Real-time monitoring of circulating pancreatic tumor cells and clusters

胰腺导管腺癌（PDA）的总五年生存率为6%， 预计将成为美国癌症相关死亡的第二大原因。80%的PDA患者 接受手术的患者可能会因诊断时无法检测到的转移而复发;然而 护理标准射线照相成像和血液测试可能是不精确的 转移负荷循环肿瘤细胞（CTC）被认为介导了大部分转移过程， 虽然FDA批准的测试是建立在对乳房流入血液的CTC计数的基础上的， 前列腺和结肠直肠肿瘤，但这种测试对胰腺肿瘤缺乏足够的敏感性。最近我们 结果表明：PDA CTC异质性促进了更具侵袭性的表型; CTC簇是 与更高的转移潜力相关;在远端部位接种需要一个以上的克隆， 转移性疾病进展的过程。该提案旨在开发和优化一种液体 活检用于测量单个和成簇的CTC及其分子特征， 以解决对PDA患者转移性非侵入性临床监测的迫切需求 负担、疾病进展和治疗反应。这一行业/学术合作伙伴关系将带来 结合Becton Dickinson（BD）在稀有细胞捕获和分子分析方面的优势， PDA抗原发现程序，以及Penn的临床前PDA模型，稳健的临床PDA程序，以及 专门的循环肿瘤材料实验室。第一，高效率浓缩和分选技术， 具有高纯度、回收率和活力的CTC簇将被优化，使得高质量的RNA可以被纯化。 分离用于下游分子分析（RNA-seq）。BD Focus是一种罕见的细胞富集技术， BD正在开发的一个平台，将用于预富集和分选，使用细胞 表面抗体和基于RNA的探针（即，BD独特的“nanoflare”技术）。第二， 候选CTC生物标志物将在PDA临床前小鼠模型中最终确定和验证， 转移性PDA患者。最后，我们将建立CTC生物标志物的概念证明，以预测 并监测PDA患者队列的转移负荷，从诊断开始， 治疗开始时，以及与监测CT扫描一致的时间点。单细胞和 聚集的CTC计数和分子标记将与转移负荷和存活相关。的 富集和进行CTC单细胞和簇的分子表征的能力将提供一种新的 以及检测和监测PDA患者转移的关键技术， 可以使用策略以提高总存活率。

Pancreatic ductal adenocarcinoma (PDA), which has an overall five-year survival rate of 6%, is predicted to become the second leading cause of cancer-related death in the US. 80% of PDA patients who have surgery will suffer a recurrence likely due to undetectable metastases present at diagnosis; yet standard of care radiographic imaging and blood-tests can be imprecise and are not predictive of metastatic burden. Circulating tumor cells (CTCs) are thought to mediate much of the metastatic process, and while FDA-approved tests are established for enumeration of CTCs shed into the blood by breast, prostate, and colorectal tumors, such tests lack sufficient sensitivity for pancreatic tumors. Recently, we showed that: PDA CTC heterogeneity promoted a more aggressive phenotype; CTC clusters were associated with higher metastatic potential; and seeding at distal sites required more than one clone during the course of metastatic disease progression. This proposal seeks to develop and optimize a liquid biopsy for the measurement of single and clustered CTCs and their molecular signatures, in order to address the urgent unmet need for non-invasive clinical monitoring of PDA patient metastatic burden, disease progression, and treatment response. This industry/academic partnership will bring together Becton Dickinson (BD)'s strengths in rare cell capture and molecular analysis, Johns Hopkins' PDA antigen discovery programs, and Penn's pre-clinical PDA models, robust clinical PDA programs, and dedicated Circulating Tumor Material Laboratory. First, technologies for efficient enrichment and sorting of CTC clusters with high purity, recovery and viability will be optimized so that high quality RNA can be isolated for downstream molecular analyses (RNA-seq). The BD Focus, which is a rare cell enrichment platform being developed at BD, will be used for pre-enrichment and sorting using a combination of cell surface antibody and RNA-based probes (i.e., BD's unique “nanoflare” technology). Second, a panel of candidate CTC biomarkers will be finalized and validated in PDA pre-clinical mouse models as well as patients with metastatic PDA. Finally, we will establish proof of concept for the CTC biomarkers to predict and monitor the metastatic burden of a cohort of PDA patients starting at diagnosis and continuing after commencement of therapy, and at time points coinciding with monitoring CT scans. Single cell and clustered CTC counts and molecular signature will be correlated with metastatic burden, and survival. The ability to enrich and perform molecular characterization of CTC single cells and clusters will provide a novel and crucial technology to detect and monitor metastasis in PDA patients so that the appropriate therapeutic strategies can be utilized in order to increase overall survival rates.