Mutational activation of K-ras in nonneoplastic exocrine pancreatic lesions in relation to cigarette smoking status

BACKGROUND. Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mut ations in codon 12 of the K-ras protooncogene is a frequent and early molec ular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cig arette smoking and mutational activation of K-ras in PDAC has been examined to a limited extent. The authors have examined the mutational status of K- ras in nonneoplastic pancreata in relation to cigarette smoking status. METHODS. Archival formalin fixed paraffin embedded specimens of nonneoplast ic pancreata (n = 39) were obtained from the American Cancer Society and ev aluated histopathologically. Specimens from age- and gender-matched individ uals were stratified into three groups: 1) those who never smoked cigarette s (n = 16), 2) those who smoked 1-2 packs/day for more than 20 years (n = 1 0 cases), and 3) those who smoked more than 2 packs/day for 20 or more year s (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furtherm ore, none of the patients died of primary PDAC or had evidence of pancreati c metastases from an extrapancreatic primary tumor. Tissue sections were mi crodissected and deparaffinized, and genomic DNA was purified by standard p roteinase K-phenol-chloroform extraction techniques. Genomic DNA was analyz ed for mutations in codon 12 of the K-rns protooncogene by two mutant-allel e-enriched polymerase chain reaction (PCR)-restriction fragment length poly morphism (RFLP) assays and by multiplex PCR-based ligase chain reaction (LC R) analyses. RESULTS. Analyses of multiple microdissected pancreata specimens from 39 ca ses revealed wild-type K-ras codon 12 sequences in both nonsmoking individu als and those who smoked 1-2 packs/day for 20 or more years. R-ms codon 12 mutations were confirmed by PCR-RFLP and PCR-LCR assays in 5 of 13 pancreat a cases (39%) obtained from individuals who smoked more than 2 packs of cig arettes/day for 20 years or more (P < 0.005). The K-ras mutation spectra re vealed two G-->T transversions, one G --> C transversion and two G-->A tran sitions. There was no clear relation between the incidence or spectra of mu tations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atyp ia revealed reproducible K-ms alterations. Similarly, among those 34 cases in which a wild-type K-rns sequence was revealed by both approaches, a simi lar histopathologic profile was evident. CONCLUSIONS. Mutational activation of codon 12 of the K-ms protooncogene wa s confirmed reproducibly by mutant allele-enriched PCR-RFLP and multiplex P CR-LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from a ge- and gender-matched individuals who smoked more than 2 packs of cigarett es/day for 20 or more years. The presence of a mutated or wild-type or K- m s was independent of the histopathologic profile of the 39 cases examined. The data provide further suggestive molecular epidemiologic evidence of an association between a major and unequivocal risk factor for PDAC (heavy cig arette smoking) and mutations in a molecular target (K-ras), the activation of which is an important and early event both in the pathogenesis of PDAC and in the development of a variety of nonneoplastic ductal pancreatic lesi ons.(C) 1999 American Cancer Society.