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Sample records for kras mutation detection

  1. Comparative analysis of dideoxy sequencing, the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

    PubMed Central

    Gao, Jing; Li, Yan-Yan; Sun, Ping-Nai; Shen, Lin

    2010-01-01

    AIM: To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients. METHODS: Formalin-fixed, paraffin-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS StripAssay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%, 50%, 20%, 15%, 10%, 5%, 1%, 0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing, the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector for KRAS StripAssay and the pyrosequencing PyroMarkTM Q24 system, respectively. RESULTS: Among 100 patients, KRAS mutations were identified in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS StripAssay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12Asp (GGT>GAT), Gly12Val (GGT>GTT) and Gly13Asp (GGC>GAC). Thirty-three patients were identified to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identified by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was confirmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identified by dideoxy sequencing were

  2. 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.

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

    PubMed

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

    2016-07-01

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

  4. 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

  5. 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

  6. Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer

    PubMed Central

    Buim, Marcilei EC; Fanelli, Marcello F; Souza, Virgilio S; Romero, Juliana; Abdallah, Emne A; Mello, Celso AL; Alves, Vanessa; Ocea, Luciana MM; Mingues, Natália B; Barbosa, Paula NVP; Tyng, Chiang J; Chojniak, Rubens; Chinen, Ludmilla TD

    2015-01-01

    Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results: KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available. PMID:26252055

  7. Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer.

    PubMed

    Buim, Marcilei Ec; Fanelli, Marcello F; Souza, Virgilio S; Romero, Juliana; Abdallah, Emne A; Mello, Celso Al; Alves, Vanessa; Ocea, Luciana Mm; Mingues, Natália B; Barbosa, Paula Nvp; Tyng, Chiang J; Chojniak, Rubens; Chinen, Ludmilla Td

    2015-01-01

    Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.

  8. Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.

    PubMed

    Kristensen, Lasse S; Daugaard, Iben L; Christensen, Mariann; Hamilton-Dutoit, Stephen; Hager, Henrik; Hansen, Lise Lotte

    2010-12-01

    Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. © 2010 Wiley-Liss, Inc.

  9. Nanofluidic digital PCR for KRAS mutation detection and quantification in gastrointestinal cancer.

    PubMed

    Azuara, Daniel; Ginesta, Mireia M; Gausachs, Mireia; Rodriguez-Moranta, Francisco; Fabregat, Joan; Busquets, Juli; Pelaez, Nuria; Boadas, Jaume; Galter, Sara; Moreno, Victor; Costa, Jose; de Oca, Javier; Capellá, Gabriel

    2012-09-01

    Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutation-detection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples. We assessed 2 groups of patients (colorectal and pancreatic disease): Group 1 consisted of 27 patients with colorectal carcinomas, 14 patients with adenomas, and 5 control individuals; group 2 consisted of 42 patients with pancreatic carcinoma, 4 with adenocarcinomas of the ampulla, and 6 with chronic pancreatitis). Digital PCR was performed with the Digital Array Chip (Fluidigm). Nanofluidic digital PCR detected mutant alleles at 0.05% to 0.1%, depending on the variant analyzed. For the colorectal disease group, conventional PCR detected 9 (64%) of 14 adenomas that were positive for KRAS mutants, whereas digital PCR increased this number to 11 (79%) of 14. Sixteen (59%) of 27 carcinomas showed KRAS mutation with conventional PCR. Two additional cases were detected with digital PCR. In 5 cases (3 adenomas, 2 carcinomas), the total number of mutant alleles changed. For the pancreatic disease group, digital PCR increased the number of positive cases from 26 to 34 (81%) and identified ≥ 2 mutant alleles in 25 cases, compared with conventional PCR, which identified multiple KRAS mutant alleles in only 12 cases. A good correlation was observed between results obtained with tumor biopsies and those obtained with pancreatic juice. Digital PCR provides a robust, quantitative measure of the proportion of KRAS mutant alleles in routinely obtained samples. It also allows a better classification of tumors, with potential clinical relevance.

  10. KRAS Mutation Detection in Paired Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Colorectal Cancer Tissues

    PubMed Central

    Solassol, Jérome; Ramos, Jeanne; Crapez, Evelyne; Saifi, Majda; Mangé, Alain; Vianès, Evelyne; Lamy, Pierre-Jean; Costes, Valérie; Maudelonde, Thierry

    2011-01-01

    KRAS mutation has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies of KRAS mutation analysis have been performed using homogenously archived CRC specimens, and studies that compare freshly frozen specimens and formalin-fixed paraffin-embedded (FFPE) specimens of CRC are lacking. The aim of the present study was to evaluate the impact of tissue preservation on the determination of KRAS mutational status. A series of 131 mCRC fresh-frozen tissues were first analyzed using both high-resolution melting (HRM) and direct sequencing. KRAS mutations were found in 47/131 (35.8%) using both approaches. Out of the 47 samples that were positive for KRAS mutations, 33 had available matched FFPE specimens. Using HRM, 2/33 (6%) demonstrated suboptimal template amplification, and 2/33 (6%) expressed an erroneous wild-type KRAS profile. Using direct sequencing, 6/33 (18.1%) displayed a wild-type KRAS status, and 3/33 (9.1%) showed discordant mutations. Finally, the detection of KRAS mutations was lower among the FFPE samples compared with the freshly frozen samples, demonstrating that tissue processing clearly impacts the accuracy of KRAS genotyping. PMID:21686179

  11. A Commercial Real-Time PCR Kit Provides Greater Sensitivity than Direct Sequencing to Detect KRAS Mutations

    PubMed Central

    Angulo, Bárbara; García-García, Elena; Martínez, Rebeca; Suárez-Gauthier, Ana; Conde, Esther; Hidalgo, Manuel; López-Ríos, Fernando

    2010-01-01

    KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post- PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%). PMID:20203003

  12. 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.

  13. KRAS and BRAF mutations and MSI status in precursor lesions of colorectal cancer detected by colonoscopy.

    PubMed

    Yamane, L S; Scapulatempo-Neto, C; Alvarenga, L; Oliveira, C Z; Berardinelli, G N; Almodova, E; Cunha, T R; Fava, G; Colaiacovo, W; Melani, A; Fregnani, J H; Reis, R M; Guimarães, D P

    2014-10-01

    Colorectal cancer (CRC) is one of the most frequent cancers worldwide. Adenoma is the main precursor lesion and, recently, the serrated polyps were described as a group of colorectal lesions with malignant potential. The morphologic and biologic characterizations of serrated polyps remain limited. The aim of the present study was to determine the frequency of KRAS and BRAF mutations and microsatellite instability (MSI) in CRC precursor lesions, to evaluate the association between molecular, pathologic and morphologic alterations in precursor lesions and to compare with the alterations detected in CRC. A series of 342 precursor lesions were removed from 155 patients during colonoscopy. After morphologic classification, molecular analysis was performed in 103 precursor lesions, and their genetic profile compared with 47 sporadic CRCs. Adenomas were the main precursor lesions (70.2%). Among the serrated polyps, the main precursor lesion was hyperplastic polyps (HPs) (82.4%), followed by sessile serrated adenomas (12.7%) and traditional serrated adenomas (2.0%). KRAS mutations were detected in 13.6% of the precursor lesions, namely in adenomas and in HPs, but in no serrated adenoma. BRAF mutations were found in 9 (8.7%) precursor lesions, mainly associated with serrated polyps and absent in adenomas (P<0.001). High MSI (MSI-H) was absent in precursor lesions. In the 47 CCR cases, 46.8% exhibited KRAS mutation, 6.5% BRAF mutations and 10.6% MSI-H. This study confirms the role of KRAS and BRAF mutations in CRC carcinogenesis, a crucial step in implementing CRC screening strategies.

  14. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

    PubMed Central

    Do, Hongdo; Krypuy, Michael; Mitchell, Paul L; Fox, Stephen B; Dobrovic, Alexander

    2008-01-01

    Background Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. Methods We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. Results All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. Conclusion This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings. PMID:18495026

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed

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

    2016-06-15

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

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

    PubMed Central

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

    2016-01-01

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

  19. 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.

  20. A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers

    PubMed Central

    Magnin, Sandrine; Viel, Erika; Baraquin, Alice; Valmary-Degano, Severine; Kantelip, Bernadette; Pretet, Jean-Luc; Mougin, Christiane; Bigand, Marthe; Girardo, Benoît; Borg, Christophe; Ferrand, Christophe

    2011-01-01

    The analysis of KRAS mutations has become a prerequisite for anti-epidermal growth factor receptor therapy in patients with metastatic colorectal cancers. KRAS mutations are associated with resistance to treatment by monoclonal antibodies such as cetuximab and panitumumab and thus are correlated with a shorter progression-free survival. BRAF mutations also may play a role in treatment decisions. The widespread use of these targeted therapies has generated the need to develop cost-effective methods for routine KRAS and BRAF analysis. The aim of this study was to compare a multiplex SNaPshot assay with DNA sequencing and high-resolution melting analysis for identifying KRAS codons 12 and 13 and BRAF codon 600 mutations. Thus 110 routinely formalin-fixed and paraffin-embedded tissue blocks were tested by each method. The SNaPshot analysis detected KRAS and BRAF codon 600 mutations in, respectively, 34.5% (n = 38) and 10% (n = 11) of these tissue blocks. These results were confirmed by direct DNA sequencing and by high-resolution melting analysis. The costs and time constraints of each detection method were compared at the same time. In conclusion, our newly designed multiplex SNaPshot assay is a fast, inexpensive, sensitive, and robust technique for molecular diagnostic practices and patient selection. PMID:21742054

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

    PubMed Central

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

    2017-01-01

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

  2. 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.
    <...

  3. 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.
    <...

  4. [Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].

    PubMed

    Zhang, Xun; Wang, Yuehua; Gao, Ning; Wang, Jinfen

    2014-02-01

    To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas. Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed. KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058). (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost

  5. The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer

    PubMed Central

    Mancini, Irene; Santucci, Claudio; Sestini, Roberta; Simi, Lisa; Pratesi, Nicola; Cianchi, Fabio; Valanzano, Rosa; Pinzani, Pamela; Orlando, Claudio

    2010-01-01

    Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30–50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAFV600E detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAFV600E in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAFV600E in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAFV600E), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAFV600E. Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures. PMID:20616366

  6. Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

    PubMed

    Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

    2014-08-01

    Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

  7. 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.

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

    PubMed

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

    2015-12-15

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

  9. 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

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

    PubMed Central

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

    2017-01-01

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

  11. SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

    PubMed Central

    Fariña Sarasqueta, Arantza; Moerland, Elna; de Bruyne, Hanneke; de Graaf, Henk; Vrancken, Tamara; van Lijnschoten, Gesina; van den Brule, Adriaan J.C.

    2011-01-01

    Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2–specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives. PMID:21354055

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

    PubMed

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

    2013-03-01

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

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

    PubMed

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

    2013-02-01

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

  14. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  17. Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

    PubMed

    da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R

    2010-12-25

    The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.

  18. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

    PubMed

    Ward, R; Hawkins, N; O'Grady, R; Sheehan, C; O'Connor, T; Impey, H; Roberts, N; Fuery, C; Todd, A

    1998-08-01

    The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

  19. K-ras oncogene mutation in pterygium.

    PubMed

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

    2017-03-01

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

  20. KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer: a cohort study

    PubMed Central

    Negru, Serban; Papadopoulou, Eirini; Apessos, Angela; Stanculeanu, Dana Lucia; Ciuleanu, Eliade; Volovat, Constantin; Croitoru, Adina; Kakolyris, Stylianos; Aravantinos, Gerasimos; Ziras, Nikolaos; Athanasiadis, Elias; Touroutoglou, Nikolaos; Pavlidis, Nikolaos; Kalofonos, Haralabos P; Nasioulas, George

    2014-01-01

    Objectives Treatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations. Setting Diagnostic molecular laboratory located in Athens, Greece. Participants 2425 patients with colorectal cancer participated in the study. Primary and secondary outcome measures 2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing. Results KRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method. Conclusions The HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer. PMID:24859998

  1. Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Seo, Si Hyung; Park, Won Sang; Nam, Suk Woo; Song, Sang Yong; Han, Joung Ho; Park, Cheol Keun; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2005-05-01

    Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, chi2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2013-12-01

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

  4. KRAS-mutated plasma DNA as predictor of outcome from irinotecan monotherapy in metastatic colorectal cancer

    PubMed Central

    Spindler, K G; Appelt, A L; Pallisgaard, N; Andersen, R F; Jakobsen, A

    2013-01-01

    Background: We investigated the clinical implications of KRAS and BRAF mutations detected in both archival tumor tissue and plasma cell-free DNA in metastatic colorectal cancer patients treated with irinotecan monotherapy. Methods: Two hundred and eleven patients receiving second-line irinotecan (350 mg m−2 q3w) were included in two independent cohorts. Plasma was obtained from pretreatment EDTA blood-samples. Mutations were detected in archival tumour and plasma with qPCR methods. Results: Mutation status in tumor did not correlate to efficacy in either cohort, whereas none of the patients with mutations detectable in plasma responded to therapy. Response rate and disease control rate in plasma KRAS wt patients were 19 and 66% compared with 0 and 37%, in patients with pKRAS mutations, (P=0.04 and 0.01). Tumor KRAS status was not associated with PFS but with OS in the validation cohort. Plasma BRAF and KRAS demonstrated a strong influence on both PFS and OS. The median OS was 13.0 mo in pKRAS wt patients and 7.8 in pKRAS-mutated, (HR=2.26, P<0.0001). PFS was 4.6 and 2.7 mo, respectively (HR=1,69, P=0.01). Multivariate analysis confirmed the independent prognostic value of pKRAS status but not KRAS tumor status. Conclusion: Tumor KRAS has minor clinical impact, whereas plasma KRAS status seems to hold important predictive and prognostic information. PMID:24263065

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

    PubMed Central

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

    2012-01-01

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

  6. Enteric-type adenocarcinoma of the lung harbouring a novel KRAS Q22K mutation with concomitant KRAS polysomy: a case report

    PubMed Central

    Metro, Giulio; Valtorta, Emanuele; Siggillino, Annamaria; Lauricella, Calogero; Cenci, Matteo; Ludovini, Vienna; Minenza, Elisa; Prosperi, Enrico; Ricciuti, Biagio; Rebonato, Alberto; Bassetti, Alessandra; Crinò, Lucio

    2015-01-01

    This case describes a novel KRAS Q22K mutation with simultaneous KRAS polysomy in a patient with advanced, enteric-type, adenocarcinoma of the lung. Despite the administration of systemic chemotherapy, the disease underwent rapid progression and led to the patient’s death in a short period of time. Such an aggressive clinical course suggests that, in this specific case, KRAS dependency was the major genetic driver of poor prognosis. Direct deoxy ribonucleic acid (DNA) sequencing of the KRAS gene allows for the detection of novel KRAS mutations, and it might be advocated in patients with advanced non-small cell lung cancer in view of the emerging role of KRAS as a potential therapeutic target. PMID:26284123

  7. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2017-02-08

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

  10. Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

    PubMed

    Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

    2014-01-01

    The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

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

    PubMed

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

    2013-01-01

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

  12. 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

  13. A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue.

    PubMed

    Li, Guoqiang; Luo, Xiaodi; He, Jiaying; Zhu, Zeyao; Yu, Gang; Qin, Huijuan; Zeng, Tao; Liu, Zhiming; Wu, Shiyang; Xu, Jiasen; Ren-Heidenreich, Lifen

    2011-02-01

    DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex®-xTAG70plex-EGFR liquidchip. This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized. A total of 70 alleles of somatic mutations of EGFR, KRAS, BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was < 8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%-5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected. A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR, KRAS, BRAF and PIK3CA gene mutations from FFPE tumor slides.

  14. 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

  15. 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

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

    PubMed

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

    2013-12-01

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

  17. 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.

  18. Associations between mutations and histologic patterns of mucin in lung adenocarcinoma: invasive mucinous pattern and extracellular mucin are associated with KRAS mutation

    PubMed Central

    Kadota, Kyuichi; Yeh, Yi-Chen; D’Angelo, Sandra P.; Moreira, Andre L.; Kuk, Deborah; Sima, Camelia S.; Riely, Gregory J.; Arcila, Maria E.; Kris, Mark G.; Rusch, Valerie W.; Adusumilli, Prasad S.; Travis, William D.

    2015-01-01

    Multiple reports indicate that epidermal growth factor receptor (EGFR) mutations are associated with lepidic-pattern lung adenocarcinoma, and that KRAS mutations are associated with invasive mucinous adenocarcinoma. We sought to investigate the association between EGFR and KRAS mutations and specific morphologic characteristics, such as predominant histologic subtype and mucinous features. Clinical data for 864 patients with resected lung adenocarcinoma that underwent molecular testing for EGFR and KRAS mutations were collected. Histologic subtyping was performed according to the IASLC/ATS/ERS lung adenocarcinoma classification, with attention given to signet-ring cell feature and extracellular mucin. EGFR mutations were detected using a polymerase chain reaction–based sizing assay, KRAS mutations were detected using Sanger sequencing, and ALK expression was detected using immunohistochemistry. Invasive mucinous adenocarcinoma was associated with KRAS mutation (P<0.001). Among invasive mucinous adenocarcinomas with KRAS mutation, a pure mucinous pattern was more common than a mixed mucinous/nonmucinous pattern (P=0.002). Invasive mucinous adenocarcinoma was associated with KRAS transition mutations (G→A) but not transversion mutations (G→T or G→C) compared to non-mucinous tumors (P=0.009). The lepidic-predominant group was associated with EGFR mutation compared to nonlepidic-predominant tumors (P=0.011). Extracellular mucin was associated with KRAS mutation (P<0.001), whereas signet-ring cell feature was not associated with EGFR or KRAS mutation (P=0.517). ALK expression was associated with signet-ring cell feature (P=0.001) but not with extracellular mucin (P=0.089). Our study shows that histologic patterns of mucin in lung adenocarcinoma - including invasive mucinous adenocarcinoma and extracellular mucin - are associated with KRAS mutation. PMID:25029118

  19. KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancer.

    PubMed

    Franklin, Wilbur A; Haney, Jerry; Sugita, Michio; Bemis, Lynne; Jimeno, Antonio; Messersmith, Wells A

    2010-01-01

    Treatment of colon carcinoma with the anti-epidermal growth factor receptor antibody Cetuximab is reported to be ineffective in KRAS-mutant tumors. Mutation testing techniques have therefore become an urgent concern. We have compared three methods for detecting KRAS mutations in 59 cases of colon carcinoma: 1) high resolution melting, 2) the amplification refractory mutation system using a bifunctional self-probing primer (ARMS/Scorpion, ARMS/S), and 3) direct sequencing. We also evaluated the effects of the methods of sectioning and coring of paraffin blocks to obtain tumor DNA on assay sensitivity and specificity. The most sensitive and specific combination of block sampling and mutational analysis was ARMS/S performed on DNA derived from 1-mm paraffin cores. This combination of tissue sampling and testing method detected KRAS mutations in 46% of colon tumors. Four samples were positive by ARMS/S, but initially negative by direct sequencing. Cloned DNA samples were retested by direct sequencing, and in all four cases KRAS mutations were identified in the DNA. In six cases, high resolution melting abnormalities could not be confirmed as specific mutations either by ARMS/S or direct sequencing. We conclude that coring of the paraffin blocks and testing by ARMS/S is a sensitive, specific, and efficient method for KRAS testing.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2017-01-01

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

  3. Emergence of KRAS mutations and acquired resistance to anti EGFR therapy in colorectal cancer

    PubMed Central

    Misale, Sandra; Yaeger, Rona; Hobor, Sebastijan; Scala, Elisa; Janakiraman, Manickam; Liska, David; Valtorta, Emanuele; Schiavo, Roberta; Buscarino, Michela; Siravegna, Giulia; Bencardino, Katia; Cercek, Andrea; Chen, Chin-Tung; Veronese, Silvio; Zanon, Carlo; Sartore-Bianchi, Andrea; Gambacorta, Marcello; Gallicchio, Margherita; Vakiani, Efsevia; Boscaro, Valentina; Medico, Enzo; Weiser, Martin; Siena, Salvatore; Di Nicolantonio, Federica; Solit, David; Bardelli, Alberto

    2014-01-01

    Summary A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers1. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug2. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood3-8. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK. Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6/10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance. PMID:22722830

  4. 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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2000-02-01

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

  7. 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

  8. KRAS and BRAF mutation analysis in metastatic colorectal cancer: a cost-effectiveness analysis from a Swiss perspective.

    PubMed

    Blank, Patricia R; Moch, Holger; Szucs, Thomas D; Schwenkglenks, Matthias

    2011-10-01

    Monoclonal antibodies against the epidermal growth factor receptor (EGFR), such as cetuximab, have led to significant clinical benefits for metastatic colorectal cancer (mCRC) patients but have also increased treatment costs considerably. Recent evidence associates KRAS and BRAF mutations with resistance to EGFR antibodies. We assessed the cost-effectiveness of predictive testing for KRAS and BRAF mutations, prior to cetuximab treatment of chemorefractory mCRC patients. A life-long Markov simulation model was used to estimate direct medical costs (€) and clinical effectiveness [quality-adjusted life-years (QALY)] of the following strategies: KRAS testing, KRAS testing with subsequent BRAF testing of KRAS wild-types (KRAS/BRAF), cetuximab treatment without testing. Comparison was against no cetuximab treatment (reference strategy). In the testing strategies, cetuximab treatment was initiated if no mutations were detected. Best supportive care was given to all patients. Survival times/utilities were derived from published randomized clinical trials. Costs were assessed from the perspective of the Swiss health system. Average remaining lifetime costs ranged from €3,983 (no cetuximab) to €38,662 (no testing). Cetuximab treatment guided by KRAS/BRAF achieved gains of 0.491 QALYs compared with the reference strategy. The KRAS testing strategy achieved an additional gain of 0.002 QALYs compared with KRAS/BRAF. KRAS/BRAF testing was the most cost-effective approach when compared with the reference strategy (incremental cost-effectiveness ratio: €62,653/QALY). New predictive tests for KRAS and BRAF status are currently being introduced in pathology. Despite substantial costs of predictive testing, it is economically favorable to identify patients with KRAS and BRAF wild-type status. ©2011 AACR

  9. 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

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

    PubMed

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

    2017-02-01

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

  11. 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.

  12. Clinicopathologic characteristics and gene expression analyses of non-KRAS 12/13, RAS-mutated metastatic colorectal cancer.

    PubMed

    Morris, V K; Lucas, F A San; Overman, M J; Eng, C; Morelli, M P; Jiang, Z-Q; Luthra, R; Meric-Bernstam, F; Maru, D; Scheet, P; Kopetz, S; Vilar, E

    2014-10-01

    KRAS mutations in codons 12 and 13 are present in ∼40% of all colorectal cancers (CRC). Activating mutations in codons 61 and 146 of KRAS and in codons 12, 13, and 61 of NRAS also occur but are less frequent. The clinicopathologic features and gene expression profiles of this latter subpopulation of RAS-mutant colorectal tumors have not yet been clearly defined but in general are treated similarly to those with KRAS 12 or 13 mutations. Records of patients with metastatic CRC (mCRC) treated at MD Anderson Cancer Center between December 2000 and August 2012 were reviewed for RAS (KRAS or NRAS) and BRAF mutation status, clinical characteristics, and survival outcomes. To study further with an independent cohort, data from The Cancer Genome Atlas were analyzed to define a gene expression signature for patients whose tumors feature these atypical RAS mutations and explore differences with KRAS 12/13-mutated colorectal tumors. Among the 484 patients reviewed, KRAS 12/13, KRAS 61/146, NRAS, and BRAF mutations were detected in 47.7%, 3.0%, 4.1%, and 7.4%, respectively, of patients who were tested for each of these aberrations. Lung metastases were more common in both the KRAS 12/13-mutated and atypical RAS-mutated cohorts relative to patients with RAS/BRAF wild-type tumors. Gene expression analyses revealed similar patterns regardless of the site of RAS mutation, and in silico functional algorithms predicted that KRAS and NRAS mutations in codons 12, 13, 61, and 146 alter the protein function and drive tumorgenesis. Clinicopathologic characteristics, survival outcomes, functional impact, and gene expression profiling were similar between patients with KRAS 12/13 and those with NRAS or KRAS 61/146-mutated mCRC. These clinical and bioinformatic findings support the notion that colorectal tumors driven by these RAS mutations are phenotypically similar. © The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights

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

    PubMed

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

    2013-01-01

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

  14. Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review

    PubMed Central

    2014-01-01

    Background KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. Methods We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse’s Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. Results KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence

  15. Analyses of clinicopathological, molecular, and prognostic associations of KRAS codon 61 and codon 146 mutations in colorectal cancer: cohort study and literature review.

    PubMed

    Imamura, Yu; Lochhead, Paul; Yamauchi, Mai; Kuchiba, Aya; Qian, Zhi Rong; Liao, Xiaoyun; Nishihara, Reiko; Jung, Seungyoun; Wu, Kana; Nosho, Katsuhiko; Wang, Yaoyu E; Peng, Shouyong; Bass, Adam J; Haigis, Kevin M; Meyerhardt, Jeffrey A; Chan, Andrew T; Fuchs, Charles S; Ogino, Shuji

    2014-05-31

    KRAS mutations in codons 12 and 13 are established predictive biomarkers for anti-EGFR therapy in colorectal cancer. Previous studies suggest that KRAS codon 61 and 146 mutations may also predict resistance to anti-EGFR therapy in colorectal cancer. However, clinicopathological, molecular, and prognostic features of colorectal carcinoma with KRAS codon 61 or 146 mutation remain unclear. We utilized a molecular pathological epidemiology database of 1267 colon and rectal cancers in the Nurse's Health Study and the Health Professionals Follow-up Study. We examined KRAS mutations in codons 12, 13, 61 and 146 (assessed by pyrosequencing), in relation to clinicopathological features, and tumor molecular markers, including BRAF and PIK3CA mutations, CpG island methylator phenotype (CIMP), LINE-1 methylation, and microsatellite instability (MSI). Survival analyses were performed in 1067 BRAF-wild-type cancers to avoid confounding by BRAF mutation. Cox proportional hazards models were used to compute mortality hazard ratio, adjusting for potential confounders, including disease stage, PIK3CA mutation, CIMP, LINE-1 hypomethylation, and MSI. KRAS codon 61 mutations were detected in 19 cases (1.5%), and codon 146 mutations in 40 cases (3.2%). Overall KRAS mutation prevalence in colorectal cancers was 40% (=505/1267). Of interest, compared to KRAS-wild-type, overall, KRAS-mutated cancers more frequently exhibited cecal location (24% vs. 12% in KRAS-wild-type; P < 0.0001), CIMP-low (49% vs. 32% in KRAS-wild-type; P < 0.0001), and PIK3CA mutations (24% vs. 11% in KRAS-wild-type; P < 0.0001). These trends were evident irrespective of mutated codon, though statistical power was limited for codon 61 mutants. Neither KRAS codon 61 nor codon 146 mutation was significantly associated with clinical outcome or prognosis in univariate or multivariate analysis [colorectal cancer-specific mortality hazard ratio (HR) = 0.81, 95% confidence interval (CI) = 0.29-2.26 for

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

    PubMed Central

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

    2015-01-01

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

  17. 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.

