Mutation and expression analyses reveal differential subcellular compartmentalization of PTEN in endocrine pancreatic tumors compared to normal islet cells

Abstract

The pathogenesis of sporadic endocrine pancreatic tumors (EPTs) is still primarily unknown. Comparative genomic hybridization studies revealed loss of 10q in a significant number (nine of 31) of EPTs. The tumor suppressor gene PTEN lies on 10q23, and so, is a candidate to play some role in EPT pathogenesis. Germline PTEN mutations are found in Cowden and Bannayan-Riley-Ruvalcaba syndromes, whereas somatic mutations and deletions are found in a variety of sporadic cancers. The mutation and expression status of PTEN in EPTs has not yet been examined. Mutation analysis of the entire coding region of PTEN including splice sites was performed in 33 tumors, revealing one tumor with somatic L182F (exon 6). Loss of heterozygosity of the 10q23 region was detected in eight of 15 informative malignant (53%) and in none of seven benign EPTs. PTEN expression was assessed in 24 available EPTs by immunohistochemistry using a monoclonal anti-PTEN antibody. Of these 24, 23 tumors showed strong immunoreactivity for PTEN. Only the EPTs with PTEN mutation lacked PTEN protein expression. Although normal islet cells always exhibited predominantly nuclear PTEN immunostaining, 19 of 23 EPTs had a predominantly cytoplasmic PTEN expression pattern. Exocrine pancreatic tissue was PTEN-negative throughout. PTEN mutation is a rare event in malignant EPTs and PTEN protein is expressed in most (23 of 24) EPTs. Thus, intragenic mutation or another means of physical loss of PTEN is rarely involved in the pathogenesis of EPTs. Instead, either an impaired transport system of PTEN to the nucleus or some other means of differential compartmentalization could account for impaired PTEN function. Loss of heterozygosity of the 10q23 region is a frequent event in malignant EPTs and might suggest several hypotheses: a different tumor suppressor gene in the vicinity of PTEN might be principally involved in EPT formation; alternatively, 10q loss, including PTEN, seems to be associated with malignant transformation, but the first step toward neoplasia might involve altered subcellular localization of PTEN.

Figure 1.

LOH analysis of EPT-nontumor DNA from cases 28 and 31 using microsatellite markers within and flanking PTEN. ▪, LOH; □, ROH; ni, not informative.

Figure 2.

Case 23 EPT with somatic PTEN mutation, 546A>T and loss of wild-type allele, resulting in negative PTEN immunoreactivity. Top: Negative PTEN immunostaining; note internal positive control of strongly staining neo-vessels. Single-strand conformation polymorphism and sequencing of exon 6 reveals a somatic mutation (546A>T). Note that the chromatogram shows the mutation as an apparent heterozygote although by both immunohistochemical and LOH analysis, this tumor has loss one allele and is mutant in the remaining. This pseudo-heterozygote appearance is because of some admixture with DNA from contaminating normal cells.

Figure 3.

Immunohistochemistry with anti-PTEN antibody. Original magnification, ×100. a: Normal islet (case 11). b: Cytoplasmic staining of tumor cells (case 11). c: Cytoplasmic staining of tumor cells with membrane pattern (case 25). d: Nuclear and cytoplasmic staining (case 24).