IDENTIFICATION OF H, K, AND N-RAS POINT MUTATIONS IN STAGE IB CERVICAL-CARCINOMA

Objective: The ras oncogenes, Harvey (H), Kirsten (K), and neuroblasto ma (N), are a family of genes coding for a membrane-associated protein (p21) which possesses inherent guanine triphosphatase (GTPase) activi ty. Point mutagenesis at codons 12, 13, and 61 has been implicated in ras activation and subsequent cellular transformation. Given the epide miologic relationship of HPV infection with cervical carcinoma and the tumorigenic interaction of HPV and mutated ras oncogenes, this study was undertaken to identify if mutated ras oncogenes were present in ea rly invasive cervical carcinomas. Methods: A combination of polymerase chain reaction (PCR) and dot-blot hybridization was used to determine the frequency and types of ras point mutants occurring in cervical ca rcinoma. Thirty-three patients with early-stage cervical carcinoma wer e identified. DNA was extracted from archival tumor samples. ras genes were PCR amplified using flanking primers and hybridized with a serie s of labeled allele-specific oligonucleotides corresponding to wild-ty pe forms of K-12,K-61, N-12,N-13,N-61, and H-12,H-61, as well as to al l combinations of substitution mutations (7 wild-type, 45 mutants). Re sults: ras mutations were identified in 24.2% of specimens. The detect ed mutations in H, K, and N-ras all occurred at codon 61. This was not the result of PCR or hybridization artifact in that mutations were de tected in position 12 and 13 in appropriate control samples. Conclusio ns: Mutant ras has been shown to convert HPV immortalized keratinocyte s to the tumorigenic state. Our results indicate that a significant pe rcentage (24.2%) of these early-stage cervical canters contain activat ed ras. Additional studies will be needed to evaluate whether codon 61 represents a characteristic ''hotspot'' of ras mutation in a subset o f cervical carcinoma. (C) 1997 Academic Press.