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(American Journal of Pathology. 2001;159:493-500.)
© 2001 American Society for Investigative Pathology

Technical Advances

Ganglioside GM2/GD2 Synthetase mRNA Is a Marker for Detection of Infrequent Neuroblastoma Cells in Bone Marrow

Dave S. B. Hoon^*, Christine T. Kuo^*, Shenghua Wen, Hong Wang, Leonid Metelitsa, C. Patrick Reynolds and Robert C. Seeger

From the Department of Molecular Oncology,^*
John Wayne Cancer Institute, Santa Monica; the Division of Hematology-Oncology,
Department of Pediatrics, Children’s Hospital Los Angeles, University of Southern California School of Medicine Los Angeles; and the Children’s Cancer Group,
Arcadia, California

GalNAcß1-4(NeuAc2-3)Galß1-4Glcß1-Cer (GM2)/GalNAcß1-4(NeuAc2-8NeuAc2-3)Galß1-4Glcß1-1Cer (GD2) synthetase [ß-1,4-N-acetyl-galactosaminyl transferase (GalNAc-T)] mRNA, which encodes a key glycosyltransferase for ganglioside GD2 synthesis, was assessed as a molecular marker for detecting metastatic neuroblastoma cells in bone marrow (BM). GalNAc-T mRNA expression by neuroblastoma cell lines (n = 15), primary untreated neuroblastoma tumors (n = 29), morphologically normal BM (n = 22), peripheral blood stem cells (n = 10) from patients with cancers other 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 harvested from 15 neuroblastoma patients was tested before and after ex vivo immunomagnetic bead purging, and results were compared to immunocytological analysis of the same specimens. All neuroblastoma cell lines (mean, 653 x 10³ ECL units) and primary tumors (mean, 683 x 10³ ECL units) were positive for significant expression of GalNAc-T mRNA compared to normal blood and BM cells. The RT-PCR/ECL assay could detect GalNAc-T mRNA in 100 pg of total RNA, and in a mixture of one neuroblastoma cell among 10⁷ normal BM or blood cells. Eight of 15 autologous BM cells harvested from patients with neuroblastoma had tumor cells detectable by immunocytology, and all 15 were positive for GalNAc-T mRNA. After ex vivo purging, none of the BM cells was immunocytology-positive, but six remained positive by the RT-PCR/ECL assay. GalNAc-T mRNA provides a specific and sensitive molecular marker for RT-PCR/ECL detection of infrequent neuroblastoma cells in BM.

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