Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human colorectal tumours

Abstract

Background:The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden.Methods:Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters.Results:The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype.Conclusion:Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.