Clinical utility of KRAS and BRAF mutations in a cohort of patients with colorectal neoplasms submitted for microsatellite instability testing

Abstract

Background: Molecular analysis has become important in colorectal carcinoma (CRC) evaluation. Alterations in KRAS, BRAF, or mismatch repair (MMR) genes may determine therapeutic response or define a hereditary cancer syndrome. Correlation of DNA studies with clinical findings will further clarify the clinical utility of these markers.

Patients and methods: A retrospective study was performed on 111 paraffin-embedded tumor specimens submitted for microsatellite instability (MSI) testing based on clinical history or histologic examination, or both. DNA samples were screened for 7 KRAS mutations and the BRAF p.V600E mutation using fluorescent allele-specific polymerase-chain reaction (PCR) and capillary electrophoresis. Clinical data were collected through chart review.

Results: Fifty-eight male and 53 female patients were studied. The incidence of KRAS and BRAF mutations was 49.5% and 7.2%, respectively. Dideoxy sequencing verified KRAS mutation status in 46 of 49 specimens tested. There was a trend toward significance of individual KRAS mutations on survival (P = .003). Dually positive KRAS and MSI tumors exclusively demonstrated p.G12D and p.G13D mutations (G>A transitions). BRAF-mutated tumors were predominantly right-sided and associated with a borderline worse prognosis. Forty-eight percent of tumors with MSI were present in the left colon or rectum.

Conclusion: Allele-specific PCR is an accurate and convenient method to assess KRAS and BRAF mutations and may detect mutations not identified by dideoxy sequencing. KRAS mutation status, in conjunction with morphologic or clinical parameters, may be useful in determining whether a tumor should be tested for MSI. MSI testing should not be considered exclusively in right-sided lesions. BRAF analysis may not be useful in rectal adenocarcinomas and should be evaluated in larger studies.

Keywords: Allele specific; Biomarker; Missense; Mutation; Transversion.

Figure 1

(A) KRAS Assay Design. Seven Separate Reactions are Performed, Each Containing a Fluorescently Labeled Upstream Wild-Type Primer, an Unlabeled Downstream Primer, and 1 Differentially Labeled Fluorescent Mutation-Specific Primer. Amplicons are Detected by Capillary Electrophoresis at Approximately 176 to 180 Base Pairs (Checkered circle) if the Respective Mutation is Present (Left Peak is p.G13D). Wild-Type Amplicons (Black circle) are Detected at 267 Base Pairs in All Reactions (Right peak). (B) Primer Sequences used in The Assay (Adapted from References and 24), Including 7 Mutant Allele-Specific Primers and 2 Wild-Type Primers (Upstream and Downstream). * = Fluorochrome Label; ** = Penultimate Substitution of Nucleotide to Increase Specificity

Figure 2

(A) Survival Stratified by KRAS Mutation–Positive Tumors vs. KRAS Wild-Type Tumors. (B) Survival Stratified by Individual KRAS Mutations. The P Value Indicates That the Difference in Overall Survival Among Different KRAS Mutation Groups is Statistically Significant. (C) Survival Stratified by BRAF Mutation–Positive Tumors vs. BRAF Wild-Type Tumors. NEG = Negative; POS = Positive; WT = Wild Type