Immunohistochemical labeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas : a new marker of DPC4 inactivation

Abstract

DPC4 (MADH4, SMAD4) is a tumor suppressor gene inactivated by allelic loss in approximately 55% of pancreatic adenocarcinomas. Unfortunately, it can be technically very difficult to detect the inactivation of DPC4 at the genetic level because genetic analyses require the microdissection of relatively pure samples of neoplastic and normal tissues. This is especially true for pancreatic adenocarcinomas, which elicit vigorous, non-neoplastic, stromal responses. Immunohistochemical labeling can overcome this hurdle because it preserves morphological information. We therefore studied the expression of the DPC4 gene product in 46 cancers, including 5 cancer cell lines by Western blot analysis and 41 primary periampullary adenocarcinomas by immunohistochemistry. The status of exons 1-11 of the DPC4 gene in all 46 of the cancers had been previously characterized at the molecular level, allowing us to correlate Dpc4 expression directly with gene status. Three cell lines had wild-type DPC4 genes, and Dpc4 expression was detected in all three by Western blot. The two cell lines with homozygously deleted DPC4 genes did not show Dpc4 protein by Western blot analysis. Immunohistochemical labeling revealed that 17 (94%) of the 18 primary adenocarcinomas with wild-type DPC4 genes expressed the DPC4 gene product, whereas 21 (91%) of 23 primary adenocarcinomas with inactivated DPC4 genes did not. Cases in which there was discordance between the immunohistochemical labeling and the genetic analyses were reanalyzed genetically, and we identified a deletion in exon 0 of DPC4 in one of these cases. This is the first report of a mutation in exon 0 of DPC4 in a pancreatic cancer. The contrast between the strong expression of Dpc4 by normal tissues and the loss of expression in the carcinomas was highlighted in several cases in which an infiltrating cancer was identified growing into a benign duct. These observations suggest that immunohistochemical labeling for the DPC4 gene product is an extremely sensitive and specific marker for DPC4 gene alterations in pancreatic carcinomas. The sensitivity and specificity of immunohistochemical labeling for Dpc4 in other periampullary carcinomas has yet to be determined.

Figure 1.

Western immunoblot of five cancer cell lines. The three cancers expected to express Dpc4 (HCT116, RKO, and PANC1) contain 64-kd proteins recognizable by the anti-Dpc4 antibody. The two cell lines with homozygous deletions of DPC4 (BxPC3 and MB468) do not express Dpc4. Dpc4 is a 64-kd protein.

Figure 2.

Dpc4 expression in the normal pancreas. A: This normal pancreatic duct shows strong, diffuse cytoplasmic and occasional nuclear expression of Dpc4. Slightly weaker expression is seen in the surrounding stromal cells. B: The acini and islets of Langerhans (center) of the pancreas also express the Dpc4 protein. Note the regular nuclear labeling for Dpc4 in the islet.

Figure 3.

A: An infiltrating adenocarcinoma with a wild-type DPC4 gene expresses the Dpc4 protein. B: An infiltrating adenocarcinoma with a homozygously deleted DPC4 gene (bottom) does not label with the anti-Dpc4 antibody. Note the strong labeling in the adjacent, non-neoplastic pancreatic acini (top). C: “Cancerization” of a duct, as demonstrated by Dpc4 immunolabeling. The normal cells within the duct express Dpc4 (left), whereas the infiltrating cancer cells growing into the duct do not express Dpc4 (right). Note the sharp contrast in Dpc4 expression between normal and cancer. D: An infiltrating carcinoma with two morphological patterns. The better differentiated, gland-forming adenocarcinoma (D1) expresses the Dpc4 protein, but the more poorly differentiated component (D2) does not.