The RAS gene family includes three functional genes, H-RAS, K-RAS, and N-RAS, which have been most widely studied in human tumors. Point mutations most commonly occurring at codons 12, 13, or 61 of these genes allow the RAS protooncogene to be converted to a RAS oncogene. A variety of human tumors have been studied for RAS mutations to date, however, conflicting data has been reported regarding prostate cancer. Cell line studies and two American studies of clinical material have found a low incidence of RAS mutation in prostate cancer. The few mutations found were predominantly in the H-RAS gene. Conversely, a recent study of Japanese occult autopsy specimens found an approximate 25% incidence of K-RAS mutations. In this current study, DNA was extracted from 24 archival paraffin-embedded, formalin-fixed radical prostatectomy specimens. Twenty-one of the 24 cases had pathologic stage C disease, and paraffin blocks were selected having the most concentrated area of neoplasm. Twelve, seven, and five cases demonstrated moderate, well and poorly differentiated histologic grade respectively. Polymerase chain reaction (PCR) was used to amplify the K-RAS, N-RAS, and H-RAS 12, 13, 61 codons of these specimens and mutations were detected with mutation-specific oligonucleotide probe hybridization of southern and slot blots. No definite point mutations were detected. PCR's and hybridizations were performed three separate times by three investigators to confirm these results. PCR-generated mutation-specific positive controls and known negative controls were used and found to be important to interpret oligonucleotide hybridization assays. RAS gene mutations appear to be infrequent in clinical prostate carcinomas in American males.

Preliminary studies of RAS mutational activation in human testicular germ cell neoplasms have yielded conflicting results. Whereas two studies of clinical material revealed a significant incidence of N- and KRAS mutations, two studies of a variety of germ cell lines failed to document RAS mutations. To clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), 18 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent to the tumor. Six age-matched patients had testes removed because of nonmalignant disease and were studied as controls. Polymerase chain reaction (PCR) amplified the K-, N-, and HRAS 12, 13, and 61 codons of these specimens, and mutations were detected with mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Four mutations were found in KRAS 12 (4/44;[9.1%]). One seminoma [1/18(5.6%)] contained the mutation GGT(GLY)----CGT(ARG), and three NSGCT [3/25(12%)] were found to have GGT(GLY)----GAT(ASP) mutations. One of the NSGCT mutations was detected in adjacent nonmalignant tissue, but the corresponding tumor did not contain any detectable mutation. No mutations were detected at KRAS 13 or 61, in NRAS or HRAS 12, 13, or 61, or in the control normal testes. PCR, slot blots, and hybridizations were performed twice by two separate investigators for confirmation of results. PCR-generated mutation-specific positive controls were created for all possible RAS mutations, and these along with wild-type DNA controls were integral to interpretation of the oligonucleotide mismatch hybridization assay. By using positive and negative controls, we have detected a relatively low incidence of RAS mutations in archival human testicular germ cell tumors.

A variety of human tumors have been studied for ras mutations to date. However, little is known about the prevalence and significance of ras gene activation in adrenal neoplasms. Recently, a study of 10 primary human pheochromocytomas found no evidence for ras mutations. To our knowledge no survey of ras mutations in adrenocortical neoplasms has been reported. Therefore, we analyzed deoxyribonucleic acid (DNA) from 17 archival tumors (8 adrenocortical carcinomas, 6 pheochromocytomas, 2 adrenal adenomas, 1 aldosteronoma, 2 fresh pheochromocytomas and 1 fresh benign adrenal gland) for activating mutations at the 12, 13 and 61 codons of N-ras, H-ras and K-ras. DNA was extracted from archival tissues using 3 different methods: a simplified boiling method, a proteinase-K-phenol chloroform extraction and a novel heat-stable protease Thermus rt41A technique. The boiling and heat-stable protease methods provided for more consistent polymerase chain reaction amplifications than the more laborious phenol chloroform method. This heat-stable protease Thermus rt41A method had not been reported previously for use in archival DNA extraction. Polymerase chain reaction amplified the ras gene regions of interest, and mutations were screened by mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Polymerase chain reaction-generated mutation-specific positive and negative controls were used in the hybridization protocol. With these controlled conditions no definite mutations were detected at codons 12, 13 or 61 of N, H or K-ras. Ras activation via point mutations at these codons rarely, if ever, occurs in adrenal neoplasms.

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