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Background: Although most patients with metastatic castration-resistant prostate cancer (mCRPC) initially benefit from treatment with androgen receptor signaling inhibitors (ARSi), resistance inevitably occurs. Hence, we investigated the prognostic value of automated circulating tumor cell (CTC) and tumor-derived extracellular vesicle (tdEV) enumeration and their dynamics, in patients with mCRPC in the context of the initiation of treatment with ARSi. Furthermore, we hypothesize that CTC phenotypic heterogeneity might serve as a measurable biomarker under these circumstances., Methods: Using an image analysis tool, we reanalyzed all CellSearch images previously acquired in the context of a prospective, multicenter clinical study for patients with mCRPC (n = 170) starting a new line of ARSi, for CTC and tdEV detection and enumeration. CTC (n = 19 129) phenotypic diversity was quantified by the Shannon index (SI). Progression-free survival (PFS) and overall survival (OS) were compared between groups of patients stratified according to CTC, tdEV, and SI levels., Results: Automated CTC enumeration provided similar clinical prognostication compared with operator-based counts. Patients demonstrating high CTC phenotypic heterogeneity before therapy had a shorter median PFS (4.82 vs. 8.49 months, HR 1.79; P = 0.03) and OS (12.6 months vs. not reached, HR 2.32; P = 0.03), compared to patients with low diversity, irrespective of CTC level. Multivariable analysis showed how the prognostic value of the baseline SI was lost by pretreatment chemotherapy status, CTC counts, and PSA levels., Conclusions: Automated CTC counts are a reliable substitute for reviewer-based enumeration, as they are equally informative for prognosis assessment in patients with mCRPC. Beyond enumeration, we demonstrated the added value of studying CTC phenotypic diversity for patient prognostication, warranting future investigation.

Background: Multiple therapies exist for patients with metastatic castration-resistant prostate cancer (mCRPC). However, their improvement on progression-free survival (PFS) remains modest, potentially explained by tumor molecular heterogeneity. Several prognostic molecular biomarkers have been identified for mCRPC that may have predictive potential to guide treatment selection and prolong PFS. We designed a platform trial to test this hypothesis., Methods: The Prostate-Biomarker (ProBio) study is a multi-center, outcome-adaptive, multi-arm, biomarker-driven platform trial for tailoring treatment decisions for men with mCRPC. Treatment decisions in the experimental arms are based on biomarker signatures defined as mutations in certain genes/pathways suggested in the scientific literature to be important for treatment response in mCRPC. The biomarker signatures are determined by targeted sequencing of circulating tumor and germline DNA using a panel specifically designed for mCRPC., Discussion: Patients are stratified based on the sequencing results and randomized to either current clinical practice (control), where the treating physician decides treatment, or to molecularly driven treatment selection based on the biomarker profile. Outcome-adaptive randomization is implemented to early identify promising treatments for a biomarker signature. Biomarker signature-treatment combinations graduate from the platform when they demonstrate 85% probability of improving PFS compared to the control arm. Graduated combinations are further evaluated in a seamless confirmatory trial with fixed randomization. The platform design allows for new drugs and biomarkers to be introduced in the study., Conclusions: The ProBio design allows promising treatment-biomarker combinations to quickly graduate from the platform and be confirmed for rapid implementation in clinical care., Trial Registration: ClinicalTrials.gov Identifier NCT03903835. Date of registration: April 4, 2019. Status: Recruiting.

Background: Personalized targeted treatment in metastatic breast cancer relies on accurate assessment of molecular aberrations, e.g. overexpression of Human Epidermal growth factor Receptor 2 (HER-2). Molecular interrogation of circulating tumor cells (CTCs) can provide an attractive alternative for real-time biomarker assessment. However, implementation of CellSearch-based HER-2 analysis has been limited. Immunofluorescent (IF) image interpretation is crucial, as different HER-2 categories have been described. Major questions in CTC research are how these IF categories reflect gene expression and amplification, and if we should consider 'medium' HER-2 expressing CTCs for patient selection., Methods: Tumor cells from spiked cell lines (n = 8) and CTCs (n = 116 samples) of 85 metastatic breast cancer patients were enriched using CellSearch. Comparative analysis of HER-2 expression by IF imaging (ACCEPT, DEPArray, and visual scoring) with qRT-PCR and HER-2/neu FISH was performed., Results: Automated IF HER-2-profiling by DEPArray and ACCEPT delivered comparable results. There was a 98% agreement between 17 trained observers (visual scoring) and ACCEPT considering HER-2neg and HER-2high expressing CTCs. However, 89% of HER-2med expressing CTCs by ACCEPT were scored negative by observers. HER-2high expressing tumor cells demonstrated HER-2/neu gene amplification, whereas HER-2neg and HER-2med expressing tumor cells and CTCs by ACCEPT were copy-number neutral. All patients with HER-2-positive archival tumors had ≥1 HER-2high expressing CTCs, while 80% of HER-2-negative patients did not. High relative gene expression of HER-2 measured on enriched CTC lysates correlated with having ≥1 HER-2high expressing CTCs., Conclusion: Automated images analysis has enormous potential for clinical implementation. HER-2 characterization and clinical trial design should be focused on HER-2high expressing CTCs., Competing Interests: The authors DP, PJvD, ES, and AEM are affiliated with the company HistoGeneX NV. The company HistoGeneX NV provided support in the form of salaries for authors ES and AEM. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

