ALBERT

Description: We report the results of a pilot study with a clofarabine containing conditioning regimen for allogeneic stem cell transplantation. Several studies have already shown the effectivity of clofarabine in adult patients and children with acute leukemias, resistant to standard treatment protocols. Thus, we replaced fludarabine in a melphalan based myeloablative regimen by clofarabine for pediatric patients with refractory disease after standard treatment (n=7) or after previous transplantation (n=3). A total of 10 pediatric patients with acute lymphatic leukemia (non-remission (NR) 1=3, NR 2=3), acute myeloic leukemia, (NR 1=2) and metastatic rhabdomyosarcoma/neuroblastoma (partial remission (PR) =2) received clofarabine 4×50mg/m2 (day -8 to -5), thiotepa 10mg/kg (day -4), melphalan 2×70mg/m2 (day -3 to -2) and OKT3 (0, 1 mg/kg day -8 to +14; n=9) or ATG (10mg/kg; n=1), followed by infusion of full haplotype mismatched peripheral stem cells (n= 9) or bone marrow from matched unrelated donors (n=1). In mismatched family donors, depletion of T and B cells was carried out with CD3/CD19 coated magnetic microbeads and the CliniMACS® device. A median number of 16×106/kg stem cells (5.6–28) with 90 000/kg residual T cells was infused. Primary engraftment was observed in 9/10 patients. One patient experienced graft rejection but was successfully retransplanted with stem cells from a second parental donor. Thus, sustained engraftment could be obtained in all patients. Median time to ANC 〉500/μl with G-CSF stimulation was 10 days (9–11). Independence from platelet transfusion was reached after 9 days. Median T cell count at day 100 was 138 cells/μl (n=5). 7/10 patients are disease free with a median follow up of 100 days. Single cause of death was relapse of the underlying disease (median time to relapse: 90 days). The regimen was well tolerated without severe side effects. No TRM and no toxicity grade 4 according to NCI-CTC grading system occurred apart from mucositis. The profile of toxic side effects was as follows: gastrointestinal: diarrhea grade 0–2 (n=10), stomatitis grade 3–4 (n=10); skin: none; pulmonary: hypoxia and pneumonitis grade 3 in 2 and 1 patient, respectively; cardiac: none; hepatic: GOT/GPT or bilirubin elevation grade 1–2 in 4 patients, grade 3 in 6 patients; renal: creatinine (-clearance) grade 1 in 1 patient; neurological: none; infection: grade 2 (n=8), grade 3 (n=2). Conclusions: clofarabine was well tolerated as part of a conditioning regimen for allogeneic transplantation. Compared to our standard regimen with fludarabine, thiotepa, melphalan, the introduction of clofarabine did not result in any unexpected toxicity. Immunosuppressive effects appeared to be similar to those of fludarabine, since primary engraftment was observed in 90%. Further studies are warranted to evaluate, whether clofarabine can contribute to reduce the risk of relapse in patients with refractory disease.

Description: Abstract 3548 T and B cell depleted haploidentical grafts and a melphalan based intensity reduced regimen result in low toxicity and stable event free survival in patients with leukemias in CR1-3. However, patients with active disease or with second or subsequent transplantation show unacceptable relapse rates. In an ongoing study, 36 pediatric patients with acute leukemias and advanced MDS (median age: 11 years) received melphalan (2×70mg/m2), fludarabine (4×40mg/m2) or clofarabine (4×50mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg). T and B cells were depleted by antiCD3/antiCD19 coated magnetic microbeads, whereas NK cells remained into the grafts (median number= 120×106/kg). Remission status was: CR1-3=18, NR=18, 18/36 already received previous allogeneic transplantations. Relapse rates at 2 years were 20% (CR patients) and 73% (active disease patients or 2nd trp). Thus, we investigated options to reduce the risk of relapse by increasing antileukemic activity of donor NK cells in the grafts in vitro. Over night incubation with Interleukin 15 increased NK activity most effectively (specific lysis at E:T=20:1 against K562: 28% prior to and 71% after stimulation, n=10). After additional IL2 stimulation a 22 fold increase in thymidine uptake indicated proliferation of NK cells (n=5). Due to the profound depletion, no T cell proliferation was detectable. Based on these results, we started a pilot study with ex vivo IL15 stimulated grafts in 4 patients at very high risk (ALL, 3rd relapse, active disease (n=1); ALL 2nd relapse, remission (n=1); AML 1st relapse, active disease (n=2)). All patients received a backbone of unstimulated cells at day 0 to ensure engraftment. Additionally, parts of the grafts were incubated over night, washed