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Background Allogeneic HSCT is a well-established treatment for children with AL. For pts lacking a compatible matched related or unrelated donor, HLA-haplo-HSCT represents an alternative. Promising results were reported with selective depletion of αβ T and B cells (Locatelli, Blood 2017). PX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of BPX-501 following transplant. BPX-501 provides broad virus and tumor-specific immunity; the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid. Aims Evaluate the safety and efficacy of BPX-501 in pediatric pts with AL by determining whether BPX-501 infusion can increase efficacy outcomes through an enhanced graft-versus-leukemic (GvL) effect, while maintaining a low risk of GvHD. Methods A subset of pts had high-risk ALs. BPX-501 was planned to be infused on day14±4 after the allograft with no post-transplant GvHD prophylaxis allowed. Pts who developed steroid-resistant GvHD could receive ≥1 dose of rimiducid. Results As of June 30, 2018, 100 pts with AL (described in Table 1) were efficacy evaluable. Median time for neutrophil and platelet engraftment was 16 and 12 days, respectively. Four pts (4.1%) experienced primary graft failure. Of 96 evaluable pts, 5 (3.1%) developed Grade III-IV aGvHD. Of 82 evaluable pts, 12 developed cGvHD (18.1%), with 3 moderate-severe. Rimiducid was administered to 10 pts. Best overall clinical response (CR/PR) post-rimiducid was 80% (8 pts). Among responding patients, 7 (87.5%) had a CR. Six (6.6%) pts died after transplantation. Efficacy outcomes in AL subsets are in Table 2. CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by 180 and 270 days, respectively. IgA and IgM levels achieved normal values by 180 days. Conclusion BPX-501 following αβ-T and B-cell depleted haplo-HSCT represents a highly effective transplantation strategy for pediatric pts with AL. Rimiducid was an effective treatment for pts with steroid-resistant GvHD.

Eltrombopag is an orally bioavailable, non-peptide thrombopoietin receptor agonist capable of stimulating platelet production through the differentiation of CD34+ hematopoietic progenitor cells into committed CD41+ megakaryocyte precursors and proliferation of megakaryocyte progenitor cells.This drug has been tested in several clinical trials in adult patients with chronic immune thrombocytopenia (ITP), demonstrating the ability of the drug to reduce the burden of thrombocytopenia and its associated side effects. Two multicenter trials on eltrombopag in chronic ITP of childhood have been recently completed, showing that the drug is effective also in pediatric patients. Recent studies have suggested a potential role of eltrombopag in the treatment of thrombocytopenia associated with hepatitis-C virus infection. These studies have documented that adjunct treatment with eltrombopag can help avoid either dose reductions or withdrawal of pegylated interferon due to development of thrombocytopenia. Eltrombopag has shown efficacy also in patients with acquired severe aplastic anemia and myelodysplastic syndromes.Eltrombopag plays an important therapeutic role in many different conditions characterized by persistent thrombocytopenia. A more comprehensive definition of both long-term safety and benefits deriving from the use of eltrombopag will be obtained through prolonged observation of patients already enrolled in the different studies conducted so far and from future prospective controlled trials.

Although allogeneic haematopoietic stem cell transplantation (HSCT) still represents the only consolidated possibility of cure for sickle cell disease (SCD) patients, its use has been limited by the risk of morbidity and mortality associated with conventional myeloablative therapy. The introduction of treosulfan to replace busulfan in conditioning regimens has recently been explored by virtue of its lower toxicity profile. We report our experience with a treosulfan/thiotepa/fludarabine conditioning for human leucocyte antigen (HLA)-matched sibling or unrelated donor-HSCT in 15 children with SCD, and compare patient outcomes with those of a historical cohort (15 patients) given a busulfan-based regimen. Engraftment was achieved in 28 out of 30 patients (93%), with one case of graft failure in either group. The conditioning regimen was well tolerated in both groups, with no cases of grade III-IV regimen-related toxicity. The 7-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 100% and 93%, respectively, with a 93% DFS in both busulfan and treosulfan groups. No SCD-related adverse events occurred after engraftment in patients with complete or mixed donor chimerism. This retrospective analysis suggests that a treosulfan-based conditioning regimen is able to ensure engraftment with excellent OS/DFS and low regimen-related toxicity in patients with SCD.

We report on 109 patients with hemophagocytic lymphohistiocytosis (HLH) undergoing 126 procedures of allogeneic hematopoietic stem cell transplantation (HSCT) between 2000 and 2014 in centers associated with the Italian Pediatric Hematology Oncology Association. Genetic diagnosis was FHL2 (32%), FHL3 (33%), or other defined disorders known to cause HLH (15%); in the remaining patients no genetic abnormality was found. Donor for first transplant was an HLA-matched sibling for 25 patients (23%), an unrelated donor for 73 (67%), and an HLA-partially matched family donor for 11 children (10%). Conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 (20%), and fludarabine-based for 26 children (24%). The 5-year probabilities of overall survival (OS) and event-free survival (EFS) were 71% and 60%, respectively. Twenty-six patients (24%) died due to transplant-related causes, whereas 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively. Twelve of 14 children given a second HSCT after graft failure/relapse are alive and disease-free. Use of HLA-partially matched family donors was associated with higher risk of graft failure and thus with lower EFS (but not with lower OS) in multivariable analysis. Active disease at transplantation did not significantly affect prognosis. These data confirm that HSCT can cure most HLH patients, active disease not precluding successful transplantation. Because in HLH patients HLA-haploidentical HSCT performed through CD34+ cell positive selection was found to be associated with poor sustained engraftment of donor cells, innovative approaches able to guarantee a more robust engraftment are warranted in patients given this type of allograft.

Juvenile myelomonocytic leukemia (JMML) is a clonal hematopoietic disorder of infancy/early childhood, resulting from oncogenic mutations in genes involved in the Ras pathway. As JMML often exhibits an aggressive course, the timing of diagnosis and treatment is critical to outcome. Areas covered: This review summarizes current approaches to diagnosis and treatment of JMML, highlighting most recent insights into genetic and epigenetic mechanisms underlying the disease, and providing an overview of novel potential therapeutic strategies. Expert commentary: At present, allogeneic HSCT remains the only potentially effective therapy, being able to cure more than 50% of patients, relapse representing the main cause of treatment failure. Prompt HSCT is recommended for all children with NF1, somatic PTPN11 and KRAS mutations, and for most children with somatic NRAS mutations. Conversely, a 'watch and wait' strategy should be adopted in children with germline CBL mutations, specific somatic NRAS mutation, and in Noonan syndrome patients, since spontaneous resolution has been reported to occur. Novel drugs targeting relevant nodes of JMML leukemogenesis have been explored in pre-HSCT window or at relapse. The use of 5-azacytidine, a DNA-hypomethylating agent reported to induce hematologic and molecular remission in some JMML children, is currently being investigated in clinical trials.

Hematopoietic stem cell transplantation (HSCT) is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection (GvI) effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

Objectives To describe a pediatric case of cytokine release syndrome secondary to chimeric antigen receptor-modified T cells associated with acute respiratory distress syndrome. Design Case report. Setting PICU. Patients A 14-year-old boy with refractory B cell precursor acute lymphoblastic leukemia given chimeric antigen receptor cells developed severe cytokine release syndrome 7 days after the drug product infusion with progressive respiratory failure. He was admitted to PICU with a clinical picture of acute respiratory distress syndrome, requiring mechanical ventilation, and secondary hemophagocytic lymphohistiocytosis. Interventions Hemoadsorption with cartridge column (Cytosorb) in combination with continuous renal replacement therapy was associated to the anti-cytokine therapy (tocilizumab, a monoclonal antibody targeting interleukin-6 receptor). Measurements and main results Decrease of the inflammatory biomarkers (ferritin, interleukin-6, interleukin-10) in the first 96 hours associated with a progressive improvement of acute respiratory distress syndrome (Pao2/Fio2 ratio) 7 day after the start of the multimodal treatment. Conclusions This case suggests that hemoadsorption with cartridge column in combination with continuous renal replacement therapy and tocilizumab is safe and potentially effective in pediatric patients with severe cytokine release syndrome.

