Analysis of KRAS, NRAS, BRAF and PIK3CA Mutations in Matched Primary and Metastatic Colorectal Carcinomas.
Efsevia Vakiani, Manick Janackinaram, Jinru Shia, Leonard Saltz, Nancy Kemeny, Martin Weiser, David Solit. Memorial Sloan-Kettering Cancer Center, New York City, NY
Background: Activating KRAS mutations have emerged as a major negative predictor of EGFR inhibitor efficacy in patients with metastatic colorectal carcinoma (CRC). Retrospective studies have also shown a lack of response to EGFR inhibitors among patients with BRAF and NRAS mutations, whereas preliminary studies of PIK3CA have been conflicting. However, the appropriate tumor site for genotyping studies is unclear. Some have suggested analyzing metastases, though the latter tissue is not always available. Thus, we analyzed the mutational profiles of matched primary CRC and metastases to investigate the reasons for the reported discrepancies.
Design: Fresh frozen tissue was analyzed for mutations in KRAS exons 2-4, NRAS exons 2-3, BRAF exon 15 and PIK3CA exons 9 and 20 using a Sequenome MALDI-TOF mass spectrometry-based genotyping assay. All tumors were checked for misidentification using a multiplexed PCR/MS-based genetic fingerprinting assay. Formalin-fixed paraffin embedded (FFPE) tissue from cases showing discrepant results was subjected to Sanger sequencing after microdissection.
Results: We analyzed fresh frozen tissue from 82 matched pairs of primary and metastatic CRC. Initial sequencing identified 41 (50%) KRAS (37 in exon 2, 1 in exon 3 and 3 in exon 4), 2 (2.4%) NRAS (exon 3), 5 (6.1%) BRAF (4 V600E and 1 K601E) and 15 (18.2%) PIK3CA (11 exon 9 and 4 exon 20) mutations. KRAS, NRAS and BRAF mutations were mutually exclusive. Discordant mutational profiles were found in 10 cases (12.2%); 4 of these had a relatively low tumor content in the frozen tissue and concordant mutational profiles were detected after sequencing of FFPE tissue. Three cases were found to be unrelated based on the fingerprinting assay, suggesting errors in tissue identification. One case had multiple colon primaries, raising the possibility that the metastatic focus was not derived from the genotyped primary. No clinical history was available in the remaining two discrepant cases.
Conclusions: Primary and metastatic CRC show a high rate of concordance in KRAS, NRAS, BRAF and PIK3CA mutations. The majority of discrepant results are likely to be due to technical reasons, such as sample mislabeling and low tumor content, rather than gain or loss of mutations during tumor dissemination. Barring technical limitations, genotyping of the primary carcinoma can correctly characterize the genetic lesions associated with metastatic disease. Metastatic foci should be genotyped in patients with multiple primaries, since in these cases it is unclear as to which primary tumor is associated with disease progression.
Monday, February 28, 2011 8:15 AM
Platform Session: Section E, Monday Morning