four times and afterwards infused at day +1 (median numbers: CD56+CD3- =9.4×106/kg (range 3.7–24.4); CD34=1.5×106/kg; CD14=34×106/kg, CD3=0.01×106/kg). No acute side effects occurred. All patients engrafted within 12 days. 3 patients had acute GvDH grade 0-I, 1 patient had GvHD grade III. Recovery of NK cells was remarkably fast (526 CD56+/μl at day 14 posttransplant versus 256 CD56+/μl in patients without IL15 stimulation (means)). After additional administration of IL2 in vivo (1×106 Units/m2/day s.c.) high NK activity (specific lysis〉90% against K 562, E:T=20:1) was detectable in peripheral blood. Two patients are disease free (day 154 and 674 posttransplant), 2 patients died from relapse (day 56 and 64). Conclusions: ex vivo stimulation with IL15 strongly increases cytotoxic activity of NK cells in T and B cell depleted grafts from haploidentical donors and can counterbalance G-CSF mediated inhibitory effects. Those grafts were infused without any acute side effects and resulted in a fast recovery of functional donor NK cells. Potential interactions with stem cells and stem cell derived NK reconstitution have to be investigated. Further studies have to evaluate if this approach might contribute to reduce relapse rates in high risk patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1931 Mobilization of peripheral blood stem cells (PBSC) for autologous transplant in children can be challenging especially in those with prior intensive chemotherapy and radiotherapy, and with increasing use of sequential high-dose therapy that requires high PBSC collection doses. Plerixafor is currently not licensed for use in children, but is approved for PBSC mobilization in adults. We present a series of 40 children from 19 centers worldwide who had PBSC mobilised with plerixafor off-licence, mostly on the North American or European Compassionate Use Programs. Thirteen of these cases were included in a series presented in abstract form at ASH 2010, and a further 6 were presented at EBMT 2011, and these cases are now presented with updated clinical data; the remaining 21 cases have not been presented elsewhere at the time of submission. Median age was 10 years (range 3 months–17 years); median weight was 31 kg (range 6–78 kg). Diagnoses were neuroblastoma (n=13), Ewing's sarcoma (n=10), medulloblastoma (n=5), lymphoma (n=5), PNET (n=4), multicentric Castleman's disease (n=1), Osteosarcoma (n=1) and Desmoblastic Small Round Cell Tumor (n=1). Most patients had received extensive multi-agent chemotherapy prior to plerixafor mobilization, and 13 had also received radiotherapy. For 7 patients, plerixafor had been introduced on a “rescue” basis in the course of the patient's first PBSC mobilization attempt, because of poor peripheral CD34+ counts predicting a high likelihood of mobilization failure without plerixafor use. The remaining 33 patients had undergone 45 prior failed conventional PBSC mobilization attempts: no apheresis in 31 cases; 8 were suboptimal (CD34+ dose 〈 2×10^6/kg); 5 procedures had yielded insufficient cells for planned sequential high-dose therapy. Plerixafor was used in 44 mobilization episodes (4 patients had undergone two mobilizations using plerixafor). In 29 cases, the drug was given after G-CSF 10 mcg/kg/day for 4 days, without prior mobilizing chemotherapy. In the remaining 15 cases, plerixafor was introduced to chemomobilization protocols at the time of initial neutrophil recovery, with exact protocols varying between centers. Three plerixafor mobilisation episodes were entirely unsuccessful (no apheresis); 10 episodes were suboptimal (CD34+ dose of 2×10^6/kg). For the 41 episodes where apheresis was undertaken, a median of 3.5×10^6/kg CD34+ cells (range 0.26–11.8) was collected in a median of 2 aphereses (range 1–5) after a median of 2 plerixafor doses (range 1–5). A total of 16 possible plerixafor toxicities were reported, all graded mild or moderate: these were injection site reactions (3), nausea (3), bone pain (3), vomiting (2), diarrhoea (2), insomnia (2) & headache(1). Seven patients were lost to follow-up after plerixafor due to transfer to other centers; of the remainder, 22 patients (67%) remain alive and 16 (48%) remain progression-free at a median of 9 months' follow-up post-plerixafor (range 3–63 months). Autologous PBSC transplant was performed in 27 patients & allograft in 1 patient. All transplanted patients achieved neutrophil engraftment (median of 11 days to neutrophils〉1.0×10^9/litre; range 10–23 days). There was one case of secondary AML at 〉3 years post-autograft. Plerixafor is safe and highly effective in this difficult-to-mobilize patient group. Disclosures: Douglas: Genzyme Europe: Honoraria. Duarte:Genzyme Europe: Honoraria.

