominant negative IGF-1R (IGF-1R/dn) and an IGF-1R inhibitor, picropodophyllin, on the K-RAS-MT transfectants. Overexpression of K-RAS-MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF-1R blockade suppressed cell growth, colony formation, migration, and invasion, and up-regulated chemotherapy-induced apoptosis of gastrointestinal cancer cells, even when K-RAS-MT was over-expressed. IGF-1R blockade inhibited the Akt pathway more than the extracellular signal-regulated kinase (ERK) pathway in the K-RAS-MT transfectants. IGF-1R/dn, moreover, inhibited the growth of murine xenografts expressing K-RAS-MT. Thus, K-RAS-MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF-1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K-RAS is mutated. © 2016 Wiley Periodicals, Inc.

  16. Mutation detection using Surveyor nuclease.

    PubMed

    Qiu, Peter; Shandilya, Harini; D'Alessio, James M; O'Connor, Kevin; Durocher, Jeffrey; Gerard, Gary F

    2004-04-01

    We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.

  17. Applicability of a System for fully automated nucleic acid extraction from formalin-fixed paraffin-embedded sections for routine KRAS mutation testing.

    PubMed

    Lehmann, Annika; Schewe, Christiane; Hennig, Guido; Denkert, Carsten; Weichert, Wilko; Budczies, Jan; Dietel, Manfred

    2012-06-01

    Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.

  18. Establishment and characterization of a novel uterine carcinosarcoma cell line, TU-ECS-1, with mutations of TP53 and KRAS.

    PubMed

    Chiba, Yohei; Sato, Seiya; Itamochi, Hiroaki; Suga, Yasuko; Fukagawa, Tomoyuki; Oumi, Nao; Oishi, Tetsuro; Harada, Tasuku; Sugai, Tamotsu; Sugiyama, Toru

    2017-04-01

    A new human uterine carcinosarcoma (UCS) cell line, TU-ECS-1, was established and characterized. The morphological appearance of the cultured cells was an insular of epithelial-like cells arranged in the form of a jigsaw puzzle and mesenchymal-like cells with a spindle-shaped or fibroblast-like morphology. A relatively high proliferation rate was observed with a doubling time of 18.2 h. The chromosome number ranged from 44 to 49 and had an extra chromosome 12 (trisomy 12). The respective half-maximal inhibitory concentrations of cisplatin, paclitaxel, and doxorubicin were 2.9 µM, 154 nM, and 219 ng/mL, respectively. Mutational analysis revealed that TU-ECS-1 cells have mutations of TP53 in exons 4, 6, and 8 and of KRAS at codon 12 (G12D) in exon 2, which is a mutation hot spot on this gene. Western blot analysis showed that p53 protein was overexpressed in TU-ECS-1 cells. Immunostaining of the cultured cells and in vivo tumors showed that the TU-ECS-1 cells and xenografts were positive for epithelial marker cytokeratin AE1/3 and mesenchymal marker vimentin. These results suggested that TU-ECS-1 cells might have both epithelial and mesenchymal characteristics. This cell line may be useful to study the carcinogenesis of UCS and contribute to the development of novel treatment strategies.

  19. Liquid biopsy for detection of actionable oncogenic mutations in human cancers and electric field induced release and measurement liquid biopsy (eLB).

    PubMed

    Tu, Michael; Chia, David; Wei, Fang; Wong, David

    2016-01-21

    Oncogenic activations by mutations in key cancer genes such as EGFR and KRAS are frequently associated with human cancers. Molecular targeting of specific oncogenic mutations in human cancer is a major therapeutic inroad for anti-cancer drug therapy. In addition, progressive developments of oncogene mutations lead to drug resistance. Therefore, the ability to detect and continuously monitor key actionable oncogenic mutations is important to guide the use of targeted molecular therapies to improve long-term clinical outcomes in cancer patients. Current oncogenic mutation detection is based on direct sampling of cancer tissue by surgical resection or biopsy. Oncogenic mutations were recently shown to be detectable in circulating bodily fluids of cancer patients. This field of investigation, termed liquid biopsy, permits a less invasive means of assessing the oncogenic mutation profile of a patient. This paper will review the analytical strategies used to assess oncogenic mutations from biofluid samples. Clinical applications will also be discussed.

  20. Liquid Biopsy for Detection of Actionable Oncogenic Mutations in Human Cancers and Electric Field Induced Release and Measurement Liquid Biopsy (eLB)

    PubMed Central

    Tu, Michael; Chia, David; Wei, Fang; Wong, David

    2015-01-01

    Oncogenic activations by mutations in key cancer genes such as EGFR and KRAS are frequently associated with human cancers. Molecular targeting of specific oncogenic mutations in human cancer is a major therapeutic inroad for anti-cancer drug therapy. In addition, progressive developments of oncogene mutations lead to drug resistance. Therefore, the ability to detect and continuously monitor key actionable oncogenic mutations is important to guide the use of targeted molecular therapies to improve long-term clinical outcomes in cancer patients. Current oncogenic mutation detection is based on direct sampling of cancer tissue by surgical resection or biopsy. Oncogenic mutations were recently shown to be detectable in circulating bodily fluids of cancer patients. This field of investigation, termed liquid biopsy, permits a less invasive means of assessing the oncogenic mutation profile of a patient. This paper will review the analytical strategies used to assess oncogenic mutations from biofluid samples. Clinical applications will also be discussed. PMID:26645892

  1. Miniaturized Real-Time PCR on a Q3 System for Rapid KRAS Genotyping.

    PubMed

    Guarnaccia, Maria; Iemmolo, Rosario; Petralia, Salvatore; Conoci, Sabrina; Cavallaro, Sebastiano

    2017-04-11

    Colorectal cancer (CRC) is an aggressive human malignancy with a complex genomic landscape harboring KRAS mutations. In 40%-60% of patients with CRC, constantly active KRAS proteins affect the prognosis, surgical strategy, and clinical benefit from therapy with anti-epidermal growth factor receptor (EGFR) agents. For this reason, there is a greater demand for minimally-invasive diagnostic devices to characterize the genetic pattern and prevent the acquired mechanism to drug resistance. The rapid developments in cutting-edge diagnostic techniques are expected to play a growing role in medicine and represent an attractive promise to identify potential responders to personalized medicine. Here we propose a new method to simultaneously detect the main KRAS mutations on the portable real-time PCR Q3 platform. This platform is based on hybrid silicon-plastic technology implemented in a miniaturized chip able to achieve a sample-in answer-out rapid analysis, allowing a new approach to genetic counseling and testing.

  2. Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.

    PubMed

    Hamada, Mariko; Shimase, Koji; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2014-04-01

    We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system.

  3. Alterations in the K-ras and p53 genes in rat lung tumors

    SciTech Connect

    Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E.

    1997-06-01

    Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

  4. Mutant Kras copy number defines metabolic reprogramming and therapeutic susceptibilities.

    PubMed

    Kerr, Emma M; Gaude, Edoardo; Turrell, Frances K; Frezza, Christian; Martins, Carla P

    2016-03-03

    The RAS/MAPK (mitogen-activated protein kinase) signalling pathway is frequently deregulated in non-small-cell lung cancer, often through KRAS activating mutations. A single endogenous mutant Kras allele is sufficient to promote lung tumour formation in mice but malignant progression requires additional genetic alterations. We recently showed that advanced lung tumours from Kras(G12D/+);p53-null mice frequently exhibit Kras(G12D) allelic enrichment (Kras(G12D)/Kras(wild-type) > 1) (ref. 7), implying that mutant Kras copy gains are positively selected during progression. Here we show, through a comprehensive analysis of mutant Kras homozygous and heterozygous mouse embryonic fibroblasts and lung cancer cells, that these genotypes are phenotypically distinct. In particular, Kras(G12D/G12D) cells exhibit a glycolytic switch coupled to increased channelling of glucose-derived metabolites into the tricarboxylic acid cycle and glutathione biosynthesis, resulting in enhanced glutathione-mediated detoxification. This metabolic rewiring is recapitulated in mutant KRAS homozygous non-small-cell lung cancer cells and in vivo, in spontaneous advanced murine lung tumours (which display a high frequency of Kras(G12D) copy gain), but not in the corresponding early tumours (Kras(G12D) heterozygous). Finally, we demonstrate that mutant Kras copy gain creates unique metabolic dependences that can be exploited to selectively target these aggressive mutant Kras tumours. Our data demonstrate that mutant Kras lung tumours are not a single disease but rather a heterogeneous group comprising two classes of tumours with distinct metabolic profiles, prognosis and therapeutic susceptibility, which can be discriminated on the basis of their relative mutant allelic content. We also provide the first, to our knowledge, in vivo evidence of metabolic rewiring during lung cancer malignant progression.

  5. KRAS, BRAF, and TP53 deep sequencing for colorectal carcinoma patient diagnostics.

    PubMed

    Rechsteiner, Markus; von Teichman, Adriana; Rüschoff, Jan H; Fankhauser, Niklaus; Pestalozzi, Bernhard; Schraml, Peter; Weber, Achim; Wild, Peter; Zimmermann, Dieter; Moch, Holger

    2013-05-01

    In colorectal carcinoma, KRAS (alias Ki-ras) and BRAF mutations have emerged as predictors of resistance to anti-epidermal growth factor receptor antibody treatment and worse patient outcome, respectively. In this study, we aimed to establish a high-throughput deep sequencing workflow according to 454 pyrosequencing technology to cope with the increasing demand for sequence information at medical institutions. A cohort of 81 patients with known KRAS mutation status detected by Sanger sequencing was chosen for deep sequencing. The workflow allowed us to analyze seven amplicons (one BRAF, two KRAS, and four TP53 exons) of nine patients in parallel in one deep sequencing run. Target amplification and variant calling showed reproducible results with input DNA derived from FFPE tissue that ranged from 0.4 to 50 ng with the use of different targets and multiplex identifiers. Equimolar pooling of each amplicon in a deep sequencing run was necessary to counterbalance differences in patient tissue quality. Five BRAF and 49 TP53 mutations with functional consequences were detected. The lowest mutation frequency detected in a patient tumor population was 5% in TP53 exon 5. This low-frequency mutation was successfully verified in a second PCR and deep sequencing run. In summary, our workflow allows us to process 315 targets a week and provides the quality, flexibility, and speed needed to be integrated as standard procedure for mutational analysis in diagnostics.

  6. [Clinical Research of EGFR and KRAS Mutation in Fine Needle Aspiration Cytology Specimens of Non-small Cell Lung Carcinoma].

    PubMed

    Zhang, Zhihui; Wu, Xilan; Ying, Jianming; Li, Junling; Qiu, Tian; Guo, Huiqin; Zhao, Huan; Shan, Ling; Ling, Yun

    2015-04-01

    背景与目的 肺癌患者靶向药物治疗前,需要检测表皮生长因子受体(epidermal growth factor receptor, EGFR)和KRAS基因是否突变。本研究旨在探讨细针吸取悬浮液标本检测非小细胞肺癌EGFR、KRAS基因突变的意义。方法 细胞学悬浮液标本,Real-time PCR法检测EGFR 18-21号外显子,KRAS 2号外显子12、13密码子突变。结果 检测85例肺癌转移淋巴结针吸标本,EGFR突变率37.3%,KRAS突变率7.2%。19例组织切片标本,与细胞学结果一致(kappa=1.0)。13例有EGFR突变,临床分期IV期患者, 使用酪氨酸激酶抑制剂治疗,2例完全缓解(complete remission, CR)(16.7%);8例部分缓解(partial remission, PR)(66.7%);3例最佳稳定疾病(stable disease, SD)(25.0%)。结论 细针吸取标本检测EGFR、KRAS基因突变,标本取材容易、简单、方便,具有较高的临床实用性。.

  7. An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer

    PubMed Central

    Vallejo, Adrian; Perurena, Naiara; Guruceaga, Elisabet; Mazur, Pawel K.; Martinez-Canarias, Susana; Zandueta, Carolina; Valencia, Karmele; Arricibita, Andrea; Gwinn, Dana; Sayles, Leanne C.; Chuang, Chen-Hua; Guembe, Laura; Bailey, Peter; Chang, David K.; Biankin, Andrew; Ponz-Sarvise, Mariano; Andersen, Jesper B.; Khatri, Purvesh; Bozec, Aline; Sweet-Cordero, E. Alejandro; Sage, Julien; Lecanda, Fernando; Vicent, Silve

    2017-01-01

    KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers. PMID:28220783

  8. Oncogenic KRAS signalling in pancreatic cancer.

    PubMed

    Eser, S; Schnieke, A; Schneider, G; Saur, D

    2014-08-26

    Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal. The annual number of deaths equals the number of newly diagnosed cases, despite maximal treatment. The overall 5-year survival rate of <5% has remained stubbornly unchanged over the last 30 years, despite tremendous efforts in preclinical and clinical science. There is unquestionably an urgent need to further improve our understanding of pancreatic cancer biology, treatment response and relapse, and to identify novel therapeutic targets. Rigorous research in the field has uncovered genetic aberrations that occur during PDAC development and progression. In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia. However, all attempts to target KRAS directly have failed in the clinic and KRAS is widely assumed to be undruggable. This has led to intense efforts to identify druggable critical downstream targets and nodes orchestrated by mutationally activated KRAS. This includes context-specific KRAS effector pathways, synthetic lethal interaction partners and KRAS-driven metabolic changes. Here, we review recent advances in oncogenic KRAS signalling and discuss how these might benefit PDAC treatment in the future.

  9. KRAS — EDRN Public Portal

    Cancer.gov

    The KRAS gene, a Kirsten ras oncogene homolog from the mammalian ras gene family, encodes a protein that is a member of the small GTPase superfamily. A single amino acid substitution is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma. Alternative splicing leads to variants encoding two isoforms that differ in the C-terminal region.

  10. Heterogeneity of resistance mutations detectable by next-generation sequencing in TKI-treated lung adenocarcinoma

    PubMed Central

    Belchis, Deborah A.; Tseng, Li-Hui; Gniadek, Thomas; Haley, Lisa; Lokhandwala, Parvez; Illei, Peter; Gocke, Christopher D.; Forde, Patrick; Brahmer, Julie; Askin, Frederic B.; Eshleman, James R.; Lin, Ming-Tseh

    2016-01-01

    EGFR-mutated lung adenocarcinomas routinely develop resistance to tyrosine kinase inhibitors (TKI). To better characterize the relative frequencies of the resistance mechanisms, we analyzed 48 EGFR-mutated TKI-resistant specimens from 41 patients. Next-generation sequencing of post-treatment specimens detected EGFR p.T790M in 31 (79%) of 39 patients, PIK3CA mutations in 10 (26%), EGFR p.S768_V769delinsIL in one, and KRAS p.G12C in one. Five PIK3CA mutations were outside of codons 542, 545, and 1047. Three of four pre-treatment specimens did not carry the PIK3CA mutation found in the post-treatment sample. Small cell carcinoma transformation was identified in four patients; none had p.T790M, including two where p.T790M was identified in the co-existing adenocarcinoma. In p.T790M-mutated specimens, the allele frequency was less than 5% in 24% of cases. p.T790M allele frequency was usually lower than that of the sensitizing mutation indicating that the resistance mutation was present either in a subset of cells or, if the sensitizing mutation was amplified, in a subset of the sensitizing alleles of a dominant clone. Eight patients had multiple resistance mutations, suggesting either multiple separate resistant clones or a single clone harboring multiple resistance mechanisms. PIK3CA mutations appear to be a more significant resistance mechanism than previously recognized. PMID:27304188

  11. Targeting Mutant KRAS for Anticancer Therapeutics: A Review of Novel Small Molecule Modulators

    PubMed Central

    Wang, Yuanxiang; Kaiser, Christine E.; Frett, Brendan; Li, Hong-yu

    2015-01-01

    The RAS proteins play a role in cell differentiation, proliferation, and survival. Aberrant RAS signaling has been found to play a role in 30% of all cancers. KRAS, a key member of the RAS protein family, is an attractive cancer target, as frequent point mutations in the KRAS gene render the protein constitutively active. A number of attempts have been made to target aberrant KRAS signaling by identifying small molecule compounds that (1) are synthetic lethal to mutant KRAS, (2) block KRAS/GEF interactions, (3) inhibit downstream KRAS effectors, or (4) inhibit the post-translational processing of RAS proteins. In addition, inhibition of novel targets outside the main KRAS signaling pathway, specifically the cell cycle related kinase PLK1, has been shown have an effect in cells that harbor mutant KRAS. Herein we review the use of various high-throughput screening assays utilized to identify new small-molecule compounds capable of targeting mutant KRAS-driven cancers. PMID:23566315

  12. Disruption of thioredoxin metabolism enhances the toxicity of transforming growth factor β-activated kinase 1 (TAK1) inhibition in KRAS-mutated colon cancer cells

    PubMed Central

    Hrabe, Jennifer E.; O’Leary, Brianne R.; Fath, Melissa A.; Rodman, Samuel N.; Button, Anna M.; Domann, Frederick E.; Spitz, Douglas R.; Mezhir, James J.

    2015-01-01

    Transforming growth factor β-activated kinase 1 (TAK1) is critical for survival of many KRAS mutated colorectal cancer cells, and TAK1 inhibition with 5Z-7-oxozeaenol has been associated with oxidative stress leading to tumor cell killing. When SW 620 and HCT 116 human colon cancer cells were treated with 5 µM 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10 mM N-acetylcysteine (NAC) partially reversed the growth inhibitory effects of 5Z-7-oxozeaenol. In addition, 5Z-7-oxozeaenol also increased steady-state levels of H2DCFDA oxidation as well as increased levels of total glutathione (GSH) and glutathione disulfide (GSSG). Interestingly, depletion of GSH using buthionine sulfoximine did not significantly potentiate 5Z-7-oxozeaenol toxicity in either cell line. In contrast, pre-treatment of cells with auranofin (Au) to inhibit thioredoxin reductase activity significantly increased levels of oxidized thioredoxin as well as sensitized cells to 5Z-7-oxozeaenol-induced growth inhibition and clonogenic cell killing. These results were confirmed in SW 620 murine xenografts, where treatment with 5Z-7-oxozeaenol or with Au plus 5Z-7-oxozeaenol significantly inhibited growth, with Au plus 5Z-7-oxozeaenol trending toward greater growth inhibition compared to 5Z-7-oxozeaenol alone. These results support the hypothesis that thiol-mediated oxidative stress is causally related to TAK1-induced colon cancer cell killing. In addition, these results support the hypothesis that thioredoxin metabolism is a critical target for enhancing colon cancer cell killing via TAK1 inhibition and could represent an effective therapeutic strategy in patients with these highly resistant tumors. PMID:26114584

  13. Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer

    PubMed Central

    Toulany, Mahmoud; Iida, Mari; Keinath, Simone; Iyi, Firdevs F.; Mueck, Katharina; Fehrenbacher, Birgit; Mansour, Wael Y.; Schaller, Martin; Wheeler, Deric L.; Rodemann, H. Peter

    2016-01-01

    Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome. PMID:27248324

  14. Electrochemical detection of point mutation based on surface ligation reaction and biometallization.

    PubMed

    Zhang, Peng; Chu, Xia; Xu, Xiangmin; Shen, Guoli; Yu, Ruqin

    2008-05-15

    A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5'-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90 degrees C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.

  15. Higher metastatic efficiency of KRas G12V than KRas G13D in a colorectal cancer model.

    PubMed

    Alamo, Patricia; Gallardo, Alberto; Di Nicolantonio, Federica; Pavón, Miguel Angel; Casanova, Isolda; Trias, Manuel; Mangues, María Antonia; Lopez-Pousa, Antonio; Villaverde, Antonio; Vázquez, Esther; Bardelli, Alberto; Céspedes, María Virtudes; Mangues, Ramón

    2015-02-01

    Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5- to 3.0-fold larger in KRas G12V than in KRas G13D mice (P < 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ± 1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P = 0.04), and a higher percentage of C-X-C chemokine receptor type 4 (CXCR4)-overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and β5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine-1 overexpression, whereas KRas G13D tumors showed integrin β1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing this oncogene.

  16. Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

    PubMed

    Kwon, Mi Jung; Shin, Hyung Sik; Nam, Eun Sook; Cho, Seong Jin; Lee, Min Joung; Lee, Samuel; Park, Hye-Rim

    2015-05-01

    Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs.

  17. [Detection and Analysis of EGFR and KRAS Mutation with Lung Adenocarcinoma].

    PubMed

    Zhang, Hui; Yang, Xinjie; Qin, Na; Li, Xi; Yang, Huiyi; Nong, Jingying; Lv, Jialin; Wu, Yuhua; Zhang, Quan; Zhang, Xinyong; Wang, Jinghui; Zhou, Lijuan; Zhang, Shucai

    2015-11-01

    背景与目的 表皮生长因子受体(epidermal growth factor receptor, EGFR)突变和KRAS基因突变是非小细胞肺癌(non-small cell lung cancer, NSCLC)靶向治疗的重要分子标志,与临床治疗疗效密切相关。分析肺腺癌EGFR和KRAS基因突变与临床特征的关系。方法 收集初治肺腺癌患者395例,有可供检测的肿瘤组织标本。利用突变富集液相芯片法进行EGFR和KRAS基因突变检测。结果 395例肺腺癌中,EGFR基因突变192例(48.9%),KRAS基因突变29例(7.8%),EGFR和KRAS基因同时发生突变1例(0.3%)。女性和不吸烟患者EGFR基因突变率高于男性和吸烟患者(62.0% vs 37.1%, 61.9% vs 30.3%),差异具有统计学意义(P<0.001, P<0.001);不同年龄、分期及病理取材标本之间差异均无统计学意义(P>0.05)。KRAS基因突变在EGFR基因野生型患者中发生率(13.5%, 27/200)明显高于EGFR基因突变患者(1.0%, 2/192),差异具有统计学意义(P<0.001)。结论 肺腺癌EGFR基因突变在女性和不吸烟患者中较高,KRAS基因突变只在EGFR基因野生型患者中较高。在使用TKI靶向治疗药物之前,应同时检测EGFR基因和KRAS基因的突变情况。.

  18. KRAS mutation-positive bronchial surface epithelium (BSE)-type lung adenocarcinoma with strong expression of TTF-1: a case providing a further insight as for the role of TTF-1 in the oncogenesis.

    PubMed

    Takanashi, Yusuke; Tajima, Shogo; Hayakawa, Takamitsu; Neyatani, Hiroshi; Funai, Kazuhito

    2015-01-01

    Bronchial surface epithelium (BSE)-type lung adenocarcinoma is a subtype of non-terminal respiratory unit (TRU)-type lung adenocarcinoma originating in the bronchial surface epithelium. However, there are few known cases of BSE-type adenocarcinoma with marked expression of thyroid transcription factor-1 (TTF-1). This paper describes a very rare case of KRAS mutation-positive BSE-type adenocarcinoma that exhibited strong expression of TTF-1 that was putatively involved in oncogenesis. An 84-year-old woman, a never smoker, was referred to our hospital because of an abnormal chest radiograph. Chest computed tomography (CT) showed a solid mass lesion, 15 mm × 10 mm, with a relatively smooth margin in the left upper lobe. The patient underwent partial resection of the left upper lobe for strongly suspected lung cancer with a clinical stage of cT1aN0M0. Histopathological findings showed continuous migration of papillary, hyperplastic, atypical columnar tumor cells originating from normal bronchial surface epithelium, leading to a diagnosis of BSE-type adenocarcinoma. TTF-1 was strongly expressed in almost 100% of the tumor cells, which tested positive for the KRAS mutation. TTF-1 has recently attracted attention as an oncogene, and it is purportedly involved in the carcinogenesis and survival of lung adenocarcinoma cells. There is typically an inverse correlation between the respective expressions of KRAS and TTF-1, but in the present study, they appeared simultaneously and were both putatively involved as oncogenic driver alterations. This case is important in that it sheds some light on the largely unknown pathogenic mechanism of BSE-type adenocarcinoma.

  19. Challenges in detecting pre-malignant pancreatic lesions during acute pancreatitis using a serum microRNA assay: a study based on KrasG12D transgenic mice

    PubMed Central

    Hong, Xiafei; Zhang, Jie; Wu, Qiao; Wang, Wenze; Ye, Adam Yongxin; Song, Wei; Dai, Hongmei; Wang, Xianze; Wu, Fan; You, Lei; Wu, Wenming; Zhao, Yupei

    2016-01-01

    Caerulein-induced acute pancreatitis accelerates the progression of pancreatic intraepithelial neoplasia (PanIN) lesions in a pancreas-specific KrasG12D mouse model. The purpose of this study was to explore whether serum microRNAs (miRNAs) can serve as sensitive biomarkers to detect occult PanIN in the setting of acute pancreatitis. Serum miRNA profiles were quantified by an array-based method and normalized by both Variance Stabilization Normalization (VSN) and invariant methods. Individual miRNAs were validated by TaqMan real-time PCR with synthetic spike-in C. elegans miRNAs as external controls. Serum miRNA profiles distinguished KrasG12D mice with pancreatitis from wild-type mice without pancreatitis, but failed to differentiate KrasG12D mice with pancreatitis from wild-type mice with pancreatitis. Most individual miRNAs that increased in KrasG12D mice with pancreatitis were not significantly different between KrasG12D mice without pancreatitis and wild-type mice without pancreatitis. Mechanistically, Gene Set Enrichment Analysis (GSEA) of the mRNA array data and immunohistochemical assays showed that caerulein-induced acute pancreatitis involved acinar cell loss and immune cell infiltration, which might contribute to serum miRNA profile changes. This study highlighted the challenges in using sensitive serum miRNA biomarker screening for the early detection of pancreatic malignancies during acute pancreatitis. PMID:27009811

  20. A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

    PubMed Central

    Pinzani, Pamela; Mancini, Irene; Vinci, Serena; Chiari, Marcella; Orlando, Claudio; Cremonesi, Laura; Ferrari, Maurizio

    2013-01-01

    Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. PMID:23536897

  1. Deubiquitinase USP18 Loss Mislocalizes and Destabilizes KRAS in Lung Cancer.

    PubMed

    Mustachio, Lisa Maria; Lu, Yun; Tafe, Laura J; Memoli, Vincent; Rodriguez-Canales, Jaime; Mino, Barbara; Villalobos, Pamela Andrea; Wistuba, Ignacio; Katayama, Hiroyuki; Hanash, Samir M; Roszik, Jason; Kawakami, Masanori; Cho, Kwang-Jin; Hancock, John F; Chinyengetere, Fadzai; Hu, Shanhu; Liu, Xi; Freemantle, Sarah J; Dmitrovsky, Ethan

    2017-02-27

    KRAS is frequently mutated in lung cancers and is associated with aggressive biology and chemotherapy resistance. Therefore, innovative approaches are needed to treat these lung cancers. Prior work implicated the interferon-stimulated gene 15 (ISG15) deubiquitinase (DUB) USP18 as having anti-neoplastic activity by regulating lung cancer growth and oncoprotein stability. This study demonstrates that USP18 affects the stability of the KRAS oncoprotein. Interestingly, loss of USP18 reduced KRAS expression and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells. Using the protein synthesis inhibitor cycloheximide (CHX), USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability. Intriguingly, loss of USP18 altered KRAS subcellular localization by mislocalizing KRAS from the plasma membrane. To explore the biological consequences, immunohistochemical (IHC) expression profiles of USP18 were compared in lung cancers of KrasLA2/+ versus cyclin E engineered mouse models. USP18 expression was higher in Kras-driven murine lung cancers, indicating a link between KRAS and USP18 expression in vivo. To solidify this association, loss of Usp18 in KrasLA2/+/Usp18-/- mice was found to significantly reduce lung cancers as compared to parental KrasLA2/+ mice. Lastly, translational relevance was confirmed in a human lung cancer panel by showing USP18 IHC expression was significantly higher in KRAS mutant versus wild-type lung adenocarcinomas.

  2. K-ras activation occurs frequently in mucinous adenocarcinomas and rarely in other common epithelial tumors of the human ovary.

    PubMed Central

    Enomoto, T.; Weghorst, C. M.; Inoue, M.; Tanizawa, O.; Rice, J. M.

    1991-01-01

    To explore the role of mutational activation of members of the ras family of cellular protooncogenes in the development of human ovarian neoplasms, a series of 37 ovarian tumors from Japanese patients was studied. These included 30 common epithelial tumors (1 mucinous tumor of borderline malignancy, 7 mucinous adenocarcinomas, and 22 nonmucinous carcinomas: 10 serous, 3 clear cell, 8 endometrioid, and 1 undifferentiated), 5 tumors of germ cell origin, and 2 sex cord/stromal cell tumors. Polymerase chain reaction was performed from selected areas of deparaffinized sections of formalin-fixed paraffin-embedded tissue, and the presence of activating point mutations in codons 12, 13, and 61 of the H-, N-, and K-ras genes was probed by dot-blot hybridization analysis with mutation specific oligonucleotides. Mutations in K-ras were also looked for by direct genomic sequencing. The overall frequency of ras gene mutations was 10/37 (27%). Mutations were detected only in K-ras, and were found in most of the mucinous tumors, including the one such tumor of borderline malignancy (6/8; 75%). In one mucinous adenocarcinoma, two mutations were detected in paraffin-embedded material that had not previously been found in high molecular weight DNA isolated from frozen tissue from the same case. K-ras mutations occurred significantly more frequently in mucinous tumors (6/8, 75%) than in serous carcinomas (2/10, 20%; P = 0.031) or in all nonmucinous types of epithelial ovarian tumors combined (3/22, 14%; P = 0.0031). Images Figure 1 Figure 2 PMID:1656759

  3. DNA vaccine elicits an efficient antitumor response by targeting the mutant Kras in a transgenic mouse lung cancer model.

    PubMed

    Weng, T-Y; Yen, M-C; Huang, C-T; Hung, J-J; Chen, Y-L; Chen, W-C; Wang, C-Y; Chang, J-Y; Lai, M-D

    2014-10-01

    Mutant Kras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) is observed in more than 20% of non-small-cell lung cancers; however, no effective Kras target therapy is available at present. The Kras DNA vaccine may represent as a novel immunotherapeutic agent in lung cancer. In this study, we investigated the antitumor efficacy of the Kras DNA vaccine in a genetically engineered inducible mouse lung tumor model driven by Kras(G12D). Lung tumors were induced by doxycycline, and the therapeutic effects of Kras DNA vaccine were evaluated with delivery of Kras(G12D) plasmids. Mutant Kras(G12D) DNA vaccine significantly decreased the tumor nodules. A dominant-negative mutant Kras(G12D)N17, devoid of oncogenic activity, achieved similar therapeutic effects. The T-helper 1 immune response was enhanced in mice treated with Kras DNA vaccine. Splenocytes from mice receiving Kras DNA vaccine presented an antigen-specific response by treatment with peptides of Kras but not Hras or OVA. The number of tumor-infiltrating CD8(+) T cells increased after Kras vaccination. In contrast, Kras DNA vaccine was not effective in the lung tumor in transgenic mice, which was induced by mutant L858R epidermal growth factor receptor. Overall, these results indicate that Kras DNA vaccine produces an effective antitumor response in transgenic mice, and may be useful in treating lung cancer-carrying Ras mutation.

  4. BRAF V600E mutation in metastatic colorectal cancer: Methods of detection and correlation with clinical and pathologic features.

