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

项目摘要/摘要 肌肉浸润性膀胱癌(MIBC)的最佳治疗方法是新辅助化疗 根治性膀胱切除术(RC)，其中约35%的患者将有病理完全反应(PCR)。给定 RC的病态、复杂和昂贵的性质以及公认的PCR率，出现了一股潮流 对达到聚合酶链式反应的患者避免RC感兴趣。然而，在临床上鉴定聚合酶链式反应(与 病理学上)是一个不准确的过程。在已发表的研究中，被认为是临床上的患者避免了RC 完全应答者复发、转移或膀胱癌死亡的可能性为25%-60%。更好 需要评估残留疾病状况的工具。 为了满足这一需求，我们开发了一种尿液活组织检查，我们称之为UTeRD(尿液残留测试 疾病)。在UTeRD，从尿液中提取DNA，并进行下一代测序以检测点 一组目标基因的突变。使用UTeRD，肿瘤组织中的大多数突变都可以检测到 尿液中的突变。此外，化疗结束后有无残存MU的情况强烈 分别与RC时的残留病或PCR型有关。因此，UTeRD可以在以下情况下使用 新辅助治疗，以更好地识别患者的RC回避。 尽管UTeRD在区分PCR患者和残留疾病方面做得很好，但阴性的 UTeRD的预测价值(NPV)仅为76%，部分尿样未被诊断，尿DNA 需要制定保存协议，以便该测试得到广泛采用。分析前因素 我们相信，方法的改进将增加净现值，降低非诊断率 在目标1中进行研究。在目标2中，我们将确定是否可以使用尿液防腐剂来促进运输到 集中式实验室，测试的保真度不会降低。最后，在目标3中，我们将确定是否没有突变 无论手术前的治疗如何，尿液活检与聚合酶链式反应相关。为了回答这个问题, 从多个机构获得的5项MIBC预期临床试验的样本将使用 通过本研究确定了优化的协议。 研究小组由一名泌尿科医生、一名内科肿瘤学家、一名放射肿瘤学家、一名统计学家、 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 免费，并将最终开发出一种独特而强大的生物标记物，以解决 使用独一无二的样本队列进行显著的临床需求。UTeRD可能会增强膀胱癌的治疗能力 临床医生回答高度相关的临床问题，即“此患者在治疗前有无残留病变？” 外科治疗，因此，他/她会从RC中受益吗？

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?”