Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma.

A quantitative and precise measure of treatment response is warranted in neuroblastoma patients. We compared three quantitative methods often used for detection of minimal residual disease in such patients. Specificity, sensitivity and concordance of immunocytochemistry, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were compared using experimental cell suspensions (n = 8) and clinical samples (n = 126). Neuroblastoma cells were identified by immunocytochemistry and flow cytometry using anti-GD2 (14.G2a) and anti-NCAM (5.1H11) antibodies, whereas tyrosine hydroxylase mRNA was the molecular target for quantitative RT-PCR. The sensitivity using flow cytometry was 1-2 logs less than using immunocytochemistry or quantitative RT-PCR. All control samples (n = 35) tested negative by immunocytochemistry, whereas 2/34 (6%) and 1/14 (7%) were false positive by quantitative RT-PCR and flow cytometry respectively. Concordant results were obtained in 85% of patient samples (n = 116) analyzed in parallel by quantitative RT-PCR and immunocytochemistry, whereas 71% of samples analyzed by flow cytometry and immunocytochemistry were concordant (n = 35). The correlation between tumor cell levels analyzed by quantitative RT-PCR and immunocytochemistry was high (r = 0.78, p < 0.001). Quantitative RT-PCR and immunocytochemistry both reliably detected very low levels of neuroblastoma cells in clinical samples. The agreement and correlation between these methods were high. In comparison, flow cytometry was less sensitive.