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Sample records for kras mutation detection

  1. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    PubMed

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. PMID:27116474

  2. KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

    PubMed

    Piton, Nicolas; Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

    2015-01-01

    KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

  3. A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens

    PubMed Central

    Gonzalez de Castro, D; Angulo, B; Gomez, B; Mair, D; Martinez, R; Suarez-Gauthier, A; Shieh, F; Velez, M; Brophy, V H; Lawrence, H J; Lopez-Rios, F

    2012-01-01

    Background: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. Methods: We conducted a two-site comparison of two commercial KRAS mutation kits – the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit – and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. Results: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. Conclusion: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation. PMID:22713664

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

    PubMed Central

    2016-01-01

    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. PMID:27478332

  5. 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.

  6. 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

  7. 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

  8. Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

    PubMed

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

    2016-07-01

    KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways. PMID:27289420

  9. 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. PMID:26339780

  10. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    PubMed

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

  11. A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

    PubMed

    Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

    2015-07-01

    It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency. PMID:25936694

  12. Kras gene codon 12 mutation detection enabled by gold nanoparticles conducted in a nanobioarray chip.

    PubMed

    Sedighi, Abootaleb; Li, Paul C H

    2014-03-01

    This study employs a nanobioarray (NBA) chip for multiple biodetection of single base pair mutations at the Kras gene codon 12. To distinguish between the mutant and wild-type target DNAs, current bioarray methods use high-temperature hybridization of the targets to the allele-specific probes. However, these techniques need prior temperature optimization and become harder to implement in the case of the detection of multiple mutations. We aimed to detect these mutations at a single temperature (room temperature), enabled by the use of gold nanoparticles (AuNPs) on the bioarray created within nanofluidic channels. In this method, a low amount of target oligonucleotides (5fmol) and polymerase chain reaction (PCR) products (300pg) were first loaded on the AuNP surface, and then these AuNP-bound targets were introduced into the channels of a polydimethylsiloxane (PDMS) glass chip. The targets hybridized to their complementary probes at the intersection of the target channels to the pre-printed oligonucleotide probe lines on the glass surface, creating a bioarray. Using this technique, fast and high-throughput multiple discrimination of the Kras gene codon 12 were achieved at room temperature using the NBA chip, and the specificity of the method was proved to be as high as that with the temperature stringency method.

  13. 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. PMID:26999437

  14. Diagnostic accuracy of high resolution melting analysis for detection of KRAS mutations: a systematic review and meta-analysis.

    PubMed

    Liu, Yue-Ping; Wu, Hai-Yan; Yang, Xiang; Xu, Han-Qing; Chen, Dong; Huang, Qing; Fu, Wei-Ling

    2014-01-01

    Increasing evidence points to a negative correlation between KRAS mutations and patients' responses to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo KRAS mutation detection to be eligible for treatment. High resolution melting analysis (HRM) is gaining increasing attention in KRAS mutation detection. However, its accuracy has not been systematically evaluated. We conducted a meta-analysis of published articles, involving 13 articles with 1,520 samples, to assess its diagnostic accuracy compared with DNA sequencing. The quality of included articles was assessed using the revised Quality Assessment for Studies of Diagnostic Accuracy (QUADAS-2) tools. Random effects models were applied to analyze the performance of pooled characteristics. The overall sensitivity and specificity of HRM were 0.99 (95% confidence interval [CI]: 0.98-1.00) and 0.96 (95%CI: 0.94-0.97), respectively. The area under the summary receiver operating characteristic curve was 0.996. High sensitivity and specificity, less labor, rapid turn-around and the closed-tube format of HRM make it an attractive choice for rapid detection of KRAS mutations in clinical practice. The burden of DNA sequencing can be reduced dramatically by the implementation of HRM, but positive results still need to be sequenced for diagnostic confirmation. PMID:25515911

  15. 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.
    <...

  16. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.

    PubMed

    Tsiatis, Athanasios C; Norris-Kirby, Alexis; Rich, Roy G; Hafez, Michael J; Gocke, Christopher D; Eshleman, James R; Murphy, Kathleen M

    2010-07-01

    Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively. PMID:20431034

  17. A new generation of companion diagnostics: cobas BRAF, KRAS and EGFR mutation detection tests.

    PubMed

    Angulo, Barbara; Lopez-Rios, Fernando; Gonzalez, David

    2014-06-01

    The cobas(®) (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an 'end-to-end' solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use. PMID:24844134

  18. A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas

    PubMed Central

    2012-01-01

    Background It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters. Methods We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits. Results and conclusions Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples. PMID:22995035

  19. KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography.

    PubMed

    Karim, Bougatef; Florence, Coulet; Kamel, Rouissi; Nadia, Kourda; Ines, Omrane; Raja, Marrakchi; Sarra, Ben Jileni; Florent, Soubrier; Amel, Ben Ammar-Elgaaied

    The Kirsten Rat Sarcoma (KRAS) oncogene has been introduced recently as a genetic biomarker for metastatic sporadic colorectal cancer prior to anti-EGFR treatment. Identifying patients with KRAS mutations that not respond to EGFR targeted therapies require sensitive, rapid and efficacious routine technique. We have attempted to evaluate the efficiency of three conventional methods: direct sequencing, HRM and DHPLC, to detect mutations in codon 12 and 13 of the KRAS exon2 gene. For this first Tunisian study on KRAS, we detected 45.83% of altered KRAS gene among 48 formalin-fixed paraffin-embedded sporadic colorectal adenocarcinoma patients. The use of HRM-sequencing allowed as enlarging the detected KRAS exon 2 mutations (22/48) in comparison with direct sequencing (17/48). DHPLC was used to confirm results when consensus was not observed between HRM and direct sequencing. This study brings an interesting data concerning an inter-method validation between sequencing and HRM in the investigation of sporadic colorectal cancer biomarker. It also shows that KRAS mutations occur at similar frequencies in Tunisian patients as in other populations; and suggests that the same genes are at play in sporadic CRC cancer, despite ethnic, geographical and environmental differences between countries.

  20. KRAS mutation testing in clinical practice.

    PubMed

    Perincheri, Sudhir; Hui, Pei

    2015-03-01

    Activating mutation of KRAS plays a significant role in the pathogenesis of common human malignancies and molecular testing of KRAS mutation has emerged as an essential biomarker in the current practice of clinical oncology. The presence of KRAS mutation is generally associated with clinical aggressiveness of the cancer and reduced survival of the patient. Therapeutically, KRAS mutation testing has maximum utility in stratifying metastatic colorectal carcinoma and lung cancer patients for treatment with targeted therapy. Diagnostically, KRAS mutation testing is useful in the workup of pancreaticobiliary and thyroid cancers, particularly using cytological specimens. In the era of precision medicine, the role of KRAS mutation testing is poised to expand, likely in a setting of combinatorial therapeutic strategy and requiring additional mutation testing of its upstream and/or downstream effectors.

  1. 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.

  2. 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. PMID:27225938

  3. 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

  4. Heterogeneity of KRAS Mutation Status in Rectal Cancer

    PubMed Central

    Jo, Peter; König, Alexander; Schirmer, Markus; Kitz, Julia; Conradi, Lena-Christin; Azizian, Azadeh; Bernhardt, Markus; Wolff, Hendrik A.; Grade, Marian; Ghadimi, Michael; Ströbel, Philipp; Schildhaus, Hans-Ulrich; Gaedcke, Jochen

    2016-01-01

    Introduction Anti-EGFR targeted therapy is of increasing importance in advanced colorectal cancer and prior KRAS mutation testing is mandatory for therapy. However, at which occasions this should be performed is still under debate. We aimed to assess in patients with locally advanced rectal cancer whether there is intra-specimen KRAS heterogeneity prior to and upon preoperative chemoradiotherapy (CRT), and if there are any changes in KRAS mutation status due to this intervention. Materials and Methods KRAS mutation status analyses were performed in 199 tumor samples from 47 patients with rectal cancer. To evaluate the heterogeneity between different tumor areas within the same tumor prior to preoperative CRT, 114 biopsies from 34 patients (mean 3 biopsies per patient) were analyzed (pre-therapeutic intratumoral heterogeneity). For the assessment of heterogeneity after CRT residual tumor tissue (85 samples) from 12 patients (mean 4.2 tissue samples per patient) were analyzed (post-therapeutic intratumoral heterogeneity) and assessment of heterogeneity before and after CRT was evaluated in corresponding patient samples (interventional heterogeneity). Primer extension method (SNaPshot™) was used for initial KRAS mutation status testing for Codon 12, 13, 61, and 146. Discordant results by this method were reevaluated by using the FDA-approved KRAS Pyro Kit 24, V1 and the RAS Extension Pyro Kit 24, V1 Kit (therascreen® KRAS test). Results For 20 (43%) out of the 47 patients, a KRAS mutation was detected. With 12 out of 20, the majority of these mutations affected codon 35. We did not obtained evidence that CRT results in changes of the KRAS mutation pattern. In addition, no intratumoral heterogeneity in the KRAS mutational status could be proven. This was true for both the biopsies prior to CRT and the resection specimens thereafter. The discrepancy observed in some samples when using the SNaPshot™ assay was due to insufficient sensitivity of this technique upon

  5. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    PubMed Central

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  6. KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance.

    PubMed

    Miglio, Umberto; Oldani, Alberto; Mezzapelle, Rosanna; Veggiani, Claudia; Paganotti, Alessia; Garavoglia, Marcello; Boldorini, Renzo

    2014-05-01

    Mutations of KRAS are detectable in 70-90% of pancreatic duct adenocarcinomas (PDAC), using direct sequencing. We used a highly sensitive molecular method in order to investigate: (a) the frequency and prognostic significance of different KRAS mutations and, (b) whether the presence of KRAS mutations in histologically-negative resection margins of PDAC could explain local tumor recurrence after surgery. Twenty-seven patients with histologic diagnosis of PDAC, radical pancreaticoduodenectomy and histologically-negative margins were evaluated. KRAS mutations were searched for mutant-enriched PCR in tumor and negative resection margins. KRAS mutations were detected in 85.2% of the cases; the most frequent mutation was G12D (48.1%). Shorter OS was found in patients with G12D (25 months; 95% CI, 20.5-29.5), vs patients with other mutations (31.5 months; 95% CI, 25.6-37.1) (N.S.). KRAS mutation in histologically-negative margins was detected in one patient who died of locoregional recurrence; six patients had tumor recurrence but no mutations in surgical margins. The high frequency of KRAS mutations suggests a search for KRAS status to improve the diagnosis in suspected cases; the G12D mutation could be related to poor prognosis, but without statistical significance. No correlation was found between the frequency of cancer recurrence and KRAS mutations in surgical margins.

  7. 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

  8. Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2016-02-21

    We have examined the in situ detection of a 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 the in situ mutant (MT) DNA detection specificity against the wild type (WT), detection was carried out in a flow with a flow rate of 4 mL min(-1) 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 per mL analytical sensitivity in a background of clinically-relevant 1000-fold more WT in 30 min without DNA isolation, amplification, or labeling. For validation, this 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

  9. PCR bias toward the wild-type k-ras and p53 sequences: implications for PCR detection of mutations and cancer diagnosis.

    PubMed

    Barnard, R; Futo, V; Pecheniuk, N; Slattery, M; Walsh, T

    1998-10-01

    PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work on factor IX suggests that this assumption is invalid for one case of near-sequence identity. To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For k-ras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V, cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants. PMID:9793653

  10. Monitoring KRAS mutations in circulating DNA and tumor cells using digital droplet PCR during treatment of KRAS-mutated lung adenocarcinoma.

    PubMed

    Guibert, Nicolas; Pradines, Anne; Farella, Magali; Casanova, Anne; Gouin, Sandrine; Keller, Laura; Favre, Gilles; Mazieres, Julien

    2016-10-01

    Liquid biopsies are a new non-invasive strategy to detect and monitor the biology of non-small-cell lung cancer (NSCLC) in the era of personalized medicine. KRAS is an oncogenic driver that is mutated in 30% of NSCLCs and is associated with a poor prognosis. 62 samples from 32 patients, treated for metastatic KRAS-mutated lung adenocarcinoma, had DNA extracted from plasma and circulating tumor cells (CTCs) prospectively tested for the presence of KRAS mutations using droplet digital PCR. A KRAS mutation was detected in 82% of patients. Sensitivity was 78% for circulating free DNA (cfDNA) and 34% for CTCs. The presence of a KRAS mutation in cfDNA was correlated with a poor response to chemotherapy or targeted therapy. When a KRAS-mutated-DNA was detected and then monitored in cfDNA, its variation during targeted or conventional therapy was correlated with response, according to RECIST criteria, in 87.5% of cases (n=14/16), whereas this correlation was observed in 37.5% of cases for CTCs (n=3/8). We report the usefulness of using digital droplet PCR on liquid biopsies to predict and monitor responses to treatment of KRAS-mutated lung adenocarcinoma. ctDNA was much more sensitive than CTCs in this context. PMID:27597273

  11. K-ras gene mutation in gall bladder carcinomas and dysplasia.

    PubMed Central

    Ajiki, T; Fujimori, T; Onoyama, H; Yamamoto, M; Kitazawa, S; Maeda, S; Saitoh, Y

    1996-01-01

    Epithelial dysplasia of gall bladder is an important precancerous lesion of gall bladder carcinogenesis. To investigate the frequency of K-ras gene mutation in gall bladder carcinoma and dysplasia, K-ras codon 12 mutations were investigated by the polymerase chain reaction/restriction enzyme based method following direct sequencing. Mutation was detected in 59% (30 of 51) of gall bladder carcinomas, in 73% (8 of 11) of gall bladder dysplasia in gall stone cases, and in 0% of the normal gall bladder epithelium. There was, however, no correlation between K-ras mutation and clinicopathological factors of gall bladder carcinoma. K-ras gene mutation occurs even in gall bladder dysplasia at an incidence similar to that in carcinomas, suggesting that testing for K-ras gene mutation may prove useful as an adjunct to bile cytological or biopsy analysis. Images Figure 1 Figure 2 Figure 3 PMID:8675098

  12. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    PubMed

    Cai, Xin; Sheng, Jianhui; Tang, Chuanning; Nandakumar, Vijayalakshmi; Ye, Hua; Ji, Hong; Tang, Haiying; Qin, Yu; Guan, Hongwei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Wu, Taihua; Lin, Hongli

    2014-01-01

    Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations. PMID:24760004

  13. KRAS Mutation in Stage III Colon Cancer and Clinical Outcome Following Intergroup Trial CALGB 89803

    PubMed Central

    Ogino, Shuji; Meyerhardt, Jeffrey A.; Irahara, Natsumi; Niedzwiecki, Donna; Hollis, Donna; Saltz, Leonard B.; Mayer, Robert J.; Schaefer, Paul; Whittom, Renaud; Hantel, Alexander; Benson, Al B.; Goldberg, Richard M.; Bertagnolli, Monica M.; Fuchs, Charles S.

    2009-01-01

    Purpose Alterations in the RAS and RAF pathway relate to epigenetic and epigenomic aberrations, and are important in colorectal carcinogenesis. KRAS mutation in metastatic colorectal cancer predicts resistance to anti-EGFR targeted therapy (cetuximab or panitumumab). However, it remains uncertain whether KRAS mutation predicts prognosis or clinical outcome of colon cancer patients independent of anti-EGFR therapy. Methods We conducted a study of 508 cases identified among 1264 patients with stage III colon cancer who enrolled in a randomized adjuvant chemotherapy trial (5-fluorouracil, leucovorin with or without irinotecan) in 1999–2001 (CALGB 89803). KRAS mutations were detected in 178 tumors (35%) by Pyrosequencing. Kaplan-Meier and Cox proportional hazard models assessed the prognostic significance of KRAS mutation and adjusted for potential confounders including age, sex, tumor location, tumor/node stage, performance status, adjuvant chemotherapy arm and microsatellite instability (MSI) status. Results Compared to patients with KRAS-wild-type tumors, patients with KRAS-mutated tumors did not experience any difference in disease-free (DFS), recurrence-free (RFS), or overall survival (OS). Five-year DFS, RFS and OS (KRAS-mutated vs. KRAS-wild-type patients) were: 62% vs. 63% (log-rank p=0.89); 64% vs. 66% (p=0.84); and 75% vs. 73% (p=0.56), respectively. The effect of KRAS mutation on patient survival did not significantly differ according to clinical features, chemotherapy arm or MSI status, and the effect of adjuvant chemotherapy assignment on outcome did not differ according to KRAS status. Conclusions In this large trial of chemotherapy in stage III colon cancer patients, KRAS mutational status was not associated with any significant influence on disease-free or overall survival. PMID:19934290

  14. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

    PubMed Central

    Seekhuntod, Sirirat; Thavarungkul, Paninee; Chaichanawongsaroj, Nuntaree

    2016-01-01

    Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

  15. KRAS (but not BRAF) mutations in ovarian serous borderline tumor are associated with recurrent low-grade serous carcinoma

    PubMed Central

    Tsang, Yvonne T.; Deavers, Michael T.; Sun, Charlotte C.; Kwan, Suet-Yan; Kuo, Eric; Malpica, Anais; Mok, Samuel C.; Gershenson, David M.; Wong, Kwong-Kwok

    2014-01-01

    BRAF and KRAS mutations in ovarian serous borderline tumors (OSBTs) and ovarian low-grade serous carcinomas (LGSCs) have been previously described. However, whether those OSBTs would progress to LGSCs or those LGSCs were developed from OSBT precursors in previous studies is unknown. Therefore, we assessed KRAS and BRAF mutations in tumor samples from 23 recurrent LGSC patients with known initial diagnosis of OSBT. Paraffin blocks from both OSBT and LGSC samples were available for 5 patients, and either OSBT or LGSC were available for another 18 patients. Tumor cells from paraffin-embedded tissues were dissected out for mutation analysis by conventional polymerase chain reaction (PCR) and Sanger sequencing. Tumors that appeared to have wild-type KRAS by conventional PCR–Sanger sequencing were further analyzed by full COLD (coamplification at lower denaturation temperature)-PCR and deep sequencing. Full COLD-PCR was able to enrich the amplification of mutated alleles. Deep sequencing was performed with the Ion Torrent personal genome machine (PGM). By conventional PCR–Sanger sequencing, BRAF mutation was detected only in one patient and KRAS mutations were detected in 10 patients. Full COLD-PCR deep sequencing detected low-abundance KRAS mutations in eight additional patients. Three of the five patients with both OSBT and LGSC samples available had the same KRAS mutations detected in both OSBT and LGSC samples. The remaining two patients had only KRAS mutations detected in their LGSC samples. For patients with either OSBT or LGSC samples available, KRAS mutations were detected in 7 OSBT samples and 6 LGSC samples. To our surprise, patients with the KRAS G12V mutation appeared to have shorter survival times. In summary, KRAS mutations are very common in recurrent LGSC, while BRAF mutations are rare. The findings indicate that recurrent LGSC can arise from proliferation of OSBT tumor cells with or without detectable KRAS mutations. PMID:24549645

  16. KRAS Mutations in Canine and Feline Pancreatic Acinar Cell Carcinoma.

    PubMed

    Crozier, C; Wood, G A; Foster, R A; Stasi, S; Liu, J H W; Bartlett, J M S; Coomber, B L; Sabine, V S

    2016-07-01

    Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma.

  17. Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

    PubMed

    Zopf, Agnes; Raim, Roman; Danzer, Martin; Niklas, Norbert; Spilka, Rita; Pröll, Johannes; Gabriel, Christian; Nechansky, Andreas; Roucka, Markus

    2015-03-01

    The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

  18. Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues.

    PubMed

    van Eijk, Ronald; van Puijenbroek, Marjo; Chhatta, Amiet R; Gupta, Nisha; Vossen, Rolf H A M; Lips, Esther H; Cleton-Jansen, Anne-Marie; Morreau, Hans; van Wezel, Tom

    2010-01-01

    Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.

  19. Complete surgical resection of lung tumor decreases exhalation of mutated KRAS oncogene.

    PubMed

    Kordiak, Jacek; Szemraj, Janusz; Hamara, Katarzyna; Bialasiewicz, Piotr; Nowak, Dariusz

    2012-09-01

    Exhaled breath condensate (EBC) contains extracellular DNA that may originate from pathological lesions of the respiratory tract and can be a genetic marker of pulmonary malignancy. We tested whether complete surgical excision of lung cancer will decrease exhalation of mutated KRAS oncogene. Fifty seven patients with clinical diagnosis of lung cancer and detectable KRAS mutations in pre-surgery EBC-DNA were qualified for surgical treatment. Point mutations at codon 12 of KRAS oncogene were detected using mutant-enriched PCR technique in DNA from pre-surgery blood, EBC collected before, 7 and 30 days after surgery and from specimens of resected tumor and normal pulmonary parenchyma. The ratio of mutated to wild type KRAS DNA (R mut/wild KRAS) was calculated for each specimen after electrophoresis and densitometry of the final amplification and digestion product. In 46 patients non-small cell lung cancer (NSCLC) and in 11 benign lesion (BL) were confirmed. All blood and tumor specimens were positive for KRAS mutations, while 41 specimens of normal pulmonary parenchyma were negative. In NSCLC patients pre-surgery EBC R mut/wild KRAS of 0.20 ± 0.03 decreased by 1.3- and 3.7-times (p < 0.001) at 7th and 30th day and 10 EBC specimens at day 30th became negative. The highest R mut/wild KRAS was found in NSCLC specimens - 1.36 ± 0.29 while the lowest in pulmonary parenchyma - 0.02 ± 0.03 (p < 0.001). R mut/wild KRAS in EBC did not correlate with the blood and cancer ratios. Determination of mutated KRAS oncogene in EBC can be potentially helpful in the follow-up of surgical treatment of pulmonary malignancy. PMID:22795503

  20. 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

  1. Extended KRAS and NRAS Mutation Profiling by Pyrosequencing®.

    PubMed

    Jung, Andreas

    2015-01-01

    With the advent of targeted therapies-drugs that specifically target molecules of tumor-driving signalling pathways-and the availability of biomarkers that predict the response of an individual patient on such a targeted therapy, the analysis of the status of the biomarker became an integral part of the therapy. For metastatic colorectal cancer, anti-EGFR-targeted antibodies (Cetuximab/Erbitux(®), Panitumumab/Vectibix(®)) fall into this category of drugs as it was shown in several clinical studies that oncogenic mutations in exons 2-4 of the RAS genes KRAS or NRAS result in therapeutic resistance of the metastatic colorectal cancers against the action of both targeted drugs. Therefore, mutations in the RAS genes exclude patients from this kind of targeted therapy (negative biomarker). Thus the molecular-pathological testing of the mutational status of KRAS and NRAS has become an important cornerstone in planning oncological strategies in the treatment of metastatic colorectal cancer. As the profile of mutations in the RAS genes is characterized by hotspot mutations in only a small number of codons (12, 13 in exon 2-59, 61 in exon 3-117, 146 in exon 4). Pyrosequencing(®) is an ideal and robust tool in the molecular-pathological detection. A detailed protocol for this detection procedure employing Pyrosequencing® is given here.

  2. Mutation status of somatic EGFR and KRAS genes in Chinese patients with prostate cancer (PCa).

    PubMed

    Fu, Meng; Zhang, Wei; Shan, Ling; Song, Jian; Shang, Donghao; Ying, Jianming; Zhao, Jimao

    2014-05-01

    Activating mutations of the epidermal growth factor receptor (EGFR) confers sensitivity to tyrosine kinase inhibitors (TKIs). In colorectal cancer and in lung adenocarcinomas, clinical trials have shown a lack of response to anti-EGFR therapy when KRAS gene mutations are present. In this study, the mutation status of specified exons of the EGFR and KRAS genes was profiled in patients with prostate cancer (PCa). Direct Sanger sequencing was used to screen for mutations in exons 19-21 of EGFR and in exon 2 of KRAS in 88 Chinese patients diagnosed with prostate adenocarcinomas. Mutations were detected in 11 patients. In nine cases (10 %), activating mutations in the region of EGFR encoding the tyrosine kinase (TK) domain were present. Deletions in exon 19 and the L858R substitution in exon 21 were "hotspot" mutations, together accounting for five (55 %) of nine cases. Many synonymous substitutions were also detected. KRAS mutations were found in two cases (2.3 % of 88). There were no cases with mutations in both EGFR and KRAS, suggesting that mutations in the two genes might be mutually exclusive. Although prognostic relevance of EGFR expression by immunohistochemistry (IHC) was observed in PCa patients in previous studies, we found no statistically significant association between EGFR or KRAS mutations and clinicopathological features (including age, smoking status, preoperative prostate-specific antigen, Gleason scores, and tumor stage). We contend that accurate profiling of the mutation status of EGFR and KRAS could improve prognostic stratification, and we suggest a potential anti-EGFR therapy for patients with PCa with EGFR mutations. PMID:24595526

  3. 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

  4. Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer

    PubMed Central

    2010-01-01

    Background Mutational analysis of the KRAS gene has recently been established as a complementary in vitro diagnostic tool for the identification of patients with colorectal cancer who will not benefit from anti-epidermal growth factor receptor (EGFR) therapies. Assessment of the mutation status of KRAS might also be of potential relevance in other EGFR-overexpressing tumors, such as those occurring in breast cancer. Although KRAS is mutated in only a minor fraction of breast tumors (5%), about 60% of the basal-like subtype express EGFR and, therefore could be targeted by EGFR inhibitors. We aimed to study the mutation frequency of KRAS in that subtype of breast tumors to provide a molecular basis for the evaluation of anti-EGFR therapies. Methods Total, genomic DNA was obtained from a group of 35 formalin-fixed paraffin-embedded, triple-negative breast tumor samples. Among these, 77.1% (27/35) were defined as basal-like by immunostaining specific for the established surrogate markers cytokeratin (CK) 5/6 and/or EGFR. KRAS mutational status was determined in the purified DNA samples by Real Time (RT)-PCR using primers specific for the detection of wild-type KRAS or the following seven oncogenic somatic mutations: Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val and Gly13Asp. Results We found no evidence of KRAS oncogenic mutations in all analyzed tumors. Conclusions This study indicates that KRAS mutations are very infrequent in triple-negative breast tumors and that EGFR inhibitors may be of potential benefit in the treatment of basal-like breast tumors, which overexpress EGFR in about 60% of all cases. PMID:20385028

  5. Performance of a novel KRAS mutation assay for formalin-fixed paraffin embedded tissues of colorectal cancer.

    PubMed

    Sakai, Kazuko; Yoneshige, Azusa; Ito, Akihiko; Ueda, Yoji; Kondo, Satoshi; Nobumasa, Hitoshi; Fujita, Yoshihiko; Togashi, Yosuke; Terashima, Masato; De Velasco, Marco A; Tomida, Shuta; Nishio, Kazuto

    2015-01-01

    We compared the performance of the 3D-Gene® mutation assay (3D-Gene® KRAS mutation assay kit) with the Scorpion-ARMS (therascreen® KRAS RGQ PCR Kit) and Luminex (MEBGEN™ KRAS kit) assays for the detection of KRAS mutations in formalin-fixed, paraffin-embedded tissue samples from 150 patients diagnosed with colorectal cancer. DNA was extracted from the paraffin-embedded tissue samples with or without macrodissection under hematoxylin and eosin staining and the KRAS mutation status was independently determined using these assays. Discordant results were re-analyzed by Sanger sequencing. Mutation detection analysis was successfully performed in all 150 specimens using the 3D-Gene® mutation assay without an invalid case. The concordance rate between the 3D-Gene® mutation assay and Scorpion-ARMS or Luminex was 98.7% (148/150). KRAS mutations were detected at a frequency of 35.3% (53/150) in colorectal cancer specimens. Three discrepant cases were found between the three assays. Overall, our results demonstrate a high concordance rate of between the 3D-Gene® mutation assay and the two existing in-vitro diagnostics kits. All three assays proved to be validated methods for detecting clinically significant KRAS mutations in paraffin-embedded tissue samples. PMID:25674493

  6. 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

  7. 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

  8. 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.

  9. Mutational analyses of the BRAF, KRAS, and PIK3CA genes in oral squamous cell carcinoma

    PubMed Central

    Bruckman, Karl C.; Schönleben, Frank; Qiu, Wanglong; Woo, Victoria L.; Su, Gloria H.

    2010-01-01

    OBJECTIVES The development of oral squamous cell carcinoma (OSCC) is a complex, multistep process. To date, numerous oncogenes and tumor-suppressor genes have been implicated in oral carcinogenesis. Of particular interest in this regard are genes involved in cell cycling and apoptosis, such BRAF, KRAS, and PIK3CA genes. STUDY DESIGN Mutations of BRAF, KRAS, and PIK3CA were evaluated by direct genomic sequencing of exons 1 of KRAS, 11 and 15 of BRAF, and 9 and 20 of PIK3CA in OSCC specimens. RESULTS Both BRAF and KRAS mutations were detected with a mutation frequency of 2% (1/42). PIK3CA mutations were detected at 3% (1/35). CONCLUSIONS This is the first report implicating BRAF mutation in OSCC. Our study supports that mutations in the BRAF, KRAS, and PIK3CA genes make at least a minor contribution to OSCC tumorigenesis, and pathway-specific therapies targeting these two pathways should be considered for OSCC in a subset of patients with these mutations. PMID:20813562

  10. Molecular spectrum of KRAS, BRAF, and PIK3CA gene mutation: determination of frequency, distribution pattern in Indian colorectal carcinoma.

    PubMed

    Bisht, Swati; Ahmad, Firoz; Sawaimoon, Satyakam; Bhatia, Simi; Das, Bibhu Ranjan

    2014-09-01

    Molecular evaluation of KRAS, BRAF, and PIK3CA mutation has become an important part in colorectal carcinoma evaluation, and their alterations may determine the therapeutic response to anti-EGFR therapy. The current study demonstrates the evaluation of KRAS, BRAF, and PIK3CA mutation using direct sequencing in 204 samples. The frequency of KRAS, BRAF, and PIK3CA mutations was 23.5, 9.8, and 5.9 %, respectively. Five different substitution mutations at KRAS codon 12 (G12S, G12D, G12A, G12V, and G12C) and one substitution type at codon 13 (G13D) were observed. KRAS mutations were significantly higher in patients who were >50 years, and were associated with moderate/poorly differentiated tumors and adenocarcinomas. All mutations in BRAF gene were of V600E type, which were frequent in patients who were ≤ 50 years. Unlike KRAS mutations, BRAF mutations were more frequent in well-differentiated tumors and right-sided tumors. PIK3CA-E545K was the most recurrent mutation while other mutations detected were T544I, Q546R, H1047R, G1049S, and D1056N. No significant association of PIK3CA mutation with age, tumor differentiation, location, and other parameters was noted. No concomitant mutation of KRAS and BRAF mutations was observed, while, interestingly, five cases showed concurrent mutation of KRAS and PIK3CA mutations. In conclusion, to our knowledge, this is the first study to evaluate the PIK3CA mutation in Indian CRC patients. The frequency of KRAS, BRAF, and PIK3CA was similar to worldwide reports. Furthermore, identification of molecular markers has unique strengths, and can provide insights into the pathogenic process and help optimize personalized prevention and therapy.

  11. [Research advances of K-ras mutation in the prognosis and targeted therapy of gastric cancer].

    PubMed

    Huang, Y; Wei, J; Liu, B R

    2016-02-01

    K-ras mutations have been described in 30% of human cancers with significantly different mutation frequencies. High K-ras mutation frequency is found in many cancers such as pancreas and lung cancers, whereas, gastric cancer has a relatively low K-ras mutation frequency. In recent years, numerous researches have focused on the K-ras mutation in gastric cancer. This review summarizes the K-ras mutation frequency in gastric cancer, the relationship of K-ras mutation with clinicopathologic features and prognosis of gastric cancer patients, targeted therapy for K-ras mutated gastric cancer, some small-molecular inhibitors of K-ras, and development of targeted therapy drugs for K-ras signaling pathway in gastric cancer.

  12. 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

  13. Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes.

    PubMed

    Wabuyele, Musundi B; Farquar, Hannah; Stryjewski, Wieslaw; Hammer, Robert P; Soper, Steven A; Cheng, Yu-Wei; Barany, Francis

    2003-06-11

    The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.

  14. 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

  15. Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma.

    PubMed

    Lee, Sung; Brophy, Victoria H; Cao, Jianli; Velez, Margot; Hoeppner, Corey; Soviero, Stephen; Lawrence, H Jeffrey

    2012-02-01

    KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay-the cobas® KRAS Mutation Test-designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8-6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes.

  16. 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

  17. Detection of K-ras oncogene mutations & DNA adducts in the lungs of strain A/J mice exposed to benzo[b]fluoranthene (B[b]F)

    SciTech Connect

    Abu-Shakra, A.; Roop, B.C.; Nelson, G.

    1995-11-01

    The polycyclic aromatic hydrocarbon benzo[b]fluoranthene (B[b]F) has been shown in our laboratories to induce adenomas in strain A/J mouse lungs using i.p. doses above 50 mg/kg body weight. B[b]F appears to be less potent than benzo[a]pyrene on a mg/kg basis in this tumor model. We measured the formation of B[b]F-DNA adducts in mice exposed to B[b]F after 1-21 days and analyzed B[b]F-induced tumors for K-ras oncogene mutations approximately 8 months later. The major B[b]F-DNA adduct comigrated with an adduct seen after application of 5-hydroxy-B[b]F-9,10-dihydrodiol-11,12-oxide to mouse skin. Tumor DNA was extracted and amplified by the polymerase chain reaction (PCR) using primers flanking the 111 bp region of exon 1. Samples were sequenced using the dideoxy method; those samples that failed to show a clear sequence after repetitive sequencing were subjected to single stranded conformation polymorphism analysis (SSCP). B[b]F-induced tumors with K-ras mutations in codon 12, had the following distribution: GGT {yields}GTT, 50%; GGT{yields}TGT, 40%; GGT{yields} 10%. Further characterization of these mutations and their relationship to B[b]F-DNA adducts is in progress

  18. 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.

