The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.

The C-Circle Assay for alternative-lengthening-of-telomeres activity.

The C-Circle Assay has satisfied the need for a rapid, robust and quantitative ALT assay that responds quickly to changes in ALT activity. The C-Circle Assay involves (i) extraction or simple preparation (Quick C-Circle Preparation) of the cell's DNA, which includes C-Circles (ii) amplification of the self-primed C-Circles with a rolling circle amplification reaction and (iii) sequence specific detection of the amplification products by native telomeric DNA dot blot or telomeric qPCR. Here we detail the protocols and considerations required to perform the C-Circle Assay and its controls, which include exonuclease removal of linear telomeric DNA, production of the synthetic C-Circle C96 and modulation of ALT activity by γ-irradiation.

Challenges in identifying candidate amplification targets in human cancers: chromosome 8q21 as a case study.

Detailed genomic characterization of cancer specimens is required to identify all genes whose dysregulation contributes to tumorigenesis and/or tumor progression. These include amplification target genes, whose oncogenic functions derive from their overexpression in response to increased gene copy number, and which increasingly serve as therapeutic targets and predictive markers. We propose that identifying novel amplification target genes is becoming more challenging, and may require the comparative analysis of multiple studies mapping gene copy number changes and/or defining associations between gene copy number and expression. We therefore reviewed the array comparative genomic hybridization and single nucleotide polymorphism profiling literature to identify copy number increases that were restricted to chromosome 8q21 in human cancers, which were reported most frequently in breast cancer. We determined the minimal regions of overlap between gained regions and then examined which chromosome 8q21 genes were most frequently overexpressed, or otherwise supported, in individual studies. As these combined approaches supported the previously proposed amplification targets TCEB1, TPD52, and WWP1, the comparison of multiple genomic studies may therefore effectively predict candidate gene amplification targets, and prioritize these for further study.