Optimization of urinary DNA deep sequencing tests to enhance clinical staging of bladder cancer patients

项目总结/摘要 肌肉浸润性膀胱癌（MIBC）的最佳治疗方法是新辅助化疗， 根治性化疗（RC），其中约35%的患者将有病理完全反应（pCR）。鉴于 病态的，复杂的，昂贵的性质RC和完善的pCR率，有一个风潮， 对达到pCR的患者避免RC感兴趣。然而，在临床上鉴定pCR（与 病理学上）是不准确的过程。在已发表的研究中，在被视为临床后避免RC的患者 完全反应者具有25-60%的复发、转移或膀胱癌死亡的可能性。更好 需要评估残留疾病状态的工具。 为了满足这一需求，我们开发了一种尿液活检检测，我们称之为UTeRD（尿液残留检测） 疾病）。在UTeRD中，从尿液中分离DNA并进行下一代测序以检测点 一组靶基因的突变。使用UTeRD，肿瘤组织中的大多数突变可检测为 尿液中的突变此外，化疗完成后是否存在残留MU强烈地影响着患者的预后。 分别与RC时的残留疾病或pCR相关。因此，UTeRD可以在 新辅助治疗，以更好地识别患者的RC避免。 虽然UTeRD在区分pCR患者和残留疾病方面表现良好，但阴性对照组和阴性对照组的pCR患者的pCR和残留疾病的差异不显著。 UTeRD的预测值（NPV）仅为76%，一些尿液样本无法诊断，尿DNA 需要制定保存协议，以便广泛采用该测试。分析前因子 我们认为，方法学的改进将增加净现值，降低非诊断率， 研究目标1。在目标2中，我们将确定是否可以使用尿液防腐剂，以方便运输到 集中实验室，而不会损失测试的保真度。最后，在目标3中，我们将确定是否存在突变 与pCR相关，无论术前治疗如何。为了回答这个问题， 从多个机构的5项前瞻性MIBC临床试验中获得的样本将使用 通过本研究确定的优化方案。 研究小组由泌尿科医生、医学肿瘤学家、放射肿瘤学家、统计学家组成， 一个在各自领域都是专家的计算生物学家。团队的技能和贡献是 互补的，并将最终在一个独特的和强大的生物标志物的发展，解决了 显著的临床需求。UTeRD可以增强膀胱癌的能力， 临床医生回答高度相关的临床问题，即“该患者在预处理后是否有残留疾病”。 手术治疗，因此，他/她会受益于RC吗？”

PROJECT SUMMARY/ABSTRACT Muscle-invasive bladder cancer (MIBC) is optimally treated with neoadjuvant chemotherapy followed by radical cystectomy (RC), whereby ~35% of patients will have a pathologic complete response (pCR). Given the morbid, complicated, and expensive nature of RC and the well-established pCR rate, there is a groundswell of interest in RC avoidance for patients achieving pCR. However, identifying pCR clinically (as opposed to pathologically) is an inaccurate process. In published studies, patients who avoid RC after being deemed clinical complete responders have a 25-60% likelihood of recurrence, metastasis, or bladder cancer mortality. Better tools to assess residual disease status are needed. To address this need, we developed a urine biopsy test which we call UTeRD (Urine Test for Residual Disease). In UTeRD, DNA is isolated from urine and subjected to next generation sequencing to detect point mutations in a targeted panel of genes. Using UTeRD, most mutations in tumor tissue are detectable as mutations in urine. Further, presence or absence of residual MU after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, UTeRD could be used after neoadjuvant therapy to better identify patients for RC avoidance. Although UTeRD performs well in distinguishing patients with pCR from residual disease, the negative predictive value (NPV) of UTeRD is only 76%, some urine samples were nondiagnostic, and a urinary DNA preservation protocol needs to be developed in order for the test to be widely adopted. Pre-analytical factors and methodology improvements which we believe will increase the NPV and decrease nondiagnostic rates will be studied in Aim 1. In Aim 2, we will determine if urine preservatives can be used to facilitate shipping to a centralized lab without loss of fidelity of the test. Lastly, in Aim 3, we will determine if the absence of mutations from a urine biopsy is associated with pCR regardless of the pre-surgical therapy. To answer this question, samples obtained on 5 prospective MIBC clinical trials from multiple institutions will be studied using the optimized protocols identified through this research. The research team is comprised of a urologist, medical oncologists, a radiation oncologist, a statistician, a computational biologist who are experts in their fields. The skills and contributions of the team are complimentary and will culminate in the development of a unique and robust biomarker that addresses a significant clinical need using a one-of-a-kind sample cohort. UTeRD may enhance the ability of a bladder cancer clinician to answer highly relevant clinical question, namely, “Does this patient have residual disease after pre- surgical therapy, and therefore, will he/she benefit from RC?”