  19. Decreasing relapse in colorectal cancer patients treated with cetuximab by using the activating KRAS detection chip.

    PubMed

    Huang, Ming-Yii; Liu, Hsueh-Chiao; Yen, Li-Chen; Chang, Jia-Yuan; Huang, Jian-Jhang; Wang, Jaw-Yuan; Lin, Shiu-Ru

    2014-10-01

    The KRAS oncogene was among the first genetic alterations in colorectal cancer (CRC) to be discovered. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-epidermal growth factor receptor therapeutic drugs. Because the KRAS mutations are similar in the primary CRC and/or the CRC metastasis, KRAS mutation testing can be performed on both specimen types. The purpose of this study was to investigate the clinical advantage of using a KRAS pathway-associated molecule analysis chip to analyze CRC patients treated with cetuximab. Our laboratory developed a KRAS pathway-associated molecule analysis chip and a weighted enzymatic chip array (WEnCA) technique, activating KRAS detection chip, which can detect KRAS mutation status by screening circulating cancer cells in the bloodstream. We prospectively enrolled 210 stage II-III CRC patients who received adjuvant oxaliplatin plus infusional 5-fluorouracil/leucovorin (FOLFOX)-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens with disease control status in these patients. Among the 168 CRC patients with negative chip results, 119 were treated with FOLFOX-4 plus cetuximab chemotherapy, and their relapse rate was 35.3 % (42/119). In contrast, the relapse rate was 71.4 % among the patients with negative chip results who received FOLFOX-4 treatment alone (35/49). Negative chip results were significantly correlated with better treatment outcomes in the FOLFOX-4 plus cetuximab group (P < 0.001). We suggest that the activating KRAS detection chip is a potential tool for predicting clinical outcomes in CRC patients following FOLFOX-4 treatment with or without cetuximab therapy.

  20. 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.

  1. Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells

    PubMed Central

    Yun, Jihye; Rago, Carlo; Cheong, Ian; Pagliarini, Ray; Angenendt, Philipp; Rajagopalan, Harith; Schmidt, Kerstin; Wilson, James K. V.; Markowitz, Sandy; Zhou, Shibin; Diaz, Luis A.; Velculescu, Victor; Lengauer, Christoph; Kinzler, Kenneth W.; Vogelstein, Bert; Papadopoulos, Nickolas

    2010-01-01

    Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently upregulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1 and 4% of these survivors had acquired new KRAS mutations. The glycolysis inhibitor, 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors. PMID:19661383

  2. Clinicopathologic and prognostic associations of KRAS and BRAF mutations in small intestinal adenocarcinoma.

    PubMed

    Jun, Sun-Young; Kim, Misung; Jin Gu, Mi; Kyung Bae, Young; Chang, Hee-Kyung; Sun Jung, Eun; Jang, Kee-Taek; Kim, Jihun; Yu, Eunsil; Woon Eom, Dae; Hong, Seung-Mo

    2016-04-01

    Activating KRAS and/or BRAF mutations have been identified as predictors of resistance to anti-epidermal growth factor receptor (EGFR) chemotherapy in colorectal cancer. But the status of KRAS and BRAF mutations and their clinicopathologic and prognostic significance has not been extensively evaluated in small intestinal adenocarcinomas. In this work, the KRAS and BRAF genes in 190 surgically resected small intestinal adenocarcinoma cases were sequenced and their association with various clinicopathologic variables, including survival of the patients, was analyzed. KRAS or BRAF mutations were observed in 63 (33%) cases. Sixty-one cases had KRAS mutations and 2 had BRAF mutations and the two types of mutation were mutually exclusive. The majority of KRAS mutations were G>A transition (43/61 cases, 71%) or p.G12D (31/61 cases, 51%). The patients with mutant KRAS tended to have higher pT classifications (P=0.034) and more frequent pancreatic invasion (P=0.020) than those with wild-type KRAS. Multivariate logistic regression analysis showed that certain mutated KRAS subtypes (G>A transitions and G12D mutations) were significantly correlated with higher pT classification (P=0.015 and 0.004, respectively) than wild-type KRAS and other KRAS mutations. The patients with KRAS or BRAF mutation had a tendency to shorter overall survival than those with wild-type KRAS and BRAF (P=0.148), but subgroup analysis demonstrated the patients with KRAS mutations showed worse survival (median, 46.0 months; P=0.046) than those with wild-type KRAS (85.4 months) in lower pT classification (pT1-pT3) group. In summary, KRAS and, infrequently, BRAF mutations are observed in a subset of small intestinal adenocarcinomas, and are associated with higher pT classification and more frequent pancreatic invasion. KRAS mutation is a poor prognostic predictor in patients with lower pT classification tumors. Anti-EGFR targeted therapy could be applied to about two-thirds of small intestinal

  3. KRAS and GNAS Mutations in Pancreatic Juice Collected From the Duodenum of Patients at High Risk for Neoplasia Undergoing Endoscopic Ultrasound

    PubMed Central

    Eshleman, James R.; Norris, Alexis L.; Sadakari, Yoshihiko; Debeljak, Marija; Borges, Michael; Harrington, Colleen; Lin, Elaine; Brant, Aaron; Barkley, Thomas; Almario, J. Alejandro; Topazian, Mark; Farrell, James; Syngal, Sapna; Lee, Jeffrey H.; Yu, Jun; Hruban, Ralph H.; Kanda, Mitsuro; Canto, Marcia Irene; Goggins, Michael

    2014-01-01

    BACKGROUND & AIMS Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid following secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and GNAS genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia (PanIN), intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. METHODS Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer and 78 were evaluated for pancreatic cancer (n=30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n=48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. RESULTS KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P=.0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P=.002). Among screened subjects, mutations in KRAS (but not GNAS) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients over 50 ys old had KRAS mutations (54.6%) than younger patients (36.3%) (P=.032); the older subjects also more mutations in KRAS (P=.02). CONCLUSIONS Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations are detected in pancreatic juice

  4. Notch1 Gene Mutations Target KRAS G12D-expressing CD8+ Cells and Contribute to Their Leukemogenic Transformation*

    PubMed Central

    Kong, Guangyao; Du, Juan; Liu, Yangang; Meline, Benjamin; Chang, Yuan-I; Ranheim, Erik A.; Wang, Jinyong; Zhang, Jing

    2013-01-01

    Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8+ T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/β-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells. PMID:23673656

  5. Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients

    PubMed Central

    PAPADOPOULOU, EIRINI; TSOULOS, NIKOLAOS; TSIRIGOTI, ANGELIKI; APESSOS, ANGELA; AGIANNITOPOULOS, KONSTANTINOS; METAXA-MARIATOU, VASILIKI; ZAROGOULIDIS, KONSTANTINOS; ZAROGOULIDIS, PAVLOS; KASARAKIS, DIMITRIOS; KAKOLYRIS, STYLIANOS; DAHABREH, JUBRAIL; VLASTOS, FOTIS; ZOUBLIOS, CHARALAMPOS; RAPTI, AGGELIKI; PAPAGEORGIOU, NIKI GEORGATOU; VELDEKIS, DIMITRIOS; GAGA, MINA; ARAVANTINOS, GERASIMOS; KARAVASILIS, VASILEIOS; KARAGIANNIDIS, NAPOLEON; NASIOULAS, GEORGE

    2015-01-01

    It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of

  6. 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.

  7. Are KRAS/BRAF mutations potent prognostic and/or predictive biomarkers in colorectal cancers?

    PubMed

    Yokota, Tomoya

    2012-02-01

    KRAS and BRAF mutations lead to the constitutive activation of EGFR signaling through the oncogenic Ras/Raf/Mek/Erk pathway. Currently, KRAS is the only potential biomarker for predicting the efficacy of anti-EGFR monoclonal antibodies (mAb) in colorectal cancer (CRC). However, a recent report suggested that the use of cetuximab was associated with survival benefit among patients with p.G13D-mutated tumors. Furthermore, although the presence of mutated BRAF is one of the most powerful prognostic factors for advanced and recurrent CRC, it remains unknown whether patients with BRAF-mutated tumors experience a survival benefit from treatment with anti-EGFR mAb. Thus, the prognostic or predictive relevance of the KRAS and BRAF genotype in CRC remains controversial despite several investigations. Routine KRAS/BRAF screening of pathological specimens is required to promote the appropriate clinical use of anti-EGFR mAb and to determine malignant phenotypes in CRC. The significance of KRAS/BRAF mutations as predictive or prognostic biomarkers should be taken into consideration when selecting a KRAS/BRAF screening assay. This article will review the spectrum of KRAS/BRAF genotype and the impact of KRAS/BRAF mutations on the clinicopathological features and prognosis of patients with CRC, particularly when differentiating between the mutations at KRAS codons 12 and 13. Furthermore, the predictive role of KRAS/BRAF mutations in treatments with anti-EGFR mAb will be verified, focusing on KRAS p.G13D and BRAF mutations.

  8. KRAS Mutation in Small Cell Lung Carcinoma and Extrapulmonary Small Cell Cancer

    PubMed Central

    Kodaz, Hilmi; Taştekin, Ebru; Erdoğan, Bülent; Hacıbekiroğlu, İlhan; Tozkır, Hilmi; Gürkan, Hakan; Türkmen, Esma; Demirkan, Bora; Uzunoğlu, Sernaz; Çiçin, İrfan

    2016-01-01

    Background: Lung cancer is one of the most lethal cancers. It is mainly classified into 2 groups: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Extrapulmonary small cell carcinomas (EPSCC) are very rare. The Ras oncogene controls most of the cellular functions in the cell. Overall, 21.6% of human cancers contain a Kirsten Ras (KRAS) mutation. SCLC and EPSCC have several similar features but their clinical course is different. Aims: We investigated the KRAS mutation status in SCLC and EPSCC. Study design: Mutation research. Methods: Thirty-seven SCLC and 15 EPSCC patients were included in the study. The pathological diagnoses were confirmed by a second pathologist. KRAS analysis was performed in our medical genetic department. DNA isolation was performed with primary tumor tissue using the QIAamp DNA FFPE Tissue kit (Qiagen; Hilden, Germany) in all patients. The therascreen KRAS Pyro Kit 24 V1 (Qiagen; Hilden, Germany) was used for KRAS analyses. Results: Thirty-four (91.9%) of the SCLC patients were male, while 11 (73.3%) of the EPSCC l patients were female. SCLC was more common in males, and EPSCC in females (p=0.001). A KRAS mutation was found in 6 (16.2%) if SCLC patients. The most common mutation was Q61R (CAA>CGA). Among the 15 EPSCC patients, 2 had a KRAS mutation (13.3%). When KRAS mutant and wild type patients were compared in the SCLC group, no difference was found for overall survival (p=0.6). Conclusion: In previous studies, the incidence of KRAS mutation in SCLC was 1–3%; however, it was 16.2% in our study. Therefore, there may be ethnic and geographical differences in the KRAS mutations of SCLC. As a result, KRAS mutation should not be excluded in SCLC.

  9. 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

  10. 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.

  11. Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features.

    PubMed

    Rosty, Christophe; Young, Joanne P; Walsh, Michael D; Clendenning, Mark; Walters, Rhiannon J; Pearson, Sally; Pavluk, Erika; Nagler, Belinda; Pakenas, David; Jass, Jeremy R; Jenkins, Mark A; Win, Aung Ko; Southey, Melissa C; Parry, Susan; Hopper, John L; Giles, Graham G; Williamson, Elizabeth; English, Dallas R; Buchanan, Daniel D

    2013-06-01

    KRAS-mutated carcinomas comprise 35-40% of all colorectal carcinomas but little is known about their characteristics. The aim of this study was to examine the pathological and molecular features of KRAS-mutated colorectal carcinomas and to compare them with other carcinoma subgroups. KRAS mutation testing was performed in 776 incident tumors from the Melbourne Collaborative Cohort Study. O(6)-methylguanine DNA methyltransferase (MGMT) status was assessed using both immunohistochemistry and MethyLight techniques. Microsatellite instability (MSI) phenotype and BRAF V600E mutation status were derived from earlier studies. Mutation in KRAS codon 12 or codon 13 was present in 28% of colorectal carcinomas. Compared with KRAS wild-type carcinomas, KRAS-mutated carcinomas were more frequently observed in contiguity with a residual polyp (38 vs 21%; P<0.001), demonstrated mucinous differentiation (46 vs 31%; P=0.001) and were associated with different MSI status (P<0.001) and with MGMT methylation (47 vs 21%; P=0.001). Compared with tumors demonstrating neither BRAF nor KRAS mutation, KRAS-mutated carcinomas showed more frequent location in the proximal colon (41 vs 27%; P=0.001), mucinous differentiation (46 vs 25%; P<0.001), presence of a contiguous polyp (38 vs 22%; P<0.001), MGMT methylation (47 vs 26%; P=0.01) and loss of MGMT immunohistochemical expression (27 vs 19%; P=0.02). KRAS-mutated carcinomas were distributed in a bimodal pattern along the proximal-distal axis of the colorectum. Compared with male subjects, female subjects were more likely to have KRAS-mutated carcinoma in the transverse colon and descending colon (39 vs 15%; P=0.02). No difference in overall survival was observed in patients according to their tumor KRAS mutation status. In summary, KRAS-mutated carcinomas frequently develop in contiguity with a residual polyp and show molecular features distinct from other colorectal carcinomas, in particular from tumors with neither BRAF nor KRAS mutation.

  12. Nitrative and oxidative DNA damage caused by K-ras mutation in mice

    SciTech Connect

    Ohnishi, Shiho; Saito, Hiromitsu; Suzuki, Noboru; Ma, Ning; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke

    2011-09-23

    Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

  13. BRAF, PIK3CA, and HER2 Oncogenic Alterations According to KRAS Mutation Status in Advanced Colorectal Cancers with Distant Metastasis

    PubMed Central

    Koh, Jiwon; Kwak, Yoonjin; Seo, An Na; Park, Kyoung Un; Kim, Duck-Woo; Kang, Sung-Bum; Kim, Woo Ho; Lee, Hye Seung

    2016-01-01

    Background Anti-EGFR antibody–based treatment is an important therapeutic strategy for advanced colorectal cancer (CRC); despite this, several mutations—including KRAS, BRAF, and PIK3CA mutations, and HER2 amplification—are associated with the mechanisms underlying the development of resistance to anti-EGFR therapy. The aim of our study was to investigate the frequencies and clinical implications of these genetic alterations in advanced CRC. Methods KRAS, BRAF, and PIK3CA mutations were determined by Cobas real-time polymerase chain reaction (PCR) in 191 advanced CRC patients with distant metastasis. Microsatellite instability (MSI) status was determined by a fragmentation assay and HER2 amplification was assessed by silver in situ hybridization. In addition, KRAS mutations were investigated by the Sanger sequencing method in 97 of 191 CRC cases. Results Mutations in KRAS, BRAF, and PIK3CA were found in 104 (54.5%), 6 (3.1%), and 25 (13.1%) cases of advanced CRC, respectively. MSI-high status and HER2 amplification were observed in 3 (1.6%) and 16 (8.4%) cases, respectively. PIK3CA mutations were more frequently found in KRAS mutant type (18.3%) than KRAS wild type (6.9%) (P = 0.020). In contrast, HER2 amplifications and BRAF mutations were associated with KRAS wild type with borderline significance (P = 0.052 and 0.094, respectively). In combined analyses with KRAS, BRAF and HER2 status, BRAF mutations or HER2 amplifications were associated with the worst prognosis in the wild type KRAS group (P = 0.004). When comparing the efficacy of detection methods, the results of real time PCR analysis revealed 56 of 97 (57.7%) CRC cases with KRAS mutations, whereas Sanger sequencing revealed 49 cases (50.5%). Conclusions KRAS mutations were found in 54.5% of advanced CRC patients. Our results support that subgrouping using PIK3CA and BRAF mutation or HER2 amplification status, in addition to KRAS mutation status, is helpful for managing advanced CRC patients. PMID

  14. Distribution of KRAS and BRAF mutations in Moroccan patients with advanced colorectal cancer.

    PubMed

    Marchoudi, N; Amrani Hassani Joutei, H; Jouali, F; Fekkak, J; Rhaissi, H

    2013-12-01

    Targeted therapies have an increasing importance in digestive oncology. To our knowledge, we are the first to report the distribution of KRAS and BRAF mutations in Moroccan patients with advanced colorectal cancer (CRC) in order to introduce targeted therapy in the arsenal of therapeutic modalities for management of this cancer in Morocco. In this study, 92 samples obtained from patients with CRC were tested for the presence of the nine most common mutations in the KRAS gene and BRAF gene. Among the tested patients, 76.09% of patients had wt-KRAS genotype and 23.91% were KRAS mutants and the majority of mutations would result in an amino acid substitution of glycine by aspartic acid (68.2%) The predominant mutations are G>A transitions and G>T transversions. Around 5% (5.43%) of the tested patients bore the V600E mutation in BRAF gene. Only one patient showing to have the V600E mutation in BRAF was also mutated-KRAS. Summing up the results about the KRAS and the BRAF mutation carriers from our study, the portion of potentially non responsive patients for the anti-EGFR treatment is 28.26%.

  15. Mathematical modeling of drug resistance due to KRAS mutation in colorectal cancer.

    PubMed

    Sameen, Sheema; Barbuti, Roberto; Milazzo, Paolo; Cerone, Antonio; Del Re, Marzia; Danesi, Romano

    2016-01-21

    The most challenging task in colorectal cancer research nowadays is to understand the development of acquired resistance to anti-EGFR drugs. The key reason for this problem is the KRAS mutations appearance after the treatment with monoclonal antibodies (moAb). Here we present a mathematical model for the analysis of KRAS mutations behavior in colorectal cancer with respect to moAb treatments. To evaluate the drug performance we have developed equations for two types of tumors cells, KRAS mutated and KRAS wild-type. Both tumor cell populations were treated with a combination of moAb and chemotherapy drugs. It was observed that even the minimal initial concentration of KRAS mutation before the treatment has the ability to make the tumor refractory to the treatment. Minor population of KRAS mutations has strong influence on large number of wild-type cells as well rendering them resistant to chemotherapy. Patient׳s immune responses are specifically taken into considerations and it is found that, in case of KRAS mutations, the immune strength does not affect medication efficacy. Finally, cetuximab (moAb) and irinotecan (chemotherapy) drugs are analyzed as first-line treatment of colorectal cancer with few KRAS mutated cells. Results show that this combined treatment could be only effective for patients with high immune strengths and it should not be recommended as first-line therapy for patients with moderate immune strengths or weak immune systems because of a potential risk of relapse, with KRAS mutant cells acquired resistance involved with them. PMID:26551156

  16. 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.

  17. 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

  18. 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

  19. 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

  20. G12V Kras mutations in cervical cancer under virtual microscope of molecular dynamics simulations.

    PubMed

    Chen, X P; Xu, W H; Xu, D F; Fu, S M; Ma, Z C

    2016-01-01

    Kras mutations and cancers are common and their role in the progression of cancer is well known and elucidated. The present work is searching for the most deleterious mutation of the four found at codon 12 and 13 of Kras in cervical cancers using prediction servers; different servers were used to look into different factors that govern the protein function. The in silico results predicted G12V to be the most devastating; this particular mutation was then subjected to molecular dynamics simulation (MDS) for further analysis. The authors' approach of MDSs helped them to place the native and mutant structure under virtual microscope and observe their dynamics over time. The results generated are enlightening the effect of G12V variation on the dynamics of Kras. The structural variation between the native and mutant Kras over 50 nanoseconds (ns) run varied at every parameter checked and the results are in excellent agreement with the available experimental data. PMID:27048113

  1. 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

  2. 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.

  3. Impact of genetic profiles on the efficacy of anti-EGFR antibodies in metastatic colorectal cancer with KRAS mutation.

    PubMed

    Kishiki, Tomokazu; Ohnishi, Hiroaki; Masaki, Tadahiko; Ohtsuka, Kouki; Ohkura, Yasuo; Furuse, Jyunji; Sugiyama, Masanori; Watanabe, Takashi

    2014-07-01

    Reports indicate that, even in KRAS-mutated colon cancer, there are subsets of patients who benefit from anti-EGFR monoclonal antibody (MoAb) treatment. The aim of the present study was to identify genetic profiles that contribute to the responsiveness of metastatic colorectal cancer (mCRC) to anti-EGFR MoAb. We retrospectively evaluated the efficacy of anti-EGFR MoAb in mCRC patients with KRAS mutations according to KRAS mutational subtypes, BRAF and PIK3CA mutational status and PTEN and MET expression. Among 21 patients with KRAS-mutant tumors, 8 (38%) harbored p.G13D, 7 (33%) harbored p.G12V, 5 (24%) harbored p.G12D, and 1 (5%) harbored p.G12C mutation. Patients with the p.G13D mutation exhibited a significantly higher disease control rate than patients with other KRAS mutations (P=0.042), and tended to show a longer progression-free survival (PFS) than patients with other KRAS mutations with marginal significance (P=0.074). Patients with loss of PTEN had significantly shorter PFS than those with normal PTEN expression in patients with KRAS mutations (P=0.044). MET overexpression was significantly associated with shorter PFS compared to normal MET expression in patients with KRAS mutations (P=0.016). Our data demonstrated the potential utility of alterations in PTEN and MET expression as predictive markers for response to anti-EGFR MoAbs in mCRC patients with KRAS mutations. In addition, we confirmed the predictive value of the KRAS p.G13D mutation for better response to anti-EGFR therapies in comparison with other KRAS mutations. PMID:24839940

  4. 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.

  5. 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

  6. Cost-Effectiveness Analysis of Screening for KRAS and BRAF Mutations in Metastatic Colorectal Cancer

    PubMed Central

    2012-01-01

    Background In 2009, the American Society of Clinical Oncology recommended that patients with metastatic colorectal cancer (mCRC) who are candidates for anti-epidermal growth factor receptor (EGFR) therapy have their tumors tested for KRAS mutations because tumors with such mutations do not respond to anti-EGFR therapy. Limiting anti-EGFR therapy to those without KRAS mutations will reserve treatment for those likely to benefit while avoiding unnecessary costs and harm to those who would not. Similarly, tumors with BRAF genetic mutations may not respond to anti-EGFR therapy, though this is less clear. Economic analyses of mutation testing have not fully explored the roles of alternative therapies and resection of metastases. Methods This paper is based on a decision analytic framework that forms the basis of a cost-effectiveness analysis of screening for KRAS and BRAF mutations in mCRC in the context of treatment with cetuximab. A cohort of 50 000 patients with mCRC is simulated 10 000 times, with attributes randomly assigned on the basis of distributions from randomized controlled trials. Results Screening for both KRAS and BRAF mutations compared with the base strategy (of no anti-EGFR therapy) increases expected overall survival by 0.034 years at a cost of $22 033, yielding an incremental cost-effectiveness ratio of approximately $650 000 per additional year of life. Compared with anti-EGFR therapy without screening, adding KRAS testing saves approximately $7500 per patient; adding BRAF testing saves another $1023, with little reduction in expected survival. Conclusions Screening for KRAS and BFAF mutation improves the cost-effectiveness of anti-EGFR therapy, but the incremental cost effectiveness ratio remains above the generally accepted threshold for acceptable cost effectiveness ratio of $100 000/quality adjusted life year. PMID:23197490

  7. PIK3CA mutation uncouples tumor growth and Cyclin D1 regulation from MEK/ERK and mutant KRAS signaling

    PubMed Central

    Halilovic, Ensar; She, Qing-Bai; Ye, Qing; Pagliarini, Raymond; Sellers, William R.; Solit, David B.; Rosen, Neal

    2010-01-01

    Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small molecule MEK inhibitor, decreases MEK/ERK pathway signaling, and destabilizes Cyclin D1, resulting in significant anti-cancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores Cyclin D1 expression and G1/S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and PI3K mutations are unlikely to respond to inhibition of the MEK pathway alone but will require effective inhibition of both MEK and PI3K/AKT pathway signaling. PMID:20699365

  8. Comparison of KRAS/BRAF mutations between primary tumors and serum in colorectal cancer: Biological and clinical implications.

    PubMed

    Pu, Xingxiang; Pan, Zhizhong; Huang, Ying; Tian, Ying; Guo, Hongqiang; Wu, Lin; He, Xuexing; Chen, Xinggui; Zhang, Shaodan; Lin, Tongyu

    2013-01-01

    In colorectal cancer (CRC), KRAS and BRAF mutations in primary tumors are associated with resistance to anti-epidermal growth factor receptor (anti-EGFR)-based therapies. However, the correlation between KRAS/BRAF mutation in primary tumors and serum has not been well studied. To evaluate the degree of concordance of KRAS/BRAF mutations between the primary tumors and the matched serum samples in CRC, serum and tumor tissues were collected from 115 patients with CRC and KRAS/BRAF mutations were examined by nested polymerase chain reaction (PCR) and direct sequencing. BRAF mutations were present in 3.5% (4/115) of the primary tumor tissue samples and 0.87% (1/115) of the serum samples. In the 4 primary tumors with BRAF mutations, identical mutations were not observed in the corresponding serum samples (κ=-0.016). KRAS mutations were observed in 32.2% (37/115) of the primary tumors and 11.3% (13/115) of the serum samples. Of the 37 tumor cases with KRAS mutations, 9 had identical mutations in the corresponding serum sample, with a concordance rate of 24.3% (9/37). Discordance was observed in 32 (27.8%) patients. The concordance between KRAS mutations in the primary tumors and KRAS mutations in the matched serums was low (κ=0.231). The results of the present study suggest that the possibility of differences in the mutational status of KRAS/BRAF between primary tumors and matched serum samples should be considered when patients are selected for anti-EGFR-based therapies.

  9. Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer

    PubMed Central

    Guo, Kang; Wang, Wu-Ping; Jiang, Tao; Wang, Ju-Zheng; Chen, Zhao; Li, Yong; Zhou, Yong-An; Li, Xiao-Fei

    2016-01-01

    Background The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. Methods EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients’ survival. Results A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0

  10. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples.

    PubMed

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D'Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

  11. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    PubMed Central

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID

  12. Somatic c.34G>T KRAS mutation: a new prescreening test for MUTYH-associated polyposis?

    PubMed

    Aimé, Adeline; Coulet, Florence; Lefevre, Jeremie H; Colas, Chrystelle; Cervera, Pascale; Flejou, Jean-François; Lascols, Olivier; Soubrier, Florent; Parc, Yann

    2015-01-01

    We investigated the somatic c.34G>T KRAS transversion as a marker suggestive of MUTYH-associated polyposis (MAP). We compared 86 adenomas and 19 colorectal cancers (CRCs) of 30 MAP patients to 135 adenomas and five CRCs of 47 familial adenomatous polyposis (FAP) patients. The c.34G>T mutation was investigated by DNA sequencing. Secondly, the germline MUTYH gene sequence was analyzed in patients carrying c.34G>T in CRCs diagnosed between 2008 and 2012. The c.34G>T was present in 39.7% of MAP adenomas versus 1.6% of FAP adenomas (P < 0.01). Sensitivity and specificity for detecting MAP were 39.7% and 98%, respectively. Sensitivity increased with the number of adenomas tested (P = 0.039). KRAS exon 2 analysis was performed on 2239 CRC and 2.2% harbored the c.34G>T transversion. Among 28 carriers of the c.34G>T mutation, biallelic MUTYH mutations were detected in seven patients (25%). One patient did not have any polyp or family history and did not fulfill criteria for MUTYH testing. With high specificity, the c.34G>T mutation seems to be a useful and promising test for MAP. For polyposis, it may guide genetic testing toward APC or MUTYH. If routinely performed in CRC patients, it could help to diagnose MUTYH-mutation carriers, even when they don't fulfill genetic testing criteria. PMID:26056087

  13. Treatment Rationale and Study Design for the JUNIPER Study: A Randomized Phase III Study of Abemaciclib With Best Supportive Care Versus Erlotinib With Best Supportive Care in Patients With Stage IV Non-Small-Cell Lung Cancer With a Detectable KRAS Mutation Whose Disease Has Progressed After Platinum-Based Chemotherapy.

    PubMed

    Goldman, Jonathan W; Shi, Peipei; Reck, Martin; Paz-Ares, Luis; Koustenis, Andrew; Hurt, Karla C

    2016-01-01

    This clinical trial summary provides the background and rationale for the JUNIPER study (NCT02152631). JUNIPER is a randomized study of abemaciclib (200 mg orally every 12 hours) with best supportive care (BSC) versus erlotinib (150 mg orally every 24 hours) with BSC in patients with stage IV non-small-cell lung cancer (NSCLC) whose tumors have detectable Kirsten rat sarcoma (KRAS) mutations and whose disease has progressed after platinum-based chemotherapy and 1 other previous therapy, or who are not eligible for further chemotherapy. Approximately 550 patients will be randomized in a 3:2 ratio and stratified according to number of previous chemotherapy regimens (1 vs. 2), Eastern Cooperative Oncology Group performance status (0 vs. 1), sex (male vs. female), and KRAS mutation (G12C vs. others). Erlotinib was chosen as the control arm, because it is the only agent indicated for second- and third-line therapy in advanced NSCLC. Treatment will continue until disease progression or unacceptable toxicity occurs, with assessments every 28 days, followed by short-term and long-term follow-up. The coprimary efficacy objectives of this study are progression-free survival (PFS) and overall survival (OS); secondary objectives are overall response rate, changes in patient-reported pain and disease-related symptoms, changes in health status, resource utilization, safety and tolerability, and pharmacokinetics/pharmacodynamics. This design has 80% power to detect OS hazard ratio (HR) of 0.75 (type I error 0.045) and PFS HR of 0.67 (type I error 0.005). If the coprimary objectives (OS and PFS) are achieved, this study will provide a new alternative third-line treatment option for patients with NSCLC whose tumors have detectable KRAS mutations.

  14. Concordance of KRAS/BRAF Mutation Status in Metastatic Colorectal Cancer before and after Anti-EGFR Therapy.

    PubMed

    Gattenlöhner, S; Etschmann, B; Kunzmann, V; Thalheimer, A; Hack, M; Kleber, G; Einsele, H; Germer, C; Müller-Hermelink, H-K

    2009-01-01

    Anti-EGFR targeted therapy is a potent strategy in the treatment of metastatic colorectal cancer (mCRC) but activating mutations in the KRAS gene are associated with poor response to this treatment. Therefore, KRAS mutation analysis is employed in the selection of patients for EGFR-targeted therapy and various studies have shown a high concordance between the mutation status in primary CRC and corresponding metastases. However, although development of therapy related resistance occurs also in the context of novel drugs such as tyrosine kinase-inhibitors the effect of the anti-EGFR treatment on the KRAS/BRAF mutation status itself in recurrent mCRC has not yet been clarified. Therefore, we analyzed 21 mCRCs before/after anti-EGFR therapy and found a pre-/posttherapeutic concordance of the KRAS/BRAF mutation status in 20 of the 21 cases examined. In the one discordant case, further analyses revealed that a tumor mosaicism or multiple primary tumors were present, indicating that anti-EGFR therapy has no influence on KRAS/BRAF mutation status in mCRC. Moreover, as the preselection of patients with a KRAS(wt) genotype for anti-EGFR therapy has become a standard procedure, sample sets such ours might be the basis for future studies addressing the identification of potential anti-EGFR therapy induced genetic alterations apart from KRAS/BRAF mutations.

  15. KRAS gene mutations are more common in colorectal villous adenomas and in situ carcinomas than in carcinomas

    PubMed Central

    Zauber, Peter; Marotta, Stephen; Sabbath-Solitare, Marlene

    2013-01-01

    We have evaluated the frequency of KRAS gene mutations during the critical transition from villous adenoma to colorectal carcinoma to assess whether the adenomas contain a KRAS mutation more frequently than carcinomas. We analyzed sporadic villous and tubulovillous adenomas, in situ carcinomas, and primary colorectal carcinomas from multiple patients. The cancers were further evaluated for mucinous status and microsatellite instability. Standard PCR molecular techniques were used for KRAS and microsatellite analyses. A KRAS mutation was found in 61.9% of 134 adenomas, 67.8% of 84 in situ carcinomas, and just 31.6% of 171 carcinomas. Our study clearly demonstrates that tubulovillous and villous adenomas, as well as both the benign and malignant parts of in situ carcinomas, are statistically more likely to contain a somatic KRAS gene mutation than colorectal carcinomas. This difference is confined to the non-mucinous and the microsatellite stable tumors. Our data support the possibility that non-mucinous and microsatellite stable carcinomas with wild-type KRAS gene may have had a mutation in the KRAS gene during their earlier stages, with the mutation lost during further growth. PMID:23565319

  16. 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.

  17. 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. PMID:27637917

  18. Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers.

    PubMed

    de Cuba, E M V; Snaebjornsson, P; Heideman, D A M; van Grieken, N C T; Bosch, L J W; Fijneman, R J A; Belt, E; Bril, H; Stockmann, H B A C; Hooijberg, E; Punt, C J A; Koopman, M; Nagtegaal, I D; Coupé, V H M; Carvalho, B; Meijer, G A

    2016-03-01

    Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear whether it confers worse survival in this setting. The prognostic value of KRAS mutations in both MSS and MSI CRC remains unclear. We examined the effect of BRAF and KRAS mutations on survival in stage II and III MSI colon cancer patients. BRAF exon 15 and KRAS exon 2-3 mutation status was assessed in 143 stage II (n = 85) and III (n = 58) MSI colon cancers by high resolution melting analysis and sequencing. The relation between mutation status and cancer-specific (CSS) and overall survival (OS) was analyzed using Kaplan-Meier and Cox regression analysis. BRAF V600E mutations were observed in 51% (n = 73) and KRAS mutations in 16% of cases (n = 23). Patients with double wild-type cancers (dWT; i.e., BRAF and KRAS wild-type) had a highly favourable survival with 5-year CSS of 93% (95% CI 84-100%), while patients with cancers harbouring mutations in either BRAF or KRAS, had 5-year CSS of 76% (95% CI 67-85%). In the subgroup of stage II patients with dWT cancers no cancer-specific deaths were observed. On multivariate analysis, mutation in either BRAF or KRAS vs. dWT remained significantly prognostic. Mutations in BRAF as well as KRAS should be analyzed when considering these genes as prognostic markers in MSI colon cancers.

  19. 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

  20. Mutation of NRAS but not KRAS significantly reduces myeloma sensitivity to single-agent bortezomib therapy

    PubMed Central

    Lichter, David I.; Di Bacco, Alessandra; Blakemore, Stephen J.; Berger, Allison; Koenig, Erik; Bernard, Hugues; Trepicchio, William; Li, Bin; Neuwirth, Rachel; Chattopadhyay, Nibedita; Bolen, Joseph B.; Dorner, Andrew J.; van de Velde, Helgi; Ricci, Deborah; Jagannath, Sundar; Berenson, James R.; Richardson, Paul G.; Stadtmauer, Edward A.; Orlowski, Robert Z.; Lonial, Sagar; Anderson, Kenneth C.; Sonneveld, Pieter; San Miguel, Jesús F.; Esseltine, Dixie-Lee; Schu, Matthew

    2014-01-01

    Various translocations and mutations have been identified in myeloma, and certain aberrations, such as t(4;14) and del17, are linked with disease prognosis. To investigate mutational prevalence in myeloma and associations between mutations and patient outcomes, we tested a panel of 41 known oncogenes and tumor suppressor genes in tumor samples from 133 relapsed myeloma patients participating in phase 2 or 3 clinical trials of bortezomib. DNA mutations were identified in 14 genes. BRAF as well as RAS genes were mutated in a large proportion of cases (45.9%) and these mutations were mutually exclusive. New recurrent mutations were also identified, including in the PDGFRA and JAK3 genes. NRAS mutations were associated with a significantly lower response rate to single-agent bortezomib (7% vs 53% in patients with mutant vs wild-type NRAS, P = .00116, Bonferroni-corrected P = .016), as well as shorter time to progression in bortezomib-treated patients (P = .0058, Bonferroni-corrected P = .012). However, NRAS mutation did not impact outcome in patients treated with high-dose dexamethasone. KRAS mutation did not reduce sensitivity to bortezomib or dexamethasone. These findings identify a significant clinical impact of NRAS mutation in myeloma and demonstrate a clear example of functional differences between the KRAS and NRAS oncogenes. PMID:24335104

  1. 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.

