Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line

Background: Alkylator resistance contributes to treatment failure in high-r isk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melph alan (L-PAM) for many neuroblastoma cell lines, but optimal use of this com bination needs to be defined because clinical responses have been less freq uent and not durable. Patients and Methods: The authors established and characterized a neuroblas toma cell line (CHLA-171) from a patient who died of progressive disease af ter treatment with BSO and low-dose L-PAM. Results: CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (a nti-ganglioside GD2 antibody) strongly positive) characteristic of neurobla stoma cell lines. Twenty-four hours of BSO treatment (0-1,000 mu mol/L) max imally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic resp onse of CHLA-171 to BSO and L-PAM, alone and in combination, was measured b y digital image microscopy (DIMSCAN) over a range of drug concentrations an d compared with drug levels obtained in the patient during BSO/L-PAM therap y. As single agents, CHLA-171 was highly resistant to L-PAM (LD90 = 42 mu m ol/L; peak plasma concentration in the patient equals 3.9 mu mol/L) and mod erately resistant to BSO (LD90 = 509 mu mol/L steady-state concentration in the patient equals 397 mu mol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3-4 logs of cell kil l, combination index <1) at clinically achievable levels of BSO (100-400 <m u>mol/L) and levels of L-PAM (10-40 mu mol/L) clinically achievable only wi th hematopoietic stern cell support. Conclusions: The in vitro results obtained for CHLA-171 suggest that BSO/L- PAM therapy may be optimally effective for drug-resistant neuroblastoma usi ng myeloablative doses of L-PAM.