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

    PubMed

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

    2006-07-01

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

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

    PubMed

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

    2013-01-01

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

  20. Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients11

    PubMed Central

    Taniguchi, Hiroya; Yamazaki, Kentaro; Yoshino, Takayuki; Muro, Kei; Yatabe, Yasushi; Watanabe, Toshiaki; Ebi, Hiromichi; Ochiai, Atsushi; Baba, Eishi; Tsuchihara, Katsuya

    2015-01-01

    The Japanese guidelines for the testing of KRAS mutations in colorectal cancer have been used for the past 5 years. However, new findings of RAS (KRAS/NRAS) mutations that can further predict the therapeutic effects of anti-epidermal growth factor receptor (EGFR) antibody therapy necessitated a revision of the guidelines. The revised guidelines included the following five basic requirements for RAS mutation testing to highlight a patient group in which anti-EGFR antibody therapy may be ineffective: First, anti-EGFR antibody therapy may not offer survival benefit and/or tumor shrinkage to patients with expanded RAS mutations. Thus, current methods to detect KRAS exon 2 (codons 12 and 13) mutations are insufficient for selecting appropriate candidates for this therapy. Additional testing of extended KRAS/NRAS mutations is recommended. Second, repeated tests are not required for the detection; tissue materials of either primary or metastatic lesions are applicable for RAS mutation testing. Evaluating RAS mutations prior to anti-EGFR antibody therapy is recommended. Third, direct sequencing with manual dissection or allele-specific PCR-based methods is currently applicable for RAS mutation testing. Fourth, thinly sliced sections of formalin-fixed, paraffin-embedded tissue blocks are applicable for RAS mutation testing. One section stained with H&E should be provided to histologically determine whether the tissue contains sufficient amount of tumor cells for testing. Finally, RAS mutation testing must be performed in laboratories with appropriate testing procedures and specimen management practices. PMID:25800101

  1. EGFR and KRAS mutations in lung carcinoma: molecular testing by using cytology specimens.

    PubMed

    Billah, Shahreen; Stewart, John; Staerkel, Gregg; Chen, Su; Gong, Yun; Guo, Ming

    2011-04-25

    The aim of this study was to validate clinical utilization of routinely prepared cytology specimens for molecular testing to detect EGFR or KRAS mutations in lung cancer. From September 2009 to April 2010, the authors collected 209 cytology specimens from patients with lung cancer at the MD Anderson Cancer Center Department of Pathology. The specimens included 99 cases of endobronchial ultrasound-guided (EBUS) fine-needle aspiration (FNA), 67 cases of computed tomography (CT)-guided FNA, 27 cases of body fluid, 10 cases of ultrasound-guided of superficial FNA, and 6 cases of other cytology specimens. DNA sequencing for EGFR exons 18-21 and KRAS codons 12, 13, and 61 was performed. The overall specimen insufficiency rate was low (6.2%). EBUS (4%) and body-fluid cases (3.7%) showed lower insufficiency rates than the other cases. Similar insufficiency rates were observed in smears (6.1%) and cell-block sections (6.4%). EGFR mutations were detected in 19.4% (34 of 175) of nonsmall cell lung carcinoma (NSCLC) with a significantly higher frequency in adenocarcinoma (29%, 29 of 100) than in nonadenocarcinoma (7%, 5 of 75, P = .002). KRAS mutations were detected in 23.6% (41 of 174) of NSCLCs with no statistical differences between adenocarcinoma (26%, 26 of 102) and nonadenocarcinoma (21%, 17 of 72, P = .86). Higher frequencies of EGFR mutations in exons 19 and 21 (65%) than in exons 18 and 20 were detected. Our findings support clinical utilization of routinely prepared cytology specimens, including EBUS, CT/US. FNAs and body fluid specimens, as a reliable source for molecular testing to detect EGFR or KRAS mutations in patients with NSCLC. Copyright © 2011 American Cancer Society.

  2. Missense mutations in MLH1, MSH2, KRAS, and APC genes in colorectal cancer patients in Malaysia.

    PubMed

    Abdul Murad, Nor Azian; Othman, Zulhabri; Khalid, Melati; Abdul Razak, Zuraini; Hussain, Rosniza; Nadesan, Sukumar; Sagap, Ismail; Mohamed Rose, Isa; Wan Ngah, Wan Zurinah; Jamal, Rahman

    2012-11-01

    Colorectal cancer (CRC) is the third most common cancer worldwide with approximately 1 million cases diagnosed annually. In Malaysia, CRC is the second most common cancer in women and ranked first in men. The underlying cause of CRC remains unknown. The aim of this study was to analyze the mutations in genes involved in CRC including MLH1, MSH2, KRAS, and APC genes. A total of 76 patients were recruited. We used the polymerase chain reaction-denaturing high-performance liquid chromatography for the detection of mutations in the mismatch repair (MMR) and APC genes and the PCR single-strand conformation polymorphism for screening of the KRAS gene mutations. We identified 17 types of missense mutations in 38 out of 76 patients in our patients. Nine mutations were identified in the APC gene, five mutations were detected in the KRAS gene, and two mutations were identified in the MSH2 gene. Only one mutation was identified in MLH1. Out of these 17 mutations, eight mutations (47 %) were predicted to be pathogenic. Seven patients were identified with multiple mutations (3: MSH2 and KRAS, 1: KRAS and APC, 1: MLH1 and APC, 2: APC and APC). We have established the PCR-DHPLC and PCR-SSCP for screening of mutations in CRC patients. This study has given a snapshot of the spectrum of mutations in the four genes that were analyzed. Mutation screening in patients and their family members will help in the early detection of CRC and hence will reduce mortality due to CRC.

  3. Selumetinib in advanced non small cell lung cancer (NSCLC) harbouring KRAS mutation: endless clinical challenge to KRAS-mutant NSCLC.

    PubMed

    Paolo, Maione; Assunta, Sgambato; Antonio, Rossi; Claudia, Sacco Paola; Anna, Bareschino Maria; Clorinda, Schettino; Francesca, Casaluce; Fortunato, Ciardiello; Cesare, Gridelli

    2013-06-01

    During the past few years, oncologists have witnessed the reclassification of non small cell lung cancer (NSCLC) as not one disease, but several molecularly defined subsets of disease with relevant therapeutic implications in the field of molecularly targeted therapies. Two not very common genetically defined subsets of NSCLC, including those with EGFR or ALK activating mutations, and show high sensitivity to tyrosine-kinase inhibitors such that patients frequently have sustained clinical responses to therapy. However, the largest subset harbours an activating KRAS mutation and up to now, no successful targeted therapy has been developed for RAS-mutant lung cancer, with few compounds being assessed by clinical trials. In fact, KRAS has remained an elusive target for cancer therapy for biologic reasons. The chief value of KRAS lies in providing information about the other biomarkers that are directly druggable, that is, EGFR and ALK. The presence of mutated KRAS rules out ALK and EGFR, and KRAS may therefore form part of an efficient pathway in a testing algorithm. Currently, KRAS itself remains undruggable despite decades of effort, but attention has recently focused on inhibition of the Ras-contingent downstream signalling. Selumetinib (AZD6244; ARRY-142886) is an oral, tight-binding, uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2, downstream of KRAS, with preclinical evidence of synergistic activity with docetaxel in KRAS-mutant cancers and currently in clinical development. The Ras/RAF/MEK/ERK pathway is frequently deregulated in cancer and a number of inhibitors that target this pathway are currently in clinical development. Recently, in a randomised, phase II trial selumetinib plus docetaxel has proven to improve progression free survival compared to docetaxel alone in previously treated patients with advanced KRAS-mutant NSCLC.

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

    PubMed

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

    2011-03-10

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

  5. 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

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

    PubMed

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

    2017-03-01

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

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

    PubMed Central

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

    2017-01-01

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

  8. 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.

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

    PubMed

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

    2016-12-28

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

  10. Novel Approach for Clinical Validation of the cobas KRAS Mutation Test in Advanced Colorectal Cancer.

    PubMed

    Sharma, Abha; Zhang, Guili; Aslam, Shagufta; Yu, Karen; Chee, Melody; Palma, John F

    2016-06-01

    Our objective was to assess the performance of the cobas test versus comparators for KRAS mutation status and predicting clinical response to anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). mCRC samples from 398 patients from Roche study NO16968 (XELOXA) and 82 supplemental samples were tested with the cobas(®) KRAS mutation test (cobas test), the therascreen(®) KRAS RGQ PCR kit test (therascreen test), and Sanger sequencing as the reference method for detecting mutations in codons 12/13. For 461 eligible samples, the cobas test, therascreen test, and sequencing had invalid results for 5.2, 10.8, and 2.6 % of specimens, respectively. Valid cobas and therascreen test results had similar KRAS mutation-positive rates (37.3 vs. 36.3 %, respectively); sequencing was 28.5 %. Positive and negative percent agreement (PPA/NPA) between the cobas test and sequencing was 96.9 % (95 % confidence interval [CI] 92.2-98.8), and 88.7 % (95 % CI 84.7-91.8), respectively. PPA/NPA between the cobas and therascreen tests was 93.3 % (95 % CI 88.1-96.3) and 96.5 % (95 % CI 93.5-98.1), respectively. Bridging analysis from NCIC-CO.17 and NCT00113763 using the cobas test yielded modeled hazard ratios for overall survival and progression-free survival (PFS) of 0.558 (95 % CI 0.422-0.752) and 0.413 (95 % CI 0.304-0.550), respectively, for cetuximab and 0.989 (95 % CI 0.778-1.299) and 0.471 (95 % CI 0.360-0.626), respectively, for panitumumab, demonstrating significant efficacy in the KRAS-negative population for PFS. The cobas test showed similar accuracy to the therascreen test for detecting KRAS mutations and could appropriately identify mCRC patients ineligible for anti-EGFR therapy as demonstrated by bridging analysis results.

  11. Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts

    PubMed Central

    Li, S; Li, L; Zhu, Y; Huang, C; Qin, Y; Liu, H; Ren-Heidenreich, L; Shi, B; Ren, H; Chu, X; Kang, J; Wang, W; Xu, J; Tang, K; Yang, H; Zheng, Y; He, J; Yu, G; Liang, N

    2014-01-01

    Background: Determining the somatic mutations of epidermal growth factor receptor (EGFR)-pathway networks is the key to effective treatment for non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs).The somatic mutation frequencies and their association with gender, smoking history and histology was analysed and reported in this study. Methods: Five thousand one hundred and twenty-five NSCLC patients' pathology samples were collected, and EGFR, KRAS, BRAF and PIK3CA mutations were detected by multiplex testing. The mutation status of EGFR, KRAS, BRAF and PIK3CA and their association with gender, age, smoking history and histological type were evaluated by appropriate statistical analysis. Results: EGFR, KRAS, BRAF and PIK3CA mutation rates revealed 36.2%, 8.4%, 0.5% and 3.3%, respectively, across the 5125 pathology samples. For the first time, evidence of KRAS mutations were detected in two female, non-smoking patients, age 5 and 14, with NSCLC. Furthermore, we identified 153 double and coexisting mutations and 7 triple mutations. Interestingly, the second drug-resistant mutations, T790M or E545K, were found in 44 samples from patients who had never received TKI treatments. Conclusions: EGFR exons 19, 20 and 21, and BRAF mutations tend to happen in females and non-smokers, whereas KRAS mutations were more inclined to males and smokers. Activating and resistant mutations to EGFR-TKI drugs can coexist and ‘second drug-resistant mutations', T790M or E545K, may be primary mutations in some patients. These results will help oncologists to decide candidates for mutation testing and EGFR-TKI treatment. PMID:24743704

  12. Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

    PubMed Central

    Kotoula, Vassiliki; Charalambous, Elpida; Biesmans, Bart; Malousi, Andigoni; Vrettou, Eleni; Fountzilas, George; Karkavelas, George

    2009-01-01

    Background Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. Methodology/Principal Findings Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). Conclusions/Significance Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making. PMID

  13. [KRAS Genetic Mutation Analysis Using the Clinical FFPE Samples of Colorectal Carcinomas: Comparative Study among 5 Methods].

    PubMed

    Okada, Kanae; Asano, Naoko; Kajiura, Yoko; Shimoyama, Yoshie; Matsumoto, Hiroyuki; Matsushita, Tadashi

    2015-10-01

    Targeting of epidermal growth factor receptor (EGFR) with monoclonal antibodies such as cetuximab or panitumumab inhibits the activation of downstream signaling molecules of EGFR. Since EGFR inhibitor therapy has been approved for the treatment of colorectal carcinomas in patients with tumors lacking KRAS mutations, the detection of KRAS mutation is indispensable before starting therapy. Although several laboratory procedures have been developed to detect KRAS mutations, few comparative studies of the sensitivity of mutation detection have been performed for such procedures. Here, we compared the levels of detection in 5 commercial KRAS mutation detection methods using both standard DNA samples and formalin-fixed clinical samples obtained during surgery. Scorpion-ARMS assay has been reported as the most sensitive and we compared 4 other methods with Scorpion. Both PCR-rSSO assay and F-PHFA assay showed the highest concordance. As for the detection sensitivity, there were variations between clinical samples apparently due to sample quality or assay principles and methods. It is thus suggested that routine validation is required using samples prepared in each lab and the choice of assay may depend on various factors, such as lab environment, actual needs of physicians and patients, and the quality of the formalin-fixed specimens.

  14. 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

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

    PubMed

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

    2017-03-01

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

  16. p16 Hypermethylation and KRAS Mutation Are Independent Predictors of Cetuximab Plus FOLFIRI Chemotherapy in Patients with Metastatic Colorectal Cancer.

    PubMed

    Kim, Se Hyun; Park, Kyu Hyun; Shin, Sang Joon; Lee, Kang Young; Kim, Tae Il; Kim, Nam Kyu; Rha, Sun Young; Roh, Jae Kyung; Ahn, Joong Bae

    2016-01-01

    Hypermethylation of the CpG island of p16(INK4a) occurs in a significant proportion of colorectal cancer (CRC). We aimed to investigate its predictive role in CRC patients treated with 5-fluorouracil, leucovorin, irinotecan (FOLFIRI), and cetuximab. Pyrosequencing was used to identify KRAS mutation and hypermethylation of 6 CpG island loci (p16, p14, MINT1, MINT2, MINT31, and hMLH1) in DNA extracted from formalin-fixed paraffin-embedded specimens. Logistic regression and Cox regression were performed for analysis of the relation between methylation status of CpG island methylator phenotype (CIMP) markers including p16 and clinical outcome. Hypermethylation of the p16 gene was detected in 14 of 49 patients (28.6%) and showed significant association with KRAS mutation (Fisher exact, p=0.01) and CIMP positivity (Fisher exact, p=0.002). Patients with p16-unmethylated tumors had significantly longer time to progression (TTP; median, 9.0 months vs. 3.5 months; log-rank, p=0.001) and overall survival (median, 44.9 months vs. 16.4 months; log-rank, p=0.008) than those with p16-methylated tumors. Patients with both KRAS and p16 aberrancy (n=6) had markedly shortened TTP (median, 2.8 months) compared to those with either KRAS or p16 aberrancy (n=11; median, 8.6 months; p=0.021) or those with neither (n=32; median, 9.0 months; p < 0.0001). In multivariate analysis, KRAS mutation and p16 methylation showed independent association with shorter TTP (KRAS mutation: hazard ratio [HR], 3.21; p=0.017; p16 methylation: HR, 2.97; p=0.027). Hypermethylation of p16 was predictive of clinical outcome in metastatic CRC patients treated with cetuximab and FOLFIRI, irrespective of KRAS mutation.

  17. Molecular Epidemiology of EGFR and KRAS Mutations in 3026 Lung Adenocarcinomas: Higher Susceptibility of Women to Smoking-related KRAS-mutant Cancers

    PubMed Central

    Dogan, Snjezana; Shen, Ronglai; Ang, Daphne C; Johnson, Melissa L; D’Angelo, Sandra P; Paik, Paul K; Brzostowski, Edyta B; Riely, Gregory J; Kris, Mark G; Zakowski, Maureen F; Ladanyi, Marc

    2012-01-01

    Purpose The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear. Experimental Design We genotyped 3026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical and smoking history data. Results EGFR mutations were found in 43% of never smokers (NS) and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of NS. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. NS were more likely than smokers to have KRAS G>A transition mutation (mostly G12D) (58% vs. 20%, p=0.0001). KRAS G12C, the most common G>T transversion mutation in smokers, was more frequent in women (p=0.007) and these women were younger than men with the same mutation (median 65 vs. 69, p=0.0008) and had smoked less. Conclusions The distinct types of KRAS mutations in smokers vs. NS suggest that most KRAS-mutant lung cancers in NS are not due to secondhand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens. PMID:23014527

  18. 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.

  19. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer

    PubMed Central

    Loupakis, F; Ruzzo, A; Cremolini, C; Vincenzi, B; Salvatore, L; Santini, D; Masi, G; Stasi, I; Canestrari, E; Rulli, E; Floriani, I; Bencardino, K; Galluccio, N; Catalano, V; Tonini, G; Magnani, M; Fontanini, G; Basolo, F; Falcone, A; Graziano, F

    2009-01-01

    Background: KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC. Methods: We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients. Results: Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients. Conclusion: Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs. PMID:19603018

  20. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer.

    PubMed

    Loupakis, F; Ruzzo, A; Cremolini, C; Vincenzi, B; Salvatore, L; Santini, D; Masi, G; Stasi, I; Canestrari, E; Rulli, E; Floriani, I; Bencardino, K; Galluccio, N; Catalano, V; Tonini, G; Magnani, M; Fontanini, G; Basolo, F; Falcone, A; Graziano, F

    2009-08-18

    KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC. We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients. Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients. Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.

  1. Clinicopathological features and prognostic roles of KRAS, BRAF, PIK3CA and NRAS mutations in advanced gastric cancer

    PubMed Central

    2014-01-01

    Background RAS-RAF-MEK-ERK and PI3K-AKT pathways form a significant cascade for potential molecular target therapy in advanced cancer. The clinical significance of mutations in these genes in advanced gastric cancer (AGC) is uncertain. Methods We collected formalin-fixed, paraffin-embedded and fresh frozen tumor samples from AGC patients and analyzed the KRAS, NRAS, BRAF and PIK3CA mutations by direct-sequencing. We retrospectively investigated the clinicopathological features of these mutations in AGC patients, and selected patients with metastatic gastric cancer. Results Among 167 AGC patients, mutations of KRAS codons 12/13 (N = 8/164, 4.9%), PIK3CA (N = 9/163, 5.5%), and NRAS codon 12/13(N = 3/159, 1.9%) were detected. Comparison of the clinicopathological features of the mutated KRAS, PIK3CA, NRAS genes with an all-wild type of these genes showed that the frequency of the intestinal type was significantly higher in patients whose tumor tissue contained KRAS mutations (P = 0.014). Among 125 patients with metastatic gastric cancer, patients with NRAS codon 12/13 mutations in their tumors had shorter overall survival compared with NRAS wild-type patients (MST: 14.7 vs 8.8 months, P = 0.011). By multivariate analyses, NRAS codon 12/13 mutation was an indicator for poor prognosis in patients with metastatic gastric cancer (adjusted HR 5.607, 95% CI: 1.637-19.203). Conclusions Our study indicated that mutations of KRAS, PIK3CA and NRAS were rare in AGC. NRAS mutations were likely to associate with poor prognosis in metastatic state of AGC patients, but further validation of other research is required. PMID:24774510

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

    PubMed Central

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

    2011-01-01

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

  3. 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.

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

    PubMed

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

    2014-09-01

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

  5. Cytomorphological identification of advanced pulmonary adenocarcinoma harboring KRAS mutation in lymph node fine-needle aspiration specimens: Comparative investigation of adenocarcinoma with KRAS and EGFR mutations.

    PubMed

    Song, Dae Hyun; Lee, Boram; Shin, Yooju; Choi, In Ho; Ha, Sang Yun; Lee, Jae Jun; Hong, Min Eui; Choi, Yoon-La; Han, Joungho; Um, Sang-Won

    2015-07-01

    Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutation in pulmonary adenocarcinoma is clinically important due to its association with resistance to EGFR inhibitors and poor prognosis. To our knowledge, there has not been a comparative study focusing on cytological nuclear features of pulmonary adenocarcinoma harboring KRAS mutation (KRAS-AD). Hence, we compared the cytomorphology of metastatic KRAS-AD and EGFR-positive adenocarcinoma (EGFR-AD) in aspiration specimens from lymph nodes. Forty lymph node aspiration specimens from forty KRAS-AD patients were collected at Samsung Medical Center (Seoul, Korea) from 2009 to 2013. As a control group, 40 EBUS-FNA lymph node specimens from 20 EGFR-AD patients were collected. EGFR-AD specimens were evaluated at Samsung Medical Center (Seoul, Korea) from 2012 to 2013. All 80 specimens were histologically confirmed to metastatic adenocarcinoma. Two pathologists performed a blinded review of all specimens. Compared with EGFR-AD, KRAS-AD exhibited more severe nuclear pleomorphism (P < 0.001), coarse chromatin (P = 0.001), cherry-red nucleoli (P < 0.001) and naked tumor cells (P = 0.002) with necrotic (P < 0.001) and neutrophilic (P = 0.008) background. Our study provides the first demonstration of cytomorphologic differentiation between metastatic KRAS-AD and metastatic EGFR-AD in lymph node aspiration specimens. © 2014 Wiley Periodicals, Inc.

  6. Characteristics and prevalence of KRAS, BRAF, and PIK3CA mutations in colorectal cancer by high-resolution melting analysis in Taiwanese population.

    PubMed

    Hsieh, Li-Ling; Er, Tze-Kiong; Chen, Chih-Chieh; Hsieh, Jan-Sing; Chang, Jan-Gowth; Liu, Ta-Chih

    2012-10-09

    The identification of KRAS, BRAF, and PIK3CA mutations before the administration of anti-epidermal growth factor receptor therapy of colorectal cancer has become important. The aim of the present study was to investigate the occurrence of KRAS, BRAF, and PIK3CA mutations in the Taiwanese population with colorectal cancer. This study was undertaken to identify BRAF and PIK3CA mutations in patients with colorectal cancer by high-resolution melting (HRM) analysis. HRM analysis is a new gene scan tool that quickly performs the PCR and identifies sequence alterations without requiring post-PCR treatment. In the present study, DNAs were extracted from 182 cases of formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples for clinical KRAS mutational analysis by direct sequencing. All the samples were also tested for mutations within BRAF V600E and PIK3CA (exons 9 and 20) by HRM analysis. The results were confirmed by direct sequencing. The frequency of BRAF and PIK3CA mutations is 1.1%, and 7.1%, respectively. Intriguingly, we found that nine patients (4.9%) with the KRAS mutation were coexistent with the PIK3CA mutation. Four patients (2.2%) without the KRAS mutation were existent with the PIK3CA mutation. Two patients (1.1%) without the KRAS mutation were existent with the BRAF mutation. In the current study, we suppose that HRM analysis is rapid, feasible, and powerful diagnostic tool for the detection of BRAF and PIK3CA mutations in a clinical setting. Additionally, our results indicated the prevalence of KRAS, BRAF, and PIK3CA mutational status in the Taiwanese population. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Impact of fixation artifacts and threshold selection on high resolution melting analysis for KRAS mutation screening.

    PubMed

    Pérez-Báez, Wendy; García-Latorre, Ethel A; Maldonado-Martínez, Héctor Aquiles; Coronado-Martínez, Iris; Flores-García, Leonardo; Taja-Chayeb, Lucía

    2017-10-01

    Treatment in metastatic colorectal cancer (mCRC) has expanded with monoclonal antibodies targeting epidermal growth factor receptor, but is restricted to patients with a wild-type (WT) KRAS mutational status. The most sensitive assays for KRAS mutation detection in formalin-fixed paraffin embedded (FFPE) tissues are based on real-time PCR. Among them, high resolution melting analysis (HRMA), is a simple, fast, highly sensitive, specific and cost-effective method, proposed as adjunct for KRAS mutation detection. However the method to categorize WT vs mutant sequences in HRMA is not clearly specified in available studies, besides the impact of FFPE artifacts on HRMA performance hasn't been addressed either. Avowedly adequate samples from 104 consecutive mCRC patients were tested for KRAS mutations by Therascreen™ (FDA Validated test), HRMA, and HRMA with UDG pre-treatment to reverse FFPE fixation artifacts. Comparisons of KRAS status allocation among the three methods were done. Focusing on HRMA as screening test, ROC curve analyses were performed for HRMA and HMRA-UDG against Therascreen™, in order to evaluate their discriminative power and to determine the threshold of profile concordance between WT control and sample for KRAS status determination. Comparing HRMA and HRMA-UDG against Therascreen™ as surrogate gold standard, sensitivity was 1 for both HRMA and HRMA-UDG; and specificity and positive predictive values were respectively 0.838 and 0.939; and 0.777 and 0.913. As evaluated by the McNemar test, HRMA-UDG allocated samples to a WT/mutated genotype in a significatively different way from HRMA (p > 0.001). On the other hand HRMA-UDG did not differ from Therascreen™ (p = 0.125). ROC-curve analysis showed a significant discriminative power for both HRMA and HRMA-UDG against Therascreen™ (respectively, AUC of 0.978, p > 0.0001, CI 95% 0.957-0.999; and AUC of 0.98, p > 0.0001, CI 95% 0.000-1.0). For HRMA as a screening tool, the best threshold

  8. Metastatic colorectal cancer first-line treatment with bevacizumab: the impact of K-ras mutation.

    PubMed

    Rossi, Luigi; Veltri, Enzo; Zullo, Angelo; Zoratto, Federica; Colonna, Maria; Longo, Flavia; Mottolese, Marcella; Giannarelli, Diana; Ruco, Luigi; Marchetti, Paolo; Romiti, Adriana; Barucca, Viola; Giannini, Giuseppe; Bianchi, Loredana; Tomao, Silverio

    2013-01-01

    Bevacizumab plus chemotherapy prolongs progression-free survival (PFS) and overall survival (OS) in metastatic colorectal cancer (mCRC). Although there is strong evidence to suggest that the mutational status of the K-ras oncogene has a role as a predictive factor for activity in patients treated with cetuximab and panitumumab, few data have been obtained in patients treated with bevacizumab. We conducted an additional retrospective analysis to investigate the prognostic value of K-ras mutation relative to mCRC first-line treatment with bevacizumab. A total of 108 patients were retrospectively reviewed. K-ras status was assessed in the overall population by sequencing. Statistical association for PFS and OS was analyzed using the Kaplan-Meier method, and the prognostic role of K-ras was determined using the logrank test. Median PFS was 10 months both for patients with wild-type (WT) K-ras and mutated (MT) K-ras (hazard ratio [HR] 0.94, P=0.75); neither difference in median OS was significant (27 months WT K-ras versus 26 months MT K-ras, HR 0.92; P=0.70). A further analysis was carried out in the two groups according to metastatic sites. No statistically significant difference in terms of PFS and OS was demonstrated between WT K-ras and MT K-ras with liver metastases only and in those with extrahepatic disease. Although further study is required, our results seem to confirm that K-ras mutation does not have a prognostic role in mCRC patients receiving first-line treatment with bevacizumab.

  9. [Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer].

    PubMed

    Bai, Yan-qing; Liu, Xiao-jing; Wang, Yan; Ge, Fei-jiao; Zhao, Chuan-hua; Fu, Ya-li; Lin, Li; Xu, Jian-ming

    2013-09-01

    To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC). Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed. The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P < 0.001) as compared with those of chemotherapy alone

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

    PubMed

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

    2014-08-01

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

  11. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer.

    PubMed

    Palmirotta, Raffaele; Savonarola, Annalisa; Ludovici, Giorgia; De Marchis, Maria Laura; Covello, Renato; Ettorre, Giuseppe Maria; Ialongo, Cristiano; Guadagni, Fiorella

    2011-01-01

    The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient's sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1.

  12. 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.

  13. The prognostic value of KRAS mutation by cell-free DNA in cancer patients: A systematic review and meta-analysis.

    PubMed

    Zhuang, Rongyuan; Li, Song; Li, Qian; Guo, Xi; Shen, Feng; Sun, Hong; Liu, Tianshu

    2017-01-01

    KRAS mutation has been found in various types of cancer. However, the prognostic value of KRAS mutation in cell-free DNA (cfDNA) in cancer patients was conflicting. In the present study, a meta-analysis was conducted to clarify its prognostic significance. Literature searches of Cochrane Library, EMBASE, PubMed and Web of Science were performed to identify studies related to KRAS mutation detected by cfDNA and survival in cancer patients. Two evaluators reviewed and extracted the information independently. Review Manager 5.3 software was used to perform the statistical analysis. Thirty studies were included in the present meta-analysis. Our analysis showed that KRAS mutation in cfDNA was associated with a poorer survival in cancer patients for overall survival (OS, HR 2.02, 95% CI 1.63-2.51, P<0.01) and progression-free survival (PFS, HR 1.64, 95% CI 1.27-2.13, P<0.01). In subgroup analyses, KRAS mutation in pancreatic cancer, colorectal cancer, non-small cell lung cancer and ovarian epithelial cancer had HRs of 2.81 (95% CI 1.83-4.30, P<0.01), 1.67 (95% CI 1.25-2.42, P<0.01), 1.64 (95% CI 1.13-2.39, P = 0.01) and 2.17 (95% 1.12-4.21, p = 0.02) for OS, respectively. In addition, the ethnicity didn't influence the prognostic value of KRAS mutation in cfDNA in cancer patients (p = 0.39). Prognostic value of KRAS mutation was slightly higher in plasma than in serum (HR 2.13 vs 1.65), but no difference was observed (p = 0.37). Briefly, KRAS mutation in cfDNA was a survival prognostic biomarker in cancer patients. Its prognostic value was different in various types of cancer.