  2. Rosai-Dorfman Disease Harboring an Activating KRAS K117N Missense Mutation.

    PubMed

    Shanmugam, Vignesh; Margolskee, Elizabeth; Kluk, Michael; Giorgadze, Tamara; Orazi, Attilio

    2016-09-01

    Rosai-Dorfman disease (RDD) or sinus histiocytosis with massive lymphadenopathy is a rare histiocytic proliferation that is generally considered to be reactive with a benign clinical course. The etiology of RDD is very poorly understood. Recent studies have shown frequent BRAF, NRAS, KRAS, and PIK3CA activating mutations in several histiocytic neoplasms highlighting the emerging importance of the RAF/MEK/ERK pathway in the pathogenesis of these diseases. Here we report a case of Rosai-Dorfman disease involving the submandibular salivary gland with a KRAS K117N missense mutation discovered by next-generation sequencing. These results suggest that at least a subset of RDD cases may be clonal processes. Further mutational studies on this rare histiocytic disease should be undertaken to better characterize its pathogenesis as well as open up potential avenues for therapy.

  3. Biochemical characterization of a novel KRAS insertion mutation from a human leukemia.

    PubMed

    Bollag, G; Adler, F; elMasry, N; McCabe, P C; Conner, E; Thompson, P; McCormick, F; Shannon, K

    1996-12-20

    A novel alteration in exon 1 of KRAS was detected by single strand conformational polymorphism analysis of DNA amplified from the bone marrow of a 4-year-old child with myeloid leukemia. Sequencing of this mutant allele revealed an insertion of three nucleotides between codons 10 and 11 resulting in an in-frame insertion of glycine. Expression of the mutant protein in NIH 3T3 cells caused cellular transformation, and expression in COS cells activated the Ras-mitogen-activated protein kinase signaling pathway. Surprisingly, Ras.GTP levels measured in COS cells established that this novel mutant accumulates to 90% in the GTP state, considerably higher than a residue 12 mutant. Biochemical analysis confirmed that the higher Ras.GTP levels correspond to a dramatic decrease in intrinsic GTP hydrolysis as well as resistance to GTPase-activating proteins. This mutation is the first dominant Ras mutation found in human cancer that does not involve residues 12, 13, or 61, and its biochemical properties should help elucidate the mechanism of oncogenic activation.

  4. Are high initial CEA and CA 19-9 levels associated with the presence of K-ras mutation in patients with metastatic colorectal cancer?

    PubMed

    Selcukbiricik, Fatih; Bilici, Ahmet; Tural, Deniz; Erdamar, Sibel; Soyluk, Ozlem; Buyukunal, Evin; Demirelli, Fuat; Serdengecti, Suheyla

    2013-08-01

    In certain cell culture studies, significant CEA expression was observed in K-ras mutant cells. However, the relationship between high CEA levels and K-ras status has not been sufficiently investigated. In the present study, we aimed to determine the prognostic role of initial CEA and CA 19-9 values in metastatic colorectal cancer patients according to the status of K-ras. Between 2000 and 2010, a total of 215 patients with metastatic colorectal cancer who were treated and followed up in our oncology center were analyzed. Smokers were excluded from the study. The clinicopathological findings and initial CEA and CA19-9 values were determined. K-ras mutation analysis was performed using quantitative PCR evaluation of the DNA from the tumor tissues. Eighty-two patients (38.1 %) were female and 133 (61.9 %) were male, with a median age of 59 years (range 27-83). Based on tumor localization, 127 patients (59 %) were classified as colon cancer patients and 88 patients (41 %) were classified as rectal cancer patients. The majority of patients (83.3 %) had pure adenocarcinoma histology, while 36 cases (16.7 %) had mucinous adenocarcinoma. The initial CEA levels were detected to be high (>5 ng/mL) in 108 of the patients (50.2 %), while high levels of initial CA 19-9 (>37 ng/mL) were found in 90 patients (41.8 %). K-ras mutations were detected in 99 of the patients (46 %). K-ras was found to be wild type in 116 patients (54 %). Significant differences were detected between the K-ras wild-type and mutant groups with respect to age and the initial serum CEA levels. Patients with K-ras mutations were younger (p = 0.04) and had higher initial CEA levels (p = 0.02) compared to patients with K-ras wild type. The median overall survival (OS) time and 3-year OS rate for patients with a high initial CEA level (>5 ng/mL) were significantly shorter than those of patients with a low initial CEA level (<5 ng/mL) (50.5 months and 61.8 % vs. 78.6 months and 79.1 %, p = 0.014). Furthermore

  5. Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

    PubMed Central

    Mazhab-Jafari, Mohammad T.; Marshall, Christopher B.; Smith, Matthew J.; Gasmi-Seabrook, Geneviève M. C.; Stathopulos, Peter B.; Inagaki, Fuyuhiko; Kay, Lewis E.; Neel, Benjamin G.; Ikura, Mitsuhiko

    2015-01-01

    K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery. PMID:25941399

  6. Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

    PubMed

    Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

    2015-05-26

    K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

  7. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    PubMed

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  8. 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

  9. Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

    PubMed Central

    Ardighieri, Laura; Zeppernick, Felix; Hannibal, Charlotte G; Vang, Russell; Cope, Leslie; Junge, Jette; Kjaer, Susanne K; Kurman, Robert J; Shih, Ie-Ming

    2014-01-01

    There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced-stage disease identified from a nation-wide tumour registry in Denmark. Mutational analysis for hotspots in KRAS and BRAF was successful in 55 APSTs and demonstrated KRAS mutations in 34 (61.8%) and BRAF mutations in eight (14.5%). Mutational analysis was successful in 56 peritoneal implants and revealed KRAS mutations in 34 (60.7%) and BRAF mutations in seven (12.5%). Mutational analysis could not be performed in two primary tumours and in nine implants, either because DNA amplification failed or because there was insufficient tissue for mutational analysis. For these specimens we performed VE1 immunohistochemistry, which was shown to be a specific and sensitive surrogate marker for a V600E BRAF mutation. VE1 staining was positive in one of two APSTs and seven of nine implants. Thus, among 63 implants for which mutation status was known (either by direct mutational analysis or by VE1 immunohistochemistry), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST–implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST–implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11 SBT/APST–implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST–implant (non-invasive and invasive) pairs (p < 0.00001). This study provides cogent evidence that the vast majority of peritoneal implants, non-invasive and invasive, harbour the identical KRAS or BRAF mutations that are present in the associated SBT/APST, supporting the view that peritoneal implants are derived from the primary ovarian tumour. PMID:24307542

  10. 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.

  11. 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.

  12. Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

    PubMed Central

    van der Hoeven, Dharini; Cho, Kwang-jin; Ma, Xiaoping; Chigurupati, Sravanthi; Parton, Robert G.

    2013-01-01

    Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics. PMID:23129805

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

    PubMed

    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.

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

    PubMed

    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

  15. KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer.

    PubMed

    De Roock, Wendy; De Vriendt, Veerle; Normanno, Nicola; Ciardiello, Fortunato; Tejpar, Sabine

    2011-06-01

    The discovery of mutant KRAS as a predictor of resistance to epidermal growth-factor receptor (EGFR) monoclonal antibodies brought a major change in the treatment of metastatic colorectal cancer. This seminal finding also highlighted our sparse knowledge about key signalling pathways in colorectal tumours. Drugs that inhibit oncogenic alterations such as phospho-MAP2K (also called MEK), phospho-AKT, and mutant B-RAF seem promising as single treatment or when given with EGFR inhibitors. However, our understanding of the precise role these potential drug targets have in colorectal tumours, and the oncogenic dependence that tumours might have on these components, has not progressed at the same rate. As a result, patient selection and prediction of treatment effects remain problematic. We review the role of mutations in genes other than KRAS on the efficacy of anti-EGFR therapy, and discuss strategies to target these oncogenic alterations alone or in combination with receptor tyrosine-kinase inhibition.

  16. The Prognostic Value of Microsatellite Instability, KRAS, BRAF and PIK3CA Mutations in Stage II Colon Cancer Patients

    PubMed Central

    Vogelaar, F Jeroen; N van Erning, Felice; Reimers, Marlies S; van der Linden, Hans; Pruijt, Hans; C van den Brule, Adriaan J; Bosscha, Koop

    2015-01-01

    In the era of personalized cancer medicine, identifying mutations within patient tumors plays an important role in defining high-risk stage II colon cancer patients. The prognostic role of BRAF V600E mutation, microsatellite instability (MSI) status, KRAS mutation and PIK3CA mutation in stage II colon cancer patients is not settled. We retrospectively analyzed 186 patients with stage II colon cancer who underwent an oncological resection but were not treated with adjuvant chemotherapy. KRAS mutations, PIK3CA mutation, V600E BRAF mutation and MSI status were determined. Survival analyses were performed. Mutations were found in the patients with each mutation in the following percentages: 23% (MSI), 35% (KRAS), 19% (BRAF) and 11% (PIK3CA). A trend toward worse overall survival (OS) was seen in patients with an MSI (5-year OS 74% versus 82%, adjusted hazard ratio [HR] 1.8, 95% confidence interval [CI] 0.6–4.9) and a KRAS-mutated tumor (5-year OS 77% versus 82%, adjusted HR 1.7, 95% CI 0.8–3.5). MSI and BRAF-mutated tumors tended to correlate with poorer disease-free survival (DFS) (5-year DFS 60% versus 78%, adjusted HR 1.6, 95% CI 0.5–2.1 and 5-year DFS 57% versus 77%, adjusted HR 1.1, 95% CI 0.4–2.6 respectively). In stage II colon cancer patients not treated with adjuvant chemotherapy, BRAF mutation and MSI status both tended to have a negative prognostic effect on disease-free survival. KRAS and MSI status also tended to be correlated with worse overall survival. PMID:26716438

  17. 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.

  18. Non-small Cell Lung Cancer with Concomitant EGFR, KRAS, and ALK Mutation: Clinicopathologic Features of 12 Cases

    PubMed Central

    Lee, Taebum; Lee, Boram; Choi, Yoon-La; Han, Joungho; Ahn, Myung-Ju; Um, Sang-Won

    2016-01-01

    Background: Although epidermal growth factor receptor (EGFR), v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and anaplastic lymphoma kinase (ALK) mutations in non-small cell lung cancer (NSCLC) were thought to be mutually exclusive, some tumors harbor concomitant mutations. Discovering a driver mutation on the basis of morphologic features and therapeutic responses with mutation analysis can be used to understand pathogenesis and predict resistance in targeted therapy. Methods: In 6,637 patients with NSCLC, 12 patients who had concomitant mutations were selected and clinicopathologic features were reviewed. Clinical characteristics included sex, age, smoking history, previous treatment, and targeted therapy with response and disease-free survival. Histologic features included dominant patterns, nuclear and cytoplasmic features. Results: All patients were diagnosed with adenocarcinoma and had an EGFR mutation. Six patients had concomitant KRAS mutations and the other six had KRAS mutations. Five of six EGFR-KRAS mutation patients showed papillary and acinar histologic patterns with hobnail cells. Three of six received EGFR tyrosine kinase inhibitor (TKI) and showed partial response for 7–29 months. All six EGFR-ALK mutation patients showed solid or cribriform patterns and three had signet ring cells. Five of six EGFR-ALK mutation patients received EGFR TKI and/or ALK inhibitor and four showed partial response or stable disease, except for one patient who had acquired an EGFR mutation. Conclusions: EGFR and ALK mutations play an important role as driver mutations in double mutated NSCLC, and morphologic analysis can be used to predict treatment response. PMID:27086595

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

    PubMed Central

    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. PMID:26110767

  20. KRAS G13D Mutation and Sensitivity to Cetuximab or Panitumumab in a Colorectal Cancer Cell Line Model

    PubMed Central

    Kumar, Shalini Sree; Price, Timothy J.; Mohyieldin, Omar; Borg, Matthew; Townsend, Amanda

    2014-01-01

    ABSTRACT BACKGROUND: The treatment of metastatic colorectal cancer (mCRC) includes drugs targeting the epidermal growth factor receptor (EGFR). Mutation in codon 12 or 13 in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, downstream of the EGFR, evokes constitutive activation of the RAS/RAF/MAPK signaling pathway and correlates with resistance to anti-EGFR monoclonal antibody (mAb) therapies. However, a retrospective study reported that a proportion of patients with the KRAS G13D mutation may respond to cetuximab. A similar analysis for panitumumab was not as conclusive. We sought to determine the sensitivity of CRC cell lines to cetuximab or panitumumab treatment and to investigate the correlation of the KRAS mutational status of the CRC cell lines to the responsiveness to cetuximab or panitumumab. METHODS: To determine the responsiveness of CRC cell lines to cetuximab or panitumumab, cell lines were treated with an optimized concentration of each mAb, and proliferation assays were conducted. RESULTS: After treatment with cetuximab or panitumumab, at the optimum concentration of 8 μg/well, the KRAS G13D mutant cell lines HCT-116, LoVo, and T84 showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line SW480 and the sensitive KRAS wild-type cell line LIM1215. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab (P = .02). CONCLUSION: The specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to reported clinical observations. PMID:24558511

  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. Immunophenotype and K-RAS mutation in mucinous ovarian adenocarcinoma with mural nodule of high-grade sarcoma: case report.

    PubMed

    Desouki, Mohamed M; Fadare, Oluwole; Kanbour, Anisa; Kanbour-Shakir, Amal

    2014-03-01

    Ovarian mucinous tumors with mural nodules are rare. The mural nodules are microscopically divergent neoplasms of varying sizes that may be benign (eg, sarcoma-like and carcinosarcoma-like), or malignant (eg, anaplastic carcinoma and sarcoma). The K-RAS gene mutation in ovarian mucinous neoplasms with mural nodules has not been previously reported. This is a case report of a 25-year-old female diagnosed with ovarian invasive mucinous adenocarcinoma with mural nodule of high-grade sarcoma. The mucinous tumor component demonstrated a K-RAS codon 12/13 mutation (p.G12V, c.35 G>T), whereas the sarcomatous component demonstrated a K-RAS codon 12/13 mutation (p.G12D, c.35 G>A). Although both tumor components revealed a mutation in codon 12 of K-RAS, they were of different nucleotide substitutions, indicating that these 2 tumor components were of different clonal origins. However, the fact that the 2 mutations identified in the tumor components are the most common mutations reported in mucinous tumors of the ovary, raises the possibility that sarcomatous mural nodules simply represent a form of dedifferentiation in mucinous tumors.

  3. 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

  4. Allele specific locked nucleic acid quantitative PCR (ASLNAqPCR): an accurate and cost-effective assay to diagnose and quantify KRAS and BRAF mutation.

    PubMed

    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.

  5. 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.

  6. No evidence of oncogenic KRAS mutations in squamous cell carcinomas of the anogenital tract and head and neck region independent of human papillomavirus and p16(INK4a) status.

    PubMed

    Prigge, Elena-Sophie; Urban, Katharina; Stiegler, Sandrine; Müller, Meike; Kloor, Matthias; Mai, Sabine; Ottstadt, Martine; Lohr, Frank; Wenz, Frederik; Wagner, Steffen; Wittekindt, Claus; Klussmann, Jens Peter; Hampl, Monika; von Knebel Doeberitz, Magnus; Reuschenbach, Miriam

    2014-11-01

    Carcinogenesis of squamous cell carcinomas (SCCs) in the anogenital tract and head and neck region is heterogeneous. A substantial proportion of SCC in the vulva, anus, and head and neck follows a human papillomavirus (HPV)-induced carcinogenic pathway. However, the molecular pathways of carcinogenesis in the HPV-independent lesions are not completely understood. We hypothesized that oncogenic Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations might represent a carcinogenic mechanism in a proportion of those HPV-negative cancers. Considering the repeated observation of KRAS-associated p16(INK4a) overexpression in human tumors, it was assumed that KRAS mutations might be particularly present in the group of HPV-negative, p16(INK4a)-positive cancers. To test this hypothesis, we analyzed 66 anal, vulvar, and head and neck SCC with known immunohistochemical p16(INK4a) and HPV DNA status for KRAS mutations in exon 2 (codons 12, 13, and 15). We enriched the tumor collection with HPV DNA-negative, p16(INK4a)-positive cancers. A subset of 37 cancers was also analyzed for mutations in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene. None of the 66 tumors harbored mutations in KRAS exon 2, thus excluding KRAS mutations as a common event in SCC of the anogenital and head and neck region and as a cause of p16(INK4a) expression in these tumors. In addition, no BRAF mutations were detected in the 37 analyzed tumors. Further studies are required to determine the molecular events underlying HPV-negative anal, vulvar, and head and neck carcinogenesis. Considering HPV-independent p16(INK4a) overexpression in some of these tumors, particular focus should be placed on alternative upstream activators and potential downstream disruption of the p16(INK4a) pathway.

  7. 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

    2016-01-01

    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. PMID:27070580

  8. 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...

  9. Molecular spectrum of somatic EGFR and KRAS gene mutations in non small cell lung carcinoma: determination of frequency, distribution pattern and identification of novel variations in Indian patients.

    PubMed

    Das, Bibhu Ranjan; Bhaumik, Sangeet; Ahmad, Firoz; Mandsaurwala, Aziz; Satam, Heena

    2015-07-01

    Somatic mutations of EGFR and KRAS gene represent the most common alterations currently known in NSCLC patients. This study explored the frequency, distribution pattern of EGFR and KRAS mutations in Indian patients. The frequencies of EGFR and KRAS mutations were 29 % (116/400) and 4.5 % (6/132) respectively. Both EGFR and KRAS mutations were prevalent in females, and a trend towards higher mutation frequency was seen in patients under ≥ 60 years age. The presence of EGFR and KRAS mutations were higher in adenocarcinomas in comparison to other histological subtype. Sequencing analysis of EGFR exon 18 revealed Inframe deletion (G709_T710 > A) and missense mutation (K713R). Among exon 19 positive cases, 49.3 % (37/75) were in-frame deletions, of which E746_A750del was frequent. Similarly, ~47 % (35/75) cases showed complex mutation involving indel. Among mutations in exon 20 (N = 9), 8 were substitutions, one showed duplication, while all exon 21 mutations were of the missense types with L858R as the most recurrent type. Sequencing analysis of KRAS exon 1 revealed three different types codon 12 substitutions resulting in c34G > T (G12C) (n = 4), c.35G > A (G12D) (n = 1), and c.35G > T (G12V) (n = 1). In conclusion, the present study is an example of molecular diversity of EGFR and KRAS gene in Indian patients and further confirms that the frequency of EGFR and KRAS mutations varies considerably globally. To the best of our knowledge, this is the first Indian study to evaluate KRAS mutation. The current study also served to identify novel variations that added new insights into the genetic heterogeneity of NSCLC.

  10. 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...

  11. 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...

  12. Whole genome sequencing reveals potential targets for therapy in patients with refractory KRAS mutated metastatic colorectal cancer

    PubMed Central

    2014-01-01

    Background The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients. Methods Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements. Results All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC. Conclusions Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC. PMID:24943349

  13. 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.

  14. KRAS and PIK3CA mutation frequencies in patient-derived xenograft models of pancreatic and colorectal cancer are reflective of patient tumors and stable across passages.

    PubMed

    Tignanelli, Christopher J; Herrera Loeza, Silvia G; Yeh, Jen Jen

    2014-09-01

    One obstacle in the translation of advances in cancer research into the clinic is a deficiency of adequate preclinical models that recapitulate human disease. Patient-derived xenograft (PDX) models are established by engrafting patient tumor tissue into mice and are advantageous because they capture tumor heterogeneity. One concern with these models is that selective pressure could lead to mutational drift and thus be an inaccurate reflection of patient tumors. Therefore, we evaluated if mutational frequency in PDX models is reflective of patient populations and if crucial mutations are stable across passages. We examined KRAS and PIK3CA gene mutations from pancreatic ductal adenocarcinoma (PDAC) (n = 30) and colorectal cancer (CRC) (n = 37) PDXs for as many as eight passages. DNA was isolated from tumors and target sequences were amplified by polymerase chain reaction. KRAS codons 12/13 and PIK3CA codons 542/545/1047 were examined using pyrosequencing. Twenty-three of 30 (77%) PDAC PDXs had KRAS mutations and one of 30 (3%) had PIK3CA mutations. Fifteen of 37 (41%) CRC PDXs had KRAS mutations and three of 37 (8%) had PIK3CA mutations. Mutations were 100 per cent preserved across passages. We found that the frequency of KRAS (77%) and PIK3CA (3%) mutations in PDAC PDX was similar to frequencies in patient tumors (71 to 100% KRAS, 0 to 11% PIK3CA). Similarly, KRAS (41%) and PIK3CA (8%) mutations in CRC PDX closely paralleled patient tumors (35 to 51% KRAS, 12 to 21% PIK3CA). The accurate mirroring and stability of genetic changes in PDX models compared with patient tumors suggest that these models are good preclinical surrogates for patient tumors.

  15. PIK3CA Amplification Is Common in Left Side-Tubular Adenomas but Uncommon Sessile Serrated Adenomas Exclusively with KRAS Mutation

    PubMed Central

    Lee, Hyunsu; Lee, Jae-Ho; Kim, Dae-Kwang; Choi, In-Jang; Hwang, Ilseon; Kang, Yu-Na; Kim, Shin

    2015-01-01

    Colorectal cancer is a heterogeneous disorder than arises via multiple distinct pathways, from tubular adenomas (TAs) and sessile serrated adenomas (SSAs), which are clinically, morphologically, and molecularly different. We examined PIK3CA amplification in colorectal precancerous legions, including TAs and SSAs. DNA was isolated from paired normal and tumoral tissues in 64 TAs and 32 SSAs. PIK3CA amplification, KRAS mutation, and BRAF mutation were analyzed by real-time PCR and pyrosequencing. PIK3CA amplification was found in 25% of TAs and 9.4% of SSAs, respectively. KRAS and BRAF mutations were mutually exclusive in both TAs and SSAs. In TAs, PIK3CA amplification was associated with left side and it was mutually exclusive with KRAS mutation. These results suggest that PIK3CA amplification may be early and important event in colorectal carcinogenesis and may drive the development of left-side TAs independently with KRAS mutation. PMID:26019684

  16. Clinical and metabolic parameters in non-small cell lung carcinoma and colorectal cancer patients with and without KRAS mutations.

    PubMed

    Yilmaz, Ahmet; Mohamed, Nehad; Patterson, Kara A; Tang, Yan; Shilo, Konstantin; Villalona-Calero, Miguel A; Davis, Michael E; Zhou, Xiao-Ping; Frankel, Wendy; Otterson, Gregory A; Zhao, Weiqiang

    2014-09-01

    Lung cancer (LC) and colorectal cancer (CRC) are the first and second deadliest types of cancer worldwide. EGFR-based therapy has been used in the treatment of these cancers with variable success. Presence of mutations in the KRAS driver oncogene, possibly induced by environmental factors such as carcinogens in diet and cigarette smoke, may confer worse prognosis and resistance to treatment for reasons not fully understood. Data on possible associations between KRAS mutational status and clinical and metabolic parameters, which may help in clinical management, as well as in identifying risk factors for developing these cancers, are limited in the current literature. We sequenced the KRAS gene and investigated the associations of variations in 108 patients with non-small cell lung carcinoma (NSCLC), the most common form of LC, and in 116 patients with CRC. All of the mutations originated from the guanosine nucleotide and over half of all transversions in NSCLC and CRC were c.34 G>T and c.35 G>T, respectively. c.35 G>A was the most frequent type of transition in both cancers. Excluding smoking, the clinical and metabolic parameters in patients carrying mutant and wild type KRAS were similar except that the CRC patients with transversion mutations were 8.6 years younger than those carrying the transitions (P < 0.01). Dyslipidemia, hypertension, family cancer history, and age of diagnosis older than 60 years were more frequent in NSCLC than CRC (P ≤ 0.04). These results suggest that most of the clinical and metabolic parameters investigated in this study are probably not associated with the more aggressive phenotype and differences in response to EGFR-based treatment previously reported in patients with KRAS mutations. However, the increased rates of abnormal metabolic parameters in patients with NSCLC in comparison to CRC indicate that these parameters may be more important in the management of NSCLC. CRC patients carrying transition mutations are older than those

  17. Metastatic colorectal cancer KRAS genotyping in routine practice: results and pitfalls.

    PubMed

    Lamy, Aude; Blanchard, France; Le Pessot, Florence; Sesboüé, Richard; Di Fiore, Frédéric; Bossut, Jessie; Fiant, Elodie; Frébourg, Thierry; Sabourin, Jean-Christophe

    2011-08-01

    KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.

  18. Gene methylation of SFRP2, P16, DAPK1, HIC1, and MGMT and KRAS mutations in sporadic colorectal cancer.

    PubMed

    Pehlivan, Sacide; Artac, Mehmet; Sever, Tugce; Bozcuk, Hakan; Kilincarslan, Can; Pehlivan, Mustafa

    2010-09-01

    The aim of this study was to investigate the methylation of the SFRP2, P16, DAPK1, HIC1, and MGMT genes, as well as the mutation of amino acid codons 12 and 13 of the KRAS gene in normal and tumor tissue DNA of patients diagnosed with sporadic colorectal cancer (SCRC). The methylation of gene regions and the KRAS mutations of normal (N) and tumor tissue (T) DNA obtained from 17 patients diagnosed with SCRC and 20 healthy controls were investigated using the polymerase chain reaction and reverse-hybridization methods. There was an Asp mutation in four patients, an Asp and Ser mutations in one patient in codon 12 of the KRAS gene, and an Asp mutation in codon 13 in eight patients. Overall promoter methylation (OPM) in the SFRP2 gene was observed in one N and four T, whereas partial promoter methylation (PPM) was observed in two N and five T. OPM in the P16 gene was present in one T. In the DAPK1 gene, OPM existed in seven T and five N, while PPM was present in two N. In the HIC1 gene, OPM was demonstrated in three T, while PPM was noted in two N; however, no methylation existed in N. In the MGMT gene, OPM occurred in five T and two N, and PPM was present in one T. KRAS mutations in Turkish patients with SCRC are similar to those of other population groups. Methylations in the genes, which underwent methylation analysis, were higher in T in comparison with N, and it has been suggested that significant results would be obtained by making a study with a larger population.

  19. Impact of KRAS mutation on response and outcome of patients with stage III non-squamous non-small cell lung cancer

    PubMed Central

    Yagishita, Shigehiro; Horinouchi, Hidehito; Sunami, Kuniko S; Kanda, Shintaro; Fujiwara, Yutaka; Nokihara, Hiroshi; Yamamoto, Noboru; Sumi, Minako; Shiraishi, Kouya; Kohno, Takashi; Furuta, Koh; Tsuta, Koji; Tamura, Tomohide; Ohe, Yuichiro

    2015-01-01

    The frequency and clinical profile of patients with stage III non-small cell lung cancer harboring KRAS mutations have not yet been well documented. Here, we analyzed hotspot KRAS mutations using high-resolution melting analyses in tumor specimens from patients who received chemoradiotherapy between January 2001 and December 2010 at the National Cancer Center Hospital. The associations between the presence of KRAS mutations and the response rate, relapse-free survival, first relapse sites, survival post-progression and overall survival were investigated. A total of 274 non-squamous non-small cell lung cancer patients received chemoradiotherapy at our hospital. After excluding 121 patients for whom tumor specimens were not available and 34 patients with EGFR mutations, the remaining 119 patients were included in the analysis. KRAS mutations were found at a frequency of 13%. Patients with KRAS mutations had a shorter median relapse-free survival (6.1 vs 10.9 months) and a lower response rate (63% vs 81%). As for the first relapse site, patients with KRAS mutations had fewer local relapses (8% vs 23%) and more brain metastases (46% vs 12%). After disease progression, patients with KRAS mutations had a significantly shorter median survival post-progression (2.5 vs 7.3 months, P = 0.028) and median overall survival (15.1 vs 29.1 months, P = 0.022). Our results suggested that KRAS mutation could be associated with a reduced efficacy of chemoradiotherapy and a shortened survival time. PMID:26177347

  20. 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

  1. Impact of KRAS, BRAF, PIK3CA, TP53 status and intraindividual mutation heterogeneity on outcome after liver resection for colorectal cancer metastases

    PubMed Central

    Løes, Inger Marie; Immervoll, Heike; Sorbye, Halfdan; Angelsen, Jon‐Helge; Horn, Arild; Knappskog, Stian

    2016-01-01

    We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p < 0.001, 0.002 and 0.023, respectively). KRAS and BRAF mutations also predicted a reduced median disease‐specific survival (DSS) (29 vs. 51 and 16 vs. 49 months; p <0.001 and 0.008, respectively). No effect of TP53 (60.4%) mutation status was observed. Postoperative, but not preoperative chemotherapy improved both TTR and DSS (p < 0.001 for both) with no interaction with gene mutation status. Among 94 patients harboring two or more metastatic deposits, 13 revealed mutation heterogeneity across metastatic deposits for at least one gene. Mutation heterogeneity predicted reduced median DSS compared to homogeneous mutations (18 vs. 37 months; p = 0.011 for all genes; 16 vs. 26 months; p < 0.001 analyzing BRAF or KRAS mutations separately). In multivariate analyses, KRAS or BRAF mutations consistently predicted poor TRR and DSS. Mutation heterogeneity robustly predicted DSS but not TTR, while postoperative chemotherapy improved both TTR and DSS. Our findings indicate that BRAF and KRAS mutations as well as mutation heterogeneity predict poor outcome in CRC patients subsequent to liver resections and might help guide treatment decisions. PMID:26991344

  2. Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

    PubMed Central

    Song, Yunke; Zhang, Yi; Wang, Tza-Huei

    2014-01-01

    Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. PMID:23239594

  3. K-Ras mutation-mediated IGF-1-induced feedback ERK activation contributes to the rapalog resistance in pancreatic ductal adenocarcinomas.

    PubMed

    Wei, Feng; Liu, Yan; Bellail, Anita C; Olson, Jeffrey J; Sun, Shi-Yong; Lu, Guoyue; Ding, Lijuan; Yuan, Changji; Wang, Guangyi; Hao, Chunhai

    2012-09-01

    Mammalian target of rapamycin complex 1 (mTORC1) is frequently activated in human cancers; however, clinical trials of rapalog (the mTORC1 inhibitors) have shown that pancreatic ductal adenocarcinomas (PDACs) resist to the treatment. Rapalog treatment activated the extracellular signal-regulated kinase (ERK) pathway in K-Ras mt PDAC cells. K-Ras knockdown abolished the insulin-like growth factor-1 (IGF-1)-induced ERK pathway in the K-Ras mt PDAC cells and enhanced the therapeutic efficacy of everolimus in treating K-Ras mt PDAC cells-derived mouse xenografts. The results indicate that targeting of K-Ras mutation may lead to the development of therapies that overcome rapalog resistance in PDAC.

  4. Multiplex detection of mutations.

    PubMed

    Perlin, David S; Balashov, Sergey; Park, Steven

    2008-01-01

    Rapid and reliable detection of mutations at the genetic level is an integral part of modern molecular diagnostics. These mutations can range from dominant single nucleotide polymorphisms within specific loci to codominant heterozygotic insertions and they present considerable challenges to investigators in developing rapid nucleic acid-based amplification assays that can distinguish wild-type from mutant alleles. The recent improvements of real-time polymerase chain reaction (PCR) using self-reporting fluorescence probes have given researchers a powerful tool in developing assays for mutation detection that can be multiplexed for high-throughput screening of multiple mutations and cost effectiveness. Here we describe an application of a multiplexed real-time PCR assay using Molecular Beacon probes for the detection of mutations in codon 54 of the CYP51A gene in Aspergillus fumigatus conferring triazole resistance.

  5. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers.

    PubMed

    Haley, Lisa; Tseng, Li-Hui; Zheng, Gang; Dudley, Jonathan; Anderson, Derek A; Azad, Nilofer S; Gocke, Christopher D; Eshleman, James R; Lin, Ming-Tseh

    2015-10-01

    Activating mutations in downstream genes of the epidermal growth factor receptor (EGFR) pathway may cause anti-EGFR resistance in patients with colorectal cancers. We present performance characteristics of a next-generation sequencing assay designed to detect such mutations. In this retrospective quality assessment study, we analyzed mutation detected in the KRAS, NRAS, BRAF, and PIK3CA genes by a clinically validated next-generation sequencing assay in 310 colorectal cancer specimens. Tumor cellularity and mutant allele frequency were analyzed to identify tumor heterogeneity and mutant allele-specific imbalance. Next-generation sequencing showed precise measurement of mutant allele frequencies and detected 23% of mutations with 2-20% mutant allele frequencies. Of the KRAS mutations detected, 17% were outside of codons 12 and 13. Among PIK3CA mutations, 48% were outside of codons 542, 545, and 1047. The percentage of tumors with predicted resistance to anti-EGFR therapy increased from 40% when testing for only mutations in KRAS exon 2 to 47% when testing for KRAS exons 2-4, 48% when testing for KRAS and NRAS exons 2-4, 58% when including BRAF codon 600 mutations, and 59% when adding PIK3CA exon 20 mutations. Right-sided colorectal cancers carried a higher risk of predicted anti-EGFR resistance. A concomitant KRAS mutation was detected in 51% of PIK3CA, 23% of NRAS, and 33% of kinase-impaired BRAF-mutated tumors. Lower than expected mutant allele frequency indicated tumor heterogeneity, while higher than expected mutant allele frequency indicated mutant allele-specific imbalance. Two paired neuroendocrine carcinomas and adjacent adenomas showed identical KRAS mutations, but only PIK3CA mutations in neuroendocrine carcinomas. Next-generation sequencing is a robust tool for mutation detection in clinical laboratories. It demonstrates high analytic sensitivity and broad reportable range, and it provides simultaneous detection of concomitant mutations and a quantitative

  6. Breast Cancer Heterogeneity Examined by High-Sensitivity Quantification of PIK3CA, KRAS, HRAS, and BRAF Mutations in Normal Breast and Ductal Carcinomas.

