Analytic Validation of RNA In Situ Hybridization (RISH) for AR and AR-V7 Expression in Human Prostate Cancer

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 immunohistochemistry 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 to 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.

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance

“…Among these, AR-V7 is the best-characterized due to frequent detection of the AR exon 3/CE3 splice junction by RNA-seq and RT-PCR (12, 13, 15–17), high expression of exon CE3 measured by RNA-ISH (20, 25, 43), and availability of AR-V7-specific antibodies to interrogate protein expression in tissues (19, 44, 45). In this study, we found that AR-V9 is frequently co-expressed with AR-V7 in CRPC cell lines, PDX tissue, circulating tumor cells, and biopsies of metastatic CRPC.…”

“…For example, signals from RNA-ISH assays with probes complementary to AR exon CE3 have been utilized to assess AR-V7 mRNA levels in prostate cancer tissues (20, 25, 43). Our data indicate these RNA-ISH signals would represent a composite of AR-V7 and/or AR-V9.…”

MSH2 Loss in Primary Prostate Cancer

“…An average of 67 mm 2 of tumor tissue area was selected for analysis (range: 16–152 mm 2 ). CD8+ or CD3+ cells within the selected tumor area were identified by Aperio software as previously described (42), and the ratio of CD8+ or CD3+ cells to the total tumor area analyzed was calculated for each case. PD-L1 staining was scored as positive if >1% of immune cells or tumor cells showed PD-L1 membranous positivity.…”

AR-V7 Protein Expression in Circulating Tumour Cells Is Not Predictive of Treatment Response in mCRPC

“…However, limitations and discrepancies of the studies about the predictive value of AR-V7 mRNA in CTCs due to AR-V7 expression in non-malignant cells (e.g., hematopoietic cells, peritumoural tissue, benign glands) have already been reported [28][29][30].…”