title: Molecular markers identify subtypes of stage III colon cancer associated with patient outcomes.
abstract: OBJECTIVE
  Categorization of colon cancers into distinct subtypes using a combination of pathway-based biomarkers could provide insight into stage-independent variability in outcomes.
  
  
  METHODS
  We used a polymerase chain reaction-based assay to detect mutations in BRAF (V600E) and in KRAS in 2720 stage III cancer samples, collected prospectively from patients participating in an adjuvant chemotherapy trial (NCCTG N0147). Tumors deficient or proficient in DNA mismatch repair (MMR) were identified based on detection of MLH1, MSH2, and MSH6 proteins and methylation of the MLH1 promoter. Findings were validated using tumor samples from a separate set of patients with stage III cancer (n = 783). Association with 5-year disease-free survival was evaluated using Cox proportional hazards models.
  
  
  RESULTS
  Tumors were categorized into 5 subtypes based on MMR status and detection of BRAF or KRAS mutations which were mutually exclusive. Three subtypes were MMR proficient: those with mutations in BRAF (6.9% of samples), mutations in KRAS (35%), or tumors lacking either BRAF or KRAS mutations (49%). Two subtypes were MMR deficient: the sporadic type (6.8%) with BRAF mutation and/or or hypermethylation of MLH1 and the familial type (2.6%), which lacked BRAF(V600E) or hypermethylation of MLH1. A higher percentage of MMR-proficient tumors with BRAF(V600E) were proximal (76%), high-grade (44%), N2 stage (59%), and detected in women (59%), compared with MMR-proficient tumors without BRAF(V600E) or KRAS mutations (33%, 19%, 41%, and 42%, respectively; all P < .0001). A significantly lower proportion of patients with MMR-proficient tumors with mutant BRAF (hazard ratio = 1.43; 95% confidence interval: 1.11-1.85; Padjusted = .0065) or mutant KRAS (hazard ratio = 1.48; 95% confidence interval: 1.27-1.74; Padjusted < .0001) survived disease-free for 5 years compared with patients whose MMR-proficient tumors lacked mutations in either gene. Disease-free survival rates of patients with MMR-deficient sporadic or familial subtypes was similar to those of patients with MMR-proficient tumors without BRAF or KRAS mutations. The observed differences in survival rates of patients with different tumor subtypes were validated in an independent cohort.
  
  
  CONCLUSIONS
  We identified subtypes of stage III colon cancer, based on detection of mutations in BRAF (V600E) or KRAS, and MMR status that show differences in clinical and pathologic features and disease-free survival. Patients with MMR-proficient tumors and BRAF or KRAS mutations had statistically shorter survival times than patients whose tumors lacked these mutations. The tumor subtype found in nearly half of the study cohort (MMR-proficient without BRAF(V600E) or KRAS mutations) had similar outcomes to those of patients with MMR-deficient cancers.
authors.affiliation: Department of Medicine, Mayo Clinic, Rochester, Minnesota; Department of Oncology, Mayo Clinic, Rochester, Minnesota. Electronic address: sinicrope.frank@mayo.edu.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Sinicrope
authors.affiliation: Alliance Statistics and Data Center, Mayo Clinic, Rochester, Minnesota.
authors.forename: Qian
authors.initials: Q
authors.lastname: Shi
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Smyrk
authors.affiliation: Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Stephen N
authors.initials: SN
authors.lastname: Thibodeau
authors.affiliation: Sage Bionetworks, Seattle, Washington.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Dienstmann
authors.affiliation: Sage Bionetworks, Seattle, Washington.
authors.forename: Justin
authors.initials: J
authors.lastname: Guinney
authors.affiliation: Sage Bionetworks, Seattle, Washington.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Bot
authors.affiliation: Molecular Digestive Oncology, KU Leuven, The Netherlands.
authors.forename: Sabine
authors.initials: S
authors.lastname: Tejpar
authors.affiliation: Swiss Institute of Bioinformatics, University Lausanne, Switzerland.
authors.forename: Mauro
authors.initials: M
authors.lastname: Delorenzi
authors.affiliation: Division of Medical Oncology, Ohio State University Comprehensive Cancer Center, Columbus, Ohio.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Goldberg
authors.affiliation: Alliance Statistics and Data Center, Mayo Clinic, Rochester, Minnesota.
authors.forename: Michelle
authors.initials: M
authors.lastname: Mahoney
authors.affiliation: Alliance Statistics and Data Center, Mayo Clinic, Rochester, Minnesota.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sargent
authors.affiliation: Department of Oncology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Alberts
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000235:genetics; D000230:Adenocarcinoma / Q000145:classification / Q000235:genetics* / Q000401:mortality / Q000473:pathology* / Q000628:therapy; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics*; D003110:Colonic Neoplasms / Q000145:classification / Q000235:genetics* / Q000401:mortality / Q000473:pathology* / Q000628:therapy; D019175:DNA Methylation; D053843:DNA Mismatch Repair; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins / Q000032:analysis; D018572:Disease-Free Survival; D005260:Female; D020022:Genetic Predisposition to Disease; D006801:Humans; D053208:Kaplan-Meier Estimate; D008297:Male; D008875:Middle Aged; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000032:analysis; D009154:Mutation; D009367:Neoplasm Staging; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011401:Promoter Regions, Genetic; D016016:Proportional Hazards Models; D011446:Prospective Studies; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D012307:Risk Factors; D013997:Time Factors; D016896:Treatment Outcome; D055815:Young Adult; D018631:ras Proteins / Q000235:genetics*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C117307:KRAS protein, human; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: Colorectal Cancer; Genetics; Oncogene; Prognostic Factor