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

    PubMed

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

    2016-07-01

    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. 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. 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=0.092), but esophageal cancer

  15. Different metastatic pattern according to the KRAS mutational status and site-specific discordance of KRAS status in patients with colorectal cancer

    PubMed Central

    2012-01-01

    Background We evaluated the association between a KRAS mutational status and various clinicopathologic features including the metastatic pattern in patients with metastatic or recurrent colorectal cancer (MRCRC). The concordance rates of the KRAS status between primary tumor sites and paired metastatic organs were also analyzed. Methods The KRAS mutational status in codons 12, 13, and 61 from formalin-fixed sections of both primary tumors and related metastases was determined by sequencing analysis. One hundred forty-three Korean patients with MRCRC with available tissues (resection or biopsy) from both primary tumors and related metastatic sites were consecutively enrolled. Results The KRAS mutation rate was 52.4% (75/143) when considering both the primary and metastatic sites. When the relationship between the KRAS status and initial metastatic sites at the time of diagnosis of MRCRC was analyzed, lung metastasis was more frequent as the initial metastatic site in patients with the KRAS mutation than in patients without the KRAS mutation (45.3% vs. 22.1%; P = 0.003). However, liver (37.3% vs. 70.6%; P < 0.001) or distant lymph node metastases (6.7% vs. 19.1%; P = 0.025) were less frequent as the initial metastatic organ in patients with the KRAS mutation than in patients without the KRAS mutation. The discordance rate of KRAS mutational status between primary and paired metastatic sites other than the lung was 12.3% (13/106). Compared with primary tumor sites, the KRAS discordance rate was significantly higher in matched lung metastases [32.4% (12/37)] than in other matched metastatic organs (P = 0.005). Conclusions Organs initially involved by distant metastasis were different according to the KRAS mutational status in MRCRC patients. The concordance rate (87.7%) of the KRAS mutation status at metastatic sites other than the lung was generally high compared with primary tumor sites; however, lung metastasis had a high rate of KRAS discordance (32.4%). PMID

  16. KRAS mutation testing of metastatic colorectal cancer in Australia: where are we at?

    PubMed

    Scott, Rodney J; Fox, Stephen B; Desai, Jayesh; Grieu, Fabienne; Amanuel, Benhur; Garrett, Kerryn; Harraway, James; Cheetham, Glenice; Pattle, Neville; Haddad, Afaf; Byron, Keith; Rudzki, Barney; Waring, Paul; Iacopetta, Barry

    2014-09-01

    To carry out a nationwide study of KRAS testing in metastatic colorectal cancer as reported by nine major molecular pathology service providers in Australia, including mutation frequencies and turnaround times that might impact on patient care. Participating laboratories contributed information on KRAS mutation frequencies, including the G13D mutation type, as well as turnaround times for tumor block retrieval and testing. The KRAS mutation frequency observed by nine different test sites for a total of 3688 metastatic colorectal cancers ranged from 34.4% to 40.7%, with an average across all sites of 38.8%. The average frequency of the G13D mutation type among all cases was 8.0%. The median turnaround time was 17 days (range 0-191), with 20% of cases requiring more than 4 weeks for a KRAS test result. The major contributor to long turnaround times was the time taken to retrieve archived blocks of primary tumor, particularly from sources external to the test site. The frequency of KRAS mutations in metastatic colorectal cancer reported by the major Australian test sites is very similar to that reported by other large overseas studies. More widespread introduction of routine testing at the time of initial diagnosis should eliminate the long turnaround times currently being experienced in a significant proportion of cases. Future expansion of testing to include other KRAS and NRAS mutation hotspots may spur the introduction of next-generation sequencing platforms. © 2014 Wiley Publishing Asia Pty Ltd.

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

    PubMed

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

    2008-12-01

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

  19. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer.

    PubMed

    Westcott, Peter M K; Halliwill, Kyle D; To, Minh D; Rashid, Mamunur; Rust, Alistair G; Keane, Thomas M; Delrosario, Reyno; Jen, Kuang-Yu; Gurley, Kay E; Kemp, Christopher J; Fredlund, Erik; Quigley, David A; Adams, David J; Balmain, Allan

    2015-01-22

    Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras (Kras(LA2)). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras(LA2) model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras(LA2) model. By contrast, the Kras(LA2) tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations, and underline the importance of carcinogen models for

  20. 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

  1. 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

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

    PubMed

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

    2013-11-01

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

  3. Activating KRAS mutations are characteristic of oncocytic sinonasal papilloma and associated sinonasal squamous cell carcinoma.

    PubMed

    Udager, Aaron M; McHugh, Jonathan B; Betz, Bryan L; Montone, Kathleen T; Livolsi, Virginia A; Seethala, Raja R; Yakirevich, Evgeny; Iwenofu, O Hans; Perez-Ordonez, Bayardo; DuRoss, Kathleen E; Weigelin, Helmut C; Lim, Megan S; Elenitoba-Johnson, Kojo Sj; Brown, Noah A

    2016-08-01

    Oncocytic sinonasal papillomas (OSPs) are benign tumours of the sinonasal tract, a subset of which are associated with synchronous or metachronous sinonasal squamous cell carcinoma (SNSCC). Activating EGFR mutations were recently identified in nearly 90% of inverted sinonasal papillomas (ISPs) - a related tumour with distinct morphology. EGFR mutations were, however, not found in OSP, suggesting that different molecular alterations drive the oncogenesis of these tumours. In this study, tissue from 51 cases of OSP and five cases of OSP-associated SNSCC was obtained retrospectively from six institutions. Tissue was also obtained from 50 cases of ISP, 22 cases of ISP-associated SNSCC, ten cases of exophytic sinonasal papilloma (ESP), and 19 cases of SNSCC with no known papilloma association. Using targeted next-generation and conventional Sanger sequencing, we identified KRAS mutations in 51/51 (100%) OSPs and 5/5 (100%) OSP-associated SNSCCs. The somatic nature of KRAS mutations was confirmed in a subset of cases with matched germline DNA, and four matched pairs of OSP and concurrent associated SNSCC had concordant KRAS genotypes. In contrast, KRAS mutations were present in only one (5%) SNSCC with no known papilloma association and none of the ISPs, ISP-associated SNSCCs, or ESPs. This is the first report of somatic KRAS mutations in OSP and OSP-associated SNSCC. The presence of identical mutations in OSP and concurrent associated SNSCC supports the putative role of OSP as a precursor to SNSCC, and the high frequency and specificity of KRAS mutations suggest that OSP and OSP-associated SNSCC are biologically distinct from other similar sinonasal tumours. The identification of KRAS mutations in all studied OSP cases represents an important development in our understanding of the pathogenesis of this disease and may have implications for diagnosis and therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

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

    PubMed

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

    2012-07-01

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

  5. 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

  6. 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.

  7. BRAF V600E mutation and KRAS codon 13 mutations predict poor survival in Chinese colorectal cancer patients.

    PubMed

    Chen, Jing; Guo, Fang; Shi, Xin; Zhang, Lihua; Zhang, Aifeng; Jin, Hui; He, Youji

    2014-11-03

    Mutations in KRAS, BRAF and PIK3CA are the most common somatic alterations found in the colorectal cancer (CRC) patients from Western countries; but their prevalence and prognostic value have not been adequately assessed in Asian patients. The aim of this study was to determine the mutation frequencies of these genes in Chinese CRC patients and to investigate their impact on prognosis. The sequences of exon 2 of KRAS, exon 15 of BRAF and exons 9 and 20 of PIK3CA were evaluated by PCR and direct sequencing using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues from primary CRC tumors of 214 patients (colon/rectum: 126/88). KRAS, BRAF and PIK3CA mutations were identified in 44.9% (96/214), 4.2% (9/214) and 12.3% (26/212) CRCs, respectively. The most frequent mutations in KRAS, BRAF and PIK3CA were G12D, V600E and H1047R, respectively. All BRAF and 80.8% PIK3CA mutations were from colon cancer patients. BRAF V600E was associated with advanced TNM (P < 0.001), more distant metastases (P = 0.025), and worse overall survival (OS, P < 0.001; multivariate HR = 4.2, P = 0.004) in colon cancer patients. Compared with KRAS wt/BRAF wt CRC patients (N = 109), those with KRAS codon 13 mutations (N = 25) had significantly worse OS (P = 0.016; multivariate HR = 2.7, P = 0.011), whereas KRAS codon 12-mutated cases were not significantly associated with survival. Among the three most common KRAS mutations, G13D (N = 23) showed significant association with poor OS (P = 0.024; multivariate HR = 2.6, P = 0.016) compared with KRAS wt/BRAF wt patients. Our findings indicate that PI3K/RAS-RAF signaling pathway genes are frequently mutated in Chinese CRC patients, but have different characteristics than found in Western patients. BRAF V600E is an independent prognostic factor for Chinese patients. Our finding that KRAS codon 13 mutations (in particular G13D) are associated with inferior survival in BRAF wild

  8. Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit

    PubMed Central

    2013-01-01

    Background Retrospective analyses in the West suggest that mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA are negative predictive factors for cetuximab treatment in colorectal cancer patients. We developed a novel multiplex kit detecting 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA using Luminex (xMAP) assay in a single reaction. Methods Tumor samples and clinical data from Asian colorectal cancer patients treated with cetuximab were collected. We investigated KRAS, BRAF, NRAS, and PIK3CA mutations using both the multiplex kit and direct sequencing methods, and evaluated the concordance between the 2 methods. Objective response, progression-free survival (PFS), and overall survival (OS) were also evaluated according to mutational status. Results In total, 82 of 83 samples (78 surgically resected specimens and 5 biopsy specimens) were analyzed using both methods. All multiplex assays were performed using 50 ng of template DNA. The concordance rate between the methods was 100%. Overall, 49 (59.8%) patients had all wild-type tumors, 21 (25.6%) had tumors harboring KRAS codon 12 or 13 mutations, and 12 (14.6%) had tumors harboring KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations. The response rates in these patient groups were 38.8%, 4.8%, and 0%, respectively. Median PFS in these groups was 6.1 months (95% confidence interval (CI): 3.1–9.2), 2.7 months (1.2–4.2), and 1.6 months (1.5–1.7); median OS was 13.8 months (9.2–18.4), 8.2 months (5.7–10.7), and 6.3 months (1.3–11.3), respectively. Statistically significant differences in both PFS and OS were found between patients with all wild-type tumors and those with KRAS codon 61, KRAS codon 146, BRAF, NRAS, or PIK3CA mutations (PFS: 95% CI, 0.11–0.44; P < 0.0001; OS: 95% CI, 0.15–0.61; P < 0.0001). Conclusions Our newly developed multiplex kit is practical and feasible for investigation of a range of sample types. Moreover, mutations in KRAS

  9. KRAS mutation screening by chip-based DNA hybridization--a further step towards personalized oncology.

    PubMed

    Steinbach, Christine; Steinbrücker, Carolin; Pollok, Sibyll; Walther, Katharina; Clement, Joachim H; Chen, Yuan; Petersen, Iver; Cialla-May, Dana; Weber, Karina; Popp, Jürgen

    2015-04-21

    The use of predictive biomarkers can help to improve therapeutic options for the individual cancer patient. For the treatment of colon cancer patients with anti-EGFR-based drugs, the KRAS mutation status has to be determined to pre-select responders that will benefit from this medication. Amongst others, array-based tests have been established for profiling of the KRAS mutation status. Within this article we describe an on-chip hybridization technique to screen therapeutic relevant KRAS codon 12 mutations. The DNA chip-based platform enables the reliable discrimination of selected mutations by allele-specific hybridization. Here, silver deposits represent robust endpoint signals that allow for a simple naked eye rating. With the here presented assay concept a precise identification of heterozygous and homozygous KRAS mutations, even against a background of up to 95% wild-type DNA, was realizable. The applicability of the test was successfully proven for various cancer cell lines as well as clinical tumour samples. Thus, the chip-based DNA hybridization technique seems to be a promising tool for KRAS mutation analysis to further improve personalized cancer treatment.

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

    PubMed Central

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

    2014-01-01

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

  11. Prognostic significance of KRAS gene mutations in colorectal cancer - preliminary study

    PubMed Central

    Dinu, D; Dobre, M; Panaitescu, E; Bîrlă, R; Iosif, C; Hoara, P; Caragui, A; Boeriu, M; Constantinoiu, S; Ardeleanu, C

    2014-01-01

    Objective: The prognostic significance of KRAS gene mutations, evaluated by using two methods in patients with colorectal cancer (CRC). Material and Methods: Retrospective study involving 58 patients diagnosed with CRC and treated between 2003 and 2010 in the General and Esophageal Surgery Clinic of “Sf. Maria” Hospital, Bucharest. The macroscopic and microscopic examination of the resected specimens was also processed for genetic analysis in NIRDPBS, where KRAS status was determined by using two methods: PCR-RFLP and pyrosequencing. Results: The clinical and biological parameters of the patients were assessed for 72 months in average. A relapse in 21 patients and a 5-year survival rate of 79.3% was discovered. The genetic analyses of KRAS gene found mutations in 22 cases (45.3%): 17 cases had mutations in codon 12, 5 cases in codon 13. The survival rate analyses of patients with wild KRAS gene compared with the patients carrying the mutation on codon 12 /13 revealed a superposition of the survival curve. The statistical analysis based on the TNM stage revealed different survival curves in stage I and II, shorter survival period in patients with KRAS mutation on codon 13 than in those with wild type gene (stage I - p_value=0.015; stage II - p_value=0.000). Conclusions: It was not found that KRAS gene status had any prognostic significance. Nevertheless, for stage I and II patients, the mutation found on codon 13 determined a statistic significant shorter survival rate than for those with wild type. The results obtained by using the pyrosequencing method for the determination of KRAS gene status proved that it represented a reliable and reproducible method. PMID:25713627

  12. Correlation of EGFR or KRAS mutation status with 18F-FDG uptake on PET-CT scan in lung adenocarcinoma.

    PubMed

    Takamochi, Kazuya; Mogushi, Kaoru; Kawaji, Hideya; Imashimizu, Kota; Fukui, Mariko; Oh, Shiaki; Itoh, Masayoshi; Hayashizaki, Yoshihide; Ko, Weijey; Akeboshi, Masao; Suzuki, Kenji

    2017-01-01

    18F-fluoro-2-deoxy-glucose (18F-FDG) positron emission tomography (PET) is a functional imaging modality based on glucose metabolism. The correlation between EGFR or KRAS mutation status and the standardized uptake value (SUV) of 18F-FDG PET scanning has not been fully elucidated. Correlations between EGFR or KRAS mutation status and clinicopathological factors including SUVmax were statistically analyzed in 734 surgically resected lung adenocarcinoma patients. Molecular causal relationships between EGFR or KRAS mutation status and glucose metabolism were then elucidated in 62 lung adenocarcinomas using cap analysis of gene expression (CAGE), a method to determine and quantify the transcription initiation activities of mRNA across the genome. EGFR and KRAS mutations were detected in 334 (46%) and 83 (11%) of the 734 lung adenocarcinomas, respectively. The remaining 317 (43%) patients had wild-type tumors for both genes. EGFR mutations were more frequent in tumors with lower SUVmax. In contrast, no relationship was noted between KRAS mutation status and SUVmax. CAGE revealed that 4 genes associated with glucose metabolism (GPI, G6PD, PKM2, and GAPDH) and 5 associated with the cell cycle (ANLN, PTTG1, CIT, KPNA2, and CDC25A) were positively correlated with SUVmax, although expression levels were lower in EGFR-mutated than in wild-type tumors. No similar relationships were noted with KRAS mutations. EGFR-mutated adenocarcinomas are biologically indolent with potentially lower levels of glucose metabolism than wild-type tumors. Several genes associated with glucose metabolism and the cell cycle were specifically down-regulated in EGFR-mutated adenocarcinomas.

  13. KRAS mutation testing of tumours in adults with metastatic colorectal cancer: a systematic review and cost-effectiveness analysis.

    PubMed

    Westwood, Marie; van Asselt, Thea; Ramaekers, Bram; Whiting, Penny; Joore, Manuela; Armstrong, Nigel; Noake, Caro; Ross, Janine; Severens, Johan; Kleijnen, Jos

    2014-10-01

    Bowel cancer is the third most common cancer in the UK. Most bowel cancers are initially treated with surgery, but around 17% spread to the liver. When this happens, sometimes the liver tumour can be treated surgically, or chemotherapy may be used to shrink the tumour to make surgery possible. Kirsten rat sarcoma viral oncogene (KRAS) mutations make some tumours less responsive to treatment with biological therapies such as cetuximab. There are a variety of tests available to detect these mutations. These vary in the specific mutations that they detect, the amount of mutation they detect, the amount of tumour cells needed, the time to give a result, the error rate and cost. To compare the performance and cost-effectiveness of KRAS mutation tests in differentiating adults with metastatic colorectal cancer whose metastases are confined to the liver and are unresectable and who may benefit from first-line treatment with cetuximab in combination with standard chemotherapy from those who should receive standard chemotherapy alone. Thirteen databases, including MEDLINE and EMBASE, research registers and conference proceedings were searched to January 2013. Additional data were obtained from an online survey of laboratories participating in the UK National External Quality Assurance Scheme pilot for KRAS mutation testing. A systematic review of the evidence was carried out using standard methods. Randomised controlled trials were assessed for quality using the Cochrane risk of bias tool. Diagnostic accuracy studies were assessed using the QUADAS-2 tool. There were insufficient data for meta-analysis. For accuracy studies we calculated sensitivity and specificity together with 95% confidence intervals (CIs). Survival data were summarised as hazard ratios and tumour response data were summarised as relative risks, with 95% CIs. The health economic analysis considered the long-term costs and quality-adjusted life-years associated with different tests followed by treatment

  14. 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.

  15. Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel biochips.

    PubMed

    Emelyanova, Marina; Arkhipova, Ksenia; Mazurenko, Natalia; Chudinov, Alexander; Demidova, Irina; Zborovskaya, Irina; Lyubchenko, Lyudmila; Zasedatelev, Alexander; Nasedkina, Tatiana

    2015-04-01

    Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.

  16. KRAS mutation and microsatellite instability in endometrial adenocarcinomas showing MELF-type myometrial invasion.

    PubMed

    Stewart, C J R; Amanuel, B; Grieu, F; Carrello, A; Iacopetta, B

    2010-07-01

    Some uterine endometrioid adenocarcinomas exhibit a distinctive morphological phenotype characterised by the formation of microcystic, elongated and fragmented (MELF) glands. Immunohistochemical studies have suggested that MELF-type changes represent an epithelial-mesenchymal transition which has been associated with KRAS activation in various tumours. To investigate the molecular characteristics of endometrial tumours showing MELF, with particular reference to the frequencies of KRAS and BRAF mutations and of microsatellite instability (MSI). MSI, and KRAS and BRAF mutation status, were assessed in 33 low-grade endometrial adenocarcinomas showing MELF features and the results compared with 33 control cases exhibiting a 'conventional' pattern of myometrial invasion. Standard histological parameters were also reviewed. Tumours with a MELF pattern of myometrial invasion showed more frequent vascular invasion and focal mucinous differentiation. KRAS mutations were more frequent in MELF positive than MELF negative tumours (45% vs 30%), but this difference was not statistically significant. BRAF mutations were not identified in any of the cases. MSI was identified in 20% of cases overall but did not correlate with the MELF phenotype. Mutations in KRAS and BRAF genes are not directly implicated in the development of a MELF pattern of invasion in endometrial carcinoma. However, RAS-associated signalling pathways could be activated through other genetic or epigenetic mechanisms. The characterisation of such alterations may become increasingly important as novel therapies are developed that target mediators involved in tumour invasion.

  17. 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

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

    PubMed

    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

    2015-10-13

    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. 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. 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. 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.

  19. The impact of KRAS mutations on VEGF-A production and tumour vascular network.

    PubMed

    Figueras, Agnès; Arbos, Maria Antonia; Quiles, Maria Teresa; Viñals, Francesc; Germà, Josep Ramón; Capellà, Gabriel

    2013-03-18

    The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis.

  20. Cost-effectiveness analysis of screening for KRAS and BRAF mutations in metastatic colorectal cancer.

    PubMed

    Behl, Ajay S; Goddard, Katrina A B; Flottemesch, Thomas J; Veenstra, David; Meenan, Richard T; Lin, Jennifer S; Maciosek, Michael V

    2012-12-05

    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. 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. 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. 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.

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

    PubMed Central

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

    2016-01-01

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

  2. Computational Analysis of KRAS Mutations: Implications for Different Effects on the KRAS p.G12D and p.G13D Mutations

    PubMed Central

    Liu, Yen-Yi; Hwang, Jenn-Kang; Barrio, Maria Jesus; Rodrigo, Maximiliano; Garcia-Toro, Enrique; Herreros-Villanueva, Marta

    2013-01-01

    Background The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D) on protein conformation. Methods Molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D) and c.38G>A (p.G13D). The potential of mean force (PMF) simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT) KRAS and its mutants (MT). Results Using MD simulations, we observed that the root mean square deviation (RMSD) increased as a function of time for the MT c.35G>A (p.G12D) and MT c.38G>A (p.G13D) when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D) mutant is more open than that of the WT and the c.38G>A (p.G13D) proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D) may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions). Such fluctuations may promote instability in these protein regions and hamper GTP binding. Conclusions Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions). Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC) and the KRAS c.38G>A (p.G13D) mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D) mutation in mCRC needs to be further investigated. PMID:23437064

  3. Alterations of LKB1 and KRAS and Risk of Brain Metastasis: Comprehensive Characterization by Mutation Analysis, Copy Number, and Gene Expression in Non-Small-Cell Lung Carcinoma

    PubMed Central

    Zhao, Ni; Wilkerson, Matthew D.; Shah, Usman; Yin, Xiaoying; Wang, Anyou; Hayward, Michele C.; Roberts, Patrick; Lee, Carrie B.; Parsons, Alden M.; Thorne, Leigh B.; Haithcock, Benjamin E.; Grilley-Olson, Juneko E.; Stinchcombe, Thomas E.; Funkhouser, William K.; Wong, Kwok K.; Sharpless, Norman E.; Hayes, D. Neil

    2015-01-01

    Background Brain metastases are one of the most malignant complications of lung cancer and constitute a significant cause of cancer related morbidity and mortality worldwide. Recent years of investigation suggested a role of LKB1 in NSCLC development and progression, in synergy with KRAS alteration. In this study, we systematically analyzed how LKB1 and KRAS alteration, measured by mutation, gene expression (GE) and copy number (CN), are associated with brain metastasis in NSCLC. Materials and Methods Patients treated at University of North Carolina Hospital from 1990 to 2009 with NSCLC provided frozen, surgically extracted tumors for analysis. GE was measured using Agilent 44,000 custom-designed arrays, CN was assessed by Affymetrix GeneChip Human Mapping 250K Sty Array or the Genome-Wide Human SNP Array 6.0 and gene mutation was detected using ABI sequencing. Integrated analysis was conducted to assess the relationship between these genetic markers and brain metastasis. A model was proposed for brain metastasis prediction using these genetic measurements. Results 17 of the 174 patients developed brain metastasis. LKB1 wild type tumors had significantly higher LKB1 CN (p < 0.001) and GE (p = 0.002) than the LKB1 mutant group. KRAS wild type tumors had significantly lower KRAS GE (p < 0.001) and lower CN, although the latter failed to be significant (p = 0.295). Lower LKB1 CN (p = 0.039) and KRAS mutation (p = 0.007) were significantly associated with more brain metastasis. The predictive model based on nodal (N) stage, patient age, LKB1 CN and KRAS mutation had a good prediction accuracy, with area under the ROC curve of 0.832 (p < 0.001). Conclusion LKB1 CN in combination with KRAS mutation predicted brain metastasis in NSCLC. PMID:25224251

  4. KRAS and BRAF somatic mutations in colonic polyps and the risk of metachronous neoplasia.

    PubMed

    Juárez, Miriam; Egoavil, Cecilia; Rodríguez-Soler, María; Hernández-Illán, Eva; Guarinos, Carla; García-Martínez, Araceli; Alenda, Cristina; Giner-Calabuig, Mar; Murcia, Oscar; Mangas, Carolina; Payá, Artemio; Aparicio, José R; Ruiz, Francisco A; Martínez, Juan; Casellas, Juan A; Soto, José L; Zapater, Pedro; Jover, Rodrigo

    2017-01-01

    High-risk features of colonic polyps are based on size, number, and pathologic characteristics. Surveillance colonoscopy is often recommended according to these findings. This study aimed to determine whether the molecular characteristics of polyps might provide information about the risk of metachronous advanced neoplasia. We retrospectively included 308 patients with colonic polyps. A total of 995 polyps were collected and tested for somatic BRAF and KRAS mutations. Patients were classified into 3 subgroups, based on the polyp mutational profile at baseline, as follows: non-mutated polyps (Wild-type), at least one BRAF-mutated polyp, or at least one KRAS-mutated polyp. At surveillance, advanced adenomas were defined as adenomas ≥ 10 mm and/or with high grade dysplasia or a villous component. In contrast, advanced serrated polyps were defined as serrated polyps ≥ 10 mm in any location, located proximal to the splenic flexure with any size or with dysplasia. At baseline, 289 patients could be classified as wild-type (62.3%), BRAF mutated (14.9%), or KRAS mutated (22.8%). In the univariate analysis, KRAS mutations were associated with the development of metachronous advanced polyps (OR: 2.36, 95% CI: 1.22-4.58; P = 0.011), and specifically, advanced adenomas (OR: 2.42, 95% CI: 1.13-5.21; P = 0.023). The multivariate analysis, adjusted for age and sex, also showed associations with the development of metachronous advanced polyps (OR: 2.27, 95% CI: 1.15-4.46) and advanced adenomas (OR: 2.23, 95% CI: 1.02-4.85). Our results suggested that somatic KRAS mutations in polyps represent a potential molecular marker for the risk of developing advanced neoplasia.

  5. A novel K-ras mutation in colorectal cancer. A case report and literature review.

    PubMed

    Palmirotta, Raffaele; Savonarola, Annalisa; Formica, Vincenzo; Ludovici, Giorgia; Del Monte, Girolamo; Roselli, Mario; Guadagni, Fiorella

    2009-08-01

    Activating mutations in the K-ras oncogene mainly occur in codons 12 and 13 and may be predictive of response to drugs directly linked to the K-ras signaling pathway, such as panitumumab and cetuximab. K-ras analysis was carried out on DNA extracted from paraffin-embedded tumor samples after microdissection. Exons 1 and 2 were amplified by PCR and then sequenced. A never-reported K-ras mutation (CAG>TAG) determining a premature stop signal at codon 22 (Gln22Stop) was found in a patient with metastatic colorectal cancer. BRAF and p53 were not found to be modified and microsatellite instability was not present. The patient, however, was found to be unresponsive to an anti-EGFR MAb treatment. This study is the first report of a novel K-ras truncating mutation in a patient with metastatic colorectal cancer and is also suggestive for the evaluation of alternative pathways to better identify individuals who are likely to benefit from targeted therapies.

