Assessment of K-ras Mutational Status Is Influenced by Tissue Archiving: Implications for Routine Molecular Testing.
Evelyne Crapez, Jeanne Ramos, Jerome Solassol, Valerie Costes. Centre de Lutte Contre le Cancer Val d'Aurelle, Montpellier, France; Montpellier Teaching Hospital, France
Background: K-ras mutation analysis has been unambiguously identified as a marker of resistance to cetuximab-based treatment in metastatic colorectal cancer (mCRC) patients. However, most studies have been performed using homogenously archived CRC specimens. The aim of the present study was to evaluate the impact of tissue preservation on the K-ras mutational status of mCRC patients.
Design: A series of 131 mCRC tissue specimens were cut into two equal parts. One of the halves was immediately snap-frozen, and the other half was fixed in formol and then paraffin-embedded (FFPE). The samples were first screened by high-resolution melting (HRM), followed by genotyping via subsequent direct sequencing to determine the K-ras mutational status.
Results: In frozen specimens, HRM clearly identified K-ras mutations in 47/131(35.8%) patients, which was consistent with the direct sequencing results. Among the 33 mutated FFPE-paired specimens available, 2 (6%) demonstrated suboptimal template amplification, and 2 (6%) expressed an erroneous wild-type K-ras profile by HRM. Using direct sequencing, 6/33(18.1%) samples displayed a wild-type K-ras status, 3/33 (9.1%) samples showed discordant nucleic alterations and 2/33 (6%) samples exhibited supplementary artifactual mutations in codon 12/13. Finally, by comparing the FFPE coring vs. the sampling method, no significant discrepancies in K-ras genotyping were observed.
Conclusions: Our results indicate that frozen specimen archiving should be used as often as possible in the diagnostic setting. Particular attention should be paid to the prevention of artifacts in routine molecular K-ras screening of FFPE specimens by using large amounts of template DNA, performing multiple amplifications, or by using a non-formalin-based fixative.
Wednesday, March 2, 2011 9:30 AM
Poster Session V # 51, Wednesday Morning