Antibody-based detection of ERG rearrangement-positive prostate cancer

Abstract

TMPRSS2-ERG gene fusions occur in 50% of prostate cancers and result in the overexpression of a chimeric fusion transcript that encodes a truncated ERG product. Previous attempts to detect truncated ERG products have been hindered by a lack of specific antibodies. Here, we characterize a rabbit anti-ERG monoclonal antibody (clone EPR 3864; Epitomics, Burlingame, CA) using immunoblot analysis on prostate cancer cell lines, synthetic TMPRSS2-ERG constructs, chromatin immunoprecipitation, and immunofluorescence. We correlated ERG protein expression with the presence of ERG gene rearrangements in prostate cancer tissues using a combined immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis. We independently evaluated two patient cohorts and observed ERG expression confined to prostate cancer cells and high-grade prostatic intraepithelial neoplasia associated with ERG-positive cancer, as well as vessels and lymphocytes (where ERG has a known biologic role). Image analysis of 131 cases demonstrated nearly 100% sensitivity for detecting ERG rearrangement prostate cancer, with only 2 (1.5%) of 131 cases demonstrating strong ERG protein expression without any known ERG gene fusion. The combined pathology evaluation of 207 patient tumors for ERG protein expression had 95.7% sensitivity and 96.5% specificity for determining ERG rearrangement prostate cancer. In conclusion, this study qualifies a specific anti-ERG antibody and demonstrates exquisite association between ERG gene rearrangement and truncated ERG protein product expression. Given the ease of performing IHC versus FISH, ERG protein expression may be useful for molecularly subtyping prostate cancer based on ERG rearrangement status and suggests clinical utility in prostate needle biopsy evaluation.

Figure 1

Characterization of a monoclonal anti-ERG antibody for detecting TMPRSS2-ERG gene fusion expression products. (A) Immunoblot detection of endogenous TMPRSS2-ERG gene fusion product (predicted molecular weight, 49.0 kDa) using anti-ERG monoclonal antibody. Cell lysates from benign (PrEC and RWPE) and cancerous (LNCaP, VCaP, and DU145) prostatic epithelial cell lines were immunoblotted for ERG expression using the rabbit monoclonal anti-ERG antibody. VCaP harbors a TMPRSS2-ERG gene fusion resulting in marked overexpression of ERG, LNCaP harbors a rearrangement of the entire ETV1 locus, resulting in marked overexpression of ETV1, and no ETS gene rearrangements have been identified in DU145. β-Actin was used as a loading control. (B) Schematic diagram showing different ERG deletion constructs used to identify the anti-ERG epitope. Constructs detected by the antibody are shown in red. (C) Western blot analysis of HEK293 cells transiently mock-transfected (showing endogenous ERG expression) or transfected with the constructs in panel B, and blotted with anti-ERG or anti-FLAG as loading control. (D) ChIP-quantitative PCR assays from stable RWPE-ERG and RWPE-GUS (control) cells, for ERG target genes PLAU, MMP3, and the TMPRSS2 enhancer, and the negative control gene KIAA0066. Means±SE are shown. Experiments were run in triplicate. (E) Immunofluorescence with anti-ERG on a formalin-fixed paraffin-embedded prostate tissue section with a TMPRSS2:ERG-positive tumor demonstrates strong nuclear staining in neoplastic cells. Endothelial cells in the stromal compartment also demonstrate expression of ERG.

Figure 2

ERG rearrangement by break-apart FISH is highly correlated with ERG protein expression by IHC. (A) ERG protein expression in tumors with and without the TMPRSS2-ERG gene fusion. The endothelial cells of small vessels show positive endogenous ERG expression in the context of surrounding ERG-negative cancer glands (left 20x, right 40x). (B) The box plot demonstrates a highly significant association between the automated image evaluation of ERG protein expression and the ERG gene rearrangement status for 128 cases (P < .0001, Wilcoxon rank-sum test). Of 70 ERG rearrangement-negative cases, 2 demonstrate ERG protein expression. (C) Using a threshold of 0.4 as the cut point for determining ERG status, we observed excellent ROC curve performance with an AUC of 0.98 using ERG rearrangement by FISH as the criterion standard. (D) A summary of the performance of ERG protein expression to predict ERG rearrangement status is presented for different thresholds.

Figure 3

ERG-rearranged cases express high levels of truncated ERG protein regardless of 5′ fusion partner. Representative examples of prostate cancers harboring ERG rearrangements with different 5′ partners but showing similar ERG protein expression by IHC.

Figure 4

ERG protein expression in high-grade prostatic intraepithelial neoplasia (HG PIN) adjacent to ERG-expressing cancer. (A) H&E stain demonstrates small prostatic cancerous glands on the left and a larger atypical gland on the right with a subset of epithelial cells with neoplastic features consistent with HG PIN. (B) ERG protein expression by IHC demonstrates strong expression in both cancer and HG PIN. The arrowheads indicate a discrete demarcation between HG PIN and histologically benign luminal epithelial cells (40x).

Figure 5

ERG protein expression in prostate cancer biopsy samples. A prostate needle biopsy without ERG protein expression (A) and one with intense ERG protein expression (B) demonstrates the potential of determining the ERG rearrangement status using a prostate needle biopsy.