A novel multiplex digital PCR assay for simultaneous quantification of androgen receptor amplification, mutations, and splice variants in cell-free nucleic acids from metastatic castration-resistant prostate cancer.

Somatic androgen receptor (AR) alterations including gene amplification, point mutations, and the AR-V7 splice variant occur in 50-70% of patients with metastatic castration-resistant prostate cancer (mCRPC) and drive resistance to AR-pathway inhibitors (ARPIs) in metastatic castration-resistant prostate cancer (mCRPC). However, current liquid biopsy approaches typically require separate cell-free DNA (cfDNA) and cfRNA workflows to evaluate these distinct alteration classes, increasing sample input requirements, assay complexity, and cost. Because plasma cell-free nucleic acid yield is often limited, an integrated assay capable of interrogating DNA and RNA-derived biomarkers from a single blood draw remain an unmet clinical need. Here we describe the development and proof-of-concept validation of a novel multiplex multimodal digital PCR (dPCR) assay that co-detects AR copy-number amplification, somatic point mutations (T878A), and the AR-V7 splice variant from plasma cell-free nucleic acids (cfNAs) in a single streamlined reaction. The assay employs RNase H-dependent PCR (rhPCR) primers with ultra-short amplicons (≤68 bp) that simultaneously interrogate both cfDNA and cfRNA, enabling multimodal detection without separate DNA and RNA workflows. Using prostate cancer cell lines with defined AR alteration status, the assay detected AR amplification at 2% of tumor DNA, quantified T878A mutations at a mutant allele frequency as low as 0.024%, and AR-V7 splicing variant with a sensitivity of 0.096% tumor cell fraction. In two mCRPC patient plasma samples, the assay tracked dynamic changes of these AR variants during ARPI therapy, revealing distinct patterns of clonal evolution that are consistent with treatment pressure and clinical outcomes. This multimodal dPCR platform provides a cost-effective, highly sensitive liquid biopsy tool for detection of clinically significant AR alterations and represents a foundation for a laboratory-developed test (LDT) to guide personalized therapy in mCRPC.