Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization

✍ Scribed by Dr. Judd W. Moul; Sheila M. Theune; Esther H. Chang

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: English
• Weight: 951 KB
• Volume: 5
• Category: Article
• ISSN: 1045-2257
• DOI: 10.1002/gcc.2870050204

✦ Synopsis

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 Nand KRAS mutations, two studies of a variety of germ cell lines failed t o document RAS mutations. T o clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), I8 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent t o 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, I 3, and 6 I 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 I 2 (4/44;[9. I %I). One seminoma [I/ I8(5.6%)] contained the mutation GGT(GLY) .--t CGT(ARG), and three NSGCT [3/25( I2%)] were found t o 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 I 3 or 6 I, in NRAS o r HRAS 12, 13, o r 6 I, o r 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 D N A controls were integral t o 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. Genes Chrom Cancer 5:109-1 18 (1992).