Real-time qPCR assay for detection of ALT associated telomeric C-circle DNA in bl

实时 qPCR 检测 BL 中 ALT 相关端粒 C 环 DNA

• 批准号: 8648290
• 负责人: Andrei G. Malykh
• 金额: $ 15万
• 依托单位: CAPITAL BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648290
• 关键词: Animal Model; Antineoplastic Agents; Appearance; Applications Grants; Area; Basic Science; Biological Assay; Blood; Blood specimen; Cancer Diagnostics; Cancer Patient; Cancer Prognosis; Carcinoma; Cell Line; Cell Survival; Clinical; Clinical Trials; Collection; Companions; Core Facility; DNA; Detection; Development; Diagnosis; Diagnostic; Economics; Evaluation; Exhibits; Fluorescent Probes; Genomic DNA; Goals; Growth; Health system; Hematopoietic Neoplasms; High Prevalence; In Vitro; Isotopes; Knowledge; Length; Malignant Neoplasms; Marketing; Medical; Methods; Monitor; Outcome; Outcomes Research; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phase; Plasma; Prevalence; Probability; Protocols documentation; Public Health; Publishing; Radioactive; Radioisotopes; Research; Route; Sampling; Sensitivity and Specificity; Services; Small Business Innovation Research Grant; Surrogate Endpoint; System; Telomerase; Telomere Length Maintenance; Telomere Maintenance; Testing; Time; Tissue Sample; Tumor Cell Line; Tumor Tissue; Validation; analytical tool; base; cancer cell; cancer type; cell immortalization; clinical practice; commercialization; drug development; economic value; experience; high throughput screening; laboratory facility; neoplastic cell; oncology; outcome forecast; peripheral blood; pressure; prognostic; public health relevance; research study; response; sarcoma; screening; telomere; tool; tumor

DESCRIPTION (provided by applicant): The overall goal of the proposed project is to investigate the feasibility of employing real-time qPCR method for detection of telomeric C-circle DNA in peripheral blood of patients with cancers that utilize Alternative Lengthening of Telomeres (ALT) as their Telomere length Maintenance Mechanism (TMM). Telomeric C-Circle DNA has been demonstrated to be specific for ALT, thus enabling the first quantitative test for ALT activity. In 85% of cancers, cells maintain telomere length due to increased activity of telomerase, while 5 - 15% of tumors from most cancer types rely on the ALT mechanism for continued growth and survival. It is therefore imperative to determine the prevalence of ALT[+] tumors. Initial retrospective analyses showed that ALT ranges from 25% to 60% in sarcomas and 5% to 15% in carcinomas. ALT can be also a potential 'escape route' for tumor cells to survive under pressure from treatment with anti-telomerase drugs. Recently published results of in vitro and animal model experiments confirm the possibility of such ALT induction. A clinically applicable noninvasive blood-based assay will be suitable for ALT activity detection in cancer, as companion diagnostic tool for anti-telomerase therapy and prognosis, as well as in anti-ALT and anti-telomerase drug development. However, the existing detection method is technically challenging and requires the use of a radioactive probe to achieve high levels of sensitivity. This requires highly experienced technicians and dedicated laboratory facilities for handling radioisotopes. Recently proposed non-isotope qPCR detection of CC-assay products has the potential for wide use in both academic research and in clinical practice for ALT[+] cancer diagnostics and prognosis. The first method was shown to have enough sensitivity to detect C-circle DNA in blood plasma, while the second has yet to be tested. The goal of Phase I is to determine the correlation between radioisotope and qPCR assays; first on a larger panel of tumor cell lines available from ATCC and then on peripheral blood of cancer patients. Specific aims and approaches include: 1) comparing SYBR Green and TaqMan based qPCR protocols using selected panel of ATCC tumor cell lines with expected higher chance of ALT, 2) confirming that sensitivity and linearity are maintained, 3) selecting the most promising method, and screening randomly selected panel of cell lines to define ALT[+], and 4) testing the final complete CC-assay protocol on matching tumor tissue and blood samples to confirm correlation, specificity and sensitivity of isotope and qPCR methods. Defining the ALT[+] cell lines in the ATCC collection has both fundamental and practical values, first by determining the prevalence of ALT and second by having confirmed panels of cell lines that can be used for research. Outcome of this research will allow large scale screening of tumor cell lines, tissue samples and blood samples from cancer patients. Such screening will reveal the ALT[-]/ALT[+] ratios in different cancer types and possibly the probability of spontaneous ALT activities. The latter observation will be very important for understanding the mechanisms of ALT appearance as an escape route for cancer cells to survive anti-telomerase therapy. When proven reliable as an analytical tool, services and kits can be developed and initially commercialized for research use. Application of the ALT assay for clinical diagnostics would require further clinical trials, ad could be the focus of a subsequent Phase II SBIR grant application.

