title: Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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 RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651-63. ©2016 AACR.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Liana B
authors.initials: LB
authors.lastname: Guedes
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Carlos L
authors.initials: CL
authors.lastname: Morais
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Fawaz
authors.initials: F
authors.lastname: Almutairi
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Haffner
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Qizhi
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: John T
authors.initials: JT
authors.lastname: Isaacs
authors.affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Emmanuel S
authors.initials: ES
authors.lastname: Antonarakis
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Changxue
authors.initials: C
authors.lastname: Lu
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Harrison
authors.initials: H
authors.lastname: Tsai
authors.affiliation: Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Jun
authors.initials: J
authors.lastname: Luo
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
authors.forename: Angelo M
authors.initials: AM
authors.lastname: De Marzo
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. tlotan1@jhmi.edu.
authors.forename: Tamara L
authors.initials: TL
authors.lastname: Lotan
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor; D019295:Computational Biology / Q000379:methods; D004195:Disease Models, Animal; D015870:Gene Expression; D064593:Heterografts; D006801:Humans; D061705:Image-Guided Biopsy; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D051379:Mice; D060787:Neoplasm Grading; D009362:Neoplasm Metastasis; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D059368:RNA Isoforms; D011944:Receptors, Androgen / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D014408:Biomarkers, Tumor; D059368:RNA Isoforms; D011944:Receptors, Androgen