Introduction Juvenile myelomonocytic leukemia (JMML) is a rare pediatric hematological disease, characterized by aberrant proliferation of myeloid precursors and hypersensitivity to GM-CSF stimulation. Mutations in PTPN11, N/K-RAS, CBL, or NF1 genes are found in ~90% of the cases. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for those patients harboring somatic PTPN11 mutations, while in patients with somatic NRAS mutations, transplantation need a careful evaluation, as these patients may have spontaneous resolution of the disease. Here, we show data from a deep-sequencing analysis enlightening typical molecular features of CD33+/34+ and CD14+ primary cells from NRAS and PTPN11 mutated patients compared with healthy donors (HDs). Patients and Methods Four Cord Blood samples from healthy donors (HDs) and 16 Bone Marrow (BM) samples from PTPN11 (N=8) and NRAS (n=8) mutated JMML patients were collected after approvals from International Review Boards of each Institution. CD33+/34+ and CD14+ cells were magnetically sorted following MACS protocols (Miltenyi). TruSeq and NextSeq kits (Illumina) were used for libraries preparation in order to perform the global transcriptomic and smallRNAs sequencing analyses on Illumina platform, according to the company protocols. StringTie and DESeq2 were used for differential expression analysis and REDItools python scripts for RNA editing analysis. Results In our first analysis, we screened ~60000 gene transcripts, finding different expression signatures between HDs, PTPN11 and NRAS samples. We built Expression Heat-Maps reporting the top 100 most statistically significant (Fig. 1, 2; adjusted p < 0.001) deregulated sequences in all the possible pairwise comparisons. Among the top 100 transcripts, we identified differential expression of 5 different genes: ZNF185, MRLPL30 resulted upregulated in PTPN11 vs NRAS CD14+ cells while CD36, RAG2 and CAMK2A resulted downregulated. Interestingly, comparison of PTPN11 and NRAS cohorts in CD33+/34+ subset, these 5 transcripts showed the opposite expression trend. Moreover, among the upregulated genes, we identified a subset of PRAME paralog transcripts in both NRAS and PTPN11 patients when compared to HDs. Seven transcripts (PRAMEF4, 5, 6, 9, 11, 15, 26) were shared between CD33+/34+ and CD14+ populations, while PRAMEF7, 8, 20, 21 resulted upregulated in CD33+/34+ cells and PRAME, PRAMEF22, 23, 25 were upregulated in CD14+ cells, as compared to HDs. In addition, we found aberrant upregulation of Hemoglobin γ chains 1 and 2 (HBG1, 2, i.e. those needed for fetal hemoglobin) in all JMML vs HDs in CD14+ cells, while in CD33+/34+ a downregulation of Hemoglobin α and β chains (HBA1,2 and HBB) in PTPN11 samples vs NRAS was detected. We also report an enhanced LIN28B expression in JMML samples vs HDs but only in CD33+/34+ cells. A global ADAR-dependent RNA editing analysis revealed differences in CD33+/34+ cells showing an enhanced Adenine to Inosine conversion in NRAS patients vs PTPN11 and HDs. Finally, microRNAs sequencing reveals a significant upregulation of miR-144-3p in all JMML samples and cell lineages compared to HDs, while the miR-146a-5p was upregulated only in CD14+ cells. Also the miR-22 showed decreased expression in PTPN11 vs HDs in CD33+/34+ subset, while the miR-29a-3p was downregulated in NRAS vs HDs in CD14+ cells. Discussion We report different expression and editing signatures among samples cohorts and cellular lineages, showing novel insights in JMML molecular biology. Upregulation of several PRAME paralog genes depicts an interesting array of possible targets for immunotherapies (e.g., using genetically modified T cells with a PRAME-specific T-cell receptor), although their expression have to be further validated. Differences in the expression of microRNAs subsets, LIN28B and subunits of the HbA and HbF already described in JMML, but here related to specific mutations and cell lineages, could help to better understand the pathogenic processes and peculiar clinical behaviors. Finally, the novel evidence of differential RNA editing in JMML opens new perspectives for further studies regarding differential protein isoforms expression and regulation as well as microRNA targeting. Disclosures Niemeyer: Celgene: Consultancy. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy. Merli:Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy.

Background: For children with AL candidate to receive an allograft and lacking a suitable HLA-matched donor, HLA-haplo-HSCT after abTCD may represent a valid alternative. Due to delayed recovery of adaptive T-cell immunity with abTCD-haplo-HSCT alone, we conducted a phase I/II trial testing the safety and efficacy of post-transplant infusion of a titrated number of donor-derived T cells transduced with the iC9 gene (rivogenlecleucel; ClinicalTrials.gov id: NCT02065869) in children with malignant and non-malignant diseases. Here, we report on the cohort of 70 patients with AL treated in Italy with abTCD-haplo-HSCT+rivogenlecleucel, comparing the results with those of 88 patients given abTCD-haplo-HSCT alone and previously published by our group (Blood 2018; 132:2594-2607). Patients and methods: Patients (age < 18 years) were transplanted between 2010 and 2018. Patient and disease characteristics are shown in Table 1. Median age at HSCT was 6 years (range 0.3-18), and median follow-up was 71 and 31 months for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. Compared to the control group, abTCD-haplo-HSCT+rivogenlecleucel recipients were transplanted more recently and from a younger donor, and received a higher number of CD34+ cells (Table 1). Diagnosis did not differ between the 2 groups, acute lymphoblastic leukemia (ALL) being the most frequent diagnosis. All patients were transplanted in morphological complete remission (CR1 and CR2) and received myeloablative preparation. Graft composition is reported in Table 1; notably all patients received >10x106 CD34+cells/Kg and Results: Graft failure occurred in 2% of patients in each group. Median time to neutrophil and platelet engraftment was 14 (6-23) and 11 (5-56) days, with no differences between groups (p=0.28). Rivogenlecleucel were infused at a median time of 21 days (range 11-59). Treatment was well tolerated; no infusion-related side effects were recorded. Cumulative incidence (CI) of 100-day grade II-IV acute GvHD was 18.9% vs 15.9% (p=0.77) and CI of 1-year chronic GvHD was 6.9% vs 5.7% (p=0.56) in abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. The 4-year non-relapse-mortality (NRM) was significantly lower in abTCD-haplo-HSCT+rivogenlecleucel (1.4% vs 8%, p=0.05) (Figure 1). There was no statistically significant difference in the 4-year CI of relapse (RI) (17% vs. 25%, p=0.30), respectively. Disease recurrence was the most common causes of death in both groups, viral and fungal infections being the most frequent non-relapse fatalities. The 4-year overall survival (OS) and leukemia-free survival (LFS) was 70% vs 87%, p=0.01 (Figure 2) and 67% vs 82%, p=0.05, for abTCD-haplo-HSCT and abTCD-haplo-HSCT+rivogenlecleucel, respectively. There was no difference in 4-year CI of CMV reactivation between the 2 groups (p=0.68), median time to CMV reactivation being 29 and 30 days (p=0.29), respectively. Once infused, rivogenlecleucel expanded (mainly in the CD8+ subset), reaching a peak at 9 months after infusion. At 6-months, median CD3+, CD3/CD4, CD3/CD8, CD3-/CD56 and CD20/CD19 count/microL were 820, 265, 225, 141, 171, for abTCD-haplo-HSCT and 898, 294, 288, 214, and 161 for abTCD-haplo-HSCT+rivogenlecleucel, (p=ns, p=ns, p=0.02, p=0.03, p=ns), respectively. The advantage in the recovery of CD3/CD8 and CD3-/CD56 after abTCD-haplo-HSCT+rivogenlecleucel persisted at 1 year (p=0.01, p=0.03, respectively). In multivariable analysis, abTCD-haplo-HSCT+rivogenlecleucel was associated with better OS (HR 0.27, p=0.003) and LFS (HR 0.40, p=0.001); there was also a trend for lower relapse risk (HR 0.50, p=0.098). Age below the median value at HSCT (HR 2.62, p=0.01), CR1 at HSCT (HR 0.35, p=0.03) and use of irradiation in the conditioning regimen (HR 0.32, p=0.02) were other factors correlating with OS and LFS. Conclusions: These data confirm that the infusion of donor-derived rivogenlecleucel is safe and well tolerated. Rivogenlecleucel cells infusion contributed to enhance recovery of cytotoxic T and NK cells, improving patients NRM and OS/LFS. Rivogenlecleucel (with the possibility of inducing apoptosis of donor T cells) may facilitate the cellular therapy approaches aimed at optimizing immune recovery after HSCT. Disclosures Merli: Amgen: Honoraria; Bellicum: Consultancy; Novartis: Honoraria; Sobi: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria. Woodard:Bellicum Pharmaceuticals, Inc: Employment, Other: Stock, Stock options. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy.

