Novel dual-color immunohistochemical methods for detecting ERG-PTEN and ERG-SPINK1 status in prostate carcinoma

Abstract

Identification of new molecular markers has led to the molecular classification of prostate cancer based on driving genetic lesions. The translation of these discoveries for clinical use necessitates the development of simple, reliable and rapid detection systems to screen patients for specific molecular aberrations. We developed two dual-color immunohistochemistry-based assays for the simultaneous assessment of ERG-PTEN and ERG-SPINK1 in prostate cancer. A total of 232 cases from 184 localized and 48 metastatic prostate cancers were evaluated for ERG-PTEN and 284 cases from 228 localized and 56 metastatic prostate cancers were evaluated for ERG-SPINK1. Of the 232 cases evaluated for ERG-PTEN, 81 (35%) ERG-positive and 77 (33%) PTEN-deleted cases were identified. Of the 81 ERG-positive cases, PTEN loss was confirmed in 35 (15%) cases by fluorescence in situ hybridization (FISH). PTEN status was concordant in 203 cases (sensitivity 90% and specificity 87%; P<0.0001) by both immunohistochemisty and FISH; however, immunohistochemisty could not distinguish between heterozygous and homozygous deletion status of PTEN. Of the 284 cases evaluated for ERG-SPINK1, 111 (39%) cases were positive for ERG. In the remaining 173 ERG-negative cases, SPINK1 was positive in 26 (9%) cases. SPINK1 expression was found to be mutually exclusive with ERG expression; however, we identified two cases, of which one showed concomitant expression of ERG and SPINK1 in the same tumor foci, and in the second case ERG and SPINK1 were seen in two independent foci of the same tumor nodule. Unlike the homogenous ERG staining in cancer tissues, heterogeneous SPINK1 staining was observed in the majority of the cases. Further studies are required to understand the molecular heterogeneity of cases with concomitant ERG-SPINK1 expression. Automated dual ERG-PTEN and ERG-SPINK1 immunohistochemisty assays are simple, reliable and portable across study sites for the simultaneous assessment of these proteins in prostate cancer.

Figure 1. Immunohistochemistry and FISH evaluation of ERG and PTEN in prostate cancer tissues

A. ERG-PTEN dual immunohistochemisty showing negative ERG and strong PTEN expression (blue) in a prostate cancer sample. Note the positive internal control for ERG antibody staining in the surrounding lymphocytes and endothelial cells (40X). B. Corresponding FISH image showing 2 copies of normal PTEN signal (red) and 2 copies of chromosome 10 control probe (green). C. Positive expression of ERG and negative expression of PTEN antibodies in a prostate cancer sample (40X). D. FISH analysis showing PTEN deletion (loss of red signal) in cancer tissue and adjacent benign prostatic acini showing normal PTEN signal (red) and 2 copies of chromosome 10 control probe (green). E. Prostate cancer expressing ERG antibody in the nucleus and PTEN in the cytoplasm (40X) and corresponding FISH image showing normal signal pattern for PTEN and chromosome 10 control probe (F). G. Negative ERG and PTEN expression in a prostate cancer sample (40X). H. FISH demonstrating homozygous loss of PTEN (loss of red signal). I. Image showing negative expression of ERG and PTEN (40X), however FISH confirming heterozygous PTEN deletion (J).

Figure 2. Evaluation of ERG and SPINK1 by dual immunohistochemistry in prostate cancer

Prostate cancer tissues with positive expression of ERG and no expression of SPINK1 (40X) (A&B). ERG and SPINK1 dual immunohistochemisty in a prostate cancer tissue demonstrating diffuse SPINK1 (blue) (C&D) expression and no expression of ERG (brown) (immunohistochemisty, 40X). Heterogeneous pattern of SPINK1 expression in minute cancer foci (immunohistochemisty, 40X). Note ERG staining of endothelial cells, used as positive internal control (E&F).

Figure 3. Concomitant expression of ERG and SPINK1 in prostate cancer

Hematoxylin and Eosin staining of a prostate cancer tissue (A) with expression of both ERG (red) and SPINK1 (brown) in two different adjacent foci (B). Image from the resection of the same case shown in B demonstrating ERG and SPINK1 positivity in adjacent foci with punch holes (X) for tissue microarray biopsies (C). Concomitant expression of ERG (brown) and SPINK1 (red) in the same tumor foci (D&E).