SMAD4 immunohistochemistry reflects genetic status in juvenile polyposis syndrome

Abstract

Purpose: Juvenile polyposis syndrome (JPS) can be caused by a germline defect of the SMAD4 gene. Somatic inactivation of SMAD4 occurs in pancreatic and colorectal cancers and is reflected by loss of SMAD4 immunohistochemistry. Here, SMAD4 immunohistochemistry as a marker of SMAD4 gene status and the role of SMAD4 in the adenoma-carcinoma sequence in neoplastic progression in JPS are studied.

Experimental design: Twenty polyps with a SMAD4 germline defect and 38 control polyps were studied by SMAD4 immunohistochemistry. Inactivation of the SMAD4 wild-type allele was studied in dysplastic epithelium and in areas with aberrant SMAD4 expression. APC, beta-catenin, p53, and K-ras were studied to evaluate the adenoma-carcinoma sequence.

Results: Nine of 20 polyps with a SMAD4 germline defect showed loss of epithelial SMAD4 expression. Loss of heterozygosity of SMAD4 was found in five polyps and a somatic stop codon mutation was found in two polyps without loss of heterozygosity. Remarkably, somatic inactivation of epithelial SMAD4 did not always coincide with dysplasia and aberrant p53 staining was found in four of six dysplastic polyps with normal SMAD4 staining. One K-ras mutation was found in nine juvenile polyps with dysplasia. No evidence for Wnt activation was found.

Conclusions: SMAD4 immunohistochemistry mirrors genetic status and provides a specific adjunct in the molecular diagnosis of JPS. However, epithelial SMAD4 inactivation is not required for polyp formation and is not obligatory for neoplastic progression in JPS. Instead, different routes to neoplasia in JPS caused by germline SMAD4 mutation seem to be operative, including somatic loss of SMAD4 and p53 inactivation without somatic loss of SMAD4.

Figure 1

SMAD4 Immunohistochemical scoring. SMAD4 IHC was scored as normal (A), reduced (B) or loss of SMAD4 expression (C). Nuclear staining in the epithelial cells lining normal crypts or inflammatory cells in the mesenchymal stroma on the same section served as internal control. Note loss of SMAD4 expression in non-neoplastic epithelium in C. Magnification 20X.

Figure 2

SMAD4 IHC and dysplasia. A: Dysplasia with normal epithelial SMAD4 expression. B: Dysplasia within area of reduced SMAD4 expression. C: Dysplasia extending beyond area of SMAD4 loss. E: Dysplasia with normal SMAD4 expression and non-neoplastic epithelium with loss of SMAD4 expression adjacent on one section. Magnification left panel 10X (figure 2E 5X) with 20X zoom (right panel).