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

 描述(申请人提供)：发展用于肝细胞癌筛查和液体活检的无细胞DNA分析这项建议是为了开发一组在血液或尿液中发现的非侵入性、无细胞(Cf)DNA标志物，用于临床用于肝细胞癌(肝细胞癌)的生物标志物。该面板将用于实验室改进证书法案(CLIA)的实验室设置，用于早期检测和液体活检。该小组的开发将通过与两个行业合作伙伴的合作完成，这两个合作伙伴是医疗诊断实验室(MDL)和JBS Science Inc.，MDL是一家领先的诊断公司，提供130多项CLIA认证的内部PCR诊断测试，JBS Science Inc.是一家专门检测碎片化无细胞肝癌DNA修改的公司，两家临床网站是托马斯·杰斐逊大学和约翰斯·霍普金斯大学。与其他癌症一样，肝细胞癌是一种基因组疾病；如果检测到肝细胞癌，识别导致肝细胞癌发生的DNA修饰应该提供明确的肝细胞癌检测和个性化护理。尽管已经有许多尝试开发基于cfDNA血液和液体的癌症活组织检查，但大多数都失败了。在为数不多的为肝细胞癌设计的尝试中，还没有任何用于这种能力的临床应用。这项建议是为了开发分析方法，以克服以液体活组织检查的形式将生物标记物发现带入临床使用的障碍。我们将通过检测一组DNA修饰来实现这一点，这些修饰将使我们能够筛查肝癌，识别癌症亚型，优化药物治疗计划，并监测残留疾病，以满足癌症早期发现和个性化治疗的未得到满足的需求。简而言之，在初步研究中，我们已经表明，大多数存在于血液或尿液中的cfDNA来自肿瘤细胞，是低分子量(LMW)的，因此需要专门为小DNA检测和扩增而设计的方法。在对74例肝细胞癌尿样的研究中，我们检测了77%的肝细胞癌病例，并以95%的特异度将这些病例与肝硬变(n=45)和肝炎(n=42)区分开来。这是通过检测和量化与三个cfDNA标记内的DNA修饰(突变或甲基化)相对应的DNA片段来实现的：TP53突变和甲基化的GSTP1和RASSF1a。我们相信，仅增加两个与肝癌相关的DNA修饰，即TERT和CTNNB1突变，涵盖其他主要的肝癌癌症途径，将极大地提高该小组的性能，因为几乎所有(98%，60/61)被测试的肝细胞癌组织中至少包含这五种DNA修饰(突变或甲基化)之一。检测循环中这五种DNA标志物的方法必须标准化，研究必须得到证实。因此，这项建议将开发和优化五种cfDNA检测方法，以便将其还原到实践中，并确定它们在肝细胞癌早期检测和精确处理中的临床应用。可交付的成分将是CLIA认证的分析，将确定其临床用途，这些分析将准备用于商业CLIA实验室，我们已经与之合作。

 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.