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

β-thalassemia is one of the most common monogenic blood disorders worldwide, and is highly prevalent in Mediterranean countries. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only curative treatment for transfusion-dependent β-thalassemia (TDT; the most severe disease form) for many years, but it is limited by donor availability and has a significant risk of morbidity and mortality. We conducted a chart review of patients with β-thalassemia who underwent allo-HSCT (N=80) at the Bambino Gesù Children's Hospital, Rome, Italy, between March 2011 and August 2018. Median (range) age at allo-HSCT was 5.5 (0.3-20.0) years [ In total, 18 (22.5%), 28 (35.0%), and 34 (42.5%) patients received allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors, HLA-haploidentical donors, and unrelated donors (fully matched donor: n=28, donor with a single HLA disparity: n=6), respectively. Of these donors, 42 (52.5%) were carriers for thalassemia-associated mutations. In total, 53 (66.3%) donors and 35 (43.8%) recipients were cytomegalovirus-positive. Bone marrow was the stem cell source in 51 cases (63.8%), while 28 patients received an alphabeta T-cell depleted peripheral blood haploidentical HSCT (35.0%); the remaining child (1.3%) received both bone marrow and cord blood from the same related donor. All patients continued to receive transfusions immediately after allo-HSCT; however, only 7 (8.8%) received a transfusion in the 3 to 12-month post-transplantation period (2 due to underlying disease; 5 due to other reasons including GI bleeding). Median (range) time to reach transfusion-free status was 3.8 (1.1-47.8) weeks. Median (interquartile range) hemoglobin levels at 6 and 12 months after allo-HSCT were 10.9 (10.2-11.9) and 11.9 (10.6-13.0) g/dL, respectively. The cumulative incidences of primary and secondary graft failure were 10.0% and 12.5% at 24 months (HLA-identical donor: 0% and 11.1%, haploidentical donor: 17.9% and 3.6%, unrelated donor: 8.8% and 20.6%). Eleven out of 14 patients experiencing graft failure were successfully rescued with a second allograft, while 2 patients were not retransplanted due to parental decision and 1 patient died after the engraftment of the second allograft. Eight patients developed grade II-IV acute graft-versus-host disease (GVHD) and one patient developed moderate chronic GVHD. Cumulative incidence rates of grades II-IV and III-IV acute GVHD were 12.7% and 8.0% at 24 months (HLA-identical donor: 0% and 0%, haploidentical donor: 7.3% and 0%, unrelated donor: 23.8% and 18.8%). Three patients (3.8%) died of transplant-related causes (1 case each of hemophagocytic lymphohistiocytosis, sepsis, and multi-organ failure [the patient receiving the second allograft]) with a median (range) time from transplantation to death of 8.7 (3.7-11.0) months. Of these patients, all had been transplanted from an unrelated donor and 2 had reached sustained full-donor chimerism. The probability of overall and event-free (event defined as either death or primary/secondary graft failure) survival was 96.2% and 81.2% at 24 months (HLA-identical sibling donor: 100% and 88.9%, haploidentical donor: 100% and 78.6%, unrelated donor: 91.2% and 79.4%). The probability of thalassemia-free survival (event defined as either death or primary/secondary graft failure not rescued by a second allograft) was 93.7% at 24 months (HLA-identical sibling donor: 100%, haploidentical donor: 92.9%, unrelated donor: 91.2%). In this large single-center cohort of children with predominantly TDT, allo-HSCT led to beneficial outcomes for most patients, resulting in the discontinuation of transfusions with 93.7% of patients being thalassemia-free. Nevertheless, HSCT is still associated with GVHD, graft failure, and mortality, and only 22.5% of patients had an HLA-identical sibling donor, illustrating a key limitation of allo-HSCT. Emerging research is addressing such barriers to treatment. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Algeri:Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria. Gruppioni:Bluebird bio: Employment, Equity Ownership. Kommera:Bluebird bio: Employment, Equity Ownership. Maa:Bluebird bio: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Systemic glucocorticoids are the principal treatment for acute graft-versus-host disease (GVHD), which remains the major cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). However, there are no validated biomarkers that measure a patient's response to glucocorticoid therapy, and thus response is evaluated by the change in clinical symptom severity. A major weakness in the predictive power of clinical responses is that changes to all organs are weighted equally even though the major driver of NRM is irreversible damage to the crypts of the GI tract. Recent studies from the Mount Sinai Acute GVHD International Consortium (MAGIC) have validated an algorithm probability (MAP) that combines serum concentrations of two biomarkers of GVHD (REG3α and ST2) to generate an estimated probability of 6 month NRM for individual patients. The MAP has been considered a "liquid biopsy" that estimates the damage caused by GVHD to crypts throughout the lower GI tract (Hartwell et al., JCI Insight, 2017; Major-Monfried et al., Blood, 2018). We hypothesized that the change in MAP between start of treatment and 28 days later could serve as a response biomarker for GVHD and might compare favorably to the change in clinical symptoms that measures response to GVHD treatment, which is widely used as a surrogate for long term survival and is the primary endpoint in most GVHD treatment trials (Martin et al., BBMT, 2009; MacMillan et al., Blood, 2010). We prospectively collected serum samples and clinical staging from 368 sequential HCT patients who received systemic treatment for acute GVHD in one of 20 MAGIC centers between January 2016 and February 2018. We measured the serum concentrations of REG3α and ST2 before and after systemic therapy for acute GVHD and computed MAPs, the changes in MAPs, and clinical responses for each patient. MAPs of patients who experienced 6 month NRM showed significantly greater increases than MAPs of patients who survived (p=0.0004). In patients whose MAPs at the start of treatment were low (Ann Arbor 1, MAP < 0.141) or intermediate (Ann Arbor 2, 0.141 ≤ MAP ≤ 0.290), 6 month NRM clustered among those who had the greatest increases in MAP after 28 days (Fig 1A,B). In patients with high MAPs at the start of treatment (Ann Arbor 3, MAP > 0.290), those who survived tended to have the largest decreases in MAP (Fig 1C). These changes in MAP suggested crossing a single threshold could predict risk of mortality. We found that patients whose MAPs rose above a threshold MAP of 0.290 (5% of Ann Arbor 1, 27% of Ann Arbor 2) had significantly worse survival compared to those who remained below it, whereas the large number patients with initially high MAPs that remained above the threshold (66% of Ann Arbor 3) had a large increases in mortality (Fig 2). When measured at day 28, the MAP was significantly more accurate in predicting NRM than the gold standard of the clinical response, with areas under the receiver operating characteristic curve (AUC) of 0.86 and 0.70, respectively (p We conclude that the MAP is, to our knowledge, the first validated laboratory test to serve as response biomarker for the treatment for acute GVHD and a more accurate predictor of survival than clinical response after four weeks of treatment. The MAP may serve as a novel endpoint and an important complement to changes in clinical symptom severity in future trials of GVHD treatment. Disclosures Srinagesh: National Institutes of Health: Research Funding. Ozbek:Viracor: Patents & Royalties: Biomarker Patent. Ayuk:Novartis: Honoraria, Other: Advisory Board, Research Funding. Aziz:Doris Duke Charitable Foundation: Research Funding. Defilipp:Incyte: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy; Humanigen: Consultancy. Hexner:novartis: Research Funding. Kitko:Mallinckrodt: Honoraria; Novartis: Consultancy, Honoraria. Mielke:EBMT/EHA: Other: Travel support; ISCT: Other: Travel support; Miltenyi: Consultancy, Honoraria, Other: Travel and speakers fee (via institution), Speakers Bureau; Jazz Pharma: Honoraria, Other: Travel support, Speakers Bureau; IACH: Other: Travel support; Kiadis Pharma: Consultancy, Honoraria, Other: Travel support (via institution), Speakers Bureau; DGHO: Other: Travel support; Bellicum: Consultancy, Honoraria, Other: Travel (via institution); GILEAD: Consultancy, Honoraria, Other: travel (via institution), Speakers Bureau; Celgene: Honoraria, Other: Travel support (via institution), Speakers Bureau. Merli:Sobi: Consultancy; Amgen: Honoraria; Novartis: Honoraria; Bellicum: Consultancy. Pulsipher:Amgen: Other: Lecture; Miltenyi: Research Funding; Bellicum: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Honoraria. Qayed:Bristol-Myers Squibb: Honoraria. Reshef:Pfizer: Consultancy; Magenta: Consultancy; Kite: Consultancy, Research Funding; Atara: Consultancy, Research Funding; BMS: Consultancy; Pharmacyclics: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Celgene: Research Funding; Shire: Research Funding. Levine:Incyte: Consultancy, Research Funding; Biogen: Other: non-financial support; Viracor: Patents & Royalties: biomarker patent; Ironwood: Honoraria; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Novartis: Honoraria; Kamada: Research Funding. Ferrara:National Institutes of Health: Research Funding; ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy.

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity. At Ospedale Pediatrico Bambino Gesù (OPBG) in Rome, we developed a clinical-grade, 2nd generation, CD19-specific CAR construct, including 4.1bb as costimulatory domain and the inducible caspase-9 safety switch (iC9-CD19-CAR), vehiculated by a retroviral vector, to conduct an academic, phase I/II clinical trial in patients (age 1-25 yrs) affected by BCP-ALL or B-NHL. We now report on the results of the phase I and of the first 8 patients treated in the phase II portion of the study, in terms of feasibility, toxicity, maximum tolerated/recommended dose (MTD/RD) and data on response rate and biological correlates. The phase I, dose-escalation portion of the study included 3 dose levels (DL), namely: DL1, 0.5×106; DL2, 1.5×106; DL3, 3.0×106 CAR+ T cells per kg of recipient body weight. In the phase II portion, patients were treated at the RD identified in the phase I, namely 3.0×106 CAR+cells/kg. All patients received a lymphodepleting regimen consisting of fludarabine and cyclophosphamide for 3 days and iC9-CD19-CAR T cells were subsequently administered as single infusion. Patients were monitored for toxicity, expansion and persistence of iC9-CD19-CAR T cells. Seventeen children were enrolled into the trial and received iC9-CD19-CAR T cells between January 2018 and June 2019. Data were analyzed as of July 20, 2019. The characteristics of the patients are detailed in table 1. The designed dose concentration was successfully produced for all the enrolled patients and we did not observe any production failure. The median transduction rate in the drug product was 54% (range 21-73), while the median vector copy number was 3.8 (range 2.8-6.2). During the phase I portion of the study, no dose limiting toxicities (DLTs) have been recorded, defining the MTD as 3.0×106 CAR+ T cells per kg of recipient body weight. The treatment was overall tolerated and all the toxicities were reversible, the most severe being grade 3-4 neutropenia, thrombocytopenia and/or anemia, occurring in 16/17 (94.1%) patients; in 13/16 patients (81.2%) the hematological toxicity developed before the infusion and persisted after the administration of CAR T cells. Cytokine release syndrome (CRS) occurred in 10/17 patients (58.8%) and was overall moderate, reaching grade 3 (Lee criteria) in one patient only. Notably, none of the patients developed neurotoxicity and no activation of the safety switch was required. All patients were assessed for response at 4 weeks from iC9-CD19-CAR T cell infusion and 13/15 (86.7%) patients with ALL achieved complete remission (CR) with negativity of minimal residual disease (MRD), including 2/3 patients receiving the DL1, 9 patients who had failed a previous allogeneic haematopoietic stem-cell transplantation (HSCT) and 6 patients that had previously received blinatumomab, as CD19-directed immunotherapy. The iC9-CD19-CAR T cells expanded in vivo and were detectable by both flow-cytometry and molecular biology in the blood (Fig.1), bone marrow and cerebrospinal fluid of the responders. One CD19-negative relapse 3 months after infusion was recorded, while 3 additional patients relapsed with CD19+ leukemia blasts. Four patients received HSCT while in CR with MRD negativity because of regrowth of normal CD19+ B cells. The 18-month probability of overall survival for the BCP-ALL cohort is 72.2% (Fig.2). One of the 2 B-NHL patients showed a partial response. Our data indicate that iC9-CD19-CAR T cell in an academic setting is feasible, safe and extremely effective in treating highly resistant/relapsed BCP-ALL. In our trial, no major or life-threatening toxicities were observed and, despite the moderate CRS recorded, high rates of CR were achieved, suggesting that the combination of a retroviral platform and 4.1bb as costimulation is able to mediate a potent antitumor effect Disclosures Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Objective. The aim of this study was to investigate the current use of musculoskeletal US (MSUS) and the most relevant areas of interest for this imaging modality in paediatric rheumatology. Methods. A questionnaire was developed by the paediatric subgroup of the OMERACT US task force and e-mailed to the members of the main international paediatric rheumatology networks and societies. Responses were entered in an electronic database. Results were analysed quantitatively or summarized qualitatively in the case of open questions. Results. The overall response rate was 36% (262/719). The use of MSUS varied among members of the various networks/societies. MSUS was considered of high relevance for improvement of diagnostic skills, for the guidance of joint injections and for the assessment of specific joints, namely the hip, ankle, midfoot and wrist. It was considered useful for early detection of synovitis and in determining disease activity and disease remission. Conclusion. Although at present MSUS is not widely used by paediatric rheumatologists, there is considerable interest in this imaging technology among members of the international networks. The results of this survey suggest that the next objective in the research agenda should be the standardization of the assessment of joints in healthy children. This will then help differentiate pathological (i.e. synovitic) joints from normal joints. The initial target joints should be the hip, ankle, midfoot and wrist. MSUS training focused on the assessment of paediatric patients might be very important in implementing the use of this technique in clinical practice and research.

