Abstract 4216: Novel RNA In situ hybridization method for the detection of ETV1, ETV4 and ETV5 rearrangements in prostate cancer.
Authors: Kunju Lakshmi P. , Carskadon Shannon , Bhalla Ritu , Siddiqui Javed , Tomlins Scott A. , Chinnaiyan Arul M. , Palanisamy Nallasivam 
Published: August 2014

Abstract
Prostate cancer (PCa) remains the most commonly diagnosed cancer in American men with an estimated incidence of 214,740 new cases and accounting for 28,170 deaths in 2012. The genetic basis of 50-60% of PCa is attributable to rearrangements in ETS genes (ERG, ETV1, ETV4, ETV5), BRAF, RAF1 and overexpression of SPINK1. The discovery and validation of reliable diagnostic methods are warranted to detect these molecular rearrangements. ETS gene rearrangements are typically detected by FISH and PCR methods. Recently, monoclonal antibodies against ERG have been developed which detect the truncated ERG protein and are strongly correlated with ERG rearrangement as detected by FISH. However, due to the lack of specific antibodies for ETV1, ETV4 and ETV5 genes, in situ detection of these markers is not feasible. We have developed a novel RNA in situ hybridization (RNA-ISH) based assay for in situ detection of ETV1, ETV4, and ETV5 in formalin fixed paraffin embedded (FFPE) tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using commercially available RNA-ISH probes. In order to assess the feasibility of RNA-ISH method for the reliable detection of ETS rearrangement, we compared RNA-ISH with ERG immunohistochemistry (IHC) using previously validated ERG positive cases. Of the 12 ERG IHC-positive cases, RNA-ISH was positive in all samples and no false positives were detected in the 70 ERG-negative cases, indicating a 100% concordance between IHC and RNA-ISH. Thus, we established that the RNA-ISH method is comparable to IHC for evaluation of ETS gene rearrangement in PCa We then tested ETV1 (NM_004956), ETV4 (NM_001079675), and ETV5 (NM_004454) probes on 10 known positive (ETV1-5; ETV4-4; ETV5-1) and 319 unknown cases (localized PCa N=265; Metastatic PCa N=54). All 10 known positive cases were confirmed positive by RNA-ISH. Of the 319 cases, 11 (3.4%) ETV1and 3 (1%) ETV4 positive cases were identified. No non-specific staining was observed. Positive cases with tissue available for evaluation were validated with break-apart FISH probes and found to be confirmatory in all cases. No non-specific staining was observed in rearrangement negative cases. In majority (20/24) of the positive cases the RNA-ISH staining pattern was homogeneous; however, 3 ETV1-positive cases and 1 ETV4-positive case showed heterogeneous staining pattern. ERG IHC was performed and we found that the regions negative for either ETV1 or ETV4 were positive for ERG. Thus we identified a subset of prostate cancer showing dual ETS rearrangement in two independent tumor foci. This is the first time dual ETS rearrangement in prostate cancer has been demonstrated in situ. We showed that RNA-ISH is a viable alternative method for in situ detection of ETS gene rearrangements in prostate cancer.
Citation Format: Lakshmi P. Kunju, Shannon Carskadon, Ritu Bhalla, Javed Siddiqui, Scott A. Tomlins, Arul M. Chinnaiyan, Nallasivam Palanisamy. Novel RNA In situ hybridization method for the detection of ETV1, ETV4 and ETV5 rearrangements in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2013-4216