Unlimited proliferation of cancer cells requires activation of one of two telomere maintenance mechanisms: telomerase or homologous recombination-based ALT. C-circles (CC) (extrachromosomal partially single-stranded circular telomeric DNA) are a marker of ALT activity. We ask whether CC can be detected as cell-free circulating tumor DNA in the serum of patients with CC+ neuroblastoma tumor and can therefore be used as a serum ALT biomarker.
We first examined 149 high-risk neuroblastoma tumors and found 24% (n=36) of tumor DNA to be CC+, i.e. ALT+. The outcome of ALT+ neuroblastoma (all MYCN non-amplified) was as poor as that of MYCN-amplified neuroblastoma (n=55) (5-yr OS: 32% vs. 28%). Serum was available for analysis in a subset of 35 tumors where 40% (n=14) were CC+. CC were measured in the circulating DNA extracted from the serum (referred to as serum CC level). The amount of circulating DNA ranged from 8.6 to 373 ng/100uL of serum (median 46.2) and there was no significant difference in circulating DNA level between patients with CC+ and CC- tumor (median 41.5 vs. 47.4 ng/100uL; P=0.7).
Serum CC levels ranged from 1.2 to 1556 AU (median 50 AU) and there was no correlation between serum CC and circulating DNA levels (P=0.5). However, there was significant correlation between serum and tumor CC levels (P=0.001) and serum CC level was significantly higher in the CC+ than the CC- tumor group (median 340 vs. 25 AU; P<0.001). Serum CC for the normal control (individual with no known cancer) was 0.8 AU. In the ALT+ tumor group, 12 of 14 had serum CC >100 AU. In the ALT- tumor group, 3 of 21 has serum CC >100 AU. Using a cut-off of 100 AU, the sensitivity of serum CC for detecting ALT+ tumor was 86% (12/14), with specificity of 86% (18/21) and concordance 86% (30/35). The results of this study therefore support CC as a potential serum biomarker for ALT tumor activity.