Multiplexed biomarkers dynamically detect heterogeneous residual neuroblastoma cell clone activity in the bone marrow niche

Monitoring MYCN-driven high-risk neuroblastoma presents challenges to capture dynamics of all tumor cell clones at their earliest divergence to current clinical course. Not all clones may enter the bone marrow, the most important monitoring site for minimal residual disease (MRD), causing relapse in ∼50% of patients. We developed mediator-probe PCR assays to detect up to four multiplexed patient-individual genetic alterations in 37 longitudinally collected bone marrow aspirates from 8 patients with MYCN-amplified disease. Multiplexed biomarkers, including MYCN amplicon breakpoints, detected diverse neuroblastoma clones, surpassing conventional GD2 immunocytology accuracy. We provide proof-of-principle for clonally heterogeneous MRD biomarker detection (1 tumor: 10⁶ reference cells). In selected patients, multiplexed biomarkers indicated divergent dynamics, suggesting individual tumor clones differ in their ability to disseminate to the bone marrow and escape therapy. Our pilot data support integrating multiplexed MRD detection in co-clinical trials to monitor the molecular remission state during therapy and follow-up.