Development of cell-free DNA assays for HCC screening and liquid biopsy

 描述（由申请人提供）：开发用于HCC筛查和液体活检的无细胞DNA测定本提案旨在开发一组在血液或尿液中发现的非侵入性无细胞（cf）DNA标记物，用于临床用作肝细胞癌（HCC）的生物标志物。该样本组将用于实验室改进法案（CLIA）证书实验室环境中的早期检测和液体活检。该面板的开发将通过与两个工业合作伙伴的合作来完成，一个是医疗诊断实验室（MDL），一家领先的诊断公司，拥有超过130种CLIA认证的PCR诊断测试，另一个是JBS科学公司，一家专门检测片段化无细胞HCC DNA修饰的公司，以及两个临床站点，托马斯杰斐逊大学和约翰霍普金斯大学。与其他癌症一样，HCC是一种基因组疾病;如果检测到HCC，则对HCC发展背后的DNA修饰的鉴定应提供HCC的明确检测和个性化护理。虽然已经有许多尝试开发cfDNA血液为基础的液体活检测试癌症，大多数都失败了。在为HCC设计的少数尝试中，还没有任何临床使用这种能力。该提议是为了开发将克服以液体活检形式将生物标志物发现带入临床使用的障碍的测定法。我们将通过检测一组DNA修饰来实现这一目标，这些修饰将能够筛查HCC，识别癌症亚型，优化药物治疗计划，并监测残留疾病，以满足癌症早期检测和个性化治疗的未满足需求。简而言之，在初步研究中，我们已经表明，存在于血液或尿液中并且来源于肿瘤细胞的大多数cfDNA是低分子量（LMW）的，<300 nts，因此需要专门设计用于小DNA检测和扩增的方法。在74例HCC尿液样本的研究中，我们检测到77%的HCC病例，并以95%的特异性将这些病例与肝硬化（n=45）和肝炎（n=42）区分开来。这是通过检测和定量对应于三种cfDNA标志物内的DNA修饰（突变或甲基化）的DNA片段来实现的：TP 53突变和甲基化GSTP 1和RASSF 1a。我们认为，仅添加两种HCC相关的DNA修饰，Tert和CTNNB 1突变，覆盖其他主要的HCC癌症途径，将大大提高该组的性能，因为几乎所有（98%，60/61）检测的HCC组织都含有这五种DNA修饰（突变或甲基化）中的至少一种。检测循环中这五种DNA标记物的检测方法必须标准化，研究必须得到证实。因此，该提案将开发和优化五种cfDNA测定法，以减少实践并确定其在HCC的早期检测和精确管理中的临床用途。可交付的组件将是CLIA认证的检测试剂盒，其临床有用性将被确定，这些检测试剂盒将准备用于我们合作的商业CLIA实验室。

 DESCRIPTION (provided by applicant): Development of cell-free DNA assays for HCC screening and liquid biopsy This proposal is for the development of a panel of non-invasive, cell-free (cf) DNA markers, found in the blood or urine, for clinical usage as biomarkers of hepatocellular carcinoma (HCC). This panel will be used in a Certificate for Laboratory Improvement Act (CLIA) lab setting for the early detection and liquid biopsy. The development of this panel will be accomplished by the partnership with two industrial partners, Medical Diagnostic Laboratories (MDL), a leading diagnostic company for over 130 CLIA-certified PCR diagnostic tests in-house, and JBS Science Inc., a company specialized in detecting fragmented cell-free HCC DNA modifications, and two clinical sites, Thomas Jefferson University and Johns Hopkins University. Like other cancers, HCC is a disease of the genome; identification of the DNA modifications underlying the development of HCC should provide unambiguous detection of HCC and personalized care, if HCC is detected. Although there have been numerous attempts to develop cfDNA blood-based, liquid biopsy tests for cancers, most have failed. Of the few attempts designed for HCC, there have not been any used clinically for that capacity. This proposal is for the development of assays that will overcome the obstacles of bringing biomarker discovery to clinical use in the form of liquid biopsies. We will achieve this through detection of a panel of DNA modifications that will enable screening of HCC, identifying cancer subtypes, optimizing drug treatment plans, and monitoring residual diseases to meet the unmet need in early detection and personalized treatment of the cancer. Briefly, in preliminary studies, we have shown that most cfDNA that is present in the blood or urine, and is derived from tumor cells, is low molecular weight (LMW), <300 nts, and thus requires methods specifically designed for small DNA detection and amplification. In a study of 74 HCC urine samples, we detected 77% of the HCC cases and distinguished these cases from liver cirrhosis (n=45) and hepatitis (n=42) with 95% specificity. This was accomplished by detection and quantification of DNA fragments corresponding to DNA modifications (mutations or methylation) within three cfDNA markers: TP53 mutations and methylated GSTP1 and RASSF1a. We believe that the addition of just two HCC-associated DNA modifications, Tert and CTNNB1 mutations, covering the other major HCC cancer pathways will greatly improve the performance of this panel, since almost all (98%, 60/61) HCC tissue tested contained at least one of these five DNA modifications (mutations or methylation). The assays for detecting these five DNA markers in the circulation must be standardized, and the studies must be confirmed. This proposal will therefore develop and optimize the five cfDNA assays for reduction to practice and to determine their clinical utilit in the early detection and precision management of HCC. The deliverable components will be CLIA-certified assays for which their clinical usefulness will be determined, and these assays will be ready for use in commercial CLIA labs, with which we have partnered.