Abstract Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to offset this telomere loss. Several cancer subtypes types, including neuroblastomas and sarcomas, use a telomerase-independent strategy for telomere maintenance, alternative lengthening of telomeres (ALT). The molecular event(s) through which ALT occurs in cancer remain poorly understood. Increased knowledge of these mechanism(s) is critical to our ability to effectively treat ALT(+) cancers. ALT(+) cancers harbor several hallmarks, including telomere DNA that manifests as unique, ultrabright foci by telomere-specific in situ hybridization (FISH) in histologic cancer specimens. Despite the fact that ALT-associated telomere DNA foci (ATDFs) are a reliable marker for ALT occurring in a cancer, not all cells within the cancer display them. Still, ATDFs are predicted to be the nodes for a recombination-based telomere elongation process in ALT, so may be critical for the ALT process. Despite their potential importance to the biology of ALT(+) cancers, cells containing ATDFs have not been specifically isolated and characterized. In order to study the ATDF biomarker, we have performed telomere-specific FISH on ALT(+) cancer cells in suspension, followed by isolation of the ATDF(+) cells by flow cytometry. Our approach is modified from a well-validated clinical assay (“Flow FISH”) used for measuring telomere lengths in blood. Ours is the first known application of Flow FISH to solid tumor cells. We have successfully and reproducibly isolated pure populations of ATDF(+) and ATDF(-) cells from two well-characterized ALT(+) cancer cell lines: SAOS2 (an osteosarcoma cell line) and SK-N-FI (a neuroblastoma cell line). Thus, we have overcome the technical barriers that are necessary to study ATDF(+) cancer cells in order to understand this putative biomarker and its mechanistic role in ALT-mediated telomere maintenance. Work is ongoing to study ATDF(+) versus ATDF(-) ALT-positive cancer cells through gene-expression profiling and proteomic analysis to 1) determine the existence of actionable pathways that will allow for therapeutic targeting of ATDF(+) cells, and 2) examine the mechanistic link of ATDFs to the ALT process. Therefore, for the first time, we have developed an approach to effectively and specifically study the population of cancer cells containing an ALT-specific biomarker. Through this ongoing work on multiple cancer types, we will unlock critical information about the global ALT mechanism, which will provide novel, actionable targets for ALT(+) cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Christopher M. Heaphy, Alan K. Meeker. Isolation and characterization of cancer cells containing ultrabright telomere DNA foci associated with alternative lengthening of telomeres (ALT): A novel utility for combined telomere-specific FISH and flow cytometry (Flow FISH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1467.