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

BackgroundKRAS 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.MethodsWe 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.ResultsKRAS 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 codon 61 mutation; colorectal cancer-specific mortality HR = 0.86, 95% CI = 0.42-1.78 for codon 146 mutation].ConclusionsTumors with KRAS mutations in codons 61 and 146 account for an appreciable proportion (approximately 5%) of colorectal cancers, and their clinicopathological and molecular features appear generally similar to KRAS codon 12 or 13 mutated cancers. To further assess clinical utility of KRAS codon 61 and 146 testing, large-scale trials are warranted.

[1]  R. Goody,et al.  Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction. , 1988, The Journal of biological chemistry.

[2]  Stephen McLaughlin,et al.  PIK3CA and PTEN Gene and Exon Mutation-Specific Clinicopathologic and Molecular Associations in Colorectal Cancer , 2013, Clinical Cancer Research.

[3]  R. Steele,et al.  Activating K-Ras mutations outwith ‘hotspot’ codons in sporadic colorectal tumours – implications for personalised cancer medicine , 2010, British Journal of Cancer.

[4]  W. Fang,et al.  Clinical significance of K-ras and BRAF mutations in Chinese colorectal cancer patients. , 2011, World journal of gastroenterology.

[5]  Biomarkers of benefit from cetuximab-based therapy in metastatic colorectal cancer: interaction of EGFR ligand expression with RAS/RAF, PIK3CA genotypes , 2013, BMC Cancer.

[6]  P. Laurent-Puig,et al.  Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. , 2008, Cancer research.

[7]  Sabine Tejpar,et al.  KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer. , 2011, The Lancet. Oncology.

[8]  K. Edlund,et al.  KRAS analysis in colorectal carcinoma: Analytical aspects of Pyrosequencing and allele-specific PCR in clinical practice , 2010, BMC Cancer.

[9]  V. Kosma,et al.  p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women , 2000, Molecular pathology : MP.

[10]  R. Schneider-Stock,et al.  K-ras mutation detection in colorectal cancer using the Pyrosequencing technique. , 2007, Pathology, research and practice.

[11]  J. Tabernero,et al.  Panitumumab-FOLFOX4 treatment and RAS mutations in colorectal cancer. , 2013, The New England journal of medicine.

[12]  KRAS and BRAF Mutation Analysis in Colorectal Adenocarcinoma Specimens with a Low Percentage of Tumor Cells , 2013, Molecular Diagnosis & Therapy.

[13]  A. Leroux,et al.  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 , 2013, Virchows Archiv.

[14]  J. Barrett,et al.  KRAS and BRAF mutations in advanced colorectal cancer are associated with poor prognosis but do not preclude benefit from oxaliplatin or irinotecan: results from the MRC FOCUS trial. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[15]  Mari Mino-Kenudson,et al.  Lymphocytic Reaction to Colorectal Cancer Is Associated with Longer Survival, Independent of Lymph Node Count, Microsatellite Instability, and CpG Island Methylator Phenotype , 2009, Clinical Cancer Research.

[16]  Josep Tabernero,et al.  Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[17]  M. Ilyas,et al.  The utility of diagnostic biopsy specimens for predictive molecular testing in colorectal cancer , 2012, Histopathology.

[18]  Won-Suk Jang,et al.  Coexistent mutations of KRAS and PIK3CA affect the efficacy of NVP‐BEZ235, a dual PI3K/MTOR inhibitor, in regulating the PI3K/MTOR pathway in colorectal cancer , 2013, International journal of cancer.

[19]  Y. Nakamura,et al.  Genetic alterations during colorectal-tumor development. , 1988, The New England journal of medicine.

[20]  Editors-in-Chief C. Nicot Molecular Cancer , 2009 .

[21]  L. Aaltonen,et al.  Distinct patterns of KRAS mutations in colorectal carcinomas according to germline mismatch repair defects and hMLH1 methylation status. , 2004, Human molecular genetics.