    PubMed

    Roma, Cristin; Rachiglio, Anna Maria; Pasquale, Raffaella; Fenizia, Francesca; Iannaccone, Alessia; Tatangelo, Fabiana; Antinolfi, Giuseppe; Parrella, Paola; Graziano, Paolo; Sabatino, Lina; Colantuoni, Vittorio; Botti, Gerardo; Maiello, Evaristo; Normanno, Nicola

    2016-08-02

    The screening for BRAF V600E mutation is employed in clinical practice for its prognostic and potentially predictive role in patients with metastatic colorectal carcinoma (mCRC). Little information is available on the sensitivity and specificity of the testing methods to detect this mutation in CRC. By using serial dilution of BRAF mutant DNA with wild type DNA, we found that the sensitivity of allelic discrimination-Real Time PCR was higher than PCR-Sequencing (10% vs 20%). In agreement, the Real Time PCR assay displayed increased analytical sensitivity in detecting the BRAF V600E mutation as compared with PCR-Sequencing in a cohort of 510 consecutive CRCs (21 vs 16 cases). Targeted resequencing demonstrated that all cases negative by PCR-Sequencing had an allelic frequency of the BRAF mutation <20%, thus suggesting tumor heterogeneity. The association of BRAF mutations with clinical and pathological features was assessed next in a cohort of 840 KRAS exon 2 wild type CRC patients screened with the Real Time PCR assay. The BRAF V600E mutation frequency in this cohort was 7.8% that increased to 33.4% in females over 70 y of age with right-sided tumor location. BRAF mutations were also detected in 4.4% of male patients with left-sided tumors and aged <70 y. Fourteen of 61 (22.9%) BRAF V600E mutation bearing patients exhibited microsatellite instability (MSI) as assessed by T17 mononucleotide sequence within intron 8 of HSP110. Our study indicates that Real Time PCR-based assays are more sensitive than PCR-Sequencing to detect the BRAF V600E mutation in CRC and that BRAF mutations screening should not be restricted to selected patients on the basis of the clinical-pathological characteristics.

  5. Oncogene KRAS activates fatty acid synthase, resulting in specific ERK and lipid signatures associated with lung adenocarcinoma.

    PubMed

    Gouw, Arvin M; Eberlin, Livia S; Margulis, Katherine; Sullivan, Delaney K; Toal, Georgia G; Tong, Ling; Zare, Richard N; Felsher, Dean W

    2017-04-11

    KRAS gene mutation causes lung adenocarcinoma. KRAS activation has been associated with altered glucose and glutamine metabolism. Here, we show that KRAS activates lipogenesis, and this activation results in distinct proteomic and lipid signatures. By gene expression analysis, KRAS is shown to be associated with a lipogenesis gene signature and specific induction of fatty acid synthase (FASN). Through desorption electrospray ionization MS imaging (DESI-MSI), specific changes in lipogenesis and specific lipids are identified. By the nanoimmunoassay (NIA), KRAS is found to activate the protein ERK2, whereas ERK1 activation is found in non-KRAS-associated human lung tumors. The inhibition of FASN by cerulenin, a small molecule antibiotic, blocked cellular proliferation of KRAS-associated lung cancer cells. Hence, KRAS is associated with activation of ERK2, induction of FASN, and promotion of lipogenesis. FASN may be a unique target for KRAS-associated lung adenocarcinoma remediation.

  6. Endogenous K-ras signaling in erythroid differentiation.

    PubMed

    Zhang, Jing; Lodish, Harvey F

    2007-08-15

    K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines.

  7. Mutation detection in plasmid-based biopharmaceuticals.

    PubMed

    Oliveira, Pedro H; Prather, Kristala L J; Prazeres, Duarte M F; Monteiro, Gabriel A

    2011-04-01

    As the number of applications involving therapeutic plasmid DNA (pDNA) increases worldwide, there is a growing concern over maintaining rigorous quality control through a panel of high-quality assays. For this reason, efficient, cost-effective and sensitive technologies enabling the identification of genetic variants and unwanted side products are needed to successfully establish the identity and stability of a plasmid-based biopharmaceutical. This review highlights several bioinformatic tools for ab initio detection of potentially unstable DNA regions, as well as techniques used for mutation detection in nucleic acids, with particular emphasis on pDNA.

  8. Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

    PubMed Central

    Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

    2017-01-01

    Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes. PMID:28067274

  9. Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

    NASA Astrophysics Data System (ADS)

    Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

    2017-01-01

    Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

  10. Mutational Hotspot of TET2, IDH1, IDH2, SRSF2, SF3B1, KRAS, and NRAS from Human Systemic Mastocytosis Are Not Conserved in Canine Mast Cell Tumors

    PubMed Central

    Zorzan, Eleonora; Hanssens, Katia; Giantin, Mery; Dacasto, Mauro; Dubreuil, Patrice

    2015-01-01

    Introduction Both canine cutaneous mast cell tumor (MCT) and human systemic mastocytosis (SM) are characterized by abnormal proliferation and accumulation of mast cells in tissues and, frequently, by the presence of activating mutations in the receptor tyrosine kinase V-Kit Hardy-Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog (c-KIT), albeit at different incidence (>80% in SM and 10–30% in MCT). In the last few years, it has been discovered that additional mutations in other genes belonging to the methylation system, the splicing machinery and cell signaling, contribute, with c-KIT, to SM pathogenesis and/or phenotype. In the present study, the mutational profile of the Tet methylcytosine dioxygenase 2 (TET2), the isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2), the serine/arginine-rich splicing factor 2 (SRSF2), the splicing factor 3b subunit 1 (SF3B1), the Kirsten rat sarcoma viral oncogene homolog (KRAS) and the neuroblastoma RAS viral oncogene homolog (NRAS), commonly mutated in human myeloid malignancies and mastocytosis, was investigated in canine MCTs. Methods Using the Sanger sequencing method, a cohort of 75 DNA samples extracted from MCT biopsies already investigated for c-KIT mutations were screened for the “human-like” hot spot mutations of listed genes. Results No mutations were ever identified except for TET2 even if with low frequency (2.7%). In contrast to what is observed in human TET2 no frame-shift mutations were found in MCT samples. Conclusion Results obtained in this preliminary study are suggestive of a substantial difference between human SM and canine MCT if we consider some target genes known to be involved in the pathogenesis of human SM. PMID:26562302

  11. Suppression of KRas-mutant cancer through the combined inhibition of KRAS with PLK1 and ROCK

    PubMed Central

    Wang, Jieqiong; Hu, Kewen; Guo, Jiawei; Cheng, Feixiong; Lv, Jing; Jiang, Wenhao; Lu, Weiqiang; Liu, Jinsong; Pang, Xiufeng; Liu, Mingyao

    2016-01-01

    No effective targeted therapies exist for cancers with somatic KRAS mutations. Here we develop a synthetic lethal chemical screen in isogenic KRAS-mutant and wild-type cells to identify clinical drug pairs. Our results show that dual inhibition of polo-like kinase 1 and RhoA/Rho kinase (ROCK) leads to the synergistic effects in KRAS-mutant cancers. Microarray analysis reveals that this combinatory inhibition significantly increases transcription and activity of cyclin-dependent kinase inhibitor p21WAF1/CIP1, leading to specific G2/M phase blockade in KRAS-mutant cells. Overexpression of p21WAF1/CIP1, either by cDNA transfection or clinical drugs, preferentially impairs the growth of KRAS-mutant cells, suggesting a druggable synthetic lethal interaction between KRAS and p21WAF1/CIP1. Co-administration of BI-2536 and fasudil either in the LSL-KRASG12D mouse model or in a patient tumour explant mouse model of KRAS-mutant lung cancer suppresses tumour growth and significantly prolongs mouse survival, suggesting a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers. PMID:27193833

  12. Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations.

    PubMed

    Botezatu, Irina V; Nechaeva, Irina O; Stroganova, Аnna М; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2015-12-01

    The data in this article are related to the research article entitled "Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations" Botezatu et al. [1]. Somatic mutations in the PIK3CA gene ("hot spots" in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.

  13. Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.

    PubMed

    Hamada, Mariko; Shimase, Koji; Noda, Keiichi; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2013-05-01

    We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

  14. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

    PubMed

    Luo, Feijun; Brooks, David G; Ye, Hongtao; Hamoudi, Rifat; Poulogiannis, George; Patek, Charles E; Winton, Douglas J; Arends, Mark J

    2009-10-01

    Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant

  15. Akt mediated ROS-dependent selective targeting of mutant KRAS tumors.

    PubMed

    Iskandar, Kartini; Rezlan, Majidah; Pervaiz, Shazib

    2014-10-01

    Reactive oxygen species (ROS) play a critical role in a variety of cellular processes, ranging from cell survival and proliferation to cell death. Previously, we reported the ability of a small molecule compound, C1, to induce ROS dependent autophagy associated apoptosis in human cancer cell lines and primary tumor cells (Wong C. et al. 2010). Our ongoing investigations have unraveled a hitherto undefined novel signaling network involving hyper-phosphorylation of Akt and Akt-mediated ROS production in cancer cell lines. Interestingly, drug-induced Akt activation is selectively seen in cell lines that carry mutant KRAS; HCT116 cells that carry the V13D KRAS mutation respond favorably to C1 while HT29 cells expressing wild type KRAS are relatively resistant. Of note, not only does the compound target mutant KRAS expressing cells but also induces RAS activation as evidenced by the PAK pull down assay. Corroborating this, pharmacological inhibition as well as siRNA mediated silencing of KRAS or Akt, blocked C1-induced ROS production and rescued tumor colony forming ability in HCT116 cells. To further confirm the involvement of KRAS, we made use of mutant KRAS transformed RWPE-1 prostate epithelial cells. Notably, drug-induced ROS generation and death sensitivity was significantly higher in RWPE-1-KRAS cells than the RWPE-1-vector cells, thus confirming the results obtained with mutant KRAS colorectal carcinoma cell line. Lastly, we made use of HCT116 mutant KRAS knockout cells (KO) where the mutant KRAS allele had been deleted, thus expressing a single wild-type KRAS allele. Exposure of the KO cells to C1 failed to induce Akt activation and mitochondrial ROS production. Taken together, results show the involvement of activated Akt in ROS-mediated selective targeting of mutant KRAS expressing tumors, which could have therapeutic implications given the paucity of chemotherapeutic strategies specifically targeting KRAS mutant cancers.

  16. Estrogen withdrawal, increased breast cancer risk and the KRAS-variant

    PubMed Central

    McVeigh, Terri P; Jung, Song-Yi; Kerin, Michael J; Salzman, David W; Nallur, Sunitha; Nemec, Antonio A; Dookwah, Michelle; Sadofsky, Jackie; Paranjape, Trupti; Kelly, Olivia; Chan, Elcie; Miller, Nicola; Sweeney, Karl J; Zelterman, Daniel; Sweasy, Joann; Pilarski, Robert; Telesca, Donatello; Slack, Frank J; Weidhaas, Joanne B

    2015-01-01

    The KRAS-variant is a biologically functional, microRNA binding site variant, which predicts increased cancer risk especially for women. Because external exposures, such as chemotherapy, differentially impact the effect of this mutation, we evaluated the association of estrogen exposures, breast cancer (BC) risk and tumor biology in women with the KRAS-variant. Women with BC (n = 1712), the subset with the KRAS-variant (n = 286) and KRAS-variant unaffected controls (n = 80) were evaluated, and hormonal exposures, KRAS-variant status, and pathology were compared. The impact of estrogen withdrawal on transformation of isogenic normal breast cell lines with or without the KRAS-variant was studied. Finally, the association and presentation characteristics of the KRAS-variant and multiple primary breast cancer (MPBC) were evaluated. KRAS-variant BC patients were more likely to have ovarian removal pre-BC diagnosis than non-variant BC patients (p = 0.033). In addition, KRAS-variant BC patients also appeared to have a lower estrogen state than KRAS-variant unaffected controls, with a lower BMI (P < 0.001). Finally, hormone replacement therapy (HRT) discontinuation in KRAS-variant patients was associated with a diagnosis of triple negative BC (P < 0.001). Biologically confirming our clinical findings, acute estrogen withdrawal led to oncogenic transformation in KRAS-variant positive isogenic cell lines. Finally, KRAS-variant BC patients had greater than an 11-fold increased risk of presenting with MPBC compared to non-variant patients (45.39% vs 6.78%, OR 11.44 [3.42–37.87], P < 0.001). Thus, estrogen withdrawal and a low estrogen state appear to increase BC risk and to predict aggressive tumor biology in women with the KRAS-variant, who are also significantly more likely to present with multiple primary breast cancer. PMID:25961464

  17. SIRT2 deletion enhances KRAS-induced tumorigenesis in vivo by regulating K147 acetylation status

    PubMed Central

    Song, Ha Yong; Biancucci, Marco; Kang, Hong-Jun; O'Callaghan, Carol; Park, Seong-Hoon; Principe, Daniel R.; Jiang, Haiyan; Yan, Yufan; Satchell, Karla Fullner; Raparia, Kirtee; Gius, David; Vassilopoulos, Athanassios

    2016-01-01

    The observation that cellular transformation depends on breaching a crucial KRAS activity threshold, along with the finding that only a small percentage of cellsharboring KRAS mutations are transformed, support the idea that additional, not fully uncovered, regulatory mechanisms may contribute to KRAS activation. Here we report that KrasG12D mice lacking Sirt2 show an aggressive tumorigenic phenotype as compared to KrasG12D mice. This phenotype includes increased proliferation, KRAS acetylation, and activation of RAS downstream signaling markers. Mechanistically, KRAS K147 is identified as a novel SIRT2-specific deacetylation target by mass spectrometry, whereas its acetylation status directly regulates KRAS activity, ultimately exerting an impact on cellular behavior as revealed by cell proliferation, colony formation, and tumor growth. Given the significance of KRAS activity as a driver in tumorigenesis, identification of K147 acetylation as a novel post-translational modification directed by SIRT2 in vivo may provide a better understanding of the mechanistic link regarding the crosstalk between non-genetic and genetic factors in KRAS driven tumors. PMID:27637077

  18. Methods for detection of ataxia telangiectasia mutations

    DOEpatents

    Gatti, Richard A.

    2005-10-04

    The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

  19. A Novel Technique to Detect EGFR Mutations in Lung Cancer.

    PubMed

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-05-23

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples.

  20. Master Regulators of Oncogenic KRAS Response in Pancreatic Cancer: An Integrative Network Biology Analysis

    PubMed Central

    2017-01-01

    Background KRAS is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC), but the mechanisms underlying the transcriptional response to oncogenic KRAS are still not fully understood. We aimed to uncover transcription factors that regulate the transcriptional response of oncogenic KRAS in pancreatic cancer and to understand their clinical relevance. Methods and Findings We applied a well-established network biology approach (master regulator analysis) to combine a transcriptional signature for oncogenic KRAS derived from a murine isogenic cell line with a coexpression network derived by integrating 560 human pancreatic cancer cases across seven studies. The datasets included the ICGC cohort (n = 242), the TCGA cohort (n = 178), and five smaller studies (n = 17, 25, 26, 36, and 36). 55 transcription factors were coexpressed with a significant number of genes in the transcriptional signature (gene set enrichment analysis [GSEA] p < 0.01). Community detection in the coexpression network identified 27 of the 55 transcription factors contributing to three major biological processes: Notch pathway, down-regulated Hedgehog/Wnt pathway, and cell cycle. The activities of these processes define three distinct subtypes of PDAC, which demonstrate differences in survival and mutational load as well as stromal and immune cell composition. The Hedgehog subgroup showed worst survival (hazard ratio 1.73, 95% CI 1.1 to 2.72, coxPH test p = 0.018) and the Notch subgroup the best (hazard ratio 0.62, 95% CI 0.42 to 0.93, coxPH test p = 0.019). The cell cycle subtype showed highest mutational burden (ANOVA p < 0.01) and the smallest amount of stromal admixture (ANOVA p < 2.2e–16). This study is limited by the information provided in published datasets, not all of which provide mutational profiles, survival data, or the specifics of treatment history. Conclusions Our results characterize the regulatory mechanisms underlying the transcriptional response to oncogenic

  1. Interlaboratory comparison of IDH mutation detection.

    PubMed

    van den Bent, Martin J; Hartmann, C; Preusser, Matthias; Ströbel, Thomas; Dubbink, Hendrikus J; Kros, Johan M; von Deimling, Andreas; Boisselier, Blandine; Sanson, Marc; Halling, Kevin C; Diefes, Kristin L; Aldape, Kenneth; Giannini, Caterina

    2013-04-01

    Isocitrate dehydrogenase (IDH) mutational testing is becoming increasingly important. For this, robust and reliable assays are needed. We tested the variation of results between six laboratories of testing for IDH mutations. Each laboratory received five unstained slides from 31 formalin-fixed paraffin-embedded (FFPE) glioma samples, and followed its own standard IDH diagnostic routine. All laboratories used immunohistochemistry (IHC) with an antibody against the most frequent IDH1 mutation (R132H) as a first step. Three laboratories then sequenced only IHC negative cases while the others sequenced all cases. Based on the overall analysis, 13 samples from 11 tumors had an R132H mutation and one tumor showed an R132G mutation. Results of IHC for IDH1 R132H mutations in all six laboratories were completely in agreement, and identified all R132H mutations. Upon sequencing the results of two laboratories deviated from those of the others. After a review of the entire diagnostic process, on repeat (blinded) testing one laboratory was completely in agreement with the overall result. A change in technique did only partially improve the results in the other laboratory. IHC for the IDH1 R132H mutation is very reliable and consistent across laboratories. IDH sequencing procedures yielded inconsistent results in 2 out of 6 laboratories. Quality assurance is pivotal before IDH testing is made part of clinical management of patients.

  2. XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer | Office of Cancer Genomics

    Cancer.gov

    The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity.

  3. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice

    PubMed Central

    Collins, Meredith A.; Bednar, Filip; Zhang, Yaqing; Brisset, Jean-Christophe; Galbán, Stefanie; Galbán, Craig J.; Rakshit, Sabita; Flannagan, Karen S.; Adsay, N. Volkan; Pasca di Magliano, Marina

    2012-01-01

    Pancreatic cancer is almost invariably associated with mutations in the KRAS gene, most commonly KRASG12D, that result in a dominant-active form of the KRAS GTPase. However, how KRAS mutations promote pancreatic carcinogenesis is not fully understood, and whether oncogenic KRAS is required for the maintenance of pancreatic cancer has not been established. To address these questions, we generated two mouse models of pancreatic tumorigenesis: mice transgenic for inducible KrasG12D, which allows for inducible, pancreas-specific, and reversible expression of the oncogenic KrasG12D, with or without inactivation of one allele of the tumor suppressor gene p53. Here, we report that, early in tumorigenesis, induction of oncogenic KrasG12D reversibly altered normal epithelial differentiation following tissue damage, leading to precancerous lesions. Inactivation of KrasG12D in established precursor lesions and during progression to cancer led to regression of the lesions, indicating that KrasG12D was required for tumor cell survival. Strikingly, during all stages of carcinogenesis, KrasG12D upregulated Hedgehog signaling, inflammatory pathways, and several pathways known to mediate paracrine interactions between epithelial cells and their surrounding microenvironment, thus promoting formation and maintenance of the fibroinflammatory stroma that plays a pivotal role in pancreatic cancer. Our data establish that epithelial KrasG12D influences multiple cell types to drive pancreatic tumorigenesis and is essential for tumor maintenance. They also strongly support the notion that inhibiting KrasG12D, or its downstream effectors, could provide a new approach for the treatment of pancreatic cancer. PMID:22232209

  4. Genetic aberrations in primary esophageal melanomas: molecular analysis of c-KIT, PDGFR, KRAS, NRAS and BRAF in a series of 10 cases.

    PubMed

    Langer, Rupert; Becker, Karen; Feith, Marcus; Friess, Helmut; Höfler, Heinz; Keller, Gisela

    2011-04-01

    We present a series of 10 primary esophageal melanomas of Caucasian patients characterized clinicopathologically and on the molecular level. Mutation analysis for c-Kit (exons 9, 11, 13 and 17), PDGFR (exons 12, 14 and 18), NRAS and KRAS were determined using PCR and direct sequencing. Analysis of the V600E mutation of BRAF was performed using mutation-specific PCR. Expression of c-Kit and PDGFR-A was additionally determined using immunohistochemistry. One tumor harbored a missense mutation in the c-Kit (p.F504L) and in the KRAS gene (p.G12S). A different c-Kit mutation (c.1507_1508 ins TTGCCT) was detected in another case. A third case had a V600E BRAF mutation. Using immunohistochemistry, c-Kit expression could be detected in all cases. The two cases with c-Kit mutations showed high c-Kit expression. None of the tumors showed a PDGFR mutation or expression or a NRAS mutation. We conclude that molecular analysis can identify targets for a specific therapy such as tyrosin kinase inhibitors as additional treatment option in these highly malignant tumors.

  5. Detection of mutations in health care: Strategy for retinoblastoma

    SciTech Connect

    Dunn, J.M.; Du, D.; Mostachfi, H.

    1994-09-01

    For diseases such as retinoblastoma, diagnosis of germline mutations in the RB1 gene is the only effective way to predict which members of a family will develop tumors, since each family has its own unique mutation. In order to develop a routine clinical test for mutation identification, we are using retinoblastoma as a model system for three reasons: the genetics of retinoblastoma are well understood; most of the heritable retinoblastomas are caused by new germline mutations; and the consequences of mutation identification and carrier status are clear. The mutations responsible for retinoblastoma fall into three broad classes: deletions, insertions and/or rearrangements, missense or nonsense point mutations, and translocations. Each class requires a different detection technique. Initial screening by quantitative amplification of each exon detects large and small deletions and insertions. Samples for which all exons appear normal are then directly sequenced. Samples that still appear to be normal are studied by FISH with probes flanking RB1 in order to detect translocations. Data analysis, lab coordination and patient reporting are managed using new software to efficiently handle the large amounts of data collected. The software techniques and strategy for mutation identification will be applicable to any genetic disease locus with a high proportion of new mutations, for example other tumor suppressor loci.

  6. Laser desorption mass spectrometry for point mutation detection

    SciTech Connect

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F.

    1996-10-01

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  7. Laser desorption mass spectrometry for point mutation detection

    SciTech Connect

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F.

    1996-12-31

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  8. Tilling to detect induced mutations in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world’s protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations...

  9. Different metabolic responses to PI3K inhibition in NSCLC cells harboring wild-type and G12C mutant KRAS

    PubMed Central

    Marabese, Mirko; Broggini, Massimo; Lupi, Monica; Pastorelli, Roberta

    2016-01-01

    KRAS mutations in non-small-cell lung cancer (NSCLC) patients are considered a negative predictive factor and indicate poor response to anticancer treatments. KRAS mutations lead to activation of the PI3K/akt/mTOR pathway, whose inhibition remains a challenging clinical target. Since the PI3K/akt/mTOR pathway and KRAS oncogene mutations all have roles in cancer cell metabolism, we investigated whether the activity of PI3K/akt/mTOR inhibitors (BEZ235 and BKM120) in cells harboring different KRAS status is related to their metabolic effect. Isogenic NSCLC cell clones expressing wild-type (WT) and mutated (G12C) KRAS were used to determine the response to BEZ235 and BKM120. Metabolomics analysis indicated the impairment of glutamine in KRAS-G12C and serine metabolism in KRAS-WT, after pharmacological blockade of the PI3K signaling, although the net effect on cell growth, cell cycle distribution and caspase activation was similar. PI3K inhibitors caused autophagy in KRAS-WT, but not in KRAS-G12C, where there was a striking decrease in ammonia production, probably a consequence of glutamine metabolism impairment. These findings lay the grounds for more effective therapeutic combinations possibly distinguishing wild-type and mutated KRAS cancer cells in NSCLC, exploiting their different metabolic responses to PI3K/akt/mTOR inhibitors. PMID:27283493

  10. ACB-PCR MEASUREMENT OF K-RAS CODON 12 MUTATION IN A/J MOUSE LUNG EXPOSED TO BENZO[A]PYRENE: A DOSE-RESPONSE ASSESSMENT

    EPA Science Inventory

    Benzo[a]pyrene (B[a]P) is a known human carcinogen and environmental contaminant. The direct measurement of K-Ras mutant fraction (MF) was developed as a metric with which to examine the default assumption of low dose linearity in the mutational response to B...

  11. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    PubMed

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P<0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group (P<0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group (P<0.05) but higher than the miR-143 mimic group significantly (P<0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the

  12. Early recognition of lung cancer by integrin targeted imaging in K-ras mouse model.

    PubMed

    Ermolayev, Vladimir; Mohajerani, Pouyan; Ale, Angelique; Sarantopoulos, Athanasios; Aichler, Michaela; Kayser, Gian; Walch, Axel; Ntziachristos, Vasilis

    2015-09-01

    Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex vivo and fluorescence molecular tomography-X-ray computed tomography (FMT-XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be noninvasively detected in vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

  13. Mutation detection in X-linked hydrocephalus

    SciTech Connect

    Forrest, S.M.; Balnaves, M.E.; Rosenthal, A.

    1994-09-01

    X-linked hydrocephalus (XLH), which maps to Xq28, affects about 1 in 30,000 male births. A candidate gene, L1-CAM, which codes for a neural adhesion molecule, mapped to the same region of the X chromosome. Rosenthal et al. (1992) identified a patient with XLH that had aberrant splicing of L1-CAM. A mutation at a potential branch point signal in an intron was identified. The gene has a number of exons and encodes a 4.2 kb mRNA. We isolated RNA from lymphocytes or fibroblasts from five XLH patients. cDNA was synthesized and the gene was amplified in two overlapping fragments, 2.2 kb and 1.7 kb respectively. A nested PCR approach with two rounds of PCR amplification was employed. Patient 900124 did not have a full length 5{prime} fragment and 880022 did not have a full length 3{prime} product. Restriction digestions defined the region of the alteration in the messenger RNA and sequencing in this region showed the loss of exons 10 and 21, respectively. All 5{prime} and 3{prime} products were also digested with several restriction enzymes (e.g., Msp I, Taq I), which have CG in their recognition sites, in the hope that point mutations that alter these restriction enzyme sites might be identified. A point mutation creating an Msp I site was found in patient 930067.

  14. Microarray-based mutation detection in the dystrophin gene.

    PubMed

    Hegde, Madhuri R; Chin, Ephrem L H; Mulle, Jennifer G; Okou, David T; Warren, Stephen T; Zwick, Michael E

    2008-09-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans>2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6 to 10% of males with either DMD or BMD. The remaining 30 to 35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, multiplex ligation-dependent probe amplification (MLPA), detection of virtually all mutations-SSCP (DOVAM-S), and single condition amplification/internal primer sequencing (SCAIP); however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution comparative genomic hybridization (CGH) microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin.

  15. Simple method for detection of mutations causing hereditary fructose intolerance.

    PubMed

    Kullberg-Lindh, C; Hannoun, C; Lindh, M

    2002-11-01

    Aldolase B is critical for sugar metabolism, and a catalytic deficiency due to mutations in its gene may result in hereditary fructose intolerance (HFI) syndrome, with hypoglycaemia and severe abdominal symptoms. This report describes two cases of HFI, which were identified by intravenous fructose tolerance test and a new RFLP (restriction fragment length polymorphism) test that detects the two most common mutations, A149P and A174D. The method includes PCR of a 224-base-pair segment of exon 5, a subsequent 3 h incubation with Cac8I and agarose electrophoresis, which reveals either or both of the mutations in one single reaction. The method might be useful for screening of these mutations, which may account for more than 70% of the mutations causing HFI.

  16. A novel assay to detect calreticulin mutations in myeloproliferative neoplasms

    PubMed Central

    Rosso, Valentina; Petiti, Jessica; Bracco, Enrico; Pedrola, Roberto; Carnuccio, Francesca; Signorino, Elisabetta; Carturan, Sonia; Calabrese, Chiara; Bot-Sartor, Giada; Ronconi, Michela; Serra, Anna; Saglio, Giuseppe; Frassoni, Francesco; Cilloni, Daniela

    2017-01-01

    The myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive (Ph+), chronic myeloid leukemia, or negative: polycythemia vera (PV) essential thrombocythemia (ET), and primary myelofibrosis (PMF). Most Ph negative cases have an activating JAK2 or MPL mutation. Recently, somatic mutations in the calreticulin gene (CALR) were detected in 56–88% of JAK2/MPL-negative patients affected by ET or PMF. The most frequent mutations in CARL gene are type-1 and 2. Currently, CALR mutations are evaluated by sanger sequencing. The evaluation of CARL mutations increases the diagnostic accuracy in patients without other molecular markers and could represent a new therapeutic target for molecular drugs. We developed a novel detection assay in order to identify type-1 and 2 CALR mutations by PNA directed PCR clamping. Seventy-five patients affected by myeloproliferative neoplasms and seven controls were examined by direct DNA sequencing and by PNA directed PCR clamping. The assay resulted to be more sensitive, specific and cheaper than sanger sequencing and it could be applied even in laboratory not equipped for more sophisticated analysis. Interestingly, we report here a case carrying both type 1 and type2 mutations in CALR gene. PMID:28031530

  17. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  18. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  19. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  20. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  1. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... regulator (CFTR) gene mutation detection system. 866.5900 Section 866.5900 Food and Drugs FOOD AND DRUG...) gene mutation detection system. (a) Identification. The CFTR gene mutation detection system is a device used to simultaneously detect and identify a panel of mutations and variants in the CFTR gene. It...

