Phillip Miller

Abstract 2722: Development of automated methods of in-situ hybridization for the detection of ETS gene re-arrangements in FFPE biopsy and archived prostatectomy specimens

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Abstract Prostate cancer (PCa) is the most common visceral neoplasm in men. In most cases, the disease is slowly progressive. Five year survival with organ-confined disease is 100%, while even distal spread is 32%. ETS gene re-arrangements in PCa have been shown to be associated with disease progression and have therapeutic and diagnostic implications. The goal of this study was to develop methods of automated in-situ hybridization to detect the ETS gene re-arrangements in formalin fixed, paraffin-embedded (FFPE) prostate tissue. Methods were developed on the Ventana Benchmark®XT and Discovery®XT instruments for prostate cancer cell line xenografts, including VCap, LnCaP and H660, as well as for primary biopsy and archived prostatectomy specimens. Key modules in the protocol have been evaluated and modified both in manual (offline) and automated (online) process of method development. Specific protocols were developed for deparaffinization, antigen retrieval, DNA denaturation, probe hybridization, sample washing, and detection with Quantum Dots (Qdots). Six different FISH probes specific for 3′ ERG, 5′ ERG, 5′ SLC45A3, and 3′ETV1 were used in assay development. Probe hybridization time was held to 8 hours, with overnight completion of the fully automated protocol. Assay conditions on both instruments were similar for xenografts and in archived prostectomy specimens; however increased stringency of target retrieval was required for processing of the biopsy specimens. This adjustment was necessary to compensate for the tendency of biopsy specimens to become overly fixed during procurement and routine shipping and handling. In addition we observed that the presence of commonly used detergents in washing buffers post-hybridization may increase background staining with QDots. In conclusion, we have developed fully automated protocols for the processing of clinically relevant tissue specimens. The xenograft cell line controls developed as a part of this study may also enable evaluation of assay performance in routine specimen processing for the detection of the ETS genes in biopsies and in prostatectomies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2722.