Background: We recently identified mechanisms underlying the clonal evolution of castration resistant prostate adenocarcinoma (CRPC-Adeno) to a neuroendocrine resistance phenotype (Beltran et al, Nature Med 2016). In the current study, we aimed to develop a non-invasive approach to identify patients that are developing neuroendocrine prostate cancer (NEPC).

Methods: We performed whole exome sequencing (WES) of matched circulating tumor DNA (ctDNA), germline DNA, and metastatic biopsies from patients with CRPC-Adeno and NEPC using minimum 50ng DNA, NimbleGen library prep, Illumina platform (ctDNA >300X). After applying ad hoc partial duplication filtering, we used FACETS segmentation followed by extended CLONET to calculate the fraction of tumor DNA in the circulation and assess the clonality of genomic lesions.

Results: 64 CRPC patients were prospectively enrolled. The spectrum of alterations captured by WES ctDNA was consistent with those commonly observed in CRPC validating the feasibility of the approach. The SCNA similarity between tumor tissue and ctDNA was higher in NEPC compared to CRPC-Adeno (p=0.0001) suggesting less heterogeneity in NEPC. There was enrichment of RB1 and TP53 loss in NEPC ctDNA and AR gains in CRPC-Adeno. The overall fraction of mutations shared by ctDNA and tumoral tissue was ~80%. We compared three different tumor biopsy time-points of patient PM161—CRPC-Adeno (lymph node), CRPC-Adeno (bone), and NEPC (liver). Unexpectedly the baseline ctDNA profile (at time of CRPC-Adeno) displayed genomic features most similar to the NEPC liver biopsy. These data suggest that NEPC alterations are detectable in the circulation, potentially prior to the development of NEPC clinical features. We compared the ctDNA of another patient PM0 with his 6 sites of NEPC metastases obtained 6 days later at autopsy; the relative contribution of tumor alterations in ctDNA was highest for the liver metastasis (similarity 0.59) compared with other sites suggesting differential contribution of sites of metastases in the circulation, with implications for the interpretation of single site clinical biopsies.

Conclusions: This is the first study to show that WES of ctDNA is feasible in CRPC and can help elucidate intra-patient heterogeneity and identify the spectrum and frequency of NEPC-specific genomic changes. We have incorporated our WES ctDNA discoveries into a targeted panel for larger scale validation using prospective cohorts. Our goal is to improve the earlier detection of patients transforming towards NEPC. Other data to be presented are targeted methylation of ctDNA and integrative molecular analysis of rapid autopsies (n=5) to better understand the role of intra-patient heterogeneity in NEPC.

Citation Format: Himisha Beltran, Alssandro Romanel, Nicola Casiraghi, Michael Sigouros, Matteo Benelli, Jenny Xiang, Francesca Demichelis. Tumor heterogeneity in castration resistant neuroendocrine prostate cancer from whole exome sequencing of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-122. doi:10.1158/1538-7445.AM2017-LB-122