The identification of an anti-tumor effect displayed by cells of innate immunity has opened new scenarios, not only in the field of allo-HSCT but also for nontransplanted patients with hematological malignancies or solid tumors. Donor-derived NK cells have been shown to contribute to the eradication of malignant cells after allo-HSCT, when recipients lack ligands for their inhibitory receptors. These alloreactive donor NK cells can also kill recipient APCs and CTLs, thus preventing the occurrence of GvHD and graft rejection. The role of activating receptors on the capacity of NK cells to kill leukemia targets has become evident in the last years. The adoptive infusion of ex vivo-activated NK cells has been investigated recently in Phase I/II trials on patients with hematological malignancies and solid tumors, with promising results. γδ T lymphocytes are also able to display anti-tumor activity—this providing the biological rationale for Phase I/II trials in lymphoproliferative disorders and solid tumors. Aminobisphosphonates are clinically available compounds able to boost γδ T cell function. As γδ T cells do not cause GvHD, they could also be transduced with tumor-associated chimeric antigen receptors and safely infused in allo-HSCT recipients. Basic aspects of innate immunity relevant to the field will be covered by a companion review article.

Mesenchymal stromal cells (MSCs) represent a key component of bone marrow (BM) microenvironment and display immune-regulatory properties. We performed a detailed analysis of biological/functional properties of BM-MSCs derived from 33 pediatric patients affected by primary immune-deficiencies (PID-MSCs): 7 Chronic Granulomatous Disease (CGD), 15 Wiskott-Aldrich Syndrome (WAS), 11 Severe Combined Immunodeficiency (SCID). Results were compared with MSCs from 15 age-matched pediatric healthy-donors (HD-MSCs). Clonogenic and proliferative capacity, differentiation ability, immunophenotype, immunomodulatory properties were analyzed. WB and RT-qPCR for CYBB, WAS and ADA genes were performed. All PID-MSCs displayed clonogenic and proliferative capacity, morphology and immunophenotype comparable with HD-MSCs. PID-MSCs maintained the inhibitory effect on T- and B-lymphocyte proliferation, except for decreased inhibitory ability of SCID-MSCs at MSC:PBMC ratio 1:10. While HD- and CGD-MSCs were able to inhibit monocyte maturation into immature dendritic cells, in SCID- and WAS-MSCs this ability was reduced. After Toll-like Receptor priming, PID-MSCs displayed in vitro an altered gene expression profile of pro- and anti-inflammatory soluble factors. PID-MSCs displayed lower PPARγ levels and WAS- and SCID-MSCs higher levels of key osteogenic markers, as compared with HD-MSCs. Our results indicate that PID-MSCs may be defective in some functional abilities; whether these defects contribute to disease pathophysiology deserves further investigation.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children with acute leukemia (AL) either relapsed or at high-risk of treatment failure. We developed a novel method of graft manipulation based on negative depletion of αβ T and B cells and conducted a prospective trial evaluating the outcome of children with AL transplanted with this approach. Eighty AL children, transplanted between September 2011 and September 2014, were enrolled in the trial. All children were given a fully myeloablative preparative regimen. Anti-T-lymphocyte globulin from day -5 to -3 was used for preventing graft rejection and graft-versus-host disease (GVHD); no patient received any posttransplantation GVHD prophylaxis. Two children experienced primary graft failure. The cumulative incidence of skin-only, grade 1-2 acute GVHD was 30%; no patient developed extensive chronic GVHD. Four patients died, the cumulative incidence of nonrelapse mortality being 5%, whereas 19 relapsed, resulting in a 24% cumulative incidence of relapse. With a median follow-up of 46 months for surviving patients, the 5-year probability of chronic GVHD-free, relapse-free survival (GRFS) is 71%. Total body irradiation-containing preparative regimen was the only variable favorably influencing relapse incidence and GRFS. The outcomes of these 80 patients are comparable to those of 41 and 51 children given transplantation from an HLA-identical sibling or a 10/10 allelic-matched unrelated donor in the same period. These data indicate that haplo-HSCT after αβ T- and B-cell depletion represents a competitive alternative for children with AL in need of urgent allograft. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR

Summary Background Although rabbit anti-T-lymphocyte globulin (ATLG) is largely used for the prevention of immune-mediated complications in patients given allogeneic haemopoietic stem-cell transplantation (HSCT) from an unrelated donor, the optimum dose of this drug in children is still undefined. We aimed to test whether a higher dose of ATLG was superior to a lower dose for prevention of grade II–IV acute graft-versus-host disease (GVHD). Methods We conducted a multicentre, randomised, open-label, phase 3 trial in seven Italian centres comparing two different doses of ATLG (30 mg/kg vs 15 mg/kg, given intravenously over 3 days, from day −4 to −2) in children (aged 0–18 years) with haematological malignancies transplanted from an unrelated donor, selected using high-resolution typing for HLA-class I/II loci. All patients received a myeloablative regimen and cyclosporine-A plus short-term methotrexate as post-transplantation GVHD prophylaxis. Patients were randomly assigned (1:1) to either of the two groups and were stratified by the degree of HLA-compatibility with their donor, the source of haemopoietic stem cells used (bone marrow vs peripheral blood stem cells), and the disease risk category. The randomisation was open label; all investigators were aware of the treatment allocation. The primary endpoint of the study was 100-day cumulative incidence of grade II–IV acute GVHD. Statistical analyses were done according to the per-protocol principle. Other outcomes included cumulative incidence of chronic GVHD, non-relapse mortality, disease recurrence, and probability of overall survival and event-free survival. This study was registered with ClinicalTrials.gov, number NCT00934557. Findings Between Jan 15, 2008, and Sept 25, 2012, 89 patients were randomly assigned to the 30 mg/kg ATLG group and 91 to the 15 mg/kg ATLG group; 84 patients in the 30 mg/kg ATLG group and 88 in the 15 mg/kg ATLG group were included in the analysis. The median follow-up for the whole study population was 3·4 years (IQR 1·7–5·1). The 100-day cumulative incidence of grade II–IV acute GVHD was 36% (95% CI 28–48) in the 15 mg/kg ATLG group and 29% (20–40) in the 30 mg/kg ATLG group (hazard ratio [HR] 0·74, 95% CI 0·44–1·25; p=0·26). The cumulative incidence of non-relapse mortality was 9% (5–18) in the 15 mg/kg ATLG group and 19% (12–30) in the 30 mg/kg ATLG group (HR 2·08, 0·89–4·96; p=0·092). Cumulative incidence of disease recurrence was 15% (12–24): 14% (8–23) in the 15 mg/kg ATLG group versus 20% (13–31) in the 30 mg/kg ATLG group (HR 1·54, 0·74–3·21; p=0·25). The 5-year overall survival probability was 70% (62–77) for the whole study population: 78% (69–87) in the 15 mg/kg ATLG group versus 62% (50–73) in the 30 mg/kg ATLG group (HR 1·80, 1·01–3·20; p=0·045). The 5-year event-free survival was 77% for children in the 15 mg/kg ATLG group versus 61% in the 30 mg/kg ATLG group (HR 1·87, 1·07–3·28; p=0·028). Interpretation Children with haematological malignancies transplanted from unrelated donors selected through high-resolution HLA-typing benefit from the use of a 15 mg/kg ATLG dose in comparison with a 30 mg/kg ATLG dose. ATLG at 15 mg/kg should thus be regarded as the standard serotherapy regimen for unrelated donor allogeneic HSCT in this patient population. Future randomised studies will continue to aim to optimise patient outcome and strategies to prevent acute GVHD occurrence. Funding Fresenius/Neovii Biotech.

Purpose of review Lack of a human leukocyte antigen (HLA)-matched family donor is a major obstacle limiting the use of allogeneic hematopoietic stem cell transplantation (HSCT) to cure thalassemia major. Use of alternative donors, if found well tolerated enough, may be a viable option for transplantation in this setting, allowing to expand the number of patients treatable by HSCT. This review will provide an overview of alternative HSCT approaches in thalassemia major. Recent findings With the introduction of high-resolution molecular HLA-typing techniques, the outcome of matched unrelated donor HSCT recipients has become comparable with that of patients given matched family donor HSCT. On the contrary, the use of unrelated cord blood transplantation has been limited by the risk of graft failure and slow hematopoietic recovery, although novel techniques of ex vivo graft manipulation are emerging as promising strategies for overcoming these obstacles. Although experience with haploidentical HSCT in thalassemia major is still limited, currently explored platforms hold the potential to extend the access to HSCT to thalassemia major patients lacking an HLA-matched either related or unrelated donor. Summary The significant advances achieved in transplantation techniques, both in the matched unrelated and in the haploidentical setting, are expected to significantly broaden the applicability of HSCT to patients with thalassemia major.