[22]  K. Schaefer,et al.  KRAS p.G13D mutations are associated with sensitivity to anti-EGFR antibody treatment in colorectal cancer cell lines , 2013, Journal of Cancer Research and Clinical Oncology.

[23]  C. Bokemeyer,et al.  Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[24]  Sabine Tejpar,et al.  Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. , 2010, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[25]  G. Fontanini,et al.  KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer , 2009, British Journal of Cancer.

[26]  D. Lin,et al.  Effectors of Epidermal Growth Factor Receptor Pathway: The Genetic Profiling of KRAS, BRAF, PIK3CA, NRAS Mutations in Colorectal Cancer Characteristics and Personalized Medicine , 2013, PloS one.

[27]  A. Zullo,et al.  Focus on genetic and epigenetic events of colorectal cancer pathogenesis: implications for molecular diagnosis , 2014, Tumor Biology.

[28]  R. Palmqvist,et al.  The Role of the CpG Island Methylator Phenotype in Colorectal Cancer Prognosis Depends on Microsatellite Instability Screening Status , 2010, Clinical Cancer Research.

[29]  P. Kuppen,et al.  Biomarkers in precision therapy in colorectal cancer , 2013, Gastroenterology report.

[30]  Aung Ko Win,et al.  Colorectal carcinomas with KRAS mutation are associated with distinctive morphological and molecular features , 2013, Modern Pathology.

[31]  E. Van Cutsem,et al.  Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[32]  I. Nagtegaal,et al.  PIK3CA Mutations Predict Local Recurrences in Rectal Cancer Patients , 2009, Clinical Cancer Research.

[33]  R. Malekzadeh,et al.  Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin. , 2006, Annals of oncology : official journal of the European Society for Medical Oncology.

[34]  J. Meyerhardt,et al.  KRAS Mutation in Stage III Colon Cancer and Clinical Outcome Following Intergroup Trial CALGB 89803 , 2009, Clinical Cancer Research.

[35]  M. Mäkinen,et al.  Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma , 2011, Histopathology.

[36]  S. Ogino,et al.  LINE‐1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer , 2008, International journal of cancer.

[37]  E. Oikonomou,et al.  Tumor Heterogeneity Revealed by KRAS, BRAF, and PIK3CA Pyrosequencing: KRAS and PIK3CA Intratumor Mutation Profile Differences and Their Therapeutic Implications , 2014, Human mutation.

[38]  N. Cho,et al.  Prognostic implications of CpG island hypermethylator phenotype in colorectal cancers , 2009, Virchows Archiv.

[39]  D. Lin,et al.  Direct sequencing is a reliable assay with good clinical applicability for KRAS mutation testing in colorectal cancer. , 2013, Cancer biomarkers : section A of Disease markers.

[40]  William M. Grady,et al.  Epigenetics and colorectal cancer , 2011, Nature Reviews Gastroenterology &Hepatology.

[41]  C. Tournigand,et al.  Panitumumab combined with irinotecan for patients with KRAS wild-type metastatic colorectal cancer refractory to standard chemotherapy: a GERCOR efficacy, tolerance, and translational molecular study. , 2013, Annals of oncology : official journal of the European Society for Medical Oncology.

[42]  I. Wistuba,et al.  Distinct K-ras mutation pattern characterizes signet ring cell colorectal carcinoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[43]  J. Tabernero,et al.  Open-label, multicentre expansion cohort to evaluate imgatuzumab in pre-treated patients with KRAS-mutant advanced colorectal carcinoma. , 2014, European journal of cancer.

[44]  S. Ng,et al.  Characterization of rare transforming KRAS mutations in sporadic colorectal cancer , 2014, Cancer biology & therapy.

[45]  Kebin Liu,et al.  Epigenetics and Colorectal Cancer Pathogenesis , 2013, Cancers.

[46]  T. Yamanaka,et al.  Simultaneous identification of 36 mutations in KRAS codons 61and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit , 2013, BMC Cancer.