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

    PubMed

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

    2005-04-01

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

  7. Codon 13 KRAS mutation predicts patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

    PubMed

    Margonis, Georgios A; Kim, Yuhree; Sasaki, Kazunari; Samaha, Mario; Amini, Neda; Pawlik, Timothy M

    2016-09-01

    Investigations regarding the impact of tumor biology after surgical management of colorectal liver metastasis have focused largely on overall survival. We investigated the impact of codon-specific KRAS mutations on the rates and patterns of recurrence in patients after surgery for colorectal liver metastasis (CRLM). All patients who underwent curative-intent surgery for CRLM between 2002 and 2015 at Johns Hopkins who had available data on KRAS mutation status were identified. Clinico-pathologic data, recurrence patterns, and recurrence-free survival (RFS) were assessed using univariable and multivariable analyses. A total of 512 patients underwent resection only (83.2%) or resection plus radiofrequency ablation (16.8%). Although 5-year overall survival was 64.6%, 284 (55.5%) patients recurred with a median RFS time of 18.1 months. The liver was the initial recurrence site for 181 patients, whereas extrahepatic recurrence was observed in 162 patients. Among patients with an extrahepatic recurrence, 102 (63%) had a lung recurrence. Although overall KRAS mutation was not associated with overall RFS (P = 0.186), it was independently associated with a worse extrahepatic (P = 0.004) and lung RFS (P = 0.007). Among patients with known KRAS codon-specific mutations, patients with codon 13 KRAS mutation had a worse 5-year extrahepatic RFS (P = 0.01), whereas codon 12 mutations were not associated with extrahepatic (P = 0.11) or lung-specific recurrence rate (P = 0.24). On multivariable analysis, only codon 13 mutation independently predicted worse overall extrahepatic RFS (P = 0.004) and lung-specific RFS (P = 0.023). Among patients undergoing resection of CRLM, overall KRAS mutation was not associated with RFS. KRAS codon 13 mutations, but not codon 12 mutations, were associated with a higher risk for overall extrahepatic recurrence and lung-specific recurrence. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2698-2707. © 2016

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

    PubMed

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

    2001-09-01

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

  9. New Partnership Could Help Identify Drugs to Target Cancers Driven by KRAS Mutations | FNLCR

    Cancer.gov

    More than 100,000 newly diagnosed cases of cancer each year in the United States are subsequently linked to mutations in the KRAS protein. In response to this urgent problem, a new partnership agreement involving the Frederick National Laboratory for

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

    PubMed

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

    2017-03-01

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

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

    PubMed

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

    2012-12-01

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

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

    PubMed

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

    2015-01-01

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

  13. KRAS mutations affect prognosis of non-small-cell lung cancer patients treated with first-line platinum containing chemotherapy

    PubMed Central

    Garassino, Marina C.; Shepherd, Frances A.; Piva, Sheila; Caiola, Elisa; Macerelli, Marianna; Bettini, Anna; Lauricella, Calogero; Floriani, Irene; Farina, Gabriella; Longo, Flavia; Bonomi, Lucia; Fabbri, M. Agnese; Veronese, Silvio; Marsoni, Silvia; Broggini, Massimo; Rulli, Eliana

    2015-01-01

    KRAS mutations seem to indicate a poor outcome in Non-Small-Cell Lung Cancer (NSCLC) but such evidence is still debated. The aim of this planned ancillary study within the TAILOR trial was to assess the prognostic value of KRAS mutations in advanced NSCLC patients treated with platinum-based first-line chemotherapy. Patients (N = 540), enrolled in the study in 52 Italian hospitals, were centrally genotyped twice in two independent laboratories for EGFR and KRAS mutational status. Of these, 247 patients were eligible and included in the present study. The primary endpoint was overall survival (OS) according to KRAS mutational status in patients harboring EGFR wild-type. Sixty (24.3%) out of 247 patients harbored KRAS mutations. Median OS was 14.3 months and 10.6 months in wild-type and mutated KRAS patients, respectively (unadjusted Hazard Ratio [HR]=1.41, 95%Confidence Interval [CI]: 1.03-1.94 P = 0.032; adjusted HR=1.39, 95%CI: 1.00-1.94 P = 0.050). This study, with all consecutive patients genotyped, indicates that the presence of KRAS mutations has a mild negative impact on OS in advanced NSCLC patient treated with a first-line platinum-containing regimen. Trial Registration: clinicaltrials.gov identifier NCT00637910 PMID:26416458

  14. KRAS, BRAF oncogene mutations and tissue specific promoter hypermethylation of tumor suppressor SFRP2, DAPK1, MGMT, HIC1 and p16 genes in colorectal cancer patients.

    PubMed

    Bagci, Binnur; Sari, Musa; Karadayi, Kursat; Turan, Mustafa; Ozdemir, Ozturk; Bagci, Gokhan

    2016-06-24

    Colorectal cancer is a serious disease that causes significant morbidity and mortality in developed countries. Genetic changes, such as mutations in proto-oncogenes and DNA repair genes, and loss of function in the tumor suppressor genes cause colorectal cancer development. Abnormal DNA methylation is also known to play a crucial role in colorectal carcinogenesis. In this study, frequencies of KRAS and BRAF mutations, promoter hypermethylation profiles of SFRP2, DAPK1, MGMT, HIC1 and p16 genes, and possible associations between hypermethylation of these genes and KRAS and BRAF mutations were aimed to find out. Ninety three colorectal cancer tissues and 14 normal colon mucosas were included in the study. Common twelve KRAS gene mutation were investigated with using reverse-hybridization strip assay method. BRAF V600E mutations were investigated with RFLP method. Hypermethylation status of five tumor suppressor genes were detected by using reverse-hybridization strip assay method after bisulfite modification of DNA. KRAS and BRAF mutation frequencies were determined as 54.84% and 12.9%, respectively. Promoter hypermethylation frequencies of tumor suppressor genes SFRP2, DAPK1, MGMT, HIC1 and p16 were determined as 66.7%, 45.2%, 40.9%, 40.9% and 15.1%, respectively. Statistically significant associations were found between BRAF mutation and SFRP2 and p16 tumor suppressor genes hypermethylation (SFRP2; p= 0.005, p16; p= 0.016). Compared to rectum, SFRP2 (p= 0.017) and MGMT (p= 0.013) genes have statistically significantly higher promoter hypermethylation in colon. Results of the current study have confirmed that KRAS mutations and SFRP2 hypermethylation can be used as genetic markers in colorectal cancer.

  15. Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer.

    PubMed

    Park, Hye-Jin; Park, Jeong-Bin; Lee, Sang-Jae; Song, Minjung

    2017-08-11

    Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV-fluorescein isothiocyanate/propidium iodide-stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer.

  16. Phellinus linteus Grown on Germinated Brown Rice Increases Cetuximab Sensitivity of KRAS-Mutated Colon Cancer

    PubMed Central

    Park, Hye-Jin; Park, Jeong-Bin; Lee, Sang-Jae; Song, Minjung

    2017-01-01

    Colon cancer is one of the most common types of cancer, and it has recently become a leading cause of death worldwide. Among colon cancers, the v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated form is notorious for its non-druggable features. Cetuximab, a monoclonal antibody that binds to the epidermal growth factor receptor, has been introduced as an antitumor therapy; however, secondary resistance and side effects significantly limit its effective use in these cancers. In this study, we prepared Phellinuslinteus on germinated brown rice (PBR) extracts to increase the sensitivity of KRAS-mutated colon cancers to cetuximab. The combined treatment of PBR extract and cetuximab suppressed SW480 cell viability/proliferation, with the cells exhibiting altered cellular morphology and clonogenic potential. AnnexinV–fluorescein isothiocyanate/propidium iodide–stained flow cytometry and Western blotting were performed, and PBR extract combined with cetuximab treatment increased apoptosis of the SW480 cells and suppressed their KRAS protein expression. The potential of PBR as a synergistic anticancer agent was further investigated in a tumor-xenografted mouse model. Tumor growth was significantly suppressed with PBR extract and cetuximab co-treatment. In conclusion, PBR increased the sensitivity of KRAS-mutated colon cancer cells to cetuximab, which indicates the potential use of PBR as a medical food against colon cancer. PMID:28800074

  17. 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

  18. 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.

  19. 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.

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

    PubMed Central

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

    2012-01-01

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

  1. KRAS and BRAF mutational status in primary colorectal tumors and related metastatic sites: biological and clinical implications.

    PubMed

    Italiano, Antoine; Hostein, Isabelle; Soubeyran, Isabelle; Fabas, Thibault; Benchimol, Daniel; Evrard, Serge; Gugenheim, Jean; Becouarn, Yves; Brunet, René; Fonck, Marianne; François, Eric; Saint-Paul, Marie-Christine; Pedeutour, Florence

    2010-05-01

    KRAS and BRAF mutations in primary colorectal tumors (PT) are predictive of nonresponse to anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (mCRC). The question of primary resistance to anti-EGFR treatment as a result of the presence of KRAS or BRAF mutations only in metastases has been raised but not resolved. We analyzed the mutational status of KRAS and BRAF in 64 new patients with mCRC and performed a systematic review of published data from 285 patients. A total of 285 and 95 matched PT/metastases were available for the analysis of the KRAS and the BRAF status, respectively. An identical mutational pattern of KRAS in PT and the matching metastases were reported in all the cases but 14 (5%). In six cases (2%), KRAS was mutated in the PT and wild type in the metastatic site, whereas in eight cases (3%), KRAS was wild type in the PT and mutated in the metastatic site. An identical mutational pattern of BRAF in PT and the matching metastases was reported in all but two cases (3%). In one case (1.5%), BRAF was mutated in the PT and wild type in the metastatic site, whereas in one case (1.5%), BRAF was wild type in the PT and mutated in the metastatic site. The acquisition by metastases of a KRAS or a BRAF mutation that was not present in the PT is a rare event, occurring in 5% of cases of mCRC. This is not a frequent mechanism of primary resistance to anti-EGFR treatments in mCRC.

  2. ERCC1 Induction after Oxaliplatin Exposure May Depend on KRAS Mutational Status in Colorectal Cancer Cell Line: In Vitro Veritas

    PubMed Central

    Orlandi, A.; Di Salvatore, M.; Bagalà, C.; Basso, M.; Strippoli, A.; Plastino, F.; Calegari, M.A.; Cassano, A.; Astone, A.; Barone, C.

    2015-01-01

    Introduction: Oxaliplatin (Oxa) is widely used in metastatic colorectal cancer (mCRC), but currently there are not valid predictors of response to this drug. In the control arms both of OPUS and PRIME studies Oxa seems more active in patients with mCRC with mutated (mt) KRAS than in those with wild type (wt) KRAS. Recently we have retrospectively confirmed this suggestion, therefore we have hypothesized that the mutational status of KRAS could influence the expression of ERCC1, one of the main mechanisms of Oxa resistance. Material and Methods: We used four cell lines of colorectal cancer: two KRAS wild type (wt) (HCT-8 and HT-29) and two KRAS mt (SW620 and SW480). We evaluated the sensitivity of these cell lines to Oxa by MTT-test as well the ERCC1 levels before and after 24 h exposure to Oxa by Real-Time PCR. We silenced KRAS in a KRAS mt cell line (SW620LV) to evaluate the impact on Oxa sensitivity and ERCC1 levels. Lastly, ERCC1 was also silenced in order to confirm the importance of this protein as an Oxa resistance factor. Results: The KRAS mt cell lines resulted more sensitive to Oxa (OR 2.68; IC 95% 1.511-4.757 p<0.001). The basal levels of ERCC1 did not show significant differences between KRAS mt and wt cell lines, however, after 24 h exposure to Oxa, only the wt KRAS lines showed the ability to induce ERCC1, with a statistically significant difference (OR 42.9 IC 95% 17.260-106.972 p<0.0005). By silencing KRAS, sensitivity to Oxa was reduced in mt KRAS cell lines and this effect was associated with the acquisition of ability to induce ERCC1. Silencing of ERCC1, in turn, enhanced the sensitivity to Oxa in wt KRAS cell lines and restored sensitivity to Oxa in SW620LV cell line. Conclusion: KRAS mutated cell lines were more sensitive to Oxa. This feature seems secondary to the inability of these cells to induce ERCC1 after exposure to Oxa. Thus, KRAS mutational status might be a predictor of response to Oxa in CRC surrogating the cell ability to induce ERCC

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

    PubMed Central

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

    2015-01-01

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

  4. Endometrial cancer and somatic G>T KRAS transversion in patients with constitutional MUTYH biallelic mutations.

    PubMed

    Tricarico, Rossella; Bet, Paola; Ciambotti, Benedetta; Di Gregorio, Carmela; Gatteschi, Beatrice; Gismondi, Viviana; Toschi, Benedetta; Tonelli, Francesco; Varesco, Liliana; Genuardi, Maurizio

    2009-02-18

    MUTYH-associated polyposis (MAP) is an autosomal recessive condition predisposing to colorectal cancer, caused by constitutional biallelic mutations in the base excision repair (BER) gene MUTYH. Colorectal tumours from MAP patients display an excess of somatic G>T mutations in the APC and KRAS genes due to defective BER function. To date, few extracolonic manifestations have been observed in MAP patients, and the clinical spectrum of this condition is not yet fully established. Recently, one patient with a diagnosis of endometrial cancer and biallelic MUTYH mutations has been described. We here report on two additional unrelated MAP patients with biallelic MUTYH germline mutations who developed endometrioid endometrial carcinoma. The endometrial tumours were evaluated for PTEN, PIK3CA, KRAS, BRAF and CTNNB1 mutations. A G>T transversion at codon 12 of the KRAS gene was observed in one tumour. A single 1bp frameshift deletion of PTEN was observed in the same sample. Overall, these findings suggest that endometrial carcinoma is a phenotypic manifestations of MAP and that inefficient repair of oxidative damage can be involved in its pathogenesis.

  5. High concordance rate of KRAS/BRAF mutations and MSI-H between primary colorectal cancer and corresponding metastases.

    PubMed

    Fujiyoshi, Kenji; Yamamoto, Gou; Takahashi, Akemi; Arai, Yoshiko; Yamada, Mina; Kakuta, Miho; Yamaguchi, Kensei; Akagi, Yoshito; Nishimura, Yoji; Sakamoto, Hirohiko; Akagi, Kiwamu

    2017-02-01

    Genetic testing is needed for the treatment of colorectal cancer (CRC), especially molecular-targeted therapy. The effects of anti-EGFR therapy and prognosis are affected by the presence of KRAS mutations. However, whether primary CRC or metastatic tissues are appropriate in the analysis is still unclear. In the present study, we assessed the concordance of KRAS/BRAF mutation status and microsatellite instability (MSI) in primary CRC and corresponding metastases. This study enrolled 457 patients with surgically resected primary and corresponding metastatic CRC (499 synchronous metastases and 57 metachronous metastases) and seven local recurrences, and KRAS/BRAF mutation and MSI status were analysed for these tumours. The concordance rates of KRAS mutation, BRAF mutation, wild-type, MSI-H and MSS between primary CRC and corresponding metastases were 93.9% (214/228), 100% (30/30), 99.3% (304/306), 87.5% (21/24) and 100% (137/137), respectively. These high concordance rates were not different between synchronous and metachronous metastases. In conclusion, a high concordance of KRAS/BRAF mutation status and MSI status was observed between primary CRC and corresponding metastases in this study. Either primary CRC or metastatic tissues can be used for testing KRAS/BRAF mutation status and MSI status.

  6. Loss of heterozygosity, aberrant methylation, BRAF mutation and KRAS mutation in colorectal signet ring cell carcinoma.

    PubMed

    Kakar, Sanjay; Deng, Guoren; Smyrk, Thomas C; Cun, Lisa; Sahai, Vaibhav; Kim, Young S

    2012-07-01

    The relationship of molecular abnormalities with clinicopathologic features and survival in colorectal signet ring cell carcinoma, and its comparison with mucinous and conventional adenocarcinomas, has not been well studied. High-level microsatellite instability, loss of heterozygosity (LOH) at four loci, CpG island methylation phenotype based on seven loci, BRAF V600E mutation and KRAS mutation in signet ring cell carcinoma were compared with mucinous and conventional adenocarcinomas. The relationship of these molecular features in signet ring cell carcinoma with clinicopathologic features and survival was examined. LOH was observed in 93% of signet ring cell carcinomas compared with 62 and 70% of mucinous and conventional adenocarcinomas. Also, 80% of signet ring cell carcinomas with high-level microsatellite instability showed LOH compared with 14% each of mucinous and conventional adenocarcinomas. High-level microsatellite instability, CpG island methylation phenotype-positive status and BRAF V600E mutation were more often seen in signet ring cell carcinoma and mucinous adenocarcinoma compared with conventional adenocarcinoma. BRAF V600E mutation was significantly associated with CpG island methylation phenotype-positive status. Stage and BRAF V600E mutation in microsatellite-stable cases were the only variables with an affect on survival. In conclusion, chromosomal instability manifested by LOH is nearly a universal finding in signet ring cell carcinoma, including cases with high-level microsatellite instability. This may explain the aggressive behavior of signet ring cell carcinoma irrespective of high-level microsatellite-instability status. BRAF V600E mutation and CpG island methylation phenotype-positive status are similar in signet ring cell carcinoma and mucinous adenocarcinoma but more frequent when compared with conventional adenocarcinoma. In signet ring cell carcinoma, BRAF V600E mutation adversely affects survival in microsatellite-stable tumors

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2017-02-20

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

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

    PubMed

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

    2016-02-23

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2008-05-01

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

  12. 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

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

    PubMed

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

    2016-10-04

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

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

    PubMed

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

    2015-10-12

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

  15. The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

    PubMed

    Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

    2017-06-01

    Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

  16. 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

  17. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Core Needle Lung Biopsy Specimens: Adequacy for EGFR and KRAS Mutational Analysis

    PubMed Central

    Zakowski, Maureen F.; Pao, William; Thornton, Raymond H.; Ladanyi, Marc; Kris, Mark G.; Rusch, Valerie W.; Rizvi, Naiyer A.

    2013-01-01

    OBJECTIVE The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung. SUBJECTS AND METHODS The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens. RESULTS Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy. CONCLUSION Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients. PMID:20028932

  19. 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.

  20. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2017-01-07

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

  3. Concordant analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression between primary colorectal cancer and matched metastases

    PubMed Central

    Mao, Chen; Wu, Xin-Yin; Yang, Zu-Yao; Threapleton, Diane Erin; Yuan, Jin-Qiu; Yu, Yuan-Yuan; Tang, Jin-Ling

    2015-01-01

    Current data on the concordance of KRAS, BRAF, PIK3CA mutation status or PTEN expression status between primary tumors and metastases in colorectal cancer (CRC) are conflicting. We conducted a systematic review and meta-analysis to examine concordance and discordance of the status of these four biomarkers between primary tumors and corresponding metastases in CRC patients. The biomarker status in primary tumors was used as the reference standard. Concordance data for KRAS, BRAF, PIK3CA and PTEN were provided by 43, 16, 9 and 7 studies, respectively. The pooled concordance rate was 92.0% (95% CI: 89.7%–93.9%) for KRAS, 96.8% (95% CI: 94.8%–98.0%) for BRAF, 93.9% (95% CI: 89.7%–96.5%) for PIK3CA and 71.7% (95% CI: 57.6%–82.5%) for PTEN. The pooled false positive and false negative rates for KRAS were 9.0% (95% CI: 6.5%–12.4%) and 11.3% (95% CI: 8.0%–15.8%), respectively. KRAS, BRAF and PIK3CA mutations are highly concordant between primary tumors and corresponding metastases in CRC, but PTEN loss is not. Nine percent of patients with wild-type KRAS in primary tumors who received anti-EGFR treatment had mutant KRAS in metastases, while 11.3% patients with mutant KRAS primary tumors had wild-type KRAS in the metastases. These 11.3% patients currently do not receive potentially beneficial anti-EGFR treatment. PMID:25639985

  4. Frequency of EGFR and KRAS mutations in patients with non small cell lung cancer by racial background: do disparities exist?

    PubMed

    Bauml, Joshua; Mick, Rosemarie; Zhang, Yu; Watt, Christopher D; Vachani, Anil; Aggarwal, Charu; Evans, Tracey; Langer, Corey

    2013-09-01

    Mutations in EGFR and KRAS can impact treatment decisions for patients with NSCLC. The incidence of these mutations varies, and it is unclear whether there is a decreased frequency among African Americans (AfAs). We performed a retrospective chart review of 513 NSCLC patients undergoing EGFR and KRAS mutational analysis at the Hospital of the University of Pennsylvania between May 2008 and November 2011. Clinical and pathologic data were abstracted from the patients' electronic medical record. Of 497 patients with informative EGFR mutation analyses, the frequency of EGFR mutation was 13.9%. The frequency of EGFR mutations was associated with race (p < 0.001) and was lower in AfA patients compared to Caucasian (C) patients but did not reach statistical significance (4.8% vs. 13.7%, p = 0.06). Mean Charlson Comorbidity Index and number of cigarette pack years were significantly lower in patients with EGFR mutations (p = 0.01 and p < 0.001, respectively). Multivariable logistic regression analysis showed a significant association between race and EGFR mutation (p = 0.01), even after adjusting for smoking status (p < 0.001) and gender (p = 0.03). KRAS mutation (study frequency 28.1%) was not associated with race (p = 0.08; p=0.51 for Afa vs. C patients), but was more common among smokers (p < 0.001) and females (p = 0.01). Based on multivariable analysis, even after adjusting for smoking status and gender, we found that race was statistically significantly associated with EGFR mutation, but not KRAS mutational status. To the best of our knowledge, this is the largest single institution series to date evaluating racial differences in EGFR and KRAS mutational status among patients with NSCLC. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. 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.

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

    EPA Science Inventory


    Abstract

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

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

    PubMed

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

    2015-01-01

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

  8. 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

  9. Melorheostosis: Exome sequencing of an associated dermatosis implicates postzygotic mosaicism of mutated KRAS.

    PubMed

    Whyte, Michael P; Griffith, Malachi; Trani, Lee; Mumm, Steven; Gottesman, Gary S; McAlister, William H; Krysiak, Kilannin; Lesurf, Robert; Skidmore, Zachary L; Campbell, Katie M; Rosman, Ilana S; Bayliss, Susan; Bijanki, Vinieth N; Nenninger, Angela; Van Tine, Brian A; Griffith, Obi L; Mardis, Elaine R

    2017-08-01

    Melorheostosis (MEL) is the rare sporadic dysostosis characterized by monostotic or polyostotic osteosclerosis and hyperostosis often distributed in a sclerotomal pattern. The prevailing hypothesis for MEL invokes postzygotic mosaicism. Sometimes scleroderma-like skin changes, considered a representation of the pathogenetic process of MEL, overlie the bony changes, and sometimes MEL becomes malignant. Osteopoikilosis (OPK) is the autosomal dominant skeletal dysplasia that features symmetrically distributed punctate osteosclerosis due to heterozygous loss-of-function mutation within LEMD3. Rarely, radiographic findings of MEL occur in OPK. However, germline mutation of LEMD3 does not explain sporadic MEL. To explore if mosaicism underlies MEL, we studied a boy with polyostotic MEL and characteristic overlying scleroderma-like skin, a few bony lesions consistent with OPK, and a large epidermal nevus known to usually harbor a HRAS, FGFR3, or PIK3CA gene mutation. Exome sequencing was performed to ~100× average read depth for his two dermatoses, two areas of normal skin, and peripheral blood leukocytes. As expected for non-malignant tissues, the patient's mutation burden in his normal skin and leukocytes was low. He, his mother, and his maternal grandfather carried a heterozygous, germline, in-frame, 24-base-pair deletion in LEMD3. Radiographs of the patient and his mother revealed bony foci consistent with OPK, but she showed no MEL. For the patient, somatic variant analysis, using four algorithms to compare all 20 possible pairwise combinations of his five DNA samples, identified only one high-confidence mutation, heterozygous KRAS Q61H (NM_033360.3:c.183A>C, NP_203524.1:p.Gln61His), in both his dermatoses but absent in his normal skin and blood. Thus, sparing our patient biopsy of his MEL bone, we identified a heterozygous somatic KRAS mutation in his scleroderma-like dermatosis considered a surrogate for MEL. This implicates postzygotic mosaicism of mutated KRAS

  10. Analysis of KRAS/NRAS Mutations in a Phase III Study of Panitumumab with FOLFIRI Compared with FOLFIRI Alone as Second-line Treatment for Metastatic Colorectal Cancer.

    PubMed

    Peeters, Marc; Oliner, Kelly S; Price, Timothy J; Cervantes, Andrés; Sobrero, Alberto F; Ducreux, Michel; Hotko, Yevhen; André, Thierry; Chan, Emily; Lordick, Florian; Punt, Cornelis J A; Strickland, Andrew H; Wilson, Gregory; Ciuleanu, Tudor E; Roman, Laslo; Van Cutsem, Eric; He, Pei; Yu, Hua; Koukakis, Reija; Terwey, Jan-Henrik; Jung, Andre S; Sidhu, Roger; Patterson, Scott D

    2015-12-15

    We evaluated the influence of RAS mutation status on the treatment effect of panitumumab in a prospective-retrospective analysis of a randomized, multicenter phase III study of panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) versus FOLFIRI alone as second-line therapy in patients with metastatic colorectal cancer (mCRC; ClinicalTrials.gov, NCT0039183). Outcomes were from the study's primary analysis. RAS mutations beyond KRAS exon 2 (KRAS exons 3, 4; NRAS exons 2, 3, 4; BRAF exon 15) were detected by bidirectional Sanger sequencing in wild-type KRAS exon 2 tumor specimens. Progression-free survival (PFS) and overall survival (OS) were coprimary endpoints. The RAS ascertainment rate was 85%; 18% of wild-type KRAS exon 2 tumors harbored other RAS mutations. For PFS and OS, the hazard ratio (HR) for panitumumab plus FOLFIRI versus FOLFIRI alone more strongly favored panitumumab in the wild-type RAS population than in the wild-type KRAS exon 2 population [PFS HR, 0.70 (95% confidence interval [CI], 0.54-0.91); P = 0.007 vs. 0.73 (95% CI, 0.59-0.90); P = 0.004; OS HR, 0.81 (95% CI, 0.63-1.03); P = 0.08 vs. 0.85 (95% CI, 0.70-1.04); P = 0.12]. Patients with RAS mutations were unlikely to benefit from panitumumab. Among RAS wild-type patients, the objective response rate was 41% in the panitumumab-FOLFIRI group versus 10% in the FOLFIRI group. Patients with RAS mutations were unlikely to benefit from panitumumab-FOLFIRI and the benefit-risk of panitumumab-FOLFIRI was improved in the wild-type RAS population compared with the wild-type KRAS exon 2 population. These findings support RAS testing for patients with mCRC. Clin Cancer Res; 21(24); 5469-79. ©2015 AACR.See related commentary by Salazar and Ciardiello, p. 5415. ©2015 American Association for Cancer Research.

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

    PubMed

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    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.

  16. Patient and tumor characteristics and BRAF and KRAS mutations in colon cancer, NCCTG/Alliance N0147.

    PubMed

    Gonsalves, Wilson I; Mahoney, Michelle R; Sargent, Daniel J; Nelson, Garth D; Alberts, Steven R; Sinicrope, Frank A; Goldberg, Richard M; Limburg, Paul J; Thibodeau, Stephen N; Grothey, Axel; Hubbard, Joleen M; Chan, Emily; Nair, Suresh; Berenberg, Jeffrey L; McWilliams, Robert R

    2014-07-01

    KRAS and BRAF (V600E) mutations are important predictive and prognostic markers, respectively, in colon cancer, but little is known about patient and clinical factors associated with them. Two thousand three hundred twenty-six of 3397 patients in the N0147 phase III adjuvant trial for stage III colon cancer completed a patient questionnaire. Primary tumors were assessed for KRAS and BRAF (V600E) mutations and defective mismatch repair (dMMR) status. Logistic regression models and categorical data analysis were used to identify associations of patient and tumor characteristics with mutation status. All statistical tests were two-sided. KRAS (35%) and BRAF (V600E) (14%) mutations were nearly mutually exclusive. KRAS mutations were more likely to be present in patients without a family history of colon cancer and never smokers. Tumors with KRAS mutations were less likely to have dMMR (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.15 to 0.31; P < .001) and high-grade histology (OR = 0.73; 95% CI = 0.59 to 0.92; P < .001) but were more often right-sided. Among KRAS-mutated tumors, those with a Gly13Asp mutation tended to have dMMR and high-grade histology. Tumors with BRAF (V600E) mutations were more likely to be seen in patients who were aged 70 years or older (OR = 3.33; 95% CI = 2.50 to 4.42; P < .001) and current or former smokers (OR = 1.64; 95% CI = 1.26 to 2.14; P < .001) but less likely in non-whites and men. Tumors with BRAF (V600E) mutations were more likely to be right-sided and to have four or more positive lymph nodes, high-grade histology, and dMMR. Specific patient and tumor characteristics are associated with KRAS and BRAF (V600E) mutations. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Patient and Tumor Characteristics and BRAF and KRAS Mutations in Colon Cancer, NCCTG/Alliance N0147

    PubMed Central

    Gonsalves, Wilson I.; Mahoney, Michelle R.; Sargent, Daniel J.; Nelson, Garth D.; Alberts, Steven R.; Sinicrope, Frank A.; Goldberg, Richard M.; Limburg, Paul J.; Thibodeau, Stephen N.; Grothey, Axel; Hubbard, Joleen M.; Chan, Emily; Nair, Suresh; Berenberg, Jeffrey L.

