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I I I I POINT MUTATIONS OF HA-RAS AND KI-RAS ONCOGENES IN SPUTUM SPECIMENS FROM LUNG CANCER PATIENTS H Lin , Chen Jia-kun, Yi Fei, Wu Zhong-liang and Du Ying-xiu Institute For Chemical Carcinogenesis Guangzhou Medical College, Guangzhou, China I I I I I I I I Point mutations in ras (Ha-, Ki- and N-ras) genes can be found in lung cancer. Mutations of Ki-ras genes are most frequently found in lung adenocarcinomas (30%). Because oncogene mutation may take place before cell malignant transformation, we used sputum from lung cancer patients as specimens to find the early diagnostic value of Hi and Ki-ras mutations for lung cancer and developed a simple, rapid and nonradioactive method to detect point mutations in the 12th codon of Ha-ras and Ki-ras genes. DNA was extracted from digested sputum and amplified by the polymerase chain reaction (PCR), and then digested with specific restriction enzymes (HpaII) to detect either endogenous (in Ha-ras gene) or primer-mediated(in Ki-ras gene) restriction fragment length polymorphisms (RFLPs). Fifty sputum specimens from lung cancer patients (20 adenocarcinomas, 12 squamous carcinomas, 16 small cell lung cancers and 2 large cell lung cancers) were examined for Ha-ras and Ki-ras gene mutations in codon 12. Neither Ha-ras nor Ki-ras gene mutations were detected in sputum from small cell lung cancer patients and large cell lung cancer patients; whereas, Ha-ras gene mutations were detected in 10% of the sputum from adenocarcinoma patients and 8.3% from squamous carcinomas. On the other hand, 20% of the sputum from adenocarcinoma patients contained mutations in codon 12 of the Ki-ras gene, but none of sputum samples from squamous carcinomas contained mutations in codon 12 of the Ki-ras gene. In this study, we suggest strategies for the RFLP analysis of point mutations in ras genes in sputum. This simple method is especially suitable for analyzing very small amounts of samples where only one fraction of cells may carry the activated oncogenes. We also determined that point mutations in codon 12 of the Ha-ras and Ki-ras genes are more frequently found in sputum specimens of adenocarcinomas patients than other kinds of lung cancer. This result suggests that mutations of the ras genes in sputum may be indicative of the early stages of luung cancer. O O ~ v W a O A 1V ~ I