Serially quantifying TERT rearrangement breakpoints in circulating tumor DNA enables minimal residual disease monitoring in patients with neuroblastoma.

Telomerase is reactivated by genomic TERT rearrangements in ~30% of diagnosed high-risk neuroblastomas. Dismal patient prognosis results if the RAS/MAPK/ALK signaling transduction network also harbors mutations. We present a liquid biopsy-based monitoring strategy for this particularly vulnerable pediatric patient subgroup, for whom real-time molecular diagnostic tools are limited to date. Droplet digital PCR assays quantifying patient-individualized TERT rearrangement breakpoint copies, ALK copy numbers and allelic ALK p.R1275Q mutation frequencies were applied to longitudinally collected liquid biopsies (peripheral blood and bone marrow plasma, n=44 biosamples), the mononuclear cell fraction from bone marrow and matched tumor samples. Marker detection was compared with current gold standard diagnostics. Reanalysis of whole-genome and targeted panel sequencing data from 169 patients identified 64 TERT-rearranged neuroblastoma samples collected at initial and/or relapse diagnosis from 55 patients (254 total TERT rearrangement events). Detection and quantification of unique TERT rearrangement breakpoints in as little as 1ng of total cell-free DNA in peripheral blood plasma improved therapy response assessment and early relapse detection in individual patients. Proof-of-concept is provided for minimal residual disease detection in the bone marrow niche, from which relapses frequently arise, by analyzing unique TERT rearrangement breakpoints in bone marrow plasma-derived cell-free DNA. TERT rearrangement breakpoints, as a single marker or combined with mutations in the RAS/MAPK/ALK signaling transduction network, can serve as robust and highly sensitive biomarkers for disease activity and spatially and temporally resolve disease better than current gold standard diagnostics in individual patients with TERT-driven neuroblastoma.