[47]  D. Kerr,et al.  Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. , 2011, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[48]  F. Bazzoli,et al.  Molecular Pathways Involved in Colorectal Cancer: Implications for Disease Behavior and Prevention , 2013, International journal of molecular sciences.

[49]  Jianjun Zhang,et al.  The regulation of toll-like receptor 2 by miR-143 suppresses the invasion and migration of a subset of human colorectal carcinoma cells , 2013, Molecular Cancer.

[50]  I. Alvarado,et al.  Specific c-K-ras Gene Mutations as a Tumor-Response Marker in Locally Advanced Rectal Cancer Treated With Preoperative Chemoradiotherapy , 2000, Annals of Surgical Oncology.

[51]  P. Quirke,et al.  Intra-tumoral Heterogeneity of KRAS and BRAF Mutation Status in Patients with Advanced Colorectal Cancer (aCRC) and Cost-Effectiveness of Multiple Sample Testing , 2011, Analytical cellular pathology.

[52]  Wentao Wang,et al.  miR-133b, a muscle-specific microRNA, is a novel prognostic marker that participates in the progression of human colorectal cancer via regulation of CXCR4 expression , 2013, Molecular Cancer.

[53]  Aung Ko Win,et al.  KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers , 2013, British Journal of Cancer.

[54]  N. Matsubara,et al.  Oncogenic PIK3CA mutations in colorectal cancers and polyps , 2012, International journal of cancer.

[55]  Sabine Tejpar,et al.  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. , 2010, The Lancet. Oncology.

[56]  K. Jirström,et al.  Sex differences in the prognostic significance of KRAS codons 12 and 13, and BRAF mutations in colorectal cancer: a cohort study , 2013, Biology of Sex Differences.

[57]  Tae Won Kim,et al.  KRAS mutation status and clinical outcome of preoperative chemoradiation with cetuximab in locally advanced rectal cancer: a pooled analysis of 2 phase II trials. , 2013, International journal of radiation oncology, biology, physics.

[58]  F. McCormick,et al.  Intrinsic and GTPase-activating protein-stimulated Ras GTPase assays. , 1995, Methods in enzymology.

[59]  S. Paik,et al.  A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes , 2010, BMC Cancer.

[60]  A. Viale,et al.  Comparative genomic analysis of primary versus metastatic colorectal carcinomas. , 2012, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[61]  Reiko Nishihara,et al.  Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival. , 2012, The New England journal of medicine.

[62]  T. Beißbarth,et al.  KRAS and BRAF mutations in patients with rectal cancer treated with preoperative chemoradiotherapy. , 2010, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[63]  S. Sangkhathat,et al.  Somatic mutations of K-ras and BRAF in Thai colorectal cancer and their prognostic value. , 2013, Asian Pacific Journal of Cancer Prevention.

[64]  Y. Yen,et al.  Dovitinib synergizes with oxaliplatin in suppressing cell proliferation and inducing apoptosis in colorectal cancer cells regardless of RAS-RAF mutation status , 2014, Molecular Cancer.

[65]  E. Zwarthoff,et al.  Two Multiplex Assays That Simultaneously Identify 22 Possible Mutation Sites in the KRAS, BRAF, NRAS and PIK3CA Genes , 2010, PloS one.

[66]  D. Sargent,et al.  KRAS Codon 12 and 13 Mutations in Relation to Disease-Free Survival in BRAF–Wild-Type Stage III Colon Cancers from an Adjuvant Chemotherapy Trial (N0147 Alliance) , 2014, Clinical Cancer Research.

[67]  B. Birren,et al.  Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. , 2012, Genome research.

[68]  C. Bokemeyer,et al.  Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab. , 2012, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[69]  Renzo Boldorini,et al.  Increased Detection Sensitivity for KRAS Mutations Enhances the Prediction of Anti-EGFR Monoclonal Antibody Resistance in Metastatic Colorectal Cancer , 2011, Clinical Cancer Research.

[70]  Victor Moreno,et al.  A combined oncogenic pathway signature of BRAF, KRAS and PI3KCA mutation improves colorectal cancer classification and cetuximab treatment prediction , 2012, Gut.