In children with acute myeloid leukaemia (AML), assessment of initial treatment response is an essential prognostic factor; methods more sensitive than morphology are still under evaluation. We report on the measurement of minimal residual disease (MRD), by multicolour flow-cytometry in one centralized laboratory, in 142 children with newly diagnosed AML enrolled in the Associazione Italiana di EmatoOncologia Pediatrica-AML 2002/01 trial. At the end of the first induction course, MRD was 1% in 51 patients. The 8-year disease-free survival (DFS) of 125 children in morphological complete remission and with MRD

Hematopoietic Stem Cells Transplantation (HSCT) is an effective treatment for hematological and non-hematological diseases. The main challenge in autologous HSCT is purging of malignant cells to prevent relapse. In allogeneic HSCT graft-versus-host disease (GvHD) and opportunistic infections are frequent complications. Two types of graft manipulation have been introduced: the first one in the autologous context aimed at separating malignant cells from hematopoietic stem cells (HSC), and the second one in allogeneic HSCT aimed at reducing the incidence of GvHD and at accelerating immune reconstitution. Here we describe the manipulations used for cell purging in autologous HSCT or for T Cell Depletion (TCD) and T cell selection in allogeneic HSCT. More complex manipulations, requiring a Good Manufacturing Practice (GMP) facility, are briefly mentioned.

Post-transplant lymphoproliferative disorder (PTLD) is a severe complication of solid organ transplantation that can be classified into two major subtypes, namely, early lesions and non-early lesions, based on histopathological findings. In the vast majority of cases, proliferating cells are B lymphocytes and, most frequently, proliferation is induced by Epstein–Barr virus (EBV) infection. The aim of our study was to evaluate the natural history of EBV infection and its possible evolution toward PTLD in a pediatric cohort of patients who received a renal transplant between January 2000 and December 2013. A total of 304 patients were evaluated for this study, of whom 103 tested seronegative for EBV at transplantation. Following transplantation, 50 of the 103 seronegative patients (48.5%) developed a first EBV infection, based on the results of PCR assays for EBV DNA, with 19 of these patients ultimately reverting to the negative state (

Among the novel biologic therapeutics that will increase our ability to cure human cancer in the years to come, T cell therapy is one of the most promising approaches. However, with the possible exception of tumor-infiltrating lymphocytes therapy for melanoma, clinical trials of adoptive T-cell therapy for solid tumors have so far provided only clear proofs-of-principle to build on with further development. Epstein-Barr virus (EBV)-associated malignancies offer a unique model to develop T cell-based immune therapies, targeting viral antigens expressed on tumor cells. In the last two decades, EBV-specific cytotoxic T-lymphocytes (CTL) have been successfully employed for the prophylaxis and treatment of EBV-related lymphoproliferative disorders in immunocompromised hosts. More recently, this therapeutic approach has been applied to the setting of EBV-related solid tumors, such as nasopharyngeal carcinoma. The results are encouraging, although further improvements to the clinical protocols are clearly necessary to increase anti-tumor activity. Promising implementations are underway, including harnessing the therapeutic potential of CTLs specific for subdominant EBV latent cycle epitopes, and delineating strategies aimed at targeting immune evasion mechanisms exerted by tumor cells.

Background Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) constitutes a curative treatment for children with malignant and non-malignant disorders. HLA-partially matched haploidentical (haplo) donors represent a viable alternative option for those children who lack an HLA-compatible donor. T-cell depletion approaches with positive (CD34 selection) or negative selection (alpha/beta T-cell and CD19+ B-cell-depletion) may allow engraftment of donor cells with a low risk of GvHD; however, success is limited by delayed immune recovery, increasing the risk of fatal infections. Infusion of unmanipulated donor T cells (DLI) to accelerate immune recovery is associated with high risk of fatal GVHD. In contrast, adoptive transfer of donor T cells genetically manipulated to include a safety switch can be a suitable strategy to render DLI safer and more widely applicable. BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch. The polyclonal natural of these modified T cells can provide viral immunity following stem cell transplant, with the unique ability to promptly and durably resolve GvHD symptoms following the administration of rimiducid, an inert, lipid-permeable compound that rapidly induces dimerization and activation of iC9, inducing apoptosis of the gene modified T cells. Aims To evaluate the safety and efficacy of rimiducid in the treatment of GvHD following administration of BPX-501 T cells in pediatric patients with malignant or non-malignant disorders given an αβ T-cell receptor and B-cell depleted haplo-HSCT. A key objective of this study is to assess the activity of rimiducid infusion following onset of GvHD which is refractory to standard of care therapies. Methods Two multicenter (US [NCT03301168] and EU [NCT02065869]), prospective trials utilized αβ-T-cell and B-cell-depleted haplo-HSCT followed by infusion of a titrated number of donor lymphocytes genetically modified with iC9 (BPX-501 T cells) in patients with malignant or non-malignant disorders. BPX-501 T-cells were planned to be infused on day14+/-4 after the allograft. No post-transplant pharmacological GvHD prophylaxis was employed. Patients who develop GvHD resistant to conventional steroid therapy could receive ≥1 dose of dimerizing rimiducid to activate the iC9 safety gene. The efficacy evaluable population is defined as any patient who received ≥ 1 dose of rimiducid for the treatment of GvHD and had a follow up response assessment. Results At the time of clinical cut-off (June 30th, 2018) 249 patients with a malignant (41.4%) or non-malignant disorder (58.6%) were enrolled. The conditioning regimens varied according to the original disease and were Treosulfan-based (14.9%), Busulfan-based (28.9%), TBI-based (34.9%) or other (19.3%). The donor was a parent in 229 children (92 %), a sibling in 17 (6.8 %), and a half-sibling in the remaining 3 (1.2 %). The median time to BPX-501 infusion was 18 days (10 - 66 days). Fifty-two patients developed Grade I-IV aGvHD (cumulative incidence [CI] 21.9 % [95% confidence interval (CoI): 16.7 - 27.2]). Twenty-six patients developed Grade II-IV aGvHD (CI 10.9 %). Five patients developed Grade III-IV aGvHD (CI 2.1 %). Eight patients developed cGvHD (CI of 4.6% [95% CoI: 1.3 - 7.8]). Twenty-one patients met the rimiducid efficacy evaluable population definition. An overall clinical response rate of 86% was observed. A CR or PR to rimiducid was observed in 12 and 6 patients, respectively. Median time to initial response was 2 days (1-61 days). Median number of doses received was 1 (1 - 2). At a median follow-up of 7.8 months (2.3 - 30.8 months), 77% of the initial responders were still in either complete (n=8) or partial response (n=6). Conclusion These data suggest that administration of rimiducid for treatment of steroid-refractory GvHD represents a novel and highly effective treatment approach in pediatric patients with non-malignant or malignant disorders who received a αβ-T-cell and B-cell depleted haplo-HSCT followed by infusion of BPX-501 cells. The administration of rimiducid in children given BPX-501 T cells allows for effective control of GvHD occurring after the adoptive transfer of genetically modified T cells. Disclosures Slatter: Medac: Other: Travel assistance. Merli:Neovii Biotech: Honoraria; AMGEN: Honoraria. Aldinger:Bellicum Pharmaceuticals, Inc.: Employment. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy.