    2014-01-01

    Background KRAS and BRAF V600E mutations are important predictive and prognostic markers, respectively, in colon cancer, but little is known about patient and clinical factors associated with them. Methods Two thousand three hundred twenty-six of 3397 patients in the N0147 phase III adjuvant trial for stage III colon cancer completed a patient questionnaire. Primary tumors were assessed for KRAS and BRAF V600E mutations and defective mismatch repair (dMMR) status. Logistic regression models and categorical data analysis were used to identify associations of patient and tumor characteristics with mutation status. All statistical tests were two-sided. Results KRAS (35%) and BRAF V600E (14%) mutations were nearly mutually exclusive. KRAS mutations were more likely to be present in patients without a family history of colon cancer and never smokers. Tumors with KRAS mutations were less likely to have dMMR (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.15 to 0.31; P < .001) and high-grade histology (OR = 0.73; 95% CI = 0.59 to 0.92; P < .001) but were more often right-sided. Among KRAS-mutated tumors, those with a Gly13Asp mutation tended to have dMMR and high-grade histology. Tumors with BRAF V600E mutations were more likely to be seen in patients who were aged 70 years or older (OR = 3.33; 95% CI = 2.50 to 4.42; P < .001) and current or former smokers (OR = 1.64; 95% CI = 1.26 to 2.14; P < .001) but less likely in non-whites and men. Tumors with BRAF V600E mutations were more likely to be right-sided and to have four or more positive lymph nodes, high-grade histology, and dMMR. Conclusions Specific patient and tumor characteristics are associated with KRAS and BRAF V600E mutations. PMID:24925349

  18. Is there a prognostic role of K-ras point mutations in the serum of patients with advanced non-small cell lung cancer?

    PubMed

    Camps, Carlos; Sirera, Rafael; Bremnes, Roy; Blasco, Ana; Sancho, Eva; Bayo, Pilar; Safont, Maria Jose; Sánchez, José Javier; Tarón, Miquel; Rosell, Rafael

    2005-12-01

    The purpose of this study was to investigate the prognostic significance of K-ras mutations in circulating DNA in advanced non-small lung cancer (NSCLC) patients. Serum samples were assessed prior to platinum-based chemotherapy start in 67 patients with advanced NSCLC (stage IIIB or IV), treated between April 1999 and June 2002. Patients were not previously treated with chemotherapy. K-ras oncogene mutations at codon 12 were analyzed by genomic amplification and direct sequencing of the patient's DNA present in serum. Pre-treatment serum was available in all 67 patients. Twenty patients (30%) demonstrated K-ras mutations while 47 patients (70%) had wild-type K-ras. Among K-ras mutations, the amino acid glycine was substituted by cystein in 90% and valine in 10%. When patients were grouped according to K-ras genotype, there was no significant difference for any of the baseline patient characteristics. There was a tendency towards a higher response rate for patients with K-ras mutations versus wild-type K-ras in serum, however not statistically significant (p=0.37). Median progression-free survival was 7.3 months versus 5.5 months in patients with mutations and with wild-type K-ras, respectively (p=0.23). For median overall survival time, the mutation group was comparable to the wild-type K-ras group with 12.5 and 11.4 months, respectively (p=0.28). In conclusion, there were no significant differences between the patients with K-ras mutations and those with wild-type genotype with respect to baseline patient characteristics, response rates, progression-free survival, or overall survival.

  19. Thyroid nodules with KRAS mutations are different from nodules with NRAS and HRAS mutations with regard to cytopathologic and histopathologic outcome characteristics.

    PubMed

    Radkay, Lisa A; Chiosea, Simion I; Seethala, Raja R; Hodak, Steven P; LeBeau, Shane O; Yip, Linwah; McCoy, Kelly L; Carty, Sally E; Schoedel, Karen E; Nikiforova, Marina N; Nikiforov, Yuri E; Ohori, N Paul

    2014-12-01

    Mutations in the RAS gene in the thyroid gland result in the activation of signaling pathways and are associated with a follicular growth pattern and the probability of a carcinoma outcome ranging from 74% to 87%. In the current study, the authors investigated the cytopathologic and histopathologic features of common RAS mutation subtypes. Malignant, indeterminate, and selected benign thyroid cytology cases were tested prospectively for the presence of NRAS61, HRAS61, and KRAS12/13 mutations. For each case, the Bethesda System for thyroid cytopathology diagnosis, additional cytologic descriptors, and surgical pathology outcomes were documented. The Fisher exact test and Wilcoxon 2-sample test were used for statistical comparison between the groups. A total of 204 thyroid fine-needle aspiration cases with RAS mutations (93.6% of which were associated with indeterminate cytopathology diagnoses) and corresponding surgical pathology resection specimens were identified. The KRAS12/13 mutation was associated with a significantly lower carcinoma outcome (41.7%) when compared with HRAS61 (95.5%) and NRAS61 (86.8%) mutations (P<.0001). Furthermore, oncocytic change was observed in a significantly higher percentage of cytology and resection specimens with KRAS12/13 mutations (66.7% and 75.0%, respectively) in comparison with those with HRAS61 (4.5% and 4.5%, respectively) and NRAS61 (15.4% and 14.7%, respectively) mutations (P<.0001). RAS mutations also were identified in cases of poorly differentiated carcinoma (NRAS61), anaplastic carcinoma (HRAS61), and medullary thyroid carcinoma (HRAS61 and KRAS12/13). Subclassification of RAS mutations in conjunction with cytopathologic evaluation improves presurgical risk stratification, provides better insight into lesional characteristics, and may influence patient management. In particular, KRAS12/13-mutated thyroid nodules were found to be different from HRAS61-mutated and NRAS61-mutated nodules with regard to cytopathologic and

  20. 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

  1. 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.

  2. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Detection of K-ras oncogene from the human genomic DNA using ultrasonication and a quantum dots-based microfluidic chip.

    PubMed

    Noh, Han Na; Kim, Jong Sung

    2013-09-01

    In clinical diagnostics, single-stranded DNAs (ssDNA) have been prepared from the human genomic DNA for the detection of a specific gene. In this study, the human genomic DNA was degraded via ultrasonication in solution, and K-ras oncogene was detected from the DNA fragments via the fluorescence quenching of quantum dots (QDs) by intercalating dyes after hybridization of the target, to probe DNAs in a microfluidic chip. K-ras is one of the most activated common oncogenes, and many human tumors are known to be due to the mutation of this gene. QDs are nano-sized semiconductors with a wide selection of emission wavelengths and exceptional stability against photo bleaching. In this study, probe DNA-conjugated QDs were immobilized to polystyrene microbeads, and the DNA-microbead-QDs complexes were packed through a microchannel by pillars that trap the beads in the microfluidic chip. The fluorescence of the QDs could be quenched by intercalating dye (TOTO-3) after hybridization of K-ras oncogene to the probe DNA in the channel. The fluorescence intensity decrease of the QDs can be used as an indication of the K-ras oncogene. By introducing an alkaline buffer solution, the DNAs were denatured, and the fluorescence intensity of the QDs again increased, which shows the possibility of reuse of the microfluidic chip for the detection of the K-ras gene.

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

    PubMed

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

    2016-04-07

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

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

    EPA Science Inventory


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

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

  6. 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...

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

    PubMed

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

    1993-05-08

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

  8. 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...

  9. 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...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. 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.

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

    PubMed

    van Es, Johan H; Clevers, Hans

    2015-01-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2006-04-01

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

  18. Phenotype of bone metastases of non-small cell lung cancer: epidermal growth factor receptor expression and K-RAS mutational status.

    PubMed

    Badalian, Gayane; Barbai, Tamás; Rásó, Erzsébet; Derecskei, Katalin; Szendrôi, Miklós; Tímár, József

    2007-01-01

    Bone metastasis is a frequent complication of lung cancer progression, however, studies on bone metastatic tissues are scanty. Here we have collected a small cohort of 11 non-small cell lung cancer cases where primary tumors and corresponding bone metastases were available for pathological analysis. We have tested two molecular markers: EGFR protein expression and K-RAS mutation at codon 12 using immunohistochemistry and RFLPPCR, respectively. We have shown that using improved protocols, EGFR protein (both the extracellular as well as the cytoplasmic domain) is readily detectable in decalcified bone tissue. We found that the EGFR expression status is highly similar in bone metastases compared to the primary tumors, although the expression levels may change. Individual comparison of corresponding primary and metastatic NSCLC tissues indicated that downregulation of EGFR was a rare event (2/11) compared to upregulation (4/11) in bone metastases. On the other hand, our data indicate that the K-RAS mutational status of the primary tumor does not predict the status of the bone metastatic tissue of NSCLC, since we have observed both emergence of mutant clones in metastases from wild-type (wt) primary tumors and loss of mutant clones in metastases from mutant primaries in addition to the maintained mutant status. Our data support that at least two progression models occur in NSCLC, the samegene as well as the clonal selection one. It is noteworthy that in NSCLC cases with wt- or mutant KRAS, downregulation of EGFR expression was a rare event although upregulation in bone metastases was observed more frequently in wt K-RAS cases.

  19. Response to first-line chemotherapy in patients with non-small-cell lung cancer according to epidermal growth factor receptor and K-RAS mutation status.

    PubMed

    Dong, Xiaopeng; Zhao, Xiaogang; Hao, Yingtao; Wei, Yucheng; Yin, Qiuwei; Du, Jiajun

    2013-11-01

    Epidermal growth factor receptor (EGFR)-targeted therapy has shown a favorable efficacy in patients with non-small-cell lung cancer (NSCLC). Conversely, K-RAS mutations were reported to have an adverse effect on the survival of patients with NSCLC. These studies suggest that the tumor biology of patients with EGFR or K-RAS mutations is different from that of patients with wild-type mutations. Therefore, we hypothesized that the response to cytotoxic chemotherapy may differ among patients with and without EGFR or K-RAS mutations. A total of 229 patients with advanced NSCLC who received platinum doublet chemotherapy were included in this retrospective study, and their clinical outcomes were analyzed according to EGFR and K-RAS mutation status. EGFR and K-RAS mutations were found in 52.4% and 27.9% of patients, respectively. Progression-free survival (PFS) was significantly higher in patients with EGFR mutations than in patients with wild-type EGFR (P = .008), and multivariate analysis showed that EGFR mutation was an independent factor to chemotherapy (P = .01). Among the patients with EGFR mutations, the disease control rate for docetaxel was higher than for gemcitabine-based therapy (P = .031). In addition, docetaxel or vinorelbine showed a longer PFS than gemcitabine-based chemotherapy in patients with EGFR mutations (P = .033 and P = .028). However, no similar differences were found according to the K-RAS mutations. EGFR, but not K-RAS mutation, is associated with improved survival time to platinum-based chemotherapy. In patients with EGFR mutations, PFS for docetaxel and gemcitabine was higher than for vinorelbine-based chemotherapies. The predictive meaning of EGFR mutation for chemotherapy should be further investigated. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Clinical and Metabolic Parameters in Non-Small Cell Lung Carcinoma and Colorectal Cancer Patients with and without KRAS Mutations

    PubMed Central

    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

    2014-01-01

    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

  1. Mutations of APC, K-ras, and p53 are associated with specific chromosomal aberrations in colorectal adenocarcinomas.

    PubMed

    Leslie, Amy; Pratt, Norman R; Gillespie, Karen; Sales, Mark; Kernohan, Neil M; Smith, Gillian; Wolf, C Roland; Carey, Francis A; Steele, Robert J C

    2003-08-01

    It is widely accepted that both large-scale chromosomal abnormalities and mutation of specific genes, such as APC, K-ras, and/or p53, occur in the majority of colorectal adenocarcinomas. Whether or not a relationship exists between these different forms of genetic abnormalities was previously unknown. Using comparative genomic hybridization and mutational analysis of APC, K-ras, and p53 to evaluate 50 colorectal adenocarcinomas, we have shown that mutation of p53 is significantly associated with gain of 20q, 13q, and 8q and loss of 18q (P = 0.000, 0.02, 0.044, and 0.001, respectively). Conversely, APC mutation did not associate with any of the above-mentioned aberrations but did associate significantly with gain of 7p (P = 0.01). Gain of chromosomal arm 12p, although a less common aberration, was significantly associated with K-ras mutation (P = 0.011). The associations we have described should refine the search for candidate genes underlying chromosomal aberrations and assist in the definition of distinct pathways in colorectal tumorigenesis.

  2. 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.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  5. 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

  6. A subset of lung adenocarcinomas and atypical adenomatous hyperplasia-associated foci are genotypically related: an EGFR, HER2, and K-ras mutational analysis.

    PubMed

    Sartori, Giuliana; Cavazza, Alberto; Bertolini, Federica; Longo, Lucia; Marchioni, Alessandro; Costantini, Matteo; Barbieri, Fausto; Migaldi, Mario; Rossi, Giulio

    2008-02-01

    Atypical adenomatous hyperplasia (AAH) is considered the preinvasive lesion of pulmonary adenocarcinoma, and mutations of EGFR, HER2, and K-ras are involved in the early stage of lung adenocarcinoma carcinogenesis, also predicting clinical response to anti-EGFR small molecule inhibitors. We analyzed 18 cases of primary lung adenocarcinoma with concomitant AAH foci from 13 patients for mutations of EGFR (exons 18-21), HER2 (exons 19-20), and K-ras (exon 2) by direct sequencing polymerase chain reaction. Among mutated cases, concordant mutations of EGFR or K-ras in adenocarcinoma and related AAH were observed in 5 (63%) of 8 cases. In particular, 3 of 4 adenocarcinomas with EGFR mutations (all L858R point mutations in women, never or former smokers) had a concomitant and identical mutation in AAH, and 2 of 4 adenocarcinomas with K-ras mutations (both at codon 12 in women, a never and a current smoker) showed the same mutation in concomitant AAH. All cases were wild-type for HER2. Mutations of EGFR and K-ras genes represent an early event in lung adenocarcinomagenesis, and AAH convincingly seems to be a precursor lesion in a subset of cases of adenocarcinoma.

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

    PubMed

    Løes, Inger Marie; Immervoll, Heike; Sorbye, Halfdan; Angelsen, Jon-Helge; Horn, Arild; Knappskog, Stian; Lønning, Per Eystein

    2016-08-01

    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. © 2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.

  8. K-ras, p53 mutations, and microsatellite instability (MSI) in gallbladder cancer.

    PubMed

    Saetta, Angelica A

    2006-06-15

    Despite the considerable progress in understanding the molecular pathology of carcinogenesis, the genetic mechanisms underlying the development and progression of gallbladder cancer (GC) are poorly understood. The survival of GC patients is generally poor. Therefore, it is very useful to define valuable prognostic factors. The most extensively studied oncogenes in gallbladder carcinogenesis are ras, commonly mutated in neoplasms of the gastrointestinal tract. K-ras oncogene is altered in a subset of gallbladder patients and mainly in those having anomalous junction of the pancreaticobiliary tract. Most of the studies of genetic abnormalities in GC have focused on p53 gene. p53 mutation/overexpression and/or LOH is present in more than 50% of gallbladder carcinomas, suggesting an important role in their pathogenesis. However, these results have not any predictive value yet. Moreover, the involvement of an alternative molecular pathway, that of microsatellite instability (MSI), is found in a limited group of GC patients. Additional research is necessary to establish its possible relation to defects of the mismatch repair (MMR) system and its proposed prognostic significance. Further elucidation of the molecular events specific to GC will help to identify novel molecular targets for the diagnosis and clinical management of the patients. Copyright 2006 Wiley-Liss, Inc.

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

    PubMed

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

    2015-08-01

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

  10. The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event.

    PubMed

    Jackson, P E; Cooper, D P; O'Connor, P J; Povey, A C

    1999-03-01

    In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC-->AT transitions in human tumours.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

    Xu, MengMeng; Counter, Christopher M.

    2016-01-01

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

  13. Complex karyotypes and KRAS and POT1 mutations impact outcome in CLL after chlorambucil-based chemotherapy or chemoimmunotherapy.

    PubMed

    Herling, Carmen Diana; Klaumünzer, Marion; Rocha, Cristiano Krings; Altmüller, Janine; Thiele, Holger; Bahlo, Jasmin; Kluth, Sandra; Crispatzu, Giuliano; Herling, Marco; Schiller, Joanna; Engelke, Anja; Tausch, Eugen; Döhner, Hartmut; Fischer, Kirsten; Goede, Valentin; Nürnberg, Peter; Reinhardt, Hans Christian; Stilgenbauer, Stephan; Hallek, Michael; Kreuzer, Karl-Anton

    2016-07-21

    Genetic instability is a feature of chronic lymphocytic leukemia (CLL) with adverse prognosis. We hypothesized that chromosomal translocations or complex karyotypes and distinct somatic mutations may impact outcome after first-line chemoimmunotherapy of CLL patients. We performed metaphase karyotyping and next-generation sequencing (NGS) of 85 genes in pretreatment blood samples obtained from 161 patients registered for CLL11, a 3-arm phase 3 trial comparing frontline chlorambucil (Clb) vs Clb plus rituximab (Clb-R) or Clb plus obinutuzumab in CLL patients with significant comorbidity. Chromosomal aberrations as assessed by karyotyping were observed in 68.8% of 154 patients, 31.2% carried translocations, and 19.5% showed complex karyotypes. NGS revealed 198 missense/nonsense mutations and 76 small indels in 76.4% of patients. The most frequently mutated genes were NOTCH1, SF3B1, ATM, TP53, BIRC3, POT1, XPO1, and KRAS Sole chemotherapy, treatment with Clb-R, or genetic lesions in TP53 (9.9% of patients) and KRAS (6.2% of patients) were significantly associated with nonresponse to study therapy. In multivariate models, complex karyotypes and POT1 mutations (8.1% of patients) represented significant prognostic factors for an unfavorable survival, independently of IGHV mutation status, Binet stage, and serum β-2-microglobuline. Patients with the copresence of complex karyotypes and deletions/mutations involving TP53 demonstrated a particularly short survival. In summary, this is the first prospective, controlled study in CLL patients that shows a role of complex karyotype aberrations as an independent prognostic factor for survival after front-line therapy. Moreover, the study identifies mutations in KRAS and POT1 as novel determinants of outcome after chemoimmunotherapy using chlorambucil and anti-CD20 treatment. © 2016 by The American Society of Hematology.

  14. Screening of mutations in the additional sex combs like 1, transcriptional regulator, tumor protein p53, and KRAS proto-oncogene, GTPase/NRAS proto-oncogene, GTPase genes of patients with myelodysplastic syndrome.

    PubMed

    Leite, Carolina; Delmonico, Lucas; Alves, Gilda; Gomes, Romario José; Martino, Mariana Rodrigues; da Silva, Aline Rodrigues; Moreira, Aline Dos Santos; Maioli, Maria Christina; Scherrer, Luciano Rios; Bastos, Elenice Ferreira; Irineu, Roberto; Ornellas, Maria Helena

    2017-10-01

    Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal bone marrow disorders characterized by ineffective hematopoiesis, different degrees of cellular dysplasia, and increased risk of progression to acute myeloid leukemia. International Prognostic Scoring System is the gold standard for MDS classification; however, patients exhibiting different clinical behaviors often coexist in the same group, indicating that the currently available scoring systems are insufficient. The genes that have recently been identified as mutated in MDS, including additional sex combs like 1, transcriptional regulator (ASXL1), tumor protein p53 (TP53), and KRAS proto-oncogene and GTPase (KRAS)/NRAS proto-oncogene, GTPase (NRAS), may contribute to a more comprehensive classification, as well as to the prognosis and progression of the disease. In the present study, the mutations in the ASXL1, TP53 and NRAS/KRAS genes in 50 patients were evaluated by sequencing genomic bone marrow DNA. Nine patients (18%) presented with at least one type of mutation. Mutations in TP53 were the most frequent in six patients (12%), followed by ASXL1 in two patients (4%) and NRAS in one patient (2%). The nine mutations were detected in patients with low- and high-risk MDS. The screening of mutations in MDS cases contributes to the application of personalized medicine.

  15. Effectors of epidermal growth factor receptor pathway: the genetic profiling ofKRAS, BRAF, PIK3CA, NRAS mutations in colorectal cancer characteristics and personalized medicine.

    PubMed

    Shen, Yinchen; Wang, Jianfei; Han, Xiaohong; Yang, Hongying; Wang, Shuai; Lin, Dongmei; Shi, Yuankai

    2013-01-01

    Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.

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

    PubMed

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

    2012-10-15

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

  17. Assessment of epidermal growth factor receptor and K-ras mutation status in cytological stained smears of non-small cell lung cancer patients: correlation with clinical outcomes.

    PubMed

    Lozano, Maria D; Zulueta, Javier J; Echeveste, Jose I; Gúrpide, Alfonso; Seijo, Luis M; Martín-Algarra, Salvador; Del Barrio, Anabel; Pio, Ruben; Idoate, Miguel Angel; Labiano, Tania; Perez-Gracia, Jose Luis

    2011-01-01

    Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18-21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available.

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

    PubMed Central

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

    2012-01-01

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

  19. Assessment of Epidermal Growth Factor Receptor and K-Ras Mutation Status in Cytological Stained Smears of Non-Small Cell Lung Cancer Patients: Correlation with Clinical Outcomes

    PubMed Central

    Zulueta, Javier J.; Echeveste, Jose I.; Gúrpide, Alfonso; Seijo, Luis M.; Martín-Algarra, Salvador; del Barrio, Anabel; Pio, Ruben; Idoate, Miguel Angel; Labiano, Tania; Perez-Gracia, Jose Luis

    2011-01-01

    Objective. Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. Methods. DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18–21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. Results. The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. Conclusion. Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available. PMID:21572125

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

    PubMed Central

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

    2014-01-01

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

  1. Clinical outcome of patients with non-small cell lung cancer receiving front-line chemotherapy according to EGFR and K-RAS mutation status.

    PubMed

    Kalikaki, Aristea; Koutsopoulos, Anastasios; Hatzidaki, Dora; Trypaki, Maria; Kontopodis, Emmanouel; Stathopoulos, Efstathios; Mavroudis, Dimitris; Georgoulias, Vassilis; Voutsina, Alexandra

    2010-07-01

    Somatic mutations in EGFR and K-RAS may predict for sensitivity and resistance to EGFR tyrosine kinase inhibitors (TKIs). Whether EGFR and K-RAS mutations could also predict clinical outcome of non-small cell lung cancer (NSCLC) patients following front-line chemotherapy has not yet been established. One hundred and sixty-two chemotherapy-naïve patients with locally advanced/metastatic NSCLC who received front-line chemotherapy were included in this retrospective study and their clinical outcome data was analyzed according to EGFR and K-RAS mutation status of their tumors. Classical activating EGFR and K-RAS mutations were found in 8.2 and 22.6% of patients respectively and were not associated with patients' clinicopathological characteristics. Patients with classical EGFR mutations had a higher probability of response to front-line chemotherapy as compared to those with wild type EGFR (p=0.023). Multivariate analysis showed that the presence of activating EGFR mutations was an independent factor associated with response to front-line chemotherapy (HR=4.85; 95% CI: 1.13-20.83, p=0.034). K-RAS mutation status was not associated with response to front-line chemotherapy. The presence of activating EGFR but not of K-RAS mutations was associated with a significantly higher overall survival compared to patients without mutations treated with platinum-based front-line chemotherapy (p=0.043). The data indicate that EGFR mutation status could be predictive for response to cytotoxic front-line chemotherapy in patients with NSCLC. Additional prospective studies are needed in order to validate this observation and to define whether these patients should be preferentially treated with front-line TKIs or chemotherapy. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  2. Comparative study of mutations in SNP loci of K-RAS, hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer

    PubMed Central

    Yan, Zhi-Hui; Cui, Li-Hong; Wang, Xiao-Hui; Li, Chao; He, Xing

    2014-01-01

    AIM: To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples. METHODS: A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A. RESULTS: The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ2 = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P < 0.05). The mutation rate of SNP at Va1384Asp locus of the hMLH1 gene was significantly higher in the observation group when compared to the control group II (χ2 = 10.486, 4.876, P < 0.05). The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene did not show any statistical difference from those in the control group II. CONCLUSION: There may be important clinical significance and relevance between neoplastic intestinal polyps and colorectal cancer in terms

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

    PubMed

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

    1996-12-01

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

  4. Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status.

    PubMed

    Oliveira, Carla; Westra, Jantine L; Arango, Diego; Ollikainen, Miina; Domingo, Enric; Ferreira, Ana; Velho, Sérgia; Niessen, Renee; Lagerstedt, Kristina; Alhopuro, Pia; Laiho, Paivi; Veiga, Isabel; Teixeira, Manuel R; Ligtenberg, Marjolijn; Kleibeuker, Jan H; Sijmons, Rolf H; Plukker, John T; Imai, Kohzoh; Lage, Pedro; Hamelin, Richard; Albuquerque, Cristina; Schwartz, Simo; Lindblom, Annika; Peltomaki, Päivi; Yamamoto, Hiroyuki; Aaltonen, Lauri A; Seruca, Raquel; Hofstra, Robert M W

    2004-10-01

    In sporadic colorectal tumours the BRAFV600E is associated with microsatellite instability (MSI-H) and inversely associated to KRAS mutations. Tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients carrying germline mutations in hMSH2 or hMLH1 do not show BRAFV600E, however no consistent data exist regarding KRAS mutation frequency and spectrum in HNPCC tumours. We investigated KRAS in 158 HNPCC tumours from patients with germline hMLH1, hMSH2 or hMSH6 mutations, 166 MSI-H and 688 microsatellite stable (MSS) sporadic carcinomas. All tumours were characterized for MSI and 81 of 166 sporadic MSI-H colorectal cancer (CRCs) were analysed for hMLH1 promoter hypermethylation. KRAS mutations were observed in 40% of HNPCC tumours, and the mutation frequency varied upon the mismatch repair gene affected: 48% (29/61) in hMSH2, 32% (29/91) in hMLH1 and 83% (5/6) in hMSH6 (P = 0.01). KRAS mutation frequency was different between HNPCC, MSS and MSI-H CRCs (P = 0.002), and MSI-H with hMLH1 hypermethylation (P = 0.005). Furthermore, HNPCC CRCs had more G13D mutations than MSS (P < 0.0001), MSI-H (P = 0.02) or MSI-H tumours with hMLH1 hypermethylation (P = 0.03). HNPCC colorectal and sporadic MSI-H tumours without hMLH1 hypermethylation shared similar KRAS mutation frequency, in particular G13D. In conclusion, we show that depending on the genetic/epigenetic mechanism leading to MSI-H, the outcome in terms of oncogenic activation may be different, reinforcing the idea that HNPCC, sporadic MSI-H (depending on the hMLH1 status) and MSS CRCs, may target distinct kinases within the RAS/RAF/MAPK pathway.

