Abstract B14: ALT specific C-circle detection in a whole blood of cancer patients using qPCR assay

Abstract

Abstract The goal of this study was to evaluate a real-time qPCR assay, which can be used in clinical research environments for detection of telomeric C-circle DNA in tumors and 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 cancers overall rely on the ALT mechanism for continued growth and survival. Initial retrospective analyses showed that ALT ranges from 25% to 60% in sarcomas and 5% to 15% in carcinomas. ALT is also a potential ‘escape route’ for tumor cells to survive under pressure from treatment with anti-telomerase drugs. Results of in vitro and animal model experiments confirm the possibility of such ALT induction. The existing detection method using a radioactive probe was shown to detect C-circle DNA in osteosarcoma patients' blood and plasma. However, this method requires highly experienced technicians and dedicated laboratory facilities for handling radioisotopes. Here we present data that the qPCR assay is at least 20 times more sensitive than its radioisotope predecessor and detects C-circle DNA in blood of sarcoma patients. DNA from 20 frozen whole blood and serum samples from patients with adenocarcinoma (10), sarcoma (4), desmoid fibrosarcoma (1) and melanoma (5) was isolated using QIAmp Kit. Rolling circle amplification of telomere C-circle DNA, which is the core of the assay, was done with and without Phi29 polymerase. Half of the reaction was subjected to radioisotope detection and half for qPCR. Positive results in both isotope and qPCR assay have been confirmed for 3 sarcoma samples. Two serum samples (one from adenocarcinoma and one from melanoma patient) were positive in qPCR, while negative in isotope assay. A clinically applicable qPCR C-circle assay, which can also be used as a noninvasive blood-based test, will be suitable for ALT activity detection in cancer, as a companion diagnostic tool for anti-telomerase therapy and prognosis, and for anti-ALT and anti-telomerase drug development. This study was partially supported by the NIH SBIR Grant 1R43CA168124-01A1. Citation Format: Andrei Malykh, Jonathan Doyle. ALT specific C-circle detection in a whole blood of cancer patients using qPCR assay. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B14.