Introduction. Severe acute graft-versus-host disease (GVHD) remains the most relevant complication after allogeneic HSCT. Although its incidence in the pediatric population is lower than in adults, children with severe acute GVHD and who do not respond to first-line treatment with systemic steroids still have a poor prognosis. The exact incidence of steroid-refractory acute GVHD in children is still not precisely defined, as well as the risk of non-relapse mortality (NRM) due to steroid-refractory acute GVHD. Aim of our study was to analyze the frequency of acute GVHD unresponsive to first-line steroid treatment in children and adolescents given allogenic HSCT, to describe the second line treatment employed, and the outcome of patient with this complication. Patients and methods. This retrospective study included patients younger than 18 years at the time of transplantation and given a first allogeneic HSCT between 2010 and 2015 in one of the HSCT Centers of the Italian Association for Pediatric Hematology / Oncology (AIEOP). Overall, 1608 patients (59% M and 41% F) were analyzed. Median age at HSCT was 8 years (range 0.2 - 18) 1084 (67%) were affected by malignant diseases and 524 (33%) by non-malignant disorders. The donor was an HLA-matched family donor (MFD) in 28% of cases, an unrelated donor (UD) in 52% and an HLA-haploidentical family donor in 20%. In MFD transplants Cyclosporine (CSA) was used as GVHD prophylaxis in 30% of cases and the combination of CSA + short-term methotrexate (MTX) in 48%. 75% of UD transplant recipients received CSA + MTX + anti-thymocyte globulin (ATG) as GVHD prophylaxis, and 25% other drug combinations. Ex vivo T-cell depletion of the graft was employed in most patients given a HLA-haploidentical HSCT (79% of transplants), and high-dose post-transplant cyclophosphamide in 11% of cases. Results. The cumulative incidence (CI) of grade II-IV acute GVHD was 31%, while that of grade III-IV acute GVHD was 10%. The overall incidence of chronic GVHD was 13% and that of extensive chronic GVHD was 6%. The CI of NRM was 14% for grade 0 acute GVHD patients, 9% for grade I, 11% for grade II, 26% for grade III and 68% for grade IV (P < 0.001). Of the 491 patients with grade II-IV acute GVHD, 250 (51%) required a second-line treatment after first-line steroid therapy (30% of grade II, of 75% grade III and 83% of grade IV patients). Acute GVHD requiring second-line treatment was more frequent in UD transplant recipients (21% of patients) than in matched sibling or haploidentical donor recipients (7% and 13% respectively, P < 0.01), while age at HSCT and diagnosis (malignant vs. non-malignant disease) were not associated with this complication. Second-line treatment was extracorporeal photochemotherapy in 60% of patients, mofetil mycophenolate (MMF) in 46%, mesenchymal stromal cells (MSC) in 12%, monoclonal antibodies (MoAbs) in 5% and other treatments in 28%; 32% of patients received more than one second line treatment. Overall NRM was 13% for patients with grade 0-I acute GVHD, 15% for grade II-IV responding to steroids, and 25% for grade II-IV patients requiring second-line therapy (P < 0.001). The addition of a second-line treatment partially decreased NRM only in patients with grade IV acute GVHD, but the difference was not statistically significant (66% vs. 78%; P = 0.313). In multivariable analysis, grade III (HR = 1.84; P = 0.044) and grade IV acute GVHD (HR = 7.07; P < 0.001), and the use of an UD (HR = 1.63; P = 0.007) were associated with an increased NRM, while the use of a second-line treatment did not decrease this risk (HR = 0.80; P = 0.368). Conclusions. Despite being less frequent than in adults, severe steroid-refractory acute GVHD is still associated with a very high NRM also in pediatric patients. Second or third-line treatments adopted so far have not been effective in improving control of the complication and in decreasing NRM. The prospective evaluation of acute GVHD biomarkers (such as ST2 and REG3α) could help in identifying patients at higher risk of NRM. Prospective studies are warranted to define new treatment modalities that could decrease the mortality rate associated with the most severe form of disease. Disclosures Zecca: Chimerix: Honoraria. Locatelli:bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Background Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment for children with Acute Leukemia (AL). For patients lacking a compatible matched related or unrelated donor, HLA-haploidentical HSCT (haplo-HSCT) from a relative represents a viable alternative. Promising results were reported with a novel method of selective depletion of αβ T and B cells (Locatelli, Blood 2017). This approach is associated with limitations such as suboptimal adaptive immune reconstitution, increased risk of infection and disease relapse. BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of BPX-501 following transplant. The polyclonal nature of the BPX-501 provides broad virus and tumor-specific immunity, while the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid which induces dimerization and activation of iC9, inducing apoptosis of BPX-501. Aims To evaluate the safety and efficacy of BPX-501 administered after a αβ T and B-cell depleted haplo-HSCT in pediatric patients with AL in morphological complete remission (CR). The objective was to determine whether BPX-501 infusion can increase relapse-free survival (RFS) and overall survival (OS) through an enhanced graft-versus-leukemic (GvL) effect, while maintaining a low risk of GvHD. Methods This multicenter US (NCT03301168) and EU (NCT02065869), prospective trial utilizes αβ-T and B-cell-depleted haplo-HSCT followed by infusion of donor lymphocytes genetically modified with iC9 safety switch (BPX-501) in patients with malignant or non-malignant disorders. A subset of patients had acute high-risk leukemias (AML and ALL). BPX-501 was planned to be infused on day14±4 after the allograft. No post-transplant pharmacological GvHD prophylaxis was employed. Patients who develop GvHD resistant to conventional steroid therapy could receive ≥1 dose of rimiducid to activate iC9. The efficacy-evaluable population (EEP) was defined as any patient with AL who received HSCT, BPX-501 infusion, and at least one follow-up assessment. Results At clinical cut-off (June 30, 2018), 100 patients (EU: 75, US: 25) with AL met the EEP definition. Median follow-up was 14.7 mos (1 - 40.6 mos). Key baseline characteristics are shown in Table 1. The median time for neutrophil and platelet engraftment was 16 (15 - 17) and 12 (11 - 12) days, respectively. Four patients (4.1% [95% CI: 0 - 8%]) experienced primary graft failure. Of 96 evaluable patients, 21 patients developed Grade I-IV aGvHD (21.7% [95% CI: 13.5 - 29.8%]). Five patients developed Grade III-IV aGvHD (3.1% [95% CI: 0 - 6.5%]). Of 82 evaluable patients, 12 patients developed cGvHD (18.1% [95% CI: 8.2 - 22%]), with only three cases being moderate-severe. Rimiducid was administered to 10 patients with steroid-resistant acute GvHD. Best overall clinical response of CR or PR post-rimiducid administration was seen in 8 patients (80%). Among responding patients, 7 patients (87.5%) had a CR. Six patients died after transplantation (6.6% [95% CI: 1.4 - 11.7%]). Relapse Free Survival (RFS) was 82.2% (95% CI: 74.5 - 89.7%). Overall Survival (OS) was 90.1% (95% CI: 83.9 - 96.3%). Efficacy outcomes (TRM, RFS and OS) in AL subsets (AML and ALL) are shown in Table 2. CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by 180 and 270 days, respectively. IgA and IgM levels achieved normal values by 180 days. The percentage of circulating and median absolute BPX-501 cells at Day 100 were 9.96% ± 11.6% (0 - 54.9%) and 85.58 ± 165.57 cells/ul (0 - 1001 cells/ml), respectively. Conclusion The adoptive transfer of BPX-501 following αβ-T and B-cell depleted haplo-HSCT represents a novel and highly effective transplantation strategy for pediatric patients with AL. Compared to data from children receiving only αβ T and B-cell depleted haplo-HSCT or matched unrelated donor HSCT (https://bloodcell.transplant.hrsa.gov/research/transplant_data/us_tx_data/survival_data/survival.aspx), this novel approach resulted in a comparable risk of transplant-related mortality and a lower risk of recurrence. Rimiducid was also an effective treatment for patients who developed steroid-resistant GvHD. Disclosures Locatelli: bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Qasim:Orchard: Equity Ownership; Autolus: Equity Ownership; Servier: Research Funding; Bellicum: Research Funding. Nemecek:Novartis Pharmaceuticals Corporation: Other: advisory boards.

