DETECTION OF NEUROBLASTOMA-CELLS IN CD34(-CELLS USING A COMBINATION OF TYROSINE-HYDROXYLASE NESTED RT-PCR AND ANTIGANGLIOSIDE G(D2) IMMUNOCYTOCHEMISTRY() SELECTED PERIPHERAL STEM)

A sensitive assay was developed for the detection of neuroblastoma cel l contamination in CD34(+) selected and unseparated peripheral blood s tem cells (PBSC) used for autologous transplantation in stage 4 neurob lastoma patients. Specifically, we established a non-radioactive neste d cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expr ession combined with anti-disialoganglioside G(D2) immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs co rrelated to cell line dependent basal TH gene expression levels and ra nged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytoch emistry was 1:10(5). We observed the highest PBSC contamination rate o f 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and G(D2) immunocytochemistr y in combination. Furthermore, a clinically applied purging method, CD 34(+) selection by immunoabsorption (CD34(+) purity 42.4%), was used o n 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34() selection and 56% (9/16) remained contaminated. A significant reduct ion of neuroblastoma cell contamination by CD34(+) selection was not d etectable, but the absolute amount of re-infused tumour cells was decr eased due to 100-fold smaller cell counts of CD34(+) selected grafts u sed for transplantation. 22 PBSC preparations were used for transplant ation. A Kaplan-Meier analysis showed an event-free survival probabili ty of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versu s 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamin ation. Our data suggest that the combined use of TH nPCR and G(D2) imm unocytochemistry is optimal to detect contamination and monitor purgin g strategies. (C) 1997 Elsevier Science Ltd.