Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR.
OBJECTIVE
GD2 is abundantly expressed in neuroblastoma (NB). GD2 synthesis is dependent on key enzyme beta 1,4-N-acetylgalactosaminyltransferase (GD2 synthase). We explore the potential of GD2 synthase mRNA as a molecular marker of minimal residual disease by first comparing it quantitatively with immunocytology and then testing its clinical utility.


METHODS
A real-time reverse transcription-PCR assay to quantify mRNA of GD2 synthase was developed. Quantitation was normalized to endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex PCR.


RESULTS
The upper limit of normal was defined by 31 normal marrow and blood samples, achieving a sensitivity of one NB cell in 10(6) normal mononuclear cells. When 155 bone marrows from 100 NB patients were studied, GD2 synthase mRNA levels correlated well with the number of GD2-positive cells, as measured by immunocytology using anti-GD2 antibodies (r = 0.96). This is the first demonstration of the quantitative relationship between a specific mRNA and the actual number of tumor cells. In a pilot study, the level of this transcript in sequential marrow samples of five stage 4 NB patients correlated closely with their clinical status. At 24 months after diagnosis, available remission bone marrows from patients with advanced NB diagnosed at >1 year of age initially treated with protocols N6 and N7 at Memorial Sloan-Kettering Cancer Center (n = 44) were analyzed for GD2 synthase mRNA. Positivity was strongly associated with progression-free (P < 0.005) and overall survival (P < 0.001).


CONCLUSIONS
Measurement of tumor cells by real-time quantitative reverse transcription-PCR of GD2 synthase has potential clinical utility, especially for the detection of minimal residual disease.
Cheung
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IY
Department of Pediatrics, Memorial Sloan-Kettering Cancer Center New York, New York 10021, USA. cheungi@mskcc.org
Cheung
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NK
D001854:Bone Marrow Cells / Q000378:metabolism; D019046:Bone Marrow Neoplasms / Q000175:diagnosis* / Q000556:secondary*; D018076:DNA, Complementary / Q000378:metabolism; D018450:Disease Progression; D018572:Disease-Free Survival; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis; D009447:Neuroblastoma / Q000175:diagnosis* / Q000473:pathology*; D010865:Pilot Projects; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
D018076:DNA, Complementary; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017350:N-Acetylgalactosaminyltransferases; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase