AR-E1 and AR-CE3 RISH assay digital image quantification shows minimal inter-observer variability. To examine inter-observer variability in scoring, a second, independent operator (Observer 2) circled tumor areas and performed the scoring for AR-E1 and AR-CE3 in Frida across a subset of the autopsy samples and compared with the original values created by Observer 1 for Supplementary Figure S3C. In one experiment, the second operator made their own thresholds for brown and blue intensity scoring and in another experiment, the second operator used the intensity thresholds set by the first operator. In both cases there was negligible variability between operators, with highly correlated values across all specimens.
ARTICLE ABSTRACTPurpose: 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 RISH is a prognostic or predictive biomarker in specific clinical contexts. Clin Cancer Res; 22(18); 4651–63. ©2016 AACR.