DETECTION OF P21(RAS) MUTATIONS IN COLORECTAL ADENOMAS AND CARCINOMASBY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Background. Point mutations of the ras protooncogene, primarily within codons 12 and 13, are commonly identified in colorectal carcinomas an d large adenomas. Despite data suggesting that ras genotyping may have clinical significance with respect to colorectal cancer screening and prognosis, more widespread use has been limited because of the lack o f a suitable assay system. The principal objective of this study was t o assess the feasibility and validity of a qualitative enzyme-linked i mmunosorbent assay (ELISA) for detecting the four most common ras muta tions in human colorectal tumors at the protein (p21(ras)) level. Meth ods. Tissue homogenates (11-121 mu g) from endoscopically or surgicall y resected colorectal adenomas, carcinomas, and normal mucosae were ev aluated by a commercially available ELISA (Oncogene Science, Inc. Camb ridge, MA) for mutant p21(ras) containing arginine position 12 (arg12) , valine position 12 (val12), aspartate position 12 (asp12), and aspar tate position 13 (asp13) amino acid substitutions. Portions of the sam e tissue from an initial series of 27 specimens also were subjected to mutant-enriched polymerase chain reaction (PCR) and/or PCR amplificat ion with subsequent DNA sequence analysis to validate the ELISA data. Results. Forty-seven adenomas, 9 carcinomas, and 14 normal mucosae wer e assayed. Mutations were identified in 16 (34%) of the adenomas (7-as p12, 7-val12, 2-asp13), 3 (33%) of the carcinomas (1-asp12, 1-arg12, 1 -asp13), and none of the normal mucosae by ELISA. Polymerase Chain Rea ction and DNA sequencing analyses demonstrated identical results for 2 1 of the 23 (91%) and 14 of 16 (88%) homogenates tested, respectively. The ELISA demonstrated an overall sensitivity of 80-86%, specificity of 90-99%, positive predictive value of 86-100%, and negative predicti ve value of 86-91%. Conclusions. The ELISA is a feasible and valid app roach for identifying p21(ras) mutations in human colorectal adenomas and carcinomas.