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

项目摘要/摘要前列腺癌(PCA)在美国导致超过16万例诊断 每年，基本上所有患者最终都会进展为耐去势转移性前列腺癌 (MCRPC)，这种疾病更致命的形式。MCRPC患者的早期分层至关重要，因为20%-40% 表现出对一线治疗的主要耐药，耐药者的中位存活率仅为5.5 月份。标准护理循环肿瘤细胞分析(监测AR-V7剪接变异体)强调 一种非侵入性方法监测PCA疾病进展的潜力，但它为80%-90%的患者提供了 假阴性，治疗前仅有~3%的敏感性。这项提议的长期目标是 是为了研究一种基于无细胞DNA(CfDNA)的分析方法对mCRPC患者分层的改进潜力 根据目前的护理标准。为了解决这个问题，目前的方案利用了我们发表的cfdna分析。 监测最近发现的雄激素上游增强子区域的串联复制 受体(AR)、额外的AR突变和83个额外的基因在mCRPC中通常发生突变。我们的基因小组 CfDNA分析方法(EnhanceAR-Seq)在前瞻性方面优于当前的护理标准 收集mCRPC患者队列。虽然这突显了无创监测SVS的临床实用价值，但没有 对于使用靶向cfDNA分析的SV呼叫，存在自动化管道。拷贝数变异(CNV)和SV 使用靶向测序方法分析cfDNA需要考虑未在 传统的流水线，如探头密度对读取深度的影响。这反过来又导致了研究人员 开发用于运行多个工具的定制脚本和参数以分析不同的变量类(SV， CNV，单核苷酸变异)，并最终损害临床相关工作的可重复性。这 作为以下目标的有力基础：(1)为以下目标开发标准化和强大的统一管道 使用cfDNA对所有类别的体细胞变异呼叫和(2)跨(A)评估增强AR-Seq的临床应用 一个独立的验证mCRPC患者队列，(B)跨种族和(C)早期临床环境。这个 研究将在克里斯托弗·马赫博士的指导下完成，并通过人类和统计 华盛顿大学的遗传学项目，全国排名最高的遗传学项目之一。马赫家族 实验室是这个项目的理想环境，因为他们广泛的生物信息学、软件开发、结构 变异、基因组学和前列腺癌生物学专业知识。培训将侧重于(1)开发计算能力 通过软件开发和生物信息学工具的应用提高技能，(2)提高知识 翻译肿瘤生物学，以及(3)职业发展，所有这些都将支持在 癌症研究。这项研究将通过提供标准化和可重复性的 CfDNA非侵入性检测变异体的方法，该方法将广泛应用于PCa以外的领域。 从长远来看，这项研究通过改善PCA患者的护理，具有巨大的临床翻译潜力。

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.