  2. KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2-c]quinolines

    PubMed Central

    Lavrado, João; Brito, Hugo; Borralho, Pedro M.; Ohnmacht, Stephan A.; Kim, Nam-Soon; Leitão, Clara; Pisco, Sílvia; Gunaratnam, Mekala; Rodrigues, Cecília M. P.; Moreira, Rui; Neidle, Stephen; Paulo, Alexandra

    2015-01-01

    KRAS is one of the most frequently mutated oncogenes in human cancer, yet remaining undruggable. To explore a new therapeutic strategy, a library of 5-methyl-indolo[3,2-c]quinoline derivatives (IQc) with a range of alkyldiamine side chains was designed to target DNA and RNA G-quadruplexes (G4) in the promoter and 5′-UTR mRNA of the KRAS gene. Biophysical experiments showed that di-substituted IQc compounds are potent and selective KRAS G4 stabilizers. They preferentially inhibit the proliferation of KRAS mutant cancer cell lines (0.22 < IC50 < 4.80 μM), down-regulate KRAS promoter activity in a luciferase reporter assay, and reduce both KRAS mRNA and p21KRAS steady-state levels in mutant KRAS colon cancer cell lines. Additionally, IQcs induce cancer cell death by apoptosis, explained in part by their capacity to repress KRAS expression. Overall, the results suggest that targeting mutant KRAS at the gene level with G4 binding small molecules is a promising anticancer strategy. PMID:25853628

  3. Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Zhang, Weiying; Nandakumar, Nivedita; Shi, Yuhao; Manzano, Mark; Smith, Alias; Graham, Garrett; Gupta, Swati; Vietsch, Eveline E.; Laughlin, Sean Z.; Wadhwa, Mandheer; Chetram, Mahandranauth; Joshi, Mrinmayi; Wang, Fen; Kallakury, Bhaskar; Toretsky, Jeffrey; Wellstein, Anton; Yi, Chunling

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D: Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras: Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers. PMID:24803537

  4. Association between proto-oncogene mutations and clinicopathologic characteristics and overall survival in colorectal cancer in East Azerbaijan, Iran

    PubMed Central

    Dolatkhah, Roya; Somi, Mohammad Hossein; Asvadi Kermani, Iraj; Bonyadi, Morteza; Sepehri, Bita; Boostani, Kamal; Azadbakht, Saleh; Fotouhi, Nikou; Farassati, Faris; Dastgiri, Saeed

    2016-01-01

    Background Colorectal cancer (CRC) is the third-most common cancer in Iran. The increasing incidence of CRC in the past three decades has made it a major public health burden in the country. This study aimed to determine any relationship of specific mutations in CRCs with clinicopathologic aspects and outcome of patients. Materials and methods This study was conducted on 100 CRC patients by the case-only method. Polymerase chain-reaction products were analyzed by Sanger sequencing, and sequence results were compared with the significant KRAS and BRAF gene mutations in the My Cancer Genome database. Logistic regression models were used to detect associations of clinicopathologic characteristics with each of the mutations. Kaplan–Meier and Cox regression models were constructed to estimate overall survival in patients. Results A total of 26 subjects (26%) had heterozygote-mutant KRAS, and mutations were not detected in the amplified exon of BRAF in both tumor and normal tissues of the 100 CRCs. Rectal tumors had 1.53-fold higher likelihood of KRAS mutations than colon tumors, and men had 1.37-fold higher odds than women. The presence of metastasis increased the likelihood of KRAS mutations 2.36-fold over those with nonmetastatic CRCs. Compared to patients with KRAS wild-type cancers, those with KRAS mutations had significantly higher mortality (hazard ratio 3.74, 95% confidence interval 1.44–9.68; log-rank P=0.003). Conclusion Better understanding of the causality of CRC can be established by combining epidemiology and research on molecular mechanisms of the disease. PMID:27994469

  5. Microarray-based mutation detection in the dystrophin gene

    PubMed Central

    Hegde, Madhuri R.; Chin, Ephrem L.H.; Mulle, Jennifer G.; Okou, David T.; Warren, Stephen T.; Zwick, Michael E.

    2008-01-01

    Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene affecting approximately 1 in 3,500 males. The human dystrophin gene spans > 2,200 kb, or roughly 0.1% of the genome, and is composed of 79 exons. The mutational spectrum of disease-causing alleles, including exonic copy number variations (CNVs), is complex. Deletions account for approximately 65% of DMD mutations and 85% of BMD mutations. Duplications occur in approximately 6–10% of males with either DMD or BMD. The remaining 30–35% of mutations consist of small deletions, insertions, point mutations, or splicing mutations, most of which introduce a premature stop codon. Laboratory analysis of dystrophin can be used to confirm a clinical diagnosis of DMD, characterize the type of dystrophin mutation, and perform prenatal testing and carrier testing for females. Current dystrophin diagnostic assays involve a variety of methodologies, including multiplex PCR, Southern blot analysis, MLPA, DOVAM-S, and SCAIP; however, these methods are time-consuming, laborious, and do not accurately detect duplication mutations in the dystrophin gene. Furthermore, carrier testing in females is often difficult when a related affected male is unavailable. Here we describe the development, design, validation, and implementation of a high-resolution CGH microarray-based approach capable of accurately detecting both deletions and duplications in the dystrophin gene. This assay can be readily adopted by clinical molecular testing laboratories and represents a rapid, cost-effective approach for screening a large gene, such as dystrophin. PMID:18663755

  6. Detection of BRAF mutations from solid tumors using Tumorplex™ technology

    PubMed Central

    Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

    2015-01-01

    Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

  7. Detection of BRAF mutations from solid tumors using Tumorplex™ technology.

    PubMed

    Yo, Jacob; Hay, Katie S L; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

    2015-01-01

    Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 10(7) (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. •Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal.•Tumorplex™ provides a high level of sensitivity.•Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA.

  8. Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate.

    PubMed

    Hiraoka, Kiriko; Inoue, Takahiro; Taylor, Rhys Dylan; Watanabe, Takayoshi; Koshikawa, Nobuko; Yoda, Hiroyuki; Shinohara, Ken-ichi; Takatori, Atsushi; Sugimoto, Hirokazu; Maru, Yoshiaki; Denda, Tadamichi; Fujiwara, Kyoko; Balmain, Allan; Ozaki, Toshinori; Bando, Toshikazu; Sugiyama, Hiroshi; Nagase, Hiroki

    2015-04-27

    Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.

  9. Long QT syndrome mutation detection by SNaPshot technique.

    PubMed

    Edelmann, Jeanett; Schumann, Stefanie; Nastainczyk, Marina; Husser-Bollmann, Daniela; Lessig, Rüdiger

    2012-11-01

    Long QT syndrome (LQTS) is a cardiac disorder with an abnormality of cardiac rhythm associated with sudden death especially in younger, apparently healthy individuals. If there is no clear cause of death detectable during comprehensive coroner's inquest (autopsy-negative cases), you have to consider LQTS and other heritable arrhythmia syndromes. A molecular genetic screening regarding mutations in associated genes can help to ensure the cause of death and to protect affected family members. Genetic testing of LQTS, currently performed mainly by sequencing, is still very expensive and time consuming. With this study we present a rapid and reasonable method for the simultaneously screening of some of the most common mutations associated with LQTS, focused on the KCNQ1 and KCNH2 genes. With the method of SNaPshot minisequencing, a total of 58 mutations were analyzed in four multiplex assays which were successfully established and optimized. The comparison with samples previously analyzed by direct sequencing showed concordance. Furthermore, autopsy-negative cases were tested but no mutations could be observed in any of the specimen. The presented method is well suitable for LQTS mutation screening. An enhancement to further mutations and population-based investigations regarding mutation frequencies should be the aim of prospective studies.

  10. Noninvasive detection of filaggrin gene mutations using Raman spectroscopy

    PubMed Central

    González, Francisco J.; Valdes-Rodríguez, Rodrigo; Ramírez-Elías, Miguel G.; Castillo-Martínez, Claudio; Saavedra-Alanis, Victor M.; Moncada, Benjamín

    2011-01-01

    Knowledge of the existence of filaggrin (FLG) gene mutations might be helpful for a subclassification of patients with atopic dermatitis (AD) which can be used to introduce individualized treatments. In this work the filaggrin content in the skin is assessed using Raman spectroscopy and the results are compared to FLG genotyping of Mexican-mestizo patients. Results showed that the 2282del4 and R501X mutations present in the European population but absent in people of Asian or African descent are also present in the Mexican-mestizo population. The results also showed that patients with filaggrin gene mutations presented lower filaggrin concentrations measured using the vector correlation of their skin Raman spectra and a fixed spectrum of pure human recombinant filaggrin, these results indicate that Raman spectroscopy may be used as a noninvasive tool to detect FLG gene mutations. PMID:22162825

  11. Noninvasive detection of filaggrin gene mutations using Raman spectroscopy.

    PubMed

    González, Francisco J; Valdes-Rodríguez, Rodrigo; Ramírez-Elías, Miguel G; Castillo-Martínez, Claudio; Saavedra-Alanis, Victor M; Moncada, Benjamín

    2011-12-01

    Knowledge of the existence of filaggrin (FLG) gene mutations might be helpful for a subclassification of patients with atopic dermatitis (AD) which can be used to introduce individualized treatments. In this work the filaggrin content in the skin is assessed using Raman spectroscopy and the results are compared to FLG genotyping of Mexican-mestizo patients. Results showed that the 2282del4 and R501X mutations present in the European population but absent in people of Asian or African descent are also present in the Mexican-mestizo population. The results also showed that patients with filaggrin gene mutations presented lower filaggrin concentrations measured using the vector correlation of their skin Raman spectra and a fixed spectrum of pure human recombinant filaggrin, these results indicate that Raman spectroscopy may be used as a noninvasive tool to detect FLG gene mutations.

  12. Specific inhibition of p110α subunit of PI3K: putative therapeutic strategy for KRAS mutant colorectal cancers

    PubMed Central

    Fernandes, Maria Sofia; Melo, Soraia; Velho, Sérgia; Carneiro, Patrícia; Carneiro, Fátima; Seruca, Raquel

    2016-01-01

    Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. It is often associated with activating mutations in KRAS leading to deregulation of major signaling pathways as the RAS-RAF-MAPK and PI3K-Akt. However, the therapeutic options for CRC patients harboring somatic KRAS mutations are still very limited. It is therefore urgent to unravel novel therapeutic approaches for those patients. In this study, we have awarded PI3K p110α a key role in CRC cells harboring KRAS/PIK3CA mutations or KRAS mutations alone. Specific silencing of PI3K p110α by small interfering RNA (siRNA) reduced viability and induced apoptosis or cell cycle arrest. In agreement with these cellular effects, PI3K p110α silencing led to alterations in the expression levels of proteins implicated in apoptosis and cell cycle, namely XIAP and pBad in KRAS/PIK3CA mutant cells and cyclin D1 in KRAS mutant cells. To further validate our data, a specific PI3K p110α inhibitor, BYL719, was evaluated. BYL719 mimicked the in vitro siRNA effects on cellular viability and on the alterations of apoptotic- and cell cycle-related proteins in CRC mutant cells. Overall, this study demonstrates that specific inhibition of PI3K p110α could provide an alternative therapeutic approach for CRC patients, particularly those harboring KRAS mutations. PMID:27602501

  13. Mutation analysis and carrier detection of Hunter syndrome

    SciTech Connect

    Li, P.; Thompson, J.N.; Hug, G.; Chuck, G.

    1994-09-01

    Hunter syndrome, mucopolysaccharidosis type II (MPS II, MIM 309900), is an X-linked lysosomal storage disorder due to a deficiency of iduronate-2-sulfatase (IDS, EC 3.1.6.13) activity. The IDS cDNA sequence and genomic exon/intron sequences have been characterized. In the present investigation, 8 patients with IDS deficiency enzymatically diagnosed by us were used for molecular analysis. Total cellular RNA was isolated from cultured skin fibroblasts and DNA was isolated from cultured skin fibroblasts or leukocytes of the patients and controls. A RT-PCR sequencing method and an exon-by exon PCR amplification of the IDS gene were used for mutation analysis. Six new mutations, from the 8 patients, have been identified: A346V (GCC to GTC) in exon VIII coexistent with T146T silent mutation and a two-nucleotide insertion at codon 423 (CCC to CCCCC) in exon IX coexistent with T146T were found in two patients with a mild Hunter phenotype; S71R (AGC to AGA) in exon II, L279X (TTA to TGA) in exon VI, 406delCT (CTT to --T) in exon IX, and 407delTT (TTT to T--) in exon IX were found in 4 unrelated cases. All mutations detected by RT-PCR sequencing were confirmed by restriction enzyme assay and PCR analysis of the genomic DNA sequence. The mutation L279X eliminated an Mse I site from the mutant allele. Carrier detection for this mutation revealed that the mother of the proband was a carrier; however, neither her mother nor any of her sisters were carriers and none of her brothers were affected. We postulate that the mutation probably originated from a germline mutation. Our studies support previous findings that a large proportion of MPS II families result from new mutations. The RT-PCR sequencing and exon-by-exon PCR method has proven to be a practical approach for mutation analysis and carrier detection in families with an affected MPS II.

  14. Oxidative DNA damage drives carcinogenesis in MUTYH-associated-polyposis by specific mutations of mitochondrial and MAPK genes.

    PubMed

    Venesio, Tiziana; Balsamo, Antonella; Errichiello, Edoardo; Ranzani, Guglielmina N; Risio, Mauro

    2013-10-01

    MUTYH is a DNA-base-excision-repair gene implicated in the activation of nuclear and mitochondrial cell-death pathways. MUTYH germline mutations cause an inherited polyposis, MUTYH-associated-polyposis, characterized by multiple adenomas and increased susceptibility to colorectal cancer. Since this carcinogenesis remains partially unknown, we searched for nuclear and mitochondrial gene alterations that may drive the tumorigenic process. Ninety-six adenomas and 7 carcinomas from 12 MUTYH-associated-polyposis and 13 classical/attenuated adenomatous polyposis patients were investigated by sequencing and pyrosequencing for the presence of mutations in KRAS, BRAF, MT-CO1/MT-CO2 and MT-TD genes. KRAS mutations were identified in 24% MUTYH-associated-polyposis vs 15% classical/attenuated familial polyposis adenomas; mutated MUTYH-associated-polyposis adenomas exhibited only c.34G>T transversions in codon 12, an alteration typically associated with oxidative DNA damage, or mutations in codon 13; neither of these mutations was found in classical/attenuated familial polyposis adenomas (P<0.001). Mutated MUTYH-associated-polyposis carcinomas showed KRAS c.34G>T transversions, prevalently occurring with BRAFV600E; none of the classical/attenuated familial polyposis carcinomas displayed these alterations. Comparing mitochondrial DNA from lymphocytes and adenomas of the same individuals, we detected variants in 82% MUTYH-associated-polyposis vs 38% classical/attenuated familial polyposis patients (P=0.040). MT-CO1/MT-CO2 missense mutations, which cause aminoacid changes, were only found in MUTYH-associated-polyposis lesions and were significantly associated with KRAS mutations (P=0.0085). We provide evidence that MUTYH-associated-polyposis carcinogenesis is characterized by the occurrence of specific mutations in both KRAS and phylogenetically conserved genes of mitochondrial DNA which are involved in controlling oxidative phosphorylation; this implies the existence of a

  15. Detecting gene mutations in Japanese Alzheimer's patients by semiconductor sequencing.

    PubMed

    Yagi, Ryoichi; Miyamoto, Ryosuke; Morino, Hiroyuki; Izumi, Yuishin; Kuramochi, Masahito; Kurashige, Takashi; Maruyama, Hirofumi; Mizuno, Noriyoshi; Kurihara, Hidemi; Kawakami, Hideshi

    2014-07-01

    Alzheimer's disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD.

  16. Detection of driver pathways using mutated gene network in cancer.

    PubMed

    Li, Feng; Gao, Lin; Ma, Xiaoke; Yang, Xiaofei

    2016-06-21

    Distinguishing driver pathways has been extensively studied because they are critical for understanding the development and molecular mechanisms of cancers. Most existing methods for driver pathways are based on high coverage as well as high mutual exclusivity, with the underlying assumption that mutations are exclusive. However, in many cases, mutated driver genes in the same pathways are not strictly mutually exclusive. Based on this observation, we propose an index for quantifying mutual exclusivity between gene pairs. Then, we construct a mutated gene network for detecting driver pathways by integrating the proposed index and coverage. The detection of driver pathways on the mutated gene network consists of two steps: raw pathways are obtained using a CPM method, and the final driver pathways are selected using a strict testing strategy. We apply this method to glioblastoma and breast cancers and find that our method is more accurate than state-of-the-art methods in terms of enrichment of KEGG pathways. Furthermore, the detected driver pathways intersect with well-known pathways with moderate exclusivity, which cannot be discovered using the existing algorithms. In conclusion, the proposed method provides an effective way to investigate driver pathways in cancers.

  17. Single molecule targeted sequencing for cancer gene mutation detection

    PubMed Central

    Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W.; He, Jiankui

    2016-01-01

    With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis. PMID:27193446

  18. Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

    NASA Astrophysics Data System (ADS)

    Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

    2016-11-01

    K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach –conditional time-delayed correlations (CTC) – using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras.

  19. Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

    PubMed Central

    Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

    2016-01-01

    K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach –conditional time-delayed correlations (CTC) – using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras. PMID:27845397

  20. KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated β-TrCP1 degradation.

    PubMed

    Shukla, Shirish; Allam, Uday Sankar; Ahsan, Aarif; Chen, Guoan; Krishnamurthy, Pranathi Meda; Marsh, Katherine; Rumschlag, Matthew; Shankar, Sunita; Whitehead, Christopher; Schipper, Matthew; Basrur, Venkatesha; Southworth, Daniel R; Chinnaiyan, Arul M; Rehemtulla, Alnawaz; Beer, David G; Lawrence, Theodore S; Nyati, Mukesh K; Ray, Dipankar

    2014-02-01

    Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, β-transducing repeat containing protein 1 (β-TrCP1). Conversely, β-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, β-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

  1. Enhanced MET translation and signaling sustains K-Ras driven proliferation under anchorage-independent growth conditions

    PubMed Central

    Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

    2015-01-01

    Oncogenic K-Ras mutation occurs frequently in several types of cancers including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global down-regulation of mRNA translation during anchorage independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras mediated Met expression drives “K-Ras addiction” in anchorage independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. PMID:25977330

  2. Oncogenic KRAS triggers MAPK-dependent errors in mitosis and MYC-dependent sensitivity to anti-mitotic agents

    PubMed Central

    Perera, David; Venkitaraman, Ashok R.

    2016-01-01

    Oncogenic KRAS induces cell proliferation and transformation, but little is known about its effects on cell division. Functional genetic screens have recently revealed that cancer cell lines expressing oncogenic KRAS are sensitive to interference with mitosis, but neither the mechanism nor the uniformity of anti-mitotic drug sensitivity connected with mutant KRAS expression are yet clear. Here, we report that acute expression of oncogenic KRAS in HeLa cells induces mitotic delay and defects in chromosome segregation through mitogen-activated protein kinase (MAPK) pathway activation and de-regulated expression of several mitosis-related genes. These anomalies are accompanied by increased sensitivity to anti-mitotic agents, a phenotype dependent on the transcription factor MYC and its downstream target anti-apoptotic protein BCL-XL. Unexpectedly, we find no correlation between KRAS mutational status or MYC expression levels and anti-mitotic drug sensitivity when surveying a large database of anti-cancer drug responses. However, we report that the co-existence of KRAS mutations and high MYC expression predicts anti-mitotic drug sensitivity. Our findings reveal a novel function of oncogenic KRAS in regulating accurate mitotic progression and suggest new avenues to therapeutically target KRAS-mutant tumours and stratify patients in ongoing clinical trials of anti-mitotic drugs. PMID:27412232

  3. Targeted single molecule mutation detection with massively parallel sequencing

    PubMed Central

    Gregory, Mark T.; Bertout, Jessica A.; Ericson, Nolan G.; Taylor, Sean D.; Mukherjee, Rithun; Robins, Harlan S.; Drescher, Charles W.; Bielas, Jason H.

    2016-01-01

    Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10−7 per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomyces cerevisiae genome. PMID:26384417

  4. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices.

    PubMed

    Leshchiner, Elizaveta S; Parkhitko, Andrey; Bird, Gregory H; Luccarelli, James; Bellairs, Joseph A; Escudero, Silvia; Opoku-Nsiah, Kwadwo; Godes, Marina; Perrimon, Norbert; Walensky, Loren D

    2015-02-10

    Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.

  5. Lin28-let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

    SciTech Connect

    Oh, Jee-Sun.; Kim, Jae-Jin; Byun, Ju-Yeon; Kim, In-Ah

    2010-01-15

    Purpose: To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. Methods and Materials: A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. Results: The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression and radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. Conclusions: The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.

  6. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    PubMed Central

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  7. Molecular and Functional Characteristics of Ovarian Surface Epithelial Cells Transformed by KrasG12D and loss of Pten in a Mouse Model in vivo

    PubMed Central

    Mullany, Lisa K.; Fan, Heng-Yu; Liu, Zhilin; White, Lisa D.; Marshall, Alexandra; Gunaratne, Preethi; Anderson, Matthew L.; Creighton, Chad J.; Xin, Li; Deavers, Michael; Wong, Kwong-Kwok; Richards, JoAnne S.

    2011-01-01

    Ovarian cancer is a complex and deadly disease that remains difficult to detect at an early curable stage. Furthermore, although some oncogenic (Kras, Pten/PI3K and Trp53) pathways that are frequently mutated, deleted or amplified in ovarian cancer are known, how these pathways initiate and drive specific morphological phenotypes and tumor outcomes remain unclear. We recently generated Pten fl/fl; KrasG12D;Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of KrasG12D in ovarian surface epithelial (OSE) cells. Based on histopathologic criteria, the mutant mice developed low-grade ovarian serous papillary adenocarcinomas at an early age and with 100% penetrance. This highly reproducible phenotype provides the first mouse model in which to study this ovarian cancer subtype. OSE cells isolated from ovaries of mutant mice at 5 and 10 weeks of age exhibit temporal changes in the expression of specific Mullerian epithelial marker genes, grow in soft agar and develop ectopic invasive tumors in recipient mice, indicating that the cells are transformed. Gene profiling identified specific mRNAs and microRNAs differentially expressed in purified OSE cells derived from tumors of the mutant mice compared to WT OSE cells. Mapping of transcripts or genes between the mouse OSE mutant datasets, the Kras signature from human cancer cell lines and the human ovarian tumor array datasets, documented significant overlap, indicating that KRAS is a key driver of OSE transformation in this context. Two key hallmarks of the mutant OSE cells in these mice are the elevated expression of the tumor suppressorsTrp53 (p53) and its microRNA target, miR-34a-c. We propose that elevated TRP53 and miR-34a-c may exert negatively regulatory effects that reduce the proliferative potential of OSE cells leading to the low-grade serous adenocarcinoma phenotype. PMID:21423204

  8. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  9. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  10. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  11. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  12. 21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents...

  13. Prognostic relevance of KRAS genotype in metastatic colorectal cancer patients unfit for FIr-B/FOx intensive regimen

    PubMed Central

    BRUERA, GEMMA; CANNITA, KATIA; GIORDANO, ALDO VICTOR; VICENTINI, ROBERTO; FICORELLA, CORRADO; RICEVUTO, ENRICO

    2014-01-01

    First-line triplet chemotherapy plus bevacizumab (FIr-B/FOx) can improve efficacy of metastatic colorectal cancer (MCRC), KRAS wild-type and mutant. Prognostic relevance of KRAS genotype was evaluated in patients unfit for FIr-B/FOx, treated with conventional medical treatments. Consecutive MCRC patients not eligible for FIr-B/FOx regimen due to age (≥75 years) and/or comorbidities were treated with tailored conventional first-line treatments. KRAS codon 12/13 mutations were screened by direct sequencing. Activity and efficacy were evaluated and compared according to medical treatments, age (non-elderly and elderly ≥65 years), comorbidity stage (Cumulative Illness Rating Scale), metastatic extension (liver-limited and other/multiple metastatic), and KRAS genotype, using log-rank. Selected first line treatments were medical in 37 patients (92.5%), and surgical in 3 patients (7.5%). Medical treatment regimens: triplet, 18 (45%); doublet, 15 (37.5%); mono-therapy, 4 (10%). At median follow-up of 8 months, objective response rate (ORR) was 37%, median progression-free survival (PFS) 7 months, liver metastasectomies 8% (liver-limited disease 37.5%), median overall survival (OS) 13 months. Triplet regimens failed to significantly affect clinical outcome, compared to doublet. According to KRAS genotype, ORR, PFS and OS were, respectively: wild-type 50%, 8 months, 13 months; mutant 25%, 6 months, 9 months. KRAS genotype wild-type compared to mutant significantly affected PFS, while not OS. KRAS c.35 G>A mutation (G12D) significantly affected worse PFS and OS compared to wild-type and/or other mutations. KRAS genotype, specifically the c.35 G>A KRAS mutation, may indicate poor prognosis in MCRC patients unfit for intensive medical treatments. PMID:24715238

  14. Lifetime alcohol intake is associated with an increased risk of KRAS+ and BRAF-/KRAS- but not BRAF+ colorectal cancer.

    PubMed

    Jayasekara, Harindra; MacInnis, Robert J; Williamson, Elizabeth J; Hodge, Allison M; Clendenning, Mark; Rosty, Christophe; Walters, Rhiannon; Room, Robin; Southey, Melissa C; Jenkins, Mark A; Milne, Roger L; Hopper, John L; Giles, Graham G; Buchanan, Daniel D; English, Dallas R

    2017-04-01

    Ethanol in alcoholic beverages is a causative agent for colorectal cancer. Colorectal cancer is a biologically heterogeneous disease, and molecular subtypes defined by the presence of somatic mutations in BRAF and KRAS are known to exist. We examined associations between lifetime alcohol intake and molecular and anatomic subtypes of colorectal cancer. We calculated usual alcohol intake for 10-year periods from age 20 using recalled frequency and quantity of beverage-specific consumption for 38,149 participants aged 40-69 years from the Melbourne Collaborative Cohort Study. Cox regression was performed to derive hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between lifetime alcohol intake and colorectal cancer risk. Heterogeneity in the HRs across subtypes of colorectal cancer was assessed. A positive dose-dependent association between lifetime alcohol intake and overall colorectal cancer risk (mean follow-up = 14.6 years; n = 596 colon and n = 326 rectal cancer) was observed (HR = 1.08, 95% CI: 1.04-1.12 per 10 g/day increment). The risk was greater for rectal than colon cancer (phomogeneity  = 0.02). Alcohol intake was associated with increased risks of KRAS+ (HR = 1.07, 95% CI: 1.00-1.15) and BRAF-/KRAS- (HR = 1.05, 95% CI: 1.00-1.11) but not BRAF+ tumors (HR = 0.89, 95% CI: 0.78-1.01; phomogeneity  = 0.01). Alcohol intake is associated with an increased risk of KRAS+ and BRAF-/KRAS- tumors originating via specific molecular pathways including the traditional adenoma-carcinoma pathway but not with BRAF+ tumors originating via the serrated pathway. Therefore, limiting alcohol intake from a young age might reduce colorectal cancer originating via the traditional adenoma-carcinoma pathway.

  15. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    SciTech Connect

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M.

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  16. Multipyrene Tandem Probes for Point Mutations Detection in DNA

    PubMed Central

    Kholodar, Svetlana A.; Novopashina, Darya S.; Meschaninova, Mariya I.; Venyaminova, Alya G.

    2013-01-01

    Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. PMID:24455205

  17. Targeting oncogenic KRAS in non-small cell lung cancer cells by phenformin inhibits growth and angiogenesis

    PubMed Central

    Wang, Zhi Dong; Wei, Sheng Quan; Wang, Qin Yi

    2015-01-01

    Tumors require a vascular supply to grow and can achieve this via the expression of pro-angiogenic growth factors. Many potential oncogenic mutations have been identified in tumor angiogenesis. Somatic mutations in the small GTPase KRAS are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The extracellular regulated protein kinases (ERK) signaling is known to be a major cellular target of biguanides. Based on KRAS activates several down-stream effectors leading to the stimulation of the RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAF/MEK/ERK) and phosphatidylinositol-3-kinase (PI3K) pathways, we investigated the anti-tumor effects of biguanides on the proliferation of KRAS-mutated tumor cells in vitro and on KRAS-driven tumor growth in vivo. In cancer cells harboring oncogenic KRAS, phenformin switches off the ERK pathway and inhibit the expression of pro-angiogenic molecules. In tumor xenografts harboring the KRAS mutation, phenformin extensively modifies the tumor growth causing abrogation of angiogenesis. These results strongly suggest that significant therapeutic advantage may be achieved by phenformin anti-angiogenesis for the treatment of tumor. PMID:26807315

  18. Inhibition of HSP90 by AUY922 Preferentially Kills Mutant KRAS Colon Cancer Cells by Activating Bim through ER Stress.

    PubMed

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Liu, Fen; Zhang, Yuan Yuan; Yari, Hamed; Yan, Xu Guang; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-03-01

    Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.

  19. Enhanced dependency of KRAS-mutant colorectal cancer cells on RAD51-dependent homologous recombination repair identified from genetic interactions in Saccharomyces cerevisiae.

    PubMed

    Kalimutho, Murugan; Bain, Amanda L; Mukherjee, Bipasha; Nag, Purba; Nanayakkara, Devathri M; Harten, Sarah K; Harris, Janelle L; Subramanian, Goutham N; Sinha, Debottam; Shirasawa, Senji; Srihari, Sriganesh; Burma, Sandeep; Khanna, Kum Kum

    2017-02-07

    Activating KRAS mutations drive colorectal cancer tumorigenesis and influence response to anti-EGFR-targeted therapy. Despite recent advances in understanding Ras signaling biology and the revolution in therapies for melanoma using BRAF inhibitors, no targeted agents have been effective in KRAS-mutant cancers, mainly due to activation of compensatory pathways. Here, by leveraging the largest synthetic lethal genetic interactome in yeast, we identify that KRAS-mutated colorectal cancer cells have augmented homologous recombination repair (HRR) signaling. We found that KRAS mutation resulted in slowing and stalling of the replication fork and accumulation of DNA damage. Moreover, we found that KRAS-mutant HCT116 cells have an increase in MYC-mediated RAD51 expression with a corresponding increase in RAD51 recruitment to irradiation-induced DNA double-strand breaks (DSBs) compared to genetically complemented isogenic cells. MYC depletion using RNA interference significantly reduced IR-induced RAD51 foci formation and HRR. On the contrary, overexpression of either HA-tagged wild-type (WT) MYC or phospho-mutant S62A increased RAD51 protein levels and hence IR-induced RAD51 foci. Likewise, depletion of RAD51 selectively induced apoptosis in HCT116-mutant cells by increasing DSBs. Pharmacological inhibition targeting HRR signaling combined with PARP inhibition selectivity killed KRAS-mutant cells. Interestingly, these differences were not seen in a second isogenic pair of KRAS WT and mutant cells (DLD-1), likely due to their nondependency on the KRAS mutation for survival. Our data thus highlight a possible mechanism by which KRAS-mutant-dependent cells drive HRR in vitro by upregulating MYC-RAD51 expression. These data may offer a promising therapeutic vulnerability in colorectal cancer cells harboring otherwise nondruggable KRAS mutations, which warrants further investigation in vivo.

  20. Deconstruction of Oncogenic K-RAS Signaling Reveals Focal Adhesion Kinase as a Novel Therapeutic Target in NSCLC

    DTIC Science & Technology

    2015-10-01

    SUPPLEMENTARY NOTES 14. ABSTRACT About 25% of lung adenocarcinomas express mutant KRAS (KM) often is association with co-occurring mutations that...There are no therapies that target cancers that express mutant KRAS. Thus, it is notable that inhibition of FAK causes cell death specifically in KM...lung cancer cells (KMLC) that are either CDKN2A mutant or p53 mutant . Furthermore, we found that pharmacologic inhibition of FAK causes the

  1. Absence of ras-gene hot-spot mutations in canine fibrosarcomas and melanomas.

    PubMed

    Murua Escobar, Hugo; Günther, Kathrin; Richter, Andreas; Soller, Jan T; Winkler, Susanne; Nolte, Ingo; Bullerdiek, Jörn

    2004-01-01

    Point mutations within ras proto-oncogenes, particularly within the mutational hot-spot codons 12, 13 and 61, are frequently detected in human malignancies and in different types of experimentally-induced tumours in animals. So far little is known about ras mutations in naturally occurring canine fibrosarcomas or K-ras mutations in canine melanomas. To elucidate whether ras mutations exist in these naturally occurring tumours in dogs, in the present study we screened 13 canine fibrosarcomas, 2 feline fibrosarcomas and 11 canine melanomas for point mutations, particularly within the mutational hot-spots, making this the first study to investigate a large number of canine fibrosarcomas. None of the samples showed a K- or N-ras hot spot mutation. Thus, our data strongly suggest that ras mutations at the hot-spot loci are very rare and do not play a major role in the pathogenesis of the spontaneously occurring canine tumours investigated.

