Abstract 2695: Sensitive detection of K-ras mutations reveals intratumoral heterogeneity in primary colon cancers

Abstract

Abstract BACKGROUND: Mutations in the K-ras oncogene occur in 30-50% of colorectal cancers and have been described as an early event in colorectal tumorigenesis. K-ras mutations have recently gained renewed interest due to their well-documented prediction of resistance against EGFR-directed therapy. In the diagnostic setting a single biopsy from each tumor is usually examined, assuming a homogeneous distribution of K-ras mutations in the tumor. Our aim was to investigate the distribution of K-ras mutations within primary colon tumors to determine whether intratumoral heterogeneity occurs. PATIENTS AND METHODS: Tumor biopsies from 158 prospectively recruited patients undergoing curative surgery for primary colon cancer were analysed for mutations in codon 12 and 13 of the K-ras gene by a sensitive and quantitative PNA clamp PCR assay. RESULTS: When analyzing single fresh-frozen biopsies from each tumor, K-ras mutations were detected in 67 (42%) of the 158 patients. Our K-ras mutation measure ddCt indicated low (ddCt<10) concentrations of mutated DNA in 13 (19 %) of the 67 patients, an observation that encouraged us to consider a heterogeneous distribution of K-ras mutations in these tumors. To verify this hypothesis, we analysed tissue sections from all available formalin-fixed tissue blocks (median three per patient) from the tumors of these 13 patients by the same K-ras PNA clamp PCR assay. Seven of the 13 patients with potential tumor heterogeneity had both tissue blocks positive and tissue blocks negative for K-ras mutations. Five of the patients had no mutations detected in any of their formalin-fixed tissue blocks, despite the detection of mutations in the fresh-frozen biopsy. This may partially be explained by the lower sensitivity of the assay in formalin-fixed samples due to poor DNA quality. The remaining patient had mutations detected in all analyzed formalin-fixed tissue sections. Thus, evidence supporting a heterogenous distribution of K-ras mutations within the tumor was obtained in 12 of the 13 investigated cases. The tumor cell fraction in all examined tissue sections was estimated by HES staining and microscopy of neighbouring tissue sections. It was above 10% in all cases and the median value was 40%. There was no correlation between the level of K-ras mutations and estimated tumor cell fraction in tissue sections with detected mutations (Pearson R=-0.11). CONCLUSION: Intratumoral heterogeneity of K-ras codon 12 and 13 mutations was confirmed in 12 (18%) of 67 primary colon tumors with K-ras mutations, with possible implications both for sampling routines in diagnostics and for our understanding of K-ras mutations in tumor biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2695.