Immunotherapy has emerged as an alternative strategy to treat high-risk neuroblastoma stage IV in addition to surgery and conventional radio-and chemotherapy. Today, most therapy protocols for high-risk neuroblastoma include myeloablative chemotherapy which attempts to eradicate the disease, followed by the infusion of autologous stem cells to repopulate the bone marrow. Especially for patients suffering from a relapse after autologous hematopoietic stem cell transplantation (HSCT), but also for a few patients at the early stage of disease, the transplantation of allogeneic/haploidentical stem cells has been considered. Within the last decade, these transplantation strategies have been combined with adoptive cellular therapies using donor lymphocytes with a special focus on natural killer (NK) cells. However, experience with allogeneic HSCT for neuroblastoma with and without additional cell therapies is rare. In a clinical phase I/II trial including 4 patients diagnosed with neuroblastoma stage IV, the feasibility and efficacy of haploidentical HSCT with the adoptive therapy of highly purified IL-2-activated NK cells has been shown. It is encouraging that 2 out of the 4 patients suffering from high-risk neuroblastoma stage IV are alive and well, with no evidence of disease more than 4.7 years after haploidentical HSCT and additional immunotherapy using IL-2-stimulated donor NK cells. (C) 2015 S. Karger AG, Basel

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.

Abstract Introduction: Mixed chimerism (MC) and minimal residual disease (MRD) strongly predict risk for relapse in children with acute lymphoblastic leukemia (ALL) following allogeneic stem cell transplantation (allo-SCT). Preemptive immunotherapy (IT), e.g. withdrawal of immunosuppression (WD-IS) or donor lymphocyte infusion (DLI) guided by chimerism and MRD monitoring can prevent impending relapse in allo-SCT recipients. In this study we retrospectively analyzed chimerism and MRD monitoring and the effect of preemptive IT in all pts undergoing allo-SCT for ALL in our institution. Patients: Between January 2005 and July 2014, a total of 89 children and adolescents (median age 11.5; range 2.2-26.0 years) with ALL (pB-ALL, n=63; T-ALL, n=20; biphenotypic/bilinear ALL, n=6) were transplanted in our center after remission induction treatment. 47 pts were in first, 26 pts were in second, 15 pts were in third, and 1 pt was in fourth complete remission (CR) at time of transplant. 18 pts received grafts from matched sibling donors (MSD), 61 pts from matched unrelated donors (MUD), and 10 pts were grafted from a haploidentical parent. Pts who received their graft from a matched donor (n=79) all underwent a fully myeloablative conditioning regimen consisting of TBI (12 Gy) and etoposide. Pts who were grafted from a haploidentical donor were prepared with fludarabine, thiotepa and melphalane. Methods: Serial and semi quantitative analyses of post-transplant hematopoietic chimerism in peripheral blood were performed weekly until day 200 and monthly thereafter. Bone marrow analyses were done at days +30, +60, +90, +180 and +365 post-transplant. Thereby, MRD was assessed in 56 pts, whereas no diagnostic material was available in 33 pts. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD-Group. All patients should receive preemptive IT if they develop MC after day +28. Results: 64/89 pts (72%) showed complete chimerism (CC) in all follow-up analyses, and 25/89 pts (28%) developed MC although all pts received a fully myeloablative conditioning regimen. From 56 pts in whom MRD could be assessed, 40 pts remained MRD negative and 16 developed MRD positivity after transplantation. IT (WD-IS, n=12; DLI, n=14) was initiated based on chimerism analysis in 22/25 pts with MC, and was guided by MRD detection in 4 pts. For the total cohort of pts, probability of event free (pEFS) and overall survival (pOS) were 0.67 and 0.77, respectively. Furthermore, cumulative incidence of treatment related mortality (CI-TRM) and relapse (CI-relapse) were 0.11 and 0.24 for all pts. Chimerism: pEFS was 0.74 in CC-pts and 0.51 in MC-pts (p<0.032). 22/25 MC-pts received pre-emptive IT resulting in a pEFS of 0.58. Due to rapid progression of their disease IT was not initiated in 3/25 MC-pts. All MC-pts without IT relapsed, and in part received further treatment. While CI-TRM remained low (0.10 for CC resp. 0.12 for MC, p>0.68) in both groups, CI-relapse was 0.17 in CC- and 0.41 in MC-pts (p<0.021). MRD: Besides MC, MRD level post-transplant emerged as poor prognostic factor. Pts were grouped according to their highest MRD value post-transplant in MRD negative, low positive (<10E-4), and high positive (>10E-4) pts. pEFS and pOS were 0.82 and 0.95 in MRD negative (n=40), 0.56 and 0.88 in low level MRD (n=8), and 0.25 and 0.25 in high level MRD (n=8) positive pts (p<0.001). CI-relapse was 0.14 in MRD negative, 0.36 in MRD low, and 0.75 in MRD high positive pts (p<0.001), while CI-TRM between these groups remained low (0.05 for MRD negative, 0.13 for MRD low and 0.00 for MRD high, p>0.55). Other risk factors: Furthermore, remission status at the time of transplant influenced relapse rate. CI-relapse was 0.16 in CR1, 0.26 in CR2, and 0.45 in CR>2 pts (p<0.025). Differences in pEFS, pOS and CI-TRM among CR1, CR2, and CR>2 pts were not statistically significant. Multivariate analysis: Multivariate analysis for pEFS also indicated that MC and high level MRD were independent poor prognostic factors (MC, p<0.049, RR 3.16 and high level MRD, p<0.022, RR 3.99), while remission status before transplantation, ALL lineage, donor type, graft source, T cell depletion or sex showed no significant influence. Conclusion: Our results show that analysis of chimerism and MRD allow the prediction of impending relapse in virtually all children and adolescents transplanted for ALL and that preemptive IT can improve outcome in these high-risk pts. Disclosures No relevant conflicts of interest to declare.