  2. Quantitative and sensitive detection of GNAS mutations causing mccune-albright syndrome with next generation sequencing.

    PubMed

    Narumi, Satoshi; Matsuo, Kumihiro; Ishii, Tomohiro; Tanahashi, Yusuke; Hasegawa, Tomonobu

    2013-01-01

    Somatic activating GNAS mutations cause McCune-Albright syndrome (MAS). Owing to low mutation abundance, mutant-specific enrichment procedures, such as the peptide nucleic acid (PNA) method, are required to detect mutations in peripheral blood. Next generation sequencing (NGS) can analyze millions of PCR amplicons independently, thus it is expected to detect low-abundance GNAS mutations quantitatively. In the present study, we aimed to develop an NGS-based method to detect low-abundance somatic GNAS mutations. PCR amplicons encompassing exons 8 and 9 of GNAS, in which most activating mutations occur, were sequenced on the MiSeq instrument. As expected, our NGS-based method could sequence the GNAS locus with very high read depth (approximately 100,000) and low error rate. A serial dilution study with use of cloned mutant and wildtype DNA samples showed a linear correlation between dilution and measured mutation abundance, indicating the reliability of quantification of the mutation. Using the serially diluted samples, the detection limits of three mutation detection methods (the PNA method, NGS, and combinatory use of PNA and NGS [PNA-NGS]) were determined. The lowest detectable mutation abundance was 1% for the PNA method, 0.03% for NGS and 0.01% for PNA-NGS. Finally, we analyzed 16 MAS patient-derived leukocytic DNA samples with the three methods, and compared the mutation detection rate of them. Mutation detection rate of the PNA method, NGS and PNA-NGS in 16 patient-derived peripheral blood samples were 56%, 63% and 75%, respectively. In conclusion, NGS can detect somatic activating GNAS mutations quantitatively and sensitively from peripheral blood samples. At present, the PNA-NGS method is likely the most sensitive method to detect low-abundance GNAS mutation.

  3. [Technical considerations for KRAS testing in colorectal cancer. The pathologist's point of view].

    PubMed

    Bibeau, F; Frugier, H; Denouel, A; Sabourin, J-C; Boissiere-Michot, F

    2009-12-01

    The KRAS status is now a mandatory prerequisite in order to treat metastatic colorectal patients with anti-EGFR (epidermal growth factor receptor) antibodies, such as cetuximab (Erbitux) or panitumumab (Vectibix). KRAS mutations are unambiguously linked to a lack of response to these targeted therapies and to a poor outcome. The optimal determination of the KRAS status should be based on coordination between pathologists and biologists. The pathologist must morphologically check the tumor to be analyzed and be sure that the fixatives used are valuable for molecular biology. The pathologist's involvement may also concern the DNA extraction and the KRAS mutations analyses. This involvement has to be included in a multidisciplinary setting in order to get rapid and robust tests for the clinical use. The imperative knowledge of the KRAS status in the management of metastatic disease represents a good example of this multidisciplinary coordination. In the future, the pathologist's role should be extended, considering the emergence of a more and more personalized medicine, integrating efficiency and cost-effectiveness. Thus, the pathologist may contribute to validate new molecular tests and to offer his specific techniques for translational research.

  4. Genetic Evidence for XPC-KRAS Interactions During Lung Cancer Development

    PubMed Central

    Zhang, Xiaoli; He, Nonggao; Gu, Dongsheng; Wickliffe, Jeff; Salazar, James; Boldogh, Istavan; Xie, Jingwu

    2015-01-01

    Lung cancer causes more deaths than breast, colorectal and prostate cancers combined. Despite major advances in targeted therapy in a subset of lung adenocarcinomas, the overall 5-year survival rate for lung cancer worldwide has not significantly changed for the last few decades. DNA repair deficiency is known to contribute to lung cancer development. In fact, human polymorphisms in DNA repair genes such as xeroderma pigmentosum group C (XPC) are highly associated with lung cancer incidence. However, the direct genetic evidence for the role of XPC for lung cancer development is still lacking. Mutations of the Kirsten rat sarcoma viral oncogene homolog (Kras) or its downstream effector genes occur in almost all lung cancer cells, and there are a number of mouse models for lung cancer using these mutations. Using activated Kras, KrasLA1, as a driver for lung cancer development in mice, we showed for the first time that mice with KrasLA1 and Xpc knockout had worst outcomes in lung cancer development, and this phenotype was associated with accumulated DNA damage. Using cultured cells, we demonstrated that induced expression of oncogenic KRASG12V led to increased levels of reactive oxygen species (ROS) as well as DNA damage, and both can be suppressed by anti-oxidants. Thus, it appears that XPC may help repair DNA damage caused by KRAS-mediated production of ROS. PMID:26554912

  5. Altered detoxification status and increased resistance to oxidative stress by K-ras transformation.

    PubMed

    Recktenwald, Christian V; Kellner, Roland; Lichtenfels, Rudolf; Seliger, Barbara

    2008-12-15

    Mutated K-ras is frequently found in human malignancies and plays a key role in many signal transduction processes resulting in an altered gene and/or protein expression pattern. Proteins controlled by a constitutive activated mitogen-activated protein kinase pathway are primarily related to alterations in the mitochondrial and nuclear compartments. Therefore, different K-Ras mutants and respective control cells were subjected to two-dimensional gel electrophoresis using basic pH gradients. This approach led to the identification of differentially expressed proteins, such as members of the heterogeneous ribonucleoprotein family, and enzymes involved in cellular detoxification as well as in oxidative stress. Increased expression of these enzymes was paralleled by an elevated tolerance of K-ras mutants against the cytotoxic potential of hydrogen peroxide and formaldehyde as well as an altered redox status based on enhanced intracellular glutathione (GSH) levels indicating an improved detoxification potential of defined K-ras transfectants, whereas down-regulation by RNA interference of candidate proteins reversed the tolerance against these compounds. This hypothesis is supported by an up-regulated expression of a key enzyme of the pentose phosphate pathway resulting in an increased production of NADPH required for anabolic processes as well as the rebuilding of oxidized GSH. Both the enhanced resistance against xenobiotic compounds as well as an altered oxidative pathway might confer growth advantages for tumor cells carrying dominant-positive K-ras mutations such as in lung or pancreatic adenocarcinoma.

  6. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    PubMed Central

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  7. In situ detection of specific p53 mutations in cultured cells using the amplification refractory mutation system polymerase chain reaction.

    PubMed

    Low, E O; Gibbins, J R; Walker, D M

    2000-12-01

    Accurate molecular detection of genetic mutations involved in tumorigenesis has been based predominantly on analysis of extracted DNA, but this does not provide detailed information on the location, number, type or clonal distribution of mutated cells and their precise anatomic location and clonal distribution. This study has used a sensitive and specific application of the amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) in situ, combined with in situ hybridization to localize and identify cells with defined p53 mutations. The ARMS-PCR was performed in situ in SW480 cells in suspension and in cells either cultured or cytospun onto glass slides. Amplified mutant DNA PCR products were detected in SW480 cells using digoxigenin-labeled probes, visually identifying cells harboring specific mutations in the p53 gene. In situ hybridization alone of the mutant cells without the amplification step was negative. Normal human fibroblasts or endothelial cells were refractory to in situ amplification. This reaction was mutation-specific as CEM cells with different p53 mutations reacted negatively. Mutant messenger RNA (mRNA) in tumor cells was also selectively amplified in situ by ARMS-PCR following reverse transcription (RT). This study demonstrates the potential of in situ ARMS-PCR or RT-ARMS-PCR for mutation analysis in situ and could have useful clinical applications.

  8. The incidence of PAX6 mutation in patients with simple aniridia: an evaluation of mutation detection in 12 cases.

    PubMed Central

    Axton, R; Hanson, I; Danes, S; Sellar, G; van Heyningen, V; Prosser, J

    1997-01-01

    Twelve aniridia patients, five with a family history and seven presumed to be sporadic, were exhaustively screened in order to test what proportion of people with aniridia, uncomplicated by associated anomalies, carry mutations in the human PAX6 gene. Mutations were detected in 90% of the cases. Three mutation detection techniques were used to determine if one method was superior for this gene. The protein truncation test (PTT) was used on RT-PCR products, SSCP on genomic PCR amplifications, and chemical cleavage of mismatch on both RT-PCR and genomic amplifications. For RT-PCR products, only the translated portion of the gene was screened. On genomic products exons 1 to 13 (including 740 bp of the 3' untranslated sequence and all intron/exon boundaries) were screened, as was a neuroretina specific enhancer in intron 4. Ten of the possible 12 mutations in the five familial cases and five of the sporadic patients were found, all of which conformed to a functional outcome of haploinsufficiency. Five were splice site mutations (one in the donor site of intron 4, two in the donor site of intron 6, one in each of the acceptor sites of introns 8 and 9) and five were nonsense mutations in exons 8, 9, 10, 11, and 12. SSCP analysis of individually amplified exons, with which nine of the 10 mutations were seen, was the most useful detection method for PAX6. Images PMID:9138149

  9. KRAS mutant allele-specific imbalance is associated with worse prognosis in pancreatic cancer and progression to undifferentiated carcinoma of the pancreas.

    PubMed

    Krasinskas, Alyssa M; Moser, A James; Saka, Burcu; Adsay, N Volkan; Chiosea, Simion I

    2013-10-01

    KRAS codon 12 mutations are present in about 90% of ductal adenocarcinomas and in undifferentiated carcinomas of the pancreas. The role of KRAS copy number changes and resulting KRAS mutant allele-specific imbalance (MASI) in ductal adenocarcinoma (n=94), and its progression into undifferentiated carcinoma of the pancreas (n=25) was studied by direct sequencing and KRAS fluorescence in situ hybridization (FISH). Semi-quantitative evaluation of sequencing electropherograms showed KRAS MASI (ie, mutant allele peak higher than or equal to the wild-type allele peak) in 22 (18.4%) cases. KRAS FISH (performed on 45 cases) revealed a trend for more frequent KRAS amplification among cases with KRAS MASI (7/20, 35% vs 3/25, 12%, P=0.08). KRAS amplification by FISH was seen only in undifferentiated carcinomas (10/24, 42% vs 0/21 pancreatic ductal adenocarcinoma, 0%, P=0.0007). In 6 of 11 cases with both undifferentiated and well-differentiated components, transition to undifferentiated carcinoma was associated with an increase in KRAS copy number, due to amplification and/or chromosome 12 hyperploidy. Pancreatic carcinomas with KRAS MASI (compared to those without MASI) were predominantly undifferentiated (16/22, 73% vs 9/97, 9%, P<0.001), more likely to present at clinical stage IV (5/22, 23% vs 7/97, 7%, P=0.009), and were associated with shorter overall survival (9 months, 95% confidence interval, 5-13, vs 22 months, 95% confidence interval, 17-27; P=0.015) and shorter disease-free survival (5 months, 95% confidence interval, 2-8 vs 13 months, 95% confidence interval, 10-16; P=0.02). Our findings suggest that in a subset of ductal adenocarcinomas, KRAS MASI correlates with the progression to undifferentiated carcinoma of the pancreas.

  10. The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer

    PubMed Central

    Siprashvili, Zurab; Webster, Dan E; Johnston, Danielle; Shenoy, Rajani M; Ungewickell, Alexander J; Bhaduri, Aparna; Flockhart, Ross; Zarnegar, Brian J; Che, Yonglu; Meschi, Francesca; Puglisi, Joseph D; Khavari, Paul A

    2017-01-01

    Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification1,2. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10–40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer. PMID:26595770

  11. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes

    PubMed Central

    Dou, Yongchao; Cha, Diana J.; Franklin, Jeffrey L.; Higginbotham, James N.; Jeppesen, Dennis K.; Weaver, Alissa M.; Prasad, Nripesh; Levy, Shawn; Coffey, Robert J.; Patton, James G.; Zhang, Bing

    2016-01-01

    Recent studies have shown that circular RNAs (circRNAs) are abundant, widely expressed in mammals, and can display cell-type specific expression. However, how production of circRNAs is regulated and their precise biological function remains largely unknown. To study how circRNAs might be regulated during colorectal cancer progression, we used three isogenic colon cancer cell lines that differ only in KRAS mutation status. Cellular RNAs from the parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically-matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only) were analyzed using RNA-Seq. We developed a bioinformatics pipeline to identify and evaluate circRNA candidates from RNA-Seq data. Hundreds of high-quality circRNA candidates were identified in each cell line. Remarkably, circRNAs were significantly down-regulated at a global level in DLD-1 and DKO-1 cells compared to DKs-8 cells, indicating a widespread effect of mutant KRAS on circRNA abundance. This finding was confirmed in two independent colon cancer cell lines HCT116 (KRAS mutant) and HKe3 (KRAS WT). In all three cell lines, circRNAs were also found in secreted extracellular-vesicles, and circRNAs were more abundant in exosomes than cells. Our results suggest that circRNAs may serve as promising cancer biomarkers. PMID:27892494

  12. Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis.

    PubMed

    Padanad, Mahesh S; Konstantinidou, Georgia; Venkateswaran, Niranjan; Melegari, Margherita; Rindhe, Smita; Mitsche, Matthew; Yang, Chendong; Batten, Kimberly; Huffman, Kenneth E; Liu, Jingwen; Tang, Ximing; Rodriguez-Canales, Jaime; Kalhor, Neda; Shay, Jerry W; Minna, John D; McDonald, Jeffrey; Wistuba, Ignacio I; DeBerardinis, Ralph J; Scaglioni, Pier Paolo

    2016-08-09

    KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis in vivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

  13. Endoscopic ultrasound-guided fine-needle aspirate-derived preclinical pancreatic cancer models reveal panitumumab sensitivity in KRAS wild-type tumors.

    PubMed

    Berry, William; Algar, Elizabeth; Kumar, Beena; Desmond, Christopher; Swan, Michael; Jenkins, Brendan J; Croagh, Daniel

    2017-05-15

    Pancreatic cancer (PC) is largely refractory to existing therapies used in unselected patient trials, thus emphasizing the pressing need for new approaches for patient selection in personalized medicine. KRAS mutations occur in 90% of PC patients and confer resistance to epidermal growth factor receptor (EGFR) inhibitors (e.g., panitumumab), suggesting that KRAS wild-type PC patients may benefit from targeted panitumumab therapy. Here, we use tumor tissue procured by endoscopic ultrasound-guided fine-needle aspirate (EUS-FNA) to compare the in vivo sensitivity in patient-derived xenografts (PDXs) of KRAS wild-type and mutant PC tumors to panitumumab, and to profile the molecular signature of these tumors in patients with metastatic or localized disease. Specifically, RNASeq of EUS-FNA-derived tumor RNA from localized (n = 20) and metastatic (n = 20) PC cases revealed a comparable transcriptome profile. Screening the KRAS mutation status of tumor genomic DNA obtained from EUS-FNAs stratified PC patients into either KRAS wild-type or mutant cohorts, and the engraftment of representative KRAS wild-type and mutant EUS-FNA tumor samples into NOD/SCID mice revealed that the growth of KRAS wild-type, but not mutant, PDXs was selectively suppressed with panitumumab. Furthermore, in silico transcriptome interrogation of The Cancer Genome Atlas (TCGA)-derived KRAS wild-type (n = 38) and mutant (n = 132) PC tumors revealed 391 differentially expressed genes. Taken together, our study validates EUS-FNA for the application of a novel translational pipeline comprising KRAS mutation screening and PDXs, applicable to all PC patients, to evaluate personalized anti-EGFR therapy in patients with KRAS wild-type tumors.

  14. Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations

    PubMed Central

    Kang, Suki; Kim, Baek Gil; Han, Hyun Ho; Lee, Joo Hyun; Kim, Ji Eun; Shim, Hyo Sup; Cho, Nam Hoon

    2015-01-01

    Epidermal growth factor receptor (EGFR) is an important mediator of tumor cell survival and proliferation. The detection of EGFR mutations can predict prognoses and indicate when treatment with EGFR tyrosine kinase inhibitors should be used. As such, the development of highly sensitive methods for detecting EGFR mutations is important. Targeted next-generation sequencing is an effective method for diagnosing mutations. We compared the abilities of enrichment PCR followed by ultra-deep pyrosequencing (UDP), UDP alone, and PNA-mediated RT-PCR clamping to detect low-frequency EGFR mutations in tumor cell lines and tissue samples. Using enrichment PCR-UDP, we were able to detect the E19del and L858R mutations at minimum frequencies of 0.01% and 0.05%, respectively, in the PC-9 and H197 tumor cell lines. We also confirmed the sensitivity of detecting the E19del mutation by performing a titration analysis in FFPE tumor samples. The lowest mutation frequency detected was 0.0692% in tissue samples. EGFR mutations with frequencies as low as 0.01% were detected using enrichment PCR-UDP, suggesting that this method is a valuable tool for detecting rare mutations, especially in scarce tissue samples or those with small quantities of DNA. PMID:25915533

  15. p53, erbB-2 and K-ras gene alterations are rare in spontaneous and plutonium-239-induced canine lung neoplasia

    SciTech Connect

    Tierney, L.A.; Hahn, F.F.; Lechner, J.F.

    1996-02-01

    Inhalation of high-linear energy transfer radiation in the form of radon progeny is a suspected cause of human lung cancer. To gain insight into the types of genetic derangements caused by this type of radiation, lung tumors from beagle dogs exposed to {sup 239}PuO{sub 2} and those arising in animals with no known carcinogen exposure were examined for evidence of aberrations in genes known to be altered in lung tumors. Altered expression of the p53 tumor suppressor gene and proto-oncogene erbB-2 proteins (p185{sup erbB2}) was evaluated by immunohistochemical analysis of 117 tumors representing different histological types in exposed (n = 80) and unexposed (n = 37) animals. Twenty-eight tumors were analyzed for K-ras proto-oncogene mutations by polymerase chain reaction amplification and direct sequencing. Fourteen percent (16/116) of all lung neoplasms showed elevated nuclear accumulation of p53 protein. Regardless of exposure history, adenosquamous and squamous cell cancers comprised 94% of all tumors with p53 abnormalities. Eighteen percent (21/117) of all tumors had evidence of erbB-2 protein overexpression. K-ras mutations were not detected in codons 12, 13 or 61 of tumors from unexposed (n = 9) or plutonium-exposed dogs (n = 19). These data indicate that p53 and K-ras gene abnormalities as a result of missense mutation are infrequent events in spontaneous and {sup 239}PuO{sub 2}-induced lung neoplasia in this colony of beagle dogs. Alternative mechanisms of gene alteration may be involved in canine pulmonary carcinogenesis. 45 refs., 3 figs., 2 tabs.

  16. Determination of somatic oncogenic mutations linked to target-based therapies using MassARRAY technology

    PubMed Central

    Llorca-Cardeñosa, Marta J.; Mongort, Cristina; Alonso, Elisa; Navarro, Samuel; Burgues, Octavio; Vivancos, Ana; Cejalvo, Juan Miguel; Perez-Fidalgo, José Alejandro; Roselló, Susana; Ribas, Gloria; Cervantes, Andrés

    2016-01-01

    Somatic mutation analysis represents a useful tool in selecting personalized therapy. The aim of our study was to determine the presence of common genetic events affecting actionable oncogenes using a MassARRAY technology in patients with advanced solid tumors who were potential candidates for target-based therapies. The analysis of 238 mutations across 19 oncogenes was performed in 197 formalin-fixed paraffin-embedded samples of different tumors using the OncoCarta Panel v1.0 (Sequenom Hamburg, Germany). Of the 197 specimens, 97 (49.2%) presented at least one mutation. Forty-nine different oncogenic mutations in 16 genes were detected. Mutations in KRAS and PIK3CA were detected in 40/97 (41.2%) and 30/97 (30.9%) patients respectively. Thirty-one patients (32.0%) had mutations in two genes, 20 of them (64.5%) initially diagnosed with colorectal cancer. The co-occurrence of mutation involved mainly KRAS, PIK3CA, KIT and RET. Mutation profiles were validated using a customized panel and the Junior Next-Generation Sequencing technology (GS-Junior 454, Roche). Twenty-eight patients participated in early clinical trials or received specific treatments according to the molecular characterization (28.0%). MassARRAY technology is a rapid and effective method for identifying key cancer-driving mutations across a large number of samples, which allows for a more appropriate selection for personalized therapies. PMID:26968814

  17. Glycoproteomic Approach Identifies KRAS as a Positive Regulator of CREG1 in Non-small Cell Lung Cancer Cells

    PubMed Central

    Clark, David J.; Mei, Yuping; Sun, Shisheng; Zhang, Hui; Yang, Austin J.; Mao, Li

    2016-01-01

    Protein glycosylation plays a fundamental role in a multitude of biological processes, and the associated aberrant expression of glycoproteins in cancer has made them attractive biomarkers and therapeutic targets. In this study, we examined differentially expressed glycoproteins in cell lines derived from three different states of lung tumorigenesis: an immortalized bronchial epithelial cell (HBE) line, a non-small cell lung cancer (NSCLC) cell line harboring a Kirsten rat sarcoma viral oncogene homolog (KRAS) activation mutation and a NSCLC cell line harboring an epidermal growth factor receptor (EGFR) activation deletion. Using a Triple SILAC proteomic quantification strategy paired with hydrazide chemistry N-linked glycopeptide enrichment, we quantified 118 glycopeptides in the three cell lines derived from 82 glycoproteins. Proteomic profiling revealed 27 glycopeptides overexpressed in both NSCLC cell lines, 6 glycopeptides overexpressed only in the EGFR mutant cells and 19 glycopeptides overexpressed only in the KRAS mutant cells. Further investigation of a panel of NSCLC cell lines found that Cellular repressor of E1A-stimulated genes (CREG1) overexpression was closely correlated with KRAS mutation status in NSCLC cells and could be down-regulated by inhibition of KRAS expression. Our results indicate that CREG1 is a down-stream effector of KRAS in a sub-type of NSCLC cells and a novel candidate biomarker or therapeutic target for KRAS mutant NSCLC. PMID:26722374

  18. Detected EGFR mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis

    PubMed Central

    Chunhua, Ma; Yuan, Lv; Ning, Mu; Jinduo, Li; Bin, Wang; Liwei, Sun

    2016-01-01

    Abstract Objective To discuss the application of ARMS method to detect EGFR gene mutation in cerebrospinal fluid of lung adenocarcinoma patients with meningeal metastasis. Methods 5 cases of lung adenocarcinoma were identified with meningeal metastasis that were cleared EGFR gene mutation by gene sequencing method. From each patient 5ml cerebrospinal fluid was obtained by lumbar puncture. ARMS method was used to detect EGFR mutations in cerebrospinal fluid. Results 5 samples of cerebrospinal fluid were successfully detected by ARMS method, 3 samples found that EGFR gene mutations, the mutations in line with direct sequencing method. Conclusion ARMS method can be used to detect EGFR gene mutations of cerebrospinal fluid samples in lung adenocarcinoma with meningeal metastasis. But cerebrospinal fluid specimens from histological specimens, blood samples need to be confirmed by further comparative study whether there is advantage.

  19. Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.

    PubMed

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Iannaccone, Alessia; Chicchinelli, Nicoletta; Franco, Renato; Mancini, Rita; Pisconti, Salvatore; De Luca, Antonella; Botti, Gerardo; Morabito, Alessandro; Normanno, Nicola

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

  20. A combination therapy for KRAS-driven lung adenocarcinomas using lipophilic bisphosphonates and rapamycin

    SciTech Connect

    Xia, Yifeng; Liu, Yi -Liang; Xie, Yonghua; Zhu, Wei; Guerra, Francisco; Shen, Shen; Yeddula, Narayana; Fischer, Wolfgang; Low, William; Zhou, Xiaoying; Zhang, Yonghui; Oldfield, Eric; Verma, Inder M.

    2014-11-19

    Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted. Here in this paper, we describe a combination therapy for treating these malignancies using two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growth and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant NF-κB activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Lastly, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations.

  1. A combination therapy for KRAS-driven lung adenocarcinomas using lipophilic bisphosphonates and rapamycin

    DOE PAGES

    Xia, Yifeng; Liu, Yi -Liang; Xie, Yonghua; ...

    2014-11-19

    Lung cancer is the most common human malignancy and leads to about one-third of all cancer-related deaths. Lung adenocarcinomas harboring KRAS mutations, in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted. Here in this paper, we describe a combination therapy for treating these malignancies using two agents: a lipophilic bisphosphonate and rapamycin. This drug combination is much more effective than either agent acting alone in the KRAS G12D induced mouse lung model. Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growthmore » and cell survival. Bisphosphonate treatment of cells initiated autophagy but was ultimately unsuccessful and led to p62 accumulation and concomitant NF-κB activation, resulting in dampened efficacy in vivo. However, we found that rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-κB activation and tumor cell proliferation. Lastly, these results suggest that using lipophilic bisphosphonates in combination with rapamycin may provide an effective strategy for targeting lung adenocarcinomas harboring KRAS mutations.« less

  2. Algorithms for Detecting Significantly Mutated Pathways in Cancer

    NASA Astrophysics Data System (ADS)

    Vandin, Fabio; Upfal, Eli; Raphael, Benjamin J.

    Recent genome sequencing studies have shown that the somatic mutations that drive cancer development are distributed across a large number of genes. This mutational heterogeneity complicates efforts to distinguish functional mutations from sporadic, passenger mutations. Since cancer mutations are hypothesized to target a relatively small number of cellular signaling and regulatory pathways, a common approach is to assess whether known pathways are enriched for mutated genes. However, restricting attention to known pathways will not reveal novel cancer genes or pathways. An alterative strategy is to examine mutated genes in the context of genome-scale interaction networks that include both well characterized pathways and additional gene interactions measured through various approaches. We introduce a computational framework for de novo identification of subnetworks in a large gene interaction network that are mutated in a significant number of patients. This framework includes two major features. First, we introduce a diffusion process on the interaction network to define a local neighborhood of "influence" for each mutated gene in the network. Second, we derive a two-stage multiple hypothesis test to bound the false discovery rate (FDR) associated with the identified subnetworks. We test these algorithms on a large human protein-protein interaction network using mutation data from two recent studies: glioblastoma samples from The Cancer Genome Atlas and lung adenocarcinoma samples from the Tumor Sequencing Project. We successfully recover pathways that are known to be important in these cancers, such as the p53 pathway. We also identify additional pathways, such as the Notch signaling pathway, that have been implicated in other cancers but not previously reported as mutated in these samples. Our approach is the first, to our knowledge, to demonstrate a computationally efficient strategy for de novo identification of statistically significant mutated subnetworks. We

  3. Hypoxia/Reoxygenation-Induced Mutations in Mammalian Cells Detected by the Flow Cytometry Mutation Assay and Characterized by Mutant Spectrum

    PubMed Central

    Keysar, Stephen B.; Trncic, Nadira; LaRue, Susan M.; Fox, Michael H.

    2010-01-01

    Under hypoxic conditions, cells are more resistant to cell killing by ionizing radiation by a factor of 2.5 to 3, potentially compromising the efficacy of radiotherapy. It has been shown recently that hypoxic conditions alone are sufficient to generate mutations in vitro and in vivo, likely due to the creation of reactive oxygen species (ROS) and a decrease in mismatch and homologous recombination DNA repair activity. These factors are known precursors to the onset of genetic instability and poor prognosis. We have previously characterized the flow cytometry mutation assay and its sensitivity to detect significant mutant fractions induced by genotoxic agents that are not detected by other mammalian assays. Here we measure the mutant fraction induced by hypoxia. CHO AL cells cultured at <0.1% O2 for 24 h generated a significant mutant fraction of 120 × 10−5 and had growth kinetics and survival characteristics similar to those obtained with other mutagens. We investigated the role of ROS by treating cells with the radical scavenger DMSO, which significantly reduced hypoxia toxicity and mutagenesis. Single cells were sorted from the mutant population, and the resulting clonal populations were stained for five antigens encoded by genes found along chromosome 11 to generate mutant spectra. The mutations were primarily large deletions, similar to those in background mutants, but the frequency was higher. We have demonstrated that hypoxic conditions alone are sufficient to generate mutations in mammalian cells in culture and that the spectrum of mutations is similar to background mutations. PMID:20041756

  4. Factors associated with guideline-recommended KRAS testing in colorectal cancer patients: A population-based study

    PubMed Central

    Charlton, Mary E.; Karlitz, Jordan J.; Schlichting, Jennifer A.; Chen, Vivien W.; Lynch, Charles F.

    2015-01-01

    Objectives Response to epidermal growth factor receptor inhibitors is poorer among Stage IV colorectal cancer (CRC) patients with KRAS mutations, thus KRAS testing is recommended prior to treatment. KRAS testing was collected by Surveillance, Epidemiology, and End Results (SEER) registries for 2010 CRC cases, and our goal was to provide the first population-based estimates of testing in the U.S. Methods SEER CRC cases diagnosed in 2010 were evaluated (n=30,351). Chi-square tests and logistic regression were conducted to determine patient characteristics associated with KRAS testing, stratified by Stages I-III vs. Stage IV. Log-rank tests were used to examine survival by testing status. Results KRAS testing among Stage IV cases ranged from 39% in New Mexico to 15% in Louisiana. In the model, younger age, being married, living in a metropolitan area, and having primary site surgery were associated with greater odds of receiving KRAS testing. Those who received testing had significantly better survival then those who did not (p<0.0001). Among those who received testing, there was no significant difference in survival by mutated vs. wild type KRAS. Five percent of Stage I-III cases received testing. Conclusions Wide variation in documented KRAS testing for Stage IV CRC patients exists among SEER registries. Age remained highly significant in multivariate models, suggesting it plays an independent role in the patient and/or provider decision to be tested. Further research is needed to determine drivers of variation in testing, as well as reasons for testing in Stage I-III cases where it is not recommended. PMID:25844824

  5. Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

    PubMed Central

    Grabner, Beatrice; Schramek, Daniel; Mueller, Kristina M.; Moll, Herwig P.; Svinka, Jasmin; Hoffmann, Thomas; Bauer, Eva; Blaas, Leander; Hruschka, Natascha; Zboray, Katalin; Stiedl, Patricia; Nivarthi, Harini; Bogner, Edith; Gruber, Wolfgang; Mohr, Thomas; Zwick, Ralf Harun; Kenner, Lukas; Poli, Valeria; Aberger, Fritz; Stoiber, Dagmar; Egger, Gerda; Esterbauer, Harald; Zuber, Johannes; Moriggl, Richard; Eferl, Robert; Győrffy, Balázs; Penninger, Josef M.; Popper, Helmut; Casanova, Emilio

    2015-01-01

    STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased KrasG12D-driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance. PMID:25734337

  6. Different susceptibility to lung tumorigenesis in mice with an identical Kras 2 intron 2

    SciTech Connect

    Manenti, G.; Falvella, F.S.; Gariboldi, M.; Dragani, T.A.; Pierotti, M.A.

