Robust assays to define telomere maintenance mechanisms as cancer biomarkers.

摘要 端粒是染色体末端带有TTAGGG DNA重复序列的核蛋白，它保护编码DNA不受 腐蚀和检测作为DNA损伤。端粒维持是癌细胞无限增殖所必需的 容量。大多数癌症通过激活含有催化的端粒酶的核糖核蛋白端粒酶来维持端粒。 TERT基因编码的亚基和TERC基因编码的RNA模板。端粒酶低或无端粒酶的癌症 活动经常使用另一种机制来延长端粒，在这种机制中，端粒交替延长(ALT) 端粒是通过同源端粒-DNA重组来维持的，但其机制仍然知之甚少。 ALT肿瘤含有染色体外的端粒DNA C环，它(用独特的聚合酶链式反应方法检测)提供了 特异性和敏感性的ALT生物标志物。我们已经证明，与端粒一起量化TERT mRNA的表达 DNA C环可以根据端粒维持机制(TMM)将癌症分为3组；端粒酶- 阳性(高TERT)、ALT阳性(C环)和TMM阴性。儿童癌神经母细胞瘤的分型 TMM提供的预后信息超越了目前使用的临床和生物学预后标记物，以及 我们的初步数据表明，这对其他类型的癌症也是可能的。ALT癌症一直很难治疗，但有 功能失调的端粒，创造了独特的脆弱性，可以提供新的治疗靶点。例如，我们有 最近证实在ALT神经母细胞瘤中端粒高ATMK活性导致对DNA损伤的抵抗 临床阶段的ATM抑制剂AZD0156可以逆转化疗。TMM的生物标志物既提供了 预后信息和C环分析可以潜在地作为一种伴随的诊断方法来鉴定患者 可能对新的ALT靶向治疗更敏感的肿瘤。因此，定义参数非常重要 这影响了TMM对癌症的准确评估，即TERT mRNA和DNA C环的准确定量。我们会 确定收集和存储条件的影响以及组织的最小数量和数量，以及质量 化验所需的核酸。从血液样本中识别ALT患者将具有临床实用价值，我们的 初步数据表明，在含有循环肿瘤DNA的血浆中可以检测到C环。因此，我们还建议 建立和验证血浆C-环法作为一种识别ALT癌症患者的新方法。最后，为了 确保这些分析可用于临床风险分层，并作为选择治疗的辅助诊断， 我们建议开展CAP/CLIA认证TERT mRNA qPCR和端粒DNA C-DNA所需的研究。 圆圈试验。为了实现我们的目标，我们正在整合从成人和儿童癌症中收集的样本，以及 端粒生物学和分子病理学方面的专家团队。该项目将提供有关分析的重要信息 癌症TMM表型，将对研究和临床实验室都有价值，并将使监管完成 临床实施化验所需的要求。

Abstract Telomeres are nucleoproteins with TTAGGG DNA repeats at the ends of chromosomes that protect coding DNA from erosion and detection as DNA damage. Telomere maintenance is necessary for cancer cells to have unlimited proliferation capacity. Most cancers maintain their telomeres via activation of the ribonucleoprotein telomerase containing a catalytic subunit encoded by the TERT gene and an RNA template encoded by the TERC gene. Cancers with low or no telomerase activity often use another mechanism to extend their telomeres, the Alternative Lengthening of Telomeres (ALT) in which telomeres are maintained via homologous telomeric-DNA recombination, but the mechanism is still poorly understood. ALT tumors contain extra-chromosomal telomeric DNA C-circles, which (detected with a unique PCR assay) provides a specific and sensitive ALT biomarker. We have shown that quantifying TERT mRNA expression together with telomeric DNA C-circles enables classifying cancers into 3 groups based on telomere maintenance mechanism (TMM); telomerase- positive (high TERT), ALT-positive (C-circle+), and TMM-negative. Classifying the childhood cancer neuroblastoma by TMM provides prognostic information that overrides currently employed clinical and biological prognostic markers, and our preliminary data suggest this is possible for other cancer types. ALT cancers are consistently difficult to treat but have dysfunctional telomeres, creating unique vulnerabilities that can provide novel therapeutic targets. For example, we have recently demonstrated high ATM kinase activation at telomeres in ALT neuroblastoma leads to resistance to DNA damaging chemotherapy that can be reversed with a clinical-stage ATM inhibitor AZD0156. Biomarkers of TMM provide both prognostic information and the C-circle assay can potentially serve as a companion diagnostic assay to identify patients with tumors that are likely to be more responsive to novel ALT-targeted therapies. Thus, it is important to define parameters that impact accurate TMM assessment of cancers, i.e. accurate quantitation of TERT mRNA and DNA C-circles. We will define the impact of collection and storage conditions and the minimum amounts of tissue and amounts, and quality of nucleic acids needed for the assays. Identifying ALT patients from a blood sample would have clinical utility, and our preliminary data suggest that C-circles can be detected in plasma containing circulating tumor DNA. Thus, we also propose to develop and validate a plasma C-circle assay as a novel approach to identifying patients with ALT cancers. Finally, to ensure that these assays can be used for clinical risk stratification, and as companion diagnostics for selection of therapy, we propose to carry out studies necessary for CAP/CLIA certification of TERT mRNA qPCR and the telomeric DNA C- circle assay. To accomplish our aims, we are integrating sample collection from adult and pediatric cancers together with a team of experts in telomere biology and molecular pathology. This project will provide important information on analyzing cancer TMM phenotype that will be of value to both research and clinical labs and will enable completion of regulatory requirements necessary for clinical implementation of the assays.