MOLECULAR PATHOLOGY OF PRIMARY AND METASTATIC DUCTAL PANCREATIC LESIONS - ANALYSES OF MUTATIONS AND EXPRESSION OF THE P53, MDM-2, AND P21 WAF-1 GENES IN SPORADIC AND FAMILIAL LESIONS/

BACKGROUND. The molecular pathology underlying the development and pro gression of ductal pancreatic adenocarcinoma is poorly understood rela tive to that of other major cancers in industrialized societies. The f requency, nature, and distribution of p53 abnormalities, their tempora l relationship to the metastatic and clinicopathologic phenotypes of s poradic and familial pancreatic cancer, and their consequent effects o n the genetics and expression of critical wild-type p53-regulated gene s (mdm-2 and p21/WAF-1) warrant examination in pancreatic adenocarcino ma. This molecular and immunochemical study of the p53, mdm-2, and p21 /WAF-1 genes and gene products examined the largest series of nonneopl astic, neoplastic, and metastatic ductal pancreatic lesions reported t o date in relation to clinicopathologic profile. METHODS. Histological ly confirmed specimens of primary (n=136) and metastatic (n=23) sporad ic and familial ductal pancreatic adenocarcinoma lesions were subjecte d to immunochemical analyses of p53 expression in which a panel of 3 a ntibodies was utilized. A panel of nonneoplastic but histologically ab normal pancreatic lesions (n=77) from individuals with varied historie s of cigarette smoking were subjected to similar immunohistochemical e xaminations. In addition, 3 specimens from patients with chronic pancr eatitis, 2 specimens of normal fetal pancreata, and 16 specimens of no rmal adult pancreata were examined as control tissues. Suitable frozen and archival microdissected tumor lesions were evaluated for mutation s in exons 4-9 of the p53 gene by single strand conformation polymorph ism (SSCP) and dideoxy sequencing analyses in which two distinct sets of outer and nested intron-based amplification primers were used for e ach exon. A subset of 25 tumor specimens and 18 tumor-derived cell lin es for which the p53 mutation status was known were examined for ampli fication and/or overexpression of the mdm-2 gene; amplification was de termined by Southern hybridization and overexpression by immunohistoch emical and Western blot analyses. Similarly, mutations in the coding r egion of p21/WAF-1 gene were examined by SSCP and DNA sequence analyse s, and steady-state expression of the p21/WAF-1 protein was assessed b y Western blot analysis in these subsets of tumors and tumor-derived c ell lines. RESULTS. Positive ductal nuclear p53 immunostaining was dem onstrated in 56% of primary tumors and 54% of metastatic lesions. The frequency did not differ significantly between sporadic and familial l esions, and immunostaining was not observed in ductal acinar, or islet cell elements of normal pancreata or histologically abnormal benign p ancreatic lesions from cigarette smokers. A total of 70% of tumor samp les revealed reproducible SSCP abnormalities for p53; 42% of these wer e found in exons 7 and 8. DNA sequence analysis of cases with greater than 35% epithelial cellularity (n=25) revealed 17 missense mutations, 12 of which were transitions. Seventy-five percent of these transitio ns were of G:C-A:T type. A total of 22% of the p53 mutations identifie d were microdeletions, along with one insertional mutation at exon 8. None of the normal pancreata from sporadic or familial lesions reveale d germ-line p53 alterations. Moreover, the frequency and spectra of p5 3 alterations exhibited no clear, statistically significant associatio n with tumor grade, TNM stage, or patients' cigarette-smoking historie s. The mdm-2 gene was neither amplified nor overexpressed immunochemic ally in a subset of ductal adenocarcinomas, and there was no clear rel ationship between the p53 mutation status and the status of the mdm-2 gene or protein. Similarly, SSCP and DNA sequence analysis of the p21/ WAF-1 gene revealed only 2 genetic abnormalities in a series of 25 pri mary tumors and 15 tumor-derived cell lines; 1 of the cell lines also revealed the absence of immunoreactive p21/WAF-1 protein. CONCLUSIONS. This immunochemical and molecular study of the p53 tumor suppressor g ene/protein and the p53-regulated genes mdm-2 and p21/WAF-1 in this se ries of ductal pancreatic lesions revealed that p53 mutations are a fr equent early event in pancreatic tumorigenesis not associated with met astatic progression. The authors observed complex spectra of transitio n missense mutations and microdeletions but no significant correlation with tumor grade, TNM stage, or smoking history. In contrast, amplifi cation and overexpression of the mdm-2 gene/protein and genetic abnorm alities in the p21/WAF-1 gene are infrequent events in the development of ductal pancreatic adenocarcinoma and were not clearly associated w ith the p53 mutation status of tumors examined in this study. (C) 1997 American Cancer Society.