Abstract 2218: Integrated diagnostic methods for detection of multiple gene rearrangements in prostate cancer tissue specimens
Authors: Cortez Connie C. , Svensson Maria , Nagle Raymond , Garsha Karl , Nagy Dea , Riley Janice , Sathyanarayana Ubaradka , Smith Kassie , Pozarowski Krystyn , Christopherson Kelly , Yun Steve , Otter Michael , Dittamore Ryan , Palaniswamy Nallasivam , Demichelis Francesca , Rubin Mark , Grogan Tom , Miller Phil , Pestano Gary 
Published: June 2012

Abstract
The identification of patients who will benefit from therapy is one of the more difficult questions in prostate cancer disease management. Approximately 60% of men with diagnosed prostate cancer will have an ETS rearranged tumor foci. The prostate cancer gene fusions identified thus far are characterized by the fusion of 5’ genomic regulatory elements (commonly androgen regulated), such as TMPRSS2, with the ETS family of transcription factors which include ERG, ETV1, ETV4, and ETV5. About 40-50% of prostate cancers have the TMPRSS2/ERG gene fusion which leads to the over expression of oncogenic transcription factors.
The goal of this study was to develop a novel, integrated diagnostic testing method that accounts for tissue heterogeneity including multiple gene re-arrangements in single transformed nuclei. We selected a method that sequentially reflexes from an initial H&E to ERG immunohistochemistry (IHC), and finally to a quantum dot (Life Technologies) based FISH probes for the detection of multiple gene rearrangements in prostate cancer. To this end, probes specific to the ETS gene rearrangements including, 3’ and 5’ ERG, TMPRSS2, NDRG1, ETV1, and ETV4, were detected with up to four different quantum dot bioconjugates simultaneously in single nuclei.
We have shown sensitive and specific detection of gene rearrangements with this testing method in the xenograft models, VCaP, H660, and LNCaP, as well as in samples from prostate needle biopsies and radical prostatectomies. In 6 of 88 cases, the ERG IHC was diagnostic of PIN (prostatic intraepithelial neoplasia) that was missed on initial examination of the H&E stain. Further, the four color quantum dot FISH assay inclusive of ERG gene rearrangements was confirmatory of ERG IHC reactivity. In addition, we also clearly demonstrated instances of multiple gene rearrangements in the 5’ and 3’ ends on TMPRSS2 and NDRG1 in the same cancer nuclei, but not in benign nuclei from the same tissue. The signal patterns associated with various genomic events assessed were: no rearrangement (normal), translocation through insertion, and translocation through deletion.
In summary, we propose that the ERG antibody is likely to be a key component in a diagnostic PIN IHC cocktail, that follows the initial H&E, and may be reflexed to a multiplexed quantum dot FISH assay that incorporates the detection of prevalent gene rearrangements. This method may be useful as an aid in detecting clinically relevant diagnostic markers, since multiple gene rearrangements in the same cell may be identified early in prostate cancer progression. Studies are in progress to evaluate this testing strategy for prognostic value in assessing prostate cancer progression in selected prostate cancer and biopsy cohorts. At time of submission, assay is not approved for use in the US.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2218. doi:10.1158/1538-7445.AM2011-2218