Background αβ T- and B-cell depleted HLA-haploidentical HSCT (αβ haplo-HSCT) is an alternative promptly available and virtually applicable to any children with malignant and non-malignant disorders lacking an HLA-matched donor or in need of an urgent allograft (Locatelli et al. 2017). Through this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γδ T cells, which confers protection also against infections. Nonetheless, even with this type of HSCT, the recovery of adaptive immunity is suboptimal. We have developed a further refinement of the approach based on the post-transplant infusion of a titrated number of donor T cells transduced with the inducible suicide gene iC9 (BPX-501 cells). This strategy has the potential to accelerate the recovery of adaptive immunity and to restore an efficient T-cell mediated graft-versus-leukemia (GvL) effect without the risk of inducing uncontrollable graft-versus-host disease (GvHD). Since reconstitution of a naïve T- and B-cell repertoire plays a key role in the long-term ability to respond to a broad range of pathogens, as well as to tumor antigens, we quantified T-cell receptor and kappa chain-deleting recombination excision circles (TREC and KREC), which represent reliable surrogate of T and B-cell neogenesis respectively. Study design and patients We analyzed samples of 48 children (M/F: 26/22) with acute leukemia (31 ALL, 17 AML) given an ab T-cell and B-cell depleted haplo-HSCT after a myeloablative regimen followed by the infusion of a titrated number of donor BPX-501 cells (1 million/Kg) after a median of 27 days (range: 11-87). Median age at transplantation was 8.3 years (range, 0.9-18). Thirty-two and 16 patients did or did not receive a preparative regimen containing total body irradiation (TBI). Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech, 12 mg/Kg) was administered from day -4 to -2 for preventing graft rejection and GvHD. Moreover, to reduce the risk of EBV-related PTLD, on day -1, patients received rituximab (200 mg/m2). We analyzed T-cell reconstitution by measuring the TREC, small DNA excision circles of the TCR d locus deleted during recombination of the a-locus and present in the majority of functional ab-T-cells (sjTREC), and during the TCR b-chain recombination (bTREC). We also analyzed B-cell reconstitution by measuring coding-joint (Cj), signal-joint KREC (sjKREC) which reflect newly produced naïve B cells and the mean number of B-cell division (n) using the formula n=LOG(Cj/sjKREC)/LOG2. We performed real-time quantitative PCR, as recently described (Arruda et al. 2018), on genomic DNA extracted from PBMC collected at 6 different time points (before and 1, 3, 6, 12 and 18 months after the allograft). Results Recovery of thymic function started at 3 months after transplant: sjTREC and bTREC amounts reached and overcame pre-transplant values at 6 months, continuing to increase until 18 months after HSCT. Number of B-cell divisions obtained by the measurement of cj and sjKREC found in peripheral blood matched with the newly-generated B-cells bone marrow output and showed the same kinetics of sj and bTREC, rising from the 3rd month after HSCT. All these data correlate with immunophenotyping findings, which show a progressive increase over time of both total CD3+ and ab-T-cells. Furthermore, we observed a negative impact on both T- and B-cell neogenesis played by aGvHD (occurring in 14 patients), which became significant from 3 months after HSCT for sjTREC (p=0.013) and at 6 months for sjKREC (p= 0.03). We did not observe any impact of the use of TBI during the preparative regimen on T- and B-cells generation in this cohort. Conclusions BPX-501 cell infusion aims to contribute to the acceleration of immune reconstitution after a/b haplo-HSCT. In this study, we show that naïve T- and B- cells are quickly and efficiently generated in the early post-transplant period. This finding represents an interesting result considering the fully myeloablative conditioning regimens with the large use of TBI and the Ta/b and B-cells depletion (also in vivo by the administration of Rituximab) received by the patients. We observed an expected detrimental effect of aGvHD on immune reconstitution, but we emphasize the low frequency of this complication in this transplant setting despite the absence of any post-transplant pharmacological prophylaxis. Disclosures Foster: Bellicum: Employment, Equity Ownership. Locatelli:bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Relapse of malignancy and lethal graft versus host disease (GVHD) are the principal causes of failure of allogeneic hematopoietic cell transplant (HCT). Recently we have shown that at seven days after HCT an algorithm using two serum biomarkers (ST2 and REG3α) can predict severe GVHD (Hartwell et al. JCI Insight 2017). We determined whether serial testing (in the first month following HCT) of patients with low probability biomarkers would improve the predictive accuracy of the algorithm and identify patients with different risks of relapse and lethal GVHD. Patients who received an HCT at 18 centers in the Mount Sinai Acute GVHD International Consortium (MAGIC) for hematologic malignancy and who supplied three blood samples were divided into a training set and validation set with equal numbers of lethal GVHD events, which was defined as death from GVHD or infection during treatment for GVHD. Patients in the training set (n=702) underwent HCT from January 1, 2006 until June 30, 2015, whereas patients in the validation set (n=906) underwent HCT from July 1, 2015 to May 1, 2017. Serum samples were analyzed using the previously published algorithm of two biomarkers up to three times (day 7, day 14, day 28 or GVHD onset, if onset occurred within the first 28 days). The algorithm generates a predicted probability of lethal GVHD between 0 and 1 for each patient. Patients were categorized as either low probability (LP) or high probability (HP) for lethal GVHD. HP thresholds of 0.20 and 0.16 were used to classify patients with and without GVHD symptoms, respectively (once categorized as HP, patients remained in that category and were not retested). All results were similar between training and validation sets, and we present here the validation set results. Serial testing identified 28% of patients as HP with a three-fold greater cumulative incidence of lethal GVHD at one year (13% vs 4%, p Early development of GVHD (by day 28) is a risk factor for lethal GVHD. Therefore, we next plotted RFS (dashed line), relapse (solid line), and lethal GVHD (dotted line) rates in patients who developed GVHD by day 28. 25% of patients with GVHD were categorized as HP and had a cumulative incidence of lethal GVHD more than four times higher (28%) than that of relapse (6%); however the risks were reversed for the 75% of patients who were LP, where relapse (15%) occurred twice as often as lethal GVHD (7%) (Figure 3B). In patients who did not develop GVHD in the first month, this reversal of risks was even more dramatic. Approximately half (53%) of the entire validation cohort did not develop GVHD by day 28 and was LP at all three evaluations. These patients had an exceptionally low risk of lethal GVHD and thus they relapsed (25%) much more often than they died from GVHD (3%). When malignancies were classified according to risk for relapse by the disease risk index (DRI) (Figure 3C), the probability of relapse was three fold higher than lethal GVHD in malignancies with a low DRI (12%), six fold higher for intermediate DRI (20%), and eleven fold higher for high/very high DRI (33%). We conclude that a serial monitoring strategy using GVHD biomarkers for one month after HCT is able to identify two groups of patients with very different risks of lethal GVHD and relapse. For these patients, the intensity of immunosuppression after day 28 could be tailored according to the probabilities of developing lethal GVHD and relapse in the context of clinical trials. Disclosures Aziz: Doris Duke Charitable Foundation: Research Funding. Ayuk:Therakos (Mallinckrodt): Honoraria; Novartis: Honoraria; Celgene: Consultancy; Gilead: Consultancy. Chen:REGiMMUNE: Consultancy; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Merli:Neovii Biotech: Honoraria; AMGEN: Honoraria. Roesler:Sanofi: Other: Travel, Accommodations, Expenses; Amgen: Equity Ownership; Jazz Pharmaceuticals: Other: Travel, Accommodations, Expenses; Immunomedics: Equity Ownership; Biogen: Equity Ownership; Merck: Consultancy; Pfizer: Consultancy. Kitko:Novartis: Consultancy, Honoraria; Mallinckrodt: Honoraria, Other: Travel, Accommodations, Expenses. Qayed:Novartis: Consultancy. Wölfl:Bristol-myers Squibb: Equity Ownership; Novartis: Equity Ownership; Taheda: Equity Ownership; Juno: Equity Ownership; Neovii: Other: Travel, Accommodations, Expenses. Mielke:Celgene: Speakers Bureau; DGHO: Speakers Bureau; EHA: Speakers Bureau; Kiadis Pharma: Speakers Bureau; Miltenyi: Speakers Bureau. Wudhikarn:Takeda Oncology: Other: Travel, Accommodations, Expenses. Nakamura:Celgene: Honoraria; Molmed: Honoraria; Merck: Consultancy; Pharmacyclics: Consultancy; Atara: Consultancy; Jazz Pharmaceuticals: Consultancy. Pulsipher:CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria. Reshef:Pfizer: Consultancy; Atara Biotherapeutics: Consultancy; Kite Pharma: Consultancy; Takeda Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy. Levine:Therakos: Consultancy; Novartis: Consultancy; Bluebird: Consultancy; Incyte: Consultancy; Kamada: Research Funding; Viracor: Patents & Royalties. Ferrara:Incyte: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Xenikos: Consultancy, Other: Travel, Accommodations, Expenses; Kamada: Consultancy, Research Funding; Viracor: Consultancy, Patents & Royalties.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-haploidentical relative (haplo-HSCT) is a suitable option for children/young adults with acute leukemia (AL) either relapsed or at high-risk of treatment failure and in urgent need of an allograft. A novel method of graft manipulation based on the selective, negative depletion of αβ T and B cells has been recently developed. We published the results of a prospective trial (ClinicalTrial.gov identifier: NCT01810120) enrolling 80 children with AL transplanted until September/2014 using this approach (Locatelli, Blood 2017). In the present analysis, we update those results, evaluating also additional patients given haplo-HSCT after that date. Patients and methods: Analyzed are 111 children with AL enrolled in the trial; median age is 10 years (range 0.9-22.2). Eighty-two (74%) and 29 (26%) patients had acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), respectively; they were transplanted between 09/2011 and 05/2018. All children were transplanted in complete morphological remission and received a fully myeloablative preparative regimen. Details on patients' characteristics, as well as on the number of HSC and lymphocyte subsets infused, are shown in Table 1. The donor was mainly chosen according to immunological criteria, giving priority to NK-cell alloreactivity, evaluated according to the killer immunoglobulin-like receptor (KIR)/KIR-Ligand mismatch in graft-versus-host direction model, KIR B haplotype, higher B-content score and size of NK alloreactive subset. Anti-T lymphocyte globulin (ATLG Grafalon®, Neovii Biotech) was administered at a dose of 12 mg/Kg from day -5 to -3 for preventing graft rejection and graft-versus-host disease (GvHD). Moreover, to reduce the risk of EBV-related post-transplant lymphoproliferative disorder (PTLD), on day -1, patients received rituximab (200 mg/m2) for in vivo depletion of both donor and recipient B cells. No patient was given any post-transplantation pharmacological GvHD prophylaxis. Results: Median follow-up of surviving patients is 47 months (range: 2 months - 7.7 years). All patients but two successfully engrafted and the median time to neutrophil and platelet recovery was 13 (range 9-22) and 11 (range 8-20) days, respectively. Acute GvHD occurred in 28 patients; it was of grade I and grade II severity in 9 and 19 patients, respectively. Skin was the sole organ involved in all patients but one, who had gut involvement. The cumulative incidence of grade I-II acute GvHD was 25% (95% confidence interval, CI, 17-33). Four out of the 91 patients at risk developed chronic GvHD, in all cases of limited severity, the cumulative incidence of this complication being 5% (95% CI, 1-9). Six patients died for transplant-related complications, this resulting into a 5-year cumulative incidence of transplant-related mortality (TRM) of 6% (95% CI, 2-11). Twenty-three patients relapsed at a median time of 186 days (range 60-1012) after transplantation, the 5-year cumulative incidence of relapse being 24% (95% CI, 16-33). The 5-year probability of overall and leukemia free survival (LFS) were both above 70%, as shown in Figure 1A and 1B, respectively. The 5-year probability of LFS in children with ALL and AML was 69% (95% CI, 57-79) and 73% (95% CI, 52-86), respectively (Figure 1C). Use of total body irradiation (TBI) during the preparative regimen was associated with better patient's outcome (Figure 1D), since it protected against the risk of leukemia recurrence [18% (95% CI, 10-28) vs. 45% (95% CI, 22-66) in patients who did or did not receive TBI, respectively, p Conclusions: This study, reporting long-term outcome of a large population of children/young adults with AL, confirms that αβ T- and B-cell depleted haplo-HSCT is an effective option for patients in need of an urgent allograft and lacking an HLA-identical donor. While TRM is impressively low, the main cause of treatment failure is leukemia recurrence, whose incidence can be lowered by the use of TBI during the conditioning regimen. The remarkably low risk of chronic GvHD renders the approach attractive also in terms of patient's quality of life. Figure 1 Figure 1. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

