Allelic loss is often the first hit in the biallelic inactivation of the p53 and DPC4 genes during pancreatic carcinogenesis

Abstract

The presumed precursor lesions of pancreatic ductal adenocarcinoma were recently classified according to their increasing grade of dysplasia and were designated as pancreatic intraepithelial neoplasia (PanIN) 1 through 3. In this study, we tested whether molecular genetic alterations can be correlated with this classification and may help to further categorize the various PanIN grades. We determined the frequencies of allelic loss at chromosomal arms 9p, 17p, and 18q in 81 microdissected duct lesions of various PanIN grades, using a combination of whole genome amplification and microsatellite analysis. In addition we examined the p53 and Dpc4 protein expression patterns by immunohistochemical analysis. In PanIN-1, we did not detect allelic losses. In PanIN-2, allelic losses were found in increasing frequency, and were particularly high in those lesions with moderate-grade dysplasia (low grade, 20, 33, and 17%, loss at 9p, 17p, and 18q, respectively; moderate grade, 46, 77, and 58%). PanIN-3 and invasive carcinomas exhibited abundant losses. Abnormal p53 and Dpc4 protein expression was only rarely identified in PanIN-2 lesions, but occurred frequently in PanIN-3 lesions and invasive carcinomas. The combined genetic and protein expression data support a model in which allelic loss is the first hit in the biallelic inactivation of the p53 and DPC4 tumor suppressor genes. In addition, our data indicate that allelic loss analysis may be useful in separating PanIN-2 lesions with low-grade dysplasia from those PanIN-2 lesions with moderate-grade dysplasia, each potentially representing a distinct progression step toward invasive carcinoma.

Figure 1.

Example of microsatellite analysis of DOP-PCR templates derived from various PanINs. Amplification products were separated on a standard sequencing gel and visualized by silver staining. A: Microsatellite marker D9S304. B: Marker D17S786. C: Marker D18S363. Arrows, samples with allelic loss; brackets, pairs of microsatellite PCRs generated from the same DOP-PCR template; asterisks, examples of discordant PCR amplification patterns. N, normal DNA; C, negative control.

Figure 2.

Microsatellite analyses of various grades of PanIN. A: Microdissected lesions were derived from specimens from 21 pancreatic carcinoma patients and from one patient with noncancerous pancreatic disease (case 13). Allelic loss at chromosomal regions 9p, 17p, and 18q, was determined with 13 microsatellite markers. For each microsatellite marker, a data set (squares) of four independent PCRs was generated per lesion. Denotation of the color-coded squares: yellow, heterozygosity; red, allelic loss of the upper allele; green, allelic loss of the lower allele; white, noninformative; gray, excluded data set, black, not done. B: Detailed microsatellite analysis data on two representative cases (cases 19 and 20). Data sets (see A) are represented in data pairs (in uppercase and lowercase) from two microsatellite PCRs that were performed on each of two DOP-PCR templates. 2/L, PanIN-2 lesion with low-grade dysplasia; 2/m, PanIN-2 lesion with moderate-grade dysplasia; U/u, allelic loss of the upper allele (PCR1/PCR2); L/L, allelic loss of the lower allele (PCR1/PCR2); H/h, heterozygosity (PCR1/PCR2); Nd, not done; Ni, noninformative; (−), failed PCR reaction.

Figure 3.

Molecular analyses of various grades of PanIN. A: Frequencies of allelic loss at chromosomal regions 9p, 17p, and 18q together with frequencies of nuclear p53 expression and lack of Dpc4 protein expression. B: Accumulation of genetic alterations. The number of alterations refers to the number of allelic losses at three chromosomal regions (9p, 17p, and 18q) in those lesions that were informative for at least one marker at each of the three loci. PanIN-2/L, PanIN-2 lesion with low-grade dysplasia; PanIN-2/m, PanIN-2 lesion with moderate-grade dysplasia.

Figure 4.

Examples of various grades of duct lesions associated with pancreatic carcinoma. A: Papillary duct lesion, grade PanIN-2 with low-grade dysplasia: slight nuclear enlargement and nuclear crowding. H&E; original magnification, ×250. B: Papillary duct lesion, grade PanIN-2 with moderate-grade dysplasia: moderate nuclear enlargement, nuclear hyperchromasia and loss of polarity. H&E; original magnification, ×250. C: Papillary duct lesion, grade PanIN-3: loss of polarity and structure and increasing branching of the papillae. H&E; original magnification, ×250. D–F: Immunohistochemical staining for Dpc4. D: Duct lesion, mainly PanIN-2 with low-grade dysplasia and partially PanIN-1A, both with positive staining for Dpc4 protein. Original magnification, ×125. E: Duct lesion, grade PanIN-2 with moderate-type dysplasia with nuclear Dpc4 staining, and a few negative nuclei in the adjacent intraductal carcinoma. Original magnification, ×125. F: Duct lesion, mainly grade PanIN-3 without Dpc4 protein expression, but retained Dpc4 expression in adjacent hyperplastic epithelium. Original magnification, ×250.