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

Cigarette smoking is among the few unequivocal risk factors for the development of pancreatic ductal adenocarcinoma (PDAC). Activating mutations in codon 12 of the K-ras protooncogene is a frequent and early molecular event in the pathogenesis of PDAC and a variety of nonmalignant ductal pancreatic lesions. The molecular epidemiologic relation between heavy cigarette 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.

Archival formalin fixed paraffin embedded specimens of nonneoplastic pancreata (n = 39) were obtained from the American Cancer Society and evaluated histopathologically. Specimens from age- and gender-matched individuals were stratified into three groups: 1) those who never smoked cigarettes (n = 16), 2) those who smoked 1-2 packs/day for more than 20 years (n = 10 cases), and 3) those who smoked more than 2 packs/day for 20 or more years (n = 13). Cases were preselected from 77 specimens based on the quality, suitability, and cellularity of the archival tissues for analyses. Furthermore, none of the patients died of primary PDAC or had evidence of pancreatic metastases from an extrapancreatic primary tumor. Tissue sections were microdissected and deparaffinized, and genomic DNA was purified by standard proteinase K-phenol-chloroform extraction techniques. Genomic DNA was analyzed for mutations in codon 12 of the K-ras protooncogene by two mutant-allele-enriched polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays and by multiplex PCR-based ligase chain reaction (LCR) analyses.

Analyses of multiple microdissected pancreata specimens from 39 cases revealed wild-type K-ras codon 12 sequences in both nonsmoking individuals and those who smoked 1-2 packs/day for 20 or more years. K-ras codon 12 mutations were confirmed by PCR-RFLP and PCR-LCR assays in 5 of 13 pancreata cases (39%) obtained from individuals who smoked more than 2 packs of cigarettes/day for 20 years or more (P < 0.005). The K-ras mutation spectra revealed two G-->T transversions, one G-->C transversion and two G-->A transitions. There was no clear relation between the incidence or spectra of mutations and pancreatic histopathology, as overtly normal pancreata as well as pancreata with squamous metaplasia, periductal fibrosis, and ductal atypia revealed reproducible K-ras alterations. Similarly, among those 34 cases in which a wild-type K-ras sequence was revealed by both approaches, a similar histopathologic profile was evident.

Mutational activation of codon 12 of the K-ras protooncogene was confirmed reproducibly by mutant allele-enriched PCR-RFLP and multiplex PCR-LCR analyses in 39% (5 of 13) of archival nonneoplastic pancreata from age- and gender-matched individuals who smoked more than 2 packs of cigarettes/day for 20 or more years. The presence of a mutated or wild-type or K-ras 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 cigarette 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 lesions.