Abstract
Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. However, the absence of a uniform CTC isolation platform and consensus on an analytical assay has prevented the incorporation of these measurements in routine clinical practice. Here, we present a single-CTC sensitive digital droplet PCR (ddPCR) assay for the quantitation of the two most common AR-Vs, AR-V7, and AR-v567es, using antigen agnostic CTC enrichment. In a cohort of 29 mCRPC patients, we identify AR-V7 in 66% and AR-v567es in 52% of patients. These results are corroborated using another gene expression platform (NanoStringTM) and by analysis of RNA-Seq data from patients with mCRPC (SU2C- PCF Dream Team). We next quantify AR-V expression in matching EpCAM-positive vs EpCAM-negative CTCs, as EpCAM-based CTC enrichment is commonly used. We identify lower AR-V prevalence in the EpCAM-positive fraction, suggesting that EpCAM-based CTC enrichment likely underestimates AR-V prevalence. Lastly, using single CTC analysis we identify enrichment for AR-v567es in patients with neuroendocrine prostate cancer (NEPC) indicating that AR-v567es may be involved in lineage plasticity, which warrants further mechanistic interrogation.
Highlights
Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer has great potential for treatment customization
Two of the most investigated AR splice variants are AR-V7 and AR-v567es, which are transcriptionally active in the absence of androgens
The AR-V digital droplet PCR (ddPCR) assay analytical specificity is demonstrated in experiments where cells AR-V negative are transduced with low levels of plasmids encoding each AR transcript (AR-FL, AR-V7, and AR-v567es)
Summary
Quantitation of androgen receptor variant (AR-V) expression in circulating tumor cells (CTCs) from patients with metastatic castration-resistant prostate cancer (mCRPC) has great potential for treatment customization. Our recent results from a prospective multiinstitutional clinical trial of patients with mCRPC receiving taxane chemotherapy, showed that AR-V7 and AR-v567es expression in patient CTCs, was associated with lower biochemical response rates and shorter progression free survial (PFS)[9] implicating both variants in taxane resistance. As both variants are co-expressed in patient samples where bulk tumor or the entire CTC fraction is used, the relative impact of each variant alone on disease progression and treatment response has not been yet determined. As EpCAM can be downregulated during epithelial-to-mesenchymal transition (EMT), a biological process that precedes metastatic dissemination, EpCAM-based CTC enrichment may not capture the heterogeneous pool of CTCs13
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