    PubMed

    Myers, Meagan B; Banda, Malathi; McKim, Karen L; Wang, Yiying; Powell, Michael J; Parsons, Barbara L

    2016-04-01

    Mutant cancer subpopulations have the potential to derail durable patient responses to molecularly targeted cancer therapeutics, yet the prevalence and size of such subpopulations are largely unexplored. We employed the sensitive and quantitative Allele-specific Competitive Blocker PCR approach to characterize mutant cancer subpopulations in ductal carcinomas (DCs), examining five specific hotspot point mutations (PIK3CA H1047R, KRAS G12D, KRAS G12V, HRAS G12D, and BRAF V600E). As an approach to aid interpretation of the DC results, the mutations were also quantified in normal breast tissue. Overall, the mutations were prevalent in normal breast and DCs, with 9/9 DCs having measureable levels of at least three of the five mutations. HRAS G12D was significantly increased in DCs as compared to normal breast. The most frequent point mutation reported in DC by DNA sequencing, PIK3CA H1047R, was detected in all normal breast tissue and DC samples and was present at remarkably high levels (mutant fractions of 1.1 × 10(-3) to 4.6 × 10(-2)) in 4/10 normal breast samples. In normal breast tissue samples, PIK3CA mutation levels were positively correlated with age. However, the PIK3CA H1047R mutant fraction distributions for normal breast tissues and DCs were similar. The results suggest PIK3CA H1047R mutant cells have a selective advantage in breast, contribute to breast cancer susceptibility, and drive tumor progression during breast carcinogenesis, even when present as only a subpopulation of tumor cells. PMID:27108388

  7. Bexarotene plus erlotinib suppress lung carcinogenesis independent of KRAS mutations in two clinical trials and transgenic models.

    PubMed

    Dragnev, Konstantin H; Ma, Tian; Cyrus, Jobin; Galimberti, Fabrizio; Memoli, Vincent; Busch, Alexander M; Tsongalis, Gregory J; Seltzer, Marc; Johnstone, David; Erkmen, Cherie P; Nugent, William; Rigas, James R; Liu, Xi; Freemantle, Sarah J; Kurie, Jonathan M; Waxman, Samuel; Dmitrovsky, Ethan

    2011-06-01

    The rexinoid bexarotene represses cyclin D1 by causing its proteasomal degradation. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib represses cyclin D1 via different mechanisms. We conducted a preclinical study and 2 clinical/translational trials (a window-of-opportunity and phase II) of bexarotene plus erlotinib. The combination repressed growth and cyclin D1 expression in cyclin-E- and KRAS/p53-driven transgenic lung cancer cells. The window-of-opportunity trial in early-stage non-small-cell lung cancer (NSCLC) patients (10 evaluable), including cases with KRAS mutations, repressed cyclin D1 (in tumor biopsies and buccal swabs) and induced necrosis and inflammatory responses. The phase II trial in heavily pretreated, advanced NSCLC patients (40 evaluable; a median of two prior relapses per patient (range, 0-5); 21% with prior EGFR-inhibitor therapy) produced three major clinical responses in patients with prolonged progression-free survival (583-, 665-, and 1,460-plus days). Median overall survival was 22 weeks. Hypertriglyceridemia was associated with an increased median overall survival (P = 0.001). Early PET (positron emission tomographic) response did not reliably predict clinical response. The combination was generally well tolerated, with toxicities similar to those of the single agents. In conclusion, bexarotene plus erlotinib was active in KRAS-driven lung cancer cells, was biologically active in early-stage mutant KRAS NSCLC, and was clinically active in advanced, chemotherapy-refractory mutant KRAS tumors in this study and previous trials. Additional lung cancer therapy or prevention trials with this oral regimen are warranted.

  8. Low prevalence of K-RAS, EGF-R and BRAF mutations in sinonasal adenocarcinomas. Implications for anti-EGFR treatments.

    PubMed

    Franchi, Alessandro; Innocenti, Duccio Rossi Degli; Palomba, Annarita; Miligi, Lucia; Paiar, Fabiola; Franzese, Ciro; Santucci, Marco

    2014-07-01

    We have previously shown that a subset of sinonasal intestinal-type adenocarcinomas (ITAC) shows activation of the epidermal growth factor-receptor (EGFR) pathway. In this study we examine the status of the EGFR, KRAS and BRAF genes in a series of sinonasal intestinal (ITAC) and non-intestinal type adenocarcinomas (non-ITAC). Eighteen ITACs and 12 non-ITACs were studied immunohistochemically for EGFR expression. Point mutations were analyzed for EGFR exons 19 and 21, KRAS exon 2 and BRAF exon 15 by direct sequencing. Non-ITACs showed significantly higher expression of EGFR (p = 0.015). Mutation analysis revealed one ITAC with EGFR and one ITAC with KRAS mutation, while two non-ITACs presented mutation of BRAF. We conclude that a subset of sinonasal adenocarcinomas shows overexpression of EGFR, while activating mutations of the signaling cascade downstream of EGFR are rare, suggesting that these tumors could be good candidates for anti-EGFR therapies.

  9. Molecular spectrum of KRAS, NRAS, BRAF and PIK3CA mutations in Chinese colorectal cancer patients: analysis of 1,110 cases

    PubMed Central

    Zhang, Jing; Zheng, Jianming; Yang, Yinghong; Lu, Junliang; Gao, Jie; Lu, Tao; Sun, Jian; Jiang, Hui; Zhu, Yan; Zheng, Yuhui; Liang, Zhiyong; Liu, Tonghua

    2015-01-01

    Mutations in genes such as KRAS, NRAS, BRAF and PIK3CA have become an important part of colorectal carcinoma evaluation. The aim of this study was to screen for mutations in these genes in Chinese patients with colorectal cancer (CRC) and to explore their correlations with certain clinicopathological parameters. We tested mutations in the KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4), PIK3CA (exon 20) and BRAF (exon 15) genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing in a large cohort of 1,110 Chinese CRC patients who underwent surgical resection at one of three major teaching hospitals located in different regions of China. The prevalence rates of KRAS, NRAS, BRAF and PIK3CA mutations were 45.4%, 3.9%, 3.1% and 3.5%, respectively. Mutant KRAS was associated with the mucinous subtype and greater differentiation, while mutant BRAF was associated with right-sided tumors and poorer differentiation. Our results revealed differences in the genetic profiles of KRAS, NRAS, PIK3CA and BRAF at mutation hotspots between Chinese CRC patients and those of Western countries, while some of these gene features were shared among patients from other Asian countries. PMID:26691448

  10. EGFR and KRAS mutational analysis in a large series of Italian non-small cell lung cancer patients: 2,387 cases from a single center.

    PubMed

    Giannini, Riccardo; Lupi, Cristiana; Sensi, Elisa; Alì, Greta; Proietti, Agnese; Boldrini, Laura; Servadio, Adele; Giordano, Mirella; Macerola, Elisabetta; Bruno, Rossella; Borrelli, Nicla; Chella, Antonio; Melfi, Franca; Lucchi, Marco; Ribechini, Alessandro; Vasile, Enrico; Cappuzzo, Federico; Mussi, Alfredo; Fontanini, Gabriella

    2016-08-01

    Activating EGFR mutations are important genetic alterations that have strong therapeutic implications for non-small cell lung cancer (NSCLC) patients. However, the role of KRAS mutations in this process is still under evaluation. Here, we report on the feasibility of a large‑scale EGFR and KRAS mutation analysis in the daily routine of a single center. NSCLCs from 2,387 patients were screened for EGFR and KRAS mutations from January 2010 to September 2015. Mutational analyses were performed in a single laboratory using single strand conformation polymorphism (SSCP)-Sanger sequencing and matrix‑assisted laser desorption ionization‑time of flight (MALDI‑TOF) on Sequenom platform for EGFR and pyrosequencing for KRAS. Activating EGFR mutations were found in 14.1% of all tumors, whereas KRAS mutations were found in 30.5% of all tumors. Direct sequencing showed analyzable cytological, small biopsy and surgical specimen percentages of 90.3, 90.9 and 98.1%, respectively, whereas the MALDI‑TOF platform showed analyzable cytological samples, small biopsies and surgical specimens percentages of 94.6, 95.7 and 96.9%, respectively. The mean analytical turnaround times (TAT) were 4 and 3 days for direct sequencing and the MALDI‑TOF platform, respectively. Our results confirm that small biopsy or cytological samples can be used for reliable EGFR and KRAS mutation testing and indicate that adopting the MALDI‑TOF platform reduces the rate of missed samples among the samples. Moreover, the 3-day analytical TAT of the MALDI-TOF multi-target technique is appropriate for clinical management and reduces the overall treatment decision time. PMID:27373829

  11. 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.

  12. 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

  13. 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

  14. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

    PubMed Central

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  15. Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

    PubMed

    Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian

    2015-01-01

    Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

  16. Deep Sequence Analysis of Non-Small Cell Lung Cancer: Integrated Analysis of Gene Expression, Alternative Splicing, and Single Nucleotide Variations in Lung Adenocarcinomas with and without Oncogenic KRAS Mutations

    PubMed Central

    Kalari, Krishna R.; Rossell, David; Necela, Brian M.; Asmann, Yan W.; Nair, Asha; Baheti, Saurabh; Kachergus, Jennifer M.; Younkin, Curtis S.; Baker, Tiffany; Carr, Jennifer M.; Tang, Xiaojia; Walsh, Michael P.; Chai, High-Seng; Sun, Zhifu; Hart, Steven N.; Leontovich, Alexey A.; Hossain, Asif; Kocher, Jean-Pierre; Perez, Edith A.; Reisman, David N.; Fields, Alan P.; Thompson, E. Aubrey

    2012-01-01

    KRAS mutations are highly prevalent in non-small cell lung cancer (NSCLC), and tumors harboring these mutations tend to be aggressive and resistant to chemotherapy. We used next-generation sequencing technology to identify pathways that are specifically altered in lung tumors harboring a KRAS mutation. Paired-end RNA-sequencing of 15 primary lung adenocarcinoma tumors (8 harboring mutant KRAS and 7 with wild-type KRAS) were performed. Sequences were mapped to the human genome, and genomic features, including differentially expressed genes, alternate splicing isoforms and single nucleotide variants, were determined for tumors with and without KRAS mutation using a variety of computational methods. Network analysis was carried out on genes showing differential expression (374 genes), alternate splicing (259 genes), and SNV-related changes (65 genes) in NSCLC tumors harboring a KRAS mutation. Genes exhibiting two or more connections from the lung adenocarcinoma network were used to carry out integrated pathway analysis. The most significant signaling pathways identified through this analysis were the NFκB, ERK1/2, and AKT pathways. A 27 gene mutant KRAS-specific sub network was extracted based on gene–gene connections from the integrated network, and interrogated for druggable targets. Our results confirm previous evidence that mutant KRAS tumors exhibit activated NFκB, ERK1/2, and AKT pathways and may be preferentially sensitive to target therapeutics toward these pathways. In addition, our analysis indicates novel, previously unappreciated links between mutant KRAS and the TNFR and PPARγ signaling pathways, suggesting that targeted PPARγ antagonists and TNFR inhibitors may be useful therapeutic strategies for treatment of mutant KRAS lung tumors. Our study is the first to integrate genomic features from RNA-Seq data from NSCLC and to define a first draft genomic landscape model that is unique to tumors with oncogenic KRAS mutations. PMID:22655260

  17. Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer.

    PubMed

    Hutton, Josiah E; Wang, Xiaojing; Zimmerman, Lisa J; Slebos, Robbert J C; Trenary, Irina A; Young, Jamey D; Li, Ming; Liebler, Daniel C

    2016-09-01

    Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein. In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at ftp://MSV000079486@massive.ucsd.edu. PMID:27340238

  18. Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

    PubMed Central

    Schrumpf, Melanie; Talebian Yazdi, Mehrdad; Ruano, Dina; Forte, Giusi I.; Nederlof, Petra M.; Veselic, Maud; Rabe, Klaus F.; Annema, Jouke T.; Smit, Vincent; Morreau, Hans; van Wezel, Tom

    2011-01-01

    Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients. PMID:21408138

  19. k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

    PubMed Central

    Lu, Hui-Yu

    2016-01-01

    Aim of the study Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitions (TKIs) in advanced non-small cell lung cancer (NSCLC) treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR) was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results We identified 12 studies with 1193 patients, including 196 patients (16.4%) with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764) in patients with smoke expose vs. 5.4% (23/429) in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000). No publication bias was found (Begg's test: z = 1.09, p = 0.274 and Egger's test: t = 1.38, p = 0.201). In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000) in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083), but the sample size was underpowered (0.465). Conclusions The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs. PMID:27358590

  20. Involvement of KRAS G12A mutation in the IL-2-independent growth of a human T-LGL leukemia cell line, PLT-2.

    PubMed

    Mizutani, Naoki; Ito, Hiromi; Hagiwara, Kazumi; Kobayashi, Misa; Hoshikawa, Asuka; Nishida, Yayoi; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Osawa, Yosuke; Ohnishi, Kazunori; Daibata, Masanori; Murate, Takashi

    2012-08-01

    Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share beta- and gamma-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression.

  1. Clinicopathologic characteristics of EGFR, KRAS, and ALK alterations in 6,595 lung cancers

    PubMed Central

    Lee, Boram; Lee, Taebum; Lee, Se-Hoon; Choi, Yoon-La; Han, Joungho

    2016-01-01

    Background EGFR, KRAS, and ALK alterations are major genetic changes found in non-small cell lung cancers (NSCLCs). Testing advanced lung adenocarcinoma tumors for these three genes is now standard care. The purpose of this study was to investigate the clinicopathologic expression pattern of these three genes in East Asian NSCLC patients. Patients and methods We conducted a retrospective study of all patients tested for mutations of these three genes at a single institute in Korea between 2006 and 2014. Study data were extracted from electronic medical records. Univariate and multivariate logistic regression analyses were used to measure associations between clinicopathologic features and alterations of EGFR, KRAS, and ALK. Results We detected 12 EGFR-mutated tumors with additional mutations in KRAS (N=6, 0.1%) or ALK (N=6, 0.1%). General clinicopathologic characteristics of tumors with EGFR, KRAS, or ALK mutations were similar to previous reports. Patients having EGFR L858R point mutations were older than patients having EGFR exon 19 deletions. EGFR G719X point mutations were more common in men and smokers than exon 19 deletions or L858R point mutations. Tumors having KRAS G12C mutations were less often of mucinous type than those with G12D or G12V, mutations. Conclusions This is the largest three gene molecular epidemiology study in East Asian NSCLC patients. Each genetic alteration was associated with distinct clinicopathologic characteristics. Furthermore, different age and sex are associated with different subtypes of EGFR and KRAS mutations. PMID:26992209

  2. Detection of K-Ras oncogene using magnetic beads-quantum dots in microfluidic chip.

    PubMed

    Noh, Han Na; Kim, Jong Sung

    2013-08-01

    Recently quantum dots (QDs) have been extensively used in the field of biotechnology. QDs have merits of wide selection of emission wavelength and exceptional stability against photo bleaching over conventional organic fluorophores and are used in cell imaging, biomarker, and fluorescence resonance energy transfer (FRET) sensor. Magnetic beads have been used as solid support in microfluidic devices to trace bio-molecules. In this study, Polydimethylsiloxane (PDMS) based microfluidic chips were prepared for the detection of K-Ras oncogene by using QDs-DNA conjugate. K-Ras oncogene can be detected by fluorescence quenching in microfluidic chip. Carboxylated CdSe/ZnS QDs (emission wavelength: 605 nm) could bind to magnetic beads of polystyrene/divinyl benzene via EDC/NHS crosslinking reaction. The fluorescence from QDs could be quenched by intercalating dye (thiazol orange dimers: TOTO-3) after hybridization with target DNA and probe DNA in the channel of microfluidic chip. The fluorescence intensity change of QDs after hybridization in microfluidic chip has been studied. PMID:23882748

  3. Driver Gene Mutations in Stools of Colorectal Carcinoma Patients Detected by Targeted Next-Generation Sequencing.

    PubMed

    Armengol, Gemma; Sarhadi, Virinder K; Ghanbari, Reza; Doghaei-Moghaddam, Masoud; Ansari, Reza; Sotoudeh, Masoud; Puolakkainen, Pauli; Kokkola, Arto; Malekzadeh, Reza; Knuutila, Sakari

    2016-07-01

    Detection of driver gene mutations in stool DNA represents a promising noninvasive approach for screening colorectal cancer (CRC). Amplicon-based next-generation sequencing (NGS) is a good option to study mutations in many cancer genes simultaneously and from a low amount of DNA. Our aim was to assess the feasibility of identifying mutations in 22 cancer driver genes with Ion Torrent technology in stool DNA from a series of 65 CRC patients. The assay was successful in 80% of stool DNA samples. NGS results showed 83 mutations in cancer driver genes, 29 hotspot and 54 novel mutations. One to five genes were mutated in 75% of cases. TP53, KRAS, FBXW7, and SMAD4 were the top mutated genes, consistent with previous studies. Of samples with mutations, 54% presented concomitant mutations in different genes. Phosphatidylinositol 3-kinase/mitogen-activated protein kinase pathway genes were mutated in 70% of samples, with 58% having alterations in KRAS, NRAS, or BRAF. Because mutations in these genes can compromise the efficacy of epidermal growth factor receptor blockade in CRC patients, identifying mutations that confer resistance to some targeted treatments may be useful to guide therapeutic decisions. In conclusion, the data presented herein show that NGS procedures on stool DNA represent a promising tool to detect genetic mutations that could be used in the future for diagnosis, monitoring, or treating CRC. PMID:27155048

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

    PubMed Central

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

    2016-01-01

    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. PMID:26840022

  5. 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

  6. Fluorescence detection of KRAS2 mRNA hybridization in lung cancer cells with PNA-peptides containing an internal thiazole orange.

    PubMed

    Sonar, Mahesh V; Wampole, Matthew E; Jin, Yuan-Yuan; Chen, Chang-Po; Thakur, Mathew L; Wickstrom, Eric

    2014-09-17

    We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5' end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5' terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5-6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA.

  7. Fluorescence Detection of KRAS2 mRNA Hybridization in Lung Cancer Cells with PNA-Peptides Containing an Internal Thiazole Orange

    PubMed Central

    2015-01-01

    We previously developed reporter-peptide nucleic acid (PNA)-peptides for sequence-specific radioimaging and fluorescence imaging of particular mRNAs in cells and tumors. However, a direct test for PNA-peptide hybridization with RNA in the cytoplasm would be desirable. Thiazole orange (TO) dye at the 5′ end of a hybridization agent shows a strong increase in fluorescence quantum yield when stacked upon a 5′ terminal base pair, in solution and in cells. We hypothesized that hybridization agents with an internal TO could distinguish a single base mutation in RNA. Thus, we designed KRAS2 PNA-IGF1 tetrapeptide agents with an internal TO adjacent to the middle base of the 12th codon, a frequent site of cancer-initiating mutations. Our molecular dynamics calculations predicted a disordered bulge with weaker hybridization resulting from a single RNA mismatch. We observed that single-stranded PNA-IGF1 tetrapeptide agents with an internal TO showed low fluorescence, but fluorescence escalated 5–6-fold upon hybridization with KRAS2 RNA. Circular dichroism melting curves showed ∼10 °C higher Tm for fully complementary vs single base mismatch TO-PNA-peptide agent duplexes with KRAS2 RNA. Fluorescence measurements of treated human lung cancer cells similarly showed elevated cytoplasmic fluorescence intensity with fully complementary vs single base mismatch agents. Sequence-specific elevation of internal TO fluorescence is consistent with our hypothesis of detecting cytoplasmic PNA-peptide:RNA hybridization if a mutant agent encounters the corresponding mutant mRNA. PMID:25180641

  8. Performance and Cost Efficiency of KRAS Mutation Testing for Metastatic Colorectal Cancer in Routine Diagnosis: The MOKAECM Study, a Nationwide Experience

    PubMed Central

    Chatellier, Gilles; Côté, Jean-François; Pages, Jean-Christophe; de Fraipont, Florence; Boyer, Jean-Christophe; Merlio, Jean Philippe; Morel, Alain; Gorisse, Marie-Claude; de Cremoux, Patricia; Leroy, Karen; Milano, Gérard; Ouafik, L’Houcine; Merlin, Jean-Louis; Le Corre, Delphine; Aucouturier, Pascaline; Sabourin, Jean-Christophe; Nowak, Frédérique; Frebourg, Thierry; Emile, Jean-François; Durand-Zaleski, Isabelle; Laurent-Puig, Pierre

    2013-01-01

    Purpose Rapid advances in the understanding of cancer biology have transformed drug development thus leading to the approval of targeted therapies and to the development of molecular tests to select patients that will respond to treatments. KRAS status has emerged as a negative predictor of clinical benefit from anti-EGFR antibodies in colorectal cancer, and anti-EGFR antibodies use was limited to KRAS wild type tumors. In order to ensure wide access to tumor molecular profiling, the French National Cancer Institute (INCa) has set up a national network of 28 regional molecular genetics centers. Concurrently, a nationwide external quality assessment for KRAS testing (MOKAECM) was granted to analyze reproducibility and costs. Methods 96 cell-line DNAs and 24 DNA samples from paraffin embedded tumor tissues were sent to 40 French laboratories. A total of 5448 KRAS results were collected and analyzed and a micro-costing study was performed on sites for 5 common methods by an independent team of health economists. Results This work provided a baseline picture of the accuracy and reliability of KRAS analysis in routine testing conditions at a nationwide level. Inter-laboratory Kappa values were >0.8 for KRAS results despite differences detection methods and the use of in-house technologies. Specificity was excellent with only one false positive in 1128 FFPE data, and sensitivity was higher for targeted techniques as compared to Sanger sequencing based methods that were dependent upon local expertise. Estimated reagent costs per patient ranged from €5.5 to €19.0. Conclusion The INCa has set-up a network of public laboratories dedicated to molecular oncology tests. Our results showed almost perfect agreements in KRAS testing at a nationwide level despite different testing methods ensuring a cost-effective equal access to personalized colorectal cancer treatment. PMID:23935912

  9. 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. PMID:12211696

  10. 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.

  11. 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

  12. Detection of endogenous K-ras mRNA in living cells at a single base resolution by a PNA molecular beacon.

    PubMed

    Kam, Yossi; Rubinstein, Abraham; Nissan, Aviram; Halle, David; Yavin, Eylon

    2012-03-01

    Detection of mRNA alterations is a promising approach for identifying biomarkers as means of differentiating benign from malignant lesions. By choosing the KRAS oncogene as a target gene, two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (TO-PNA-MB, where TO = thiazole orange) were synthesized and compared in vitro and in vivo. Their specificity was examined in wild-type KRAS (HT29) or codon 12 point mutation (Panc-1, SW480) cells. Incubation of both beacons with total RNA extracted from the Panc-1 cell line (fully complementary sequence) showed a fluorescent signal for both beacons. Major differences were observed, however, for single mismatch mRNA transcripts in cell lines HT29 and SW480. PS-DNA-MB weakly discriminated such single mismatches in comparison to TO-PNA-MB, which was profoundly more sensitive. Cell transfection of TO-PNA-MB with the aid of PEI resulted in fluorescence in cells expressing the fully complementary RNA transcript (Panc-1) but undetectable fluorescence in cells expressing the K-ras mRNA that has a single mismatch to the designed TO-PNA-MB (HT29). A weaker fluorescent signal was also detected in SW480 cells; however, these cells express approximately one-fifth of the target mRNA of the designed TO-PNA-MB. In contrast, PS-DNA-MB showed no fluorescence in all cell lines tested post PEI transfection. Based on the fast hybridization kinetics and on the single mismatch discrimination found for TO-PNA-MB we believe that such molecular beacons are promising for in vivo real-time imaging of endogenous mRNA with single nucleotide polymorphism (SNP) resolution.

  13. GNAS and KRAS mutational analyses of intraductal papillary neoplasms of the pancreas and bile duct developing in the same individual: A case report.

    PubMed

    Date, Kenjiro; Ohtsuka, Takao; Fujimoto, Takaaki; Gotoh, Yoshitaka; Nakashima, Yohei; Kimura, Hideyo; Matsunaga, Taketo; Mori, Yasuhisa; Mochidome, Naoki; Miyazaki, Tetsuyuki; Oda, Yoshinao; Tanaka, Masao; Nakamura, Masafumi

    2015-01-01

    Intraductal papillary mucinous neoplasm (IPMN) of the pancreas and intraductal papillary neoplasm of the bile duct (IPNB) are considered as counterparts of each other, and it is suggested that these two entities have similar molecular alteration pathways. However, the occurrence of IPMN of the pancreas and IPNB in the same patient is rare. We report a surgical case of a 69-year-old woman who developed invasive IPMN of the pancreas and underwent pancreatectomy, 6 months after hepatic resection of invasive IPNB. Molecular analysis revealed GNAS/KRAS mutation in both invasive IPMN of the pancreas and IPNB. This is believed to be the first case report investigating GNAS/KRAS mutational status in both IPMN of the pancreas and IPNB developing in the same patient, and these two entities may show similar molecular alternations.

  14. 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.

  15. Lauric acid can improve the sensitization of Cetuximab in KRAS/BRAF mutated colorectal cancer cells by retrievable microRNA-378 expression.

    PubMed

    Weng, Wen-Hui; Leung, Wai-Hung; Pang, Yeu-Jye; Hsu, Hsi-Hsien

    2016-01-01

    EGFR-inhibitor (Cetuximab) is one of the main targeted drugs used for metastatic colorectal carcinoma (CRC). The benefit from Cetuximab appears to be limited to a subtype of patients, not for the patients with tumors harboring mutated BRAF or KRAS genes; unfortunately, it accounts for ~40-50% of CRC cases. Previous studies have connected higher expression levels of miR-378 to be commonly presented in patients without BRAF or KRAS mutants than in mutated CRCs. The microRNA-378 (miR-378) is coexpressed with PGC-1β and can be easily induced by fatty acid, for example lauric acid. Therefore, we hypothesized that elevation of miR-378 expression in mutated CRCs may stimulate the cell response to Cetuximab. Herein, seven CRC cell lines with confirmed mutation status were involved in two parallel experiments; directly in vitro transfected miR-378 mimics, and using lauric acid to indirectly induce the level of miR-378 in cells. After the increase of miR-378 in cells by either direct or indirect approaches, sensitivity to Cetuximab was restored in all BRAF mutants (p-value <0.0001-0.0003), and half of KRAS mutants CRC (p-value 0.039-0.007). Further evidence was gained by decreasing expression of MEK and ERK2 proteins after transfection with miR-378; it was similar to the indirect induction by lauric acid approach. In conclusion, the present study demonstrated that lauric acid may efficiently induce miR-378 expression in CRC mutants, and both BRAF and a subtype of KRAS mutants presented significantly improved sensitivity to Cetuximab. Notably, BRAF mutants could even be inhibited in cell proliferation after elevated concentration of miR-378 in cells without combining with targeted therapy. This new approach may shed new light on BRAF or KRAS mutation in CRC patients for clinical trial, since lauric acid may easily be obtain from natural food, and it is supposed to be harmless to the cardiovascular system. PMID:26496897

  16. Colon tumors with the simultaneous induction of driver mutations in APC, KRAS, and PIK3CA still progress through the adenoma-to-carcinoma sequence

    PubMed Central

    Hadac, Jamie N.; Leystra, Alyssa A.; Olson, Terrah J. Paul; Maher, Molly E.; Payne, Susan N; Yueh, Alexander E.; Schwartz, Alexander R.; Albrecht, Dawn M.; Clipson, Linda; Pasch, Cheri A.; Matkowskyj, Kristina A.; Halberg, Richard B.; Deming, Dustin A.

    2015-01-01

    Human colorectal cancers often possess multiple mutations, including 3–6 driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present prior to the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence. PMID:26276752

  17. A multicenter blinded study evaluating EGFR and KRAS mutation testing methods in the clinical non-small cell lung cancer setting--IFCT/ERMETIC2 Project Part 1: Comparison of testing methods in 20 French molecular genetic National Cancer Institute platforms.

    PubMed

    Beau-Faller, Michèle; Blons, Hélène; Domerg, Caroline; Gajda, Dorota; Richard, Nicolas; Escande, Fabienne; Solassol, Jérôme; Denis, Marc G; Cayre, Anne; Nanni-Metellus, Isabelle; Olschwang, Sylviane; Lizard, Sarab; Piard, Fabienne; Pretet, Jean-Luc; de Fraipont, Florence; Bièche, Ivan; de Cremoux, Patricia; Rouquette, Isabelle; Bringuier, Pierre-Paul; Mosser, Jean; Legrain, Michèle; Voegeli, Anne-Claire; Saulnier, Patrick; Morin, Franck; Pignon, Jean-Pierre; Zalcman, Gérard; Cadranel, Jacques

    2014-01-01

    Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non-small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%-16% versus 40%; 95% CI, 39%-42%; P < 0.001]. Mutated cases increased from 42% (95% CI, 31%-54%) to 53% (95% CI, 41%-64%), with three supplementary EGFR mutations (p.G179A at exon 18 and p.L858R and p.L861Q at exon 21) and five supplementary KRAS mutations, when using alternative testing instead of direct sequencing. False-positive results were observed when using a PCR-based sizing assay, high-resolution melting, or pyrosequencing. Concordance analysis returned good kappa test scores for EGFR exon 19 and KRAS analysis when comparing sequencing with alternative methods and revealed no difference between alternative techniques themselves.

  18. 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

  19. Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to pro-apoptotic agents.

    PubMed

    Klingbeil, Olaf; Lesche, Ralf; Gelato, Kathy A; Haendler, Bernard; Lejeune, Pascale

    2016-01-01

    Non-small cell lung cancer (NSCLC) has the highest incidence of cancer-related death worldwide and a high medical need for more effective therapies. Small-molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1, I-BET762 and OTX-015 are active in a wide range of different cancer types, including lung cancer. Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. Here we evaluated the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptosis response. Using a panel of 12 KRAS-mutated NSCLC models, we found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with downregulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. In the H1373 xenograft model, treatment with JQ1 significantly reduced tumor growth and downregulated the expression of c-Myc. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and we found the expression of the two key apoptosis regulators FLIP and XIAP to be highly BET dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Further we showed that combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether, these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment. They also demonstrate that BET inhibition primes NSCLC cells for induction of apoptosis and that a combination with pro

  20. Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to pro-apoptotic agents

    PubMed Central

    Klingbeil, Olaf; Lesche, Ralf; Gelato, Kathy A; Haendler, Bernard; Lejeune, Pascale

    2016-01-01

    Non-small cell lung cancer (NSCLC) has the highest incidence of cancer-related death worldwide and a high medical need for more effective therapies. Small-molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1, I-BET762 and OTX-015 are active in a wide range of different cancer types, including lung cancer. Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. Here we evaluated the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptosis response. Using a panel of 12 KRAS-mutated NSCLC models, we found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with downregulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. In the H1373 xenograft model, treatment with JQ1 significantly reduced tumor growth and downregulated the expression of c-Myc. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and we found the expression of the two key apoptosis regulators FLIP and XIAP to be highly BET dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Further we showed that combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether, these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment. They also demonstrate that BET inhibition primes NSCLC cells for induction of apoptosis and that a combination with pro

  1. Heterogeneity of Colorectal Cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology

    PubMed Central

    Perez, K; Walsh, R; Brilliant, K; Noble, L; Yakerivich, E; Breese, V; Jackson, C; Chatterjee, D; Pricolo, V; Roth, L; Shah, N; Cataldo, T; Safran, H; Hixson, D; Quesenberry, P

    2014-01-01

    Background New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10–20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. Methods In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (High performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for presence of KRAS proto-oncogene mutations at codon 12 and 13. Results Initially, sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by

  2. MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

    PubMed

    Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

    2016-10-01

    Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (P<0.001). Both MLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P<0.001). There was no significant difference in mucinous differentiation, tumor-infiltrating lymphocytes, Crohn-like reaction, and medullary differentiation between the 3 tumor groups. Using Kaplan-Meier survival functions, there was no significant difference in disease-specific survival between the 3 patient groups (P>0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas

  3. Cell type-specific targeted mutations of Kras and Pten document proliferation arrest in granulosa cells versus oncogenic insult to ovarian surface epithelial cells.

    PubMed

    Fan, Heng-Yu; Liu, Zhilin; Paquet, Marilene; Wang, Jinrong; Lydon, John P; DeMayo, Francesco J; Richards, JoAnne S

    2009-08-15

    The small G-protein KRAS is crucial for mediating gonadotropin-induced events associated with ovulation. However, constitutive expression of KrasG12D in granulosa cells disrupted normal follicle development leading to the persistence of abnormal follicle-like structures containing nonmitotic cells. To determine what factors mediate this potent effect of KrasG12D, gene profiling analyses were done. We also analyzed KrasG12D;Cyp19-Cre and KrasG12;Pgr-Cre mutant mouse models that express Cre prior to or after the initiation of granulosa cell differentiation, respectively. KrasG12D induced cell cycle arrest in granulosa cells of the KrasG12D;Cyp19-Cre mice but not in the KrasG12D;Pgr-Cre mice, documenting the cell context-specific effect of KrasG12D. Expression of KrasG12D silenced the Kras gene, reduced cell cycle activator genes, and impaired the expression of granulosa cell and oocyte-specific genes. Conversely, levels of PTEN and phosphorylated p38 mitogen-activated protein kinase (MAPK) increased markedly in the mutant granulosa cells. Because disrupting Pten in granulosa cells leads to increased proliferation and survival, Pten was disrupted in the KrasG12D mutant mice. The Pten/Kras mutant mice were infertile but lacked granulosa cell tumors. By contrast, the Ptenfl/fl;KrasG12D;Amhr2-Cre mice developed aggressive ovarian surface epithelial cell tumors that did not occur in the Ptenfl/fl;KrasG12D;Cyp19-Cre or Ptenfl/fl;KrasG12D;Pgr-Cre mouse strains. These data document unequivocally that Amhr2-Cre is expressed in and mediates allelic recombination of oncogenic genes in ovarian surface epithelial cells. That KrasG12D/Pten mutant granulosa cells do not transform but rather undergo cell cycle arrest indicates that they resist the oncogenic insults of Kras/Pten by robust self-protecting mechanisms that silence the Kras gene and elevate PTEN and phosphorylated p38 MAPK.

  4. Discordant cellular response to presurgical letrozole in bilateral synchronous ER+ breast cancers with a KRAS mutation or FGFR1 gene amplification.

