Minimal residual disease (MRD) is derived from tumor-initiating cells (TICs) and is responsible for tumor relapse. Neuroblastoma is characterized by extreme tumor heterogeneity, and more than half of high-risk patients experience tumor relapse. To overcome tumor heterogeneity and achieve more sensitive detection of MRD, several sets of real-time RT-PCR markers have been reported for MRD monitoring in neuroblastoma patients from different centers. However, these markers vary across centers and are still being validated. In the present study, we validated the ability of 14 commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11 real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range. By using this protocol, we prospectively monitored MRD in 73 bone marrow (BM), 12 peripheral blood stem cell and 8 peripheral blood samples from 14 neuroblastoma patients treated at a single center. We scored 100, 56, 56 and 57% BM cytology-positive, elevated vanillylmandelic acid (VMA), elevated homovanillic acid (HVA) and elevated neuron-specific enolase (NSE) samples as MRD-positive, respectively. MRD was also positive in 48, 45, 46 and 43% of the BM cytology-negative and normal VMA, normal HVA and normal NSE samples, respectively. These results suggest that the present MRD detection protocol based on the expression of a set of 11 real-time RT-PCR markers in neuroblastoma TICs achieves sensitive MRD monitoring in neuroblastoma patients.

Abstract. Neuroblastoma is an aggressive pediatric tumor accounting for ~15% of cancer-associated mortalities in children. Despite the current intensive therapy, >50% of high-risk patients experience tumor relapse or regrowth caused by the activation of minimal residual disease (MRD). Although several MRD detection protocols using various reverse transcription-quantitative polymerase chain reaction (RT-qPCR) markers have been reported to evaluate the therapeutic response and disease status of neuroblastoma patients, their clinical significance remains elusive. The present study reports two high-risk neuroblastoma patients, whose MRD was consecutively monitored using 11 RT-qPCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) during their course of treatment. The two patients initially responded to the induction therapy and reached MRD-negative status. The patients' MRD subsequently became positive with no elevation of their urinary homovanillic acid, urinary vanillylmandelic acid and serum neuron-specific enolase levels at 13 or 19 weeks prior to the clinical diagnosis of tumor relapse or regrowth. The present cases highlight the possibility of consecutive MRD monitoring using 11 markers to enable an early detection of tumor relapse or regrowth in high-risk neuroblastoma patients.