Description: Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease. Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach 〉 100 CD3+/µl, 〉 200 CD19+ cells/µl and 〉 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial. Disclosures No relevant conflicts of interest to declare.

Description: Refractory B-precursor acute lymphoblastic leukemia (ALL) remains an unsolved therapeutic challenge. Various T-cell immunotherapies are promising options in relapsed/refractory B-ALL, like the CD19/CD3-bispecific T-cell engaging antibody Blinatumomab. Until now it has not been possible to determine critical factors for T-cell attack against leukemia that decide on in vivo response or non-response to treatment. Immune-checkpoint molecules regulate immune escape of malignant cells and antibody blockade of these inhibitory pathways enhances antitumor immune responses. Therefore, we investigated the role of co-stimulatory and co-inhibitory molecules for effector-target cell interactions and influence on T-cell attack against leukemia. CD19+ lymphoblast lines, primary pediatric B-ALL bone marrow blasts (n=10) and physiologic CD19+ CD10+ pre-B bone marrow precursors from healthy bone marrow were screened for surface expression of 20 different co-signaling molecules. Surface expression of PD-L1, PD-1, LAG3, CD40, CD86, CD27, CD70 and HVEM revealed differences in stimulatory and inhibitory profiles of pediatric ALL blasts as compared to physiologic cells. Pediatric ALL patients refractory to Blinatumomab-treatment (n=4) as well as patients with relapsed leukemia (n=7) showed increased expression of PD-L1 on blasts. Expression of exhaustion markers PD-1 and TIM-3 was significantly higher on patients' T cells as compared to healthy donors and is induced by T-cell attack against blasts. Blinatumomab-mediated T-cell function was examined in healthy donors as compared to pediatric patients with ALL through analysis of proliferation and effector function. Significant differences in Blinatumomab-induced T-cell function were found to be target-cell dependent and correlated to expression of co-signaling molecules on target cells. Blockade of inhibitory PD-1-PD-L and CTLA-4-CD80/CD86 interactions could further enhance effector T-cell function of healthy donors and patients whereas blockade of co-stimulatory CD28-CD80/86 interactions resulted in reduced T-cell effector and proliferation potential. Combined treatment with Blinatumomab and PD-1 blocking antibody Pembrolizumab was feasible and induced an anti-leukemic immune response in a 12 year old patient with refractory ALL. In conclusion, we show that regulation of T-cell activation and inhibition by co-signaling molecules guides the efficacy of T-cell attack against ALL. Inhibitory interactions between leukemia-induced checkpoint molecules on T cells and their counterparts on ALL regulate in vivo resistance to T-cell immunotherapy and will guide future therapeutic interventions. Disclosures Off Label Use: Pembrolizumab.

Description: We introduced bioluminescence in vivo imaging as a novel, sensitive and reliable readout parameter for preclinical treatment trials in the individualized model of patients' primary AML cells growing in mice. Novel treatment approaches require preclinical in vivo evaluation. While the individualized xenograft mouse model of individual patients AML cells growing in mice inherits the advantage of mimicking the broad genetic heterogeneity of AML, disease monitoring remained challenging so far due to the lack of appropriate readout parameters. In the individualized mouse model of AML, primary patients' AML cells are xenotransplanted into immuno-compromised mice. Here, we aimed at increasing sensitivity and reliability of disease monitoring in the individualized mouse model of patient-derived AML. Towards this aim, we engrafted primary tumor cells from 16 adult patients with AML. 8/16 (50%) samples allowed serial transplantation and thereby generation of stable patient-derived xenograft (PDX) cells with constant characteristics regarding growth and immunophenotype. PDX cells were derived from genetically distinct patient samples, mimicking the known heterogeneity of AML. Targeted re-sequencing of 43 genes important for AML leukemogenesis revealed identical mutations in primary and PDX cells after initial or serial transplantation, except the loss of two minor subclones within two samples. Lentiviral transduction was established to genetically manipulate PDX cells and introduce stable expression of transgenes which was feasible in 7/8 PDX AML samples tested. Transgenic PDX cells were enriched by flow cytometry gating on a co-expressed fluorochrome. Recombinant expression of luciferase enabled bioluminescence in vivo imaging for reliable follow up of PDX cell leukemia growth in mice. Imaging was highly sensitive and detected a single PDX cell within 10,000 normal mouse bone marrow cells covering the clinically important situation of minimal disease. Furthermore, imaging facilitated reliable analysis of preclinical treatment trials, visualizing drug effects in single mice over time. Novel treatment approaches aim at eliminating AML propagating cells, and the limiting dilution transplantation assay represents the gold standard for determining frequency of AML propagating cells. Bioluminescence in vivo imaging facilitated quantifying AML propagating cells by determining engraftment as early as 5 weeks after cell transplantation. Taken together, we advanced the individualized mouse model of AML by introducing serial transplantation, lentiviral transduction and in vivo imaging. These improvements now allow sensitive and reliable preclinical treatment trials in patient-derived AML cells of various different genetic subgroups including AML propagating cells. Disclosures No relevant conflicts of interest to declare.

