αβ T-cell-depleted haploidentical hematopoietic cell transplantation and zoledronate/interleukin-2 therapy in children with relapsed, high-risk neuroblastoma

Outcomes in patients with relapsed, high-risk neuroblastoma have an extremely poor prognosis and novel treatment options are required [1]. For those patients, haploidentical hematopoietic cell transplantation (haplo-HCT) can be a potential option as it is expected to induce graft-versus-tumor (GVT) effects on neuroblastoma [2, 3]. Limited reports investigating the use of haplo-HCT for neuroblastoma suggested a potential GVT effect, but this was not potent enough to prevent tumor progression in patients with a high tumor burden prior to transplant [4,5,6].

Haplo-HCT using αβ T-cell depletion provides grafts containing many γδ T lymphocytes and other effector cells. γδ T cells can kill tumor cells in an MHC-unrestricted manner without significant risk of graft-versus-host disease (GVHD) [7, 8]. In addition, it can provide a platform for further post-transplant immunotherapy to maximize the GVT effect. Zoledronate, a potent aminobisphosphonate, augments the cytolytic activity of γδ T cells [9]. Interleukin (IL)-2 is also required for the expansion of γδ T cells [10]. Therefore, zoledronate in combination with IL-2 can potentially augment the anti-neuroblastoma activity of γδ T cells, which can be enriched after αβ T-cell-depleted haplo-HCT.

In this prospective pilot study of patients with relapsed, high-risk neuroblastoma, we aimed to use high-dose 131I-metaiodobenzylguanidine (MIBG) to reduce pre-transplant tumor burden, followed by αβ T-cell-depleted haplo-HCT using RIC to utilize alloimmunity with minimal transplant-related toxicity. In addition, post-transplant immunotherapy using a combination of zoledronate and IL-2 was adopted to enhance the GVT effect of γδ T cells. The study was approved by the Institutional Review Board at the Asan Medical Center, Seoul, Korea.

Between June 2015 and March 2016, four patients with relapsed, high-risk neuroblastoma after HDCT/ASCT, who showed at least partial response (PR) to salvage treatment, were enrolled and assigned a unique patient number (UPN 1–4). Patient characteristics are summarized in Table 1. Patients had received a median of six (range, 5–6) cycles of salvage chemotherapy after relapse prior to haplo-HCT. The median intervals between initial diagnosis and haplo-HCT, and relapse and haplo-HCT were 36.5 (range, 29–48) months, and 7 (range, 6–9) months, respectively.

Table 1 Patient characteristics and transplant outcome

At 21 days prior to haplo-HCT, all patients received a single intravenous infusion of 131I-MIBG at a median dose of 11.8 (range, 10.5–11.9) mCi/kg. No immediate adverse events were observed. The RIC regimen comprised fludarabine (40 mg/m2/day, days −8 to −5), thiotepa (10 mg/kg/day, day −4), melphalan (70 mg/m2/day, days −3 to −2) and rabbit anti-thymocyte globulin (ATG, ThymoglobulinTM, 2 mg/kg at day −9 and 1 mg/kg at day −8). RIC was well-tolerated and caused no serious regimen-related adverse events during the first 100 days post transplant.

Peripheral blood stem cells were processed for αβ T-cell depletion using the CliniMACSTM system (Miltenyi Biotec). The target dose of CD34+ cells were 5 × 106/kg and the minimum required dose was 3 × 106/kg. The efficacy of depletion was expected to be a 3- to 4-log reduction in the αβ T cells. Graft manipulation and composition data are shown in Table 1. Tacrolimus and mycophenolate mofetil were used for GVHD prophylaxis. Since UPN-4, prophylactic immunosuppressants were not routinely used to maximize the GVT effect. Patients except UPN-1 received rituximab to deplete CD20+ cells 28 days post transplant. All patients achieved neutrophil engraftment at a median of 10 (range, 10–11) days and platelet engraftment at a median of 17 (range, 16–18) days. All patients achieved complete donor chimerism at 30 days post transplant, which was sustained thereafter.

Zoledronate and IL-2 were administered once engraftments were stable. Zoledronate (ZolenicTM) 2.3 mg/m2/dose was administered intravenously on day 0; IL-2 (ProleukinTM) was administered subcutaneously inpatient on days 0–4 and 14–18 every 28 days. Three patients received zoledronate and IL-2 post-transplant, while UPN-4 did not receive them because permission for administration was refused. UPN-1 received two rounds of zoledronate and IL-2 (days 52–102), it was then withheld due to EBV-associated post-transplant lymphoproliferative disease (PTLD); one round was administered after completion of PTLD treatment, at which point the patient relapsed. UPN-2 completed seven rounds of treatment (days 55–349). UPN-3 received six rounds of treatment (days 76–281) at the end of which the patient relapsed. All patients developed spiking, remittent fever during the IL-2 period. UPN-2 experienced severe itching without apparent skin rash during the IL-2 period, prompting a 50% dose reduction. Reversible laboratory abnormalities were observed, including hypocalcemia (one case grade 2, two cases grade 3), hypophosphatemia (three cases grade 3), hypokalemia (one case grade 3), transaminasemia (three cases grade 2) and elevation of serum creatinine (two cases grade 2).