    PubMed

    Balko, Justin M; Mayer, Ingrid A; Sanders, Melinda E; Miller, Todd W; Kuba, Maria G; Meszoely, Ingrid M; Wagle, Nikhil; Garraway, Levi A; Arteaga, Carlos L

    2012-10-01

    We describe herein a patient presenting with bilateral estrogen-receptor-positive (ER+) breast tumors who was enrolled in a clinical trial exploring molecular aberrations associated with hormone-refractory tumor cell proliferation. Short-term (two week) hormonal therapy with the aromatase inhibitor letrozole substantially reduced proliferation as measured by Ki67 immunohistochemistry in one tumor, whereas the second was essentially unchanged. Extensive molecular and genetic work-up of the two tumors yielded divergent lesions in the two tumors: an activating KRAS mutation in the responsive tumor and an amplification of the fibroblast growth factor receptor-1 (FGFR1) locus in the treatment-refractory tumor. These findings provide an insight to possible mechanisms of resistance to antiestrogen therapy in ER+ breast cancers. First, they illustrate the necessity of clinically approved assays to identify FGFR1 gene amplification, which occur in approximately 5% of breast tumors and have been linked to antiestrogen resistance. It is quite possible that the addition of FGFR inhibitors to ER-targeted therapy will yield a superior antitumor effect and improved patient outcome. Second, they suggest that the role of activating mutations in RAS, although rare in breast cancer, may need to be explored in the context of ER+ breast tumors.

  5. Atorvastatin overcomes gefitinib resistance in KRAS mutant human non-small cell lung carcinoma cells.

    PubMed

    Chen, J; Bi, H; Hou, J; Zhang, X; Zhang, C; Yue, L; Wen, X; Liu, D; Shi, H; Yuan, J; Liu, J; Liu, B

    2013-09-26

    The exact influence of statins on gefitinib resistance in human non-small cell lung cancer (NSCLC) cells with KRAS mutation alone or KRAS/PIK3CA and KRAS/PTEN comutations remains unclear. This work found that transfection of mutant KRAS plasmids significantly suppressed the gefitinib cytotoxicity in Calu3 cells (wild-type KRAS). Gefitinib disrupted the Kras/PI3K and Kras/Raf complexes in Calu3 cells, whereas not in Calu3 KRAS mutant cells. These trends were corresponding to the expression of pAKT and pERK in gefitinib treatment. Atorvastatin (1 μM) plus gefitinib treatment inhibited proliferation, promoted cell apoptosis, and reduced the AKT activity in KRAS mutant NSCLC cells compared with gefitinib alone. Atorvastatin (5 μM) further enhanced the gefitinib cytotoxicity through concomitant inhibition of AKT and ERK activity. Atorvastatin could interrupt Kras/PI3K and Kras/Raf complexes, leading to suppression of AKT and ERK activity. Similar results were also obtained in comutant KRAS/PTEN or KRAS/PIK3CA NSCLC cells. Furthermore, mevalonate administration reversed the effects of atorvastatin on the Kras/Raf and Kras/PI3K complexes, as well as AKT and ERK activity in both A549 and Calu1 cells. The in vivo results were similar to those obtained in vitro. Therefore, mutant KRAS-mediated gefitinib insensitivity is mainly derived from failure to disrupt the Kras/Raf and Kras/PI3K complexes in KRAS mutant NSCLC cells. Atorvastatin overcomes gefitinib resistance in KRAS mutant NSCLC cells irrespective of PIK3CA and PTEN statuses through inhibition of HMG-CoA reductase-dependent disruption of the Kras/Raf and Kras/PI3K complexes.

  6. Ultra-sensitive biosensor for K-ras gene detection using enzyme capped gold nanoparticles conjugates for signal amplification.

    PubMed

    Fang, Xian; Bai, Lijuan; Han, Xiaowei; Wang, Jiao; Shi, Anqi; Zhang, Yuzhong

    2014-09-01

    In this study, an ultra-sensitive hairpin DNA-based electrochemical DNA biosensor for K-ras gene detection is described. Gold nanoparticles (Au-NPs) and horseradish peroxidase (HRP)-streptavidin capped Au-NPs (HAS) conjugates are used for signal amplification. Initially, hairpin DNA dually labeled with thiol at its 5' end and with biotin at its 3' end is immobilized on the surface of Au-NPs modified electrode, and the hairpin DNA is in a "closed" state; hence, the HAS conjugates are shielded from being approached by the biotin due to steric hindrance. However, in the presence of target DNA, the target DNA hybridizes with the loop structure of hairpin DNA and causes conformational change, resulting in biotin forced away from the electrode surface, thereby becoming accessible for the HAS conjugates. Thus, the HAS conjugates are linked to the electrode surface via the specific interaction between biotin and streptavidin. Electrochemical detection was performed in phosphate-buffered saline (PBS) containing tetramethylbenzidine (TMB) and H2O2. Under optimal conditions, the peak current differences (ΔI) are linear with the target DNA in the range from 0.1 fM to 1 nM with a detection limit of 0.035 fM. Furthermore, this biosensor also demonstrates its excellent specificity for single-base mismatched DNA. PMID:24939462

  7. 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.

  8. [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。.

  9. High accuracy mutation detection in leukemia on a selected panel of cancer genes.

    PubMed

    Kalender Atak, Zeynep; De Keersmaecker, Kim; Gianfelici, Valentina; Geerdens, Ellen; Vandepoel, Roel; Pauwels, Daphnie; Porcu, Michaël; Lahortiga, Idoya; Brys, Vanessa; Dirks, Willy G; Quentmeier, Hilmar; Cloos, Jacqueline; Cuppens, Harry; Uyttebroeck, Anne; Vandenberghe, Peter; Cools, Jan; Aerts, Stein

    2012-01-01

    With the advent of whole-genome and whole-exome sequencing, high-quality catalogs of recurrently mutated cancer genes are becoming available for many cancer types. Increasing access to sequencing technology, including bench-top sequencers, provide the opportunity to re-sequence a limited set of cancer genes across a patient cohort with limited processing time. Here, we re-sequenced a set of cancer genes in T-cell acute lymphoblastic leukemia (T-ALL) using Nimblegen sequence capture coupled with Roche/454 technology. First, we investigated how a maximal sensitivity and specificity of mutation detection can be achieved through a benchmark study. We tested nine combinations of different mapping and variant-calling methods, varied the variant calling parameters, and compared the predicted mutations with a large independent validation set obtained by capillary re-sequencing. We found that the combination of two mapping algorithms, namely BWA-SW and SSAHA2, coupled with the variant calling algorithm Atlas-SNP2 yields the highest sensitivity (95%) and the highest specificity (93%). Next, we applied this analysis pipeline to identify mutations in a set of 58 cancer genes, in a panel of 18 T-ALL cell lines and 15 T-ALL patient samples. We confirmed mutations in known T-ALL drivers, including PHF6, NF1, FBXW7, NOTCH1, KRAS, NRAS, PIK3CA, and PTEN. Interestingly, we also found mutations in several cancer genes that had not been linked to T-ALL before, including JAK3. Finally, we re-sequenced a small set of 39 candidate genes and identified recurrent mutations in TET1, SPRY3 and SPRY4. In conclusion, we established an optimized analysis pipeline for Roche/454 data that can be applied to accurately detect gene mutations in cancer, which led to the identification of several new candidate T-ALL driver mutations.

  10. High-Throughput DNA Array for SNP Detection of KRAS Gene Using a Centrifugal Microfluidic Device.

    PubMed

    Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    Here, we describe detection of single nucleotide polymorphism (SNP) in genomic DNA samples using a NanoBioArray (NBA) chip. Fast DNA hybridization is achieved in the chip when target DNAs are introduced to the surface-arrayed probes using centrifugal force. Gold nanoparticles (AuNPs) are used to assist SNP detection at room temperature. The parallel setting of sample introduction in the spiral channels of the NBA chip enables multiple analyses on many samples, resulting in a technique appropriate for high-throughput SNP detection. The experimental procedure, including chip fabrication, probe array printing, DNA amplification, hybridization, signal detection, and data analysis, is described in detail.

  11. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  12. A randomized Phase II clinical study of combining panitumumab and bevacizumab, plus irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) compared with FOLFIRI alone as second-line treatment for patients with metastatic colorectal cancer and KRAS mutation

    PubMed Central

    Liu, Yuguo; Luan, Lijuan; Wang, Xingli

    2015-01-01

    Background This study investigated the efficacy and safety of a new treatment strategy of combining panitumumab and bevacizumab, plus irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) versus FOLFIRI alone as second-line chemotherapy for metastatic colorectal cancer (mCRC) patients with known V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) mutation status. Methods Patients with mCRC who had known KRAS tumor status and unsuccessful previous oxaliplatin-based chemotherapy were included in the study. They were randomly assigned to two groups to receive panitumumab and bevacizumab plus FOLFIRI, or FOLFIRI alone. In panitumumab and bevacizumab plus FOLFIRI group, patients were given 4 mg/kg panitumumab and bevacizumab plus FOLFIRI every 2 weeks. Results In all, 65 patients were assigned to panitumumab and bevacizumab plus FOLFIRI group, and 77 to FOLFIRI alone group. For WT KRAS patients, the median progression-free survival (PFS) was 5.7 months (95% confidence interval [CI], 2.4–7.5 months) for panitumumab and bevacizumab plus FOLFIRI and 3.8 months (95% CI, 3.0–6.7 months) for FOLFIRI alone; median overall survival (OS) was 15.2 months (95% CI, 8.9–19.7 months) for panitumumab and bevacizumab plus FOLFIRI and 11.0 months (95% CI, 8.2–15.4 months) for FOLFIRI alone. For MU KRAS patients, median PFS was 5.1 months (95% CI, 2.7–10.2 months) for panitumumab and bevacizumab plus FOLFIRI and 4.1 months (95% CI, 2.5–8.4 months) for FOLFIRI alone; median OS was 12.8 months (95% CI, 7.8–15.8 months) for panitumumab and bevacizumab plus FOLFIRI and 10.5 months (95% CI, 6.1–15.3 months) for FOLFIRI alone. Grade 3 and 4 adverse events were associated with panitumumab and bevacizumab plus FOLFIRI but tolerable among patients. Conclusion Patients with mCRC can be safely and efficiently treated with second-line chemotherapy of combining panitumumab and bevacizumab plus FOLFIRI, despite their KRAS mutation status. PMID:25999741

  13. 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.

  14. Mutation detection by Southern blotting.

    PubMed

    Mellars, Gillian; Gomez, Keith

    2011-01-01

    Following the discovery of the structure of DNA in 1953, it became clear that scientists needed to be able to distinguish different DNA sequences. In 1975, Edward Southern published details of a new method for detecting DNA fragments based upon their specific sequence [corrected]. An indication of the importance of his work is that the technique was eponymously named after him and that subsequent methods based loosely on similar principles were named using a play on his surname (western and northern blot). The simplicity and effectiveness of the technique led to its universal acceptance as a standard method for identification of DNA sequences. In the modern laboratory where turn-around times assume ever greater importance, the process can seem relatively time-consuming. In some cases, this has led to its replacement by more rapid techniques such as long-range PCR. Nevertheless, more than 30 years after its invention, the Southern blot remains a cornerstone of molecular biology. PMID:20938846

  15. [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。.

  16. 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.

  17. 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

  18. Colorectal cancer-related mutant KRAS alleles function as positive regulators of autophagy

    PubMed Central

    Alves, Sara; Castro, Lisandra; Fernandes, Maria Sofia; Francisco, Rita; Castro, Paula; Priault, Muriel; Chaves, Susana Rodrigues; Moyer, Mary Pat; Oliveira, Carla; Seruca, Raquel; Côrte-Real, Manuela

    2015-01-01

    The recent interest to modulate autophagy in cancer therapy has been hampered by the dual roles of this conserved catabolic process in cancer, highlighting the need for tailored approaches. Since RAS isoforms have been implicated in autophagy regulation and mutation of the KRAS oncogene is highly frequent in colorectal cancer (CRC), we questioned whether/how mutant KRAS alleles regulate autophagy in CRC and its implications. We established two original models, KRAS-humanized yeast and KRAS-non-cancer colon cells and showed that expression of mutated KRAS up-regulates starvation-induced autophagy in both. Accordingly, KRAS down-regulation inhibited autophagy in CRC-derived cells harboring KRAS mutations. We further show that KRAS-induced autophagy proceeds via up-regulation of the MEK/ERK pathway in both colon models and that KRAS and autophagy contribute to CRC cell survival during starvation. Since KRAS inhibitors have proven difficult to develop, our results suggest using autophagy inhibitors as a combined/alternative therapeutic approach in CRCs with mutant KRAS. PMID:26418750

  19. 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

  20. Analysis of the K-ras and p53 pathways in x-ray-induced lung tumors in the rat

    SciTech Connect

    Belinsky, S.A.; Middleton, S.K.; Hahn, F.F.; Nikula, K.J.; Picksley, S.M.

    1996-04-01

    The risk from exposure to low-dose radiation in conjunction with cigarette smoking has not been estimated due in part to lmited knowledge surrounding the molecular mechanisms underlying radiation-induced cancers. The purpose of this investigation was to determine the frequency for alterations in genes within the K-ras and p53 signal and cell cycle regulatory pathways, respectively, in X-ray-induced lung tumors in the F344/N rat. These tumors were examined for genetic alterations in the K-ras, c-raf-1, p53, mdm2 and cip1 genes. No K-ras mutations were detected by sequencing in 18 squamous cell carcinomas (SCCs) or 17 adenocarcinomas. However, using a K-ras codon 12 mutation selection assay, a codon 12 GGT {r_arrow} GAT mutation was detected in one SCC, suggesting that activation of the K-ras proto-oncogene is both a rare and late event. Single-strand conformation polymorphism (SSCP) analysis of the kinase-binding domain of the c-raf-1 gene did not detect any polymorphisms. Three of 18 SCCs but none of the adenocarcinomas showed p53 nuclear immunoreactivity. Single-strand conformation polymorphism analysis of exons 4-9 of the p53 gene detected only an exon 9 mutation in one SCC. Mutations were not detected in the three SCCs with immunoreactive p53 protein. No amplification of the mdm2 gene was detected; however, nuclear mdm2 immunoreactivity was present in one of the three SCCs that stained positive for the p53 protein. The complete cDNA of the rat cip1 gene comprising 810 bases was cloned and sequenced. The frequency of somatic mutations in exon 2 of the cip1 gene was determined by SSCP analysis. No alterations in electrophoretic mobility were detected. The results of this investigation indicate that alterations in the K-ras and p53 pathways do not play a major role in the genesis of X-ray-induced lung tumors in the rat. 49 refs., 5 figs.

  1. Establishment and application of a multiplex genetic mutation-detection method of lung cancer based on MassARRAY platform

    PubMed Central

    Tian, Hong-Xia; Zhang, Xu-Chao; Wang, Zhen; Chen, Jian-Guang; Chen, Shi-Liang; Guo, Wei-Bang; Wu, Yi-Long

    2016-01-01

    Objective: This study aims to establish a method for highly parallel multiplexed detection of genetic mutations in Chinese lung cancer samples through Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on MassARRAY mass spectrometry platform. Methods: We reviewed the related literature and data on lung cancer treatments. We also identified 99 mutation hot spots in 13 target genes closely related to the pathogenesis, drug resistance, and metastasis of lung cancer. A total of 297 primers, composed of 99 paired forward and reverse amplification primers and 99 matched extension primers, were designed using Assay Design software. The detection method was established by analyzing eight cell lines and six lung cancer specimens. The proposed method was then validated through comparisons by using a LungCartaTM kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results: The proposed method was able to detect multiplex genetic mutations in lung cancer cell lines. This finding was consistent with the observations on previously reported mutations. The proposed method can also detect such mutations in clinical lung cancer specimens. This result was consistent with the observations with LungCartaTM kit. However, an FGFR2 mutation was detected only through the proposed method. The measured sensitivity and specificity were 100% and 96.3%, respectively. Conclusions: The proposed MassARRAY technology-based multiplex method can detect genetic mutations in Chinese lung cancer patients. Therefore, the proposed method can be applied to detect mutations in other cancer tissues. PMID:27144063

  2. 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.

  3. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.

    PubMed

    Lochhead, Pamela A; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R; Wedge, Stephen R; Cook, Simon J

    2016-01-01

    ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

  4. 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

  5. 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

  6. Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation

    PubMed Central

    Lochhead, Pamela A.; Clark, Jonathan; Wang, Lan-Zhen; Gilmour, Lesley; Squires, Matthew; Gilley, Rebecca; Foxton, Caroline; Newell, David R.; Wedge, Stephen R.; Cook, Simon J.

    2016-01-01

    Abstract ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRASG12C/G13D or BRAFV600E. Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target. PMID:26959608

  7. Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human colorectal tumours

    PubMed Central

    Weidlich, S; Walsh, K; Crowther, D; Burczynski, M E; Feuerstein, G; Carey, F A; Steele, R J C; Wolf, C R; Miele, G; Smith, G

    2011-01-01

    Background: The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden. Methods: Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters. Results: The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype. Conclusion: Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies. PMID:21712828

  8. DNA mutation detection with chip-based temperature gradient capillary electrophoresis using a slantwise radiative heating system.

    PubMed

    Zhang, Hui-Dan; Zhou, Jing; Xu, Zhang-Run; Song, Jin; Dai, Jing; Fang, Jin; Fang, Zhao-Lun

    2007-09-01

    A simple and robust chip-based temperature gradient capillary electrophoresis (TGCE) system was developed for DNA mutation/single-nucleotide polymorphism (SNP) analysis using a radiative heating system. Reproducible, stable and uniform temperature gradients were established along a 3 cm length of the electrophoretic separation channel using a single thermostated aluminium heater plate. The heater was slightly slanted relative to the plane of the glass chip at 0.2-1.3 degrees by inserting thin spacers between the plate and chip at one end to produce differences in radiative heating that created the temperature gradient. On-chip TGCE analyses of 4 mutant DNA model samples amplified from plasmid templates, each containing a single base substitution, with a wide range of melting temperatures, showed that mutations were successfully detected under a wide temperature gradient of 10 degrees C and within a short gradient region of about 3 cm (3.3 degrees C cm(-1) gradient). The radiative heating system was able to establish stable spatial temperature gradients along short microfluidic separation channels using simple peripheral equipment and manipulation while ensuring good resolution for detecting a wide range of mutations. Effectiveness of the system was demonstrated by the successful detection of K-ras gene mutations in 6 colon cancer cell lines.

  9. [Mutation detection by PCR-TGGE].

    PubMed

    Shirakawa, T; Nishiyama, K; Matsuo, M

    1995-07-01

    The variants in enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) gene were detected by temperature gradient gel electrophoresis (TGGE). 15 clinical strains isolated from patients and a wild type strain (B2C) were analyzed after the conventional PCR. Although all PCR products (707bp) corresponded to A-subunit of LT, three strains were the different electrophoretic patterns after silver staining as compared to the wild type. For further electrophoretic analyses, the 707bp region was divided into 4 parts. The different ones were localized in the downstream part (183bp), but each those DNA bands was not so clear than DNA bands in 707bp. The clearer patterns were obtained by using a primer attached GC-clamp. The hetero-duplex assays in TGGE were proceeded by a series of procedures in mixing with the equal quantity of PCR products derived from a variant and a wild type, heat-denaturation and then annealing. TGGE of those mixed samples had 4 bands that were 2 front bands as homo-duplex and 2 slower migration bands as hetero-duplex. To Confirm the site of the mutations, the nucleotide sequences in each 183bp PCR products were decided by dideoxynucleotide-fluorescent dye method. Indeed, two variants were recognized four one-base substitutions without deletions and the one was five. Thus, the difference of migration in TGGE depended on the number and the localization in mutation sites.

  10. MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Alam, Maroof; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal. PMID:25245423

  11. Overview of KRAS-Driven Genetically Engineered Mouse Models of Non-Small Cell Lung Cancer.

    PubMed

    Sheridan, Clare; Downward, Julian

    2015-01-01

    KRAS, the most frequently mutated oncogene in non-small cell lung cancer, has been utilized extensively to model human lung adenocarcinomas. The results from such studies have enhanced considerably an understanding of the relationship between KRAS and the development of lung cancer. Detailed in this overview are the features of various KRAS-driven genetically engineered mouse models (GEMMs) of non-small cell lung cancer, their utilization, and the potential of these models for the study of lung cancer biology.

  12. 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

  13. 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

  14. Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy

    PubMed Central

    Dai, Xin; Jiang, Ying; Tan, Chalet

    2015-01-01

    KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors. PMID:25946136

  15. 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.

  16. KRAS as a Therapeutic Target

    PubMed Central

    McCormick, Frank

    2015-01-01

    KRAS proteins play a major role in human cancer, but have not yielded to therapeutic attack. New technologies in drug discovery and insights into signaling pathways that KRAS controls have promoted renewed efforts to develop therapies, either through direct targeting of KRAS itself, new ways of blocking KRAS processing, or by identifying targets that KRAS cancers depend on for survival. While drugs that block the well-established downstream pathways, RAF-MAPK and PI 3 kinase, are being tested in the clinic, new efforts are underway to exploit previously unrecognized vulnerabilities, such as altered metabolic networks, or novel pathways identified through synthetic lethal screens. Furthermore, new ways of suppressing KRAS gene expression and of harnessing the immune system offer further hope that new ways of treating KRAS are finally coming into view. These issues are discussed in this edition of CCR Focus. PMID:25878360

  17. 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.

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

    PubMed

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

    2016-01-01

    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. PMID:27223277

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

    PubMed Central

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

    2016-01-01

    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. PMID:27223277

  20. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability

    PubMed Central

    Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D.; Silkworth, Whitney; Ross, Nathan T.; Scherer, Christina A.; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A.; Stern, Andrew M.; Schreiber, Stuart L.; Golub, Todd R.

    2012-01-01

    Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33’s kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33’s kinase activity does not represent a promising anti-KRAS therapeutic strategy. PMID:22323609

  1. Panitumumab Use in Metastatic Colorectal Cancer and Patterns of KRAS Testing: Results from a Europe-Wide Physician Survey and Medical Records Review

    PubMed Central

    Trojan, Jörg; Mineur, Laurent; Tomášek, Jiří; Rouleau, Etienne; Fabian, Pavel; de Maglio, Giovanna; García-Alfonso, Pilar; Aprile, Giuseppe; Taylor, Aliki; Kafatos, George; Downey, Gerald; Terwey, Jan-Henrik; van Krieken, J. Han

    2015-01-01

    Background From 2008–2013, the European indication for panitumumab required that patients’ tumor KRAS exon 2 mutation status was known prior to starting treatment. To evaluate physician awareness of panitumumab prescribing information and how physicians prescribe panitumumab in patients with metastatic colorectal cancer (mCRC), two European multi-country, cross-sectional, observational studies were initiated in 2012: a physician survey and a medical records review. The first two out of three planned rounds for each study are reported. Methods The primary objective in the physician survey was to estimate the prevalence of KRAS testing, and in the medical records review, it was to evaluate the effect of test results on patterns of panitumumab use. The medical records review study also included a pathologists’ survey. Results In the physician survey, nearly all oncologists (299/301) were aware of the correct panitumumab indication and the need to test patients’ tumor KRAS status before treatment with panitumumab. Nearly all oncologists (283/301) had in the past 6 months of clinical practice administered panitumumab correctly to mCRC patients with wild-type KRAS status. In the medical records review, 97.5% of participating oncologists (77/79) conducted a KRAS test for all of their patients prior to prescribing panitumumab. Four patients (1.3%) did not have tumor KRAS mutation status tested prior to starting panitumumab treatment. Approximately one-quarter of patients (85/306) were treated with panitumumab and concurrent oxaliplatin-containing chemotherapy; of these, 83/85 had confirmed wild-type KRAS status prior to starting panitumumab treatment. All 56 referred laboratories that participated used a Conformité Européenne-marked or otherwise validated KRAS detection method, and nearly all (55/56) participated in a quality assurance scheme. Conclusions There was a high level of knowledge amongst oncologists around panitumumab prescribing information and the

  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. BRAF Mutation Is Rare in Advanced-Stage Low-Grade Ovarian Serous Carcinomas

    PubMed Central

    Wong, Kwong-Kwok; Tsang, Yvonne T.M.; Deavers, Michael T.; Mok, Samuel C.; Zu, Zhifei; Sun, Charlotte; Malpica, Anais; Wolf, Judith K.; Lu, Karen H.; Gershenson, David M.

    2010-01-01

    Low-grade ovarian serous carcinomas are believed to arise via an adenoma-serous borderline tumor-serous carcinoma sequence. In this study, we found that advanced-stage, low-grade ovarian serous carcinomas both with and without adjacent serous borderline tumor shared similar regions of loss of heterozygosity. We then analyzed 91 ovarian tumor samples for mutations in TP53, BRAF, and KRAS. TP53 mutations were not detected in any serous borderline tumors (n = 30) or low-grade serous carcinomas (n = 43) but were found in 73% of high-grade serous carcinomas (n = 18). BRAF (n = 9) or KRAS (n = 5) mutation was detected in 47% of serous borderline tumors, but among the low-grade serous carcinomas (39 stage III, 2 stage II, and 2 stage I), only one (2%) had a BRAF mutation and eight (19%) had a KRAS mutation. The low frequency of BRAF mutations in advanced-stage, low-grade serous carcinomas, which contrasts with previous findings, suggests that aggressive, low-grade serous carcinomas are more likely derived from serous borderline tumors without BRAF mutation. In addition, advanced-stage, low-grade carcinoma patients with BRAF or KRAS mutation have a better apparent clinical outcome. However, further investigation is needed. PMID:20802181

  4. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients.

    PubMed

    Kim, Seung Tae; Lee, Won-Suk; Lanman, Richard B; Mortimer, Stefanie; Zill, Oliver A; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-11-24

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.

  5. Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer

    PubMed Central

    Hiraki, Masayuki; Nishimura, Junichi; Takahashi, Hidekazu; Wu, Xin; Takahashi, Yusuke; Miyo, Masaaki; Nishida, Naohiro; Uemura, Mamoru; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Soh, Jae-Won; Doki, Yuichiro; Mori, Masaki; Yamamoto, Hirofumi

    2015-01-01

    KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC. PMID:25756961

  6. Prognostic impact of mutation profiling in patients with stage II and III colon cancer

    PubMed Central

    Shen, Yinchen; Han, Xiaohong; Wang, Jianfei; Wang, Shuai; Yang, Hongying; Lu, Shih-Hsin; Shi, Yuankai

    2016-01-01

    Development of colorectal cancer (CRC) associates with accumulation of genetic mutations include the epidermal growth factor receptor (EGFR) signaling pathway. However, whether mutations in KRAS together with downstream factors BRAF, PIK3CA and NRAS impact prognosis is still unclear for stage II-III colon cancer. In the present study a total of 228 stage II-III colon cancer samples were retrospectively collected, KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons 12, 13 and 61) status was detected by Sanger sequencing, 37.89% (86/227) tumors harbored a KRAS mutation, 7.02% (16/228) harbored a BRAF mutation, 13.18% (29/220) harbored a PIK3CA mutation and 0.89% (2/224) harbored a NRAS mutation. NRAS mutations existed only in stage II colon cancer. Older groups harbored a higher KRAS and BRAF mutation (P < 0.05), PIK3CA (exon9) mutations appeared more common in worse differentiation tumors (P = 0.032). Moreover, PIK3CA (E545K) mutation was significantly associated with tumor recurrence (P = 0.031) and acted independently prognostic for poor OS (P = 0.044), while only in stage III colon cancer. KRAS, BRAF and NRAS mutations do not have major prognostic value in stage II and III colon cancer, subtypes of gene mutations should be further investigated for a better understanding in CRC. PMID:27074743

  7. 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.

  8. 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.

  9. Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

    PubMed

    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

    2015-11-02

    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.

  10. RAS Mutations as Predictive Biomarkers in Clinical Management of Metastatic Colorectal Cancer.

    PubMed

    Waring, Paul; Tie, Jeanne; Maru, Dipen; Karapetis, Christos S

    2016-06-01

    The use of anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies in patients with metastatic colorectal cancer is guided by the presence of activating point mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS and NRAS genes in the primary tumor. Although these mutations have been incorporated into the prescribing information for both cetuximab and panitumumab, highlighted in the National Comprehensive Cancer Network Guidelines, and routinely tested, a number of controversial issues and unanswered questions related to these mutations and their clinical significance remain. In the present review, we explored the contradictory data related to the prognostic value of KRAS mutations, the reported frequent discordance of KRAS mutations, and the reported nonequivalence of some of these mutations. We also considered the issues related to incorporating additional mutations into the already accredited and approved assays and the challenges created by changing an assay's analytical and clinical limits of detection. We also discuss the lack of biologic data supporting the pathogenicity of newly described clinically actionable mutations and explore the uncertainty regarding the clinical significance of low-frequency mutations, highlighting the importance of correcting allele frequencies for tumor purity. We also considered the importance of distinguishing the significance of low-frequency RAS mutations in tumors previously not treated or treated with anti-EGFR therapies and explore new technologies capable of detecting emerging polyclonal RAS mutations that appear to confer drug resistance. PMID:26952655

  11. 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.

  12. 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...

  13. 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.

  14. EGFR, KRAS, BRAF, and HER-2 molecular status in brain metastases from 77 NSCLC patients

    PubMed Central

    Villalva, Claire; Duranton-Tanneur, Valérie; Guilloteau, Karline; Burel-Vandenbos, Fanny; Wager, Michel; Doyen, Jérôme; Levillain, Pierre Marie; Fontaine, Denys; Blons, Hélène; Pedeutour, Florence; Karayan-Tapon, Lucie

    2013-01-01

    The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern. PMID:23930206

  15. Oncogenic K-Ras promotes proliferation in quiescent intestinal stem cells.

    PubMed

    Gierut, Jessica J; Lyons, Jesse; Shah, Manasvi S; Genetti, Casie; Breault, David T; Haigis, Kevin M

    2015-07-01

    K-Ras is a monomeric GTPase that controls cellular and tissue homeostasis. Prior studies demonstrated that mutationally activated K-Ras (K-Ras(G12D)) signals through MEK to promote expansion and hyperproliferation of the highly mitotically active transit-amplifying cells (TACs) in the intestinal crypt. Its effect on normally quiescent stem cells was unknown, however. Here, we have used an H2B-Egfp transgenic system to demonstrate that K-Ras(G12D) accelerates the proliferative kinetics of quiescent intestinal stem cells. As in the TAC compartment, the effect of mutant K-Ras on the quiescent stem cell is dependent upon activation of MEK. Mutant K-Ras is also able to increase self-renewal potential of intestinal stem cells following damage. These results demonstrate that mutant K-Ras can influence intestinal homeostasis on multiple levels.

  16. 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...

  17. 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...

  18. 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...

  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. Extended RAS and BRAF Mutation Analysis Using Next-Generation Sequencing.

    PubMed

    Sakai, Kazuko; Tsurutani, Junji; Yamanaka, Takeharu; Yoneshige, Azusa; Ito, Akihiko; Togashi, Yosuke; De Velasco, Marco A; Terashima, Masato; Fujita, Yoshihiko; Tomida, Shuta; Tamura, Takao; Nakagawa, Kazuhiko; Nishio, Kazuto

    2015-01-01

    Somatic mutations in KRAS, NRAS, and BRAF genes are related to resistance to anti-EGFR antibodies in colorectal cancer. We have established an extended RAS and BRAF mutation assay using a next-generation sequencer to analyze these mutations. Multiplexed deep sequencing was performed to detect somatic mutations within KRAS, NRAS, and BRAF, including minor mutated components. We first validated the technical performance of the multiplexed deep sequencing using 10 normal DNA and 20 formalin-fixed, paraffin-embedded (FFPE) tumor samples. To demonstrate the potential clinical utility of our assay, we profiled 100 FFPE tumor samples and 15 plasma samples obtained from colorectal cancer patients. We used a variant calling approach based on a Poisson distribution. The distribution of the mutation-positive population was hypothesized to follow a Poisson distribution, and a mutation-positive status was defined as a value greater than the significance level of the error rate (α = 2 x 10(-5)). The cut-off value was determined to be the average error rate plus 7 standard deviations. Mutation analysis of 100 clinical FFPE tumor specimens was performed without any invalid cases. Mutations were detected at a frequency of 59% (59/100). KRAS mutation concordance between this assay and Scorpion-ARMS was 92% (92/100). DNA obtained from 15 plasma samples was also analyzed. KRAS and BRAF mutations were identified in both the plasma and tissue samples of 6 patients. The genetic screening assay using next-generation sequencer was validated for the detection of clinically relevant RAS and BRAF mutations using FFPE and liquid samples.

  1. Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

    PubMed

    Rössle, Matthias; Sigg, Michèle; Rüschoff, Jan H; Wild, Peter J; Moch, Holger; Weber, Achim; Rechsteiner, Markus P

    2013-11-01

    The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma.

  2. Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

    PubMed

    Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

    2014-09-15

    Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples.

  3. Detecting negative selection on recurrent mutations using gene genealogy

    PubMed Central

    2013-01-01

    Background Whether or not a mutant allele in a population is under selection is an important issue in population genetics, and various neutrality tests have been invented so far to detect selection. However, detection of negative selection has been notoriously difficult, partly because negatively selected alleles are usually rare in the population and have little impact on either population dynamics or the shape of the gene genealogy. Recently, through studies of genetic disorders and genome-wide analyses, many structural variations were shown to occur recurrently in the population. Such “recurrent mutations” might be revealed as deleterious by exploiting the signal of negative selection in the gene genealogy enhanced by their recurrence. Results Motivated by the above idea, we devised two new test statistics. One is the total number of mutants at a recurrently mutating locus among sampled sequences, which is tested conditionally on the number of forward mutations mapped on the sequence genealogy. The other is the size of the most common class of identical-by-descent mutants in the sample, again tested conditionally on the number of forward mutations mapped on the sequence genealogy. To examine the performance of these two tests, we simulated recurrently mutated loci each flanked by sites with neutral single nucleotide polymorphisms (SNPs), with no recombination. Using neutral recurrent mutations as null models, we attempted to detect deleterious recurrent mutations. Our analyses demonstrated high powers of our new tests under constant population size, as well as their moderate power to detect selection in expanding populations. We also devised a new maximum parsimony algorithm that, given the states of the sampled sequences at a recurrently mutating locus and an incompletely resolved genealogy, enumerates mutation histories with a minimum number of mutations while partially resolving genealogical relationships when necessary. Conclusions With their

  4. KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

    PubMed Central

    Rahman, Mohammed Tanjimur; Nakayama, Kentaro; Rahman, Munmun; Katagiri, Hiroshi; Katagiri, Atsuko; Ishibashi, Tomoka; Ishikawa, Masako; Sato, Emi; Iida, Kouji; Nakayama, Naomi; Ishikawa, Noriyuki; Miyazaki, Kohji

    2013-01-01

    In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications. PMID:23820584

  5. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

    PubMed Central

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  6. CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours.

    PubMed

    Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J; Ashworth, Alan

    2016-01-01

    Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

  7. Electrical detection of single-base DNA mutation using functionalized nanoparticles

    NASA Astrophysics Data System (ADS)

    Noor, Mohammud R.; Goyal, Swati; Christensen, Shawn M.; Iqbal, Samir M.

