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.

ERG immunohistochemistry is not predictive for PSA recurrence, local recurrence or overall survival after radical prostatectomy for prostate cancer

“…10 The description of a novel ERG antibody in 2010 highly facilitates detection of ERG genomic fusions in clinical specimens. 9 Nuclear staining by the EPR3864 antibody showed excellent concordance with the presence of TMPRSS2-ERG fusion detected by FISH or QPCR. 9,10 Although crossreactivity of the EPR3864 antibody with ETS family member FLI-1 has been reported by the manufacturer, we demonstrated lack of FLI-1 expression in prostate cancer, indicating that the antibody actually detects ERG protein in this disease.…”

“…9 Nuclear staining by the EPR3864 antibody showed excellent concordance with the presence of TMPRSS2-ERG fusion detected by FISH or QPCR. 9,10 Although crossreactivity of the EPR3864 antibody with ETS family member FLI-1 has been reported by the manufacturer, we demonstrated lack of FLI-1 expression in prostate cancer, indicating that the antibody actually detects ERG protein in this disease. 10 In this study, we found concordance of nuclear ERG expression and TMPRSS2-ERG fusion in seven human prostate cancer cell lines and 22 xenografts, with exception of xenografts PC133, PC324 and PC339, which have breakpoint in intron 1 of TMPRSS2.…”

“…Nuclear reactivity of the antibody in endothelial cells (ERG) and in stromal cells (androgen receptor) was used as internal control for the staining procedure. 9 In case of staining heterogeneity, the highest level was used for statistical analysis. All cores were scored by two investigators (MH, GvL) in a blinded setting.…”

ERG protein expression and genomic rearrangement status in primary and metastatic prostate cancer—a comparative study of two monoclonal antibodies

“…By IHC using a rabbit ERG-MAb, Park et al and van Leenders et al have provided a comprehensive data showing that virtually all ERG rearranged PCa exhibit an ERG overexpression on the transcriptional and translational level. 20,21 He et al 22 confirmed the feasibility of the ERG IHC on an independent cohort containing a broader spectrum of prostatic lesions.…”

“…IHC was conducted with the Ventana Discovery XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA) using Ventana reagents. The following clones and primary antibodies were used: anti-ERG rabbit monoclonal antibody (1:100 dilution, clone EPR3864, Abcam, Cambridge, MA, USA) 21 and anti-ERG mouse monoclonal antibody (1:1000 dilution, CPDR ERG-MAb, clone 9FY, CPDR, Rochville, MD, USA). 19 Dilution was Figure 1.…”