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.
Accurate diagnosis and prognosis of cancer is an area of intense medical need, and is of great economic importance for the public health system. High-throughput screening of tumor samples and blood from oncology patients using the C-circles assay will identify ALT[+] cancers. This knowledge can be used to predict and monitor anti-cancer drug response. A high-throughput ALT assay can also be used to discover new drugs to inhibit ALT activity and thereby suppress a significant pathway for cancer cell survival. The ALT assay can potentially be used as a surrogate endpoint in clinical trials, as well as a diagnostic / prognostic tool. The goal of this project is to demonstrate feasibility of employing real-time qPCR for detection of telomeric C- circle DNA in peripheral blood of cancer patients.
