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.

Project Narrative In order to continually grow, cancer cells need to maintain telomeres which if not maintained by some form of telomere maintenance mechanism (TMM) done by telomerase, an enzyme that adds telomeric repeats to the ends of chromosomes or by the alternative lengthening of telomeres (ALT) mechanism. This project will define parameters that impact on two assays needed to define cancer TMM as prognostic markers and companion diagnostics for novel drugs: TERT mRNA expression qPCR and the unique PCR assay for telomeric DNA C-circles, which provides a specific marker for ALT cancers. We will also develop an assay to detect C-circles in patient blood samples and complete regulatory steps needed for clinical implementation of these assays.