    2009-08-01

    We report an electrical scheme to detect specific DNA. Engineered hairpin probe DNA are immobilized on a silicon chip between gold nanoelectrodes. Hybridization of target DNA to the hairpin melts the stem nucleotides. Gold nanoparticle-conjugated universal reporter sequence detects the open hairpins by annealing to the exposed stem nucleotides. The gold nanoparticles increase charge conduction between the electrodes. Specifically, we report on a hairpin probe designed to detect a medically relevant mutant form of the K-ras oncogene. Direct current measurements show three orders of magnitude increase in conductivity for as low as 2fmol of target molecules.

  8. Electrical detection of single-base DNA mutation using functionalized nanoparticles

    NASA Astrophysics Data System (ADS)

    Noor, Mohammud R.; Goyal, Swati; Christensen, Shawn M.; Iqbal, Samir M.

    2009-08-01

    We report an electrical scheme to detect specific DNA. Engineered hairpin probe DNA are immobilized on a silicon chip between gold nanoelectrodes. Hybridization of target DNA to the hairpin melts the stem nucleotides. Gold nanoparticle-conjugated universal reporter sequence detects the open hairpins by annealing to the exposed stem nucleotides. The gold nanoparticles increase charge conduction between the electrodes. Specifically, we report on a hairpin probe designed to detect a medically relevant mutant form of the K-ras oncogene. Direct current measurements show three orders of magnitude increase in conductivity for as low as 2 fmol of target molecules.

  9. Resveratrol prevents tumorigenesis in mouse model of Kras activated sporadic colorectal cancer by suppressing oncogenic Kras expression

    PubMed Central

    Saud, Shakir M.; Li, Weidong; Morris, Nicole L.; Matter, Matthias S.; Colburn, Nancy H.; Kim, Young S.; Young, Matthew R.

    2014-01-01

    Sporadic and non-hereditary mutations account for the majority of colorectal cancers (CRC). After the loss of adenomatous polyposis coli (APC) function and activation of the β-catenin/LEF signaling pathway, activating mutations in Kras are major drivers of sporadic CRC. Preventing the outgrowth of cells that develop sporadic mutations will decrease CRC. Resveratrol, a naturally occurring polyphenolic compound has anti-inflammatory, anti-oxidant and anti-cancer activities. We used a genetically engineered mouse model for sporadic CRC where the APC locus is knocked out and Kras is activated specifically in the distal colon to determine the effects of resveratrol on preventing and treating CRC. Feeding mice a diet supplemented with 150 or 300 ppm resveratrol (105 and 210mg daily human equivalent dose, respectively) before tumors were visible by colonoscopy resulted in a 60% inhibition of tumor production. In the 40% of mice that did develop tumors Kras expression was lost in the tumors. In a therapeutic assay where tumors were allowed to develop prior to treatment, feeding tumor bearing mice with resveratrol resulted in a complete remission in 33% of the mice and a 97% decrease in tumor size in the remaining mice. Analysis of miRNA expression in non-tumoral and tumoral colonic tissue of resveratrol treated mice showed an increased expression of miR-96, a miRNA previously shown to regulate Kras translation. These data indicate that resveratrol can prevent the formation and growth of colorectal tumors by downregulating Kras expression. PMID:25280562

  10. Single molecule targeted sequencing for cancer gene mutation detection.

    PubMed

    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

  11. 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

  12. Quantification of Kras mutant fraction in the lung DNA of mice exposed to aerosolized particulate vanadium pentoxide by inhalation.

    PubMed

    Banda, Malathi; McKim, Karen L; Haber, Lynne T; MacGregor, Judith A; Gollapudi, B Bhaskar; Parsons, Barbara L

    2015-08-01

    This study investigated whether Kras mutation is an early event in the development of lung tumors induced by inhalation of particulate vanadium pentoxide (VP) aerosols. A National Toxicology Program tumor bioassay of inhaled particulate VP aerosols established that VP-induced alveolar/bronchiolar carcinomas of the B6C3F1 mouse lung carried Kras mutations at a higher frequency than observed in spontaneous mouse lung tumors. Therefore, this study sought to: (1) characterize any Kras mutational response with respect to VP exposure concentration, and (2) investigate the possibility that amplification of preexisting Kras mutation is an early event in VP-induced mouse lung tumorigenesis. Male Big Blue B6C3F1 mice (6 mice/group) were exposed to aerosolized particulate VP by inhalation, 6h/day, 5 days/week for 4 or 8 weeks, using VP exposure concentrations of 0, 0.1, and 1 mg/m(3). The levels of two different Kras codon 12 mutations [GGT → GAT (G12D) and GGT → GTT (G12V)] were measured in lung DNAs by Allele-specific Competitive Blocker PCR (ACB-PCR). For both exposure concentrations (0.1 and 1.0mg/m(3)) and both time points (4 and 8 weeks), the mutant fractions observed in VP-exposed mice were not significantly different from the concurrent controls. Given that 8 weeks of inhalation of a tumorigenic concentration of particulate aerosols of VP did not result in a significant change in levels of lung Kras mutation, the data do not support either a direct genotoxic effect of VP on Kras or early amplification of preexisting mutation as being involved in the genesis of VP-induced mouse lung tumors under the exposure conditions used. Rather, the data suggest that accumulation of Kras mutation occurs later with chronic VP exposure and is likely not an early event in VP-induced mouse lung carcinogenesis. PMID:26232258

  13. Direct identification of all oncogenic mutants in KRAS exon 1 by cycling temperature capillary electrophoresis.

    PubMed

    Bjørheim, Jens; Gaudernack, Gustav; Giercksky, Karl-Erik; Ekstrøm, Per O

    2003-01-01

    Over the past few decades, advances in genetics and molecular biology have revolutionized our understanding of cancer initiation and progression. Molecular progression models outlining genetic events have been developed for many solid tumors, including colon cancer. Previous reports in the literature have shown a relationship between different KRAS mutations and prognosis and response to medical treatment in colon cancer patients. Furthermore, the presence of a mutated KRAS has been correlated with different clinicopathological variables including age and gender of patients and tumor location. To our knowledge, few institutions screen for KRAS mutations on regular basis in colon cancer patients despite such evidence that knowledge of KRAS exon 1 status is informative. Here, we report on a mutation analysis method adapted to a 96-capillary electrophoresis instrument that allows identification of all 12 oncogenic mutations in KRAS exon 1 under denaturing conditions. To determine the optimal parameters, a series of DNA constructs generated by site-directed mutagenesis was analyzed and the migration times of all mutant peaks were measured. A classification tree was then made based on the differences in migration time between the mutants and an internal standard. A randomized series of 500 samples constructed with mutagenesis as well as 60 blind samples from sporadic colon carcinomas was analyzed to test the method. No wild-type samples were scored as mutants and all mutants were correctly identified. Post polymerase chain reaction (PCR) analysis time of 96 samples was performed within 40 min. PMID:12652573

  14. A mouse model of melanoma driven by oncogenic KRAS

    PubMed Central

    Milagre, Carla; Dhomen, Nathalie; Geyer, Felipe C; Hayward, Robert; Lambros, Maryou; Reis-Filho, Jorge S; Marais, Richard

    2010-01-01

    The small G-protein NRAS is mutated in 22% of human melanomas, whereas the related proteins, KRAS and HRAS are mutated in only 2% and 1% of melanomas respectively. We have developed a mouse models of melanoma in which Cre recombinase/loxP technology is used to drive inducible expression of G12VKRAS in the melanocytic lineage. The mice develop skin hyper-pigmentation, nevi and tumors that bear many of the cardinal histopathology features and molecular characteristics of human melanoma. These tumors invade and destroy the underlying muscles and cells derived from them can grow as subcutaneous tumors and colonise the lungs of nude mice. These data establish that oncogenic KRAS can be a founder event in melanomagenesis. PMID:20516123

  15. Sensitive quantification of somatic mutations using molecular inversion probes.

    PubMed

    Hirani, Rena; Connolly, Ashley R; Putral, Lisa; Dobrovic, Alexander; Trau, Matt

    2011-11-01

    Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.

  16. 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

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

    PubMed

    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

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

    PubMed

    Perera, David; Venkitaraman, Ashok R

    2016-07-14

    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.

  19. Inhibition of KRAS-driven tumorigenicity by interruption of an autocrine cytokine circuit.

    PubMed

    Zhu, Zehua; Aref, Amir R; Cohoon, Travis J; Barbie, Thanh U; Imamura, Yu; Yang, Shenghong; Moody, Susan E; Shen, Rhine R; Schinzel, Anna C; Thai, Tran C; Reibel, Jacob B; Tamayo, Pablo; Godfrey, Jason T; Qian, Zhi Rong; Page, Asher N; Maciag, Karolina; Chan, Edmond M; Silkworth, Whitney; Labowsky, Mary T; Rozhansky, Lior; Mesirov, Jill P; Gillanders, William E; Ogino, Shuji; Hacohen, Nir; Gaudet, Suzanne; Eck, Michael J; Engelman, Jeffrey A; Corcoran, Ryan B; Wong, Kwok-Kin; Hahn, William C; Barbie, David A

    2014-04-01

    Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)-related kinases Tank-binding kinase-1 (TBK1) and IKKε promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKε inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKε promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKε, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS. PMID:24444711

  20. 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

  1. 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.

  2. Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

    PubMed Central

    Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

    2014-01-01

    The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

  3. Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway

    PubMed Central

    Conti, Annalisa; Majorini, Maria Teresa; Elliott, Richard; Ashworth, Alan; Lord, Christopher J.; Cancelliere, Carlotta; Bardelli, Alberto; Seneci, Pierfausto; Walczak, Henning; Delia, Domenico; Lecis, Daniele

    2015-01-01

    KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation. PMID:26028667

  4. IDH1 mutation detection by droplet digital PCR in glioma.

    PubMed

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation.

  5. IDH1 mutation detection by droplet digital PCR in glioma

    PubMed Central

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; Tony To, Shing-shun; Chen, Zhong-ping

    2015-01-01

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  6. Molecular and functional characteristics of ovarian surface epithelial cells transformed by KrasG12D and loss of Pten in a mouse model in vivo.

    PubMed

    Mullany, L K; Fan, H-Y; Liu, Z; White, L D; Marshall, A; Gunaratne, P; Anderson, M L; Creighton, C J; Xin, L; Deavers, M; Wong, K-K; Richards, J S

    2011-08-11

    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); Kras(G12D); Amhr2-Cre mice to disrupt the Pten gene and express a stable mutant form of Kras(G12D) in ovarian surface epithelial (OSE) cells. On the basis of 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 with wild-type OSE cells. Mapping of transcripts or genes between the mouse OSE mutant data sets, the Kras signature from human cancer cell lines and the human ovarian tumor array data sets, 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-suppressor Trp53 (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.

  7. 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...

  8. 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...

  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, 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...

  11. 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...

  12. IKK is a therapeutic target in KRAS-Induced lung cancer with disrupted p53 activity

    PubMed Central

    Bassères, Daniela S.; Ebbs, Aaron; Cogswell, Patricia C.; Baldwin, Albert S.

    2014-01-01

    Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. These results argue that targeting downstream effectors of RAS will be an alternative route for blocking RAS-driven oncogenic pathways. We and others have shown that oncogenic RAS activates the NF-κB transcription factor pathway and that KRAS-induced lung tumorigenesis is suppressed by expression of a degradation-resistant form of the IκBα inhibitor or by genetic deletion of IKKβ or the RELA/p65 subunit of NF-κB. Here, genetic and pharmacological approaches were utilized to inactivate IKK in human primary lung epithelial cells transformed by KRAS, as well as KRAS mutant lung cancer cell lines. Administration of the highly specific IKKβ inhibitor Compound A (CmpdA) led to NF-κB inhibition in different KRAS mutant lung cells and siRNA-mediated knockdown of IKKα or IKKβ reduced activity of the NF-κB canonical pathway. Next, we determined that both IKKα and IKKβ contribute to oncogenic properties of KRAS mutant lung cells, particularly when p53 activity is disrupted. Based on these results, CmpdA was tested for potential therapeutic intervention in the Kras-induced lung cancer mouse model (LSL-KrasG12D) combined with loss of p53 (LSL-KrasG12D/p53fl/fl). CmpdA treatment was well tolerated and mice treated with this IKKβ inhibitor presented smaller and lower grade tumors than mice treated with placebo. Additionally, IKKβ inhibition reduced inflammation and angiogenesis. These results support the concept of targeting IKK as a therapeutic approach for oncogenic RAS-driven tumors with altered p53 activity. PMID:24955217

  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. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

    PubMed Central

    Chattopadhyay, Nibedita; Berger, Allison J.; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  15. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

    PubMed

    Chattopadhyay, Nibedita; Berger, Allison J; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  16. Improved detection of mutated human cytomegalovirus UL97 by pyrosequencing.

    PubMed

    Schindele, Birgit; Apelt, Luise; Hofmann, Jörg; Nitsche, Andreas; Michel, Detlef; Voigt, Sebastian; Mertens, Thomas; Ehlers, Bernhard

    2010-12-01

    Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.

  17. KRAS Testing for Anti-EGFR Therapy in Advanced Colorectal Cancer

    PubMed Central

    2010-01-01

    Executive Summary In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario. Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenomics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports. The following reports can be publicly accessed at the MAS website at: www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.html Gene Expression Profiling for Guiding Adjuvant Chemotherapy Decisions in Women with Early Breast Cancer: An Evidence-Based and Economic Analysis Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based and Economic Analysis K-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based and Economic Analysis. Objective The objective of this systematic review is to determine the predictive value of KRAS testing in the treatment of metastatic colorectal cancer (mCRC) with two anti-EGFR agents, cetuximab and panitumumab. Economic analyses are also being conducted to evaluate the cost-effectiveness of KRAS testing. Clinical Need: Condition and Target Population Metastatic colorectal cancer (mCRC) is usually defined as stage IV disease according to the American Joint Committee on Cancer

  18. KRAS and the Reality of Personalized Medicine in Non-Small Cell Lung Cancer

    PubMed Central

    Kilgoz, Havva O; Bender, Guzide; Scandura, Joseph M; Viale, Agnes; Taneri, Bahar

    2016-01-01

    Lung cancer is the leading cause of mortality among all cancer types worldwide. The latest available global statistics of the World Health Organization report 1.59 million casualities in 2012. Worldwide, 1 in 5 cancer deaths are caused by lung cancer. In 2016, in the United States alone, there are an estimated 224,390 new cases of lung cancer, of which 158,080 are expected to result in death, as reported by the National Cancer Institute. Non-small cell lung cancer (NSCLC), a histological subtype, comprises about 85% of all cases, which is nearly 9 out of 10 lung cancer patients. Efforts are under way to develop and improve targeted therapy strategies. Certain mutations are being clinically targeted, such as those in EGFR and ALK genes. However, one of the most frequently mutated genes in NSCLC is the Kirsten rat sarcoma viral oncogene homolog (KRAS), which is currently not targetable. Approximately 25% of all types of NSCLC tumors contain KRAS mutations, which remain as an undruggable challenge. These mutations are indicative of poor prognosis and show negative response to standard chemotherapy. Furthermore, tumors harboring KRAS mutations are unlikely to respond to currently available targeted treatments such as tyrosine kinase inhibitors. Therefore, there is a definitive, urgent need to generate new targeted therapy approaches for KRAS mutations. Current strategies have major limitations and revolve around targeting molecules upstream and downstream of KRAS. Direct targeting is not available in the clinic. Combination therapies using multiple agents are being sought. Concentrated efforts are needed to accelerate basic research and consecutive clinical trials to achieve effective targeting of KRAS. PMID:27447490

  19. Combined targeting of EGFR/HER promotes anti-tumor efficacy in subsets of KRAS mutant lung cancer resistant to single EGFR blockade

    PubMed Central

    Umelo, Ijeoma Adaku; De Wever, Olivier; Kronenberger, Peter; Van Deun, Jan; Noor, Alfiah; Singh, Kshitiz; Teugels, Erik; Chen, Gang; Bracke, Marc; De Grève, Jacques

    2015-01-01

    KRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers. PMID:25992771

  20. MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms

    PubMed Central

    Amankwatia, E B; Chakravarty, P; Carey, F A; Weidlich, S; Steele, R J C; Munro, A J; Wolf, C R; Smith, G

    2015-01-01

    Background: Colorectal cancers arise from benign adenomas, although not all adenomas progress to cancer and there are marked interpatient differences in disease progression. We have previously associated KRAS mutations with disease progression and reduced survival in colorectal cancer patients. Methods: We used TaqMan low-density array (TLDA) qRT–PCR analysis to identify miRNAs differentially expressed in normal colorectal mucosa, adenomas and cancers and in isogeneic KRAS WT and mutant HCT116 cells, and used a variety of phenotypic assays to assess the influence of miRNA expression on KRAS activity, chemosensitivity, proliferation and invasion. Results: MicroRNA-224 was differentially expressed in dysplastic colorectal disease and in isogeneic KRAS WT and mutant HCT116 cells. Antagomir-mediated miR-224 silencing in HCT116 KRAS WT cells phenocopied KRAS mutation, increased KRAS activity and ERK and AKT phosphorylation. 5-FU chemosensitivity was significantly increased in miR-224 knockdown cells, and in NIH3T3 cells expressing KRAS and BRAF mutant proteins. Bioinformatics analysis of predicted miR-224 target genes predicted altered cell proliferation, invasion and epithelial–mesenchymal transition (EMT) phenotypes that were experimentally confirmed in miR-224 knockdown cells. Conclusions: We describe a novel mechanism of KRAS regulation, and highlight the clinical utility of colorectal cancer-specific miRNAs as disease progression or clinical response biomarkers. PMID:25919696

  1. 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.

  2. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33

    PubMed Central

    Azoitei, Ninel; Hoffmann, Christopher M.; Ellegast, Jana M.; Ball, Claudia R.; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A.; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno

    2012-01-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential. PMID:22451720

  3. Targeting of KRAS mutant tumors by HSP90 inhibitors involves degradation of STK33.

    PubMed

    Azoitei, Ninel; Hoffmann, Christopher M; Ellegast, Jana M; Ball, Claudia R; Obermayer, Kerstin; Gößele, Ulrike; Koch, Britta; Faber, Katrin; Genze, Felicitas; Schrader, Mark; Kestler, Hans A; Döhner, Hartmut; Chiosis, Gabriela; Glimm, Hanno; Fröhling, Stefan; Scholl, Claudia

    2012-04-01

    Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS, the most commonly mutated human oncogene, have not been successful. Cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, identifying STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33 are not yet available. Here, using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex binds to and stabilizes STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon tumor-initiating cells harboring mutant KRAS. These findings provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, and identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential.

  4. K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling.

    PubMed

    Wang, Man-Tzu; Holderfield, Matthew; Galeas, Jacqueline; Delrosario, Reyno; To, Minh D; Balmain, Allan; McCormick, Frank

    2015-11-19

    K-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca(2+) signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-Ras(V12)-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this "undruggable" protein.

  5. Oncogenic KRAS regulates BMP4 expression in colon cancer cell lines.

    PubMed

    Duerr, Eva-Maria; Mizukami, Yusuke; Moriichi, Kentaro; Gala, Manish; Jo, Won-Seok; Kikuchi, Hirotoshi; Xavier, Ramnik J; Chung, Daniel C

    2012-05-15

    Activating mutations in the KRAS oncogene are common in colorectal cancer. However, the complete spectrum of KRAS targets that mediate its tumorigenic effect has not yet been fully delineated. We identified bone morphogenetic protein 4 (Bmp4), a transforming growth factor-β family member that regulates development and tissue homeostasis, as a new target of KRAS. In SW480, Hela, and 293 cells, oncogenic KRAS(V12) downregulated BMP4 RNA levels, a BMP4 promoter luciferase construct, and Bmp4 protein levels. The MEK inhibitor PD98059 but not the phosphatidylinositol 3-kinase inhibitor LY294002 blocked this downregulation of BMP4. To identify the region of the BMP4 promoter that mediated this regulation by KRAS, serial 5'-deletions of the promoter were generated. An inhibitory region was identified between -3,285 and -3,258 bp in the Bmp4 promoter. In summary, oncogenic KRAS can downregulate Bmp4 through a transcriptional pathway that depends on ERK. These findings point to a unique link between two pathways that are frequently altered in colon cancer.

  6. EGFR-mediated chromatin condensation protects KRAS-mutant cancer cells against ionizing radiation.

    PubMed

    Wang, Meng; Kern, Ashley M; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H; Efstathiou, Jason A; Settleman, Jeff; Willers, Henning

    2014-05-15

    Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in vitro and in vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like colocalization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is codependent upon EGFR and protein kinase C α (PKCα). PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC.

  7. EGFR-Mediated Chromatin Condensation Protects KRAS-Mutant Cancer Cells Against Ionizing Radiation

    PubMed Central

    Wang, Meng; Kern, Ashley M.; Hülskötter, Marieke; Greninger, Patricia; Singh, Anurag; Pan, Yunfeng; Chowdhury, Dipanjan; Krause, Mechthild; Baumann, Michael; Benes, Cyril H.; Efstathiou, Jason A.; Settleman, Jeff; Willers, Henning

    2014-01-01

    Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non-small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in-vitro and in-vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like co-localization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is co-dependent upon EGFR and PKCα. PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. PMID:24648348

  8. Safety and efficacy of the addition of simvastatin to panitumumab in previously treated KRAS mutant metastatic colorectal cancer patients.

    PubMed

    Baas, Jara M; Krens, Lisanne L; Bos, Monique M; Portielje, Johanneke E A; Batman, Erdogan; van Wezel, Tom; Morreau, Hans; Guchelaar, Henk-Jan; Gelderblom, Hans

    2015-09-01

    Panitumumab has proven efficacy in patients with metastatic or locally advanced colorectal cancer patients, provided that they have no activating KRAS mutation in their tumour. Simvastatin blocks the mevalonate pathway and thereby interferes with the post-translational modification of KRAS. We hypothesize that the activity of the RAS-induced pathway in patients with a KRAS mutation might be inhibited by simvastatin. This would theoretically result in increased sensitivity to panitumumab, potentially comparable with tumours with wild-type KRAS. A Simon two-stage design single-arm, phase II study was designed to test the safety and efficacy of the addition of simvastatin to panitumumab in colorectal cancer patients with a KRAS mutation after failing fluoropyrimidine-based, oxaliplatin-based and irinotecan-based therapy. The primary endpoint of this study was the proportion of patients alive and free from progression 11 weeks after the first administration of panitumumab, aiming for at least 40%, which is comparable with, although slightly lower than, that in KRAS wild-type patients in this setting. If this 40% was reached, then the study would continue into the second step up to 46 patients. Explorative correlative analysis for mutations in the KRAS and related pathways was carried out. One of 14 patients was free from progression at the primary endpoint time. The median progression-free survival was 8.4 weeks and the median overall survival status was 19.6 weeks. We conclude that the concept of mutant KRAS phenotype expression modulation with simvastatin was not applicable in the clinic.

  9. Detecting ultralow-frequency mutations by Duplex Sequencing

    PubMed Central

    Kennedy, Scott R; Schmitt, Michael W; Fox, Edward J; Kohrn, Brendan F; Salk, Jesse J; Ahn, Eun Hyun; Prindle, Marc J; Kuong, Kawai J; Shen, Jiang-Cheng; Risques, Rosa-Ana; Loeb, Lawrence A

    2014-01-01

    Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 107 wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence ‘families’ that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1–3 d. PMID:25299156

  10. 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

  11. EVI1 oncogene promotes KRAS pathway through suppression of microRNA-96 in pancreatic carcinogenesis.

    PubMed

    Tanaka, M; Suzuki, H I; Shibahara, J; Kunita, A; Isagawa, T; Yoshimi, A; Kurokawa, M; Miyazono, K; Aburatani, H; Ishikawa, S; Fukayama, M

    2014-05-01

    Despite frequent KRAS mutation, the early molecular mechanisms of pancreatic ductal adenocarcinoma (PDAC) development have not been fully elucidated. By tracking a potential regulator of another feature of PDAC precursors, acquisition of foregut or gastric epithelial gene signature, we herein report that aberrant overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein, which is usually absent in normal pancreatic duct, is a widespread marker across the full spectrum of human PDAC precursors and PDAC. In pancreatic cancer cells, EVI1 depletion caused remarkable inhibition of cell growth and migration, indicating its oncogenic roles. Importantly, we found that EVI1 upregulated KRAS expression through suppression of a potent KRAS suppressor, miR-96, in pancreatic cancer cells. Collectively, the present findings suggest that EVI1 overexpression and KRAS mutation converge on activation of the KRAS pathway in early phases of pancreatic carcinogenesis and propose EVI1 and/or miR-96 as early markers and therapeutic targets in this dismal disease.

  12. Novel CDKN2A mutation detected in Spanish melanoma pedigree.

    PubMed

    de Torre, Carlos; Martínez-Escribano, Jorge

    2010-08-01

    We have examined alterations in the cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase 4 (CDK4), major melanoma predisposing genes, in a Spanish melanoma-prone population comprising 61 patients from 45 families. Using an extensive genetic analysis of these genes, including sequence analysis and multiplex ligation-dependent probe amplification, we have found four different CDKN2A alterations in cases from seven melanoma kindred. Three of them are CDKN2A mutations previously described in the Mediterranean population (p.G101W, p.V59G and c.358delG) in addition to an undescribed deletion (p. M54del) which has been detected in a melanoma kindred. This codon deletion affects an essential residue in the interaction of p16INK4A with cdk6 and has not been reported in melanoma patients and other cancers. PMID:20653773

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

    PubMed

    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

    2016-01-01

    Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification. 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.

  14. Practicability of detecting somatic point mutation from RNA high throughput sequencing data.

    PubMed

    Sheng, Quanhu; Zhao, Shilin; Li, Chung-I; Shyr, Yu; Guo, Yan

    2016-05-01

    Traditionally, somatic mutations are detected by examining DNA sequence. The maturity of sequencing technology has allowed researchers to screen for somatic mutations in the whole genome. Increasingly, researchers have become interested in identifying somatic mutations through RNAseq data. With this motivation, we evaluated the practicability of detecting somatic mutations from RNAseq data. Current somatic mutation calling tools were designed for DNA sequencing data. To increase performance on RNAseq data, we developed a somatic mutation caller GLMVC based on bias reduced generalized linear model for both DNA and RNA sequencing data. Through comparison with MuTect and Varscan we showed that GLMVC performed better for somatic mutation detection using exome sequencing or RNAseq data. GLMVC is freely available for download at the following website: https://github.com/shengqh/GLMVC/wiki.

  15. 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

    2016-08-01

    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. PMID:27477280

  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. 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

  18. Insights into K-Ras 4B regulation by post-translational lysine acetylation.

    PubMed

    Knyphausen, Philipp; Lang, Franziska; Baldus, Linda; Extra, Antje; Lammers, Michael

    2016-10-01

    Ras is a molecular switch cycling between an active, GTP-bound and an inactive, GDP-bound state. Mutations in Ras, mostly affecting the off-switch, are found in many human tumours. Recently, it has been shown that K-Ras 4B is targeted by lysine acetylation at K104. Based on results obtained for an acetylation mimetic Ras mutant (K104Q), it was hypothesised that K104-acetylation might interfere with its oncogenicity by impairing SOS-catalysed guanine-nucleotide exchange. We prepared site-specifically K104-acetylated K-Ras 4B and the corresponding oncogenic mutant protein G12V using the genetic-code expansion concept. We found that SOS-catalysed nucleotide exchange, also of allosterically activated SOS, was neither affected by acetylation of K104 in wildtype K-Ras 4B nor in the G12V mutant, suggesting that glutamine is a poor mimetic for acetylation at this site. In vitro, the lysine-acetyltransferases CBP and p300 were able to acetylate both, wildtype and G12V K-Ras 4B. In addition to K104 we identified further acetylation sites in K-Ras 4B, including K147, within the important G5/SAK-motif. However, the intrinsic and the SOS-catalysed nucleotide exchange was not affected by K147-acetylation of K-Ras 4B. Finally, we show that Sirt2 and HDAC6 do neither deacetylate K-Ras 4B if acetylated at K104 nor if acetylated at K147 in vitro.

  19. High Resolution Melting Analysis: A Rapid and Accurate Method to Detect CALR Mutations

    PubMed Central

    Moreno, Melania; Torres, Laura; Santana-Lopez, Gonzalo; Rodriguez-Medina, Carlos; Perera, María; Bellosillo, Beatriz; de la Iglesia, Silvia; Molero, Teresa; Gomez-Casares, Maria Teresa

    2014-01-01

    Background The recent discovery of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN). We tested the feasibility of high-resolution melting (HRM) as a screening method for rapid detection of CALR mutations. Methods CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET. Results Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34), 14% of persistent thrombocytosis suggestive of MPN (3/21) and none of the secondary thrombocytosis (0/98). Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%. Conclusions This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations. PMID:25068507

  20. 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.

  1. 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.

  2. Mutations associated with carcinomas arising from pleomorphic adenomas of the salivary glands.

    PubMed

    Yamamoto, Y; Kishimoto, Y; Virmani, A K; Smith, A; Vuitch, F; Albores-Saavedra, J; Gazdar, A F

    1996-08-01

    Pleomorphic adenoma (PA) is the most common benign tumor of salivary glands. Carcinomas in pleomorphic adenomas (CPAs) may arise by malignant transformation of the epithelial components of PAs. Occasionally, transitional zones containing cells with histological features intermediate between those of the benign PA and carcinomatous components of CPA are identified. After careful microdissection of archival microslides, the authors studied 12 cases of CPAs and their attendant adenomatous and transitional areas for mutations in the p53, RB, and K-ras genes, and at chromosomal loci 5q and 9p. The authors failed to find mutations in the K-ras gene or 9p locus. A relatively high rate of mutations (loss of heterozygosity [LOH] and microsatellite alterations) at the p53 gene were detected in CPAs (58%), and at somewhat lower frequencies at the RB gene (33%) and chromosomal location 5q (17%). Mutational frequency in the associated transitional and adenomatous areas were slightly lower than in the corresponding CPAs. No mutations were detected in adenomatous or transitional areas unless they also were present in the corresponding CPAs. Mutations of these three genes were absent in four cases of CPA, and in seven PAs without malignant change. These findings indicate that most CPAs arise from adenomas as the result of mutations in the three genes, especially p53. In addition, other, as yet unidentified genes may also be involved both in the development of PA and in its malignant progression to CPA. Mutational analysis of PAs may provide information of prognostic importance.

  3. Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis.

    PubMed

    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

    2015-03-03

    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 Kras(G12D)-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.

  4. 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

  5. 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

  6. Sensitivity of conformation sensitive gel electrophoresis in detecting mutations in Marfan syndrome and related conditions

    PubMed Central

    Korkko, J; Kaitila, I; Lonnqvist, L; Peltonen, L; Ala-Kokko, L

    2002-01-01

    Design: Setting up CSGE analysis for the FBN1 gene and testing the method first by screening coded samples from 17 MFS patients with previously detected FBN1 mutations. We then used a test set consisting of 46 coded samples representing MFS, related phenotypes, and controls. Results: Sixteen of the 17 known mutations were detected. Altogether 23 mutations were detected in a test set consisting of 46 coded samples representing MFS, related phenotypes, and controls. Nineteen of the mutations were novel. The mutation was detected in 18 of the 20 MFS patients and in one patient with familial EL, but not in a patient with sporadic MASS syndrome, any of the five sporadic annuloaortic ectasia (AAE) patients, or any of the 15 controls. A FBN1 mutation was detected in four members of a multigeneration family with AAE, however. Conclusions: These results indicate that CSGE is highly sensitive for the detection of mutations in FBN1, and that molecular diagnostics is a useful means of confirming clinical diagnoses of MFS and related disorders. Further careful investigations are needed, however, in order to correlate the interfamilial and intrafamilial clinical variabilities of fibrillinopathies and mutations in FBN1. PMID:11826022

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

    PubMed

    Lee, Sojung; Kang, Jungwoo; Cho, Minchul; Seo, Eunhee; Choi, Heesook; Kim, Eunjin; Kim, Junghee; Kim, Heejong; Kang, Gum Yong; Kim, Kwang Pyo; Park, Young-Ho; Yu, Dae-Yeul; Yum, Young Na; Park, Sue-Nie; Yoon, Do-Young

    2009-01-01

    The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as

  12. Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

    PubMed

    Lee, Sojung; Kang, Jungwoo; Cho, Minchul; Seo, Eunhee; Choi, Heesook; Kim, Eunjin; Kim, Junghee; Kim, Heejong; Kang, Gum Yong; Kim, Kwang Pyo; Park, Young-Ho; Yu, Dae-Yeul; Yum, Young Na; Park, Sue-Nie; Yoon, Do-Young

    2009-01-01

    The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as

  13. External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds

    PubMed Central

    Bellon, Ellen; Ligtenberg, Marjolijn J.L.; Tejpar, Sabine; Cox, Karen; de Hertogh, Gert; de Stricker, Karin; Edsjö, Anders; Gorgoulis, Vassilis; Höfler, Gerald; Jung, Andreas; Kotsinas, Athanassios; Laurent-Puig, Pierre; López-Ríos, Fernando; Hansen, Tine Plato; Rouleau, Etienne; Vandenberghe, Peter; van Krieken, Johan J.M.

    2011-01-01

    The use of epidermal growth factor receptor–targeting antibodies in metastatic colorectal cancer has been restricted to patients with wild-type KRAS tumors by the European Medicines Agency since 2008, based on data showing a lack of efficacy and potential harm in patients with mutant KRAS tumors. In an effort to ensure optimal, uniform, and reliable community-based KRAS testing throughout Europe, a KRAS external quality assessment (EQA) scheme was set up. The first large assessment round included 59 laboratories from eight different European countries. For each country, one regional scheme organizer prepared and distributed the samples for the participants of their own country. The samples included unstained sections of 10 invasive colorectal carcinomas with known KRAS mutation status. The samples were centrally validated by one of two reference laboratories. The laboratories were allowed to use their own preferred method for histological evaluation, DNA isolation, and mutation analysis. In this study, we analyze the setup of the KRAS scheme. We analyzed the advantages and disadvantages of the regional scheme organization by analyzing the outcome of genotyping results, analysis of tumor percentage, and written reports. We conclude that only 70% of laboratories correctly identified the KRAS mutational status in all samples. Both the false-positive and false-negative results observed negatively affect patient care. Reports of the KRAS test results often lacked essential information. We aim to further expand this program to more laboratories to provide a robust estimate of the quality of KRAS testing in Europe, and provide the basis for remedial measures and harmonization. PMID:21441573

  14. Enhanced ratio of signals enables digital mutation scanning for rare allele detection.

    PubMed

    Castellanos-Rizaldos, Elena; Paweletz, Cloud; Song, Chen; Oxnard, Geoffrey R; Mamon, Harvey; Jänne, Pasi A; Makrigiorgos, G Mike

    2015-05-01

    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.

