[553] K-RAS Mutational Analysis in 562 Consecutive Colon Carcinomas

KJ Bloom, P Choppa, TA Bloom, M Hibbard. Clarient, Aliso Viejo, CA

Background: Oncogenic mutations in K-RAS, the human homolog of the Kirsten rat sarcoma-2 virus oncogene, results in a constitutively active protein in approximately 30% to 50% of colorectal carcinomas (CRC) and has also been described in carcinomas of the pancreas, lung, head and neck and ovary. KRAS encodes a small GTP binding protein that acts as a self-inactivating signal transducer by cycling from GDP- to GTP-bound states in response to stimulation of cell surface receptors, including EGFR. Several recent studies have shown that the presence of a KRAS mutation in CRC correlates with poor prognosis, and is associated with lack of response to EGFR inhibitors. The purpose of this study was to confirm the frequency of mutations reported in the literature as well as to determine the distribution of the specific mutations identified by PCR.
Design: Formalin-fixed, paraffin-embedded tumor sections were deparaffinized and cells of interest were identified and removed using a PALM laser capture microdissection system (Zeiss, Jena, Germany). DNA was isolated from the captured cells using the DNA mini kit (Qiagen, Valencia, CA). Mutant KRAS was detected using a validated KRAS mutation kit (DxS Ltd, Manchester, United Kingdom) that identifies seven somatic mutations located in codons 12 and 13 (Gly12Asp, Gly12Ala, Gly12Val, Gly12Ser, Gly12Arg, Gly12Cys, and Gly13Asp) using allele-specific real-time polymerase chain reaction. Amplification and detection were performed on an Applied Biosytems 7900HT (ABI, Foster City, CA).
Results: Five hundred sixty-two consecutive colon carcinomas (primary and metastatic) were evaluated. Four samples failed to yield a result. Of the 558 analyzed samples, 370 (66%) revealed wild-type K-RAS while 188 (34%) revealed a mutation. The distribution of mutations was as follows Gly12Asp (GGT->GAT) 77 tumors (13%), Gly12Val (GGT->GTT) 51 tumors (9%), Gly13Asp (GGC->GAC) 33 tumors (6%), Gly12Ala (GGT->GCT) 12 tumors (2%), Gly12Cys (GGT->TGT) 9 tumors (1.6%), Gly12Ser (GGT->AGT) 4 tumors (0.7%) and Gly12Arg (GGT->CGT) 2 tumors (0.4%).
Conclusions: The detection rate of K-RAS mutations was consistent with the 30-40% described in the literature. Although all seven mutations known to be associated with lack of response to EGFR inhibitors were identified, five mutations, Gly12Asp (GGT->GAT), Gly12Val (GGT->GTT), Gly13Asp (GGC->GAC), Gly12Ala (GGT->GCT), and Gly12Cys (GGT->TGT), accounted for 97% of the mutations detected.
Category: Gastrointestinal

Tuesday, March 10, 2009 11:30 AM

Platform Session: Section C, Tuesday Morning