Received on October 7, 2008
Accepted on March 24, 2009

Cancer Diagnostics

Detecting Minimal Residual Disease in Neuroblastoma: the Superiority of a Panel of Real-Time Quantitative PCR Markers

¹ Department of Pediatric Oncology, Emma Children's Hospital, Academic Medical Center, Amsterdam, the Netherlands, and Department of Immunohematology, Sanquin-AMC Landsteiner Laboratory, Amsterdam, the Netherlands
² Department of Immunohematology, Sanquin-AMC Landsteiner Laboratory, Amsterdam, the Netherlands
³ Department of Pediatric Oncology, Hacettepe University Faculty of Medicine, Ankara, Turkey
⁴ Department of Pediatric Oncology/Hematology, Sophia Children's Hospital, Erasmus Medical Center, Rotterdam, the Netherlands
⁵ Department of Pediatric Oncology and Hematology, Children's Hospital, University of Cologne, Cologne, Germany
⁶ Department of Human Genetics, Academic Medical Center, Amsterdam, the Netherlands
⁷ Department of Pediatric Oncology, Emma Children's Hospital, Academic Medical Center, Amsterdam, the Netherlands

^* To whom correspondence should be addressed. E-mail: e.vanderschoot{at}sanquin.nl.

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: one for analysis of BM samples (PHOX2B, TH, DDC, CHRNA3, and GAP43) and one 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 one 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.