Immune reconstitution data are shown in Fig. 1. Median CD3+ cell counts at days 14, 30, 60, 90, 180, and 365 were 287 (range, 150–460), 196 (range, 103–808), 137 (range, 105–353), 394 (range, 206–61010), 416 (range, 277–523), and 539 (range, 428–778) cells/μL, respectively. CD4+ cells tended to recover to >100 cells/μL after 90 days post transplant. NK cell counts reached a median of 141 cells/μL at day 30. B-cell recovery was delayed until 3 months due to the use of rituximab. Prior to transplant, αβ T cells were predominant among CD3+ cells (median 96%); at day 14 post transplant, CD3+ cells predominantly comprised γδ T cells (median 82%, range 77–92%). The γδ T-cell population gradually decreased to a median of 66% (range, 46–80%), 45% (range, 16–55%), 22% (range, 15–42%), and 13% (range, 9–20%) at 30, 90, 180, and 365 days, respectively. The percentage of γδ T cells appeared higher in the first 6 months post transplant in the three patients who received zoledronate and IL-2 (UPN-1, 2, 3) than in UPN-4 who did not (Fig. 1c). Treg cells were consistently low without significant change.

Fig. 1
figure1

Immune reconstitution after transplant. a Change in CD3+ cells, CD4+ cells, CD19+ cells, NK cells and Treg cells. Median values for cell counts are presented. b Change in αβ T and γδ T-cell counts and percentage of γδ T cells among CD3+ cells. Data are presented as median values and standard errors. c Change in percentage of γδ T cells among CD3+ cells in individual patients

Transplant-associated complications are summarized in Table 1. Post-transplant clinical courses and outcomes are depicted in Fig. 2. Overall, two of the four patients remained in CR without chronic GVHD (Table 1). UPN-1 relapsed at multiple CNS sites 5.5 months post transplant; the patient showed CR after salvage treatment but relapsed again at multiple CNS sites 14 months post transplant and died of progressive disease 33 months post transplant. UPN-2 relapsed at an isolated CNS site 15.5 months post transplant; the patient underwent resection and systemic chemotherapy, achieved CR and remained alive in CR 33 months post transplant. UPN-3 received haplo-HCT in PR and lesions improved after transplant, but developed multiple lymph node, bone, and liver metastases at 10 months post transplant and died of progressive disease at 21 months post transplant. UPN-4 received haplo-HCT in PR and the lesions resolved after transplant without immunotherapy; the patient remained alive in CR 24 months post transplant.

Fig. 2
figure2

Post-transplant clinical courses and outcomes. Abbreviations: CMV cytomegalovirus; CR complete response; 13-CRA 13-cis-retinoic acid; CTx chemotherapy; DOD died of disease; HCT hematopoietic cell transplantation; IL-2 interleukin-2; Op operation; PR partial response; PTLD post-transplant lymphoproliferative disease; UPN unique patient number; Zol zoledronate

Our study shows that high-dose 131I-MIBG followed by αβ T-cell-depleted haplo-HCT using RIC was well-tolerated in patients with relapsed, high-risk neuroblastoma, who had been heavily treated with HDCT/ASCT. In addition, post-transplant immunotherapy using a combination of zoledronate and IL-2 was feasible without serious adverse events.

Our study showed a rapid reconstitution of NK cells and γδ T cells, which are possible immune effector cells against neuroblastoma. γδ T cells were predominant, accounting for >50% of all T cells by 3 months post transplant. Numbers gradually decreased to 20% at 6 months but remained >10% until 1 year, which was higher than the pretransplant proportion of <5%. The presumed GVT effect of γδ T cells could not be evaluated due to small patient numbers. Further studies are needed to determine whether higher γδ T-cell counts are associated with better outcomes.

Immunotherapy using γδ T cells stimulated by zoledronate and IL-2 has been evaluated in pre-clinical and early clinical studies of various solid tumors (prostate cancer, renal cell carcinoma, and breast cancer), with data suggesting some benefit in limited patients [11,12,13]. In previous studies, γδ T-cell immunotherapy using zoledronate and IL-2 can be categorized as either in vivo expansion of γδ T-cells by phosphoantigens and IL-2 or ex vivo expansion of γδ T cells and systemic/local administration of expanded cells with zoledronate and IL-2 [11]. Both methods were performed in non-transplant settings. Recently, zoledronate infusion after αβ T-cell-depleted haplo-HCT is reported to be feasible for children with leukemia [14], but there are no studies on zoledronate and IL-2 use after αβ T-cell-depleted haplo-HCT for solid tumors. The present pilot study suggests that this approach is a feasible immunotherapeutic option for neuroblastoma and is not associated with significant risk of serious adverse events.

The overall tolerability of αβ T-cell-depleted haplo-HCT using RIC allows this strategy to serve as a platform for further post-transplant therapy, because the donor will be readily available for cellular therapy and no or minimal immunosuppression is required after transplant. Such post-transplant interventions can include donor lymphocyte infusion, NK cell infusion, anti-GD2 antibody, or immune checkpoint inhibitors, as well as zoledronate in combination with IL-2.

In conclusion, our study shows that αβ T-cell-depleted haplo-HCT is a feasible therapeutic option for patients with relapsed high-risk neuroblastoma. In addition, post-transplant immunotherapy using zoledronate and IL-2 was well-tolerated and contributed to maintaining a high level of γδ T cells. Further accrual is required to assess the efficacy of this strategy in the ongoing trial.

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Funding

This study was supported by grants from the National R&D Program for Cancer Control, Ministry of Health, Welfare and Family Affairs, Republic of Korea Government (grant No. 1520060).

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Correspondence to Kyung-Nam Koh.

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Koh, KN., Im, H.J., Kim, H. et al. αβ T-cell-depleted haploidentical hematopoietic cell transplantation and zoledronate/interleukin-2 therapy in children with relapsed, high-risk neuroblastoma. Bone Marrow Transplant 54, 348–352 (2019). https://doi.org/10.1038/s41409-018-0305-3

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