Extracellular vesicles (EVs) secreted by tumors are abundant in plasma, but their potential for interrogating the molecular features of tumors through multi-omic profiling remains widely unexplored. Genomic and transcriptomic profiling of circulating EV-DNA and EV-RNA isolated from a range of in-vitro and in-vivo models of metastatic prostate cancer (mPC) revealed a high contribution of tumor material to EV-loaded DNA/RNA. Findings were validated in a cohort of longitudinal plasma samples collected from mPC patients during androgen receptor signaling inhibitor (ARSI) therapy. EV-DNA genomic features recapitulated matched-patient biopsies and associated with clinical progression. We developed a novel approach to enable the transcriptomic profiling of EV-RNA (RExCuE). We report how the transcriptomic profile in mPC EV-RNA is enriched for tumor-associated transcripts when compared to same patient blood RNA and healthy individuals EV-RNA, and reflect early on-therapy tumor adaptation changes. Altogether, we show that EV profiling enables longitudinal transcriptomic and genomic profiling of mPC in liquid biopsy.

Background: Liquid biopsies enable minimally-invasive molecular characterization of tumors, allowing the study of tumor evolution, and are valuable in supporting clinical decisions in metastatic prostate cancer. Extracellular vesicles (EVs) secreted by tumor cells are abundant in the plasma of cancer patients, but their potential as a source of tumor material for multi-omic profiling remains widely unexplored. Design: We present a comprehensive analysis of plasma-derived tumor EV-DNA and EV-RNA by next-generation sequencing (NGS), in metastatic castration resistant prostate cancer (mCRPC). First, we used a range of in vitro (PC3, LnCap, 22Rv1 and C4-2) and in vivo models, including patient-derived tumor xenografts (PDXs), to optimize genomic and transcriptomic analysis of DNA and RNA contained in tumoral EVs. Next, we validated our findings in a cohort of 35 mCRPC patients with longitudinal plasma samples (n=92) collected before, 4-weeks after treatment and at progression to androgen receptor signaling inhibitor (ARSI) therapy. Tumor copy number alterations (CNAs) obtained by low-pass whole genome sequencing (lpWGS) were used to infer tumor fraction (TF). In addition, transcriptomic signatures were obtained from EV-RNA sequencing. Results: We demonstrate that circulating EV-DNA copy-number (CN) profiling matches tumor CN profiles in same-patient mCRPC biopsies (r: 0.47-0.85, p<0.001) and ctDNA, confirming its tumoral origin. EV-DNA tumor fraction (TF) was prognostic, identifying patients with shorter time to progression (HR 2.29, p=0.04). In addition, we developed a novel approach for the analysis of mRNA Expression in Circulating EVs (RExCuE), showing high correlation between EV-RNA and tumor biopsy RNA in vivo and in patient samples for the whole transcriptome, tumor-associated transcripts and the Hallmark androgen response gene set (all r>0.70, p<0.001). EV-RNAseq was able to detect and monitor transcriptomic signatures of response and resistance to ARSI in clinical samples as early as after 4 weeks of therapy. Last, we derived a specific signature of ARSI response in EV-RNA in vivo that predicts patient response to therapy. Conclusion: Our work represents the first evidence for circulating EVs as a biomarker to monitor evolution of tumor genomics and transcriptomics in mCRPC on therapy, allowing the study of drug response and resistance mechanisms and with a unique potential to be developed as clinical biomarker. Citation Format: Irene Casanova-Salas, Sarai Córdoba-Terreros, Daniel Aguilar, Laura Agúndez, Julián Brandariz, Nicolás Herranz, Alexandre Sierra, Pablo Cresta, Maria del Mar Suanes, Mario Soriano, Elena Castellano, Javier Hernández-Losa, Héctor Peinado, Joan Carles, Joaquin Mateo. Circulating tumor extracellular vesicles to monitor metastatic prostate cancer genomic and transcriptomic evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2304.