    1995-09-20

    The A/J mouse strain is genetically susceptible to pulmonary tumorigenesis. We have performed a genetic linkage analysis to map pulmonary adenoma susceptibility (Pas) loci in an urethane-treated (A/J X Mus spretus) x C57BL/6J (ASB) interspecific testcross. In this interspecific cross we have confirmed our previous results in AC3F2 mice on the mapping of the Pas1 locus to the digital region of chromosome 6, near Kras2. The A/J and M. spretus strains differed as the Pas1 locus, with the M. spretus providing the resistant allele. In the latter strain, we studied the nucleotide sequence of a portion of the second intron of Kras2 that contains polymorphisms associated with lung tumor susceptibility in several inbred strains. The lung tumor-resistant M. spretus strain had the same specific nucleotide sequence of susceptible strains. Mutations in codon 61 of Kras2 in urethane-induced lung tumors from ASF1 hybrids involved the A/J allele in all cases, while the M. spretus allele was never affected. Our results indicate that the M. spretus and A/J mice have an identical structure of the second intron of the Kras2 gene, but they differ in genetic susceptibility to pulmonary tumorigenesis and in mutability of their Kras2 allele. 30 refs., 4 figs., 3 tabs.

  7. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  8. A multiplexed fragment analysis-based assay for detection of JAK2 exon 12 mutations.

    PubMed

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-09-01

    Mutations within exon 12 of the JAK2 gene occur in most cases of JAK2 V617F-mutation negative polycythemia vera. Several methods have been developed to identify exon 12 mutations, with both Sanger sequencing and high resolution melting (HRM) being widely used. However, mutations can occur at allelic levels lower than 15%, which may hamper detection by these methods. We developed a novel fragment analysis-based assay capable of detecting nearly all JAK2 exon 12 mutations associated with polycythemia vera down to a sensitivity of 2% mutant allele. Test results were reviewed from a set of 20 reference cases and 1731 consecutive specimens that were referred to our laboratory for testing. Assay performance was compared to sequencing and HRM across a series of 27 specimens with JAK2 exon 12 mutations. Positive cases consisted of 22 with deletion mutations, four with duplications, and one with K539L. Nine cases had mutation levels between 6% and 15% that may not be reliably detected by sequencing or HRM. All cases were easily interpreted in the fragment analysis assay. Sequencing, HRM, and fragment analysis each represent viable platforms for detection of JAK2 exon 12 mutations. Our method performed favorably by providing a simple, robust, and highly sensitive solution for JAK2 exon 12 mutation testing.

  9. Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations

    SciTech Connect

    Hodgdon, A.L.; Marcus, A.H.; Arenaz, P.; Rosichan, J.L.; Bogyo, T.P.; Nilan, R.A.

    1981-01-01

    Clustering of mutant pollen grains in a population of normal pollen due to premeiotic mutational events complicates translating mutation frequencies into rates. Embryo ontogeny in barley will be described and used to illustrate the formation of such mutant clusters. The nature of the statistics for mutation frequency will be described from a study of the reversion frequencies of various waxy mutants in barley. Computer analysis by a ''jackknife'' method of the reversion of a waxy mutant treated with the mutagen sodium azide showed a significantly higher reversion frequency than untreated material. Problems of the computer analysis suggest a better experimental design for pollen mutation experiments. Preliminary work on computer modeling for pollen development and mutation will be described.

  10. Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations

    SciTech Connect

    Hodgdon, A.L.; Marcus, A.H.; Arenaz, P.; Rosichan, J.L.; Bogyo, T.P.; Nilan, R.A.

    1980-05-29

    Clustering of mutant pollen grains in a population of normal pollen due to premeiotic mutational events complicates translating mutation frequencies into rates. Embryo ontogeny in barley will be described and used to illustrate the formation of such mutant clusters. The nature of the statistics for mutation frequency will be described from a study of the reversion frequencies of various waxy mutants in barley. Computer analysis by a jackknife method of the reversion frequencies of a waxy mutant treated with the mutagen sodium azide showed a significantly higher reversion frequency than untreated material. Problems of the computer analysis suggest a better experimental design for pollen mutation experiments. Preliminary work on computer modeling for pollen development and mutation will be described.

  11. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions

    PubMed Central

    Gillette, William K.; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H.; Grose, Carissa; Jones, Jane E.; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V.; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G.

    2015-01-01

    Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer’s disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5–10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ. PMID:26522388

  12. Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

    NASA Astrophysics Data System (ADS)

    Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

    2016-10-01

    Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

  13. Coupled mutation finder: A new entropy-based method quantifying phylogenetic noise for the detection of compensatory mutations

    PubMed Central

    2012-01-01

    Background The detection of significant compensatory mutation signals in multiple sequence alignments (MSAs) is often complicated by noise. A challenging problem in bioinformatics is remains the separation of significant signals between two or more non-conserved residue sites from the phylogenetic noise and unrelated pair signals. Determination of these non-conserved residue sites is as important as the recognition of strictly conserved positions for understanding of the structural basis of protein functions and identification of functionally important residue regions. In this study, we developed a new method, the Coupled Mutation Finder (CMF) quantifying the phylogenetic noise for the detection of compensatory mutations. Results To demonstrate the effectiveness of this method, we analyzed essential sites of two human proteins: epidermal growth factor receptor (EGFR) and glucokinase (GCK). Our results suggest that the CMF is able to separate significant compensatory mutation signals from the phylogenetic noise and unrelated pair signals. The vast majority of compensatory mutation sites found by the CMF are related to essential sites of both proteins and they are likely to affect protein stability or functionality. Conclusions The CMF is a new method, which includes an MSA-specific statistical model based on multiple testing procedures that quantify the error made in terms of the false discovery rate and a novel entropy-based metric to upscale BLOSUM62 dissimilar compensatory mutations. Therefore, it is a helpful tool to predict and investigate compensatory mutation sites of structural or functional importance in proteins. We suggest that the CMF could be used as a novel automated function prediction tool that is required for a better understanding of the structural basis of proteins. The CMF server is freely accessible at http://cmf.bioinf.med.uni-goettingen.de. PMID:22963049

  14. Heteroduplex analysis of the dystrophin gene: application to point mutation and carrier detection.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Sedra, M S; Western, L M; Bartolo, C; Moxley, R T; Mendell, J R

    1994-03-01

    Approximately one-third of the Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, we identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. We conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing.

  15. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    SciTech Connect

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R.; Moxley, R.T.

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  16. Direct detection of a BRAF mutation in total RNA from melanoma cells using cantilever arrays

    NASA Astrophysics Data System (ADS)

    Huber, F.; Lang, H. P.; Backmann, N.; Rimoldi, D.; Gerber, Ch.

    2013-02-01

    Malignant melanoma, the deadliest form of skin cancer, is characterized by a predominant mutation in the BRAF gene. Drugs that target tumours carrying this mutation have recently entered the clinic. Accordingly, patients are routinely screened for mutations in this gene to determine whether they can benefit from this type of treatment. The current gold standard for mutation screening uses real-time polymerase chain reaction and sequencing methods. Here we show that an assay based on microcantilever arrays can detect the mutation nanomechanically without amplification in total RNA samples isolated from melanoma cells. The assay is based on a BRAF-specific oligonucleotide probe. We detected mutant BRAF at a concentration of 500 pM in a 50-fold excess of the wild-type sequence. The method was able to distinguish melanoma cells carrying the mutation from wild-type cells using as little as 20 ng µl-1 of RNA material, without prior PCR amplification and use of labels.

  17. Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.

    PubMed

    Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

    2015-12-15

    In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.

  18. One-Step Ligation on RNA Amplification for the Detection of Point Mutations.

    PubMed

    Zhang, Lei; Wang, Jingjing; Coetzer, Mia; Angione, Stephanie; Kantor, Rami; Tripathi, Anubhav

    2015-11-01

    The detection of point mutations is required in the diagnosis of many human diseases. The conformal specificity of DNA ligases was elegantly used to distinguish single-nucleotide mismatches. However, to detect point mutations in RNA retroviruses, conventional ligase-mediated approaches require the reverse transcription of viral genomes before separate ligation and amplification steps. We developed one-step ligation on RNA amplification (LRA) for the direct detection of RNA point mutations. The process combines the ligase-mediated joining of two oligonucleotides and subsequent hot start amplification into a single-tube reaction. We report that modifications to the structure of the oligonucleotide ligation probes improve the rate of ligation and the specificity of mutation detection on RNA. We applied LRA to the detection of a common, clinically relevant HIV-1 reverse transcriptase drug-resistant point mutation, K103N, and compared it with allele-specific PCR and pyrosequencing. LRA achieved a limit of specific quantitation of 1:100 (1%), and a limit of specific detection for mutant K103N RNA transcripts among excess wild-type strands of 1:10,000 (0.01%). LRA also exhibited good detection threshold of 5 × 10(2) copies/μL K103N RNA transcripts. LRA is a novel point mutation detection method, with potential utilization in HIV drug resistance detection and early diagnostics of genetic disorders associated with other infectious diseases and cancer.

  19. Enhanced Ratio of Signals Enables Digital Mutation Scanning for Rare Allele Detection

    PubMed Central

    Castellanos-Rizaldos, Elena; Paweletz, Cloud; Song, Chen; Oxnard, Geoffrey R.; Mamon, Harvey; Jänne, Pasi A.; Makrigiorgos, G. Mike

    2016-01-01

    The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence. PMID:25772705

  20. Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

    PubMed Central

    Eng, Christina H.; Wang, Zuncai; Tkach, Diane; Toral-Barza, Lourdes; Ugwonali, Savuth; Liu, Shanming; Fitzgerald, Stephanie L.; George, Elizabeth; Frias, Elizabeth; Cochran, Nadire; De Jesus, Rowena; McAllister, Gregory; Hoffman, Gregory R.; Bray, Kevin; Lemon, LuAnna; Lucas, Judy; Fantin, Valeria R.; Abraham, Robert T.; Murphy, Leon O.; Nyfeler, Beat

    2016-01-01

    Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy. PMID:26677873

  1. Kras is required for adult hematopoiesis

    PubMed Central

    Damnernsawad, Alisa; Kong, Guangyao; Wen, Zhi; Liu, Yangang; Rajagopalan, Adhithi; You, Xiaona; Wang, Jinyong; Zhou, Yun; Ranheim, Erik A.; Luo, Hongbo R.; Chang, Qiang; Zhang, Jing

    2017-01-01

    Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its rolein adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced TPO signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while SCF-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias towards myeloid differentiation in MPPs. Although GM-CSF-evoked ERK1/2 activation is only moderately decreased in Kras−/− myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9–12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras−/− bone marrow cells is greatly compromised, which is attributable to defects in the self-renewal of Kras−/− HSCs and defects in differentiated hematopietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. PMID:26972179

  2. A novel mouse model of intrahepatic cholangiocarcinoma induced by liver-specific Kras activation and Pten deletion

    PubMed Central

    Ikenoue, Tsuneo; Terakado, Yumi; Nakagawa, Hayato; Hikiba, Yohko; Fujii, Tomoaki; Matsubara, Daisuke; Noguchi, Rei; Zhu, Chi; Yamamoto, Keisuke; Kudo, Yotaro; Asaoka, Yoshinari; Yamaguchi, Kiyoshi; Ijichi, Hideaki; Tateishi, Keisuke; Fukushima, Noriyoshi; Maeda, Shin; Koike, Kazuhiko; Furukawa, Yoichi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and its incidence is increasing worldwide. Recently, several types of cells have been considered as the origin of ICC, namely cholangiocytes, liver progenitor cells, and hepatocytes. Here, we have established a novel mouse model of ICC by liver-specific Kras activation and Pten deletion. An activating mutation of Kras in combination with deletion of Pten was introduced in embryonic hepatic bipotential progenitor cells (so-called hepatoblasts) and mature hepatocytes using the Cre-loxP system. As a result, liver-specific Kras activation and homozygous Pten deletion cooperated to induce ICCs exclusively. In contrast, Kras activation in combination with heterozygous Pten deletion induced both ICCs and HCCs, whereas Kras activation alone resulted in HCCs but not ICCs. Furthermore, a cell-lineage visualization system using tamoxifen-inducible Cre-loxP demonstrated that the ICCs did not originate from hepatocytes but from cholangiocytes. Our data suggest that mice carrying liver-specific Kras activation in combination with homozygous Pten deletion should be useful for the investigation of therapeutic strategies for human ICC. PMID:27032374

  3. High frequency of codon 61 K-ras A-->T transversions in lung and Harderian gland neoplasms of B6C3F1 mice exposed to chloroprene (2-chloro-1,3-butadiene) for 2 years, and comparisons with the structurally related chemicals isoprene and 1,3-butadiene.

    PubMed

    Sills, R C; Hong, H L; Melnick, R L; Boorman, G A; Devereux, T R

    1999-04-01

    Chloroprene is the 2-chloro analog of 1,3-butadiene, a potent carcinogen in laboratory animals. Following 2 years of inhalation exposure to 12.8, 32 or 80 p.p.m. chloroprene, increased incidences of lung and Harderian gland (HG) neoplasms were observed in B6C3F1 mice at all exposure concentrations. The present study was designed to characterize genetic alterations in the K- and H-ras proto-oncogenes in chloroprene-induced lung and HG neoplasms. K-ras mutations were detected in 80% of chloroprene-induced lung neoplasms (37/46) compared with only 30% in spontaneous lung neoplasms (25/82). Both K- and H-ras codon 61 A-->T transversions were identified in 100% of HG neoplasms (27/27) compared with a frequency of 56% (15/27) in spontaneous HG neoplasms. The predominant mutation in chloroprene-induced lung and HG neoplasms was an A-->T transversion at K-ras codon 61. This mutation has not been detected in spontaneous lung tumors of B6C3F1 mice and was identified in only 7% of spontaneous HG neoplasms. In lung neoplasms, greater percentages (80 and 71%) of A-->T transversions were observed at the lower exposures (12.8 and 32 p.p.m.), respectively, compared with 18% at the high exposure. In HG neoplasms, the percentage of A-->T transversions was the same at all exposure concentrations. The chloroprene-induced ras mutation spectra was similar to that seen with isoprene, where the predominant base change was an A-->T transversion at K-ras codon 61. This differed from 1,3-butadiene, where K-ras codon 13 G-->C transitions and H-ras codon 61 A-->G transitions were the predominant mutations. The major finding of K-ras A-->T transversions in lung and Harderian gland neoplasms suggests that this mutation may be important for tumor induction by this class of carcinogens.

  4. Potential actionable targets in appendiceal cancer detected by immunohistochemistry, fluorescent in situ hybridization, and mutational analysis

    PubMed Central

    Millis, Sherri Z.; Kimbrough, Jeffery; Doll, Nancy; Von Hoff, Daniel; Ramanathan, Ramesh K.

    2017-01-01

    Background Appendiceal cancers are rare and consist of carcinoid, mucocele, pseudomyxoma peritonei (PMP), goblet cell carcinoma, lymphoma, and adenocarcinoma histologies. Current treatment involves surgical resection or debulking, but no standard exists for adjuvant chemotherapy or treatment for metastatic disease. Methods Samples were identified from approximately 60,000 global tumors analyzed at a referral molecular profiling CLIA-certified laboratory. A total of 588 samples with appendix primary tumor sites were identified (male/female ratio of 2:3; mean age =55). Sixty-two percent of samples were adenocarcinomas (used for analysis); the rest consisted of 9% goblet cell, 15% mucinous; 6% pseudomyxoma, and less than 5% carcinoids and 2% neuroendocrine. Tests included sequencing [Sanger, next generation sequencing (NGS)], protein expression/immunohistochemistry (IHC), and gene amplification [fluorescent in situ hybridization (FISH) or CISH]. Results Profiling across all appendiceal cancer histological subtypes for IHC revealed: 97% BRCP, 81% MRP1, 81% COX-2, 71% MGMT, 56% TOPO1, 5% PTEN, 52% EGFR, 40% ERCC1, 38% SPARC, 35% PDGFR, 35% TOPO2A, 25% RRM1, 21% TS, 16% cKIT, and 12% for TLE3. NGS revealed mutations in the following genes: 50.4% KRAS, 21.9% P53, 17.6% GNAS, 16.5% SMAD4, 10% APC, 7.5% ATM, 5.5% PIK3CA, 5.0% FBXW7, and 1.8% BRAF. Conclusions Appendiceal cancers show considerable heterogeneity with high levels of drug resistance proteins (BCRP and MRP1), which highlight the difficulty in treating these tumors and suggest an individualized approach to treatment. The incidence of low TS (79%) could be used as a backbone of therapy (using inhibitors such as 5FU/capecitabine or newer agents). Therapeutic options includeTOPO1 inhibitors (irinotecan/topotecan), EGFR inhibitors (erlotinib, cetuximab), PDGFR antagonists (regorafenib, axitinib), MGMT (temozolomide). Clinical trials targeting pathways involving KRAS, p53, GNAS, SMAD4, APC, ATM, PIK3CA, FBXW7, and

  5. Utility of comprehensive genomic sequencing for detecting HER2-positive colorectal cancer.

    PubMed

    Shimada, Yoshifumi; Yagi, Ryoma; Kameyama, Hitoshi; Nagahashi, Masayuki; Ichikawa, Hiroshi; Tajima, Yosuke; Okamura, Takuma; Nakano, Mae; Nakano, Masato; Sato, Yo; Matsuzawa, Takeaki; Sakata, Jun; Kobayashi, Takashi; Nogami, Hitoshi; Maruyama, Satoshi; Takii, Yasumasa; Kawasaki, Takashi; Homma, Kei-Ichi; Izutsu, Hiroshi; Kodama, Keisuke; Ring, Jennifer E; Protopopov, Alexei; Lyle, Stephen; Okuda, Shujiro; Akazawa, Kohei; Wakai, Toshifumi

    2017-02-21

    HER2-targeted therapy is considered effective for KRAS codon 12/13 wild-type, HER2-positive metastatic colorectal cancer (CRC). In general, HER2 status is determined by the use of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Comprehensive genomic sequencing (CGS) enables the detection of gene mutations and copy number alterations including KRAS mutation and HER2 amplification; however, little is known about the utility of CGS for detecting HER2-positive CRC. To assess its utility, we retrospectively investigated 201 patients with stage I-IV CRC. The HER2 status of the primary site was assessed using IHC and FISH, and HER2 amplification of the primary site was also assessed using CGS, and the findings of these approaches were compared in each patient. CGS successfully detected alterations in 415 genes including KRAS codon 12/13 mutation and HER2 amplification. Fifty-nine (29%) patients had a KRAS codon 12/13 mutation. Ten (5%) patients were diagnosed as HER2-positive because of HER2 IHC 3+, and the same 10 (5%) patients had HER2 amplification evaluated using CGS. The results of HER2 status and HER2 amplification were completely identical in all 201 patients (P < 0.001). Nine of the 10 HER2-positive patients were KRAS 12/13 wild-type and were considered possible candidates for HER2-targeted therapy. CGS has the same utility as IHC and FISH for detecting HER2-positive patients who are candidates for HER2-targeted therapy, and facilitates precision medicine and tailor-made treatment.

  6. Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids

    NASA Astrophysics Data System (ADS)

    Wen, Li-Na; Xie, Meng-Xia

    2017-01-01

    KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol- 1, (0.93 ± 0.21) × 106 L mol- 1 and (1.16 ± 0.45) × 106 L mol- 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

  7. BRAF mutations and phosphorylation status of mitogen-activated protein kinases in the development of flat and depressed-type colorectal neoplasias.

    PubMed

    Konishi, K; Takimoto, M; Kaneko, K; Makino, R; Hirayama, Y; Nozawa, H; Kurahashi, T; Kumekawa, Y; Yamamoto, T; Ito, H; Yoshikawa, N; Kusano, M; Nakayama, K; Rembacken, B J; Ota, H; Imawari, M

    2006-01-30

    Although some molecular differences between flat-depressed neoplasias (FDNs) and protruding neoplasias (PNs) have been reported, it is uncertain if the BRAF mutations or the status of phosphorylated mitogen-activated protein kinase (p-MAPK) are different between theses two groups. We evaluated the incidence of BRAF and KRAS mutations, high-frequency microsatellite instability (MSI-H), and the immunohistochemical status of p-MAPK in the nonserrated neoplasias (46 FDNs and 57 PNs). BRAF mutations were detected in four FDNs (9%) and none of PNs (P=0.0369 by Fisher's exact test). KRAS mutations were observed in none of FDNs and in 14 PNs (25%; P=0.0002 by Fisher's exact test). MSI-H was detected in seven out of 44 FDNs (16%) and in one out of 52 of PNs (2%) (P=0.022 by Fisher's exact test). Type B and C immunostaining for p-MAPK was observed in 34 out of 46 FDNs (72%), compared with 24 out of 55 PNs (44%; P=0.0022 by chi(2) test). There was no significant difference in the type B and C immunostaining of p-MAPK between FDNs with and without BRAF mutations. BRAF and KRAS mutations are mutually exclusive in the morphological characteristics of colorectal nonserrated neoplasia. Abnormal accumulation of p-MAPK protein is more likely to be implicated in the tumorigenesis of FDNs than of PNs. However, this abnormality in FDNs might occur via the genetic alteration other than BRAF or KRAS mutation.

  8. Identification of candidate genes for lung cancer somatic mutation test kits.

    PubMed

    Chen, Yong; Shi, Jian-Xin; Pan, Xu-Feng; Feng, Jian; Zhao, Heng

    2013-09-01

    Over the past three decades, mortality from lung cancer has sharply and continuously increased in China, ascending to the first cause of death among all types of cancer. The ability to identify the actual sequence of gene mutations may help doctors determine which mutations lead to precancerous lesions and which produce invasive carcinomas, especially using next-generation sequencing (NGS) technology. In this study, we analyzed the latest lung cancer data in the COSMIC database, in order to find genomic "hotspots" that are frequently mutated in human lung cancer genomes. The results revealed that the most frequently mutated lung cancer genes are EGFR, KRAS and TP53. In recent years, EGFR and KRAS lung cancer test kits have been utilized for detecting lung cancer patients, but they presented many disadvantages, as they proved to be of low sensitivity, labor-intensive and time-consuming. In this study, we constructed a more complete catalogue of lung cancer mutation events including 145 mutated genes. With the genes of this list it may be feasible to develop a NGS kit for lung cancer mutation detection.

  9. Dnmt3a haploinsufficiency cooperates with oncogenic Kras to promote an early-onset T-cell acute lymphoblastic leukemia

    PubMed Central

    Chang, Yuan-I; Kong, Guangyao; Ranheim, Erik A; Tu, Po-Shu; Yu, Yi-Shan; Zhang, Jing

    2017-01-01

    Mutations in DNA methyltransferase 3A (DNMT3A) are prevalent in various myeloid and lymphoid malignancies. The most common DNMT3A R882 mutations inhibit methyltransferase activity of the remaining wild-type DNMT3A proteins at a heterozygous state due to their dominant-negative activity. Reports and COSMIC database analysis reveal significantly different frequencies of R882 mutations in myeloid versus T-cell malignancies, inspiring us to investigate whether downregulation of DNMT3A regulates malignancies of different lineages in a dose-dependent manner. In a competitive transplant setting, the survival of recipients with KrasG12D/+; Dnmt3a+/- bone marrow (BM) cells was significantly shortened than that of recipients with KrasG12D/+ cells. Moreover, all of the recipients with KrasG12D/+; Dnmt3a+/- cells developed a lethal T-cell acute lymphoblastic leukemia (T-ALL) without significant myeloproliferative neoplasm (MPN) phenotypes, while ~20% of recipients with KrasG12D/+ cells developed MPN with or without T-ALL. This is in sharp contrast to the recipients with KrasG12D/+; Dnmt3a-/- cells, in which ~60% developed a lethal myeloid malignancy (MPN or acute myeloid leukemia [AML]). Our data suggest that in the context of oncogenic Kras, loss of Dnmt3a promotes myeloid malignancies, while Dnmt3a haploinsufficiency induces T-ALL. This dose-dependent phenotype is highly consistent with the prevalence of DNMT3A R882 mutations in AML versus T-ALL in human. PMID:28386358

  10. Accurate detection and quantitation of heteroplasmic mitochondrial point mutations by pyrosequencing.

    PubMed

    White, Helen E; Durston, Victoria J; Seller, Anneke; Fratter, Carl; Harvey, John F; Cross, Nicholas C P

    2005-01-01

    Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations.

  11. [Detection of a founder mutation in an Argentine family with hereditary non polyposis colorectal cancer].

    PubMed

    Gómez, Laura; Adi, José; Ibarra, Jorge; Roqué, María

    2010-01-01

    Hereditary non polyposis colorectal cancer (HNPCC) has been related to mutations in the DNA mismatch repair genes (MLH1, MSH2 y MSH6). Mutation detection analysis requires the complete sequencing of these genes, given the high frequency of family-specific alterations. A point mutation (2269-2270insT) in the last codon of the MLH1 gene has been detected in families from a northern region of Italy (Reggio Emilia).Given that this alteration was registered only in people from this region, it has been considered a founder mutation. In this work, we present an Argentine HNPCC family whose ancestors were natives from the Reggio Emilia, Italy, and who were carriers for this mutation. In order to detect the genetic alteration, a PCR was developed followed by a restriction enzyme incubation assay. The mutation was detected in 3 family members, two of them without clinical symptoms. The PCR/restriction enzyme methodology has been sensitive and specific for the detection of this mutation. It has allowed the performance of a pre-symptomatic genetic diagnosis in the Argentine HNPCC family, avoiding sending samples abroad. It is worth mentioning that pre-symptomatic diagnosis of hereditary cancers allows enhanced surveillance and support for the affected families when it is performed by a multidisciplinary group.

  12. Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis*

    PubMed Central

    Sumita, Kazutaka; Yoshino, Hirofumi; Sasaki, Mika; Majd, Nazanin; Kahoud, Emily Rose; Takahashi, Hidenori; Takeuchi, Koh; Kuroda, Taruho; Lee, Susan; Charest, Pascale G.; Takeda, Kosuke; Asara, John M.; Firtel, Richard A.; Anastasiou, Dimitrios; Sasaki, Atsuo T.

    2014-01-01

    Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers. PMID:24338482

  13. Digenic mutations involving both the BSND and GJB2 genes detected in Bartter syndrome type IV.

    PubMed

    Wang, Hong-Han; Feng, Yong; Li, Hai-Bo; Wu, Hong; Mei, Ling-Yun; Wang, Xing-Wei; Jiang, Lu; He, Chu-Feng

    2017-01-01

    Bartter syndrome type IV, characterized by salt-losing nephropathies and sensorineural deafness, is caused by mutations of BSND or simultaneous mutations of both CLCNKA and CLCNKB. GJB2 is the primary causative gene for non-syndromic sensorineural deafness and associated with several syndromic sensorineural deafness. Owing to the rarity of Bartter syndrome, only a few mutations have been reported in the abovementioned causative genes. To investigate the underlying mutations in a Chinese patient with Bartter syndrome type IV, genetic analysis of BSND, CLCNKA, CLCNKB and GJB2 were performed by polymerase chain reaction and direct sequencing. Finally, double homozygous mutations c.22C > T (p.Arg8Trp) and c.127G > A (Val43Ile) were detected in exon 1 of BSND. Intriguingly, compound heterozygous mutations c.235delC (p.Leu79CysfsX3) and c.109G > A (p.Val37Ile) were also revealed in exon 2 of GJB2 in the same patient. No pathogenic mutations were found in CLCNKA and CLCNKB. Our results indicated that the homozygous mutation c.22C > T was the key genetic reason for the proband, and a digenic effect of BSND and GJB2 might contributed to sensorineural deafness. To our knowledge, it was the first report showing that the GJB2 gene mutations were detected in Bartter syndrome.

  14. Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform

    PubMed Central

    Péterfia, Bálint; Kalmár, Alexandra; Patai, Árpád V.; Csabai, István; Bodor, András; Micsik, Tamás; Wichmann, Barnabás; Egedi, Krisztina; Hollósi, Péter; Kovalszky, Ilona; Tulassay, Zsolt; Molnár, Béla

    2017-01-01

    Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes (APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations (FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation (APC). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument. PMID:28243320

  15. DNA detection on transistor arrays following mutation-specific enzymatic amplification

    NASA Astrophysics Data System (ADS)

    Pouthas, F.; Gentil, C.; Côte, D.; Bockelmann, U.

    2004-03-01

    An integrated array of silicon field-effect transistor structures is used for electronic detection of label-free DNA. Measurements of the dc current-voltage characteristics of the transistors gives us access to reproducible detection of single- and double-stranded DNA, locally adsorbed on the surface of the device. We combine this approach with allele-specific polymerase chain reaction, to test for the 35delG mutation, a frequent mutation related to prelingual nonsyndromic deafness.

  16. Sensitivity of KRAS-Mutant Colorectal Cancers to Combination Therapy that Co-Targets MEK and CDK4/6

    PubMed Central

    Ziemke, Elizabeth K.; Dosch, Joseph S.; Maust, Joel D.; Shettigar, Amrith; Sen, Ananda; Welling, Theodore H.; Hardiman, Karin M.; Sebolt-Leopold, Judith S.

    2015-01-01

    Purpose The emerging need for rational combination treatment approaches led us to test the concept that co-targeting MEK and CDK4/6 would prove efficacious in KRAS mutant (KRASmt) colorectal cancers, where upregulated CDK4 and hyperphosphorylated retinoblastoma (RB) typify the vast majority of tumors. Experimental Design Initial testing was carried out in the HCT-116 tumor model, which is known to harbor a KRAS mutation. Efficacy studies were then performed with five RB+ patient-derived colorectal xenograft models, genomically diverse with respect to KRAS, BRAF, and PIK3CA mutational status. Tolerance, efficacy, and pharmacodynamic evaluation of target modulation were evaluated in response to daily dosing with either agent alone or concurrent co-administration. Results Synergy was observed in vitro when HCT-116 cells were treated over a broad range of doses of trametinib and palbociclib. Subsequent in vivo evaluation of this model showed a higher degree of antitumor activity resulting from the combination compared to that achievable with single agent treatment. Testing of colorectal patient-derived xenograft (PDX) models further showed that combination of trametinib and palbociclib was well tolerated and resulted in objective responses in all KRASmt models tested. Stasis was observed in a KRAS/BRAF wild type and a BRAFmt model. Conclusions Combination of trametinib and palbociclib was well tolerated and highly efficacious in all three KRAS mutant CRC PDX models tested. Promising preclinical activity seen here supports clinical evaluation of this treatment approach to improve therapeutic outcome for metastatic colorectal cancer patients. PMID:26369631

  17. Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

    PubMed Central

    Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

    2017-01-01

    Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare

  18. Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia

    PubMed Central

    de Smith, Adam J.; Ojha, Juhi; Francis, Stephen S.; Sanders, Erica; Endicott, Alyson A.; Hansen, Helen M.; Smirnov, Ivan; Termuhlen, Amanda M.; Walsh, Kyle M.; Metayer, Catherine; Wiemels, Joseph L.