[71]  E. Van Cutsem,et al.  Massively Parallel Tumor Multigene Sequencing to Evaluate Response to Panitumumab in a Randomized Phase III Study of Metastatic Colorectal Cancer , 2013, Clinical Cancer Research.

[72]  E. Giovannucci,et al.  Molecular pathological epidemiology of colorectal neoplasia: an emerging transdisciplinary and interdisciplinary field , 2010, Gut.

[73]  Yu Zhou,et al.  MicroRNA-32 (miR-32) regulates phosphatase and tensin homologue (PTEN) expression and promotes growth, migration, and invasion in colorectal carcinoma cells , 2013, Molecular Cancer.

[74]  J. Feliu,et al.  Prognostic and predictive biomarkers for epidermal growth factor receptor-targeted therapy in colorectal cancer: beyond KRAS mutations. , 2013, Critical reviews in oncology/hematology.

[75]  Brian C. Netzel,et al.  Companion-diagnostic testing limited to KRAS codons 12 and 13 misses 17% of potentially relevant RAS mutations in colorectal cancer. , 2013, Clinica chimica acta; international journal of clinical chemistry.

[76]  Shuji Ogino,et al.  Editorial Lifestyle Factors and Microsatellite Instability in Colorectal Cancer: the Evolving Field of Molecular Pathological Epidemiology , 2022 .

[77]  H. Lee,et al.  Different metastatic pattern according to the KRAS mutational status and site-specific discordance of KRAS status in patients with colorectal cancer , 2012, BMC Cancer.

[78]  B. Vogelstein,et al.  Prevalence of ras gene mutations in human colorectal cancers , 1987, Nature.

[79]  Larissa V Furtado,et al.  Frequency of KRAS, BRAF, and NRAS mutations in colorectal cancer , 2011, Genes, chromosomes & cancer.

[80]  P. Laird,et al.  Genome-scale analysis of aberrant DNA methylation in colorectal cancer. , 2012, Genome research.

[81]  Susan Richman,et al.  Panitumumab and irinotecan versus irinotecan alone for patients with KRAS wild-type, fluorouracil-resistant advanced colorectal cancer (PICCOLO): a prospectively stratified randomised trial , 2013, The Lancet. Oncology.

[82]  P. Febbo,et al.  NCCN Task Force report: Evaluating the clinical utility of tumor markers in oncology. , 2011, Journal of the National Comprehensive Cancer Network : JNCCN.

[83]  Sarah Edkins,et al.  Recurrent KRAS codon 146 mutations in human colorectal cancer , 2006, Cancer biology & therapy.

[84]  M. Loda,et al.  Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. , 2005, The Journal of molecular diagnostics : JMD.

[85]  Semra Ozdemir,et al.  The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population. , 2013, Genetic testing and molecular biomarkers.

[86]  A. Bardelli,et al.  Association of KRAS p.G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. , 2010, JAMA.

[87]  T. Winder,et al.  Different types of K-Ras mutations are conversely associated with overall survival in patients with colorectal cancer. , 2009, Oncology reports.

[88]  F. Sinicrope,et al.  Aspirin and Colorectal Cancer: Back to the Future , 2013, Clinical Cancer Research.

[89]  Steven J. M. Jones,et al.  Comprehensive molecular characterization of human colon and rectal cancer , 2012, Nature.

[90]  Jan-Gowth Chang,et al.  Fast simultaneous detection of K-RAS mutations in colorectal cancer , 2009, BMC Cancer.

[91]  R. Palmqvist,et al.  The prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer , 2013, British Journal of Cancer.

[92]  Levi Waldron,et al.  Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum , 2012, Gut.

[93]  Barry S Taylor,et al.  Genomic and biological characterization of exon 4 KRAS mutations in human cancer. , 2010, Cancer research.

[94]  M. Loda,et al.  CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations. , 2006, The Journal of molecular diagnostics : JMD.