Detecting minimal residual disease in neuroblastoma: The superiority of a panel of real-time quantitative PCR markers

BACKGROUND: PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) patients can be used for initial staging and monitoring therapy response in bone marrow (BM) and peripheral blood (PB). PHOX2B has been identified as a sensitive and specific MRD marker; however, its expression varies between tumors. Therefore, a panel of markers could increase sensitivity. METHODS: To identify additional MRD markers for NB, we selected genes by comparing SAGE (serial analysis of gene expression) libraries of healthy and NB tissues followed by extensive real-time quantitative PCR (RQ-PCR) testing in samples of tumors (n = 56), control BM (n = 51), PB (n = 37), and cell subsets. The additional value of a panel was determined in 222 NB samples from 82 Dutch stage 4 NB patients (54 diagnosis BM samples, 143 BM samples during/after treatment, and 25 PB samples). RESULTS: We identified 2 panels of specific RQ-PCR markers for MRD detection in NB patients: 1 for analysis of BM samples (PHOX2B, TH, DDC, CHRNA3, and GAP43) and 1 for analysis of PB samples (PHOX2B, TH, DDC, DBH, and CHRNA3). These markers all showed high expression in NB tumors and no or low expression in control BM or PB samples. In patients' samples, the PHOX2B marker detected most positive samples. In PB samples, however, 3 of 7 PHOX2B-negative samples were positive for 1 or more markers, and in BM examinations during treatment, 7% (6 of 86) of the PHOX2B-negative samples were positive for another marker. CONCLUSIONS: Because of differences in the sensitivities of the markers in BM and PB, we advise the use of 2 different panels to detect MRD in these compartments.