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Metastatic castration-resistant prostate cancer (mCRPC) has a highly complex genomic landscape. With the recent development of novel treatments, accurate stratification strategies are needed. Here we present the whole-genome sequencing (WGS) analysis of fresh-frozen metastatic biopsies from 197 mCRPC patients. Using unsupervised clustering based on genomic features, we define eight distinct genomic clusters. We observe potentially clinically relevant genotypes, including microsatellite instability (MSI), homologous recombination deficiency (HRD) enriched with genomic deletions and BRCA2 aberrations, a tandem duplication genotype associated with CDK12-/- and a chromothripsis-enriched subgroup. Our data suggests that stratification on WGS characteristics may improve identification of MSI, CDK12-/- and HRD patients. From WGS and ChIP-seq data, we show the potential relevance of recurrent alterations in non-coding regions identified with WGS and highlight the central role of AR signaling in tumor progression. These data underline the potential value of using WGS to accurately stratify mCRPC patients into clinically actionable subgroups.

BACKGROUNDContinued androgen receptor (AR) signaling constitutes a key target for treatment in metastatic castration-resistant prostate cancer (CRPC). Studies have identified 11-ketotestosterone (11KT) as a potent AR agonist, but it is unknown if 11KT is present at physiologically relevant concentrations in patients with CRPC to drive AR activation. The goal of this study was to investigate the circulating steroid metabolome including all active androgens in patients with CRPC.METHODSPatients with metastatic CRPC (n = 29) starting a new line of systemic therapy were included. Sequential plasma samples were obtained for measurement of circulating steroid concentrations by multisteroid profiling employing liquid chromatography-tandem mass spectrometry. Metastatic tumor biopsy samples were obtained at baseline and subjected to RNA sequencing.RESULTS11KT was the most abundant circulating active androgen in 97% of patients with CRPC (median 0.39 nmol/L, range: 0.03-2.39 nmol/L), constituting 60% (IQR 43%-79%) of the total active androgen (TA) pool. Treatment with glucocorticoids reduced 11KT by 84% (49%-89%) and testosterone by 68% (38%-79%). Circulating TA concentrations at baseline were associated with a distinct intratumor gene expression signature comprising AR-regulated genes.CONCLUSIONThe potent AR agonist 11KT is the predominant circulating active androgen in patients with CRPC and, therefore, one of the potential drivers of AR activation in CRPC. Assessment of androgen status should be extended to include 11KT, as current clinical approaches likely underestimate androgen abundance in patients with CRPC.TRIAL REGISTRATIONNetherlands Trial Register: NL5625 (NTR5732).FUNDINGDaniel den Hoed Foundation and Wellcome Trust (Investigator Award WT209492/Z/17/Z).

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell-free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked-in synthetic plant DNA. In addition, a β-actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild-type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the 'gold standard', and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high-throughput cfDNA isolation is needed., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)