KRAS Mutation Detection in Colorectal Cancer,
Direct sequencing
Clinical Significance:KRAS mutations have been known to be present in a variety of human cancers, including lung cancer, colorectal cancer, and pancreatic cancer and is associated with a poor prognosis. More recent data suggest that KRAS mutations are a useful biomarker of resistance to epidermal growth factor receptor (EGFR)-based therapeutics, especially colorectal carcinomas. Cetuximab is used alone and in combination with irinotecan for patients with advanced colorectal cancer based on demonstrated improvements in overall survival. Thus KRAS mutations have recently emerged as a useful negative predictive biomarker, predicting when therapy with this class of targeted agents is unlikely to work. As a result, determining the KRAS mutational status of a tumor may guide therapeutic decision making for patients with colorectal cancers.
Major types of KRAS mutations: Current estimates of the prevalence of KRAS mutation in colorectal adenocarcinomas range from 30-50%. Overall, according to the COSMIC mutation database, over 99% of KRAS mutations in lung cancers are in codons 12 and 13.
Methodology: The activating mutations in KRAS codons 12 and 13 are detected by standard direct DNA sequencing procedures using an ABI 3100 DNA sequencer. PCR amplification of a 332bp fragment including the entire coding region of exon 2 is performed and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). All PCR amplifications are performed in duplicate for each sample. One PCR product is sequenced with forward primer and the second PCR product is sequenced with the reverse primer and concordant results are expected in both directions (both strands).
Turnaround Time: 3-5 business days
Sample requirements:
The presence of adequate tumor in the material submitted for analysis should be confirmed by a surgical pathologist. A section from archival paraffin material or frozen surgical biopsies should be confirmed to contain >50% tumor by a surgical pathologist. If the submitted material for analysis contains less than 50% of tumor, areas of predominant tumor will be macrodissected using a scalpel to trim away non-neoplastic areas.
Fresh Tissue
Curls from FFPE Samples - 10uM thick, 5-6 sections
