Abstract
BACKGROUND. The molecular pathology underlying the development and progression of ductal pancreatic adenocarcinoma is poorly understood relative to that of other major cancers industrialized societies. The frequency, nature, and distribution of p53 abnormalities, their temporal relationship to the metastatic and clinicopathologic phenotypes of sporadic and familial pancreatic cancer, and their consequent effects on the genetics and expression of critical wild-type p53-regulated genes (mdm-2 and p21/WAF-1) warrant examination in pancreatic adenocarcinoma. This molecular and immunochemical study of the p53, mdm-2, and p21/WAF-1 genes and gene products examined the largest series of nonneoplastic, neoplastic, and metastatic ductal pancreatic lesions reported to date in relation to clinicopathologic profile. METHODS. Histologically confirmed specimens of primary (n = 136) and metastatic (n = 23) sporadic and familial ductal pancreatic adenocarcinoma lesions were subjected to immunochemical analysis of p53 expression in which a panel of 3 antibodies was utilized. A panel of nonneoplastic but histologically abnormal pancreatic lesions (n = 77) from individuals with varied histories of cigarette smoking were subjected to similar immunohistochemical examinations. In addition, 3 specimens from patients with chronic pancreatitis, 2 specimens of normal fetal pancreata, and 16 specimens of normal adult pancreata were examined as control tissues. Suitable frozen and archival microdissected tumor lesions were evaluated for mutations in exons 4-9 of the p53 gene by single strand conformation polymorphism (SSCP) and dideoxy sequencing analyses in which two distinct sets of outer and nested intron-based amplification primers were used for each exon. A subset of 25 tumor specimens and 18 tumor-derived cells lines for which the p53 mutation status was known were examined for amplification and/or overexpression of the mdm-2 gene; amplification was determined by Southern hybridization and overexpression by immunohistochemical and Western blot analyses. Similarly, mutations in the coding region of p21/WAF-1 gene were examined by SSCP and DNA sequence analyses, and steady-sate expression of the p21/WAF-1 protein was assessed by Western blot analysis in these subsets of tumors and tumor-derived cell lines. RESULTS. Positive ductal nuclear p53 immunostaining was demonstrated in 56%, of primary tumors and 54% of metastatic lesions. The frequency did not differ significantly between sporadic and familial lesions, and immunostaining was not observed in ductal, acinar, or islet cell elements of normal pancreata or histologically abnormal benign pancreatic lesions from cigarette smokers. A total of 70% of tumor samples revealed reproducible SSCP abnormalities of p53; 42% of these were found in exons 7 and 8. DNA sequence analysis of cases with greater than 35% epithelial celluliarity (n = 25) revealed 17 missense mutations, 12 of which were transitions. Seventy-five percent of these transitions were G:C→A:T type. A total of 22% of the p53 mutations identified were microdeletions, along with one insertional mutation at exon 8. None of the normal pancreata from sporadic or familial lesions revealed germ-line p53 alterations. Moreover, the frequency and spectra of p53 alterations exhibited no clear, statistically significant association with tumor grade, TNM stage, or patients' cigarette-smoking histories. The mdm-2 gene was neither amplified nor overexpressed immunochemically in a subset of ductal adenocarcinomas, and there was no clear relationship 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 primary 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 gene/protein and the p53-regulated genes mdm-2 and p21/WAF-1 in this series of ductal pancreatic lesions revealed that p53 mutations area frequent early event in pancreatic tumorigenesis not associated with metastatic progression. The authors observed complex spectra of transition missense mutations and microdeletions but no significant correlation with tumor grade, TNM stage, or smoking history. In contrast, amplification and overexpression of the mdm-2 gene/protein and genetic abnormalities in the p21/WAF-1 gene are infrequent events in the development of ductal pancreatic adenocarcinoma and were not clearly associated with the p53 mutation status of tumors examined in this study.