    2016-01-01

    High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8×10−4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches. PMID:27683039

  19. Defining New Treatment Approaches for KRAS-Mutant Lung Cancer

    DTIC Science & Technology

    2014-10-01

    AWARD NUMBER: W81XWH-13-1-0225 TITLE: Defining New Treatment Approaches for KRAS- Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Eric Collisson...TITLE AND SUBTITLE Defining New Treatment Approaches for KRAS- Mutant Lung Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13-1-0225 5c...one RAS-dependent. This Aim is underway and has verified that KRAS is indeed essential in the KRAS mutant mouse cell lines. Specific Aim 2. To

  20. An Overview of Mutation Detection Methods in Genetic Disorders

    PubMed Central

    Mahdieh, Nejat; Rabbani, Bahareh

    2013-01-01

    Genetic disorders are traditionally categorized into three main groups: single-gene, chromosomal, and multifactorial disorders. Single gene or Mendelian disorders result from errors in DNA sequence of a gene and include autosomal dominant (AD), autosomal recessive (AR), X-linked recessive (XR), X-linked dominant and Y-linked (holandric) disorders. Chromosomal disorders are due to chromosomal aberrations including numerical and structural damages. Molecular and cytogenetic techniques have been applied to identify genetic mutations leading to diseases. Accurate diagnosis of diseases is essential for appropriate treatment of patients, genetic counseling and prevention strategies. Characteristic features of patterns of inheritance are briefly reviewed and a short description of chromosomal disorders is also presented. In addition, applications of cytogenetic and molecular techniques and different types of mutations are discussed for genetic diagnosis of the pediatric genetic diseases. The purpose is to make pediatricians familiar with the applications of cytogenetic and molecular techniques and tools used for genetic diagnosis. PMID:24427490

  1. MicroRNA-143 replenishment re-sensitizes colorectal cancer cells harboring mutant, but not wild-type, KRAS to paclitaxel treatment.

    PubMed

    Fei, Bing-Yuan; Wang, Xiu-Ying; Fang, Xue-Dong

    2016-05-01

    Colorectal cancer (CRC) global incidence is one of the highest among cancers. The KRAS gene has been shown as a robust biomarker for poor prognosis and drug resistance. MicroRNA-143 (miR-143) and let-7 are families of tumor suppressor microRNAs that are often downregulated in CRC, especially with coexistent KRAS mutations. In order to evaluate if miR-143 and/or let-7b replenishment would re-sensitize CRC cells to paclitaxel treatment, we investigated in effect of miR-143 and let-7b replenishments on sensitivity to paclitaxel treatment in KRAS mutant LoVo and wild-type SW48 CRC cell lines. Our results showed that miR-143, but not let-7b, increased sensitization of KRAS mutant tumor cells to paclitaxel. Furthermore, transfection of miR-143, but not let-7b, mimic negatively regulated the expression of mutant but not wild-type KRAS. Combination of miR-143 mimic and paclitaxel induced the onset of apoptosis, and reverted in vitro metastatic properties (migration and invasion) in KRAS mutant tumor cells. MiR-143 thus can be used as a chemosensitizer for the treatment of KRAS mutant tumors and warrants further investigations in in vitro and pre-clinical in vivo models.

  2. Frederick National Lab and the Pancreatic Cancer Action Network Award Fellowships for KRAS Research | Poster

    Cancer.gov

    By Nancy Parrish, Staff Writer The Frederick National Laboratory for Cancer Research (FNLCR) recently formed a partnership with the Pancreatic Cancer Action Network (PanCAN) to award a one-year fellowship to two scientists whose research will help lead to new therapies for pancreatic cancer. The scientists will focus on KRAS, a gene in the RAS family that is mutated in 95 percent of pancreatic cancers, according to the National Cancer Institute (NCI).

  3. microRNA classifiers are powerful diagnostic/prognostic tools in ALK-, EGFR-, and KRAS-driven lung cancers.

    PubMed

    Gasparini, Pierluigi; Cascione, Luciano; Landi, Lorenza; Carasi, Stefania; Lovat, Francesca; Tibaldi, Carmelo; Alì, Greta; D'Incecco, Armida; Minuti, Gabriele; Chella, Antonio; Fontanini, Gabriella; Fassan, Matteo; Cappuzzo, Federico; Croce, Carlo M

    2015-12-01

    microRNAs (miRNAs) can act as oncosuppressors or oncogenes, induce chemoresistance or chemosensitivity, and are major posttranscriptional gene regulators. Anaplastic lymphoma kinase (ALK), EGF receptor (EGFR), and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) are major drivers of non-small cell lung cancer (NSCLC). The aim of this study was to assess the miRNA profiles of NSCLCs driven by translocated ALK, mutant EGFR, or mutant KRAS to find driver-specific diagnostic and prognostic miRNA signatures. A total of 85 formalin-fixed, paraffin-embedded samples were considered: 67 primary NSCLCs and 18 matched normal lung tissues. Of the 67 primary NSCLCs, 17 were echinoderm microtubule-associated protein-like 4-ALK translocated (ALK(+)) lung cancers; the remaining 50 were not (ALK(-)). Of the 50 ALK(-) primary NSCLCs, 24 were EGFR and KRAS mutation-negative (i.e., WT; triple negative); 11 were mutant EGFR (EGFR(+)), and 15 were mutant KRAS (KRAS(+)). We developed a diagnostic classifier that shows how miR-1253, miR-504, and miR-26a-5p expression levels can classify NSCLCs as ALK-translocated, mutant EGFR, or mutant KRAS versus mutation-free. We also generated a prognostic classifier based on miR-769-5p and Let-7d-5p expression levels that can predict overall survival. This classifier showed better performance than the commonly used classifiers based on mutational status. Although it has several limitations, this study shows that miRNA signatures and classifiers have great potential as powerful, cost-effective next-generation tools to improve and complement current genetic tests. Further studies of these miRNAs can help define their roles in NSCLC biology and in identifying best-performing chemotherapy regimens.

  4. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing

    PubMed Central

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T.; Scarpa, Aldo

    2016-01-01

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

  5. A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing

    PubMed Central

    Williams, Richard D.; Side, Lucy; Hubank, Mike; West, Rebecca; Pearson, Katie; Sebire, Neil; Tarpey, Patrick; Futreal, Andrew; Brooks, Tony; Stratton, Michael R.; Anderson, John

    2014-01-01

    Background Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described. Methods and Findings We describe a 2 years old child presenting with three separate cancers over a 6 month period; two soft tissue mesenchymal tumors and an aggressive metastatic neuroblastoma. As conventional testing of blood DNA by Sanger sequencing for mutations in TP53, ALK, and SDH was negative, whole exome sequencing of the blood DNA of the patient and both parents was performed to screen more widely for cancer predisposing mutations. In the patient's but not the parents' DNA we found a c.743 G>A, p.Arg248Gln (CCDS11118.1) TP53 mutation in 3–20% of sequencing reads, a level that would not generally be detectable by Sanger sequencing. Homozygosity for this mutation was detected in all tumor samples analyzed, and germline mosaicism was demonstrated by analysis of the child's newborn blood spot DNA. The occurrence of separate tumors derived from different germ layers suggests that this de novo mutation occurred early in embryogenesis, prior to gastrulation. Conclusion The case demonstrates pathogenic mosaicim, detected by next generation deep sequencing, that arose in the early stages of embryogenesis. PMID:24810334

  6. BRCA somatic and germline mutation detection in paraffin embedded ovarian cancers by next-generation sequencing.

    PubMed

    Mafficini, Andrea; Simbolo, Michele; Parisi, Alice; Rusev, Borislav; Luchini, Claudio; Cataldo, Ivana; Piazzola, Elena; Sperandio, Nicola; Turri, Giona; Franchi, Massimo; Tortora, Giampaolo; Bovo, Chiara; Lawlor, Rita T; Scarpa, Aldo

    2016-01-12

    BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue.

  7. Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

    PubMed

    Amary, Maria Fernanda C; Pauwels, Patrick; Meulemans, Els; Roemen, Guido M; Islam, Lily; Idowu, Bernadine; Bousdras, Konstantinos; Diss, Timothy C; O'Donnell, Paul; Flanagan, Adrienne M

    2007-09-01

    Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

  8. Mutation profiling in chinese patients with metastatic colorectal cancer and its correlation with clinicopathological features and anti-EGFR treatment response

    PubMed Central

    Wang, Fang; Zhao, Qi; Zhang, Dong-Sheng; Wang, Feng-Hua; Wang, Zhi-Qiang; Luo, Hui-Yan; He, Ming-Ming; Wang, De-Shen; Jin, Ying; Ren, Chao; Qiu, Miao-Zhen; Ren, Jian; Pan, Zhi-Zhong; Li, Yu-Hong; Shao, Jiao-Yong; Xu, Rui-Hua

    2016-01-01

    An increasing number of studies reveal the significance of genetic markers in guiding target treatment and refining prognosis. This retrospective observational study aims to assess the mutation profile of metastatic colorectal cancer (mCRC) in Chinese population with the help of MassARRAY® technique platform and OncoCarta™ Panel. 322 Chinese patients with mCRC who received clinical molecular testing as part of their standard care were investigated. 80 patients received cetuximab palliative treatment. 238 common hot-spot mutations of 19 cancer related genes in the OncoCarta™ Panel were tested. 44 mutations in 11 genes were detected in 156 cases (48.4%). At least one mutation was identified in 38.5% (124/322) of all tested cases, two concomitant mutations in 9.0% (29/322) and three mutations in 3 cases (<1%). KRAS was the most frequently mutated gene (34.8%), followed by PIK3CA (9.6%), NRAS (4.3%), BRAF (3.4%), EGFR (2.5%) and HRAS (1.2%). Less frequent mutations were detected in PDGFRA, RET, AKT1, FGFR1, and ERBB2. Co-mutation of RAS family subtypes was observed in 5 patients, and KRAS and BRAF concurrent mutation in 1 patient. KRAS, NRAS, BRAF and PIK3CA mutations had association with some clinicopathological features statistically. Patients identified as wild-type in all 19 genes had better objective response rate when treated with cetuximab. The clinical molecular testing with OncoCarta™ Panel supplemented the limited data of mCRC in Chinese population, and offered a clearer landscape of multiple gene mutational profile in not only clinically prognostic KRAS, NRAS, BRAF and PIK3CA genes, but also less frequent mutated genes. Knowledge of these multiple gene mutation patterns may give clues in exploring interesting accompanying co-occurrence relationship or mutually exclusive relationship between mutated genes, as well as in predicting benefit of all-wild-type patients from anti-EGFR treatment. PMID:27050078

  9. USP39 Deubiquitinase Is Essential for KRAS Oncogene-driven Cancer.

    PubMed

    Fraile, Julia M; Manchado, Eusebio; Lujambio, Amaia; Quesada, Víctor; Campos-Iglesias, Diana; Webb, Thomas R; Lowe, Scott W; López-Otín, Carlos; Freije, José M P

    2017-03-10

    KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

  10. ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

    PubMed Central

    Wang, Lidong; Yang, Huibin; Abel, Ethan V.; Ney, Gina M.; Palmbos, Phillip L.; Bednar, Filip; Zhang, Yaqing; Leflein, Jacob; Waghray, Meghna; Owens, Scott; Wilkinson, John E.; Prasad, Jayendra; Ljungman, Mats; Rhim, Andrew D.; Pasca di Magliano, Marina

    2015-01-01

    The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT. PMID:25593307

  11. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    PubMed Central

    Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample (n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers. PMID:26498688

  12. Methods in molecular cardiology: DHPLC mutation detection analysis

    PubMed Central

    Jongbloed, R.J.E.; Smeets, H.; Doevendans, P.A.; van den Wijngaard, A.

    2005-01-01

    An increasing number of mutations have been identified in genes involved in cardiac disorders which has led to novel insights in the pathophysiology of inherited cardiac diseases. As a result of these findings, techniques specialised in automated high-throughput analysis are implemented to handle the increasing number of diagnostic genetic requests. Denaturing high-performance liquid chromatography (DHPLC) is one such novel technique that fulfils the criteria of speed, sensitivity and accuracy. This issue focuses on the basic principle of the technique and illustrates how genetic alterations can be identified. ImagesFigure 1AFigure 2Figure 4 PMID:25696406

  13. Efficiency of BRCAPRO and Myriad II mutation probability thresholds versus cancer history criteria alone for BRCA1/2 mutation detection.

    PubMed

    van Harssel, J J T; van Roozendaal, C E P; Detisch, Y; Brandão, R D; Paulussen, A D C; Zeegers, M; Blok, M J; Gómez García, E B

    2010-06-01

    Considerable differences exist amongst countries in the mutation probability methods and thresholds used to select patients for BRCA1/2 genetic screening. In order to assess the added value of mutation probability methods, we have retrospectively calculated the BRCAPRO and Myriad II probabilities in 306 probands who had previously been selected for DNA-analysis according to criteria based on familial history of cancer. DNA-analysis identified 52 mutations (16.9%) and 11 unclassified variants (UVs, 3.6%). Compared to cancer history, a threshold > or = 10% with BRCAPRO or with Myriad II excluded about 40% of the patients from analysis, including four with a mutation and probabilities <10% with both programs. All four probands had a BRCA2 mutation. BRCAPRO and Myriad II showed similar specificity at 10% threshold, overall BRCAPRO was more sensitive than Myriad II for the detection of mutations. Only two of the probands with an UV had probabilities >20% with BRCAPRO and Myriad II. In summary, BRCAPRO and Myriad II are more efficient than cancer history alone to exclude patients without a mutation. BRCAPRO performs better for the detection of BRCA1 mutations than of BRCA2 mutations. The Myriad II scores provided no additional information than the BRCAPRO scores alone for the detection of patients with a mutation. The use of thresholds excluded from analysis the majority of patients carrying an UV.

  14. Germline p53 mutations are frequently detected in young children with rhabdomyosarcoma.

    PubMed Central

    Diller, L; Sexsmith, E; Gottlieb, A; Li, F P; Malkin, D

    1995-01-01

    We investigated the possibility that a proportion of children with sporadic rhabdomyosarcoma (RMS) carry constitutional mutations of the p53 tumor suppressor gene. 33 patients with sporadic RMS at two large outpatient pediatric oncology clinics submitted blood samples. Genomic DNA was extracted from peripheral blood leukocytes and PCR was used to amplify exons 2-11 of the p53 gene. Amplified genomic DNA was screened for the presence of germline p53 mutations using single-strand conformation polymorphism (SSCP) analysis. The DNA sequence of those samples that showed aberrant migration of bands on SSCP analysis was determined to identify the precise nature of the gene mutations. Patient records were reviewed to assess clinical correlates of the mutant p53 carrier state. Heterozygous constitutional mutations were detected in 3/33 patient samples screened. Two of these missense mutations are located in exon 7 and one in exon 8 of the p53 gene. The presence of mutations was not correlated with tumor histology, stage, or site. However, an association between young age at diagnosis and presence of a constitutional p53 mutation was noted: 3/13 children under the age of 3 yr at diagnosis carried mutations, whereas none of 20 children over 3 yr of age at diagnosis harbored a detectable constitutional mutation. These results in children with RMS corroborates previous findings in other clinical settings suggesting that the mutant p53 carrier state may predispose individuals to malignancy at an early age. Although this study did not assess whether the mutations were preexisting or new germline alterations, assessment of close relatives of RMS patients for cancer risk and predictive genetic testing may be indicated. Images PMID:7706467

  15. Worse prognosis of KRAS c.35 G > A mutant metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx)

    PubMed Central

    2013-01-01

    Background Prognosis of KRAS wild-type and mutant metastatic colorectal cancer (MCRC) patients (pts) treated with bevacizumab (BEV)-containing chemotherapy is not significantly different. Since specific KRAS mutations confer different aggressive behaviors, the prognostic role of prevalent KRAS mutations was retrospectively evaluated in MCRC pts treated with first line FIr-B/FOx, associating BEV to triplet chemotherapy. Methods Tumor samples were screened for KRAS codon 12, 13 and BRAF V600E mutations by SNaPshot and/or direct sequencing. MCRC pts <75-years-old were consecutively treated with FIr-B/FOx: weekly 12 hour-timed-flat-infusion/5-fluorouracil (900 mg/m2 on days 1,2, 8, 9, 15, 16,22, 23), irinotecan plus BEV (160 mg/m2 and 5 mg/kg, respectively, on days 1,15); and oxaliplatin (80 mg/m2, on days 8,22). Pts were classified as liver-limited (L-L) and other/multiple metastatic (O/MM). Progression-free survival (PFS) and overall survival (OS) were compared using the log-rank test. Results Fifty-nine pts were evaluated at a median follow-up of 21.5 months. KRAS mutant pts: c.35 G > A, 15 (25.4%); c.35 G > T, 7 (11.8%); c.38 G > A, 3 (5%); other, 3 (5%). KRAS wild-type, 31 pts (52.7%). The objective response rate (ORR), PFS and OS were, respectively: c.35 G > A mutant, 71%, 9 months, 14 months; other than c.35 G > A mutants, 61%, 12 months, 39 months. OS was significantly worse in c.35 G > A pts compared to KRAS wild-type (P = 0.002), KRAS/BRAF wild-type (P = 0.03), other MCRC patients (P = 0.002), other than c.35 G > A (P = 0.05), other codon 12 (P = 0.03) mutant pts. OS was not significantly different compared to c.35 G > T KRAS mutant (P = 0.142). Conclusions KRAS c.35 G > A mutant status may be significantly associated with a worse prognosis of MCRC pts treated with first line FIr-B/FOx intensive regimen compared to KRAS/BRAF wild type and other than c.35 G > A mutant pts. PMID:23497191

  16. Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.).

    PubMed

    Wang, I-C; Ustiyan, V; Zhang, Y; Cai, Y; Kalin, T V; Kalinichenko, V V

    2014-11-13

    Lung cancer is the leading cause of deaths in cancer patients in the United States. Identification of new molecular targets is clearly needed to improve therapeutic outcomes of this devastating human disease. Activating mutations in K-Ras oncogene and increased expression of FOXM1 protein are associated with poor prognosis in patients with non-small-cell lung cancer. Transgenic expression of activated Kras(G12D) in mouse respiratory epithelium is sufficient to induce lung adenocarcinomas; however, transcriptional mechanisms regulated by K-Ras during the initiation of lung cancer remain poorly understood. Foxm1 transcription factor, a downstream target of K-Ras, stimulates cellular proliferation during embryogenesis, organ repair and tumor growth, but its role in tumor initiation is unknown. In the present study, we used transgenic mice expressing Kras(G12D) under control of Sftpc promoter to demonstrate that Foxm1 was induced in type II epithelial cells before the formation of lung tumors. Conditional deletion of Foxm1 from Kras(G12D)-expressing respiratory epithelium prevented the initiation of lung tumors in vivo. The loss of Foxm1 inhibited expression of K-Ras target genes critical for the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, including Ikbkb, Nfkb1, Nfkb2, Rela, Jnk1, N-Myc, Pttg1 and Cdkn2a. Transgenic overexpression of activated FOXM1 mutant was sufficient to induce expression of these genes in alveolar type II cells. FOXM1 directly bound to promoter regions of Ikbkb, Nfkb2, N-Myc, Pttg1 and Cdkn2a, indicating that these genes are direct FOXM1 targets. FOXM1 is required for K-Ras-mediated lung tumorigenesis by activating genes critical for the NF-κB and JNK pathways.

  17. Detecting novel genetic mutations in Chinese Usher syndrome families using next-generation sequencing technology.

    PubMed

    Qu, Ling-Hui; Jin, Xin; Xu, Hai-Wei; Li, Shi-Ying; Yin, Zheng-Qin

    2015-02-01

    Usher syndrome (USH) is the most common cause of combined blindness and deafness inherited in an autosomal recessive mode. Molecular diagnosis is of great significance in revealing the molecular pathogenesis and aiding the clinical diagnosis of this disease. However, molecular diagnosis remains a challenge due to high phenotypic and genetic heterogeneity in USH. This study explored an approach for detecting disease-causing genetic mutations in candidate genes in five index cases from unrelated USH families based on targeted next-generation sequencing (NGS) technology. Through systematic data analysis using an established bioinformatics pipeline and segregation analysis, 10 pathogenic mutations in the USH disease genes were identified in the five USH families. Six of these mutations were novel: c.4398G > A and EX38-49del in MYO7A, c.988_989delAT in USH1C, c.15104_15105delCA and c.6875_6876insG in USH2A. All novel variations segregated with the disease phenotypes in their respective families and were absent from ethnically matched control individuals. This study expanded the mutation spectrum of USH and revealed the genotype-phenotype relationships of the novel USH mutations in Chinese patients. Moreover, this study proved that targeted NGS is an accurate and effective method for detecting genetic mutations related to USH. The identification of pathogenic mutations is of great significance for elucidating the underlying pathophysiology of USH.

  18. A Novel Intronic Splice Site Tafazzin Gene Mutation Detected Prenatally in a Family with Barth Syndrome

    PubMed Central

    Bakšienė, M; Benušienė, E; Morkūnienė, A; Ambrozaitytė, L; Utkus, A; Kučinskas, V

    2016-01-01

    Abstract Barth syndrome (BTHS) is a rare X-linked disease characterized by dilated cardiomyopathy, proximal skeletal myopathy and cyclic neutropenia. It is caused by various mutations in the tafazzin (TAZ) gene located on Xq28 that results in remodeling of cardiolipin and abnormalities in mitochondria stability and energy production. Here we report on a novel c.285-1G>C splice site mutation in intron 3 of the TAZ gene that was detected prenatally. PMID:28289596

  19. Validation of a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection.

    PubMed

    Zhang, Guanbin; Li, Caixia; Lu, Yuan; Hu, Hua; Xiang, Guangxin; Liang, Zhiqing; Liao, Pu; Dai, Pu; Xing, Wanli; Cheng, Jing

    2011-08-15

    We have established a mobile phone-assisted microarray decoding platform for signal-enhanced mutation detection. A large amount of single-stranded DNA (ssDNA) was obtained by combining symmetric PCR and magnetic isolation, and ssDNA prepared with magnetic bead as label was further allowed to hybridize against the tag-array for decoding purpose. High sensitivity and specificity was achieved with the detection of genomic DNA. When simultaneously genotyping nine common mutations associated with hereditary hearing loss, the detection limit of 1 ng genomic DNA was achieved. Significantly, a mobile phone was also used to record and decode the genotyping results through a custom-designed imaging adaptor and a dedicated mobile phone software. A total of 51 buccal swabs from patients probably with deafness-related mutations were collected and analyzed. The genotyping results were all confirmed by fluorescence-based laser confocal scanning and direct DNA sequencing. This mobile phone-assisted decoding platform provides an effective but economic mutation detection alternative for the future quicker and sensitive detection of virtually any mutation-related diseases in developing and underdeveloped countries.

  20. DNA mutation detection and analysis using miniaturized microfluidic systems.

    PubMed

    Handal, Maria I; Ugaz, Victor M

    2006-01-01

    Identification of genetic sequence variations occurring on a population-wide scale is key to unraveling the complex interactions that are the underlying cause of many medical disorders and diseases. A critical need exists, however, for advanced technology to enable DNA mutation analysis to be performed with significantly higher throughput and at significantly lower cost than is currently attainable. Microfluidic systems offer an attractive platform to address these needs by combining the ability to perform rapid analysis with a simplified device format that can be inexpensively mass-produced. This paper will review recent progress toward developing these next-generation systems and discuss challenges associated with adapting these technologies for routine laboratory use.

  1. Analysis of PTEN, BRAF and PI3K status for determination of benefit from cetuximab therapy in metastatic colorectal cancer patients refractory to chemotherapy with wild-type KRAS.

    PubMed

    Tural, Deniz; Batur, Sebnem; Erdamar, Sibel; Akar, Emre; Kepil, Nuray; Mandel, Nil Molinas; Serdengeçti, Süheyla

    2014-02-01

    We investigated predictive values of BRAF, PI3K and PTEN in cetuximab responses in KRAS wild-type (+) chemotherapy refractory, metastatic colorectal cancer (CRC) patients. Primary tumour tissues of 41 KRAS wild-type mCRC patients receiving cetuximab-based chemotherapy were investigated for PI3K, PTEN, KRAS and BRAF mutations. Progression-free survival (PFS) and overall survival (OS) periods were calculated with Kaplan-Meier method and the Cox proportional hazards model was used. PTEN and PI3K expressions were 63 and 42 %, respectively. BRAF mutation was observed as 9.8 % among patients. Tumours with BRAF mutation had statistically lower response rates (RR) for cetuximab-based treatment than tumours with BRAF wild type (0 vs. 58 %, p = 0.02). PTEN expressing tumours had statistically higher RR for cetuximab-based treatment than tumours with PTEN loss (42 vs. 12 %, p = 0.04). PI3K expression had worse significant effect on cetuximab RR than PI3K non-expressed tumours (15 vs. 44 %, p = 0.023). Median PFS was significantly longer in patients with PTEN expression (14 months) than in patients with PTEN loss (5 months) (HR, 0.4; p = 0.028). Median PFS was significantly longer in patients with PI3K non-expression (15.2 months) than in patients with PI3K expression (4.1 months) (HR, 0.31; p = 0.001). Significant difference in PFS and OS between patients with BRAF mutated and BRAF wild-type tumours was not detected. However, patients with PTEN expression had significantly longer OS (15.1 months) than patients with PTEN loss tumour (9.9 months) (HR, 0.34; p = 0.008). Patients without PI3K expression had significantly longer OS (18.2 months) than patients with PI3K expression (10.1 months) (HR, 0.27; p = 0.001). Multivariate analyses revealed that PTEN expression (HR, 0.48; p = 0.02) and absence of PI3K expression (HR, 0.2; p = 0.001) were independent prognostic factors for increased PFS. Similarly, PTEN overexpression (HR, 0.62; p = 0.03) and absence of PI3K expression (HR, 0

  2. Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia.

    PubMed

    Scholl, Sebastian; Krause, Claudia; Loncarevic, Ivan F; Müller, Rouven; Kunert, Christa; Wedding, Ulrich; Sayer, Herbert G; Clement, Joachim H; Höffken, Klaus

    2005-06-01

    Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.

  3. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis.

    PubMed

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F; Gorfe, Alemayehu A

    2016-03-08

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15-20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1-3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.

  4. An extra copy of p53 suppresses development of spontaneous Kras-driven but not radiation-induced cancer

    PubMed Central

    Moding, Everett J.; Min, Hooney D.; Castle, Katherine D.; Ali, Moiez; Woodlief, Loretta; Williams, Nerissa; Ma, Yan; Kim, Yongbaek; Lee, Chang-Lung

    2016-01-01

    The tumor suppressor p53 blocks tumor progression in multiple tumor types. Radiation-induced cancer following exposure to radiation therapy or space travel may also be regulated by p53 because p53 has been proposed to respond to DNA damage to suppress tumorigenesis. Here, we investigate the role of p53 in lung carcinogenesis and lymphomagenesis in LA-1 KrasG12D mice with wild-type p53 or an extra copy of p53 (super p53) exposed to fractionated total body irradiation with low linear energy transfer (low-LET) X-rays or high-LET iron ions and compared tumor formation in these mice with unirradiated controls. We found that an additional copy of p53 suppressed both Kras-driven lung tumor and lymphoma development in the absence of radiation. However, an additional copy of p53 did not affect lymphoma development following low- or high-LET radiation exposure and was unable to suppress radiation-induced expansion of thymocytes with mutated Kras. Moreover, radiation exposure increased lung tumor size in super p53 but not wild-type p53 mice. These results demonstrate that although p53 suppresses the development of spontaneous tumors expressing KrasG12D, in the context of exposure to ionizing radiation, an extra copy of p53 does not protect against radiation-induced lymphoma and may promote KrasG12D mutant lung cancer. PMID:27453951

  5. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma

    PubMed Central

    Aguirre, Andrew J.; Bardeesy, Nabeel; Sinha, Manisha; Lopez, Lyle; Tuveson, David A.; Horner, James; Redston, Mark S.; DePinho, Ronald A.

    2003-01-01

    Pancreatic ductal adenocarcinoma ranks among the most lethal of human malignancies. Here, we assess the cooperative interactions of two signature mutations in mice engineered to sustain pancreas-specific Cre-mediated activation of a mutant Kras allele (KrasG12D) and deletion of a conditional Ink4a/Arf tumor suppressor allele. The phenotypic impact of KrasG12D alone was limited primarily to the development of focal premalignant ductal lesions, termed pancreatic intraepithelial neoplasias (PanINs), whereas the sole inactivation of Ink4a/Arf failed to produce any neoplastic lesions in the pancreas. In combination, KrasG12D expression and Ink4a/Arf deficiency resulted in an earlier appearance of PanIN lesions and these neoplasms progressed rapidly to highly invasive and metastatic cancers, resulting in death in all cases by 11 weeks. The evolution of these tumors bears striking resemblance to the human disease, possessing a proliferative stromal component and ductal lesions with a propensity to advance to a poorly differentiated state. These findings in the mouse provide experimental support for the widely accepted model of human pancreatic adenocarcinoma in which activated KRAS serves to initiate PanIN lesions, and the INK4A/ARF tumor suppressors function to constrain the malignant conversion of these PanIN lesions into lethal ductal adenocarcinoma. This faithful mouse model may permit the systematic analysis of genetic lesions implicated in the human disease and serve as a platform for the identification of early disease markers and for the efficient testing of novel therapies. PMID:14681207

  6. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    PubMed

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases.

  7. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    PubMed

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

  8. Detection of JAK2 V617F mutation increases the diagnosis of myeloproliferative neoplasms

    PubMed Central

    ZHANG, SHU-PENG; LI, HUI; LAI, REN-SHENG

    2015-01-01

    The Janus kinase (JAK)2 gene, which is located on chromosome 9p24, is involved in the signaling transduction pathways of the hematopoietic and immune system. Mutations in the JAK2 gene have served as disease markers for myeloproliferative neoplasms (MPNs). The aim of the present study was to investigate the occurrence of the JAK2 gene mutation in 140 clinical samples, and to evaluate its clinical significance in MPNs and other hematological diseases. Genomic DNA was extracted from the peripheral blood leukocytes or bone marrow karyocytes of 140 clinical samples, which included 130 patients with various types of hematological disease and 10 control patients. In addition, exons 12 and 14 of the JAK2 gene were analyzed by direct sequencing and the mutation rates of various MPN subtypes were evaluated. Of the 140 samples, exons 12 and 14 were tested in 74 samples, however, exon 14 only was tested in 66 samples. No mutations were identified in exon 12. The V617F mutation rate in polycythemia vera was 82.1% (23/28), and the mutation rates in essential thrombocythemia histiocytosis, primary myelofibrosis and other MPNs were 53.1% (17/32), 40.0% (4/10) and 60.0% (6/10), respectively. Therefore, the total mutation rate of the JAK2 gene in MPN was 62.5% (50/80). For non-MPN hematological diseases, four V617F mutations were detected in samples of leukocytosis of unknown origin (4/12), however, no JAK2 V617F mutations were identified in the 10 controls. Therefore, JAK2 V617F mutations may present a novel marker for diagnosis of MPNs. Furthermore, the direct sequencing method appeared to be satisfactory for the clinical gene testing of hematological samples. PMID:25624900

  9. Mutation Detection in Patients with Retinal Dystrophies Using Targeted Next Generation Sequencing

    PubMed Central

    Weisschuh, Nicole; Mayer, Anja K.; Strom, Tim M.; Kohl, Susanne; Glöckle, Nicola; Schubach, Max; Andreasson, Sten; Bernd, Antje; Birch, David G.; Hamel, Christian P.; Heckenlively, John R.; Jacobson, Samuel G.; Kamme, Christina; Kellner, Ulrich; Kunstmann, Erdmute; Maffei, Pietro; Reiff, Charlotte M.; Rohrschneider, Klaus; Rosenberg, Thomas; Rudolph, Günther; Vámos, Rita; Varsányi, Balázs; Weleber, Richard G.; Wissinger, Bernd

    2016-01-01

    Retinal dystrophies (RD) constitute a group of blinding diseases that are characterized by clinical variability and pronounced genetic heterogeneity. The different nonsyndromic and syndromic forms of RD can be attributed to mutations in more than 200 genes. Consequently, next generation sequencing (NGS) technologies are among the most promising approaches to identify mutations in RD. We screened a large cohort of patients comprising 89 independent cases and families with various subforms of RD applying different NGS platforms. While mutation screening in 50 cases was performed using a RD gene capture panel, 47 cases were analyzed using whole exome sequencing. One family was analyzed using whole genome sequencing. A detection rate of 61% was achieved including mutations in 34 known and two novel RD genes. A total of 69 distinct mutations were identified, including 39 novel mutations. Notably, genetic findings in several families were not consistent with the initial clinical diagnosis. Clinical reassessment resulted in refinement of the clinical diagnosis in some of these families and confirmed the broad clinical spectrum associated with mutations in RD genes. PMID:26766544

  10. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA).

