Integrated analysis of HCC CTCs for Liver Transplant Candidate Selection

项目总结 肝细胞癌是全球癌症相关死亡的第二大常见原因。肝 移植(LT)是治疗不能切除的、非转移性肝癌患者的唯一根治方法。 符合严格的放射学肿瘤大小和数量标准(米兰标准)。符合条件的候选人要经过有限的 允许对肿瘤生物学进行评估的观察期(&gt；6个月)，但这本身就存在肝细胞癌的风险 15%-20%的患者在1年后进展和等待名单退出。尽管有这些选择做法，但在LT之后 肝细胞癌复发困扰高达20%的患者，是造成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 等待名单退出和肝移植后复发的风险，并允许改进肝癌患者接受治疗的优先顺序 稀有的同种异体肝移植。这项提议旨在开发一种基于血液的综合分析(即纳米尼龙搭扣 VimCTC法检测Vimentin+循环肿瘤细胞[CTCs]和肝细胞癌CTC-RNA检测肝细胞癌 特定的RNA签名)，以确定最适合肝移植的患者。集成的 本实验为研究肝细胞癌CTCs的表型和分子特性提供了一种新的方法。 使用肝癌特异性多标记捕获鸡尾酒和优化的免疫细胞化学(ICC)染色 协议，所提出的纳米魔术贴VimCTC检测能够识别肝癌CTC亚群与 波形蛋白表达(命名为vimCTC，DAPI+/CK+/CD45-/vientin+)。这个亚群与 接受治疗意向治疗的临床无法区分的早期患者的亚组中复发增加 治疗。CTC-RNA检测是通过CTC分离和Click芯片相结合而建立的，具有以下特点 Click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 利用纳米串的nCounter平台对纯化的CTCs进行图谱分析。这样就可以准确地量化 一组肝癌CTC衍生的非侵入性的mRNA标记物。由此产生的vimCTC计数和mRNA Profile具有极大的潜力来增强当前LT候选选择算法的能力。 拟议的研究将通过具体目标1a实施：在BANKED进行回溯性研究 使用NanoVelcro vimCTC分析血液样本以细化vimCTC计数与后... 复发/等待名单退出；具体目标1b：对新鲜采集的血液进行前瞻性研究 样本，以确定VimCTC计数与LT后复发/等待名单辍学之间的关联；以及 具体目标2：对新采集的血液样本进行前瞻性研究，以确定两者之间的联系 肝细胞癌CTC-RNA检测与肝移植术后复发/等待名单退出之间的差异。被评估的中心假说将 VmCTC和侵略性RNA签名的基线和纵向变化将显著改善 现行临床放射学肝移植选择标准在预测肝移植术后复发和等待名单退出方面的能力 为基于肿瘤生物学的肝细胞癌移植候选选择实践铺平道路。

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.