Development of personalized surgical algorithms for muscle-invasive bladder cancer patients

项目总结 根治性膀胱切除术是肌肉浸润性膀胱癌治疗的基石 是美国进行的最复杂的肿瘤学手术之一。与.一起 通过新辅助化疗，这两种方式构成了目前治疗该病的标准。 在新辅助化疗后接受根治性膀胱切除术的患者中，约有三分之一的患者将没有 残留在膀胱里的残留病。回顾文献支持在患者中使用膀胱切除术延期 接受内窥镜活检未发现残留疾病的患者。然而，该算法容易产生较高的误码率 阴性率需要行保留膀胱切除术，延迟手术可能导致较差的生存结果。 该提案的第一个目标将探讨突变清除和持续存在于 尿液来源的无细胞DNA分别作为内窥镜检查后疾病消失或持续的标志 切除和化疗。在初步研究中，单分子标签和下一代标签的结合 测序导致对以前在整个肿瘤中发现的体细胞变体的高灵敏度检测 化疗前尿样中的外显子。至少有一种变异在化疗后尿样中持续存在 来自两个无反应者中的两个，并且在两个完整的尿样中没有检测到变异 响应者。初步数据表明，该方法可行、可靠，值得进一步探索。 该分析将用于在之前完成的试验(NCT01031420)和 来自福克斯·蔡斯癌症中心现在开始的一项预期临床试验的样本。 局限性膀胱癌表现出较高的病理完全化疗反应率， 但化疗敏感性的机制仍不完全清楚。这里描述的数据表明 免疫渗入是由CD8+细胞和高密度的新抗原驱动的。新抗原是非我的 由体细胞错义或移码突变引起的可被免疫识别的多肽 系统。该提案的目标2试图通过确定免疫系统在这方面的作用 识别肿瘤新抗原的肿瘤特异性细胞毒T细胞。表达的新抗原将被鉴定 使用肿瘤的整个外显子组测序和RNA-SEQ以及可用的计算算法。这些 然后将合成突变多肽，并用于筛选活的患者来源的CD8+细胞的新抗原- 特异性细胞毒反应。这项研究中使用的样本将从前瞻性临床试验中获得。 现在在福克斯·蔡斯癌症中心开业。这种假设驱动的分子和细胞生物学研究可以 潜在地确定了一种新的、意想不到的由免疫系统介导的化学反应机制。 如果成功，这些目标将在未来携手并进，以前瞻性和回溯性地确定 并提供一个框架来操纵免疫系统以实现化学反应 避免安全的根治性膀胱切除术的长期目标。

PROJECT SUMMARY Radical cystectomy is the cornerstone of treatment for patients with muscle-invasive bladder cancer and is among the most complicated oncologic procedures performed in the United States. Along with neoadjuvant chemotherapy, these two modalities comprise the current standard of care for the disease. Approximately 1/3 of patients who undergo radical cystectomy after neoadjuvant chemotherapy will have no residual disease left in the bladder. Retrospective literature supports the use of cystectomy deferral in patients who undergo endoscopic biopsy showing no residual disease. However, this algorithm is prone to a high false negative rate necessitating salvage cystectomy, delay of which may result in worse survival outcomes. The first aim of the proposal will explore the phenomenon of mutation clearance and persistence in urine-derived cell-free DNA as a marker for disease absence or persistence, respectively, after endoscopic resection and chemotherapy. In preliminary studies, combination of single molecule tags and next generation sequencing resulted in high sensitivity detection of somatic variants previously identified in tumor whole exomes in prechemotherapy urine samples. At least one variant persisted in postchemotherapy urine samples from two of two nonresponders, and no variants were detected in urine samples from two of two complete responders. This preliminary data shows that the assay is feasible, reliable, and merits further exploration. The assay will be used on banked samples collected on a previously completed trial (NCT01031420) and on samples from a prospective clinical trial opening now at Fox Chase Cancer Center. Localized bladder cancer demonstrates a relatively high rate of pathological complete chemoresponse, but the mechanism of chemosensitivity remains incompletely understood. Data described herein suggests that the immune infiltrate is driven by CD8+ cells and high neoantigen density. Neoantigens are the non-self peptides that result from somatic missense or frameshift mutations that can be recognized by the immune system. Aim 2 of the proposal seeks to clinch a role for the immune system in this context by identifying tumor-specific cytotoxic T-cells which recognize tumor neoantigens. Expressed neoantigens will be identified using whole exome sequencing and RNA-seq of tumors and available computational algorithms. These mutant peptides will then be synthesized and used to screen live patient-derived CD8+ cells for neoantigen- specific cytotoxic responses. Samples to be used in this study will be obtained from a prospective clinical trial opening now at Fox Chase Cancer Center. This hypothesis-driven molecular and cellular biological study can potentially identify a new and unexpected mechanism for chemoresponse mediated by the immune system. If successful, these aims will go hand-in-hand in the future to prospectively and retrospectively identify responders and provide a framework to manipulate the immune system to achieve chemoresponse with the long term goal of safe radical cystectomy avoidance.