  5. Mutant KRAS promotes malignant pleural effusion formation.

    PubMed

    Agalioti, Theodora; Giannou, Anastasios D; Krontira, Anthi C; Kanellakis, Nikolaos I; Kati, Danai; Vreka, Malamati; Pepe, Mario; Spella, Magda; Lilis, Ioannis; Zazara, Dimitra E; Nikolouli, Eirini; Spiropoulou, Nikolitsa; Papadakis, Andreas; Papadia, Konstantina; Voulgaridis, Apostolos; Harokopos, Vaggelis; Stamou, Panagiota; Meiners, Silke; Eickelberg, Oliver; Snyder, Linda A; Antimisiaris, Sophia G; Kardamakis, Dimitrios; Psallidas, Ioannis; Marazioti, Antonia; Stathopoulos, Georgios T

    2017-05-16

    Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

  6. Clinical Significance of EML4-ALK Fusion Gene and Association with EGFR and KRAS Gene Mutations in 208 Chinese Patients with Non-Small Cell Lung Cancer

    PubMed Central

    Wei, Sen; Wang, Jing; Wang, Min; Wang, Yuli; Zhou, Qinghua; Liu, Hongyu; Chen, Jun

    2013-01-01

    The EML4-ALK fusion gene has been recently identified in a small subset of non-small cell lung cancer (NSCLC) patients who respond positively to ALK inhibitors. The characteristics of the EML4-ALK fusion gene in Chinese patients with NSCLC are poorly understood. Here, we report on the prevalence of EML4-ALK, EGFR status and KRAS mutations in 208 Chinese patients with NSCLC. EGFR mutations were found in 24.5% (51/208) of patients. In concordance with previous reports, these mutations were identified at high frequencies in females (47.5% vs 15.0% in males; P<0.05); never-smokers (42.3% vs 13.9% in smokers; P<0.05), and adenocarcinoma patients (44.2% vs 8.0% in non-adenocarcinoma patients; P<0.05). There were only 2.88% (6/208) patients with KRAS mutations in our study group. We identified 7 patients who harbored the EML4-ALK fusion gene (3.37%, 7/208), including 4 cases with variant 3 (57.1%), 2 with variant 1, and 1 with variant 2. All positive cases corresponded to female patients (11.5%, 7/61). Six of the positive cases were non-smokers (7.69%, 6/78). The incidence of EML4-ALK translocation in female, non-smoking adenocarcinoma patients was as high as 15.2% (5/33). No EGFR/KRAS mutations were detected among the EML4-ALK positive patients. Pathological analysis showed no difference between solid signet-ring cell pattern (4/7) and mucinous cribriform pattern (3/7) in ALK-positive patients. Immunostaining showed intratumor heterogeneity of ALK rearrangement in primary carcinomas and 50% (3/6) of metastatic tumors with ALK-negative staining. Meta-analysis demonstrated that EML4-ALK translocation occurred in 4.84% (125/2580) of unselected patients with NSCLC, and was also predominant in non-smoking patients with adenocarcinoma. Taken together, EML4-ALK translocations were infrequent in the entire NSCLC patient population, but were frequent in the NSCLC subgroup of female, non-smoker, adenocarcinoma patients. There was intratumor heterogeneity of ALK rearrangement in

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

    PubMed

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

    2017-02-01

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

  8. Colorectal cancer metastatic to the brain: analysis of prognostic factors and impact of KRAS mutations on presentation and outcome.

    PubMed

    Nieder, C; Hintz, M; Grosu, A L

    2016-01-01

    Treatment concepts for metastatic colorectal cancer continue to evolve. While the presence of RAS mutations impacts systemic therapy, little is known about the influence of such mutations in patients with brain metastases. Pooled retrospective analysis was conducted of 57 patients with brain metastases from colorectal cancer treated in two different institutions (2005-2013). The only mutations analyzed in a relatively large subgroup were KRAS mutations (14 wild type, 12 mutated). Mutation status was not associated with baseline characteristics such as number or location of metastases, and did not impact prognosis. Three factors were significantly associated with survival in multivariate analysis: Karnofsky Performance Status (KPS), management strategy, and systemic treatment. Median survival was 0.6 months with best supportive care, 3.0 months with initial whole-brain radiotherapy (WBRT), and 12.7 months if initial treatment included surgery or stereotactic radiosurgery (SRS), p = 0.0001. The survival difference between the WBRT and surgery/SRS groups was largest in patients with KPS 80-100. Effective local treatment was a prerequisite for improved survival. The only significant prognostic baseline factor was KPS, which forms the basis of the diagnosis-specific graded prognostic assessment (DS-GPA) score. Thus, our results validate the DS-GPA in this patient population. So far, neither this nor other studies suggest a clinically important impact of KRAS mutations beyond their previously reported association with development of brain metastases. Studies focusing on patients who develop brain metastases early during the course of metastatic disease might be warranted, because the influence of different systemic therapies might be larger in this subgroup.

  9. Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma.

    PubMed

    Rasmussen, Thomas; Haaber, Jacob; Dahl, Inger Marie; Knudsen, Lene M; Kerndrup, Gitte B; Lodahl, Marianne; Johnsen, Hans E; Kuehl, Michael

    2010-10-01

    Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Cells with the phenotype of tumor plasma cells (CD38(++)CD19(-)CD45(-/+)CD56(-/+/++)) or memory B cells (CD38(-)/CD19(+)/CD27(+)) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified. The increased expression of a specific "early" oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack "late" oncogene (K-RAS) mutations but express the "early" oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-04-01

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

  12. 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.

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

    PubMed

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

    2015-08-01

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

  14. 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

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

    PubMed Central

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

    2016-01-01

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

  16. KRAS Testing: A Tool for the Implementation of Personalized Medicine.

    PubMed

    Shackelford, Rodney E; Whitling, Nicholas A; McNab, Patricia; Japa, Shanker; Coppola, Domenico

    2012-07-01

    Activating point mutations in codons 12, 13, and 61 of the KRAS proto-oncogene are common in colorectal, non-small cell lung, pancreatic, and thyroid cancers. Constitutively activated KRAS mutations are strongly associated with a resistance to anti-epidermal growth factor receptor (EGFR) therapies, such as panitumumab and cetuximab used for treating metastatic colorectal carcinoma and EGFR tyrosine inhibitors used for advanced non-small cell lung cancers. Since anti-EGFR therapies are costly and may exert deleterious effects on individuals without activating mutations, KRAS mutation testing is recommended prior to the initiation of anti-EGFR therapy for these malignancies. The goal of this review is to summarize the KRAS mutation testing methods. Testing is now routinely requested in the clinical practice to provide data to assign the most appropriate anticancer chemotherapy for each given patient. Review of the most relevant literature was performed. Several areas were considered: ordering of the test, selection of the sample to be tested, and review of the testing methodologies. We found that several different methods are used for clinical KRAS mutation testing. Each of the methodologies is described, and information is provided about their performance, cost, turnaround times, detection limits, sensitivities, and specificities. We also provided "tips" for the appropriate selection and preparation of the sample to be tested. This is an important aspect of KRAS testing for clinical use, as the results of the test will affect clinical decisions with consequences for the patient.

  17. Mutational status of NRAS, KRAS, and PTPN11 genes is associated with genetic/cytogenetic features in children with B-precursor acute lymphoblastic leukemia.

    PubMed

    Liang, Der-Cherng; Chen, Shih-Hsiang; Liu, Hsi-Che; Yang, Chao-Ping; Yeh, Ting-Chi; Jaing, Tang-Her; Hung, Iou-Jih; Hou, Jen-Yin; Lin, Tung-Huei; Lin, Chun-Hui; Shih, Lee-Yung

    2017-08-29

    We aimed to investigate the frequencies and the association with genetic/cytogenetic abnormalities as well as prognostic relevance of RAS pathway mutations in Taiwanese children with B-precursor acute lymphoblastic leukemia (ALL), the largest cohort in Asians. Between 1995 and 2012, marrow samples at diagnosis from 535 children were studied for NRAS, KRAS, and PTPN11 mutations. The mutational status of each gene was correlated with the clinico-hematological features, recurrent genetic abnormalities, and outcomes for those treated with TPOG-ALL-2002 protocol (n = 346). The frequencies of NRAS, KRAS, and PTPN11 mutations were 10.8% (57/530), 10.2% (54/530), and 3.0% (16/526), respectively. NRAS mutations were associated with a higher frequency of hyperdiploidy (P = 0.01) and lower frequency of ETV6-RUNX1 (P < 0.01), whereas KRAS mutations were associated with younger age (P < 0.01), a higher frequency of KMT2A rearranged (P < 0.01) but no significant difference if infants with ALL were excluded, and inferior event-free survival (66.6% vs. 80.5%, P = 0.04). None of patients with TCF3-PBX1 had KRAS mutation (P = 0.02). Our study showed that the frequency of KRAS mutations in Taiwan was significantly higher than that reported in Caucasians. The occurrence of RAS pathway mutations was associated with recurrent genetic/cytogenetic abnormalities in pediatric B-precursor ALL. © 2017 Wiley Periodicals, Inc.

  18. 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

  19. Enumeration and targeted analysis of KRAS, BRAF and PIK3CA mutations in CTCs captured by a label-free platform: Comparison to ctDNA and tissue in metastatic colorectal cancer

    PubMed Central

    Triboulet, Melanie M.; Che, James; Ramani, Vishnu C.; Visser, Brendan C.; Poultsides, George A.; Longacre, Teri A.; Marziali, Andre; Vysotskaia, Valentina; Wiggin, Matthew; Heirich, Kyra; Hanft, Violet; Keilholz, Ulrich; Tinhofer, Ingeborg; Norton, Jeffrey A.; Lee, Mark; Sollier-Christen, Elodie; Jeffrey, Stefanie S.

    2016-01-01

    Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles. PMID:27863403

  20. Enumeration and targeted analysis of KRAS, BRAF and PIK3CA mutations in CTCs captured by a label-free platform: Comparison to ctDNA and tissue in metastatic colorectal cancer.

    PubMed

    Kidess-Sigal, Evelyn; Liu, Haiyan E; Triboulet, Melanie M; Che, James; Ramani, Vishnu C; Visser, Brendan C; Poultsides, George A; Longacre, Teri A; Marziali, Andre; Vysotskaia, Valentina; Wiggin, Matthew; Heirich, Kyra; Hanft, Violet; Keilholz, Ulrich; Tinhofer, Ingeborg; Norton, Jeffrey A; Lee, Mark; Sollier-Christen, Elodie; Jeffrey, Stefanie S

    2016-12-20

    Treatment of advanced colorectal cancer (CRC) requires multimodal therapeutic approaches and need for monitoring tumor plasticity. Liquid biopsy biomarkers, including CTCs and ctDNA, hold promise for evaluating treatment response in real-time and guiding therapeutic modifications. From 15 patients with advanced CRC undergoing liver metastasectomy with curative intent, we collected 41 blood samples at different time points before and after surgery for CTC isolation and quantification using label-free Vortex technology. For mutational profiling, KRAS, BRAF, and PIK3CA hotspot mutations were analyzed in CTCs and ctDNA from 23 samples, nine matched liver metastases and three primary tumor samples. Mutational patterns were compared. 80% of patient blood samples were positive for CTCs, using a healthy baseline value as threshold (0.4 CTCs/mL), and 81.4% of captured cells were EpCAM+ CTCs. At least one mutation was detected in 78% of our blood samples. Among 23 matched CTC and ctDNA samples, we found a concordance of 78.2% for KRAS, 73.9% for BRAF and 91.3% for PIK3CA mutations. In several cases, CTCs exhibited a mutation that was not detected in ctDNA, and vice versa. Complementary assessment of both CTCs and ctDNA appears advantageous to assess dynamic tumor profiles.

  1. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

    PubMed Central

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-01-01

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

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

    PubMed

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

    2014-05-01

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

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

    PubMed

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

    2014-01-27

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

  4. Regulation of uptake of 18F-FDG by a follicular human thyroid cancer cell line with mutation-activated K-ras.

    PubMed

    Prante, Olaf; Maschauer, Simone; Fremont, Valerie; Reinfelder, Julia; Stoehr, Robert; Szkudlinski, Mariusz; Weintraub, Bruce; Hartmann, Arndt; Kuwert, Torsten

    2009-08-01

    Dedifferentiation of thyroid carcinoma is accompanied by increased accumulation of the PET tracer (18)F-FDG. The molecular mechanisms responsible for this phenomenon are poorly understood. Therefore, we studied the regulation of (18)F-FDG uptake by the human follicular thyroid carcinoma cell line ML-1 and the as-yet-unknown oncogene expression of that cell line. The data obtained in ML-1 were compared with those of a well-differentiated thyroid cell line of rat origin (FRTL-5). The expression of the thyroid-stimulating hormone (TSH) receptor was investigated by immunocytochemistry, and the expression of the glucose transporters (GLUTs) was determined by Western blotting. Mutation analysis of ML-1 was performed for K-ras codons 12 and 13. The effect of TSH on intracellular cAMP levels was determined by a competitive enzyme immunoassay. Cells were incubated with (18)F-FDG (0.5-1.0 MBq/mL) for 1 h, and tracer uptake was related to protein concentration. The effects of bovine TSH, the cAMP analog (Bu)(2)cAMP, and the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor LY294002 on (18)F-FDG uptake were investigated. The TSH receptor was present in both cell lines. FRTL-5 clearly expressed GLUT-1 and also GLUT-4. In ML-1 only, the expression of GLUT-3 was detected. TSH and (Bu)(2)cAMP had a significant effect on (18)F-FDG uptake or GLUT-1 expression in FRTL-5, but not in ML-1 cells. PI3-kinase inhibition by LY294002 downregulated (18)F-FDG uptake in FRTL-5 by 58% +/- 9% (n = 6) and in ML-1 by 26% +/- 5% (n = 42, both P < 0.05). Mutation analysis of ML-1 cells revealed a Gly12Ser point mutation at codon 12 of the K-ras gene. (18)F-FDG uptake in the thyroid carcinoma cell line ML-1 is no longer regulated by TSH or cAMP or mediated by GLUT-1. However, in this cell line, this variable is still governed to some extent by PI3-kinase located downstream to the constitutively active K-ras in the Ras-PI3-kinase-Akt pathway. These data suggest that increases in (18)F-FDG uptake in

  5. Concurrent PEDF deficiency and Kras mutation induce invasive pancreatic cancer and adipose-rich stroma in mice.

    PubMed

    Grippo, Paul J; Fitchev, Philip S; Bentrem, David J; Melstrom, Laleh G; Dangi-Garimella, Surabhi; Krantz, Seth B; Heiferman, Michael J; Chung, Chuhan; Adrian, Kevin; Cornwell, Mona L; Flesche, Jan B; Rao, Sambasiva M; Talamonti, Mark S; Munshi, Hidayatullah G; Crawford, Susan E

    2012-10-01

    Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

  6. [Effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity].

    PubMed

    Li, Jing-nan; Li, Xiao; Qian, Jia-ming; Lu, Xin-qing; Yang, Hong

    2010-02-01

    To explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity. K-ras wild-type colon cancer cell line Caco-2 was transiently transfected by phr-GFP vector (control group), transfected by mutant K-ras gene phr-K-ras (Val12) vector (transfection group), transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific MAPK pathway inhibitor PD98059 (MAPK inhibition group), or transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific PI-3K pathway inhibitor LY294002 (PI-3K inhibition group), respectively. Cell migration was tested by Transwell experiment. E-cadherin and beta-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. beta-catenin protein level which combined with E-cadherin was detected by immunoprecipitation. RhoA activity was analyzed by Pull-down assay. The Caco-2 cell migration rate was (19.8 +/- 5.6) % in transfection group, which was significantly higher than that in control group [(14.0 +/- 4.2) %] (P = 0.001) and in MAPK inhibition group [(15.8 +/- 1.2) %] (P = 0.044), but was not significantly different from that in PI-3K inhibition group [(17.5 +/- 2.8) %] (P = 0.095). Immunofluorescence method showed that the E-cadherin and beta-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 protein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that beta-catenin protein level combined with E-cadherin decreased in transfection group and PI-3K group. Pull-down test showed that RhoA protein activity was up-regulated in transfection group. K-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E

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

    PubMed Central

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

    2016-01-01

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

  8. Analysis of p53, K-ras gene mutation & Helicobacter pylori infection in patients with gastric cancer & peptic ulcer disease at a tertiary care hospital in north India.

    PubMed

    Saxena, Ashish; Shukla, Sanket Kumar; Prasad, Kashi Nath; Ghoshal, Uday Chand

    2012-10-01

    Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients.

  9. Analysis of p53, K-ras gene mutation & Helicobacter pylori infection in patients with gastric cancer & peptic ulcer disease at a tertiary care hospital in north India

    PubMed Central

    Saxena, Ashish; Shukla, Sanket Kumar; Prasad, Kashi Nath; Ghoshal, Uday Chand

    2012-01-01

    Background & objectives: Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. Methods: In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. Results: Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. Interpretation & conclusions: Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients. PMID:23168708

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

    PubMed

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

    2016-12-01

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

  11. Modulation of EZH2 expression by MEK-ERK or PI3K-AKT signaling in lung cancer is dictated by different KRAS oncogene mutations

    PubMed Central

    Riquelme, Erick; Behrens, Carmen; Lin, Heather Y.; Simon, George; Papadimitrakopoulou, Vassiliki; Izzo, Julie; Moran, Cesar; Kalhor, Neda; Lee, J. Jack; Minna, John D.; Wistuba, Ignacio I.

    2016-01-01

    EZH2 overexpression promotes cancer by increasing histone methylation to silence tumor suppressor genes, but how EZH2 levels become elevated in cancer is not understood. In this study, we investigated the mechanisms by which EZH2 expression is regulated in non-small cell lung carcinoma cells by oncogenic KRAS. In cells harboring KRASG12C and KRASG12D mutations, EZH2 expression was modulated by MEK-ERK and PI3K/AKT signaling, respectively. Accordingly, MEK-ERK depletion decreased EZH2 expression in cells harboring the KRASG12C mutation, whereas PI3K/AKT depletion decreased EZH2 expression, EZH2 phosphorylation, and STAT3 activity in KRASG12D mutant cell lines. Combined inhibition of EZH2 and MEK-ERK or PI3K/AKT increased the sensitivity of cells with specific KRAS mutations to MEK-ERK and PI3K/AKT targeted therapies. Our work define EZH2 as a downstream effector of KRAS signaling and offer a rationale for combining EZH2 inhibitory strategies with MEK-ERK- or PI3K/AKT-targeted therapies to treat lung cancer patients, as stratified into distinct treatment groups based on specific KRAS mutations. PMID:26676756

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

    PubMed

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

    2008-01-01

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

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

    PubMed Central

    Jia, Xiangbo; Qian, Rulin; Zhang, Binbin

    2016-01-01

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

  14. The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status.

    PubMed

    Ii, Masanori; Li, Hua; Adachi, Yasushi; Yamamoto, Hiroyuki; Ohashi, Hirokazu; Taniguchi, Hiroaki; Arimura, Yoshiaki; Carbone, David P; Imai, Kohzoh; Shinomura, Yasuhisa

    2011-08-01

    Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown successful targeting therapy for colorectal, pancreatic, gastric, and esophageal carcinomas using recombinant adenoviruses expressing dominant negative IGF-IR. Mutation in k-ras is one of key factors in gastrointestinal cancers. In this study, we sought to evaluate the effect of a new monoclonal antibody for IGF-IR, figitumumab (CP-751,871), on the progression of human gastrointestinal carcinomas with/without k-ras mutation. We assessed the effect of figitumumab on signal transduction, proliferation, and survival in six gastrointestinal cancer cell lines with/without k-ras mutation, including colorectal and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. Combination effects of figitumumab and chemotherapy were also studied. Then figitumumab was evaluated in the treatment of xenografts in nude mice. Figitumumab blocked autophosphorylation of IGF-IR and its downstream signals. The antibody suppressed proliferation and tumorigenicity in all cell lines. Figitumumab inhibited survival by itself and up-regulated chemotherapy (5-FU and gemcitabine) induced apoptosis. Moreover, the combination of this agent and chemotherapy was effective against tumors in mice. The effect of figitumumab was not influenced by the mutation status of k-ras. Figitumumab reduced expression of IGF-IR but not insulin receptor in these xenografted tumors. The drug did not affect murine body weight or blood concentrations of glucose, insulin, IGF binding protein 3, and growth hormone. IGF-IR might be a good molecular therapeutic target and figitumumab may thus have therapeutic value in human gastrointestinal malignancies even in the presence of k-ras mutations. ©2011 AACR.

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

    PubMed

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

    2012-12-01

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

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

    PubMed Central

    Langer, Corey J.

    2011-01-01

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

  17. Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

    PubMed Central

    2012-01-01

    Background To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. Methods Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). Results 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. Conclusions Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. Trial registration number 2002-04-017 PMID:22876876

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

    PubMed

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

    2016-02-16

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

  19. 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

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

    PubMed Central

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

    2016-01-01

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

  1. Detection of low abundant mutations in DNA using single-molecule FRET and ligase detection reactions

    NASA Astrophysics Data System (ADS)

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

    2003-06-01

    New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled 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 MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated 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. Real time analyses of point mutations were also performed in PMMA microfluidic device.

  2. 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.

  3. 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

  4. Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

    PubMed

    De Roock, Wendy; Claes, Bart; Bernasconi, David; De Schutter, Jef; Biesmans, Bart; Fountzilas, George; Kalogeras, Konstantine T; Kotoula, Vassiliki; Papamichael, Demetris; Laurent-Puig, Pierre; Penault-Llorca, Frédérique; Rougier, Philippe; Vincenzi, Bruno; Santini, Daniele; Tonini, Giuseppe; Cappuzzo, Federico; Frattini, Milo; Molinari, Francesca; Saletti, Piercarlo; De Dosso, Sara; Martini, Miriam; Bardelli, Alberto; Siena, Salvatore; Sartore-Bianchi, Andrea; Tabernero, Josep; Macarulla, Teresa; Di Fiore, Frédéric; Gangloff, Alice Oden; Ciardiello, Fortunato; Pfeiffer, Per; Qvortrup, Camilla; Hansen, Tine Plato; Van Cutsem, Eric; Piessevaux, Hubert; Lambrechts, Diether; Delorenzi, Mauro; Tejpar, Sabine

    2010-08-01

    Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types

  5. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy.

    PubMed

    Allegra, Carmen J; Jessup, J Milburn; Somerfield, Mark R; Hamilton, Stanley R; Hammond, Elizabeth H; Hayes, Daniel F; McAllister, Pamela K; Morton, Roscoe F; Schilsky, Richard L

    2009-04-20

    An American Society of Clinical Oncology (ASCO) provisional clinical opinion (PCO), offers timely clinical direction to ASCO's oncologists following publication or presentation of potentially practice-changing data from major studies. This PCO addresses the utility of KRAS gene mutation testing in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MoAb) therapy with cetuximab or panitumumab (see Note). Recent results from phase II and III clinical trials demonstrate that patients with metastatic colorectal cancer benefit from therapy with monoclonal antibodies directed against the EGFR, when used either as monotherapy or combined with chemotherapy. Retrospective subset analyses of the data from these trials strongly suggest that patients who have KRAS mutations detected in codon 12 or 13 do not benefit from this therapy. Five randomized controlled trials of cetuximab or panitumumab have evaluated outcomes for patients with metastatic colorectal carcinoma in relation to KRAS mutational status as no mutation detected (wild type) or abnormal (mutated). Another five single-arm studies have retrospectively evaluated tumor response according to KRAS status. Based on systematic reviews of the relevant literature, all patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations in a CLIA-accredited laboratory. If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment. ASCO's provisional clinical opinions (PCOs) reflect expert consensus based on clinical evidence and literature available at the time they are written, and are intended to assist physicians in clinical decision-making and identify questions and settings for further research. Due to the rapid flow of scientific information in

  6. KRAS discordance between primary and metastatic tumor in patients with metastatic colorectal carcinoma.

    PubMed

    Siyar Ekinci, Ahmet; Demirci, Umut; Cakmak Oksuzoglu, Berna; Ozturk, Ayse; Esbah, Onur; Ozatli, Tahsin; Celik, Burcin; Budakoglu, Burcin; Turker, Ibrahim; Bal, Oznur; Turan, Nedim

    2015-01-01

    Adding targeted therapies to chemotherapy in metastatic colorectal cancer (CRC) improves response rates and survival. KRAS is a predictive indicator for anti-epidermal growth factor receptor (EGFR) treatments. The most important reasons for KRAS discordance are intratumoral heterogeneity and incorrect mutation analysis. Evaluating the status of KRAS in primary and metastatic lesions becomes even more crucial to ensure efficient usage of anti-EGFR treatments. Patients with metastatic CRC, whose primary disease and liver and/or lung metastases were operated, were retrospectively evaluated, and KRAS assessment was performed on 31 patients who were suitable for DNA analysis. Pyrosequencing with polymerase chain reaction (PCR) was used for KRAS analysis. The median age of 31 patients diagnosed with rectal cancer (N=13) and colon cancer (N=18) was 63 years (range 33-73). Metastasectomy locations included the liver (N=27), lung (N=3), and both lung and liver (N=1). KRAS discordance was detected in 22% (7/31) of the patients. While 3 patients with detected discordance had mutated KRAS in the primary material, wild type KRAS was detected in their liver or lung lesions. On the other hand, while 4 patients had wild type KRAS in the primary material, mutated KRAS was determined in their liver or lung lesions. The McNemar test revealed no significant discordance between primary and metastatic disease (p=1.00). No progression free survival (PFS) difference was detected between patients with determined discordance and patients with undetermined discordance (10.6 vs 14.7 months, p=0.719). This is the first study to evaluate KRAS discordance between primary and metastasis in CRC patients, who underwent metastasectomy, together with survival data. In the literature and recent studies with large patient numbers in which modern KRAS tests were used, the KRAS discordance rate varies between 3-12%. In our study, a higher KRAS discordance (22%) was detected, and no survival difference

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

    PubMed

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

    2004-04-01

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

  8. 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.

  9. 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

  10. SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate.

    PubMed

    Wong, Chi Chun; Qian, Yun; Li, Xiaona; Xu, Jiaying; Kang, Wei; Tong, Joanna H; To, Ka-Fai; Jin, Ye; Li, Weilin; Chen, Huarong; Go, Minnie Y Y; Wu, Jian-Lin; Cheng, Ka Wing; Ng, Simon S M; Sung, Joseph J Y; Cai, Zongwei; Yu, Jun

    2016-11-01

    Many colorectal cancer (CRC) cells contain mutations in KRAS. Analyses of CRC cells with mutations in APC or CTNNB1 and KRAS identified SLC25A22, which encodes mitochondrial glutamate transporter, as a synthetic lethal gene. We investigated the functions of SLC25A22 in CRC cells with mutations in KRAS. We measured levels of SLC25A22 messenger RNA and protein in paired tumor and nontumor colon tissues collected from 130 patients in Hong Kong and 17 patients in China and compared protein levels with patient survival times. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. DLD1 and HCT116 cells with SLC25A22 knockdown were grown as xenograft tumors in nude mice; tumor growth and metastasis were measured. SLC25A22 was expressed ectopically in HCT116 cells, which were analyzed in vitro and grown as xenograft tumors in nude mice. Levels of SLC25A22 messenger RNA and protein were increased in colorectal tumor tissues compared with matched nontumor colon tissues; increased protein levels were associated with shorter survival times of patients (P = .01). Knockdown of SLC25A22 in KRAS mutant CRC cells reduced their proliferation, migration, and invasion in vitro, and tumor formation and metastasis in mice, compared with cells without SLC25A22 knockdown. Knockdown of SLC25A22 reduced aspartate biosynthesis, leading to apoptosis, decreased cell proliferation in KRAS mutant CRC cells. Incubation of KRAS mutant CRC cells with knockdown of SLC25A22 with aspartate increased proliferation and reduced apoptosis, which required GOT1, indicating that oxaloacetate is required for cell survival. Decreased levels of oxaloacetate in cells with knockdown of SLC25A22 reduced

  11. A phase II study of sorafenib in patients with platinum-pretreated, advanced (Stage IIIb or IV) non-small cell lung cancer with a KRAS mutation.

    PubMed

    Dingemans, Anne-Marie C; Mellema, Wouter W; Groen, Harry J M; van Wijk, Atie; Burgers, Sjaak A; Kunst, Peter W A; Thunnissen, Erik; Heideman, Danielle A M; Smit, Egbert F

    2013-02-01

    Sorafenib inhibits the Ras/Raf pathway, which is overactive in cancer patients with a KRAS mutation. We hypothesized that patients with non-small cell lung cancer (NSCLC) with KRAS mutation will benefit from treatment with sorafenib. In this phase II study, patients with KRAS-mutated, stage IIIb or IV NSCLC that progressed after at least one platinum-containing regimen were treated with sorafenib. Treatment consisted of sorafenib 400 mg twice daily until disease progression or unacceptable toxicity. Pretreatment serum from each patient was obtained to predict outcome using a proteomic assay (VeriStrat). Primary endpoint was disease control rate (DCR) at 6 weeks. Fifty-nine patients were entered between May 2010 and February 2011. Fifty-seven patients started sorafenib. Mean age was 58.5 (SD = ±8.1) years, 16 male/41 female, Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0/1/2 24/30/3. At 6 weeks, 5 partial response, 25 stable disease, and 27 progressive disease were observed; DCR was 52.6%. Median duration of treatment was 9 weeks. The median progression-free survival (PFS) was 2.3 months and median overall survival (OS) was 5.3 months. Patients with a prediction of good prognosis according to VeriStrat serum proteomics assay showed a significantly superior PFS [HR, 1.4; 95% confidence interval (CI), 1.0-1.9] but not OS (HR, 1.3; 95% CI, 0.9-1.7). Sorafenib-related grade III/IV toxicity was reported in 10 patients (17.5%); all but one patient experienced grade III skin toxicity (14.0%) or grade III gastrointestinal toxicity (8.8%). Treatment with sorafenib has relevant clinical activity in patients with NSCLC harboring KRAS mutations. Further randomized study with this agent is warranted as single-agent or combination therapy.

