A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent/Refractory High-Risk Neuroblastoma.

BACKGROUND: Myeloablative therapy for high-risk neuroblastoma commonly includes melphalan. Increased cellular glutathione (GSH) can mediate melphalan resistance. Buthionine sulfoximine (BSO), a GSH synthesis inhibitor, enhances melphalan activity against neuroblastoma cell lines, providing the rationale for a Phase 1 trial of BSO-melphalan. PROCEDURES: Patients with recurrent/resistant high-risk neuroblastoma received BSO (3 gram/m(2) bolus, then 24 grams/m(2) /day infusion days -4 to -2), with escalating doses of intravenous melphalan (20-125 mg/m(2) ) days -3 and -2, and autologous stem cells day 0 using 3 + 3 dose escalation. RESULTS: Among 28 patients evaluable for dose escalation, one dose-limiting toxicity occurred at 20 mg/m(2) melphalan (grade 3 aspartate aminotransferase/alanine aminotransferase) and one at 80 mg/m(2) (streptococcal bacteremia, grade 4 hypotension/pulmonary/hypocalcemia) without sequelae. Among 25 patients evaluable for response, there was one partial response (PR) and two mixed responses (MRs) among eight patients with prior melphalan exposure; one PR and three MRs among 16 patients without prior melphalan; one stable disease with unknown melphalan history. Melphalan pharmacokinetics with BSO were similar to reports for melphalan alone. Melphalan Cmax for most patients was below the 10 µM concentration that showed neuroblastoma preclinical activity with BSO. CONCLUSIONS: BSO (75 gram/m(2) ) with melphalan (125 mg/m(2) ) is tolerable with stem cell support and active in recurrent/refractory neuroblastoma. Further dose escalation is feasible and may increase responses.

Pilot study of intravenous melphalan combined with continuous infusion L-S,R-buthionine sulfoximine for children with recurrent neuroblastoma.

PURPOSE: To evaluate BSO-mediated glutathione (GSH) depletion in combination with L-PAM for children with recurrent or refractory high-risk neuroblastoma (NB) as a means to enhance alkylator sensitivity. PROCEDURE: This pilot study (NCI #T95-0092) administered L-S,R-buthionine sulfoximine (BSO) as a bolus followed by 72 hr continuous infusion of either 0.75 g/m(2)/hr (level 1) or 1.0 g/m(2)/hr (level 2) and melphalan (L-PAM) (15 mg/m(2) bolus at hour 48 of BSO infusion). GSH in blood mononuclear cells and bone marrow was measured by enzymatic assay, BSO in plasma by HPLC. RESULTS: Thirty two patients received 58 courses of therapy (median 1, range 1-4 courses). Blood mononuclear cell GSH decreased (48 hr) to 47% ± 15.7%. Level 2 mean steady-state concentration (Css) for BSO = 524 ± 207 µM and peak L-PAM concentration = 3.32 ± 1.2 µM. Grade 3-4 leukopenia and thrombocytopenia were common. There were two deaths from CNS toxicity and acute tubular necrosis; one had a large, intracranial mass, both were receiving cephalosporin antibiotics. No other significant toxicities were seen. There were six responses (five partial and, one mixed) representing an 18% response rate; four/six responses occurred in patients that relapsed following myeloablative therapy and included a 98% reduction in volume (cm(3)) of a pelvic mass, and three/five patients with >3 log reduction of tumor in marrow as measured by immunocytology (sensitivity 1/10(5)). CONCLUSIONS: BSO/L-PAM has activity against recurrent high-risk NB. Exclusion of cephalosporin antibiotics in future clinical trials of BSO may diminish the potential for serious renal and CNS toxicity.

Long-Term Follow-up of High-Dose Chemotherapy with Autologous Stem Cell Transplantation in Children and Young Adults with Metastatic or Relapsed Ewing Sarcoma: A Single-Institution Experience.

Forty-seven patients with metastatic disease at diagnosis or recurrent Ewing sarcoma (EWS) received high-dose chemotherapy (HDC) followed by tandem (n = 20, from February 13, 1997, to October 24, 2002) or single (n = 27, from October 1, 2004, to September 5, 2018) autologous hematopoietic stem cell transplantation (ASCT). To our knowledge, this is the largest single-institution study with sustained long-term follow-up exceeding 10 years. All patients who underwent single ASCT received a novel conditioning regimen with busulfan, melphalan, and topotecan. The overall survival (OS) and disease-free survival (DFS) were 46% and 37% at 10 years and 42% and 37% at 15 years, respectively. Disease status at transplant and the time to disease relapse prior to ASCT were identified as important prognostic factors in OS, DFS, and risk of relapse. At 10 years, patients who underwent transplantation in first complete response (1CR) had an excellent outcome (OS 78%), patients in 1CR/second complete response (2CR)/first partial response (1PR) had an OS of 66%, and patients at third or more complete response, second or more partial response, or advanced disease had an OS of 26%. Ten-year OS for patients without a history of relapse, with late relapse (≥2 years from diagnosis), or with early relapse (<2 years from diagnosis) was 75%, 50%, and 18%, respectively. Selected patients in 1CR, 2CR, 1PR, and with late relapse had excellent, sustained 10- and 15-year OS and DFS.

Antagonism of buthionine sulfoximine cytotoxicity for human neuroblastoma cell lines by hypoxia is reversed by the bioreductive agent tirapazamine.

Relapse of neuroblastoma (NB) commonly occurs in hypoxic tissues. Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, is cytotoxic for NB cell lines in atmospheric oxygen (20% O(2)). Tirapazamine (TPZ) is a bioreductive agent that forms a toxic-free radical in hypoxia. We determined in four NB cell lines cytotoxicity using the DIMSCAN digital imaging fluorescence assay, glutathione (GSH) levels by the DTNB-GSSG reductase method, apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (Delta psi(m)) by flow cytometry. Hypoxia (2% O(2)) antagonized BSO-mediated ROS, apoptosis, and cytotoxicity but not GSH depletion. TPZ synergistically enhanced BSO cytotoxicity in hypoxia for all four NB cell lines, achieving 2-4 logs of cell kill. BSO depleted GSH (8-42% of controls) in 20 and 2% O(2), whereas TPZ only decreased GSH in hypoxia. Maximal GSH depletion was induced by BSO + TPZ. N-acetylcysteine abrogated GSH depletion caused by TPZ but not by BSO. BSO increased ROS, decreased Delta psi(m), and caused apoptosis in 20% O(2) (but not in 2% O(2)). TPZ elevated ROS in 2% O(2) (but not in 20% O(2)), whereas BSO + TPZ increased ROS both in 20 and 2% O(2). In hypoxia, TPZ alone or TPZ + BSO caused an 80% decrease of Delta psi(m) at 24 h, preceding apoptosis in 74-86% of cells at 48 h. Thus, hypoxia significantly antagonizes BSO-mediated cytotoxicity for NB cell lines, but TPZ reversed the inhibition of BSO-mediated cytotoxicity in hypoxia, causing increased ROS, Delta psi(m) decrease, GSH depletion, apoptosis, and synergistic cytotoxicity. These data additionally define the role of ROS in BSO-mediated cytotoxicity and suggest that combining BSO with TPZ could have clinical activity against NB in hypoxic sites.