Quantitation of marrow disease in neuroblastoma by real-time reverse transcription-PCR

Purpose: GD2 is abundantly expressed in neuroblastoma (NB). GD2 synthesis i s dependent on key enzyme beta 1,4-N-acetylgalactosaminyltransferase (GD2 s ynthase), We explore the potential of GD2 synthase mRNA as a molecular mark er of minimal residual disease by first comparing it quantitatively with im munocytology and then testing ifs clinical utility, Experimental Design: A real-time reverse transcription-PCR assay to quantif y mRNA of GD2 synthase was developed. Quantitation was normalized to endoge nous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex PCR, Results: The upper limit of normal was defined by 31 normal marrow and bloo d samples, achieving a sensitivity of one NB cell in 10(6) normal mononucle ar tells. When 155 bone marrows from 100 NE patients were studied, GD2 synt hase mRNA levels correlated well with the number of GD2-positive cells, as measured by immunocytology using anti-GD2 antibodies (r = 0.96). This is th e first demonstration of the quantitative relationship between a specific m RNA and the actual number of tumor cells. In a pilot study, the level of th is transcript in sequential marrow samples of five stage 4 NE patients corr elated closely with their clinical status. At 24 months after diagnosis, av ailable remission bone marrows from patients with advanced NE 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. Posi tivity was strongly associated with progression-free (P < 0.005) and overal l survival (P < 0.001). Conclusions: Measurement of tumor cells by real-time quantitative reverse t ranscription-PCR of GD2 synthase has potential clinical utility, especially for the detection of minimal residual disease.