  12. Radiation Resistance in KRAS-Mutated Lung Cancer Is Enabled by Stem-like Properties Mediated by an Osteopontin-EGFR Pathway.

    PubMed

    Wang, Meng; Han, Jing; Marcar, Lynnette; Black, Josh; Liu, Qi; Li, Xiangyong; Nagulapalli, Kshithija; Sequist, Lecia V; Mak, Raymond H; Benes, Cyril H; Hong, Theodore S; Gurtner, Kristin; Krause, Mechthild; Baumann, Michael; Kang, Jing X; Whetstine, Johnathan R; Willers, Henning

    2017-04-15

    Lung cancers with activating KRAS mutations are characterized by treatment resistance and poor prognosis. In particular, the basis for their resistance to radiation therapy is poorly understood. Here, we describe a radiation resistance phenotype conferred by a stem-like subpopulation characterized by mitosis-like condensed chromatin (MLCC), high CD133 expression, invasive potential, and tumor-initiating properties. Mechanistic investigations defined a pathway involving osteopontin and the EGFR in promoting this phenotype. Osteopontin/EGFR-dependent MLCC protected cells against radiation-induced DNA double-strand breaks and repressed putative negative regulators of stem-like properties, such as CRMP1 and BIM. The MLCC-positive phenotype defined a subset of KRAS-mutated lung cancers that were enriched for co-occurring genomic alterations in TP53 and CDKN2A. Our results illuminate the basis for the radiation resistance of KRAS-mutated lung cancers, with possible implications for prognostic and therapeutic strategies. Cancer Res; 77(8); 2018-28. ©2017 AACR. ©2017 American Association for Cancer Research.

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

    PubMed Central

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

    2017-01-01

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

  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. Proteomic Analysis of Exosomes from Mutant KRAS Colon Cancer Cells Identifies Intercellular Transfer of Mutant KRAS*

    PubMed Central

    Demory Beckler, Michelle; Higginbotham, James N.; Franklin, Jeffrey L.; Ham, Amy-Joan; Halvey, Patrick J.; Imasuen, Imade E.; Whitwell, Corbin; Li, Ming; Liebler, Daniel C.; Coffey, Robert J.

    2013-01-01

    Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression. PMID:23161513

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

    PubMed Central

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

    2016-01-01

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

  17. Prognostic value of the KRAS G12V mutation in 841 surgically resected Caucasian lung adenocarcinoma cases.

    PubMed

    Renaud, Stéphane; Falcoz, Pierre-Emmanuel; Schaëffer, Mickaël; Guenot, Dominique; Romain, Benoit; Olland, Anne; Reeb, Jérémie; Santelmo, Nicola; Chenard, Marie-Pierre; Legrain, Michèle; Voegeli, Anne-Claire; Beau-Faller, Michèle; Massard, Gilbert

    2015-10-20

    Identifying patients who will experience lung cancer recurrence after surgery remains a challenge. We aimed to evaluate whether mutant forms of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (mEGFR and mKRAS) are useful biomarkers in resected non-small cell lung cancer (NSCLC). We retrospectively reviewed data from 841 patients who underwent surgery and molecular testing for NSCLC between 2007 and 2012. mEGFR was observed in 103 patients (12.2%), and mKRAS in 265 (31.5%). The median overall survival (OS) and time to recurrence (TTR) were significantly lower for mKRAS (OS: 43 months; TTR: 19 months) compared with mEGFR (OS: 67 months; TTR: 24 months) and wild-type patients (OS: 55 months; disease-free survival (DFS): 24 months). Patients with KRAS G12V exhibited worse OS and TTR compared with the entire cohort (OS: KRAS G12V: 26 months vs 60 months; DFS: KRAS G12V: 15 months vs 24 months). These results were confirmed using multivariate analyses (non-G12V status, hazard ratio (HR): 0.43 (confidence interval: 0.28-0.65), P<0.0001 for OS; HR: 0.67 (0.48-0.92), P=0.01 for TTR). Risk of recurrence was significantly lower for non-KRAS G12V (HR: 0.01, (0.001-0.08), P<0.0001). mKRAS and mEGFR may predict survival and recurrence in early stages of NSCLC. Patients with KRAS G12V exhibited worse OS and higher recurrence incidences.

  18. Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

    PubMed

    Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

    2016-12-01

    A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC50: 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC50: 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC50: 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. A Novel Fully Automated Molecular Diagnostic System (AMDS) for Colorectal Cancer Mutation Detection

    PubMed Central

    Kitano, Shiro; Myers, Jamie; Nakamura, Junko; Yamane, Akio; Yamashita, Mami; Nakayama, Masato; Tsukahara, Yusuke; Ushida, Hiroshi; Liu, Wanqing; Ratain, Mark J.; Amano, Masahiko

    2013-01-01

    Background KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC). In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC). For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS) for point of care testing (POCT). Here we report the results of a comparison study between AMDS and direct sequencing (DS) in the detection of KRAS, BRAF and PI3KCA somatic mutations. Methodology/Principal Finding DNA was extracted from a slice of either frozen (n = 89) or formalin-fixed and paraffin-embedded (FFPE) CRC tissue (n = 70), and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples) detected by DS were also successfully (100%) detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous “hot spot” mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89) and 74.3% (52/70) in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41) were successfully detected all mutations within 70 minutes. Conclusions/Significance AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis. PMID:23671647

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

    PubMed Central

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

    2016-01-01

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

  1. Pyrosequencing data analysis software: a useful tool for EGFR, KRAS, and BRAF mutation analysis

    PubMed Central

    2012-01-01

    Background Pyrosequencing is a new technology and can be used for mutation tests. However, its data analysis is a manual process and involves sophisticated algorithms. During this process, human errors may occur. A better way of analyzing pyrosequencing data is needed in clinical diagnostic laboratory. Computer software is potentially useful for pyrosequencing data analysis. We have developed such software, which is able to perform pyrosequencing mutation data analysis for epidermal growth factor receptor, Kirsten rat sarcoma viral oncogene homolog and v-raf murine sarcoma viral oncogene homolog B1. The input data for analysis includes the targeted nucleotide sequence, common mutations in the targeted sequence, pyrosequencing dispensing order, pyrogram peak order and peak heights. The output includes mutation type and percentage of mutant gene in the specimen. Results The data from 1375 pyrosequencing test results were analyzed using the software in parallel with manual analysis. The software was able to generate correct results for all 1375 cases. Conclusion The software developed is a useful molecular diagnostic tool for pyrosequencing mutation data analysis. This software can increase laboratory data analysis efficiency and reduce data analysis error rate. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1348911657684292. PMID:22640803

  2. Pyrosequencing data analysis software: a useful tool for EGFR, KRAS, and BRAF mutation analysis.

    PubMed

    Shen, Shanxiang; Qin, Dahui

    2012-05-28

    Pyrosequencing is a new technology and can be used for mutation tests. However, its data analysis is a manual process and involves sophisticated algorithms. During this process, human errors may occur. A better way of analyzing pyrosequencing data is needed in clinical diagnostic laboratory. Computer software is potentially useful for pyrosequencing data analysis. We have developed such software, which is able to perform pyrosequencing mutation data analysis for epidermal growth factor receptor, Kirsten rat sarcoma viral oncogene homolog and v-raf murine sarcoma viral oncogene homolog B1. The input data for analysis includes the targeted nucleotide sequence, common mutations in the targeted sequence, pyrosequencing dispensing order, pyrogram peak order and peak heights. The output includes mutation type and percentage of mutant gene in the specimen. The data from 1375 pyrosequencing test results were analyzed using the software in parallel with manual analysis. The software was able to generate correct results for all 1375 cases. The software developed is a useful molecular diagnostic tool for pyrosequencing mutation data analysis. This software can increase laboratory data analysis efficiency and reduce data analysis error rate. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1348911657684292.

  3. The Presence of Telomere Fusion in Sporadic Colon Cancer Independently of Disease Stage, TP53/KRAS Mutation Status, Mean Telomere Length, and Telomerase Activity

    PubMed Central

    Tanaka, Hiromi; Beam, Matthew J.; Caruana, Kevin

    2014-01-01

    Defects in telomere maintenance can result in telomere fusions that likely play a causative role in carcinogenesis by promoting genomic instability. However, this proposition remains to be fully understood in human colon carcinogenesis. In the present study, the temporal sequence of telomere dysfunction dynamics was delineated by analyzing telomere fusion, telomere length, telomerase activity, hotspot mutations in KRAS or BRAF, and TP53 of tissue samples obtained from 18 colon cancer patients. Our results revealed that both the deficiency of p53 and the shortening of mean telomere length were not necessary for producing telomere fusions in colon tissue. In five cases, telomere fusion was observed even in tissue adjacent to cancerous lesions, suggesting that genomic instability is initiated in pathologically non-cancerous lesions. The extent of mean telomere attrition increased with lymph node invasiveness of tumors, implying that mean telomere shortening correlates with colon cancer progression. Telomerase activity was relatively higher in most cancer tissues containing mutation(s) in KRAS or BRAF and/or TP53 compared to those without these hotspot mutations, suggesting that telomerase could become fully active at the late stage of colon cancer development. Interestingly, the majority of telomere fusion junctions in colon cancer appeared to be a chromatid-type containing chromosome 7q or 12q. In sum, this meticulous correlative study not only highlights the concept that telomere fusion is present in the early stages of cancer regardless of TP53/KRAS mutation status, mean telomere length, and telomerase activity, but also provides additional insights targeting key telomere fusion junctions which may have significant implications for colon cancer diagnoses. PMID:25379018

  4. EGFR and KRAS mutations do not enrich for the activation of IL-6, JAK1 or phosphorylated STAT3 in resected lung adenocarcinoma.

    PubMed

    Clay, Timothy D; Russell, Prudence A; Do, Hongdo; Sundararajan, Vijaya; Conron, Matthew; Wright, Gavin M; Solomon, Benjamin; Dobrovic, Alexander; McLachlan, Sue-Anne; Moore, Melissa M

    2017-09-06

    Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) against EGFR mutant lung adenocarcinoma develops after a median of nine to thirteen months. Upregulation of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway may be a potential source of resistance to EGFR TKIs. We undertook a detailed assessment of the IL-6/JAK1/phosphorylated STAT3 (pSTAT3) pathway in resected lung adenocarcinoma specimens, with special interest in whether the presence of an EGFR mutation enriched for pSTAT3 positivity. Tumours from 143 patients with resected lung adenocarcinoma were assessed. EGFR and KRAS mutation status were scanned for with high-resolution melting and confirmed by polymerase chain reaction. Immunohistochemisty (IHC) was performed for IL-6, gp130, JAK1 and pSTAT3. Two methods for assigning IHC positivity were assessed (the presence of any positivity, and the presence of positivity at an H score >40). We found statistically significant associations between IL-6, JAK1 and pSTAT3 measured by IHC, consistent with the activation of the pathway in clinical specimens. No relationship was demonstrated between members of this pathway and oncogenic mutations in EGFR or KRAS. However, a proportion of tumours with EGFR mutations showed staining for IL-6, JAK1 and pSTAT3. No correlations with clinicopathologic features or survival outcomes were found for IL-6, JAK1 or pSTAT3 staining. The presence of EGFR or KRAS mutations did not enrich for the activation of IL-6, JAK1 or pSTAT3. pSTAT3 may still play a role in resistance to EGFR TKIs in clinical practice.

  5. Pooled Analysis of the Prognostic and Predictive Effects of TP53 Comutation Status Combined With KRAS or EGFR Mutation in Early-Stage Resected Non-Small-Cell Lung Cancer in Four Trials of Adjuvant Chemotherapy.

    PubMed

    Shepherd, Frances A; Lacas, Benjamin; Le Teuff, Gwénaël; Hainaut, Pierre; Jänne, Pasi A; Pignon, Jean-Pierre; Le Chevalier, Thierry; Seymour, Lesley; Douillard, Jean-Yves; Graziano, Stephen; Brambilla, Elizabeth; Pirker, Robert; Filipits, Martin; Kratzke, Robert; Soria, Jean-Charles; Tsao, Ming-Sound

    2017-06-20

    Purpose Our previous work evaluated individual prognostic and predictive roles of TP53, KRAS, and EGFR in non-small-cell lung cancer (NSCLC). In this analysis, we explore the prognostic and predictive roles of TP53/KRAS and TP53/EGFR comutations in randomized trials of adjuvant chemotherapy versus observation. Patients and Methods Mutation analyses (wild-type [WT] and mutant) for TP53, KRAS, and EGFR were determined in blinded fashion in multiple laboratories. Primary and secondary end points of pooled analysis were overall survival and disease-free survival. We evaluated the role of TP53/KRAS comutation in all patients and in the adenocarcinoma subgroup as well as the TP53/EGFR comutation in adenocarcinoma only through a multivariable Cox proportional hazards model stratified by trial. Results Of 3,533 patients with NSCLC, 1,181 (557 deaths) and 404 (170 deaths) were used for TP53/KRAS and TP53/EGFR analyses. For TP53/KRAS mutation status, no prognostic effect was observed ( P = .61), whereas a borderline predictive effect ( P = .04) was observed with a deleterious effect of chemotherapy with TP53/KRAS comutations versus WT/WT (hazard ratio, 2.49 [95% CI, 1.10 to 5.64]; P = .03). TP53/EGFR comutation in adenocarcinoma was neither prognostic ( P = .83), nor significantly predictive ( P = .86). Similar results were observed for both groups for disease-free survival. Conclusion We could identify no prognostic effect of the KRAS or EGFR driver and TP53 tumor suppressor comutation. Our observation of a potential negative predictive effect of TP53/KRAS comutation requires validation.

  6. 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.

  7. Adenocarcinoma arising from intracranial recurrent mature teratoma and featuring mutated KRAS and wild-type BRAF genes.

    PubMed

    Kim, Eun Soo; Kwon, Mi Jung; Song, Joon Ho; Kim, Dong Hoon; Park, Hye-Rim

    2015-02-01

    Malignant transformation or recurrence of intracranial mature teratoma is an extremely rare occurrence, compared to the usual ovarian counterpart. Previously, yolk sac tumor elements have been considered to be selective progenitors of enteric-type adenocarcinoma arising from intracranial germ cell tumors. However, the present case demonstrates the occurrence of enteric-type adenocarcinoma in recurrent intracranial mature cystic teratoma 12 years after gross total removal, a case of which has not previously been documented in the literature. The 11.5-cm long, dura mater-based tumor on the right fronto-temporal lobe displaced the brain; however, the patient had no neurologic symptoms or discomfort other than pus-like discharge on the scalp. Microscopic examinations revealed a small focus of adenocarcinoma and dysplastic colonic mucosa in the mature cystic teratoma. No immature elements were seen. The cystic wall was almost denuded and showed an exuberant xanthogranulomatous reaction with foreign-body type giant cells engulfing keratin materials and cholesterol clefts, suggesting that chronic inflammation due to repeated cyst wall rupture and the previous resection may contribute to malignant transformation. The adenocarcinoma showed strong immunohistochemical expression of CK20 and p53, but CK7 in patches. The molecular profile of the adenocarcinoma showed a mutation in KRAS and wild-type BRAF, which might be associated with malignant transformation of intracranial mature teratomas. In conclusion, the intracranial mature teratomas should require long-term follow-up, and clinicians, radiologists and pathologists should be aware of the potential for malignant progression of recurrent intracranial mature cystic teratoma despite gross total resection and no neurologic symptoms. © 2014 Japanese Society of Neuropathology.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2008-07-01

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

  11. High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort.

    PubMed

    Sorbye, Halfdan; Dragomir, Anca; Sundström, Magnus; Pfeiffer, Per; Thunberg, Ulf; Bergfors, Monica; Aasebø, Kristine; Eide, Geir Egil; Ponten, Fredrik; Qvortrup, Camilla; Glimelius, Bengt

    2015-01-01

    RAS and BRAF mutations impact treatment and prognosis of metastatic colorectal cancer patients (mCRC), but the knowledge is based on trial patients usually not representative for the general cancer population. Patient characteristics, treatment and efficacy according to KRAS, BRAF and MSI status were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue micro array (TMA) (42%) had worse prognostic factors and inferior survival (all patients; 7m vs 11m, chemotherapy-treated;12m vs 17m). The 92 patients (21%) with BRAF mutation had a poor prognosis regardless of microsatellite instability, but receipt of 1-2nd chemotherapy was similar to wildtype BRAF patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients <75 years in good PS. TMA availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF mutation incidence is higher than the previously and repeatedly reported incidence of 5-12% in mCRC. Screening for BRAF mutations before selection of treatment is relevant for many patients, especially outside clinical trials. A BRAF mutation only partly explained the very poor prognosis of many mCRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA studies and implies that we lack information on the biologically worst mCRC cases. Lack of available tissue is an important underexposed issue which introduces sample bias, and this should

  12. High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

    PubMed Central

    Kießling, Michael K.; Oberholzer, Patrick A.; Mondal, Chandrani; Karpova, Maria B.; Zipser, Marie C.; Lin, William M.; Girardi, Michael; MacConaill, Laura E.; Kehoe, Sarah M.; Hatton, Charlie; French, Lars E.; Garraway, Levi A.; Polier, Gernot; Süss, Dorothee; Klemke, Claus-Detlev; Krammer, Peter H.

    2011-01-01

    Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non–mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRASG13D mutation; one Sézary syndrome and one CD30+ CTCL harbored a NRASQ61K amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRASQ61K mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRASQ61K mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors. PMID:21209378

  13. Thyroid Metastasis as First Manifestation of a Colon Adenocarcinoma With KRAS Mutation: Usefulness of 18F-FDG PET/CT.

    PubMed

    Lecumberri, Beatriz; López, Elena; Coronado, Mónica; Rodado, Sonia; Castelo, Beatriz

    2017-07-29

    The synchronous diagnosis of a thyroid metastasis and of the primary colon adenocarcinoma that produces it is very rare, with only 5 cases reported to date, all of them treated with thyroid surgery showing a mean survival of 7 months. An ¹⁸F-FDG PET/CT in an asymptomatic 74-year-old woman with a thyroid cytology suggestive of malignancy but uncertain about the origin of the tumor revealed an stage IV colon adenocarcinoma with KRAS mutation and multiple metastasis (thyroid, lung, and liver). A prompt therapeutic planning with chemotherapy allowed 21 months of survival.

  14. Thyroid Metastasis as First Manifestation of a Colon Adenocarcinoma With KRAS Mutation: Usefulness of 18F-FDG PET/CT.

    PubMed

    Lecumberri, Beatriz; López, Elena; Coronado, Mónica; Rodado, Sonia; Castelo, Beatriz

    2017-10-01

    The synchronous diagnosis of a thyroid metastasis and of the primary colon adenocarcinoma that produces it is very rare, with only 5 cases reported to date, all of them treated with thyroid surgery showing a mean survival of 7 months. An F-FDG PET/CT in an asymptomatic 74-year-old woman with a thyroid cytology suggestive of malignancy but uncertain about the origin of the tumor revealed an stage IV colon adenocarcinoma with KRAS mutation and multiple metastasis (thyroid, lung, and liver). A prompt therapeutic planning with chemotherapy allowed 21 months of survival.

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

    PubMed

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

    2013-06-01

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

  16. KRAS analysis in colorectal carcinoma: Analytical aspects of Pyrosequencing and allele-specific PCR in clinical practice

    PubMed Central

    2010-01-01

    Background Epidermal growth factor receptor inhibitor therapy is now approved for treatment of metastatic colorectal carcinomas (CRC) in patients with tumors lacking KRAS mutations. Several procedures to detect KRAS mutations have been developed. However, the analytical sensitivity and specificity of these assays on routine clinical samples are not yet fully characterised. Methods The practical aspects and clinical applicability of a KRAS-assay based on Pyrosequencing were evaluated in a series of 314 consecutive CRC cases submitted for diagnostic KRAS analysis. The performance of Pyrosequencing compared to allele-specific, real-time PCR was then explored by a direct comparison of CE-IVD-marked versions of Pyrosequencing and TheraScreen (DxS) KRAS assays for a consecutive subset (n = 100) of the 314 clinical CRC samples. Results Using Pyrosequencing, 39% of the 314 CRC samples were found KRAS-mutated and several of the mutations (8%) were located in codon 61. To explore the analytical sensitivity of the Pyrosequencing assay, mutated patient DNA was serially diluted with wild-type patient DNA. Dilutions corresponding to 1.25-2.5% tumor cells still revealed detectable mutation signals. In clinical practice, our algorithm for KRAS analysis includes a reanalysis of samples with low tumor cell content (< 10%, n = 56) using an independent assay (allele-specific PCR, DxS). All mutations identified by Pyrosequencing were then confirmed and, in addition, one more mutated sample was identified in this subset of 56 samples. Finally, a direct comparison of the two technologies was done by re-analysis of a subset (n = 100) of the clinical samples using CE-IVD-marked versions of Pyrosequencing and TheraScreen KRAS assays in a single blinded fashion. The number of samples for which the KRAS codon 12/13 mutation status could be defined using the Pyrosequencing or the TheraScreen assay was 94 and 91, respectively, and both assays detected the same number of codon 12 and 13

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-02-28

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

  19. p16INK4A, p53, EGFR expression and KRAS mutation status in squamous cell cancers of the anus: correlation with outcomes following chemo-radiotherapy.

    PubMed

    Gilbert, Duncan C; Williams, Anthony; Allan, Kimberley; Stokoe, Joanna; Jackson, Tim; Linsdall, Suzanne; Bailey, Charles Mh; Summers, Jeff

    2013-10-01

    Squamous cell carcinomas of the anal canal are associated with infection with Human Papilloma Viruses (HPVs). Chemo-radiotherapy (CRT) gives 70% 3-year relapse-free survival. Improved predictive markers and therapeutic options are required. Tumours from 153 patients treated with radical chemo-radiotherapy (50.4 Gy in 28# with concurrent Mitomycin and 5-Fluorouracil between 2004 and 2009) were retrieved and immunohistochemistry performed for p16(INK4A), p53 and EGFR and correlated with outcome. Primary and relapsed samples were analysed for mutations in KRAS. 137/153 (89.5%) stained moderately or strongly for p16(INK4A). p16(INK4A) correlated strongly with outcome. 37/137 patients demonstrating moderate/strong p16(INK4A) expression relapsed (27.0%), as opposed to 10/16 (62.5%) with absent/weak staining (log rank test p<0.001). p16 and p53 expression were inversely correlated. p16(INK4A) negative tumours were more frequent in men. p16(INK4A) negative patients had significantly worse overall survival (p<0.001). No mutations in KRAS were identified in primary tumours or relapses following treatment. p16(INK4A) is strongly associated with relapse in SCC of the anus and identifies patients with very poor rates of relapse-free and overall survival. Primary and recurrent anal cancer expresses wild type KRAS, unaffected by treatment, supporting trials targeting EGFR in poor risk/recurrent anal cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  20. 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

    2016-09-08

    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. Analysis of TSC cortical tubers by deep sequencing of TSC1, TSC2, and KRAS demonstrates that small second hit mutations in these genes are rare events

    PubMed Central

    Qin, Wei; Chan, Jennifer A.; Vinters, Harry V.; Mathern, Gary W.; Franz, David Neal; Taillon, Bruce E.; Bouffard, Pascal; Kwiatkowski, David J.

    2010-01-01

    Tuberous sclerosis complex (TSC) is an often severe neurocutaneous syndrome. Cortical tubers are the predominant neuropathological finding in TSC, and their number and location has been shown to correlate roughly with the severity of neurologic features in TSC. Past studies have shown that genomic deletion events in TSC1 or TSC2 are very rare in tubers, and suggested the potential involvement of the MAPK pathway in their pathogenesis. We used deep sequencing to assess all coding exons of TSC1 and TSC2, and the activating mutation hot spots within KRAS in 46 tubers from TSC patients. Germline heterozygous mutations were identified in 81% of tubers. The same secondary mutation in TSC2 was identified in 6 tuber samples from one individual. Further study showed that this second hit mutation was widely distributed in the cortex from one cerebral hemisphere of this individual at frequencies up to 10%. No other secondary mutations were found in the other 40 tubers analyzed. These data indicate that small second hit mutations in any of these three genes are very rare in TSC tubers. However, in one TSC individual, a second hit TSC2 point mutation occurred early during brain development, and likely contributed to tuber formation. PMID:20633017

  2. SMAD4--molecular gladiator of the TGF-beta signaling is trampled upon by mutational insufficiency in colorectal carcinoma of Kashmiri population: an analysis with relation to KRAS proto-oncogene.

    PubMed

    Sameer, A Syed; Chowdri, Nissar A; Syeed, Nidda; Banday, Mujeeb Z; Shah, Zaffar A; Siddiqi, Mushtaq A

    2010-06-17

    The development and progression of colorectal cancer has been extensively studied and the genes responsible have been well characterized. However the correlation between the SMAD4 gene mutations with KRAS mutant status has not been explored by many studies so far. Here, in this study we aimed to investigate the role of SMAD4 gene aberrations in the pathogenesis of CRC in Kashmir valley and to correlate it with various clinicopathological variables and KRAS mutant genotype. We examined the paired tumor and normal tissue specimens of 86 CRC patients for the occurrence of aberrations in MCR region of SMAD4 and exon 1 of KRAS by PCR-SSCP and/or PCR-Direct sequencing. The overall mutation rate of mutation cluster region (MCR) region of SMAD4 gene among 86 patients was 18.6% (16 of 86). 68.75% (11/16) of the SMAD4 gene mutants were found to have mutations in KRAS gene as well. The association between the KRAS mutant genotype with SMAD4 mutants was found to be significant (P = or < 0.05). Further more, we found a significant association of tumor location, tumor grade, node status, occupational exposure to pesticides and bleeding PR/Constipation with the mutation status of the SMAD4 gene (P = or < 0.05). Our study suggests that SMAD4 gene aberrations are the common event in CRC development but play a differential role in the progression of CRC in higher tumor grade (C+D) and its association with the KRAS mutant status suggest that these two molecules together are responsible for the progression of the tumor to higher/advanced stage.

  3. Detection of oncogenic mutations in cervical carcinoma using method High Resolution Melting (HRM).

    PubMed

    Wayhelova, M; Mikulasova, A; Smetana, J; Vallova, V; Blazkova, D; Filkova, H; Moukova, L; Kuglik, P

    2016-01-01

    Oncogenic mutations in proto-oncogenes and tumor suppressor genes represent one of key events in cancerogenesis. In this study, we analysed mutation status in PIK3CA, KRAS and EGFR proto-oncogenes and TP53 tumor suppressor gene in a cohort of twenty-four patients diagnosed with squamous cell carcinoma or adenocarcinoma using the screening method "High Resolution Melting" (HRM). Positive findings were confirmed and identified by Sanger sequencing. Totally, we detected DNA sequence changes in targeted regions in seven patients (7/24, 29.2%). In PIK3CA gene, we found six sequence changes in four patients (4/24, 16.7%) and four of them were confirmed as oncogenic mutations. In KRAS gene, we detected sequence changes in four patients (4/24, 16.7%). Conversely, we identified pathogenic or potentially pathogenic sequence changes neither in EGFR nor TP53 genes. Our results suggest that sequence changes are specific neither for a certain histological subtype, clinical stage nor lymph node involvement and they appear independently on the presence of HPV (human papillomavirus) infection since early clinical stages. We observed the correlation between the presence of DNA sequence changes and hTERC gene amplification, but we did not find a significant relationship between the identified DNA sequence changes and detected copy-number alterations using the technique of array-CGH (array-based comparative genomic hybridization). Regardless our results confirmed an important role of oncogenic mutations in PIK3CA and KRAS genes in the neoplastic transformation process in the cervical carcinoma pathogenesis. Their identification in the early clinical stages should encourage further studies to better understand these mutations and exploit them for more detailed diagnostics.

