Custom assays for personalized, longitudinal monitoring of circulating tumor DNA using PIPSenSeq

项目总结/摘要 脱落到患者血液中的无细胞肿瘤DNA（循环肿瘤DNA，ctDNA）的靶向DNA测序 为癌症的检测、诊断和监测带来了巨大的希望。鉴于所有肿瘤细胞都具有 利用现代下一代测序方法和数字PCR进行靶向检测， 可以提供对患者肿瘤负荷的整体了解。因此，ctDNA是一种有吸引力的生物标志物， 通过非侵入性外周血抽取对癌症患者进行诊断和监测。然而，这样的测定， 需要超灵敏的测序保真度、灵活的靶多重和均匀的靶扩增， 高效、定量和成本效益高的测试。此外，在执行《行动纲领》方面也出现了一些挑战， 用于鉴定和监测从血液中提取的肿瘤特异性突变的测定。首先，来自 与正常DNA的背景相比，癌细胞是罕见的，要求检测灵敏度低于0.1%。 一些临床应用。其次，从血浆中提取的游离DNA的质量有限（<100 ng / 5 ml 样品），并且通常降解为非常短的片段长度，需要有效捕获和扩增 感兴趣的目标。第三，癌症诊断和监测可能需要监测大量肿瘤细胞， 或患者特异性突变，需要多重扩增以有效利用样品。 与我们在波士顿大学和加州旧金山弗朗西斯科的学术合作伙伴合作， 我们开发了一种新的下一代测序样品制备平台， PIPSenSeq（Pre-templated Instant Partitions for Sensitive Sequencing）这种方法 利用分子索引进行共有阅读测序， 泊松分布的纳米级分区中的扩增。此外，PIPSenSeq提供了一种简单、快速的 文库制备不需要复杂、昂贵的仪器或微流体消耗品。在 我们将开发PIPSenSeq作为癌症监测的商业平台， 在60 - 70名头颈部鳞状细胞癌患者的研究中证明了临床实用性。这些 术后将使用个性化的PIPSenSeq面板监测患者的肿瘤复发， 纵向收集的ctDNA样品。

PROJECT SUMMARY / ABSTRACT Targeted DNA sequencing of cell-free tumor DNA (circulating tumor DNA, ctDNA) shed into a patient’s blood holds great promise for detection, diagnosis, and monitoring of cancer. Given that all tumor cells have the potential to shed ctDNA, targeted assays using modern next-generation sequencing methods and digital PCR can provide insight into the entirety of a patient’s tumor burden. ctDNA is therefore an attractive biomarker for diagnosis and monitoring of cancer patients via non-invasive peripheral blood draw. Such assays, however, require ultra-sensitive sequencing fidelity, flexible target multiplexing, and uniform target amplification for efficient, quantitative, and cost-effective testing. In addition, several challenges arise in the implementation of assays to identify and monitor tumor-specific mutations extracted from blood. First, the mutant DNA from cancer cells is rare compared to the background of normal DNA, requiring detection sensitivities below 0.1% for some clinical applications. Second, cell-free DNA extracted from plasma is limited in mass (<100 ng / 5 ml sample), and typically degraded to very short fragment lengths, requiring efficient capture and amplification of the targets of interest. Third, cancer diagnosis and monitoring may require surveillance of a multitude of tumor or patient-specific mutations, requiring multiplex amplification for efficient sample utilization. In collaboration with our academic partners at Boston University and the University of California San Francisco, we have developed a novel next-generation sequencing sample preparation platform that addresses these critical challenges - PIPSenSeq (Pre-templated Instant Partitions for Sensitive Sequencing). This approach takes advantage of molecular indexing for consensus-read sequencing in combination with single-molecule amplification in Poisson-distributed nanoscale partitions. Furthermore, PIPSenSeq provides a simple, rapid library preparation that does not require complex, expensive instrumentation or microfluidic consumables. In this proposal we will develop PIPSenSeq as a commercial-ready platform for cancer monitoring, and demonstrate clinical utility in a study of 60 - 70 head and neck squamous cell carcinoma patients. These patients will be monitored post-operatively for tumor recurrence using personalized PIPSenSeq panels on longitudinally collected ctDNA samples.