BACKGROUNG: Bone marrow (BM) evaluation is one of the crucial steps of the clinical staging in neuroblastoma (NB) patients. Additionally, it is believed that the most likely cause of the treatment failure is the presence of NB cells persistent after therapy. Unfortunately, a limit level of tumor cells in BM or their molecular markers that would be clinically significant have not been yet well established.
AIMS: The detection of NB cells in BM samples before and during therapy, and the evaluation of diagnostic accuracy of designed method.
MATERIALS AND METHODS: We employed study samples: 145 BM (bilateral, before and during treatment) from 40 NB patients in all stage and control samples: 32 BM from non-NB cancer patients, 10 peripheral bloods (PB) from healthy volunteers, 3 NB cell lines. BM samples were obtained from patients treated at the Department3 between 2010 and 2015 year. The QRT-PCR was used to evaluate transcripts of TH and PHOX2B as marker genes and B2M as a control gene. The relative quantification was used to indicate metastases (ΔCtmeancontrol-ΔCtsample≥3). The absolute quantification was measured with plasmid cDNA templates for TH, PHOX2B and B2M genes for establishing standard curves. The absolute quantification was also used to trace the ROC curve with anti-GD2 immunostaining as a reference. The specificity and the sensitivity of selected marker genes were adequately checked by relative evaluation of its expression in control samples and in serial dilutions of NB cell lines in PB samples.
RESULTS: Positive results were obtained for metastatic disease. The ROC curves determined cut-points for true-positive samples (TH=1,32 PHOX2B=3,67 transcripts per 105B2M). The PHOX2B gene was the most specific NB cells marker. The sensitivity of QRT-PCR was confirmed at 10-6.
CONCLUSION: It was showed the utility and the high sensitivity of designed method for the detection and monitoring metastases in BM of NB patients. Longitudinal studies are needed to definitely establish its clinical usefulness.