Quantitation of GD2 synthase mRNA by real‐time reverse transcription‐polymerase chain reaction

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Abstract
BACKGROUND: Antigen ganglioside GD2 is expressed abundantly on neuroblastoma (NB) cells. Anti‐GD2 monoclonal antibody (MoAb) 3F8 kills NB cells by complement‐dependent cytotoxicity and antibody‐dependent cellular cytotoxicity. Its utility in bone marrow (BM) purging is evaluated by a real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assay to quantify the mRNA of GD2 synthase, the key enzyme in GD2 synthesis.METHODS: From 1990 to 1993, 10 patients with relapsed/refractory Stage 4 NB participated in a pilot study. In these patients, MoAb 3F8 was used to purge tumor cells from harvested BM that had 5% or less tumor content by immunofluorescence (IF). Subsequently, 31 Stage 4 NB patients who underwent treatment on the N7 protocol (1994–1999) had their BM, which was in remission, purged by 3F8 before 131I‐3F8 myeloablative radioimmunotherapy. GD2‐positive tumor cells before and after purging were quantified by real‐time quantitative RT‐PCR of GD2 synthase.RESULTS: GD2 positivity by IF was found before purging in six of eight patients in the pilot study. Five of six patients became negative postpurging. Of 31 patients on the N7 protocol, the more sensitive real‐time quantitative RT‐PCR detected GD2 synthase mRNA in the BM samples of 7 patients even though the prepurge BM samples were negative by histology and IF. Six of the seven BM samples became negative after 3F8 purging. Marker positivity before purging was statistically significant in predicting overall survival (P = 0.04), but not progression‐free survival (P = 0.1). In vitro hematopoietic stem cell recovery and the median time to engraftment were acceptable.CONCLUSION: Tumor cell depletion quantified by real‐time RT‐PCR demonstrated efficacy of MoAb 3F8 in BM purging. Cancer 2002;94:3042–8. © 2002 American Cancer Society.DOI 10.1002/cncr.10519

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