  15. 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. PMID:26322949

  16. 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

  17. 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

  18. Immunohistochemistry is highly sensitive and specific for the detection of NRASQ61R mutation in melanoma.

    PubMed

    Massi, Daniela; Simi, Lisa; Sensi, Elisa; Baroni, Gianna; Xue, Gongda; Scatena, Cristian; Caldarella, Adele; Pinzani, Pamela; Fontanini, Gabriella; Carobbio, Alessandra; Urso, Carmelo; Mandalà, Mario

    2015-04-01

    Testing for NRAS is now integral part in the assessment of metastatic melanoma patients because there is evidence that NRAS-mutated patients may be sensitive to MEK inhibitors, and RAS mutation is a common mechanism of acquired resistance during treatment with BRAF inhibitors. This study evaluated the sensitivity and specificity of immunohistochemical analysis using an N-Ras (Q61R) antibody to detect the presence of the NRASQ61R mutation in melanoma patients. A total of 98 primary cutaneous melanomas that have undergone examination of NRAS mutation were retrieved from a multicentric database. Formalin-fixed and paraffin-embedded melanoma tissues were analyzed for BRAF and NRAS mutations by independent, blinded observers using both conventional DNA molecular techniques and immunohistochemistry with the novel anti-human N-Ras (Q61R) monoclonal antibody (clone SP174). The antibody showed a sensitivity of 100% (14/14) and a specificity of 100% (83/83) for detecting the presence of an NRASQ61R mutation. Of the NRAS-mutated cases, none of the non-Q61R cases stained positive with the antibody (0/7). There were three cases with discordant NRAS mutational results. Additional molecular analysis confirmed the immunohistochemically obtained NRAS result in all cases, suggesting that a multiple analytical approach can be required to reach the correct sample classification. The reported immunohistochemical method is an accurate, rapid, and cost-effective method for detecting NRASQ61R mutation in melanoma patients, and represents a valuable supplement to traditional mutation testing. If validated in further studies, genetic testing would only be required for immunohistochemistry-negative patients to detect non-Q61R mutations.

  19. Loss of Dnmt3a and endogenous KrasG12D/+ cooperate to regulate hematopoietic stem and progenitor cell functions in leukemogenesis

    PubMed Central

    Chang, Yuan-I; You, Xiaona; Kong, Guangyao; Ranheim, Erik A.; Wang, Jinyong; Du, Juan; Liu, Yangang; Zhou, Yun; Ryu, Myung-Jeom; Zhang, Jing

    2015-01-01

    Oncogenic NRAS and KRAS mutations are prevalent in human juvenile and chronic myelomonocytic leukemia (JMML/CMML). However, additional genetic mutations cooperating with oncogenic RAS in JMML/CMML progression and/or their transformation to acute myeloid leukemia (AML) remain largely unknown. Here, we tested the potential genetic interaction of DNMT3A mutations and oncogenic RAS mutations in leukemogenesis. We found that Dnmt3a−/− induces multiple hematopoietic phenotypes after a prolonged latency, including T cell expansion in peripheral blood, stress erythropoiesis in spleen, and myeloid malignancies in liver. Dnmt3a−/− significantly promoted JMML/CMML progression and shortened the survival of KrasG12D/+ mice in a cell-autonomous manner. Similarly, downregulating Dnmt3a also promoted myeloid malignancies in NrasG12D/+ mice. Further studies show that Dnmt3a deficiency rescues KrasG12D/+-mediated depletion of hematopoietic stem cells and increases self-renewal of KrasG12D/+ myeloid progenitors. Moreover, ~33% of animals developed an AML-like disease, which is driven by KrasG12D/+; Dnmt3a−/− myeloid progenitors. Consistent with our result, COSMIC database mining demonstrates that the combination of oncogenic RAS and DNMT3A mutations exclusively occurred in patients with JMML, CMML, or AML. Our results suggest that DNMT3A mutations and oncogenic RAS cooperate to regulate hematopoietic stem and progenitor cells and promote myeloid malignancies. PMID:25801914

  20. Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China.

    PubMed

    Zhang, Min; Yue, Jun; Yang, Yan-Ping; Zhang, Hong-Mei; Lei, Jian-Qiang; Jin, Rui-Liang; Zhang, Xue-Lian; Wang, Hong-Hai

    2005-11-01

    Nine structural genes (furA, katG, inhA, kasA, Rv0340, iniB, iniA, iniC, and efpA) and two regulatory regions (the oxyR-ahpC intergenic region and the promoter of mabA-inhA) in 87 isoniazid (INH)-monoresistant and 50 INH-susceptible Mycobacterium tuberculosis isolates collected from five provinces of China were analyzed by sequencing. Eighty-two (94.3%) INH-resistant isolates had mutations in the katG gene, with the katG Ser315Thr mutation predominant (55.2%). No mutation at codon 463 of katG was detected among the 50 INH-susceptible isolates with different IS6110 fingerprints. In addition, there were 35 (40.2%) INH-resistant isolates that had a mutation at codon 463 of katG. Of the INH-resistant strains, 20 (23.0%) isolates harbored double mutations at two separate loci of katG. Mutations in the inhA promoter region occurred in 13 (14.9%) isolates; 4.6% of the isolates had inhA structural gene mutations, and 11.5% harbored mutations in the oxyR-ahpC intergenic region. Drug resistance-associated mutations were detected in the iniBAC region and efpA. PMID:16272473

  1. 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.

  2. Use of monoclonal antibodies to detect specific mutations in formalin-fixed, paraffin-embedded tissue sections.

    PubMed

    Guo, Zhenying; Lloyd, Ricardo V

    2016-07-01

    Treatment options for cancer patients have changed considerably in recent years with the introduction of variable gene mutation and targeted therapy. Although molecular testing for gene mutations remains the gold standard in assessing biopsy tissues for specific mutations and for subsequent therapy, recent developments have led to the use of highly specific monoclonal antibodies to detect mutated genes in tissue sections. Some of the early developments included antibodies against EGFR, but have expanded to include antibodies detecting mutated RAS, BRAF, and SDHx. Immunohistochemical detection of gene mutations using mutation-specific antibodies has the advantage of allowing the detailed visualization of protein distributions in situ and provides direct visualization of the heterogeneity in the distribution of targeted proteins. This review will discuss the use of selected mainly monoclonal antibodies targeting specific mutated molecules and indicate how the detection of these proteins can be used for chemotherapeutic purposes in targeting mutated genes. PMID:27083401

  3. Copy number variants and rasopathies: germline KRAS duplication in a patient with syndrome including pigmentation abnormalities.

    PubMed

    Gilbert-Dussardier, Brigitte; Briand-Suleau, Audrey; Laurendeau, Ingrid; Bilan, Frédéric; Cavé, Hélène; Verloes, Alain; Vidaud, Michel; Vidaud, Dominique; Pasmant, Eric

    2016-01-01

    RAS/MAPK pathway germline mutations were described in Rasopathies, a class of rare genetic syndromes combining facial abnormalities, heart defects, short stature, skin and genital abnormalities, and mental retardation. The majority of the mutations identified in the Rasopathies are point mutations which increase RAS/MAPK pathway signaling. Duplications encompassing RAS/MAPK pathway genes (PTPN11, RAF1, MEK2, or SHOC2) were more rarely described. Here we report, a syndromic familial case of a 12p duplication encompassing the dosage sensitive gene KRAS, whose phenotype overlapped with rasopathies. The patient was referred because of a history of mild learning disabilities, small size, facial dysmorphy, and pigmentation abnormalities (café-au-lait and achromic spots, and axillar lentigines). This phenotype was reminiscent of rasopathies. No mutation was identified in the most common genes associated with Noonan, cardio-facio-cutaneous, Legius, and Costello syndromes, as well as neurofibromatosis type 1. The patient constitutional DNA exhibited a ~10.5 Mb duplication at 12p, including the KRAS gene. The index case's mother carried the same chromosome abnormality and also showed development delay with short stature, and numerous café-au-lait spots. Duplication of the KRAS gene may participate in the propositus phenotype, in particular of the specific pigmentation abnormalities. Array-CGH or some other assessment of gene/exon CNVs of RAS/MAPK pathway genes should be considered in the evaluation of individuals with rasopathies. PMID:27450488

  4. Combination therapy with zoledronic acid and cetuximab effectively suppresses growth of colorectal cancer cells regardless of KRAS status

    PubMed Central

    Kato, Junko; Kanematsu, Masako; Gaowa, Siqin; Mori, Ryutaro; Tanahashi, Toshiyuki; Matsuhashi, Nobuhisa; Yoshida, Kazuhiro

    2016-01-01

    Targeted molecular therapy is an effective anticancer strategy. Anti‐EGFR monoclonal antibodies such as cetuximab (CTX) have been approved for the treatment of various malignancies, including colorectal cancer (CRC) with wild‐type KRAS. However, their efficacy in patients with KRAS mutations has not been established. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. CRC cell lines SW48 (wild‐type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX and a combination of both drugs. Cytotoxicity was measured using the MTT assay. Changes in the levels of intracellular signaling proteins were evaluated using western blot analysis. Finally, we evaluated the efficacy of the combination treatment in an in vivo xenograft model. We observed that ZOL apparently inhibited growth in both cell lines, whereas CTX showed little effect. ZOL also increased the levels of unprenylated RAS. Combined ZOL and CTX treatment was synergistic in both cell lines and was associated with inhibition of the RAS‐MAPK and AKT‐mTOR signaling pathways. Furthermore, the combination treatment was more effective in suppressing the growth of xenografts derived from both SW48 and LS174T cells; this effect was associated with increased apoptosis. These results demonstrate that ZOL inhibits the growth of colon cancer cells regardless of KRAS status, and combination therapy using ZOL and CTX enhances this growth suppression. These findings suggest a novel strategy for the treatment of CRC independent of KRAS mutational status. PMID:26437179

  5. 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

  6. Deciphering intra-tumor heterogeneity of lung adenocarcinoma confirms that dominant, branching, and private gene mutations occur within individual tumor nodules.

    PubMed

    Pelosi, Giuseppe; Pellegrinelli, Alessio; Fabbri, Alessandra; Tamborini, Elena; Perrone, Federica; Settanni, Giulio; Busico, Adele; Picciani, Benedetta; Testi, Maria Adele; Militti, Lucia; Maisonneuve, Patrick; Valeri, Barbara; Sonzogni, Angelica; Proto, Claudia; Garassino, Marina; De Braud, Filippo; Pastorino, Ugo

    2016-06-01

    While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance.

  7. Deciphering intra-tumor heterogeneity of lung adenocarcinoma confirms that dominant, branching, and private gene mutations occur within individual tumor nodules.

    PubMed

    Pelosi, Giuseppe; Pellegrinelli, Alessio; Fabbri, Alessandra; Tamborini, Elena; Perrone, Federica; Settanni, Giulio; Busico, Adele; Picciani, Benedetta; Testi, Maria Adele; Militti, Lucia; Maisonneuve, Patrick; Valeri, Barbara; Sonzogni, Angelica; Proto, Claudia; Garassino, Marina; De Braud, Filippo; Pastorino, Ugo

    2016-06-01

    While pulmonary adenocarcinoma (ADC) is morphologically heterogeneous, little is known about intra-tumor gene mutation heterogeneity (ITH). We therefore subjected 20 ADC nodules, 5 mutated for EGFR and 5 for KRAS, 5 with an ALK translocation, and 5 wild type (WT) for these alterations, to unsupervised next-generation sequencing of tumor regions from diverse architectural patterns. When 2 or more different gene mutations were found in a single tumor, this fulfilled the criteria for ITH. In the 84 studied tumor regions with diverse architecture, 71 gene mutations and 34 WT profiles were found. ITH was observed in 9/15 (60 %) ADC, 3 with an EGFR, 3 with a KRAS, and 3 with an ALK aberration, as reflected in 5, 6, and 9 additional mutations, respectively, detected in these tumors. EGFR mutations were observed in 21/22 and KRAS mutations in 18/22 tumor regions, suggesting that they appear early and have a driver role (dominant or trunk mutations). Branching mutations (in EZH2, PIK3CA, TP53, and EGFR exon 18) occurred in two or more regions, while private mutations (in ABL1, ALK, BRAF, HER2, KDR, LKB1, PTEN, MET, SMAD4, SMARCB1, and SRC) were confined to unique tumor samples of individual lesions, suggesting that they occurred later on during tumor progression. Patients with a tumor showing branching mutations ran a worse clinical course, independent of confounding factors. We conclude that in ADC, ITH exists in a pattern suggesting spatial and temporal hierarchy with dominant, branching, and private mutations. This is consistent with diverse intra-tumor clonal evolution, which has potential implications for patient prognosis or development of secondary therapy resistance. PMID:27056568

  8. 5-Fluorouracil preferentially sensitizes mutant KRAS non-small cell lung carcinoma cells to TRAIL-induced apoptosis.

    PubMed

    Wang, Haizhen; Yang, Tao; Wu, Xiangwei

    2015-11-01

    Mutations in the KRAS gene are very common in non-small cell lung cancer (NSCLC), but effective therapies targeting KRAS have yet to be developed. Interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent inducer of cell death, has increased following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and in xenograft models. However, results from clinical trials of TRAIL-based therapy are disappointingly modest at best and many have demonstrated a lack of therapeutic benefit. Current research has focused on selecting a subpopulation of cancer patients who may benefit from TRAIL-based therapy and identifying best drugs to work with TRAIL. In the current study, we found that NSCLC cells with a KRAS mutation were highly sensitive to treatment with TRAIL and 5-fluorouracil (5FU). Compared with other chemotherapeutic agents, 5FU displayed the highest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells. We also found that, on a mechanistic level, 5FU preferentially repressed survivin expression and induced expression of TRAIL death receptor 5 to sensitize NSCLC cells to TRAIL. The combination of low-dose 5FU and TRAIL strongly inhibited xenograft tumor growth in mice. Our results suggest that the combination of TRAIL and 5FU may be beneficial for patients with mutant KRAS NSCLC.

  9. 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

  10. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing

    PubMed Central

    Walsh, Tom; Lee, Ming K.; Casadei, Silvia; Thornton, Anne M.; Stray, Sunday M.; Pennil, Christopher; Nord, Alex S.; Mandell, Jessica B.; Swisher, Elizabeth M.; King, Mary-Claire

    2010-01-01

    Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, “next-generation” sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer. PMID:20616022

  11. Megalencephaly Syndromes: Exome Pipeline Strategies for Detecting Low-Level Mosaic Mutations

    PubMed Central

    Tapper, William J.; Foulds, Nicola; Cross, Nicholas C. P.; Aranaz, Paula; Score, Joannah; Hidalgo-Curtis, Claire; Robinson, David O.; Gibson, Jane; Ennis, Sarah; Temple, I. Karen; Collins, Andrew

    2014-01-01

    Two megalencephaly (MEG) syndromes, megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyriapolydactyly-hydrocephalus (MPPH), have recently been defined on the basis of physical and neuroimaging features. Subsequently, exome sequencing of ten MEG cases identified de-novo postzygotic mutations in PIK3CA which cause MCAP and de-novo mutations in AKT and PIK3R2 which cause MPPH. Here we present findings from exome sequencing three unrelated megalencephaly patients which identified a causal PIK3CA mutation in two cases and a causal PIK3R2 mutation in the third case. However, our patient with the PIK3R2 mutation which is considered to cause MPPH has a marked bifrontal band heterotopia which is a feature of MCAP. Furthermore, one of our patients with a PIK3CA mutation lacks syndactyly/polydactyly which is a characteristic of MCAP. These findings suggest that the overlap between MCAP and MPPH may be greater than the available studies suggest. In addition, the PIK3CA mutation in one of our patients could not be detected using standard exome analysis because the mutation was observed at a low frequency consistent with somatic mosaicism. We have therefore investigated several alternative methods of exome analysis and demonstrate that alteration of the initial allele frequency spectrum (AFS), used as a prior for variant calling in samtools, had the greatest power to detect variants with low mutant allele frequencies in our 3 MEG exomes and in simulated data. We therefore recommend non-default settings of the AFS in combination with stringent quality control when searching for causal mutation(s) that could have low levels of mutant reads due to post-zygotic mutation. PMID:24497998

  12. BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens.

    PubMed

    Thomas, Carla; Amanuel, Benhur; Finlayson, Jill; Grieu-Iacopetta, Fabienne; Spagnolo, Dominic V; Erber, Wendy N

    2015-06-01

    Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

  13. Mutation detection in autosomal dominant Hirschsprung disease: SSCP analysis of the RET proto-oncogene

    SciTech Connect

    Angrist, M.; Bolk, S.; Chakravarti, A.

    1994-09-01

    Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction, with an incidence of 1 in 5000. Recently, linkage of an incompletely penetrant, dominant form of HSCR to the pericentromeric region of chromosome 10 was reported, followed by identification of mutations in the RET proto-oncogene in HSCR patients. RET mutations have also been reported in both sporadic and familial forms of three neuroendrocrine tumor syndromes. Unlike the clustered RET mutations observed in these syndromes, the 18 reported HSCR mutations are distributed throughout the extracellular and tryosine kinase domains of RET. In an effort to determine the frequency of RET mutations in HSCR and correlate genotype with phenotype, we have begun to screen for mutations among 80 HSCR probands representing a wide range of phenotypes and pedigree structures. Non-isotopic single strand conformation of polymorphism (SSCP) analysis was carried out using the Pharmacia PhastSystem{trademark}. Initial screening of exons 2 through 6 detected variants in 11 patients not seen in 24 controls. One additional band shift in exon 6 has been observed in both patients and controls. Preliminary sequence analysis has revealed two putative familial mutations in exon 2: a single base pair deletion (49Pro del C 296) and a point mutation that leads to a conservative amino acid substitution (93Gly{r_arrow}Ser). These results suggest that HSCR may be associated with a range of alterations in the coding sequence of the RET extracellular domain. Additional mutations will be described.

  14. Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

    PubMed

    Bockstahler, L E; Li, Z; Nguyen, N Y; Van Houten, K A; Brennan, M J; Langone, J J; Morris, S L

    2002-03-01

    The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h. PMID:11926172

  15. 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

  16. Mutation profiling in chinese patients with metastatic colorectal cancer and its correlation with clinicopathological features and anti-EGFR treatment response.

    PubMed

    Li, Zhe-Zhen; Wang, Feng; Zhang, Zi-Chen; 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

    2016-05-10

    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

  17. 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

  18. 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.

  19. [Glandular papilloma of the right main bronchus. Detection of an exon 2 mutation of the KRAS gene (c.35G>A)].

    PubMed

    Wohlschläger, J; Welter, S; Stamatis, G; Theegarten, D; Hager, T; Mairinger, F; Worm, K; Schmid, K W; Müller, K M

    2013-07-01

    Benign epithelial tumors of the tracheobronchial system and the lungs are exceedingly rare. These entities encompass squamous and glandular papillomas (as well as their mixed forms) and adenomas (alveolar adenoma, papillary adenoma, salivary gland-like pleomorphic and mucinous adenomas and mucinous cystadenomas). These tumors are considered to be biologically benign neoplasms; however, they can pose considerable diagnostic difficulties, especially during frozen section evaluation, as they can mimic malignant tumors and in particular they can resemble well differentiated papillary adenocarcinomas. As a result of the extreme rarity of these tumors only a few descriptive diagnostic series exist and a systematic investigation including molecular data does not exist. This article presents the case of a 64-year-old patient with a glandular papilloma of the right main bronchus including the immunohistochemical and molecular work-up as well as a review of the current literature.

  20. Genetic events that limit the efficacy of MEK and RTK inhibitor therapies in a mouse model of KRAS-driven pancreatic cancer.

    PubMed

    Pettazzoni, Piergiorgio; Viale, Andrea; Shah, Parantu; Carugo, Alessandro; Ying, Haoqiang; Wang, Huamin; Genovese, Giannicola; Seth, Sahil; Minelli, Rosalba; Green, Tessa; Huang-Hobbs, Emmet; Corti, Denise; Sanchez, Nora; Nezi, Luigi; Marchesini, Matteo; Kapoor, Avnish; Yao, Wantong; Francesco, Maria E Di; Petrocchi, Alessia; Deem, Angela K; Scott, Kenneth; Colla, Simona; Mills, Gordon B; Fleming, Jason B; Heffernan, Timothy P; Jones, Philip; Toniatti, Carlo; DePinho, Ronald A; Draetta, Giulio F

    2015-03-15

    Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS.

  1. Genetic events that limit the efficacy of MEK and RTK inhibitor therapies in a mouse model of KRAS-driven pancreatic cancer

    PubMed Central

    Pettazzoni, Piergiorgio; Viale, Andrea; Shah, Parantu; Carugo, Alessandro; Ying, Haoqiang; Wang, Huamin; Genovese, Giannicola; Seth, Sahil; Minelli, Rosalba; Green, Tessa; Huang-Hobbs, Emmet; Corti, Denise; Sanchez, Nora; Nezi, Luigi; Marchesini, Matteo; Kapoor, Avnish; Yao, Wantong; Di Francesco, Maria Emilia; Petrocchi, Alessia; Deem, Angela K.; Scott, Kenneth; Colla, Simona; Mills, Gordon B.; Fleming, Jason B.; Heffernan, Timothy P.; Jones, Philip; Toniatti, Carlo; DePinho, Ronald A.; Draetta, Giulio F.

    2014-01-01

    Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction to pharmacological inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis while MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K-AKT-MTOR pathway and by the activation of AXL, PDGFRa, and HER1-2 receptor tyrosine kinases (RTKs) expressed in a large proportion of human PDAC samples analyzed. While single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three co-activated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. Additionally, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. PMID:25736685

  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. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  4. [Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer].

    PubMed

    Shuai, Sun; Yuliang, Deng

    2015-12-01

    Circulating tumor cells (CTCs) are cells that shed from a primary tumor and enter the peripheral blood circulation. The CTCs are closely associated with tumor development and metastasis because of its high heterogeneity. However, there are still no effective methods to detect single-cell heterogeneity of the CTCs. To this end, we developed a method to detect gene mutation in CTCs at the single-cell level and applied it to the detection of EGFR gene mutation in single lung cancer CTC. Specifically, the rare CTCs were captured from blood using an integrated microfluidic system, and then were released into a microchip with thousands of nanoliter wells to isolate single CTC. The single CTC was then transferred into a PCR tube under the microscope for single-cell genome amplification and detection of EGFR gene mutation. We firstly modified chip and capillary and optimized PCR conditions (annealing temperature, number of cycles) using non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H1650 and NCI-H1975 as samples, which showed maximal amplification after 30 cycles with an annealing temperature at 59℃. We then successfully detected blood samples from NSCLC patients using this method. 5 CTCs were obtained from 2 mL patient's blood and the sequencing of EGFR exons 18, 19, 20 and 21 showed no mutations. Our results demonstrated that this method is sensitive enough to detect gene mutation in single CTC and has guiding significance in clinic research. PMID:26704950

  5. ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis.

    PubMed

    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; Simeone, Diane M

    2015-01-15

    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.

  6. Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis

    SciTech Connect

    Gayther, S.A.; Harrington, P.; Russell, P.

    1996-03-01

    Germ-line mutations of the BRCA1 gene are responsible for a substantial proportion of families with multiple cases of early-onset breast and/or ovarian cancer. Since the isolation of BRCA1 last year, >65 distinct mutations scattered throughout the coding region have been detected, making analysis of the gene time consuming and technically challenging. We have developed a multiplex heteroduplex analysis that is designed to analyze one-quarter of the coding sequence in a single-step screening procedure and that will detect {approximately}50% of all BRCA1 mutations so far reported in breast/ovarian cancer families. We have used this technique to analyze BRCA1 in 162 families with a history of breast and/or ovarian cancer and identified 12 distinct mutations in 35 families. 20 refs., 2 figs., 2 tabs.

  7. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. PMID:26867649

  8. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers.

  9. Novel technique for rapid detection of alpha-globin gene mutations and deletions.

    PubMed

    Liu, Jingzhong; Jia, Xingyuan; Tang, Ning; Zhang, Xu; Wu, Xiaoyi; Cai, Ren; Wang, Lirong; Liu, Quanzhang; Xiao, Bai; Zhu, Jim; Wang, Qingtao

    2010-03-01

    Populations in Southeast Asia and South China have high frequencies of alpha-thalassemia caused by alpha-globin gene mutations and/or deletions. This study was designed to find an efficient and simple diagnostic test for the mutations and deletions. A duplex polymerase chain reaction (PCR)/denaturing high-pressure liquid chromatography (DHPLC) was used to detect the mutations and deletions. A blinded study of 110 samples, which included 92 alpha-thalassemia samples with various genotypes and 18 normal DNA samples, was carried out by the methods. The duplex PCR products of the sample with known Constand spring mutation (CS)/alphaalpha, Quonsze mutation (QS)/alphaalpha, and Weastmead mutation (WS)/alphaalpha DNA showed significantly different profiles, which suggests that DHPLC analysis at 63.8 degrees C can detect potential mutations directly. The DHPLC at 50 degrees C analysis can distinguish the --SEA and nondeletional alleles. The new assay is 100% concordant with the original genotype. In conclusion, the technique including the duplex PCR assay followed by DHPLC analysis can be used to diagnose alpha-thalassemia; this methodology is simple, rapid, accurate, semiautomatic, and high output, and thus, it is suitable for large-scale screening.

  10. BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma.

    PubMed

    Affolter, Kajsa; Samowitz, Wade; Tripp, Sheryl; Bronner, Mary P

    2013-08-01

    The serine/threonine-protein kinase B-raf (BRAF) is an oncogene mutated in various neoplasms, including 5-15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor-only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti-EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E-mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti-BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild-type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods.

  11. 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

  12. 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

  13. Mutation detection of E6 and LCR genes from HPV 16 associated with carcinogenesis.

    PubMed

    Mosmann, Jessica P; Monetti, Marina S; Frutos, Maria C; Kiguen, Ana X; Venezuela, Raul F; Cuffini, Cecilia G

    2015-01-01

    Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from Cordoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions. PMID:25735347

  14. Mutational analysis of primary and metastatic colorectal cancer samples underlying the resistance to cetuximab-based therapy

    PubMed Central

    Nemecek, Radim; Berkovcova, Jitka; Radova, Lenka; Kazda, Tomas; Mlcochova, Jitka; Vychytilova-Faltejskova, Petra; Slaby, Ondrej; Svoboda, Marek

    2016-01-01

    Purpose Although several molecular markers predicting resistance to cetuximab- or panitumumab-based therapy of metastatic colorectal cancer were described, mutations in RAS proto-oncogenes remain the only predictors being used in daily clinical practice. However, 35%–45% of wild-type RAS patients still do not respond to this anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody-based therapy, and therefore the definition of other predictors forms an important clinical need. The aim of the present retrospective single-institutional study was to evaluate potential genes responsible for resistance to anti-EGFR therapy in relation to mutational analysis of primary versus metastatic lesions. Patients and methods Twenty-four paired primary and corresponding metastatic tissue samples from eight nonresponding and four responding metastatic colorectal cancer patients treated with cetuximab-based therapy were sequenced using a next-generation sequencing panel of 26 genes involved in EGFR signaling pathway and colorectal carcinogenesis. Results Mutational status of primary tumors and metastatic lesions was highly concordant in TP53, APC, CTNNB1, KRAS, PIK3CA, PTEN, and FBXW7 genes. Metastatic samples harbor significantly more mutations than primary tumors. Potentially negative predictive value of FBXW7 mutations in relationship to anti-EGFR treatment outcomes was confirmed. Finally, new occurrences of activating KRAS mutations were identified in a group of patients initially determined as wild-type RAS by routinely used qPCR-based RAS mutational tests. All newly detected activating KRAS mutations most likely led to cetuximab treatment failure. Conclusion The results of the present study suggest a need of careful consideration of previously published results of anti-EGFR-targeted therapy with regard to potentially inaccurate diagnostic tools used in the past. Based on our findings, we recommend more extensive use of next-generation sequencing testing in daily

  15. Hypothesis: Obesity Is Associated with a Lower Mutation Threshold in Colon Cancer

    PubMed Central

    Bordonaro, Michael; Lazarova, Darina

    2015-01-01

    Neoplastic progression requires accumulation of several mutations (mutation threshold). We hypothesize that obesity raises the risk of microsatellite stable (MSS) colon cancer (CC) at least in part by decreasing the mutation threshold. Thus, we posit that obese patients require fewer mutations, particularly driver mutations, compared to their normal BMI counterparts. Further, we suggest that the reduced number of required mutations in obese patients could be due to several factors, including the high levels of cytokines that accompany obesity. Cytokine-activated ERK, AKT, and JAK/STAT signaling could synergize with CC-initiating mutations to promote intestinal neoplastic development. Therefore, driver mutations that induce these specific pathways may not be “required” for neoplastic development in obesity; alteration in cell signaling consequent to obesity can substitute for some driver mutations in neoplastic progression. This hypothesis is supported by preliminary analyses of data from The Cancer Genome Atlas (TCGA). Thus, we observed that, compared to normal weight patients, cancer genomes of obese MSS CC patients exhibit fewer somatic mutations, and correspondingly lower numbers of mutations in driver genes (P = 0.026).The most striking observation was the lower number of KRAS mutations detected in patients with high body-mass index (BMI). These intriguing observations require further validation with increased number of patients, taking into account all possible confounding factors. If the hypothesis is confirmed, future studies should also address several possible explanations for the observed lower mutation threshold in obese MSS CC patients. PMID:26284133

  16. 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

  17. 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.

  18. 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

  19. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA).

    PubMed

    Housden, Benjamin E; Perrimon, Norbert

    2016-01-01

    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. PMID:27587781

  20. A novel mutation in Von Hippel-Lindau disease detected by SSCP of dideoxynucleotide sequence products

    SciTech Connect

    Vance, J.M.; Loeb, D.B.; Stajich, J.M.

    1994-09-01

    VHL is an inherited tumor disease in which the defect has recently been partially cloned on chromosome 3p25. Only about 15-20% of mutations are currently known. Using SSCP analysis, we detected a new C to T transition in exon 3 of the VHL tumor-suppressor gene, which produces an umber stop codon. To enhance the sensitivity of the mutation search, we employed the modification of Liu, Q. et al. that applies SSCP to the various-sized products of the standard dideoxynucleotide sequence analysis. This allows the analysis of larger DNA fragments by SSCP. All 5 affected individuals tested in this family have the same point mutation. The new mutation reported here parallels previous observations of transcript truncation produced in this disorder.

  1. The impact of TP53 and RAS mutations on cerebellar glioblastomas.

    PubMed

    Milinkovic, Vedrana P; Skender Gazibara, Milica K; Manojlovic Gacic, Emilija M; Gazibara, Tatjana M; Tanic, Nikola T

    2014-10-01

    Cerebellar glioblastoma (cGBM) is a rare, inadequately characterized disease, without detailed information on its molecular basis. This is the first report analyzing both TP53 and RAS alterations in cGBM. TP53 mutations were detected in more than half of the samples from our cohort, mainly in hotspot codons. There were no activating mutations in hotspot codons 12/13 and 61 of KRAS and HRAS genes in cGBM samples but we detected alterations in other parts of exons 2 and 3 of these genes, including premature induction of STOP codon. This mutation was present in 3 out of 5 patients. High incidence of RAS mutations, as well as significantly longer survival of cGBM patients compared to those with supratentorial GBM suggest that cGBM may have different mechanisms of occurrence. Our results suggest that inactivation of TP53 and RAS may play an important role in the progression of cerebellar GBM. PMID:25036404

  2. Comparison of techniques for the successful detection of BRCA1 mutations in fixed paraffin-embedded tissue.

    PubMed

    Bernstein, Jonine L; Thompson, W Douglas; Casey, Graham; DiCioccio, Richard A; Whittemore, Alice S; Diep, Anh T; Thakore, Seema S; Vaziri, Susan; Xue, Shanyan; Haile, Robert W

    2002-09-01

    Genomic DNA isolated from archived paraffin-embedded tissues (PETs) has important applicability in genetic epidemiological studies. To determine the accuracy of the sequence data, using DNA derived from PET among patients with known mutations characterized from blood, we conducted a blinded factorial experiment to simultaneously examine the influence of mutation type, age of the PET, PCR product type, and Taq DNA polymerase on BRCA1 gene mutation detection. The probability of detecting sequencing artifacts was also investigated. We found that: (a) gene detection was most accurate for newer PET; (b) high fidelity Taq with shorter PCR amplicon length yielded the highest mutation detection success rate and lowest artifact rate; and (c) base substitutions were more often correctly identified than frameshift mutations or wild-type sequences. We concluded that DNA derived from PET that archived for less than 18 years can be used successfully for detecting BRCA1 gene mutations if quality control is strictly maintained.

  3. 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.

  4. 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.

  5. 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

  6. Precise Classification of Cervical Carcinomas Combined with Somatic Mutation Profiling Contributes to Predicting Disease Outcome

    PubMed Central

    Spaans, Vivian M.; Trietsch, Marjolijn D.; Peters, Alexander A. W.; Osse, Michelle; ter Haar, Natalja; Fleuren, Gert J.; Jordanova, Ekaterina S.

    2015-01-01

    Introduction Squamous cell carcinoma (SCC), adenocarcinoma (AC), and adenosquamous carcinoma (ASC) are the most common histological subtypes of cervical cancer. Differences in the somatic mutation profiles of these subtypes have been suggested. We investigated the prevalence of somatic hot-spot mutations in three well-defined cohorts of SCC, AC, and ASC and determined the additional value of mutation profiling in predicting disease outcome relative to well-established prognostic parameters. Materials and Methods Clinicopathological data were collected for 301 cervical tumors classified as SCC (n=166), AC (n=55), or ASC (n=80). Mass spectrometry was used to analyze 171 somatic hot-spot mutations in 13 relevant genes. Results In 103 (34%) tumors, 123 mutations were detected (36% in SCC, 38% in AC, and 28% in ASC), mostly in PIK3CA (20%) and KRAS (7%). PIK3CA mutations occurred more frequently in SCC than AC (25% vs. 11%, P=0.025), whereas KRAS mutations occurred more frequently in AC than SCC (24% vs. 3%, P<0.001) and ASC (24% vs. 3%, P<0.001). A positive mutation status correlated with worse disease-free survival (HR 1.57, P=0.043). In multivariate analysis, tumor diameter, parametrial infiltration, and lymph node metastasis, but not the presence of a somatic mutation, were independent predictors of survival. Conclusion Potentially targetable somatic mutations occurred in 34% of cervical tumors with different distributions among histological subtypes. Precise classification of cervical carcinomas in combination with mutation profiling is valuable for predicting disease outcome and may guide the development and selection of tumor-specific treatment approaches. PMID:26197069

  7. 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

  8. Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

    PubMed

    Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

    2016-01-01

    We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent. PMID:26960620

  9. Next-generation sequencing is highly sensitive for the detection of beta-catenin mutations in desmoid-type fibromatoses.