描述（由申请方提供）：拟议项目的总体目标是研究采用实时qPCR方法检测利用端粒替代性延长（ALT）作为其端粒长度维持机制（TMM）的癌症患者外周血中端粒C环DNA的可行性。端粒C环DNA已被证明对ALT具有特异性，从而使ALT活性的第一个定量测试成为可能。在85%的癌症中，由于端粒酶活性增加，细胞保持端粒长度，而大多数癌症类型的5 - 15%的肿瘤依赖于ALT机制持续生长和存活。因此，必须确定ALT[+]肿瘤的患病率。最初的回顾性分析显示，ALT在肉瘤中的范围为25%至60%，在癌中为5%至15%。ALT也可能是肿瘤细胞在抗端粒酶药物治疗的压力下生存的潜在“逃逸途径”。最近发表的体外和动物模型实验结果证实了这种ALT诱导的可能性。临床上可应用的非侵入性基于血液的测定将适合于癌症中的ALT活性检测，作为抗端粒酶治疗和预后的伴随诊断工具，以及抗ALT和抗端粒酶药物开发。然而，现有的检测方法在技术上具有挑战性，并且需要使用放射性探针来实现高水平的灵敏度。这 需要经验丰富的技术人员和专门的实验室设施来处理放射性同位素。最近提出的CC检测产品的非同位素qPCR检测在学术研究和ALT[+]癌症诊断和预后的临床实践中具有广泛应用的潜力。第一种方法已被证明具有足够的灵敏度来检测血浆中的C环DNA，而第二种方法尚未进行测试。第一阶段的目标是确定放射性同位素和qPCR测定之间的相关性;首先在ATCC提供的更大的肿瘤细胞系组上，然后在癌症患者的外周血上。具体目标和办法包括：1）使用具有预期更高的ALT机会的选定的ATCC肿瘤细胞系组比较基于SYBR绿色和TaqMan的qPCR方案，2）确认保持了灵敏度和线性，3）选择最有希望的方法，并筛选随机选择的细胞系组以确定ALT[+]，和4）在匹配的肿瘤组织和血液样品上测试最终的完整CC-测定方案，以确认同位素和qPCR方法的相关性、特异性和灵敏度。确定ATCC保藏中的ALT[+]细胞系具有基本和实用价值，首先是通过确定ALT的患病率，其次是通过确认可用于研究的细胞系组。这项研究的结果将允许大规模筛选肿瘤细胞系，组织样本和癌症患者的血液样本。这种筛查将揭示不同癌症类型中的ALT[-]/ALT[+]比率，并可能揭示自发ALT活动的概率。后一种观察对于理解ALT作为癌细胞在抗端粒酶治疗中存活的逃逸途径的机制将是非常重要的。当证明作为一种分析工具是可靠的时，可以开发服务和成套工具，并初步商业化用于研究。ALT检测在临床诊断中的应用需要进一步的临床试验，这可能是随后的II期SBIR资助申请的重点。