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Quantitation of Marrow Disease in Neuroblastoma by Real-Time Reverse Transcription-PCR¹

Department of Pediatrics, Memorial Sloan-Kettering Cancer Center New York, New York 10021

Purpose: GD2 is abundantly expressed in neuroblastoma (NB). GD2 synthesis is dependent on key enzyme ß 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.

Experimental Design: 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 10⁶ 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.

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