Abstract / Summary
Abstract Alternative lengthening of telomeres (ALT) is a recombination-based telomere maintenance mechanism that sustains replicative immortality in approximately 10–15% of human cancers, particularly those of mesenchymal and neuroepithelial origin. As precision oncology increasingly demands biomarkers that accurately define telomere maintenance mechanisms, extrachromosomal telomeric C-circles - partially single-stranded, C-rich telomeric circular DNAs - have emerged as robust, quantifiable indicators of ALT activity with growing diagnostic, prognostic, and therapeutic relevance. Here, we summarize recent mechanistic and translational advances that establish C-circles as both functional readouts of ALT biology and clinically actionable biomarkers. We integrate emerging evidence into a unified model in which replication stress drives C-circle biogenesis through two complementary replication-coupled pathways that are dynamically shaped by telomeric DNA structures and DNA repair factors, linking the molecular origins of C-circle formation to specific vulnerabilities with direct therapeutic implications. We further critically evaluate current detection technologies, spanning the gold-standard C-circle assay and its quantitative PCR and high-throughput adaptations and single-cell native FISH, which collectively enable sensitive and scalable detection across tissue specimens and liquid biopsies, including exosome-protected C-circles. Clinically, C-circles outperform surrogate genomic markers such as ATRX/DAXX loss for defining ALT status, improve prognostic stratification across pediatric brain tumors, diffuse gliomas, sarcomas, and other ALT-associated malignancies, and show considerable promise as dynamic pharmacodynamic biomarkers for treatment monitoring, minimal residual disease assessment, and longitudinal disease surveillance. Therapeutically, the molecular circuitry underlying C-circle biogenesis exposes selective ALT dependencies on replication stress and DNA repair regulators, including the BLM/BTR and FANCM pathways. Perturbation of these nodes reshapes C-circle dynamics while revealing synthetic lethal vulnerabilities that can be exploited through helicase-, nuclease-, chromatin-, replication stress-, and G-quadruplex-targeted strategies. We also discuss the important caveat that telomerase-positive tumors with hyperextended telomeres can generate C-circles, emphasizing the need for cautious interpretation of C-circle measurements. Collectively, C-circles have evolved from experimental hallmarks of ALT into mechanistically informative and clinically actionable biomarkers. Continued assay standardization and integration into tissue and liquid biopsy workflows will facilitate their application in diagnosis, patient stratification, therapeutic monitoring, and biomarker-driven clinical studies.