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

    PubMed

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

    2017-03-01

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

  5. Impact of Epidermal Growth Factor Receptor and KRAS Mutations on Clinical Outcomes in Previously Untreated Non–Small Cell Lung Cancer Patients: Results of an Online Tumor Registry of Clinical Trials

    PubMed Central

    Jackman, David M.; Miller, Vincent A.; Cioffredi, Leigh-Anne; Yeap, Beow Y.; Jänne, Pasi A.; Riely, Gregory J.; Ruiz, Marielle Gallegos; Giaccone, Giuseppe; Sequist, Lecia V.; Johnson, Bruce E.

    2011-01-01

    Purpose The impact of epidermal growth factor receptor (EGFR) and KRAS genotypes on outcomes with erlotinib or gefitinib therapy continues to be debated. This study combines patient data from five trials in predominantly Western populations to assess the impact of EGFR and KRAS mutations on first-line therapy with an EGFR–tyrosine kinase inhibitor (TKI) and compare clinical versus molecular predictors of sensitivity. Experimental Design Chemotherapy-naïve patients with advanced non–small cell lung cancer and known EGFR mutation status treated with erlotinib or gefitinib monotherapy as part of a clinical trial were eligible for inclusion. Patients received daily erlotinib (150 mg) or gefitinib (250 mg) until disease progression or unacceptable toxicity. Data were collected in a password-protected web database. Clinical outcomes were analyzed to look for differences based on EGFR and KRAS genotypes, as well as clinical characteristics. Results Patients (223) from five clinical trials were included. Sensitizing EGFR mutations were associated with a 67% response rate, time to progression (TTP) of 11.8 months, and overall survival of 23.9 months. Exon 19 deletions were associated with longer median TTP and overall survival compared with L858R mutations. Wild-type EGFR was associated with poorer outcomes (response rate, 3%; TTP, 3.2 months) irrespective of KRAS status. No difference in outcome was seen between patients harboring KRAS transition versus transversion mutations. EGFR genotype was more effective than clinical characteristics at selecting appropriate patients for consideration of first-line therapy with an EGFR-TKI. Conclusion EGFR mutation status is associated with sensitivity to treatment with an EGFR-TKI in patients with advanced non–small cell lung cancer. Patients harboring sensitizing EGFR mutations should be considered for first-line erlotinib or gefitinib. PMID:19671843

  6. Mutation detection using Surveyor nuclease.

    PubMed

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

    2004-04-01

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

  7. Phase II study of selumetinib (AZD6244, ARRY-142886) plus irinotecan as second-line therapy in patients with K-RAS mutated colorectal cancer.

    PubMed

    Hochster, H S; Uboha, N; Messersmith, W; Gold, P J; ONeil, B H; Cohen, D; Denlinger, C; Cohen, S; Leichman, C G; Leichman, L; Lenz, H-J

    2015-01-01

    More than half of colorectal tumors harbor activating mutations in RAS/RAF proteins. Selumetinib (AZD6244, ARRY-142886) is a small molecule kinase inhibitor targeting MEK kinase, downstream of RAS. We examined the efficacy and safety of selumetinib with irinotecan in second-line therapy. Patients with K-RAS mutated colorectal cancer, progressing on first-line oxaliplatin-based chemotherapy with bevacizumab, were eligible for this multicenter open-label phase I/II trial. In part A, a dose was determined using a standard "3 + 3" design; in part B, efficacy was determined. The primary endpoint was RECIST response rate. Historical data for irinotecan were used as reference. Secondary endpoints included progression-free survival and overall survival. Thirty-two patients entered the study, and 31 were treated. All had K-RAS exon 2 mutated tumors. In phase I, the recommended oral dose of selumetinib was 75 mg twice per day with intravenous (IV) irinotecan, 180 mg/m² every 2 weeks. Three patients (9.7 %) had partial response . Sixteen patients (51.6 %) had stable disease for ≥4 weeks, including three >1 year. The most common grade 3 adverse events included diarrhea, neutropenia, fatigue, anemia, nausea, and dehydration. The study was terminated before a pre-planned accrual of 45 subjects. Despite termination before full accrual, the point estimates of RR and median PFS show promising results, suggesting that further investigations of MEK inhibition in the treatment of metastatic colorectal cancer are warranted. Studies combining MEK inhibitors with cytotoxics or other targeted agents may lead to improved clinical activity based on the emerging preclinical data.

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

    PubMed

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

    2017-02-01

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

  9. High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort

    PubMed Central

    Sorbye, Halfdan; Dragomir, Anca; Sundström, Magnus; Pfeiffer, Per; Thunberg, Ulf; Bergfors, Monica; Aasebø, Kristine; Eide, Geir Egil; Ponten, Fredrik; Qvortrup, Camilla; Glimelius, Bengt

    2015-01-01

    RAS and BRAF mutations impact treatment and prognosis of metastatic colorectal cancer patients (mCRC), but the knowledge is based on trial patients usually not representative for the general cancer population. Patient characteristics, treatment and efficacy according to KRAS, BRAF and MSI status were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue micro array (TMA) (42%) had worse prognostic factors and inferior survival (all patients; 7m vs 11m, chemotherapy-treated;12m vs 17m). The 92 patients (21%) with BRAF mutation had a poor prognosis regardless of microsatellite instability, but receipt of 1-2nd chemotherapy was similar to wildtype BRAF patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients <75 years in good PS. TMA availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF mutation incidence is higher than the previously and repeatedly reported incidence of 5-12% in mCRC. Screening for BRAF mutations before selection of treatment is relevant for many patients, especially outside clinical trials. A BRAF mutation only partly explained the very poor prognosis of many mCRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA studies and implies that we lack information on the biologically worst mCRC cases. Lack of available tissue is an important underexposed issue which introduces sample bias, and this should

  10. A lethal course of hypertrophic cardiomyopathy in Noonan syndrome due to a novel germline mutation in the KRAS gene: case study

    PubMed Central

    Nosan, Gregor; Bertok, Sara; Vesel, Samo; Yntema, Helger G.; Paro-Panjan, Darja

    2013-01-01

    Noonan syndrome is a relatively common and heterogeneous genetic disorder, including congenital heart defect in more than half of the cases. If the defect is not large, life expectancy is normal. Here we report on a case of an infant with Noonan syndrome and rapidly progressive hypertrophic cardiomyopathy with lethal outcome, in whom we identified a novel mutation in the KRAS gene. This heterozygous unclassified missense variant in exon 3: c.179G>T (p.Gly60Val) might be associated with a lethal form of Noonan syndrome. The malignant clinical course of the disease and the lethal outcome in an infant only a few months old might be connected to RAS-mitogen-activated protein kinase pathway hyperactivation, consequently promoting cell growth and proliferation, leading to rapidly progressive hypertrophic cardiomyopathy. Further biochemical and functional studies are needed to confirm this hypothesis. PMID:24382853

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

    PubMed

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

    2012-06-01

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

  12. Bevacizumab plus chemotherapy continued beyond first progression in patients with metastatic colorectal cancer previously treated with bevacizumab plus chemotherapy: ML18147 study KRAS subgroup findings.

    PubMed

    Kubicka, S; Greil, R; André, T; Bennouna, J; Sastre, J; Van Cutsem, E; von Moos, R; Osterlund, P; Reyes-Rivera, I; Müller, T; Makrutzki, M; Arnold, D

    2013-09-01

    ML18147 evaluated continued bevacizumab with second-line chemotherapy for patients with metastatic colorectal cancer (mCRC) progressing after the standard first-line bevacizumab-containing therapy. Evaluating outcomes according to tumor Kirsten rat sarcoma virus oncogene (KRAS) status was an exploratory analysis. KRAS data were collected from local laboratories (using their established methods) and/or from a central laboratory (mutation-specific Scorpion amplification-refractory mutation system). No adjustment was made for multiplicity; analyses were not powered to detect statistically significant differences. Of 820 patients, 616 (75%) had unambiguous KRAS data; 316 (51%) had KRAS wild-type tumors and 300 (49%) had mutant KRAS tumors. The median progression-free survival (PFS) was 6.4 months for bevacizumab plus chemotherapy and 4.5 months for chemotherapy [P < 0.0001; HR = 0.61; 95% confidence interval (CI): 0.49-0.77] for wild-type KRAS and 5.5 and 4.1 months, respectively (P = 0.0027; HR = 0.70; 95% CI: 0.56-0.89) for mutant KRAS. The median overall survival (OS) was 15.4 and 11.1 months, respectively (P = 0.0052; HR = 0.69; 95% CI: 0.53-0.90) for wild-type KRAS and 10.4 versus 10.0 months, respectively (P = 0.4969; HR = 0.92; 95% CI: 0.71-1.18) for mutant KRAS. In both analyses, no treatment interaction by KRAS status was observed (PFS, P = 0.4436; OS, P = 0.1266). Bevacizumab beyond first progression represents an option for patients with mCRC treated with bevacizumab plus standard first-line chemotherapy, independent of KRAS status.

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

    PubMed

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

    2017-04-01

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

  14. Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS.

    PubMed

    Ross, Sarah J; Revenko, Alexey S; Hanson, Lyndsey L; Ellston, Rebecca; Staniszewska, Anna; Whalley, Nicky; Pandey, Sanjay K; Revill, Mitchell; Rooney, Claire; Buckett, Linda K; Klein, Stephanie K; Hudson, Kevin; Monia, Brett P; Zinda, Michael; Blakey, David C; Lyne, Paul D; Macleod, A Robert

    2017-06-14

    Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl-containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  15. The genetics and biology of KRAS in lung cancer.

    PubMed

    Westcott, Peter M K; To, Minh D

    2013-02-01

    Mutational activation of KRAS is a common oncogenic event in lung cancer and other epithelial cancer types. Efforts to develop therapies that counteract the oncogenic effects of mutant KRAS have been largely unsuccessful, and cancers driven by mutant KRAS remain among the most refractory to available treatments. Studies undertaken over the past decades have produced a wealth of information regarding the clinical relevance of KRAS mutations in lung cancer. Mutant Kras-driven mouse models of cancer, together with cellular and molecular studies, have provided a deeper appreciation for the complex functions of KRAS in tumorigenesis. However, a much more thorough understanding of these complexities is needed before clinically effective therapies targeting mutant KRAS-driven cancers can be achieved.

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

    PubMed

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

    2016-01-21

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2017-04-11

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

  20. Discordant cellular response to pre-surgical letrozole in bilateral synchronous ER+ breast cancers with a KRAS mutation or FGFR1 gene amplification

    PubMed Central

    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.

    2013-01-01

    We describe herein a patient presenting with bilateral ER+ breast tumors who was enrolled in a clinical trial exploring molecular aberrations associated with hormone-refractory tumor cell proliferation. Short-term (2-week) hormonal therapy with the aromatase inhibitor letrozole substantially reduced proliferation as measured by Ki67 immunohistochemistry in one tumor, while the second was essentially unchanged. Extensive molecular and genetic workup of the two tumors yielded divergent lesions in the two tumors: an activating KRAS mutation in the responsive tumor, and an amplification of the 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 ~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. PMID:22879364

  1. Screening of KRAS Mutation in Pre- and Post-Surgery Serum of Patients Suffering from Colon Cancer by COLD-PCR HRM.

    PubMed

    Trujillo-Arribas, Elena; Macher, Hada C; Jiménez-Arriscado, Pilar; de la Portilla, Fernando; Molinero, Patrocinio; Guerrero, Juan M; Rubio, Amalia

    2016-01-01

    Genomic characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics throughout the course of a patient's illness. The existence of KRAS driver mutations in colon cancer patients is determinant to decide their treatment and to predict their outcome. DNA is extracted automatically from 400 μL of serum using the MagNa Pure Compact with the Nucleic Acid Isolation Kit I. DNA amplification, COLD-PCR and HRM were performed in the same run in the Light Cycler 480.We found three different situations: pre- and post-surgical samples grouped with the negative control, pre-surgical samples appear to group with the positive control and the post-surgical samples appear to group with the negative control and finally both samples, pre- and post-surgical ones, appear to be grouped with the positive control. COLD-PCR HRM is a cost-effective way for screening one of the most common driver mutations to predict the worst prognosis in colorectal cancer.

  2. Differences of protein expression profiles, KRAS and BRAF mutation, and prognosis in right-sided colon, left-sided colon and rectal cancer.

    PubMed

    Gao, Xian Hua; Yu, Guan Yu; Gong, Hai Feng; Liu, Lian Jie; Xu, Yi; Hao, Li Qiang; Liu, Peng; Liu, Zhi Hong; Bai, Chen Guang; Zhang, Wei

    2017-08-11

    To compare protein expression levels, gene mutation and survival among Right-Sided Colon Cancer (RSCC), Left-Sided Colon Cancer (LSCC) and rectal cancer patients, 57 cases of RSCC, 87 LSCC and 145 rectal cancer patients were included retrospectively. Our results demonstrated significant differences existed among RSCC, LSCC and rectal cancer regarding tumor diameter, differentiation, invasion depth and TNM stage. No significant difference was identified in expression levels of MLH1, MSH2, MSH6, PMS2, β-Tubulin III, P53, Ki67 and TOPIIα, and gene mutation of KRAS and BRAF among three groups. Progression Free Survival (PFS) of RSCC was significantly lower than that of LRCC and rectal cancer. In univariate analyses, RSCC, preoperative chemoradiotherapy, poor differentiation, advanced TNM stage, elevated serum CEA and CA19-9 level, tumor deposit, perineural and vascular invasion were found to be predictive factors of shorter PFS. In multivariate analyses, only differentiation and TNM stages were found to be independent predictors of PFS. In conclusion, compared with LSCC and rectal cancer, RSCC has larger tumor size, poor differentiation, advanced TNM stage and shorter survival. The shorter survival in RSCC might be attributed to the advanced tumor stage caused by its inherent position feature of proximal colon rather than genetic difference.

  3. Evaluation of KRAS Gene Expression and LCS6 Variant in Genomic and Cell-Free DNA of Iranian Women With Endometriosis

    PubMed Central

    Farahani, Maryam Shahrabi; Moghaddam, Soheila Amini; Mahdian, Reza

    2015-01-01

    Since the activation of KRAS results in de novo endometriosis in mice, KRAS is regarded as a crucial gene in ectopic endometrial implantation. Recently, it has been reported that 31% of women with endometriosis have KRAS let-7 complementary binding site 6 single-nucleotide polymorphism (LCS6 SNP). This study addresses the correlation between KRAS LCS6 SNP and endometriosis in a case–control study. To detect probable somatic mutation in ectopic endometrial tissue, we evaluated LCS6 SNP in cell-free DNA samples. Quantitative real-time reverse transcription-polymerase chain reaction was performed to determine the expression of KRAS transcripts in eutopic endometrial tissue. Our results suggest that the variant is not associated with the development of endometriosis in Iranian women. We observed higher levels of KRAS messenger RNA (mRNA) expression in eutopic endometrium of patients with endometriosis compared to controls. Although, the KRAS LCS6 is neither constitutional nor somatic biomarker for endometriosis, increased expression ratio of KRAS mRNA indicates its role in the implantation of endometrial tissue outside the uterine cavity. PMID:25361550

  4. 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.

  5. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    PubMed

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui

    2014-07-01

    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC.

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

    PubMed

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

    2016-03-03

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

  7. Molecular methods for the detection of mutations.

    PubMed

    Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

    2000-01-01

    We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

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

    PubMed

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

    2013-05-01

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

  10. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

    PubMed Central

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

  11. Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

    PubMed

    Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

    2017-01-01

    The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

  12. Updated Frequency of EGFR and KRAS Mutations in NonSmall-Cell Lung Cancer in Latin America: The Latin-American Consortium for the Investigation of Lung Cancer (CLICaP).

    PubMed

    Arrieta, Oscar; Cardona, Andrés F; Martín, Claudio; Más-López, Luis; Corrales-Rodríguez, Luis; Bramuglia, Guillermo; Castillo-Fernandez, Omar; Meyerson, Matthew; Amieva-Rivera, Eduardo; Campos-Parra, Alma Delia; Carranza, Hernán; Gómez de la Torre, Juan Carlos; Powazniak, Yanina; Aldaco-Sarvide, Fernando; Vargas, Carlos; Trigo, Mariana; Magallanes-Maciel, Manuel; Otero, Jorge; Sánchez-Reyes, Roberto; Cuello, Mauricio

    2015-05-01

    Previously, we reported the frequency of epidermal growth factor receptor (EGFR) and KRAS mutations in nonsmall-cell lung cancer (NSCLC) patients in Latin America. The EGFR mutation frequency was found between Asian (40%) and Caucasian (15%) populations. Here, we report the updated distribution of NSCLC mutations. A total of 5738 samples from NSCLC patients from Argentina (1713), Mexico (1417), Colombia (1939), Peru (393), Panama (174), and Costa Rica (102) were genotyped for EGFR and KRAS. The median patient age was 62.2 ± 12.3 years; 53.5% were women, 46.7% had a history of smoking, and 95.2% had adenocarcinoma histology. The frequency of EGFR mutations was 26.0% (95% confidence interval [CI], 24.9-27.1; Argentina, 14.4% [12.8-15.6]; México, 34.3% [31.9-36.7]; Colombia, 24.7% [22.8-26.6]; Peru, 51.1% [46.2-55.9]; Panamá, 27.3 [20.7-33.9]; and Costa Rica, 31.4% [22.4-40.4]). The frequency of KRAS mutations was 14.0% (9.1-18.9). In patients with adenocarcinoma, EGFR mutations were independently associated with gender (30.7% females vs. 18.4% males; p < 0.001), nonsmoker status (27.4% vs. 17.1%, p < 0.001), ethnicity (mestizo/indigenous, 35.3% vs. Caucasian, 13.7%, p < 0.001), and the absence of KRAS mutation (38.1% vs. 4.7%; p < 0.001). The overall response rate to EGFR tyrosine kinase inhibitors was 60.6% (95% CI, 52-69), with a median progression-free survival and overall survival of 15.9 (95% CI, 12.420.6) and 32 months (95% CI, 26.5-37.6), respectively. Our findings support the genetic heterogeneity of NSCLC in Latin America, confirming that the frequency of EGFR mutations is intermediate between that observed in the Asian and Caucasian populations.

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

    PubMed Central

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

    2017-01-01

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

  14. Oncogenic KRAS signalling in pancreatic cancer.

    PubMed

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

    2014-08-26

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

  15. KRAS — EDRN Public Portal

    Cancer.gov

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

  16. Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line

    PubMed Central

    Tokunaga, T; Tsuchida, T; Kijima, H; Okamoto, K; Oshika, Y; Sawa, N; Ohnishi, Y; Yamazaki, H; Miura, S; Ueyama, Y; Nakamura, M

    2000-01-01

    Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The β-actin promoter-driven KrasRz sequence (pHβ/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett’s test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher’s exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett’s test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression. © 2000 Cancer Research Campaign PMID:10952790

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

    PubMed Central

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

    2016-01-01

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

  18. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon.

    PubMed

    Calcagno, Shelly R; Li, Shuhua; Colon, Migdalisel; Kreinest, Pamela A; Thompson, E Aubrey; Fields, Alan P; Murray, Nicole R

    2008-06-01

    Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-ras(G12D) allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-ras(G12D) throughout the colon (LSL-K-ras(G12D)/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-ras(G12D) induces increased expression of procarcinogenic protein kinase C beta II (PKC beta II), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-ras(G12D) inhibits expression of procarcinogenic PKC beta II and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice. (c) 2008 Wiley-Liss, Inc.

  19. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

    PubMed Central

    Calcagno, Shelly R.; Li, Shuhua; Colon, Migdalisel; Kreinest, Pamela A.; Thompson, E. Aubrey; Fields, Alan P.; Murray, Nicole R.

    2014-01-01

    Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-rasG12D allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-rasG12D throughout the colon (LSL-K-rasG12D/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-rasG12D induces increased expression of procarcinogenic protein kinase CβII (PKCβII), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-rasG12D inhibits expression of procarcinogenic PKCβII and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice. PMID:18271008

  20. Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung—importance of mouse strain

    PubMed Central

    Calvert, Richard J.; Tepper, Shirley; Kammouni, Wafa; Anderson, Lucy M.; Kritchevsky, David

    2007-01-01

    Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For three weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25 mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After three weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione-S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in 2 of 3 mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control (p = 0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold (p = 0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type. PMID:17005160

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

    PubMed Central

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

    2015-01-01

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

  2. EGF Receptor is Required for KRAS-induced Pancreatic Tumorigenesis

    PubMed Central

    Ardito, Christine M.; Grüner, Barbara M.; Takeuchi, Kenneth K.; Lubeseder-Martellato, Clara; Teichmann, Nicole; Mazur, Pawel K.; DelGiorno, Kathleen E.; Carpenter, Eileen S.; Halbrook, Christopher J.; Hall, Jason C.; Pal, Debjani; Briel, Thomas; Herner, Alexander; Trajkovic-Arsic, Marija; Sipos, Bence; Liou, Geou-Yarh; Storz, Peter; Murray, Nicole R.; Threadgill, David W.; Sibilia, Maria; Washington, M. Kay; Wilson, Carole L.; Schmid, Roland M.; Raines, Elaine W.; Crawford, Howard C.; Siveke, Jens T.

    2012-01-01

    SUMMARY Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent upon the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation. PMID:22975374

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

    PubMed

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

    2008-05-15

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

  4. Oncogenic Kras drives invasion and maintains metastases in colorectal cancer.

    PubMed

    Boutin, Adam T; Liao, Wen-Ting; Wang, Melody; Hwang, Soyoon Sarah; Karpinets, Tatiana V; Cheung, Hannah; Chu, Gerald C; Jiang, Shan; Hu, Jian; Chang, Kyle; Vilar, Eduardo; Song, Xingzhi; Zhang, Jianhua; Kopetz, Scott; Futreal, Andrew; Wang, Y Alan; Kwong, Lawrence N; DePinho, Ronald A

    2017-02-15

    Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the KRAS gene. To understand the role of oncogenic KRAS in CRC, we engineered a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele (Kras(mut) ) and conditional null alleles of Apc and Trp53 (iKAP). The iKAP model recapitulates tumor progression from adenoma through metastases. Whole-exome sequencing revealed that the Kras(mut) allele was heterogenous in primary tumors yet homogenous in metastases, a pattern consistent with activated Kras(mut) signaling being a driver of progression to metastasis. System-level and functional analyses revealed the TGF-β pathway as a key mediator of Kras(mut) -driven invasiveness. Genetic extinction of Kras(mut) resulted in specific elimination of the Kras(mut) subpopulation in primary and metastatic tumors, leading to apoptotic elimination of advanced invasive and metastatic disease. This faithful CRC model provides genetic evidence that Kras(mut) drives CRC invasion and maintenance of metastases. © 2017 Boutin et al.; Published by Cold Spring Harbor Laboratory Press.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. KRAS Genotyping of Paraffin-Embedded Colorectal Cancer Tissue in Routine Diagnostics

    PubMed Central

    Weichert, Wilko; Schewe, Christiane; Lehmann, Annika; Sers, Christine; Denkert, Carsten; Budczies, Jan; Stenzinger, Albrecht; Joos, Hans; Landt, Olfert; Heiser, Volker; Röcken, Christoph; Dietel, Manfred

    2010-01-01

    KRAS mutation testing before anti-epidermal growth factor receptor therapy of metastatic colorectal cancer has become mandatory in Europe. However, considerable uncertainty exists as to which methods for detection can be applied in a reproducible and economically sound manner in the routine diagnostic setting. To answer this question, we examined 263 consecutive routine paraffin slide specimens. Genomic DNA was extracted from microdissected tumor tissue. The DNA was analyzed prospectively by Sanger sequencing and array analysis as well as retrospectively by melting curve analysis and pyrosequencing; the results were correlated to tissue characteristics. The methods were then compared regarding the reported results, costs, and working times. Approximately 40% of specimens contained KRAS mutations, and the different methods reported concordant results (κ values >0.9). Specimens harboring fewer than 10% tumor cells showed lower mutation rates regardless of the method used, and histoanatomical variables had no influence on the frequency of the mutations. Costs per assay were higher for array analysis and melting curve analysis when compared with the direct sequencing methods. However, for sequencing methods equipment costs were much higher. In conclusion, Sanger sequencing, array analysis, melting curve analysis, and pyrosequencing were equally effective for routine diagnostic KRAS mutation analysis; however, interpretation of mutation results in conjunction with histomorphologic tissue review and on slide tumor tissue dissection is required for accurate diagnosis. PMID:20007841

  8. 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.

  9. [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。.

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

    PubMed

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

    2015-05-01

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

  11. 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

  12. 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.

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

    PubMed Central

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

    2013-01-01

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

  14. Exploratory biomarker analysis for treatment response in KRAS wild type metastatic colorectal cancer patients who received cetuximab plus irinotecan.

    PubMed

    Kim, Seung Tae; Ahn, Tae Jin; Lee, Eunjin; Do, In-Gu; Lee, Su Jin; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Kim, Suk Hyeong; Lee, Jeeyun; Kim, Hee Cheol

    2015-10-20

    More than half of the patients selected based on KRAS mutation status fail to respond to the treatment with cetuximab in metastatic colorectal cancer (mCRC). We designed a study to identify additional biomarkers that could act as indicators for cetuximab treatment in mCRC. We investigated 58 tumor samples from wild type KRAS CRC patients treated with cetuximab plus irinotecan (CI). We conducted the genotyping for mutations in either BRAF or PIK3CA and profiled comprehensively the expression of 522 kinase genes. BRAF mutation was detected in 5.1 % (3/58) of patients. All 50 patients showed wild type PIK3CA. Gene expression patterns that categorized patients with or without the disease control to CI were compared by supervised classification analysis. PSKH1, TLK2 and PHKG2 were overexpressed significantly in patients with the disease control to IC. The higher expression value of PSKH1 (r = 0.462, p < 0.001) and TLK2 (r = 0.361, p = 0.005) had the significant correlation to prolonged PFS. The result of this work demonstrated that expression nature of kinase genes such as PSKH1, TLK2 and PHKG2 may be informative to predict the efficacy of CI in wild type KRAS CRC. Mutations in either BRAF or PIK3CA were rare subsets in wild type KRAS CRC.

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

    PubMed

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

    2017-02-27

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

  16. Co-dependency of PKCδ and K-Ras: Inverse association with cytotoxic drug sensitivity in KRAS mutant lung cancer

    PubMed Central

    Ohm, Angela M.; Tan, Aik-Choon; Heasley, Lynn E.; Reyland, Mary E.

    2017-01-01

    Recent studies suggest that the presence of a KRAS mutation may be insufficient for defining a clinically homogenous molecular group, as many KRAS mutant tumors lose reliance on K-Ras for survival. Identifying pathways that support K-Ras dependency may define clinically relevant KRAS sub-groups and lead to the identification of new drug targets. We have analyzed a panel of 17 KRAS mutant lung cancer cell lines classified as K-Ras dependent or independent, for co-dependency on PKCδ. We show that functional dependency on K-Ras and PKCδ co-segregate, and that dependency correlates with a more epithelial-like phenotype. Furthermore, we show that the pro-apoptotic and pro-tumorigenic functions of PKCδ also segregate based on K-Ras dependency, as K-Ras independent cells are more sensitive to topoisomerase inhibitors, and depletion of PKCδ in this sub-group suppresses apoptosis through increased activation of ERK. In contrast, K-Ras dependent lung cancer cells are largely insensitive to topoisomerase inhibitors, and depletion of PKCδ can increase apoptosis and decrease activation of ERK in this sub-group. We have previously shown that nuclear translocation of PKCδ is necessary and sufficient for pro-apoptotic signaling. Our current studies show that K-Ras dependent cells are refractive to PKCδ driven apoptosis. Analysis of this sub-group showed increased PKCδ expression and an increase in the nuclear:cytoplasmic ratio of PKCδ. In addition, targeting PKCδ to the nucleus induces apoptosis in K-Ras independent, but not K-Ras dependent NSCLC cells. Our studies provide tools for identification of the subset of patients with KRAS mutant tumors most amenable to targeting of the K-Ras pathway, and identify PKCδ as a potential target in this tumor population. These sub-groups are likely to be of clinical relevance, as high PKCδ expression correlates with increased overall survival and a more epithelial tumor phenotype in patients with KRAS mutant lung adenocarcinomas

  17. Mu