    PubMed

    Housden, Benjamin E; Perrimon, Norbert

    2016-09-01

    Although CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer.

  11. Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

    PubMed

    Pilch, Jacek; Asman, Marek; Jamroz, Ewa; Kajor, Maciej; Kotrys-Puchalska, Elżbieta; Goss, Małgorzata; Krzak, Maria; Witecka, Joanna; Gmiński, Jan; Sieroń, Aleksander L

    2010-11-01

    Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy.

  12. Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide-nanoparticle conjugates.

    PubMed

    Murphy, Deirdre; Redmond, Gareth

    2005-03-01

    Novel methods for application of oligonucleotide-gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide-gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for Delta F 508, M470V, R74W and R75Q mutations.

  13. Document forgery detection using distortion mutation of geometric parameters in characters

    NASA Astrophysics Data System (ADS)

    Shang, Shize; Kong, Xiangwei; You, Xingang

    2015-03-01

    Tampering related to document forgeries is often accomplished by copy-pasting or add-printing. These tampering methods introduce character distortion mutation in documents. We present a method of exposing document forgeries using distortion mutation of geometric parameters. We estimate distortion parameters, which consist of translation and rotation distortions, through image matching for each character. Detection of tampered characters with distortion mutation occurs based on a distortion probability, which is calculated from character distortion parameters. The introduction of a visualized probability map describes the degree of distortion mutation for a full page. The proposed method exposes the forgeries based on individual characters and applies to English and Chinese document examinations. Experimental results demonstrate the effectiveness of our method on low JPEG compression quality and low resolution.

  14. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling.

    PubMed

    Siljamäki, Elina; Abankwa, Daniel

    2016-10-15

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.

  15. KRAS Mutant Pancreatic Cancer: No Lone Path to an Effective Treatment

    PubMed Central

    Zeitouni, Daniel; Pylayeva-Gupta, Yuliya; Der, Channing J.; Bryant, Kirsten L.

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers with a dismal 7% 5-year survival rate and is projected to become the second leading cause of cancer-related deaths by 2020. KRAS is mutated in 95% of PDACs and is a well-validated driver of PDAC growth and maintenance. However, despite comprehensive efforts, an effective anti-RAS drug has yet to reach the clinic. Different paths to inhibiting RAS signaling are currently under investigation in the hope of finding a successful treatment. Recently, direct RAS binding molecules have been discovered, challenging the perception that RAS is an “undruggable” protein. Other strategies currently being pursued take an indirect approach, targeting proteins that facilitate RAS membrane association or downstream effector signaling. Unbiased genetic screens have identified synthetic lethal interactors of mutant RAS. Most recently, metabolic targets in pathways related to glycolytic signaling, glutamine utilization, autophagy, and macropinocytosis are also being explored. Harnessing the patient’s immune system to fight their cancer is an additional exciting route that is being considered. The “best” path to inhibiting KRAS has yet to be determined, with each having promise as well as potential pitfalls. We will summarize the state-of-the-art for each direction, focusing on efforts directed toward the development of therapeutics for pancreatic cancer patients with mutated KRAS. PMID:27096871

  16. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

    PubMed Central

    Siljamäki, Elina

    2016-01-01

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner. PMID:27503857

  17. Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress

    PubMed Central

    Shaw, Alice T.; Winslow, Monte M.; Magendantz, Margaret; Ouyang, Chensi; Dowdle, James; Subramanian, Aravind; Lewis, Timothy A.; Maglathin, Rebecca L.; Tolliday, Nicola; Jacks, Tyler

    2011-01-01

    Activating K-RAS mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream signaling pathways have been shown to be deregulated by oncogenic K-ras. However, to date there are still no effective targeted therapies for this genetically defined subset of patients. Here we report the results of a small molecule, synthetic lethal screen using mouse embryonic fibroblasts derived from a mouse model harboring a conditional oncogenic K-rasG12D allele. Among the >50,000 compounds screened, we identified a class of drugs with selective activity against oncogenic K-ras–expressing cells. The most potent member of this class, lanperisone, acts by inducing nonapoptotic cell death in a cell cycle- and translation-independent manner. The mechanism of cell killing involves the induction of reactive oxygen species that are inefficiently scavenged in K-ras mutant cells, leading to oxidative stress and cell death. In mice, treatment with lanperisone suppresses the growth of K-ras–driven tumors without overt toxicity. Our findings establish the specific antitumor activity of lanperisone and reveal oxidative stress pathways as potential targets in Ras-mediated malignancies. PMID:21555567

  18. Toward PCR-free mutation detection based on surface-enhanced Raman scattering

    NASA Astrophysics Data System (ADS)

    Culha, Mustafa; Karatas, Omer F.; Aydin, Omer; Kahraman, Mehmet; Keseroğlu, Kemal; Sayin, Ismail; Bayrak, Omer F.

    2009-02-01

    The development of an assay for the detection of gene mutations has been attempted based on surface-enhanced Raman scattering (SERS). Using multiplexing property and high sensitivity of SERS technique, the detection of all mutation possibilities on one given spot is achievable. To test the feasibility of approach, SNPs and other types of mutations such as insertion and deletion are investigated. The PCR amplified and isolated genomic DNA without PCR amplification is immobilized on poly-L/D-lysine coated glass surface after denaturing with heating. The SERS probes are prepared by simultaneous attachment of oligonucleotides complementary to the target mutation regions and Raman active dyes to 13 nm gold nanoparticles (GNPs). After the hybridization of SERS probes on the poly-L/D-lysine surfaces, it was stained with silver colloidal nanoparticles for further enhancement of Raman scattering. In the second approach, Raman active dyes are chemically attached on gold nanoparticles and a thin layer of silver film is deposited on top of it to prepare core-shell nanoparticles. The complementary oligonucleotides to the target regions of the gene are chemically attached to silver surfaces of the nanoparticles. The promising results indicate that it is possible to detect certain mutation types without PCR amplification using the approach.

  19. Rapid and Reliable Detection of Nonsyndromic Hearing Loss Mutations by Multicolor Melting Curve Analysis

    PubMed Central

    Wang, Xudong; Hong, Yongjun; Cai, Peihong; Tang, Ning; Chen, Ying; Yan, Tizhen; Liu, Yinghua; Huang, Qiuying; Li, Qingge

    2017-01-01

    Hearing loss is a common birth defect worldwide. The GJB2, SLC26A4, MT-RNR1 and MT-TS1 genes have been reported as major pathogenic genes in nonsyndromic hearing loss. Early genetic screening is recommended to minimize the incidence of hearing loss. We hereby described a multicolor melting curve analysis (MMCA)-based assay for simultaneous detection of 12 prevalent nonsyndromic hearing loss-related mutations. The three-reaction assay could process 30 samples within 2.5 h in a single run on a 96-well thermocycler. Allelic types of each mutation could be reproducibly obtained from 10 pg ~100 ng genomic DNA per reaction. For the mitochondrial mutations, 10% ~ 20% heteroplasmic mutations could be detected. A comparison study using 501 clinical samples showed that the MMCA assay had 100% concordance with both SNaPshot minisequencing and Sanger sequencing. We concluded that the MMCA assay is a rapid, convenient and cost-effective method for detecting the common mutations, and can be expectedly a reliable tool in preliminary screening of nonsyndromic hearing loss in the Chinese Han population. PMID:28225033

  20. Newborn Screening Quality Assurance Program for CFTR Mutation Detection and Gene Sequencing to Identify Cystic Fibrosis

    PubMed Central

    Hendrix, Miyono M.; Foster, Stephanie L.; Cordovado, Suzanne K.

    2016-01-01

    All newborn screening laboratories in the United States and many worldwide screen for cystic fibrosis. Most laboratories use a second-tier genotyping assay to identify a panel of mutations in the CF transmembrane regulator (CFTR) gene. Centers for Disease Control and Prevention’s Newborn Screening Quality Assurance Program houses a dried blood spot repository of samples containing CFTR mutations to assist newborn screening laboratories and ensure high-quality mutation detection in a high-throughput environment. Recently, CFTR mutation detection has increased in complexity with expanded genotyping panels and gene sequencing. To accommodate the growing quality assurance needs, the repository samples were characterized with several multiplex genotyping methods, Sanger sequencing, and 3 next-generation sequencing assays using a high-throughput, low-concentration DNA extraction method. The samples performed well in all of the assays, providing newborn screening laboratories with a resource for complex CFTR mutation detection and next-generation sequencing as they transition to new methods. PMID:28261631

  1. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  2. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  3. EGF RECEPTOR SIGNALING IS ESSENTIAL FOR K-RAS ONCOGENE-DRIVEN PANCREATIC DUCTAL ADENOCARCINOMA

    PubMed Central

    Navas, Carolina; Hernández-Porras, Isabel; Schuhmacher, Alberto J; Sibilia, Maria; Guerra, Carmen; Barbacid, Mariano

    2013-01-01

    Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with the presence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53–defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic. PMID:22975375

  4. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    PubMed Central

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-01-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines. PMID:28262827

  5. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    NASA Astrophysics Data System (ADS)

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-03-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

  6. Growth Regulation via Insulin-Like Growth Factor Binding Protein-4 and -2 in Association with Mutant K-ras in Lung Epithelia

    PubMed Central

    Sato, Hanako; Yazawa, Takuya; Suzuki, Takehisa; Shimoyamada, Hiroaki; Okudela, Koji; Ikeda, Masaichi; Hamada, Kenji; Yamada-Okabe, Hisafumi; Yao, Masayuki; Kubota, Yoshinobu; Takahashi, Takashi; Kamma, Hiroshi; Kitamura, Hitoshi

    2006-01-01

    Gain-of-function point mutations in K-ras affect early events in pulmonary bronchioloalveolar carcinoma. We investigated altered mRNA expression on K-Ras activation in human peripheral lung epithelial cells (HPL1A) using oligonucleotide microarrays. Mutated K-Ras stably expressed in HPL1A accelerated cell growth and induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-4 and IGFBP-2, which modulate cell growth via IGF. Other lung epithelial cell lines (NHBE and HPL1D) revealed the same phenomena as HPL1A by mutated K-ras transgene. Lung cancer cell growth was also accelerated by mutated K-ras gene transduction, whereas IGFBP-4/2 induction was weaker compared with mutated K-Ras-expressing lung epithelial cells. To understand the differences in IGFBP-4/2 inducibility via K-Ras-activated signaling between nonneoplastic lung epithelia and lung carcinoma, we addressed the mechanisms of IGFBP-4/2 transcriptional activation. Our results revealed that Egr-1, which is induced on activation of Ras-mitogen-activated protein kinase signaling, is crucial for transactivation of IGFBP-4/2. Furthermore, IGFBP-4 and IGFBP-2 promoters were often hypermethylated in lung carcinoma, yielding low basal expression/weak induction of IGFBP-4/2. These findings suggest that continuous K-Ras activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth. Moreover, this growth regulation is disrupted in lung cancers because of promoter hypermethylation of IGFBP-4/2 genes. PMID:17071580

  7. Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection.

    PubMed

    Larsen, Lars Allan; Jespersgaard, Cathrine; Andersen, Paal Skytt

    2007-01-01

    This protocol describes capillary array electrophoresis single-strand conformation polymorphism (CAE-SSCP), a screening method for detection of unknown and previously identified mutations. The method detects 98% of mutations in a sample material and can be applied to any organism where the goal is to determine genetic variation. This protocol describes how to screen for mutations in 192 singleplex or up to 768 multiplex samples over 3 days. The protocol is based on the principle of sequence-specific mobility of single-stranded DNA in a native polymer, and covers all stages in the procedure, from initial DNA purification to final CAE-SSCP data analysis, as follows: DNA is purified, followed by PCR amplification using fluorescent primers. After PCR amplification, double-stranded DNA is heat-denatured to separate the strands and subsequently cooled on ice to avoid reannealing. Finally, samples are analyzed by capillary electrophoresis and appropriate analysis software.

  8. Predictive and Prognostic Analysis of PIK3CA Mutation in Stage III Colon Cancer Intergroup Trial

    PubMed Central

    Liao, Xiaoyun; Imamura, Yu; Yamauchi, Mai; McCleary, Nadine J.; Ng, Kimmie; Niedzwiecki, Donna; Saltz, Leonard B.; Mayer, Robert J.; Whittom, Renaud; Hantel, Alexander; Benson, Al B.; Mowat, Rex B.; Spiegelman, Donna; Goldberg, Richard M.; Bertagnolli, Monica M.; Meyerhardt, Jeffrey A.; Fuchs, Charles S.

    2013-01-01

    Background Somatic mutations in PIK3CA (phosphatidylinositol-4,5-bisphosphonate 3-kinase [PI3K], catalytic subunit alpha gene) activate the PI3K-AKT signaling pathway and contribute to pathogenesis of various malignancies, including colorectal cancer. Methods We examined associations of PIK3CA oncogene mutation with relapse, survival, and treatment efficacy in 627 stage III colon carcinoma case subjects within a randomized adjuvant chemotherapy trial (5-fluorouracil and leucovorin [FU/LV] vs irinotecan [CPT11], fluorouracil and leucovorin [IFL]; Cancer and Leukemia Group B 89803 [Alliance]). We detected PIK3CA mutation in exons 9 and 20 by polymerase chain reaction and pyrosequencing. Cox proportional hazards model was used to assess prognostic and predictive role of PIK3CA mutation, adjusting for clinical features and status of routine standard molecular pathology features, including KRAS and BRAF mutations and microsatellite instability (mismatch repair deficiency). All statistical tests were two-sided. Results Compared with PIK3CA wild-type cases, overall status of PIK3CA mutation positivity or the presence of PIK3CA mutation in either exon 9 or 20 alone was not statistically significantly associated with recurrence-free, disease-free, or overall survival (log-rank P > .70; P > .40 in multivariable regression models). There was no statistically significant interaction between PIK3CA and KRAS (or BRAF) mutation status in survival analysis (P interaction > .18). PIK3CA mutation status did not appear to predict better or worse response to IFL therapy compared with FU/LV therapy (P interaction > .16). Conclusions Overall tumor PIK3CA mutation status is not associated with stage III colon cancer prognosis. PIK3CA mutation does not appear to serve as a predictive tumor molecular biomarker for response to irinotecan-based adjuvant chemotherapy. PMID:24231454

  9. Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

    PubMed Central

    Aydin, Selda; Dekairelle, Anne-France; Ambroise, Jérôme; Durant, Jean-François; Heusterspreute, Michel; Guiot, Yves

    2014-01-01

    In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5–8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a

  10. Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer

    PubMed Central

    Wong; Lin, David T; Huang, Chien-Chung; Wei, Wei-Lun; Su, Fang; Wu-Chou

    2016-01-01

    Summary Advances in target therapies for lung cancer have enabled detection of gene mutations, specifically those of EGFR. Assays largely depend on the acquisition of tumor tissue biopsy, which is invasive and may not reflect the genomic profile of the tumor at treatment due to tumor heterogeneity or changes that occur during treatment through acquired resistance. Liquid biopsy, a blood test that detects evidence of cancer cells or tumor DNA, has generated considerable interest for its ability to detect EGFR mutations, however, its clinical application is limited by complicated collection methods and the need for technique-dependent platforms. Recently, simpler techniques for EGFR mutant detection in in urine or saliva samples have been developed. This review focuses on advances in liquid biopsy and discusses its potential for clinical implementation in lung cancer. PMID:26420338

  11. Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer.

    PubMed

    Lin, Chien-Chung; Huang, Wei-Lun; Wei, Fang; Su, Wu-Chou; Wong, David T

    2015-01-01

    Advances in target therapies for lung cancer have enabled detection of gene mutations, specifically those of EGFR. Assays largely depend on the acquisition of tumor tissue biopsy, which is invasive and may not reflect the genomic profile of the tumor at treatment due to tumor heterogeneity or changes that occur during treatment through acquired resistance. Liquid biopsy, a blood test that detects evidence of cancer cells or tumor DNA, has generated considerable interest for its ability to detect EGFR mutations. However, its clinical application is limited by complicated collection methods and the need for technique-dependent platforms. Recently, simpler techniques for EGFR mutant detection in urine or saliva samples have been developed. This review focuses on advances in liquid biopsy and discusses its potential for clinical implementation in lung cancer.

  12. Screening for EGFR Mutations in Patients with Head and Neck Cancer Treated with Gefitinib on a Compassionate-Use Program: A Hellenic Cooperative Oncology Group Study

    PubMed Central

    Murray, Samuel; Bobos, Mattheos; Angouridakis, Nikolaos; Nikolaou, Angelos; Linardou, Helena; Razis, Evangelia; Fountzilas, George

    2010-01-01

    Background and Aim. EGFR is commonly expressed in cancers of the head and neck (H and N), and anti-EGFR agents have demonstrated improvements in outcomes (TTP and OS). The aim of this study was to determine EGFR gene status in H and N cancer patients treated with gefitinib and to correlate mutational status with clinico-pathological data and response. Patients and Methods. Patients with histologically confirmed H and N cancer having failed prior treatment for advanced disease entered this compassionate-use-program. Nineteen patients received gefitinib. EGFR expression was assessed by IHC, gene copy number by FISH, and mutation analysis was conducted for EGFR (18-21), KRAS, BRAF (V600E), and HER-2 exon 20. An additional TKI naive cohort of 73 patients was also screened. Results. Mutations were detected in 6/19 patients (3× EGFR, 1× KRAS, and 2× HER2-exon 20). There were no significant differences in TTP or OS for patients with somatic EGFR mutations. No BRAF mutations were detected. Conclusions. The incidence of EGFR mutations in H and N cancer in this study was 5.3%. No statistically relevant correlations between mutation or gene gain and response or survival were observed. Due to the limited number of patients and low incidence of genetic aberrations in the genes analyzed, additional studies are warranted. PMID:21274259

  13. Diagnostic Value of RAS Mutations in Indeterminate Thyroid Nodules.

    PubMed

    Clinkscales, William; Ong, Adrian; Nguyen, Shaun; Harruff, Elizabeth Emily; Gillespie, Marion Boyd

    2017-03-01

    Objectives To determine the diagnostic value of HRAS, KRAS, and NRAS mutations in fine-needle aspiration biopsies of thyroid nodules that are nondiagnostic on cytology. Data Sources PubMed, Scopus, Embase, CINAHL. Review Methods Two authors independently searched the data sources. To be included, studies reported the RAS mutational status and postoperative histopathologic diagnosis of nodules that exhibited indeterminate cytology after fine-needle aspiration biopsy. Data were extracted to calculate sensitivity, specificity, and positive/negative predictive values of any HRAS, KRAS, or NRAS mutation. A meta-analysis was performed to generate pooled values for each parameter. Results A total of 7 studies with a combined 1025 patients met inclusion criteria. The pooled sensitivity of a RAS mutation for detecting cancer was 0.343 (95% confidence interval [95% CI], 0.198-0.506), while the pooled specificity was 0.935 (95% CI, 0.882-0.973). The weighted averages for positive predictive value and negative predictive value were 78.0% and 64.0%, respectively, with 68.0% accuracy. The positive likelihood ratio was 4.235 (95% CI, 1.506-11.910), and the negative likelihood ratio was 0.775 (95% CI, 0.630-0.953). Conclusion Our data suggest that testing for any RAS mutation is unlikely to change the clinical management of thyroid nodules that have indeterminate cytology. While a RAS mutation may rule in malignancy, the sensitivity of testing is low enough to merit further mutational analysis, repeat fine-needle aspiration, or surgical excision, even in the presence of a negative test.

  14. Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

    PubMed

    Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

    2015-08-01

    Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

  15. Detection of mutations in the ALD gene (ABCD1) in seven Italian families: description of four novel mutations.

    PubMed

    Lira, M G; Mottes, M; Pignatti, P F; Medica, I; Uziel, G; Cappa, M; Bertini, E; Rizzuto, N; Salviati, A

    2000-09-01

    The study describes the mutations causing adrenoleukodystrophy in seven Italian families. Four missense mutations leading to amino acid substitutions, two frameshift mutations leading to a premature termination signal, and a splicing mutation were identified. Mutations 2014C>T (P543L), 2053A>G (Q556A), 673-674insCC, and 1874+1G>A are described for the first time in this report. Mutations 1638C>T (R418W), 1588G>A(R401Q), and 1801-1802delAG are already known to be link to ALD.

  16. Detection of eight BRCA1 mutations in 10 breast/ovarian cancer families, including 1 family with male breast cancer

    SciTech Connect

    Sruewing, J.P.; Brody, L.C.; Erdos, M.R.

    1995-07-01

    Genetic epidemiological evidence suggests that mutations in BRCA1 may be responsible for approximately one half of early onset familial breast cancer and the majority of familial breast/ovarian cancer. The recent cloning of BRCA1 allows for the direct detection of mutations, but the feasibility of presymptomatic screening for cancer susceptibility is unknown. We analyzed genomic DNA from one affected individual from each of 24 families with at least three cases of ovarian or breast cancer, using SSCP assays. Variant SSCP bands were subcloned and sequenced. Allele-specific oligonucleotide hybridization was used to verify sequence changes and to screen DNA from control individuals. Six frameshift and two missense mutations were detected in 10 different families. A frameshift mutation was detected in a male proband affected with both breast and prostate cancer. A 40-bp deletion was detected in a patient who developed intra-abdominal carcinomatosis 1 year after prophylactic oophorectomy. Mutations were detected throughout the gene, and only one was detected in more than a single family. These results provide further evidence that inherited breast and ovarian cancer can occur as a consequence of a wide array of BRCA1 mutations. These results suggests that development of a screening test for BRCA1 mutations will be technically challenging. The finding of a mutation in a family with male breast cancer, not previously thought to be related to BRCA1, also illustrates the potential difficulties of genetic counseling for individuals known to carry mutations. 37 refs., 1 fig., 1 tab.

  17. Identification of common cystic fibrosis mutations in African-Americans with cystic fibrosis increases the detection rate to 75%.

    PubMed Central

    Macek, M; Mackova, A; Hamosh, A; Hilman, B C; Selden, R F; Lucotte, G; Friedman, K J; Knowles, M R; Rosenstein, B J; Cutting, G R

    1997-01-01

    Cystic fibrosis (CF)--an autosomal recessive disorder caused by mutations in CF transmembrane conductance regulator (CFTR) and characterized by abnormal chloride conduction across epithelial membranes, leading to chronic lung and exocrine pancreatic disease--is less common in African-Americans than in Caucasians. No large-scale studies of mutation identification and screening in African-American CF patients have been reported, to date. In this study, the entire coding and flanking intronic sequence of the CFTR gene was analyzed by denaturing gradient-gel electrophoresis and sequencing in an index group of 82 African-American CF chromosomes to identify mutations. One novel mutation, 3120+1G-->A, occurred with a frequency of 12.3% and was also detected in a native African patient. To establish frequencies, an additional group of 66 African-American CF chromosomes were screened for mutations identified in two or more African-American patients. Screening for 16 "common Caucasian" mutations identified 52% of CF alleles in African-Americans, while screening for 8 "common African" mutations accounted for an additional 23%. The combined detection rate of 75% was comparable to the sensitivity of mutation analysis in Caucasian CF patients. These results indicate that African-Americans have their own set of "common" CF mutations that originate from the native African population. Inclusion of these "common" mutations substantially improves CF mutation detection rates in African-Americans. PMID:9150159

  18. Gel-based nonradioactive single-strand conformational polymorphism and mutation detection: limitations and solutions.

    PubMed

    Gupta, Vibhuti; Arora, Reetakshi; Gochhait, Sailesh; Bairwa, Narendra K; Bamezai, Rameshwar N K

    2014-01-01

    Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every polymerase chain reaction product and assisting in choosing only the amplicons of interest with expected mutations. The principle of detection of small changes in DNA sequences is based on changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence dependent. The limitations faced in SSCP range from routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems can be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP remains the method of choice to analyze mutations and SNPs in order to understand genomic variations, both spontaneous and induced, and the genetic basis of diseases.

  19. Method for detecting point mutations in DNA utilizing fluorescence energy transfer

    DOEpatents

    Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

    2001-01-01

    A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

  20. A combinatorial strategy for treating KRAS-mutant lung cancer.

    PubMed

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T; Gainor, Justin F; Corcoran, Ryan B; Engelman, Jeffrey A; Rudin, Charles M; Rosen, Neal; Lowe, Scott W

    2016-06-30

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proved difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, a MEK inhibitor approved by the US Food and Drug Administration, which acts downstream of KRAS to suppress signalling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signalling rebound and adaptive drug resistance. As a consequence, genetic or pharmacological inhibition of FGFR1 in combination with trametinib enhances tumour cell death in vitro and in vivo. This compensatory response shows distinct specificities: it is dominated by FGFR1 in KRAS-mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patients’ tumours treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer.

  1. A combinatorial strategy for treating KRAS mutant lung cancer

    PubMed Central

    Manchado, Eusebio; Weissmueller, Susann; Morris, John P.; Chen, Chi-Chao; Wullenkord, Ramona; Lujambio, Amaia; de Stanchina, Elisa; Poirier, John T.; Gainor, Justin F.; Corcoran, Ryan B.; Engelman, Jeffrey A.; Rudin, Charles M.; Rosen, Neal; Lowe, Scott W.

    2016-01-01

    Therapeutic targeting of KRAS-mutant lung adenocarcinoma represents a major goal of clinical oncology. KRAS itself has proven difficult to inhibit, and the effectiveness of agents that target key KRAS effectors has been thwarted by activation of compensatory or parallel pathways that limit their efficacy as single agents. Here we take a systematic approach towards identifying combination targets for trametinib, an FDA-approved MEK inhibitor that acts downstream of KRAS to suppress signaling through the mitogen-activated protein kinase (MAPK) cascade. Informed by a short-hairpin RNA (shRNA) screen, we show that trametinib provokes a compensatory response involving the fibroblast growth factor receptor 1 (FGFR1) that leads to signaling rebound and adaptive drug resistance. As a consequence, genetic or pharmacologic inhibition of FGFR1 in combination with trametinib enhances tumor cell death in vitro and in vivo. This compensatory response shows distinct specificities – it is dominated by FGFR1 in KRAS mutant lung and pancreatic cancer cells, but is not activated or involves other mechanisms in KRAS wild-type lung and KRAS-mutant colon cancer cells. Importantly, KRAS-mutant lung cancer cells and patient tumors treated with trametinib show an increase in FRS2 phosphorylation, a biomarker of FGFR activation; this increase is abolished by FGFR1 inhibition and correlates with sensitivity to trametinib and FGFR inhibitor combinations. These results demonstrate that FGFR1 can mediate adaptive resistance to trametinib and validate a combinatorial approach for treating KRAS-mutant lung cancer. PMID:27338794

  2. Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation

    PubMed Central

    2011-01-01

    Introduction Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation. Methods IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated. Results The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked

  3. Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans

    PubMed Central

    Dudiuk, Catiana; Gamarra, Soledad; Jimenez-Ortigosa, Cristina; Leonardelli, Florencia; Macedo, Daiana; Perlin, David S.

    2015-01-01

    A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory. PMID:25878347

  4. An ECL-PCR method for quantitative detection of point mutation

    NASA Astrophysics Data System (ADS)

    Zhu, Debin; Xing, Da; Shen, Xingyan; Chen, Qun; Liu, Jinfeng

    2005-04-01

    A new method for identification of point mutations was proposed. Polymerase chain reaction (PCR) amplification of a sequence from genomic DNA was followed by digestion with a kind of restriction enzyme, which only cut the wild-type amplicon containing its recognition site. Reaction products were detected by electrochemiluminescence (ECL) assay after adsorption of the resulting DNA duplexes to the solid phase. One strand of PCR products carries biotin to be bound on a streptavidin-coated microbead for sample selection. Another strand carries Ru(bpy)32+ (TBR) to react with tripropylamine (TPA) to emit light for ECL detection. The method was applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than 3 orders of magnitude, thus, make quantitative analysis possible. The genotype can be clearly discriminated. Results of the study suggest that ECL-PCR is a feasible quantitative method for safe, sensitive and rapid detection of point mutation in human genes.

  5. Prognostic value of KRAS genotype in metastatic colorectal cancer (MCRC) patients treated with intensive triplet chemotherapy plus bevacizumab (FIr-B/FOx) according to extension of metastatic disease

    PubMed Central

    2012-01-01

    Background Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of metastatic colorectal cancer (MCRC), particularly integrated with secondary liver surgery in liver-limited (L-L) patients. The prognostic value of the KRAS genotype in L-L and other or multiple metastatic (O/MM) MCRC patients treated with the FIr-B/FOx regimen was retrospectively evaluated. Methods Tumoral and metastatic samples were screened for KRAS codon 12 and 13 and BRAF mutations by SNaPshot and/or direct sequencing. Fit MCRC patients <75 years were consecutively treated with FIr-B/FOx regimen: weekly 12-h timed flat-infusion/5-fluorouracil (TFI 5-FU) 900 mg/m2, days 1, 2, 8, 9, 15, 16, 22 and 23; irinotecan (CPT-11) 160 mg/m2 plus BEV 5 mg/kg, days 1, 15; oxaliplatin (OXP) 80 mg/m2, days 8, 22; every 4 weeks. MCRC patients were classified as L-L and O/MM. Activity and efficacy were evaluated and compared using log-rank test. Results In all, 59 patients were evaluated: 31 KRAS wild-type (53%), 28 KRAS mutant (47%). At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were not significantly different. PFS and OS were significantly different in L-L compared to O/MM evaluable patients. In KRAS wild-type patients, clinical outcome of 12 L-L compared to 18 O/MM was significantly different: PFS 21 versus 12 months and OS 47 versus 28 months, respectively. In KRAS mutant patients, the clinical outcome of 13 L-L compared to 14 O/MM was not significantly different: PFS 11 months equivalently and OS 39 versus 19 months, respectively. Conclusions The KRAS genotype wild-type and mutant does not significantly affect different clinical outcomes for MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve a significantly

  6. Frequent PIK3CA Mutations in Colorectal and Endometrial Cancer with Double Somatic Mismatch Repair Mutations

    PubMed Central

    Cohen, Stacey A.; Turner, Emily H.; Beightol, Mallory B.; Jacobson, Angela; Gooley, Ted A.; Salipante, Stephen J.; Haraldsdottir, Sigurdis; Smith, Christina; Scroggins, Sheena; Tait, Jonathan F.; Grady, William M.; Lin, Edward H.; Cohn, David E.; Goodfellow, Paul J.; Arnold, Mark W.; de la Chapelle, Albert; Pearlman, Rachel; Hampel, Heather; Pritchard, Colin C.

