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Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (<1%), with high concordance observed between the in-house and commercial assays. The optimized protocol dramatically improved the efficiency of the assay and enabled the detection of low-frequency somatic variants from plasma cell-free DNA (cfDNA).

Background: Metastatic prostate cancer is a clonally heterogeneous disease state characterized by progressive somatic perturbations. The aim of this study was to identify cell free DNA- (cfDNA-) based alterations and their associations with outcomes in progressive metastatic prostate cancer., Methods: In this longitudinal prospective cohort study plasma cfDNA/circulating tumor DNA (ctDNA) was analyzed before, during, and after androgen deprivation therapy (ADT) in 4 independent patient groups ranging from untreated metastatic hormone sensitive prostate cancer (mHSPC) to metastatic castrate resistant prostate cancer (mCRPC). Next generation sequencing was performed on ctDNA and germline DNA to characterize alterations and associations with clinical outcomes were determined for each group., Findings: cfDNA yields were different in progressive mHSPC and mCRPC states (P < .001). In mHSPC, a higher than median ctDNA fraction was predictive of shorter time to ADT failure (HR, 2.29 [95% CI, 1.13-4.65]; Log-Rank P  =  .02). cfDNA, ctDNA taken with volume of metastatic disease in mHSPC and with alkaline phosphatase levels prognosticated survival better than clinical factors alone in mHSPC and mCRPC states (Log Rank P = 0.03). ctDNA-based AR, APC mutations were increased in mCRPC compared to mHSPC (P < ·05).TP53 mutations, RB1 loss, and AR gene amplifications correlated with poorer survival in mCRPC. Mutations in multiple DNA repair genes (ATM, BRCA1, BRCA2, CHEK2) were associated with time to ADT treatment failure and survival in mHSPC., Interpretation: ctDNA fraction can further refine clinical prognostic factors in metastatic prostate cancer. Somatic ctDNA alterations have potential prognostic, predictive, and therapeutic implications in metastatic prostate cancer management., Funding: Several funding sources have supported this study. A full list is provided in the Acknowledgments. No funding was received from Predicine, Inc. during the conduct of the study., Competing Interests: Declaration of Competing Interest Manish Kohli received travel/accommodation from Celgene; Tiantian Zheng, Amy Wang, Carlos Montesinos, Calven Wong, Pan Du, Shidong Jia, and Jianjun Yu are stockholders in Predicine, Inc.; Lisa Horvath received research funding from Astellas Pharma, travel/accommodation from Astellas Pharma and Pfizer, and is on the Scientific Advisory Board for Imagion; Kate Mahon received travel/accommodation from Astellas Pharma; Edmond M Kwan received honoraria from Janssen, research funding from Astellas Pharma and AstraZeneca, and travel /accommodations from Astellas Pharma, Pfizer, and Ipsen; Arun A Azad is a consultant for Astellas Pharma, Janssen, and Novartis, is on the speakers bureau for Astellas Pharma, Janssen, Novartis and Amgen received honoraria from Astellas Pharma, Janssen, Novartis, Tolmar, Amgen, Pfizer, and Telix, is on the Scientific Advisory Board for Astellas Pharma, Novartis, Sanofi, AstraZeneca, Tolmar, Pfizer, and Telix, and received research funding from Astellas Pharma, and Merck Serono., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)