    PubMed

    Aitken, Sarah J; Presneau, Nadège; Kalimuthu, Sangeetha; Dileo, Palma; Berisha, Fitim; Tirabosco, Roberto; Amary, M Fernanda; Flanagan, Adrienne M

    2015-08-01

    Desmoid-type fibromatoses are locally aggressive and frequently recurrent tumours, and an accurate diagnosis is essential for patient management. The majority of sporadic lesions harbour beta-catenin (CTNNB1) mutations. We used next-generation sequencing to detect CTNNB1 mutations and to compare the sensitivity and specificity of next-generation sequencing with currently employed mutation detection techniques: mutation-specific restriction enzyme digestion and polymerase chain reaction amplification. DNA was extracted from formalin-fixed paraffin-embedded needle biopsy or resection tissue sections from 144 patients with sporadic desmoid-type fibromatoses, four patients with syndrome-related desmoid-type fibromatoses and 11 morphological mimics. Two primer pairs were designed for CTNNB1 mutation hotspots. Using ≥10 ng of DNA, libraries were generated by Fluidigm and sequenced on the Ion Torrent Personal Genome Machine. Next-generation sequencing had a sensitivity of 92.36 % (133/144, 95 % CIs: 86.74 to 96.12 %) and a specificity of 100 % for the detection of CTNNB1 mutations in desmoid-type fibromatoses-like spindle cell lesions. All mutations detected by mutation-specific restriction enzyme digestion were identified by next-generation sequencing. Next-generation sequencing identified additional mutations in 11 tumours that were not detected by mutation-specific restriction enzyme digestion, two of which have not been previously described. Next-generation sequencing is highly sensitive for the detection of CTNNB1 mutations. This multiplex assay has the advantage of detecting additional mutations compared to those detected by mutation-specific restriction enzyme digestion (sensitivity 82.41 %). The technology requires minimal DNA and is time- and cost-efficient.

  10. Clinical investigation of EGFR mutation detection by pyrosequencing in lung cancer patients

    PubMed Central

    KIM, HEE JOUNG; OH, SEO YOUNG; KIM, WAN SEOP; KIM, SUN JONG; YOO, GWANG HA; KIM, WON DONG; LEE, KYE YOUNG

    2013-01-01

    Direct sequencing is the standard method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer, however, its relatively low sensitivity limits its clinical use. Pyrosequencing is a bioluminometric, real-time non-electrophoretic DNA sequencing technique with a number of advantages compared with direct sequencing, including higher sensitivity, speed, automation and cost-effectiveness. Clinical specimens from 202 lung cancer patients were analyzed for EGFR mutations in exons 18, 19, 20 and 21 using the pyrosequencing method following genomic DNA extraction from paraffin-embedded tissue specimens. All clinical data and tumor specimens were obtained from the Konkuk University Hospital (Korea) between July 2006 and December 2008. The results and clinical responses to EGFR-tyrosine kinase inhibitors (TKIs) were compared. Overall, EGFR mutation-positive rate was 26.7% (54/202). Activating EGFR mutations were observed more frequently in female (52.1 vs. 13.0%), non-smoking (47.8 vs. 15.8%) and adenocarcinoma (35.2 vs. 5.2%) patients. However, significant numbers of EGFR mutation-positive patients were identified as male, former or current smokers and non-adenocarcinoma patients. The combinations of favorable clinicopathological factors, including female, non-smoking and adenocarcinoma, were not identified to significantly increase the positive EGFR mutation rate (female, 52.1%; female and non-smoker, 52.6%; female, non-smoker and adenocarcinoma, 51.9%). The present findings indicate that EGFR mutation analysis is a highly useful method for the prediction of response to EGFR-TKI and the use of favorable clinicopathological factors to perform this analysis is not suitable. Exon 19 deletion was the most common mutation (63.6%) and exon 21 L858R substitution was measured at 32.7%. The exon 20 T790M mutation was identified in 1 patient prior to EGFR-TKI treatment. EGFR mutation status is associated with response to EGFR-TKI and the overall

  11. Denaturation fingerprinting: two related mutation detection methods especially advantageous for high G + C regions.

    PubMed

    Liu, Q; Weinshenker, B G; Wingerchuk, D M; Sommer, S S

    1998-01-01

    Two versions of denaturation fingerprinting (dnF2R and dnF1R are described for detecting mutations. DnF2R is a sensitive screening method in which fingerprints are generated by performing denaturing gel electrophoresis on bidirectional "cycle-sequencing" reactions with each of two dideoxy terminators, e.g. ddATP and ddCTP. When the fingerprints generated by ddATP and ddCTP are combined, all sequence changes are expected to result in one extra and one absent segment. DnF2R was performed on 246- and 318-bp segments of the human factor IX gene, and the products were electrophoresed through a 6% Long Ranger gel with 7 M urea. All 32 single-base mutations were detected in hemizygous males with hemophilia B. DnF2R has been applied to detect a total of seven heterozygous sequence changes in large-scale screening and was found to be especially suitable for high G+C regions. In a blinded analysis, all of twenty-four additional single-base mutations were detected, but 7 of 31 heterozygous mutations were missed (23%). To reduce the effort of dnF2R by almost twofold while retaining the ability to detect all types of single-base changes, one fingerprint (dnF1R) was generated by performing a single reaction with ddATP and a second chemically modified terminator (e.g., ROX-conjugated ddCTP), which retards the mobility of the same termination products. The sensitivity and specificity of dnF1R equaled that of dnF2R, with the exception that the blinded analysis of heterozygotes in the 318-bp segment, which revealed the presence of an additional mutation. DnF under partially denaturing conditions may have optimal sensitivity for the detection of heterozygotes.

  12. A Novel Targeted Approach for Noninvasive Detection of Paternally Inherited Mutations in Maternal Plasma.

    PubMed

    van den Oever, Jessica M E; van Minderhout, Ivonne J H M; Harteveld, Cornelis L; den Hollander, Nicolette S; Bakker, Egbert; van der Stoep, Nienke; Boon, Elles M J

    2015-09-01

    The challenge in noninvasive prenatal diagnosis for monogenic disorders lies in the detection of low levels of fetal variants in the excess of maternal cell-free plasma DNA. Next-generation sequencing, which is the main method used for noninvasive prenatal testing and diagnosis, can overcome this challenge. However, this method may not be accessible to all genetic laboratories. Moreover, shotgun next-generation sequencing as, for instance, currently applied for noninvasive fetal trisomy screening may not be suitable for the detection of inherited mutations. We have developed a sensitive, mutation-specific, and fast alternative for next-generation sequencing-mediated noninvasive prenatal diagnosis using a PCR-based method. For this proof-of-principle study, noninvasive fetal paternally inherited mutation detection was performed using cell-free DNA from maternal plasma. Preferential amplification of the paternally inherited allele was accomplished through a personalized approach using a blocking probe against maternal sequences in a high-resolution melting curve analysis-based assay. Enhanced detection of the fetal paternally inherited mutation was obtained for both an autosomal dominant and a recessive monogenic disorder by blocking the amplification of maternal sequences in maternal plasma. PMID:26162331

  13. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  14. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  15. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  16. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... addition to the reporting recommendations as specified under 40 CFR part 792, subpart J the following... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell...

  17. Optimization of competitively differentiated polymerase chain reaction in detection of HBV basal core promoter mutation

    PubMed Central

    Peng, Xiao-Mou; Gu, Lin; Chen, Xue-Juan; Li, Jian-Guo; Huang, Yang-Su; Gao, Zhi-Liang

    2005-01-01

    AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe-ndent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles. PMID:15962387

  18. 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.

  19. 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.

  20. Rapid Detection of rpoB Gene Mutations Conferring Rifampin Resistance in Mycobacterium tuberculosis

    PubMed Central

    Ao, Wanyuan; Aldous, Stephen; Woodruff, Evelyn; Hicke, Brian; Rea, Larry; Kreiswirth, Barry

    2012-01-01

    Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute. PMID:22518852

  1. Mutations in mammalian tolloid-like 1 gene detected in adult patients with ASD

    PubMed Central

    Stańczak, Paweł; Witecka, Joanna; Szydło, Anna; Gutmajster, Ewa; Lisik, Małgorzata; Auguściak-Duma, Aleksandra; Tarnowski, Maciej; Czekaj, Tomasz; Czekaj, Hanna; Sieroń, Aleksander L

    2009-01-01

    Atrial septal defect (ASD) is an incomplete septation of atria in human heart causing circulatory problems. Its frequency is estimated at one per 10 000. Actions of numerous genes have been linked to heart development. However, no single gene defect causing ASD has yet been identified. Incomplete heart septation similar to ASD was reported in transgenic mice with both inactive alleles of gene encoding mammalian zinc metalloprotease a mammalian tolloid-like 1 (tll1). Here, we have screened 19 ASD patients and 15 healthy age-matched individuals for mutations in TLL1 gene. All 22 exons were analyzed exon by exon for heteroduplex formation. Subsequently, DNA fragments forming heteroduplexes were sequenced. In four nonrelated patients, three missense mutations in coding sequence, and one single base change in the 5′UTR have been detected. Two mutations (Met182Leu, and Ala238Val) were detected in ASD patients with the same clinical phenotype. As the second mutation locates immediately upstream of the catalytic zinc-binding signature, it might change the enzyme substrate specificity. The third change, Leu627Val in the CUB3 domain, has been found in an ASD patient with interatrial septum aneurysm in addition to ASD. The CUB3 domain is important for substrate-specific recognition. In the remaining 15 patients as well as in 15 reference samples numerous base substitutions, deletions, and insertions have been detected, but no mutations changing the coding sequence have been found. Lack of mutations in relation to ASD of these patients could possibly be because of genetic heterogeneity of the syndrome. PMID:18830233

  2. Rapid detection of drug-resistant mutations in hepatitis B virus by the PCR-Invader assay.

    PubMed

    Tadokoro, Kenichi; Suzuki, Fumitaka; Kobayashi, Mariko; Yamaguchi, Toshikazu; Nagano, Makoto; Egashira, Toru; Kumada, Hiromitsu

    2011-01-01

    Early detection of resistant mutations of hepatitis B virus (HBV) is important for patients on nucleos(t)ide analog therapy. An assay based on the PCR-Invader technology was developed to detect resistant mutations with high sensitivity. The assay specifically detects mutations at codons 180, 181, 184, 202, 204, and 250 of the HBV polymerase reverse transcriptase domain. These mutations result in resistance to lamivudine and entecavir. In mixtures of plasmids containing wild-type and resistant mutants, fold-over-zero values for resistant mutations were detected in 2% of the total. Seventy-five serum samples from patients, whose treatment had been switched from lamivudine to entecavir, were examined by the PCR-Invader assay and direct sequencing. The PCR-Invader assay detected all resistant mutations that were detected by direct sequencing and even detected the presence of mutants that direct sequencing could not. Cloning sequencing confirmed those mutations found by the PCR-Invader assay and not by direct sequencing. The PCR-Invader assay is a useful tool for the early detection of drug-resistant mutations. PMID:20950650

  3. 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.

  4. 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

  5. Detection of filaggrin gene mutation (2282del4) in Pakistani Ichthyosis vulgaris families.

    PubMed

    Naz, Naghma; Samdani, Azam Jah

    2011-06-01

    The aim of this study was to detect an 811 bp filaggrin (FLG) gene fragment known to carry a mutation 2282del4 which causes ichthyosis vulgaris. Seven clinically examined ichthyosis vulgaris families were included in this study. An 811 bp FLG gene fragment was targeted in the genomic DNA of all the members of the seven families by PCR amplification using known primers RPT1P7 and RPT2P1. Successful amplification of an 811 bp FLG gene fragment in all the families suggested the possible role of the 2282del4 mutation in causing ichthyosis vulgaris in Pakistani population.

  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. A Likelihood-Based Framework for Variant Calling and De Novo Mutation Detection in Families

    PubMed Central

    Li, Bingshan; Chen, Wei; Zhan, Xiaowei; Busonero, Fabio; Sanna, Serena; Sidore, Carlo; Cucca, Francesco; Kang, Hyun M.; Abecasis, Gonçalo R.

    2012-01-01

    Family samples, which can be enriched for rare causal variants by focusing on families with multiple extreme individuals and which facilitate detection of de novo mutation events, provide an attractive resource for next-generation sequencing studies. Here, we describe, implement, and evaluate a likelihood-based framework for analysis of next generation sequence data in family samples. Our framework is able to identify variant sites accurately and to assign individual genotypes, and can handle de novo mutation events, increasing the sensitivity and specificity of variant calling and de novo mutation detection. Through simulations we show explicit modeling of family relationships is especially useful for analyses of low-frequency variants and that genotype accuracy increases with the number of individuals sequenced per family. Compared with the standard approach of ignoring relatedness, our methods identify and accurately genotype more variants, and have high specificity for detecting de novo mutation events. The improvement in accuracy using our methods over the standard approach is particularly pronounced for low-frequency variants. Furthermore the family-aware calling framework dramatically reduces Mendelian inconsistencies and is beneficial for family-based analysis. We hope our framework and software will facilitate continuing efforts to identify genetic factors underlying human diseases. PMID:23055937

  8. Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection

    NASA Astrophysics Data System (ADS)

    Su, Qiang; Zhou, Xiaoming

    2008-12-01

    Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

  9. 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.

  10. Detection of rarely identified multiple mutations in MECP2 gene do not contribute to enhanced severity in Rett syndrome.

    PubMed

    Chapleau, Christopher A; Lane, Jane; Kirwin, Susan M; Schanen, Carolyn; Vinette, Kathy M B; Stubbolo, Danielle; MacLeod, Patrick; Glaze, Daniel G; Motil, Kathleen J; Neul, Jeffrey L; Skinner, Steven A; Kaufmann, Walter E; Percy, Alan K

    2013-07-01

    The objective of our study was to characterize the influence of multiple mutations in the MECP2 gene in a cohort of individuals with Rett syndrome. Further analysis demonstrated that nearly all resulted from de novo in cis mutations, where the disease severity was indistinguishable from single mutations. Our methods involved enrolling participants in the RTT Natural History Study (NHS). After providing informed consent through their parents or principal caretakers, additional molecular assessments were performed in the participants and their parents to assess the presence and location of more than one mutation in each. Clinical severity was assessed at each visit in those participants in the NHS. Non-contiguous MECP2 gene variations were detected in 12 participants and contiguous mutations involving a deletion and insertion in three participants. Thirteen of 15 participants had mutations that were in cis; four (of 13) had three MECP2 mutations; two (of 15) had mutations that were both in cis and in trans (i.e., on different alleles). Clinical severity did not appear different from NHS participants with a single similar mutation. Mutations in cis were identified in most participants; two individuals had mutations both in cis and in trans. The presence of multiple mutations was not associated with greater severity. Nevertheless, multiple mutations will require greater thought in the future, if genetic assignment to drug treatment protocols is considered.

  11. 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

  12. Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

    PubMed

    Dudiuk, Catiana; Gamarra, Soledad; Jimenez-Ortigosa, Cristina; Leonardelli, Florencia; Macedo, Daiana; Perlin, David S; Garcia-Effron, Guillermo

    2015-07-01

    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

  13. 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.

  14. Contribution of dfrA and inhA mutations to the detection of isoniazid-resistant Mycobacterium tuberculosis isolates.

    PubMed

    Ho, Yu Min; Sun, Yong-Jiang; Wong, Sin-Yew; Lee, Ann S G

    2009-09-01

    Screening of 127 isoniazid (INH)-resistant Mycobacterium tuberculosis isolates from Singapore for mutations within the dfrA and inhA genes revealed mutations in 0 and 5 (3.9%) isolates respectively, implying that mutations in dfrA do not contribute to the detection of INH-resistant M. tuberculosis and that mutations within inhA are rare. Thirty-seven (29%) of the 127 isolates had no mutations in any of the genes implicated in INH resistance (katG, kasA, and ndh; inhA and ahpC promoters), suggesting that there are new INH targets yet to be discovered. PMID:19581462

  15. 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

  16. A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing.

    PubMed

    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

  17. 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.

  18. 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. PMID:27065589

  19. Oncogene Mutations in Colorectal Polyps Identified in the Norwegian Colorectal Cancer Prevention (NORCCAP) Screening Study

    PubMed Central

    Lorentzen, Jon A.; Grzyb, Krzysztof; De Angelis, Paula M.; Hoff, Geir; Eide, Tor J.; Andresen, Per Arne

    2016-01-01

    Data are limited on oncogene mutation frequencies in polyps from principally asymptomatic participants of population-based colorectal cancer screening studies. In this study, DNA from 204 polyps, 5 mm or larger, were collected from 176 participants of the NORCCAP screening study and analyzed for mutations in KRAS, BRAF, and PIK3CA including the rarely studied KRAS exons 3 and 4 mutations. KRAS mutations were identified in 23.0% of the lesions and were significantly associated with tubulovillous adenomas and large size. A significantly higher frequency of KRAS mutations in females was associated with mutations in codon 12. The KRAS exon 3 and 4 mutations constituted 23.4% of the KRAS positive lesions, which is a larger proportion compared to previous observations in colorectal cancer. BRAF mutations were identified in 11.3% and were associated with serrated polyps. None of the individuals were diagnosed with de novo or recurrent colorectal cancer during the follow-up time (median 11.2 years). Revealing differences in mutation-spectra according to gender and stages in tumorigenesis might be important for optimal use of oncogenes as therapeutic targets and biomarkers. PMID:27656095

  20. Oncogene Mutations in Colorectal Polyps Identified in the Norwegian Colorectal Cancer Prevention (NORCCAP) Screening Study.

    PubMed

    Lorentzen, Jon A; Grzyb, Krzysztof; De Angelis, Paula M; Hoff, Geir; Eide, Tor J; Andresen, Per Arne

    2016-01-01

    Data are limited on oncogene mutation frequencies in polyps from principally asymptomatic participants of population-based colorectal cancer screening studies. In this study, DNA from 204 polyps, 5 mm or larger, were collected from 176 participants of the NORCCAP screening study and analyzed for mutations in KRAS, BRAF, and PIK3CA including the rarely studied KRAS exons 3 and 4 mutations. KRAS mutations were identified in 23.0% of the lesions and were significantly associated with tubulovillous adenomas and large size. A significantly higher frequency of KRAS mutations in females was associated with mutations in codon 12. The KRAS exon 3 and 4 mutations constituted 23.4% of the KRAS positive lesions, which is a larger proportion compared to previous observations in colorectal cancer. BRAF mutations were identified in 11.3% and were associated with serrated polyps. None of the individuals were diagnosed with de novo or recurrent colorectal cancer during the follow-up time (median 11.2 years). Revealing differences in mutation-spectra according to gender and stages in tumorigenesis might be important for optimal use of oncogenes as therapeutic targets and biomarkers. PMID:27656095

  1. Oncogene Mutations in Colorectal Polyps Identified in the Norwegian Colorectal Cancer Prevention (NORCCAP) Screening Study

    PubMed Central

    Lorentzen, Jon A.; Grzyb, Krzysztof; De Angelis, Paula M.; Hoff, Geir; Eide, Tor J.; Andresen, Per Arne

    2016-01-01

    Data are limited on oncogene mutation frequencies in polyps from principally asymptomatic participants of population-based colorectal cancer screening studies. In this study, DNA from 204 polyps, 5 mm or larger, were collected from 176 participants of the NORCCAP screening study and analyzed for mutations in KRAS, BRAF, and PIK3CA including the rarely studied KRAS exons 3 and 4 mutations. KRAS mutations were identified in 23.0% of the lesions and were significantly associated with tubulovillous adenomas and large size. A significantly higher frequency of KRAS mutations in females was associated with mutations in codon 12. The KRAS exon 3 and 4 mutations constituted 23.4% of the KRAS positive lesions, which is a larger proportion compared to previous observations in colorectal cancer. BRAF mutations were identified in 11.3% and were associated with serrated polyps. None of the individuals were diagnosed with de novo or recurrent colorectal cancer during the follow-up time (median 11.2 years). Revealing differences in mutation-spectra according to gender and stages in tumorigenesis might be important for optimal use of oncogenes as therapeutic targets and biomarkers.

  2. Prognostic and Predictive Significance of MYC and KRAS Alterations in Breast Cancer from Women Treated with Neoadjuvant Chemotherapy

    PubMed Central

    de Souza, Carolina Rosal Teixeira; Montenegro, Raquel Carvalho; Rey, Juan Antonio; Carvalho, Antônio Alberto; Assumpção, Paulo Pimentel; Khayat, André Salim; Pinto, Giovanny Rebouças; Demachki, Sâmia; de Arruda Cardoso Smith, Marília; Burbano, Rommel Rodríguez

    2013-01-01

    Breast cancer is a complex disease, with heterogeneous clinical evolution. Several analyses have been performed to identify the risk factors for breast cancer progression and the patients who respond best to a specific treatment. We aimed to evaluate whether the hormone receptor expression, HER2 and MYC genes and their protein status, and KRAS codon 12 mutations may be prognostic or predictive biomarkers of breast cancer. Protein, gene and mutation status were concomitantly evaluated in 116 breast tumors from women who underwent neoadjuvant chemotherapy with doxorubicin plus cyclophosphamide. We observed that MYC expression was associated with luminal B and HER2 overexpression phenotypes compared to luminal A (p<0.05). The presence of MYC duplication or polysomy 8, as well as KRAS mutation, were also associated with the HER2 overexpression subtype (p<0.05). MYC expression and MYC gain were more frequently observed in early-onset compared to late-onset tumors (p<0.05). KRAS mutation was a risk factor of grade 3 tumors (p<0.05). A multivariate logistic regression demonstrated that MYC amplification defined as MYC/nucleus ratio of ≥2.5 was a protective factor for chemotherapy resistance. On the other hand, age and grade 2 tumors were a risk factor. Additionally, luminal B, HER2 overexpression, and triple-negative tumors presented increased odds of being resistant to chemotherapy relative to luminal A tumors. Thus, breast tumors with KRAS codon 12 mutations seem to present a worse prognosis. Additionally, MYC amplification may help in the identification of tumors that are sensitive to doxorubicin plus cyclophosphamide treatment. If confirmed in a large set of samples, these markers may be useful for clinical stratification and prognosis. PMID:23555992

  3. The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

    PubMed

    Andersson, Anna K; Ma, Jing; Wang, Jianmin; Chen, Xiang; Gedman, Amanda Larson; Dang, Jinjun; Nakitandwe, Joy; Holmfeldt, Linda; Parker, Matthew; Easton, John; Huether, Robert; Kriwacki, Richard; Rusch, Michael; Wu, Gang; Li, Yongjin; Mulder, Heather; Raimondi, Susana; Pounds, Stanley; Kang, Guolian; Shi, Lei; Becksfort, Jared; Gupta, Pankaj; Payne-Turner, Debbie; Vadodaria, Bhavin; Boggs, Kristy; Yergeau, Donald; Manne, Jayanthi; Song, Guangchun; Edmonson, Michael; Nagahawatte, Panduka; Wei, Lei; Cheng, Cheng; Pei, Deqing; Sutton, Rosemary; Venn, Nicola C; Chetcuti, Albert; Rush, Amanda; Catchpoole, Daniel; Heldrup, Jesper; Fioretos, Thoas; Lu, Charles; Ding, Li; Pui, Ching-Hon; Shurtleff, Sheila; Mullighan, Charles G; Mardis, Elaine R; Wilson, Richard K; Gruber, Tanja A; Zhang, Jinghui; Downing, James R

    2015-04-01

    Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

  4. Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis.

    PubMed

    Richards, J S; Fan, H-Y; Liu, Z; Tsoi, M; Laguë, M-N; Boyer, A; Boerboom, D

    2012-03-22

    WNT, RAS or phosphoinositide 3-kinase signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector β-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRAS(G12D) dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRAS(G12D) in granulosa cells, we sought to determine whether KRAS(G12D) would block precancerous lesion and tumor formation in follicles of the CTNNB1-mutant mice. Unexpectedly, transgenic Ctnnb1;Kras-mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten-mutant mice. Microarray and reverse transcription-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, they may be related to juvenile GCTs or to tumors in other tissues where CTNNB1 is mutated. Importantly, the results provide strong evidence that CTNNB1 is the driver in these contexts and that KRAS(G12D) and Pten loss promote the program set

  5. 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.

  6. A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations.

    PubMed

    Cupples, C G; Cabrera, M; Cruz, C; Miller, J H

    1990-06-01

    We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites. PMID:2199309

  7. Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-PCR coupled with sanger sequencing.

    PubMed

    Liu, Can; Lin, Jinpiao; Chen, Huijuan; Shang, Hongyan; Jiang, Ling; Chen, Jing; Ye, Yang; Yang, Bin; Ou, Qishui

    2014-08-01

    Mutations in the reverse transcriptase (rt) region of the DNA polymerase gene are the primary cause of hepatitis B virus (HBV) drug resistance. In this study, we established a novel method that couples coamplification at lower denaturation temperature (COLD)-PCR and Sanger sequencing, and we applied it to the detection of known and unknown HBV mutations. Primers were designed based on the common mutations in the HBV rt sequence at positions 180 to 215. The critical denaturation temperature (Tc) was established as a denaturing temperature for both fast and full COLD-PCR procedures. For single mutations, when a melting temperature (Tm)-reducing mutation occurred (e.g., C-G → T-A), the sensitivities of fast and full COLD-PCR for mutant detection were 1% and 2%, respectively; when the mutation caused no change in Tm (e.g., C-G → G-C) or raised Tm (e.g., T-A → C-G), only full COLD-PCR improved the sensitivity for mutant detection (2%). For combination mutations, the sensitivities of both full and fast COLD-PCR were increased to 0.5%. The limits of detection for fast and full COLD-PCR were 50 IU/ml and 100 IU/ml, respectively. In 30 chronic hepatitis B (CHB) cases, no mutations were detected by conventional PCR, whereas 18 mutations were successfully detected by COLD-PCR, including low-prevalence mutations (<10%), as confirmed by ultradeep pyrosequencing. In conclusion, COLD-PCR provides a highly sensitive, simple, inexpensive, and practical tool for significantly improving amplification efficacy and detecting low-level mutations in clinical CHB cases. PMID:24899029

  8. A new monoclonal antibody (CAL2) detects CALRETICULIN mutations in formalin-fixed and paraffin-embedded bone marrow biopsies.

    PubMed

    Stein, H; Bob, R; Dürkop, H; Erck, C; Kämpfe, D; Kvasnicka, H-M; Martens, H; Roth, A; Streubel, A

    2016-01-01

    Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method.

  9. 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.

  10. 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.

  11. Detection of inherited mutations for hereditary cancer using target enrichment and next generation sequencing.

    PubMed

    Guan, Yanfang; Hu, Hong; Peng, Yin; Gong, Yuhua; Yi, Yuting; Shao, Libin; Liu, Tengfei; Li, Gairui; Wang, Rongjiao; Dai, Pingping; Bignon, Yves-Jean; Xiao, Zhe; Yang, Ling; Mu, Feng; Xiao, Liang; Xie, Zeming; Yan, Wenhui; Xu, Nan; Zhou, Dongxian; Yi, Xin

    2015-03-01

    Hereditary cancers occur because of inherited gene mutations. Genetic testing has been approved to provide information for risk assessment and rationale for appropriate intervention. Testing methods currently available for clinical use have some limitations, including sensitivity and testing throughput, etc. Next generation sequencing (NGS) has been rapidly evolving to increase testing sensitivity and throughput. It can be potentially used to identify inherited mutation in clinical diagnostic setting. Here we develop an effective method employing target enrichment and NGS platform to detect common as well as rare mutations for all common hereditary cancers in a single assay. Single base substitution across 115 hereditary cancer related genes using YH (the first Asian genome) was characterized to validate our method. Sensitivity, specificity and accuracy of 93.66, 99.98 and 99.97 %, were achieved, respectively. In addition, we correctly identified 53 SNVs and indels of BRCA1 and BRCA2 in two breast cancer specimens, all confirmed by Sanger sequencing. Accuracy in detecting copy number variation (CNV) was corroborated in 4 breast cancer specimens with known CNVs in BRAC1. Application of the method to 85 clinical cases revealed 22 deleterious mutations, 11 of which were novel. In summary, our studies demonstrate that the target enrichment combined with NGS method provides the accuracy, sensitivity, and high throughput for genetic testing for patients with high risk of hereditary or familial cancer.

  12. Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas

    PubMed Central

    Wakimoto, Hiroaki; Tanaka, Shota; Curry, William T.; Loebel, Franziska; Zhao, Dan; Tateishi, Kensuke; Chen, Juxiang; Klofas, Lindsay K.; Lelic, Nina; Kim, James C.; Dias-Santagata, Dora; Ellisen, Leif W.; Borger, Darrell R.; Fendt, Sarah-Maria; Heiden, Matthew G. Vander; Batchelor, Tracy T.; Iafrate, A. John; Cahill, Daniel P.; Chi, Andrew S.

    2014-01-01

    PURPOSE Isocitrate dehydrogenase (IDH) gene mutations occur in low-grade and high-grade gliomas. We sought to identify the genetic basis of malignant phenotype heterogeneity in IDH-mutant gliomas. METHODS We prospectively implanted tumor specimens from 20 consecutive IDH1-mutant glioma resections into mouse brains and genotyped all resection specimens using a CLIA-certified molecular panel. Gliomas with cancer driver mutations were tested for sensitivity to targeted inhibitors in vitro. Associations between genomic alterations and outcomes were analyzed in patients. RESULTS By 10 months, 8 of 20 IDH1-mutant gliomas developed intracerebral xenografts. All xenografts maintained mutant IDH1 and high levels of 2-hydroxyglutarate on serial transplantation. All xenograft-producing gliomas harbored “lineage-defining” mutations in CIC (oligodendroglioma) or TP53 (astrocytoma), and 6 of 8 additionally had activating mutations in PIK3CA or amplification of PDGFRA, MET or N-MYC. Only IDH1 and CIC/TP53 mutations were detected in non-xenograft-forming gliomas (P=.0007). Targeted inhibition of the additional alterations decreased proliferation in vitro. Moreover, we detected alterations in known cancer driver genes in 13.4% of IDH-mutant glioma patients, including PIK3CA, KRAS, AKT or PTEN mutation or PDGFRA, MET or N-MYC amplification. IDH/CIC mutant tumors were associated with PIK3CA/KRAS mutations while IDH/TP53 tumors correlated with PDGFRA/MET amplification. Presence of driver alterations at progression was associated with shorter subsequent progression-free survival (median 9.0 vs. 36.1 months, P=.0011). CONCLUSION A subset of IDH-mutant gliomas with mutations in driver oncogenes has a more malignant phenotype in patients. Identification of these alterations may provide an opportunity for use of targeted therapies in these patients. PMID:24714777

  13. Activation-Induced Cytidine Deaminase Contributes to Pancreatic Tumorigenesis by Inducing Tumor-Related Gene Mutations.

    PubMed

    Sawai, Yugo; Kodama, Yuzo; Shimizu, Takahiro; Ota, Yuji; Maruno, Takahisa; Eso, Yuji; Kurita, Akira; Shiokawa, Masahiro; Tsuji, Yoshihisa; Uza, Norimitsu; Matsumoto, Yuko; Masui, Toshihiko; Uemoto, Shinji; Marusawa, Hiroyuki; Chiba, Tsutomu

    2015-08-15

    Pancreatic ductal adenocarcinoma (PDAC) develops via an accumulation of various gene mutations. The mechanism underlying the mutations in PDAC development, however, is not fully understood. Recent insight into the close association between the mutation pattern of various cancers and specific mutagens led us to investigate the possible involvement of activation-induced cytidine deaminase (AID), a DNA editing enzyme, in pancreatic tumorigenesis. Our immunohistochemical findings revealed AID protein expression in human acinar ductal metaplasia, pancreatic intraepithelial neoplasia, and PDAC. Both the amount and intensity of the AID protein expression increased with the progression from precancerous to cancerous lesions in human PDAC tissues. To further assess the significance of ectopic epithelial AID expression in pancreatic tumorigenesis, we analyzed the phenotype of AID transgenic (AID Tg) mice. Consistent with our hypothesis that AID is involved in the mechanism of the mutations underlying pancreatic tumorigenesis, we found precancerous lesions developing in the pancreas of AID Tg mice. Using deep sequencing, we also detected Kras and c-Myc mutations in our analysis of the whole pancreas of AID Tg mice. In addition, Sanger sequencing confirmed the presence of Kras, c-Myc, and Smad4 mutations, with the typical mutational footprint of AID in precancerous lesions in AID Tg mice separated by laser capture microdissection. Taken together, our findings suggest that AID contributes to the development of pancreatic precancerous lesions by inducing tumor-related gene mutations. Our new mouse model without intentional manipulation of specific tumor-related genes provides a powerful system for analyzing the mutations involved in PDAC.

  14. Detection of mutation by allele-specific loop-mediated isothermal amplification (AS-LAMP).

    PubMed

    Aonuma, Hiroka; Badolo, Athanase; Okado, Kiyoshi; Kanuka, Hirotaka

    2013-01-01

    For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes. PMID:24026691

  15. 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).

  16. Comprehensive detection of diverse exon 19 deletion mutations of EGFR in lung Cancer by a single probe set.

    PubMed

    Bae, Jin Ho; Jo, Seong-Min; Kim, Hak-Sung

    2015-12-15

    Detection of exon 19 deletion mutation of EGFR, one of the most frequently occurring mutations in lung cancer, provides the crucial information for diagnosis and treatment guideline in non-small-cell lung cancer (NSCLC). Here, we demonstrate a simple and efficient method to detect various exon 19 deletion mutations of EGFR using a single probe set comprising of an oligo-quencher (oligo-Q) and a molecular beacon (MB). While the MB hybridizes to both the wild and mutant target DNA, the oligo-Q only binds to the wild target DNA, leading to a fluorescent signal in case of deletion mutation. This enables the comprehensive detection of the diverse exon 19 deletion mutations using a single probe set. We demonstrated the utility and efficiency of the approach by detecting the frequent exon 19 deletion mutations of EGFR through a real-time PCR and in situ fluorescence imaging. Our approach enabled the detection of genomic DNA as low as 0.02 ng, showing a detection limit of 2% in a heterogeneous DNA mixture, and could be used for detecting mutations in a single cell level. The present MB and oligo-Q dual probe system can be used for diagnosis and treatment guideline in NSCLC.

  17. 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.

  18. 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 approa