Immunohistochemical Labeling for Dpc4 Mirrors Genetic Status in Pancreatic Adenocarcinomas

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.

Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis

“…Of particularly interest, 47% of PDC showed negative staining for the protein. DPC4 immunohistochemistry has been recently shown to correlate with inactivation of the gene in more than 90% of cases (Wilentz et al, 2000). This would provide additional evidence that homozygous deletion is a major mechanism of DPC4 inactivation in ductal cancers.…”

“…The inactivation of the DPC4 gene by additional mechanisms such as homozygous deletion was studied using immunohistochemistry, which has been shown to correlate with inactivation of the gene (Wilentz et al, 2000). All tumours were also presently or previously analysed for K-ras and p53 mutations and allelic loss at chromosomal arms 9p, 17p and 18q at sites linked to p16, p53 and DPC4 genes.…”

Chromosome 18 deletions are common events in classical midgut carcinoid tumors

“…22,23 Although the deletion pattern suggested a tumor suppressor telomeric of the gene cluster at 18q21, we chose to analyze the Smad4/DPC4 gene in more detail since exonspecific PCR primers were available and the described anti-serum for immunohistochemical labeling of Smad4/DPC4 has proven specificity and sensitivity for homozygous gene inactivation of 94% and 91%, respectively. 25 For Smad2/MADR2/JV18-1 and DCC, the correlation of immunohistochemical labeling and gene inactivation is unknown. In our series of classical midgut carcinoids, we did not identify mutations in exons 8 -11 of the Smad4/DPC4 gene, including the intron-exon boundaries.…”

Allelic Analysis of Serous Ovarian Carcinoma Reveals Two Putative Tumor Suppressor Loci at 18q22-q23 Distal to SMAD4, SMAD2, and DCC

“…19 If one allele of these genes is inactivated by mutation and the other allele by allelic loss, one would expect to see decreased or lost protein expression. We found a tendency toward a higher amount of lost expression of SMAD4, SMAD2, and DCC in tumors showing LOH compared with tumors showing no LOH at 18q21.1.…”