Ganglioside GM2/GD2 synthetase mRNA is a marker for detection of infrequent neuroblastoma cells in bone marrow

GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-Cer (GM2)/GalNAc beta1-4 (NeuAc alpha2-8NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer (GD2) synthetase [b eta -1,4-N-acetyl-galactosaminyl transferase (GalNAc-T)] mRNA, which encode s a key glycosyltransferase for ganglioside GD2 synthesis, was assessed as a molecular marker for detecting metastatic neuroblastoma cells in bone mar row (BM). GalNAc-T mRNA expression by neuroblastoma cell lines (n = 15), pr imary untreated neuroblastoma rumors (n = 29), morphologically normal BM (n = 22), peripheral blood stem cells (n = 10) from patients with cancers oth er than neuroblastoma, and blood mononuclear cells from normal donors (n = 17) was assessed by using reverse transcriptase-polymerase chain reaction ( RT-PCR) and electrochemiluminescence detection assay (RT-PCR/ECL). BM harve sted from 15 neuroblastoma patients was tested before and after ex vivo imm unomagnetic bead purging, and results were compared to immunocytological an alysis of the same specimens. All neuroblastoma cell lines (mean, 653 X 10( 3) ECL units) and primary tumors (mean, 683 X 10(3) ECL units) were positiv e for significant expression of GalNAc-T mRNA compared to normal blood and BM cells. The RT-PCR/ECL assay could detect GaINAc-T mRNA in 100 pg of tota l RNA, and in a mixture of one neuroblastoma cell among 10(7) normal BM or blood cells. Eight of 15 autologous BM cells harvested from patients with n euroblastoma had tumor cells detectable by immunocytology, and all 15 were positive for GaINAc-T mRNA. After ex vivo purging, none of the BM cells was immunocytology-positive, but six remained positive by the RT-PCR/ECL assay . GaINAc-T mRNA provides a specific and sensitive molecular marker for RT-P CR/ECL detection of infrequent neuroblastoma cells in BM.