The third International Conference on Immunotherapy in Pediatric Oncology was held in Frankfurt/Main, Germany, October 1-2, 2012. Major topics of the conference included (i) cellular therapies using antigen-specific and gene-modified T cells for targeting leukemia and pediatric solid tumors; (ii) overcoming hurdles and barriers with regard to immunogenicity, immune escape, and the role of tumor microenvironment; (iii) vaccine strategies and antigen presentation; (iv) haploidentical transplantation and innate immunity; (v) the role of immune cells in allogeneic transplantation; and (vi) current antibody/immunoconjugate approaches for the treatment of pediatric malignancies. During the past decade, major advances have been made in improving the efficacy of these modalities and regulatory hurdles have been taken. Nevertheless, there is still a long way to go to fully exploit the potential of immunotherapeutic strategies to improve the cure of children and adolescents with malignancies. This and future meetings will support new collaborations and insights for further translational and clinical immunotherapy studies.

Allogeneic Natural Killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity and T cell depletion during GMP-grade NK cell selection. 40 NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T cell depletion, either the depletion 2.1 program in single or double procedure (D2.1 1depl, n=18; D2.1 2depl, n=13) or the faster depletion 3.1 (D3.1, n=9) was used on the CliniMACS instrument. 17 purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59x10e8 CD56+CD3- cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.1 1depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p<0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.1 1depl/2depl with regard to recovery of CD56+CD3- NK cells (68% vs 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9-14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/-) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(-) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/-)CD69(+)NCR(high)CD62L(-) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.

Regulatory T cells (Tregs) are of crucial importance to suppress graft versus host disease (GvHD) post allogeneic stem cell transplantation (SCT), but are also known to impair antitumor immunity. However, Treg longitudinal studies are rare and in this respect advanced flowcytometric approaches for Treg characterization are necessary. To investigate the relation of both the percentage and the absolute numbers of Tregs on GvHD or relapse we measured CD4(+)CD25(+/hi)CD127(lo/-) Tregs in 239 peripheral blood (PB) samples of 16 patients during the first two years post-SCT. A 10-color flowcytometric panel was established to evaluate Treg subpopulations and has been tested in ten healthy individuals. In patients we demonstrated a decrease in CD127 expression on T cells early post-SCT which increases during the first year. Moreover, Tregs reached higher absolute numbers in patients with GvHD≤grade I compared to those with GvHD grades II-IV. In contrast, the percentage of Tregs was significantly higher in patients with GvHD grades II-IV or disease relapse compared to those without GvHD. These patients fit into the range of healthy individuals where a median value of 7.5% and 6.4% of T helper cells were characterized as CD4(+)CD25(+/hi)CD127(lo/-) and CD4(+)CD25(+/hi) Tregs, respectively. Furthermore, Tregs could be further subdivided into 40% naïve, 51% central memory and 9% effector memory Tregs. Our results showed for the first time a downregulation of CD127 expression on T cells including Tregs in patients early post-SCT. Additionally, new insights into the recovery of Tregs regarding GvHD and relapse were provided.