Purpose: To infer the prognostic value of simultaneous androgen receptor ( AR ) and TP53 profiling in liquid biopsies from patients with metastatic castration-resistant prostate cancer (mCRPC) starting a new line of AR signaling inhibitors (ARSi). Experimental Design: Between March 2014 and April 2017, we recruited patients with mCRPC ( n = 168) prior to ARSi in a cohort study encompassing 10 European centers. Blood samples were collected for comprehensive profiling of CellSearch-enriched circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). Targeted CTC RNA sequencing (RNA-seq) allowed the detection of eight AR splice variants (ARV). Low-pass whole-genome and targeted gene-body sequencing of AR and TP53 was applied to identify amplifications, loss of heterozygosity, mutations, and structural rearrangements in ctDNA. Clinical or radiologic progression-free survival (PFS) was estimated by Kaplan-Meier analysis, and independent associations were determined using multivariable Cox regression models., Results: Overall, no single AR perturbation remained associated with adverse prognosis after multivariable analysis. Instead, tumor burden estimates (CTC counts, ctDNA fraction, and visceral metastases) were significantly associated with PFS. TP53 inactivation harbored independent prognostic value [HR 1.88; 95% confidence interval (CI), 1.18-3.00; P = 0.008], and outperformed ARV expression and detection of genomic AR alterations. Using Cox coefficient analysis of clinical parameters and TP53 status, we identified three prognostic groups with differing PFS estimates (median, 14.7 vs. 7.51 vs. 2.62 months; P < 0.0001), which was validated in an independent mCRPC cohort ( n = 202) starting first-line ARSi (median, 14.3 vs. 6.39 vs. 2.23 months; P < 0.0001)., Conclusions: In an all-comer cohort, tumor burden estimates and TP53 outperform any AR perturbation to infer prognosis.See related commentary by Rebello et al., p. 1699., (©2018 American Association for Cancer Research.)

Context: In advanced prostate cancer (PC), there is increasing investigation of circulating biomarkers, including quantitation and characterization of circulating tumour cells and cell-free nucleic acids, for therapeutic monitoring and as prognostic and predictive biomarkers. However, there is a lack of consensus and standardisation regarding analyses, reporting, and integration of results into specific clinical contexts. A consensus meeting on circulating biomarkers was held to address these topics., Objective: To present a report of the consensus statement on circulating biomarkers in advanced PC., Evidence Acquisition: Four important areas of controversy in the field of circulating biomarkers in PC management were identified: known clinical utility of circulating biomarkers; unmet clinical needs for circulating biomarkers in PC care; most pressing blood-based molecular assays required; and essential steps for developing circulating biomarker assays. A panel of 18 international PC experts in the field of circulating biomarkers developed the programme and consensus questions. The panel voted publicly but anonymously on 50 predefined questions developed following a modified Delphi process., Evidence Synthesis: Voting was based solely on panellist opinions of the predefined topics and therefore not on a standard literature review or meta-analysis. The outcomes of the voting had varying degrees of support, as reflected in the wording of this article and in the detailed voting results provided in the Supplementary material., Conclusions: The expert voting results presented can guide the future development of circulating biomarkers for PC care. Notably, the consensus meeting highlighted the importance of reproducibility and variability studies, among other significant areas in need of trials specifically designed to address them., Patient Summary: A panel of international experts met to discuss and vote on the use of different blood-based prostate cancer tests, and how they can be used to guide treatment and disease monitoring to deliver more precise and better patient care., (Copyright © 2018 European Association of Urology. Published by Elsevier B.V. All rights reserved.)