    2016-01-01

    Background & Aims Double somatic mutations in mismatch repair (MMR) genes have recently been described in colorectal and endometrial cancers with microsatellite instability (MSI) not attributable to MLH1 hypermethylation or germline mutation. We sought to define the molecular phenotype of this newly recognized tumor subtype. Methods From two prospective Lynch syndrome screening studies, we identified patients with colorectal and endometrial tumors harboring ≥2 somatic MMR mutations, but normal germline MMR testing (“double somatic”). We determined the frequencies of tumor PIK3CA, BRAF, KRAS, NRAS, and PTEN mutations by targeted next-generation sequencing and used logistic-regression models to compare them to: Lynch syndrome, MLH1 hypermethylated, and microsatellite stable (MSS) tumors. We validated our findings using independent datasets from The Cancer Genome Atlas (TCGA). Results Among colorectal cancer cases, we found that 14/21 (67%) of double somatic cases had PIK3CA mutations vs. 4/18 (22%) Lynch syndrome, 2/10 (20%) MLH1 hypermethylated, and 12/78 (15%) MSS tumors; p<0.0001. PIK3CA mutations were detected in 100% of 13 double somatic endometrial cancers (p=0.04). BRAF mutations were absent in double somatic and Lynch syndrome colorectal tumors. We found highly similar results in a validation cohort from TCGA (113 colorectal, 178 endometrial cancer), with 100% of double somatic cases harboring a PIK3CA mutation (p<0.0001). Conclusions PIK3CA mutations are present in double somatic mutated colorectal and endometrial cancers at substantially higher frequencies than other MSI subgroups. PIK3CA mutation status may better define an emerging molecular entity in colorectal and endometrial cancers, with the potential to inform screening and therapeutic decision making. PMID:27302833

  7. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    PubMed

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  8. Exon 44 nonsense mutation in two-Duchenne muscular dystrophy brothers detected by heteroduplex analysis.

    PubMed

    Prior, T W; Papp, A C; Snyder, P J; Burghes, A H; Sedra, M S; Western, L M; Bartolo, C; Mendell, J R

    1993-01-01

    Utilizing a heteroduplex method, we screened the dystrophin exon 43-45 region for point mutations, including small deletions and insertions. The method depends upon the formation of a heteroduplex between wild-type and mutant DNA PCR products. DNA specimens from one hundred and four DMD patients without detected deletions or duplications were multiplexed amplified for exons 43, 44, and 45. The PCR products were mixed with the PCR products from nonaffected controls, electrophoresed, and examined for the presence of altered mobility heteroduplex bands. An exon 44 nonsense mutation in two DMD brothers and a common intron 44 polymorphism were identified using this approach. Although the exon 44-45 region is a hotspot for deletion breakpoints, it does not appear to be prone to point mutations. The technique is extremely useful for screening several exons simultaneously and it allowed us to screen a large number of patients.

  9. A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

    PubMed Central

    Alioto, Tyler S.; Buchhalter, Ivo; Derdak, Sophia; Hutter, Barbara; Eldridge, Matthew D.; Hovig, Eivind; Heisler, Lawrence E.; Beck, Timothy A.; Simpson, Jared T.; Tonon, Laurie; Sertier, Anne-Sophie; Patch, Ann-Marie; Jäger, Natalie; Ginsbach, Philip; Drews, Ruben; Paramasivam, Nagarajan; Kabbe, Rolf; Chotewutmontri, Sasithorn; Diessl, Nicolle; Previti, Christopher; Schmidt, Sabine; Brors, Benedikt; Feuerbach, Lars; Heinold, Michael; Gröbner, Susanne; Korshunov, Andrey; Tarpey, Patrick S.; Butler, Adam P.; Hinton, Jonathan; Jones, David; Menzies, Andrew; Raine, Keiran; Shepherd, Rebecca; Stebbings, Lucy; Teague, Jon W.; Ribeca, Paolo; Giner, Francesc Castro; Beltran, Sergi; Raineri, Emanuele; Dabad, Marc; Heath, Simon C.; Gut, Marta; Denroche, Robert E.; Harding, Nicholas J.; Yamaguchi, Takafumi N.; Fujimoto, Akihiro; Nakagawa, Hidewaki; Quesada, Víctor; Valdés-Mas, Rafael; Nakken, Sigve; Vodák, Daniel; Bower, Lawrence; Lynch, Andrew G.; Anderson, Charlotte L.; Waddell, Nicola; Pearson, John V.; Grimmond, Sean M.; Peto, Myron; Spellman, Paul; He, Minghui; Kandoth, Cyriac; Lee, Semin; Zhang, John; Létourneau, Louis; Ma, Singer; Seth, Sahil; Torrents, David; Xi, Liu; Wheeler, David A.; López-Otín, Carlos; Campo, Elías; Campbell, Peter J.; Boutros, Paul C.; Puente, Xose S.; Gerhard, Daniela S.; Pfister, Stefan M.; McPherson, John D.; Hudson, Thomas J.; Schlesner, Matthias; Lichter, Peter; Eils, Roland; Jones, David T. W.; Gut, Ivo G.

    2015-01-01

    As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy. PMID:26647970

  10. Who is a carrier? Detection of unsuspected mutations in 21-hydroxylase deficiency

    SciTech Connect

    Witchel, S.S.; Lee, P.A.; Trucco, M.

    1996-01-02

    Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population. 29 refs., 3 figs., 2 tabs.

  11. SNaPshot Assay for the Detection of the Most Common CFTR Mutations in Infertile Men

    PubMed Central

    Mircevska, Marija; Plaseski, Toso; Filipovski, Vanja; Plaseska-Karanfilska, Dijana

    2014-01-01

    Congenital bilateral absence of vas deferens (CBAVD) is the most common CFTR-related disorder (CFTR-RD) that explains about 1–2% of the male infertility cases. Controversial data have been published regarding the involvement of CFTR mutations in infertile men with non-obstructive azoospermia and oligozoospermia. Here, we describe single base extension (SNaPshot) assay for detection of 11 common CFTR mutations: F508del, G542X, N1303K, 621+1G->T, G551D, R553X, R1162X, W1282X, R117H, 2184insA and 1717-1G->A and IVS8polyT variants. The assay was validated on 50 previously genotyped samples and was used to screen a total of 369 infertile men with different impairment of spermatogenesis and 136 fertile controls. Our results show that double heterozygosity of cystic fibrosis (CF) and CFTR-related disorder (CFTR-RD) mutations are found in a high percentage (22.7%) of infertile men with obstructive azoospermia, but not in other studied groups of infertile men. The SNaPshot assay described here is an inexpensive, fast and robust method for primary screening of the most common CFTR mutations both in patients with classical CF and CFTR-RD. It can contribute to better understanding of the role of CFTR mutations in impaired spermatogenesis, ultimately leading to improved management of infertile men. PMID:25386751

  12. Application of COLD-PCR for improved detection of NF2 mosaic mutations.

    PubMed

    Paganini, Irene; Mancini, Irene; Baroncelli, Marta; Arena, Guido; Gensini, Francesca; Papi, Laura; Sestini, Roberta

    2014-07-01

    Somatic mosaicism represents the coexistence of two or more cell populations with different genotypes in one person, and it is involved in >30 monogenic disorders. Somatic mosaicism characterizes approximately 25% to 33% of patients with de novo neurofibromatosis type 2 (NF2). The identification of mosaicism is crucial to patients and their families because the clinical course of the disease and its transmission risk is influenced by the degree and distribution of mutated cells. Moreover, in NF2, the capability of discriminating patients with mosaicism is especially important to make differential diagnosis with schwannomatosis. However, the identification of mosaic variants is considerably difficult, and the development of specific molecular techniques to detect low levels of unknown molecular alterations is required. Co-amplification at lower denaturation temperature (COLD)-PCR has been described as a powerful method to selectively amplify minority alleles from mixtures of wild-type and mutation-containing sequences. Here, we applied COLD-PCR to molecular analysis of patients with NF2 mosaicism. With the use of COLD-PCR, followed by direct sequencing, we were able to detect NF2 mutations in blood DNA of three patients with NF2 mosaicism. Our study has shown the capability of COLD-PCR in enriching low-represented mutated allele in blood DNA sample, making it usable for molecular diagnosis of patients with mosaicism.

  13. Detection of cystic fibrosis mutations in a GeneChip{trademark} assay format

    SciTech Connect

    Miyada, C.G.; Cronin, M.T.; Kim, S.M.

    1994-09-01

    We are developing assays for the detection of cystic fibrosis mutations based on DNA hybridization. A DNA sample is amplified by PCR, labeled by incorporating a fluorescein-tagged dNTP, enzymatically treated to produce smaller fragments and hybridized to a series of short (13-16 bases) oligonucleotides synthesized on a glass surface via photolithography. The hybrids are detected by eqifluorescence and mutations are identified by the specific pattern of hybridization. In a GeneChip assay, the chip surface is composed of a series of subarrays, each being specific for a particular mutation. Each subarray is further subdivided into a series of probes (40 total), half based on the mutant sequence and the remainder based on the wild-type sequence. For each of the subarrays, there is a redundancy in the number of probes that should hybridize to either a wild-type or a mutant target. The multiple probe strategy provides sequence information for a short five base region overlapping the mutation site. In addition, homozygous wild-type and mutant as well as heterozygous samples are each identified by a specific pattern of hybridization. The small size of each probe feature (250 x 250 {mu}m{sup 2}) permits the inclusion of additional probes required to generate sequence information by hybridization.

  14. Genetic disruption of oncogenic Kras sensitizes lung cancer cells to Fas receptor-mediated apoptosis.

    PubMed

    Mou, Haiwei; Moore, Jill; Malonia, Sunil K; Li, Yingxiang; Ozata, Deniz M; Hough, Soren; Song, Chun-Qing; Smith, Jordan L; Fischer, Andrew; Weng, Zhiping; Green, Michael R; Xue, Wen

    2017-04-04

    Genetic lesions that activate KRAS account for ∼30% of the 1.6 million annual cases of lung cancer. Despite clinical need, KRAS is still undruggable using traditional small-molecule drugs/inhibitors. When oncogenic Kras is suppressed by RNA interference, tumors initially regress but eventually recur and proliferate despite suppression of Kras Here, we show that tumor cells can survive knockout of oncogenic Kras, indicating the existence of Kras-independent survival pathways. Thus, even if clinical KRAS inhibitors were available, resistance would remain an obstacle to treatment. Kras-independent cancer cells exhibit decreased colony formation in vitro but retain the ability to form tumors in mice. Comparing the transcriptomes of oncogenic Kras cells and Kras knockout cells, we identified 603 genes that were specifically up-regulated in Kras knockout cells, including the Fas gene, which encodes a cell surface death receptor involved in physiological regulation of apoptosis. Antibodies recognizing Fas receptor efficiently induced apoptosis of Kras knockout cells but not oncogenic Kras-expressing cells. Increased Fas expression in Kras knockout cells was attributed to decreased association of repressive epigenetic marks at the Fas promoter. Concordant with this observation, treating oncogenic Kras cells with histone deacetylase inhibitor and Fas-activating antibody efficiently induced apoptosis, thus bypassing the need to inhibit Kras. Our results suggest that activation of Fas could be exploited as an Achilles' heel in tumors initiated by oncogenic Kras.

  15. Somatic mutation spectrum of non-small cell lung cancer in African Americans: a pooled analysis

    PubMed Central

    Araujo, Luiz H.; Lammers, Philip E.; Matthews-Smith, Velmalia; Eisenberg, Rosana; Gonzalez, Adriana; Schwartz, Ann G.; Timmers, Cynthia; Shilo, Konstantin; Zhao, Weiqiang; Natarajan, Thanemozhi G.; Zhang, Jianying; Yilmaz, Ayse Selen; Liu, Tom; Coombes, Kevin; Carbone, David P.

    2015-01-01

    Introduction The mutational profile of non-small cell lung cancer (NSCLC) has become an important tool in tailoring therapy to patients, with clear differences according to the population of origin. African Americans have higher lung cancer incidence and mortality than Caucasians, yet discrepant results have been reported regarding the frequency of somatic driver mutations. We hypothesized that NSCLC has a distinct mutational profile in this group. Methods We collected NSCLC samples resected from self-reported African Americans in five sites from Tennessee, Michigan, and Ohio. Gene mutations were assessed by either SNaPshot or next generation sequencing, and ALK translocations were evaluated by fluorescence in situ hybridization. Results Two hundred sixty patients were included, mostly males (62.3%) and smokers (86.6%). Eighty-one samples (31.2%) were squamous cell carcinomas. The most frequently mutated genes were KRAS (15.4%), EGFR (5.0%), PIK3CA (0.8%), BRAF, NRAS, ERBB2, and AKT1 (0.4% each). ALK translocations were detected in 2 non-squamous tumors (1.7%), totaling 61 cases (23.5%) with driver oncogenic alterations. Among 179 non-squamous samples, 54 (30.2%) presented a driver alteration. The frequency of driver alterations altogether was lower than that reported in Caucasians, while no difference was detected in either EGFR or KRAS mutations. Overall survival was longer among patients with EGFR mutations. Conclusions We demonstrated that NSCLC from African Americans has a different pattern of somatic driver mutations than from Caucasians. The majority of driver alterations in this group are yet to be described, which will require more comprehensive panels and assessment of non-canonical alterations. PMID:26301800

  16. Microelectronic DNA assay for the detection of BRCA1 gene mutations

    NASA Technical Reports Server (NTRS)

    Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

    2004-01-01

    Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

  17. Panitumumab as a radiosensitizing agent in KRAS wild-type locally advanced rectal cancer.

    PubMed

    Mardjuadi, Feby Ingriani; Carrasco, Javier; Coche, Jean-Charles; Sempoux, Christine; Jouret-Mourin, Anne; Scalliet, Pierre; Goeminne, Jean-Charles; Daisne, Jean-François; Delaunoit, Thierry; Vuylsteke, Peter; Humblet, Yves; Meert, Nicolas; van den Eynde, Marc; Moxhon, Anne; Haustermans, Karin; Canon, Jean-Luc; Machiels, Jean-Pascal

    2015-09-01

    Our goal was to optimize the radiosensitizing potential of anti-epidermal growth factor receptor (EGFR) monoclonal antibodies, when given concomitantly with preoperative radiotherapy in KRAS wild-type locally advanced rectal cancer (LARC). Based on pre-clinical studies conducted by our group, we designed a phase II trial in which panitumumab (6 mg/kg/q2 weeks) was combined with preoperative radiotherapy (45 Gy in 25 fractions) to treat cT3-4/N + KRAS wild-type LARC. The primary endpoint was complete pathologic response (pCR) (H0 = 5%, H1 = 17%, α = 0.05, β = 0.2). From 19 enrolled patients, 17 (89%) were evaluable for pathology assessment. Although no pCR was observed, seven patients (41%) had grade 3 Dworak pathological tumor regression. The regimen was safe and was associated with 95% of sphincter-preservation rate. No NRAS, BRAF, or PI3KCA mutation was found in this study, but one patient (5%) showed loss of PTEN expression. The quantification of plasma EGFR ligands during treatment showed significant upregulation of plasma TGF-α and EGF following panitumumab administration (p < 0.05). At surgery, patients with important pathological regression (grade 3 Dworak) had higher plasma TGF-α (p = 0.03) but lower plasma EGF (p = 0.003) compared to those with grade 0-2 Dworak. Our study suggests that concomitant panitumumab and preoperative radiotherapy in KRAS wild-type LARC is feasible and results in some tumor regression. However, pCR rate remained modest. Given that the primary endpoint of our study was not reached, we remain unable to recommend the use of panitumumab as a radiosensitizer in KRAS wild-type LARC outside a research setting.

  18. Detection and isolation of single tumor cells containing mutated DNA sequences

    NASA Astrophysics Data System (ADS)

    Leary, James F.; He, Feng; Reece, Lisa M.

    1999-04-01

    One of the problems in treating breast cancer patients is discovering the gene rearrangements that are occurring while the patient is in apparent remission. Spontaneous mutations in DNA sequences, particularly in tumor suppressor genes, can lead to the evolution of new clones of tumor cells that may be able to evade both clinical treatments and the patient's immune surveillance system. Isolation of these tumor clones is extremely difficult. Rare-event analysis and single-cell sorting techniques must be used to successfully detect and isolate these tumor clones. PCR amplification of selected gene sequences followed by TA cloning techniques can then be used to perform single-cell DNA sequencing in those gene regions. In this paper we present preliminary data showing successful detection and single-cell sorting of rare tumor clones from defined cell mixtures. Using TA cloning techniques and PCR we have been able to detect a single base-pair mutation in the PTEN tumor suppressor gene in single cells from a breast cancer cell line. Thus, while extremely difficult, it should in the future be possible to isolate tumor clones form a patient for subsequent molecular analyses of DNA mutations in critical gene regions.

  19. Application of a New Genetic Deafness Microarray for Detecting Mutations in the Deaf in China

    PubMed Central

    Wu, Hong; Feng, Yong; Jiang, Lu; Pan, Qian; Liu, Yalan; Liu, Chang; He, Chufeng; Chen, Hongsheng; Liu, Xueming; Hu, Chang; Hu, Yiqiao; Mei, Lingyun

    2016-01-01

    Objective The aim of this study was to evaluate the GoldenGate microarray as a diagnostic tool and to elucidate the contribution of the genes on this array to the development of both nonsyndromic and syndromic sensorineural hearing loss in China. Methods We developed a microarray to detect 240 mutations underlying syndromic and nonsyndromic sensorineural hearing loss. The microarray was then used for analysis of 382 patients with nonsyndromic sensorineural hearing loss (including 15 patients with enlarged vestibular aqueduct syndrome), 21 patients with Waardenburg syndrome, and 60 unrelated controls. Subsequently, we analyzed the sensitivity, specificity, and reproducibility of this new approach after Sanger sequencing-based verification, and also determined the contribution of the genes on this array to the development of distinct hearing disorders. Results The sensitivity and specificity of the microarray chip were 98.73% and 98.34%, respectively. Genetic defects were identified in 61.26% of the patients with nonsyndromic sensorineural hearing loss, and 9 causative genes were identified. The molecular etiology was confirmed in 19.05% and 46.67% of the patients with Waardenburg syndrome and enlarged vestibular aqueduct syndrome, respectively. Conclusion Our new mutation-based microarray comprises an accurate and comprehensive genetic tool for the detection of sensorineural hearing loss. This microarray-based detection method could serve as a first-pass screening (before next-generation-sequencing screening) for deafness-causing mutations in China. PMID:27018795

  20. Microfluidics for rapid detection of isocitrate dehydrogenase 1 mutation for intraoperative application.

    PubMed

    Aibaidula, Abudumijiti; Zhao, Wang; Wu, Jin-Song; Chen, Hong; Shi, Zhi-Feng; Zheng, Lu-Lu; Mao, Ying; Zhou, Liang-Fu; Sui, Guo-Dong

    2016-06-01

    OBJECT Conventional methods for isocitrate dehydrogenase 1 (IDH1) detection, such as DNA sequencing and immunohistochemistry, are time- and labor-consuming and cannot be applied for intraoperative analysis. To develop a new approach for rapid analysis of IDH1 mutation from tiny tumor samples, this study used microfluidics as a method for IDH1 mutation detection. METHODS Forty-seven glioma tumor samples were used; IDH1 mutation status was investigated by immunohistochemistry and DNA sequencing. The microfluidic device was fabricated from polydimethylsiloxane following standard soft lithography. The immunoanalysis was conducted in the microfluidic chip. Fluorescence images of the on-chip microcolumn taken by the charge-coupled device camera were collected as the analytical results readout. Fluorescence signals were analyzed by NIS-Elements software to gather detailed information about the IDH1 concentration in the tissue samples. RESULTS DNA sequencing identified IDH1 R132H mutation in 33 of 47 tumor samples. The fluorescence signal for IDH1-mutant samples was 5.49 ± 1.87 compared with 3.90 ± 1.33 for wild type (p = 0.005). Thus, microfluidics was capable of distinguishing IDH1-mutant tumor samples from wild-type samples. When the cutoff value was 4.11, the sensitivity of microfluidics was 87.9% and the specificity was 64.3%. CONCLUSIONS This new approach was capable of analyzing IDH1 mutation status of tiny tissue samples within 30 minutes using intraoperative microsampling. This approach might also be applied for rapid pathological diagnosis of diffuse gliomas, thus guiding personalized resection.

  1. Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

    PubMed Central

    Russo, Giancarlo; Patrignani, Andrea; Poveda, Lucy; Hoehn, Frederic; Scholtka, Bettina; Schlapbach, Ralph; Garvin, Alex M.

    2015-01-01

    Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS. We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC. PMID:27054083

  2. Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing.

    PubMed

    Russo, Giancarlo; Patrignani, Andrea; Poveda, Lucy; Hoehn, Frederic; Scholtka, Bettina; Schlapbach, Ralph; Garvin, Alex M

    2015-12-01

    Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS. We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

  3. Development of positive control materials for DNA-based detection of cystic fibrosis: Cloning and sequencing of 31 mutations

    SciTech Connect

    Iovannisci, D.; Brown, C.; Winn-Deen, E.

    1994-09-01

    The cloning and sequencing of the gene associated with cystic fibrosis (CF) now provides the opportunity for earlier detection and carrier screening through DNA-based detection schemes. To date, over 300 mutations have been reported to the CF Consortium; however, only 30 mutations have been observed frequently enough world-wide to warrant routine screening. Many of these mutations are not available as cloned material or as established tissue culture cell lines to aid in the development of DNA-based detection assays. We have therefore cloned the 30 most frequently reported mutations, plus the mutation R347H due to its association with male infertility (31 mutations, total). Two approaches were employed: direct PCR amplification, where mutations were available from patient sources, and site-directed PCR mutagenesis of normal genomic DNA to generate the remaining mutations. After amplification, products were cloned into a sequencing vector, bacterial transformants were screened by a novel method (PCR/oligonucleotide litigation assay/sequence-coded separation), and plamid DNA sequences determined by automated fluorescent methods on the Applied Biosystems 373A. Mixing of the clones allows the construction of artificial genotypes useful as positive control material for assay validation. A second round of mutagenesis, resulting in the construction of plasmids bearing multiple mutations, will be evaluated for their utility as reagent control materials in kit development.

  4. Frequent p53 mutation in brain (fetal)-type glycogen phosphorylase positive foci adjacent to human ‘de novo’olorectal carcinomas

    PubMed Central

    Shimada, S; Shiomori, K; Tashima, S; Tsuruta, J; Ogawa, M

    2001-01-01

    ‘de novo’ carcinogenesis has been advocated besides ‘adenoma carcinoma sequence’ as another dominant pathway leading to colorectal carcinoma. Our recent study has demonstrated that the distribution of brain (fetal)-type glycogen phosphorylase (BGP) positive foci (BGP foci) has a close relationship with the location of ‘de novo’ carcinoma. The aims of the present study are to investigate genetic alteration in the BGP foci and to characterize them in the ‘de novo’ carcinogenesis. 17 colorectal carcinomas without any adenoma component expressing both immunoreactive p53 and BGP protein were selected from 96 resected specimens from our previous study. Further investigations to examine the proliferating cell nuclear antigen (PCNA)-labelling index, and the p53 and the codon 12 of K-ras mutation using the polymerase chain reaction-single strand conformation polymorphism were performed in the BGP foci, BGP negative mucosa and carcinoma. The BGP foci were observed sporadically in the transitional mucosa adjacent to the carcinoma in all cases. The PCNA labelling index in the BGP foci was significantly higher than that in the BGP negative mucosa (P< 0.001). p53 mutations were observed in 8 carcinomas, but no K-ras mutation was detected. Interestingly, although none of the overexpressions of p53 protein was detected immunohistochemically in the BGP positive foci, the p53 gene frequently (41.2% of the BGP foci tested) mutated in spite of no K-ras mutation. The present study demonstrates potentially premalignant foci in the colorectal transitional mucosa with frequent p53 gene mutation. It is suggested that BGP foci are promising candidates for the further investigation of ‘de novo’ colorectal carcinogenesis. © 2001Cancer Research Campaign http://www.bjcancer.com PMID:11384100

  5. Rapid detection of mutations in the human-derived Pneumocystis carinii dihydropteroate synthase gene associated with sulfa resistance.

    PubMed

    Ma, L; Kovacs, J A

    2001-03-01

    Recent studies have shown that point mutations in the dihydropteroate synthase (DHPS) gene of human-derived Pneumocystis carinii are related to exposure to sulfa drugs and possibly represent the emergence of sulfa resistance. We developed a simple single-strand conformation polymorphism (SSCP) method to permit rapid detection of these mutations. With plasmid constructs, SSCP was able to detect as little as 10% of a minority population. The SSCP assay was compared to direct sequencing for typing the DHPS gene by examining 37 clinical isolates with known DHPS sequences and 41 clinical isolates with unknown DHPS sequences. The typing results were consistent between these two methods for all isolates except 11 in which mutations were detected by SSCP but not by direct sequencing. Sequencing of individual clones after subcloning confirmed the presence of mutations in a minority population as determined by SSCP. SSCP is a very simple and sensitive method for rapid identification of P. camii DHPS mutations.

  6. Direct electrochemical genosensing for multiple point mutation detection of Mycobacterium tuberculosis during the development of rifampin resistance.

    PubMed

    Kara, Pinar; Cavusoglu, Cengiz; Cavdar, Seda; Ozsoz, Mehmet

    2009-02-15

    We present a robust and simple method for the direct detection of multiple point mutations in the Mycobacterium tuberculosis rpoB gene during the development of rifampin (RIF) resistance using an electrochemical genosensor. The device contained five different capture probes which are designed to hybridize with several sequence segments within the bacterial rpoB gene hotspot region. Point mutations were detected by monitoring the guanine oxidation with differential pulse voltammetry after hybridization between PCR amplicons and inosine modified capture probes at graphite surface. Changes in the peak voltage corresponding to guanine oxidation provide an electrochemical signal for hybridization that can be used to determine the presence of point mutations conferring rifampin resistance. The analytical parameters (sensitivity, selectivity and reproducibility) were evaluated. High selective discrimination against point mutation of bacteria at hot-spot region was observed. Several mutations were detected at several parts of the amplicon from 21 positive samples.

  7. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  8. Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

    NASA Astrophysics Data System (ADS)

    Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

    2014-09-01

    A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

  9. Complete direct sequencing of the entire AR gene in 45 unrelated patients with androgen insensitivity syndrome: Mutations identified in 32 patients (18 novel mutations), no mutation detected in 13 other patients (29%)

    SciTech Connect

    Mebarki, F.; Forest, M.G.; Josso, N.

    1994-09-01

    The androgen insensivity syndrome (AIS) is a recessive X-linked disorder resulting from a deficient function of the androgen receptor (AR). The human AR gene has 3 functional domains: N-terminal encoded by exon 1, DNA-binding domain encoded by exons 2 and 3, and androgen-binding domain encoded by exons 4 to 8. In order to characterize the molecular defects of the AR gene in AIS, the entire coding regions and the intronic bording sequences of the AR gene were amplified by PCR before automatic direct sequencing in 45 patients. Twenty seven different point mutations were found in 32 unrelated AIS patients: 18 with a complete form (CAIS), 14 with a partial form (PAIS); 18 of these mutations are novel mutations, not published to date. Only 3 mutations were repeatedly found: R804H in 3 families; M780I in 3 families and R774C in 2 families. For 26 patients out of the 32 found to have a mutation, maternal DNA was collected and sequenced: 6 de novo mutations were detected (i.e. 23% of the cases). Finally, no mutation was detected in 13 patients (29%): 7 with CAIS and 6 familial severe PAIS. The latter all presented with perineal hypospadias, micropenis, 4 out of 6 being raised as girl. Diagnosis of AIS in these 13 families in whom no mutation was detected is supported by the following criteria: clinical data, familial history (2 or 3 index cases in the same family), familial segregation of the polymorphic CAG repeat of the AR gene. Mutations in intronic regions or the promoter of the AR gene could not explain all cases of AIS without mutations in the AR coding regions, because AR binding (performed in 9 out of 13) was normal in 6, suggesting the synthesis of an AR protein. This situation led us to speculate that another X-linked factor associated with the AR could be implicated in some cases of AIS.

  10. Detecting N-RAS Q61R Mutated Thyroid Neoplasias by Immunohistochemistry.

    PubMed

    Crescenzi, A; Fulciniti, F; Bongiovanni, M; Giovanella, L; Trimboli, Pierpaolo

    2017-03-01

    Recently, the immunohistochemistry (IHC) for N-RAS Q61R has been developed and commercialized for clinical practice. Here, we investigated the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia. A series of 24 consecutive thyroid lesions undergone surgery following indeterminate cytology were enrolled. Paraffin sections were stained for IHC using the rabbit monoclonal anti-human N-RAS Q61R, clone SP174. N-RAS mutations in codon 61 were also investigated by automated sequencing. At histology, 12 cases of follicular carcinoma, cytologically defined as follicular lesions, 1 papillary cancer, 7 follicular adenomas, and 4 hyperplastic nodules were found. Of these, 4 showed a positive IHC for anti N-RAS antibody where N-RAS expression was detected mainly at cytoplasmic level with similar intensity of reaction. The remaining cases had negative IHC. A 100% concordance between IHC and molecular analysis for N-RAS Q61R was observed. In conclusion, this study shows high reliability of IHC to identify N-RAS Q61R mutated thyroid lesions with high cost-effectiveness. These data indicate the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia and suggest to adopt this approach for a more accurate management of patients, when indicated.

  11. PCR assay confirms diagnosis in syndrome with variably expressed phenotype: mutation detection in Stickler syndrome.

    PubMed Central

    Ahmad, N N; McDonald-McGinn, D M; Dixon, P; Zackai, E H; Tasman, W S

    1996-01-01

    Stickler syndrome is an autosomal dominant disease with ocular (severe myopia, vitreal degeneration, and retinal detachment) and other systemic manifestations (hearing loss, cleft palate, epiphyseal dysplasia, and premature osteoarthritis). As with other dominantly inherited conditions, the clinical phenotype of Stickler syndrome varies considerably. To date, all mutations have been located in the type II procollagen (COL2A1) gene. Analysis of a C-->T mutation we had identified previously, in COL2A1 gene in exon 40, in a three generation pedigree showed the loss of a cleavage site for the TaqI restriction enzyme. We designed a rapid PCR based restriction enzyme assay to detect this mutation and used it to establish the diagnosis in a neonate from the same pedigree, presenting with the first occurrence of the Pierre-Robin sequence in the family and minimal ocular findings. These results underline the potential diagnostic value of many as yet undetected DNA mutations in families affected with Stickler syndrome, since the variability of the