Description: Transplantation of haploidentical, positive selected grafts with TBI or Bu based conditioning regimens can result in significant toxicity and TRM. New graft manipulation methods and a melphalan based intensity reduced regimen decreased these complications. We present our results with a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads. Melphalan (2×70mg/m2), fludarabine (4×40mg/m2), thiotepa (10mg/kg) and OKT3 (0.1mg/kg) was used as preparative regimen in 27 pediatric patients. All patients underwent intensive pretreatment according to current study protocols; 11/27 already received previous autologous or allogeneic transplantations (median time between 1st and 2nd trp: 2.3 years). Diagnoses were: AML/MDS (n=9), ALL (n=5), relapsed neuroblastoma/Ewing-/rhabdomyosarcoma (n=11), SAA (n=2); remission status: CR2=6, CR3=3, NR/PR=16. Donors were full haplotype mismatched parents. The grafts comprised a median number of 14×106/kg stem cells and 139×106/kg NK-cells with 55×103/kg residual CD3+ cells and 24×103/kg CD20+ cells. Primary engraftment occurred in 85%. After TLI based reconditioning and second haploidentical stem cell donation, final engraftment was achieved in 100%. Median time to reach 〉500 neutrophiles/μl and independence from platelet substitution was 10 (9–12) and 8 (6–188) days respectively. GvHD grade 0–1 occurred in 63%. 30% and 7% had grade II and III, respectively. Chronic GvHD was observed in 3 patients. Transplant related mortality (TRM) occurred only in 1/27 patients (4%) with a median follow up of 0.8 years (3 months-3.0 years). In general, the regimen was well tolerated without severe side effects. The profile of toxic side effects according to NCI-CTC grading system was: gastrointestinal: diarrhea grade 0–2 (n=25), grade 3 (n=2); stomatits grade 3–4 (n=27); skin: grade 0–2 (n=27); pulmonary: hypoxia grade 0 (n=24), grade 3 (n=3); pneumonitis (n=1); cardiac: lethal myocardiopathy (n=1), none (n=26); hepatic: GOT/GPT or bilirubin elevation grade 0–2 (n=19), grade 3 (n=4), grade 4 (n=4), VOD: none; renal: creatinine grade 0–2 (n=27); neurological: none; infection: grade 0–2 (n=17), grade 3 (n=7), grade 4 (n=3), EBV-LPD: none. Overall survival at 1 year was 47% for the whole group and 40% for patients with solid tumors. Causes of death were myocardiopathy (n=1) and relapse (n=13). Pretransplant disease status was predictive: 1 year EFS in patients with leukemias in CR and SAA was 83%, whereas all patients with active disease relapsed (median time to relapse: 0.3 years). Conclusions: transplantation of CD3/CD19 depleted haploidentical stem cells with our melphalan based reduced intensity regimen resulted in a fast recovery of neutrophiles and platelets. Engraftment rates similar to that of patients with standard conditioning regimens and positive selected grafts were achieved, possibly due to graft facilitating effects of cotransfused NK-cells. The regimen helped to minimize side effects and TRM. No lethal infections occurred despite delayed T cell recovery. Thus, our approach forms the basis for haploidentical transplantation with low toxicity even in intensively pretreated patients(including previous transplantations). However, relapse remains an unsolved problem in patients with active disease. In those patients, further therapeutic elements have to be evaluated, like additional cytoreductive drugs immediately before transplantation as well as posttransplant immunotherapy.