Analytic validation of RNA in situ hybridization (RISH) for AR and AR-V7 expression in human prostate cancer

Liana B. Guedes, Carlos L. Morais, Fawaz Almutairi, Michael C. Haffner, Qizhi Zheng, John Tod Isaacs, Emmanuel Antonarakis, Changxue Lu, Harrison Tsai, Jun Luo, Angelo Michael Demarzo, Tamara Lotan

Research output: Contribution to journalArticle

Abstract

Purpose: RNA expression of androgen receptor splice variants may be a biomarker of resistance to novel androgen deprivation therapies in castrate-resistant prostate cancer (CRPC). We analytically validated an RNA in situ hybridization (RISH) assay for total AR and AR-V7 for use in formalin-fixed paraffin-embedded (FFPE) prostate tumors. Experimental Design: We used prostate cell lines and xenografts to validate chromogenic RISH to detect RNA containing AR exon 1 (AR-E1, surrogate for total AR RNA species) and cryptic exon 3 (AR-CE3, surrogate for AR-V7 expression). RISH signals were quantified in FFPE primary tumors and CRPC specimens, comparing to known AR and AR-V7 status by IHC and RT-PCR. Results: The quantified RISH results correlated significantly with total AR and AR-V7 levels by RT-PCR in cell lines, xenografts, and autopsy metastases. Both AR-E1 and AR-CE3 RISH signals were localized in nuclear punctae in addition to the expected cytoplasmic speckles. Compared with admixed benign glands, AR-E1 expression was significantly higher in primary tumor cells with a median fold increase of 3.0 and 1.4 in two independent cohorts (P < 0.0001 and P = 0.04, respectively). While AR-CE3 expression was detectable in primary prostatic tumors, levels were substantially higher in a subset of CRPC metastases and cell lines, and were correlated with AR-E1 expression. Conclusions: RISH for AR-E1 and AR-CE3 is an analytically valid method to examine total AR and AR-V7 RNA levels in FFPE tissues. Future clinical validation studies are required to determine whether AR RISHis a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651-63.

Original languageEnglish (US)
Pages (from-to)4651-4663
Number of pages13
JournalClinical Cancer Research
Volume22
Issue number18
DOIs
StatePublished - Sep 15 2016

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ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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