Integrated analysis of HCC CTCs for Liver Transplant Candidate Selection

项目摘要 肝细胞癌（HCC）是全球癌症相关死亡的第二大常见原因。肝 肝移植（LT）是治疗患有不可切除的非转移性疾病的HCC患者的唯一治愈性疗法， 符合严格的放射学肿瘤大小和数量标准（米兰标准）。合格的候选人将接受有限的 观察期（>6个月），允许评估肿瘤生物学，但固有HCC风险 1年后15-20%的患者出现进展和退出等待名单。尽管有这些选择做法， HCC复发困扰高达20%的患者，并且是同种异体移植物丢失和患者死亡的主要原因。那里 迫切需要能够动态监测肿瘤生物学的非侵入性生物标志物，以更好地平衡肿瘤生物学。 等待名单脱落和LT后复发的风险，并允许改善HCC患者接受 罕见的肝脏移植该提案旨在开发一种基于血液的综合分析（即，NanoVelcro 用于检测波形蛋白+循环肿瘤细胞[CTC]的vimCTC测定和用于检测HCC- 特异性RNA标签），以鉴定最适合LT的患者。 该方法将为研究HCC CTC的表型和分子特征提供新的方法。 使用HCC特异性多标记捕获混合物和优化的免疫细胞化学（ICC）染色 根据方案，所提出的NanoVelcro vimCTC测定能够鉴定HCC CTC亚群， 波形蛋白表达（命名为vimCTC，DAPI+/CK+/CD 45-/vimentin+）。该亚群与 在接受治愈性目的治疗的临床难以区分的早期患者亚组中复发增加 治疗HCC CTC-RNA检测是通过CTC分离与Click Chip相结合开发的， 点击化学介导的细胞捕获和二硫键裂解细胞释放，以及下游RNA表达 使用NanoString的nCounter平台分析纯化的CTC。这允许精确定量 一组HCC CTC衍生的mRNA标志物。所得vimCTC计数和mRNA 简档对于增强当前LT候选选择算法的能力具有很大的希望。 拟议的研究将通过具体目标1a实施：在银行进行回顾性研究 使用NanoVelcro vimCTC测定血液样品，以细化vimCTC计数与治疗后 LT复发/等待名单脱落。具体目标1b：对新鲜采集的血液进行前瞻性研究 用于确定vimCTC计数与LT后复发/等待名单脱落之间的关联的样本;以及 具体目标2：对新鲜采集的血液样本进行前瞻性研究，以确定 HCC CTC-RNA检测与LT后复发/等待名单脱落之间的关系。评价的中心假设将 vimCTC和侵袭性RNA特征的基线和纵向变化将显著改善 当前临床放射学LT选择标准预测LT后复发和等待名单脱落的能力， 为基于肿瘤生物学的HCC LT候选选择实践铺平了道路。

PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the 2nd most common cause of cancer-related deaths worldwide. Liver transplantation (LT) is the only curative therapy for HCC patients with unresectable, non-metastatic disease who meet strict radiologic tumor size and number criteria (Milan criteria). Eligible candidates undergo a finite observation period (>6 months) that allows for an assessment of tumor biology, but which inherently risks HCC progression and wait-list dropout in 15-20% of patients after 1 year. Despite these selection practices, post-LT HCC recurrence plagues up to 20% of patients, and is a major cause of allograft loss and patient mortality. There is a dire need for non-invasive biomarkers capable of dynamic monitoring of tumor biology to better balance the risk of wait-list dropout and post-LT recurrence, and allowing for improved prioritization of HCC patients to receive scarce liver allografts. This proposal aims to develop an integrated blood-based analysis (i.e., NanoVelcro vimCTC Assay for detecting vimentin+ circulating tumor cells [CTCs] and HCC CTC-RNA Assay for HCC- specific RNA signatures) for wait-listed HCC patients, to identify patients most suitable for LT. The integrated assay will provide a novel approach to study both phenotypic and molecular characteristics of HCC CTCs. Using an HCC-specific multi-marker capture cocktail and optimized immunocytochemistry (ICC) staining protocol, the proposed NanoVelcro vimCTC Assay is capable of identifying a subpopulation of HCC CTCs with vimentin expression (named vimCTC, DAPI+/CK+/CD45-/vimentin+). This subpopulation is associated with increased recurrence in the subset of clinically indistinguishable early-stage patients undergoing curative-intent treatment. The HCC CTC-RNA Assay was developed by combining CTC isolation with Click Chip, featuring click chemistry-mediated cell capture and disulfide-cleavage cell release, with downstream RNA expression profiling of the purified CTCs with NanoString's nCounter platform. This allows for accurate quantification of a panel of HCC CTC-derived mRNA markers in a non-invasive manner. The resulting vimCTC counts and mRNA profiles hold great promise to augment the ability of the current LT candidate selection algorithm. The proposed research will be implemented via Specific Aim 1a: Conducting a retrospective study in banked blood samples using NanoVelcro vimCTC Assay to refine the association between vimCTC counts and post- LT recurrence/wait-list dropout.; Specific Aim 1b: Conducting a prospective study on freshly collected blood samples to determine association between vimCTC counts and post-LT recurrence/wait-list dropout; and Specific Aim 2: Conducting a prospective study on freshly collected blood samples to determine the association between HCC CTC-RNA Assay and post-LT recurrence/wait-list dropout. The central hypothesis evaluated will be that baseline and longitudinal changes in vimCTCs and aggressive RNA signatures will significantly improve the ability of current clinicoradiologic LT selection criteria in predicting post-LT recurrence and wait-list dropout, paving the way for tumor-biology based HCC LT candidate selection practices.