Extended KRAS and NRAS Mutation Profiling by Pyrosequencing^®

Abstract

With the advent of targeted therapies—drugs that specifically target molecules of tumor-driving signalling pathways—and the availability of biomarkers that predict the response of an individual patient on such a targeted therapy, the analysis of the status of the biomarker became an integral part of the therapy. For metastatic colorectal cancer, anti-EGFR-targeted antibodies (Cetuximab/Erbitux^®, Panitumumab/Vectibix^®) fall into this category of drugs as it was shown in several clinical studies that oncogenic mutations in exons 2–4 of the RAS genes KRAS or NRAS result in therapeutic resistance of the metastatic colorectal cancers against the action of both targeted drugs. Therefore, mutations in the RAS genes exclude patients from this kind of targeted therapy (negative biomarker). Thus the molecular-pathological testing of the mutational status of KRAS and NRAS has become an important cornerstone in planning oncological strategies in the treatment of metastatic colorectal cancer. As the profile of mutations in the RAS genes is characterized by hotspot mutations in only a small number of codons (12, 13 in exon 2–59, 61 in exon 3–117, 146 in exon 4). Pyrosequencing^® is an ideal and robust tool in the molecular-pathological detection. A detailed protocol for this detection procedure employing Pyrosequencing® is given here.