ABSENT RAS GENE-MUTATIONS IN HUMAN ADRENAL-CORTICAL NEOPLASMS AND PHEOCHROMOCYTOMAS

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.