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posted on 2025-01-28, 10:40 authored by Jan F. Hollander, Annabell Szymansky, Jasmin Wünschel, Kathy Astrahantseff, Carolina Rosswog, Anne Thorwarth, Theresa M. Thole-Kliesch, Rocío Chamorro González, Patrick Hundsdörfer, Kathrin Hauptmann, Karin Schmelz, Dennis Gürgen, Julian M.M. Rogasch, Anton G. Henssen, Matthias Fischer, Johannes H. Schulte, Cornelia Eckert, Angelika Eggert, Marco Lodrini, Hedwig E. Deubzer <p>Bone marrow (BM) plasma is sufficient to detect <i>TERT</i> breakpoint, <i>ALK</i> p.R1275Q mutation, and <i>ALK</i> copy numbers in the novel ddPCR assay. Comparative analysis of <i>TERT</i> breakpoints, <i>ALK</i> p.R1275Q mutation, and <i>ALK</i> copy numbers in cell free DNA in the separated liquid component (BM plasma) and the cellular fraction samples from BM aspirates from patients P2 (days 0, 148, and 310 after diagnosis), P3 (days 423 and 482 after diagnosis), and P4 (day 134 in hospital). Pairs from the same patient sample are connected by lines.</p>
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ARTICLE ABSTRACT
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 which 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 [BM] plasma, n = 44 biosamples), the mononuclear cell fraction from BM 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 1 ng 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 BM niche, from which relapses frequently arise, by analyzing unique TERT rearrangement breakpoints in BM 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.
Real-time molecular monitoring of TERT-rearranged high-risk neuroblastoma is an unmet clinical need. We tested liquid biopsy-based assays for patient-individualized TERT breakpoint sequences to monitor disease in pediatric patients. Our digital PCR approach provides high resolution of spatial and temporal disease quantification in individual patients and is applicable for clinical routine.
