Stability of PCR targets for monitoring minimal residual disease in neuroblastoma

In neuroblastoma (NB) patients, minimal residual disease (MRD) can be detected by real-time quantitative PCR (qPCR) using NB-specific target genes, such as PHOX2B and TH. However, it is unknown whether the mRNA levels of these targets vary either during treatment or at relapse. If marker genes are not stably expressed, estimation of MRD levels in bone marrow (BM) or peripheral blood will be hampered. We studied the stability of a panel of qPCR markers in primary tumors at diagnosis compared with i) paired metastasis (n = 7), ii) treated (n = 10), and iii) relapse (n = 6) tumors. We also compared relative expression of the targets in iv) primary tumors and BM at diagnosis (n = 17), v) BM and peripheral blood at diagnosis (n = 20), vi) BM at diagnosis and during treatment (n = 26), and vii) BM from different puncture sides (n = 110). Especially at diagnosis, PCR target expression is quite stable. Accurate quantification is possible when expression level can be related to the primary tumor; however, PCR target expression can alter on treatment and at relapse. If the median value of relative expression of a panel of PCR targets is used, most variations due to treatment and outgrowth of subclones level out, allowing for reliable application and quantification of MRD-PCR targets in NB patients.