Targeted sequencing of cell-free DNA for monitoring of prostate cancer progression

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PROJECT SUMMARY / ABSTRACT Prostate cancer (PCa) results in more than 160,000 diagnoses in the US each year with essentially all patients eventually progressing to metastatic castration-resistant prostate cancer (mCRPC), the more lethal form of the disease. Early stratification of mCRPC patients is critical since 20-40% exhibit primary resistance to first-line treatments and those with resistance have a median survival of only 5.5 months. The standard-of-care circulating tumor cell assay (monitoring the AR-V7 splice variant) highlights the potential for a non-invasive method for monitoring PCa disease progression, but it gives 80-90% of patients a false-negative and has only ~3% sensitivity when used pre-treatment. The long-term objective of this proposal is to investigate the potential of a cell-free DNA (cfDNA)-based assay for mCRPC patient stratification to improve upon the current standard-of-care. To address this, the current proposal leverages our published cfDNA assay that monitors the recently discovered tandem duplication of an enhancer region upstream of the androgen receptor (AR), additional AR mutations, and 83 additional genes commonly mutated in mCRPC. Our gene panel and cfDNA analysis method (EnhanceAR-Seq) outperformed the current standard-of-care in a prospectively collected cohort of mCRPC patients. While this highlights the clinical utility of monitoring SVs non-invasively, no automated pipelines exist for SV calling using targeted cfDNA assays. Copy number variation (CNV) and SV analysis of cfDNA using a targeted sequencing approach requires specific considerations not accounted for in traditional pipelines, such as the influence of probe density on read depth. This in turn results in researchers developing custom scripts and parameters for running multiple tools for analysis of different variant classes (SV, CNV, single nucleotide variants) and ultimately impairing the reproducibility of clinically relevant work. This serves as a strong rationale for the following aims to (1) develop a standardized and robust unified pipeline for somatic variant calling of all classes using cfDNA and (2) assess the clinical utility of EnhanceAR-Seq across (a) an independent validation mCRPC patient cohort, (b) across ethnicities and (c) in an earlier clinical setting. The research will be completed with mentorship from Dr. Christopher Maher and through the Human and Statistical Genetics program at Washington University, one of the top ranked genetics programs in the country. The Maher lab is an ideal environment for this project given their extensive bioinformatics, software development, structural variation, genomics, and prostate cancer biology expertise. Training will focus on (1) developing computational skills through software development and the application of bioinformatics tools, (2) improving knowledge of translational tumor biology, and (3) professional development, all of which will support the goal of a career in cancer research. This study will have an overall impact on the field by providing a standardized and reproducible method for variant calling with cfDNA in a non-invasive assay, which will be broadly applicable beyond PCa. Long term, this research has enormous potential for clinical translation by improving PCa patient care.