Using droplet digital PCR to analyze MYCN and ALK copy number in plasma from patients with neuroblastoma

The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for and copy number status in plasma from neuroblastoma patients. Our ddPCR protocol accurately discriminated between and amplification, gain and normal diploid status in a large panel of neuroblastoma cell lines, and discrepancies with reported and status were detected, including a high-level amplification in NB-1, a gain in SH-SY5Y, a high-level amplification in IMR-32 and gains in BE(2)-C, Kelly, SH-SY5Y and LAN-6. and status were also reliably determined from cell-free DNA derived from medium conditioned by the cell lines. and copy numbers of subcutaneous neuroblastoma xenograft tumors were accurately determined from cell-free DNA in the mouse blood plasma. In a final validation step, we accurately distinguished and copy numbers of the corresponding primary tumors using retrospectively collected blood plasma samples from 10 neuroblastoma patients. Our data justify the further development of molecular disease characterization using cell-free DNA in blood plasma from patients with neuroblastoma. This expanded molecular diagnostic palette may improve monitoring of disease progression including relapse and metastatic events as well as therapy success or failure in high-risk neuroblastoma patients.