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Background. Point mutations in codon 12 of the K-ras protooncogene occur more frequently in lung adenocarcinomas from smokers (30%) than they do in lung adenocarcinomas from nonsmokers (7%), suggesting that smoking is an important factor in the induction of these mutations. The lack of well defined 'early' premalignant or in situ glandular neoplasms of the lung, however, has not permitted direct evaluation of the chronology of ras activation in the development of lung adenocarcinomas. To circumvent the need to evaluate precursor lesions, we examined lung adenocarcinomas from former smokers for point mutations in K-ras. Methods. Mutations in codon 12 of K-ras were detected using polymerase chain reaction amplification and mutation-specific oligonucleotide probes. The types and frequencies of mutations found in adenocarcinomas obtained from 57 former smokers were compared to those found in 27 adenocarcinomas from patients who never smoked and to those found in 27 adenocarcinomas from patients who were current smokers. Results. The overall prevalence of K-ras point mutations in lung adenocarcinomas obtained from former smokers (32%) was not different from that seen in adenocarcinomas from patients who were current smokers (30%, P = 0.83), and was greater than that seen in adenocarcinomas from patients who never smoked (7%, P 0.015). This pattern was independent of the duration of abstinence from smoking. Furthermore, the predominant type of mutation found in tumors from former smokers was a guanine-to-thymine transversion, the specific type of mutation induced by benzo(a)pyrene, one of the chemical carcinogens found in tobacco smoke. Conclusions. These findings support previous findings that suggest that codon 12 of the K-ras oncogene may be a specific target of the mutagenic activity of tobacco smoke, and suggest that DNA alterations at this site can occur early and irreversibly during the development of adenocarcinomas of the lung.

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated rasoncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 μm tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated rassequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in rasgenes can be performed in a routine setting, and the results of the analyses can be available in as few as 3–4 days.