In view of the exciting results reported in patients with CD19+ malignancies given CAR T cells, it is expected that a continuously growing number of patients will be offered this treatment and, thus, will be exposed to gene-modified products. Since the techniques of gene manipulation are relatively new, some of the risks associated to CAR T therapy may be unpredictable. Recently, two patients who relapsed with CD19-, CAR-expressing leukemia were reported, this observation being interpretable in light of an inadvertent leukemic cell transduction with the second generation CAR.CD19 lentivirus during CAR T cell manufacturing (Lacey, ASH, 2016 128:281). Immunoglobulin heavy chain sequencing analysis of 17 additional infusion products also identified the leukemic clonotypes in six additional products (86%). In vitro and in vivo experiments proved that these CAR+ leukemic clones were not killed by CAR.CD19 T cells (Ruella, ASH, 2017 130:4463). Since lentiviruses proved to be superior for transduction of quiescent hematopietic stem cells due to their ability to infect non-dividing cells, we hypothesized that CAR-T cell manufacturing based on the genetic modification of T cells by gammaretroviral vector could theoretically represent a safe approach. Peripheral blood or bone marrow (BM)-derived mononuclear cells of patients with >40% of blasts at diagnosis (CD45dim+/CD34+/CD19+/CD22+/CD10+), were transduced with a retrovirus encoding for a second generation CAR.CD19.41bb.z in frame with a suicide gene (i.e., inducible caspase 9, iC9) employed in the academic Clinical Trial (NCT03373071) run at the Bambino Gesù Children's Hospital, Rome, Italy. Patient-derived CAR-T cells showed a phenotype not significantly different from that found on CAR-T cells generated by healthy-donors (data not shown). In particular, we demonstrated that both flow-cytofluorimetry and RealTime-quantitative PCR (with a sensitivity up to 10-5) failed to identify leukemic cells in the final CAR-T cell products generated from Bcp-ALL patients. To generate an in vitro model of CAR+ leukemic cells, we genetically modified CD19+ RAJI and DAUDI cell lines with the bicistronic retroviral vector carrying both second generation CAR.CD19 and the suicide gene iC9 (iC9.CAR-RAJI and iC9.CAR-DAUDI). We demonstrated the possibility of promptlyeliminating CAR+ leukemic cells, through exposure to 20nM of AP1903 of iC9.CAR-DAUDI and iC9.CAR-RAJI cells. Indeed, very early activation (6 hours) of the suicide gene iC9 resulted into a significant reduction in the percentage of CAR+ RAJI leukemic cells (Fig.A). The presence of iC9.CAR.CD19 molecule on leukemic cells precluded the detection of the CD19 antigen, whereas cells retain the expression of all other specific B-lineage markers. CD19 antigen started to be detectable 72 hours after AP1903 exposurewhen CAR negative leukemic cells become preponderant. To demonstrate that CD19 antigen was not down-regulated, but only masked by CAR molecule in iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines, we measured CD19 mRNA, showing no significant modification with respect to wild-type (WT) RAJI and DAUDI cell lines. Moreover, iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines were effectively eliminated by CAR.CD19 T cells (12.5±13.7% and 3.4±4.3% residualleukaemia, respectively) at the same extent of WT cell line (0% and 0.08±0.1%, residual leukaemia, respectively; p>0.05 Fig.B). To assess if patient-derived iC9.CAR.CD19 T cells were able to generate leukemia in vivo mouse model, NSG female mice were infused i.v. with 10x106 CAR-T cells and control NT-T cells. Mice were monitored for a total period of 250 days, by recurrent bleed. Simultaneously, another cohort of mice was infused with patient-derived BM cells (5x106) and monitored for the same time. Mice infused with Bcp-ALL BM cells developed leukemia-phenotype,with 82% of cells expressing hCD45dim and hCD19. By contrast, mice receiving patient-derived CAR-T cells showed a lowpercentage of CD45+ cells (0.1±0.01%), all CD3+. Despite the long period of observation, we did not detect any expansion of hCD19+ cells in this animal cohort. Taken together these data suggest that the use of a retroviral platform, associated with the presence of iC9 suicide gene, contributes to the genesis of a highly functional and safe CAR-T product, even when the production starts from a biological material characterized by high contamination of leukemic blasts. Disclosures Locatelli: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The interplay existing between immune reconstitution and patient outcome has been extensively demonstrated in allogeneic hematopoietic stem cell transplantation. One of the leading causes of infection-related mortality is the slow recovery of T-cell immunity due to the conditioning regimen and/or age-related thymus damage, poor naive T-cell output, and restricted T-cell receptor (TCR) repertoires. With the aim of improving posttransplantation immune reconstitution, several immunotherapy approaches have been explored. Donor leukocyte infusions are widely used to accelerate immune recovery, but they carry the risk of provoking graft-versus-host disease. This review will focus on sophisticated strategies of thymus function-recovery, adoptive infusion of donor-derived, allodepleted T cells, T-cell lines/clones specific for life-threatening pathogens, regulatory T cells, and of T cells transduced with suicide genes.

Many different studies have demonstrated that early recovery of the adaptive immune system after allogeneic hematopoietic stem cell transplantation (HSCT) is predominantly sustained by peripheral expansion of donor-derived, mature lymphocytes transferred with the graft. Different approaches based on the infusion of donor T cells after HSCT have been developed mainly to accelerate immune recovery and to treat/prevent (a) malignancy recurrence, (b) life-threatening infections, and (c) immune-mediated disorders, such as graft-versus-host disease (GVHD). For many years, donor lymphocyte infusion (DLI) has been a widely used approach to prevent and to treat leukemia recurrence, to convert mixed chimerism into complete donor chimerism, and to accelerate immune reconstitution of patients after HSCT. More sophisticated strategies of adoptive infusion of T cell lines/clones capable of mediating a graft-versus-leukemia (GVL) response, while avoiding GVHD occurrence, or specific for the most life-threatening pathogens (e.g., cytomegalovirus, Epstein-Barr virus, and adenovirus) have been envisaged and successfully tested in pilot trials in the early post-transplantation period. Also, ex vivo expanded regulatory T (Treg) cells have been shown to be beneficial for preventing GVHD post-HSCT. In this review, we will focus on DLI as well as more complex cellular therapies that require extensive cell manipulation.

We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8–10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1–2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on ‘benchtop' sequencers combining rapid turnaround times with higher manageability.

We recently described the CD56lowCD16low subset of Natural Killer (NK) cells that both mediate cytotoxic activity and produce IFNγ, being more abundant in bone marrow (BM) than in peripheral blood (PB) of pediatric normal subjects. Given the multifunctional properties of this subset, we examined its development and functional recovery in a cohort of children undergoing α/β T-cell depleted HLA-haploidentical haematopoietic stem cell transplantation (HSCT). The results obtained indicate that CD56lowCD16low NK cells are present in both PB and BM already at one month post-HSCT, with an increased frequency in BM of graft recipients as compared with normal subjects. During the first 6 months after HSCT, no difference in CD56lowCD16low NK cells distribution between PB and BM was observed. In comparison to normal subjects, CD56lowCD16low NK cells from transplanted patients show lower expression levels of CD25 and CD127 and higher levels of CD122, and accordingly, produce higher amounts of IFNγ after stimulation with IL-12 plus IL-15. The recovery of NK-cell cytotoxicity after HSCT was strictly restricted to CD56lowCD16low NK cells, and their ability to degranulate against K562 target cells or autologous leukemic blasts was completely restored only one year after HSCT. Based on the phenotypic and functional ability of reconstituted CD56lowCD16low NK cells, we suggest that they play an important role in host defense against leukemia relapse and infections after HSCT, and represent an ideal candidate for approaches of adoptive immunotherapy.

Natural killer (NK) cells are important effectors playing a relevant role in innate immunity, primarily in tumor surveillance and in defenses against viruses. Human NK cells recognize HLA class I molecules through surface receptors (KIR and NKG2A) that inhibit NK cell function and kill target cells that have lost (or underexpress) HLA class I molecules as it occurs in tumors or virus-infected cells. NK cell activation is mediated by an array of activating receptors and co-receptors that recognize ligands expressed primarily on tumors or virus-infected cells. In vivo anti-tumor NK cell activity may be suppressed by tumor or tumor-associated cells. Alloreactive NK cells (i.e. those that are not inhibited by the HLA class I alleles of the patient) derived from HSC of haploidentical donors play a major role in the cure of high-risk leukemia, by killing leukemia blasts and patient's DC, thus preventing tumor relapses and graft-versus-host disease. The expression of the HLA-C2-specific activating KIR2DS1 may also contribute to NK alloreactivity in patients expressing C2 alleles. A clear correlation has been proven between the size of the alloreactive NK cell population and the clinical outcome. Recently, haplo-HSCT has been further improved with the direct infusion, together with HSC, of donor-derived, mature alloreactive NK cells and TCRγδ+ T cells - both contributing to a prompt anti-leukemia effect together with an efficient defense against pathogens during the 6- to 8-week interval required for the generation of alloreactive NK cells from HSC.

Several studies have shown that in patients with acute leukaemia given allogeneic haematopoietic stem cell transplantation (allo-HSCT) large part of the therapeutic effect lies on the anti-tumour effect displayed by cells of both adaptive and innate immunity. This evidence has also opened new scenarios for the treatment of patients with other haematological malignancies/solid tumours. In particular, donor-derived natural killer (NK) cells play a crucial role in the eradication of cancer cells in patients given an allograft from an HLA-haploidentical relative, especially when there is a killer inhibitory-receptor (KIR)-KIR ligand mismatched in the donor-recipient direction. Alloreactive donor-derived NK cells have been also demonstrated to kill recipient antigen-presenting cells and cytotoxic T lymphocytes, thus preventing graft-versus-host disease (GvHD) and graft rejection and to largely contribute to the defence against cytomegalovirus infection in the early post-transplant period. Several clinical studies have recently focused also on the influence of NK-cell activating receptors on the outcome of allo-HSCT recipients; in particular, B/x haplotype donors offer clinical advantages compared with A/A donors, even when the donor is an HLA-identical volunteer. Altogether, these data have provided the rationale for implementing phase I/II clinical trials based on adoptive infusion of either selected or ex vivo activated NK cells from an HLA-mismatched donor. This review summarizes the biological and clinical data on the role played by NK cells in patients with high-risk acute leukaemia, focusing also on the still unsolved issues and the future perspectives related to the approaches of adoptive NK cell therapy.

Twenty-three children with nonmalignant disorders received HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) after ex vivo elimination of αβ(+) T cells and CD19(+) B cells. The median number of CD34(+), αβ(+)CD3(+), and B cells infused was 16.8 × 10(6), 40 × 10(3), and 40 × 10(3) cells/kg, respectively. No patient received any posttransplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD). All but 4 patients engrafted, these latter being rescued by a second allograft. Three patients experienced skin-only grade 1 to 2 acute GVHD. No patient developed visceral acute or chronic GVHD. Cumulative incidence of transplantation-related mortality was 9.3%. With a median follow-up of 18 months, 21 of 23 children are alive and disease-free, the 2-year probability of disease-free survival being 91.1%. Recovery of γδ(+) T cells was prompt, but αβ(+) T cells progressively ensued over time. Our data suggest that this novel graft manipulation strategy is safe and effective for haplo-HSCT. This trial was registered at www.clinicaltrials.gov as #NCT01810120.

Objectives To investigate whether children with juvenile idiopathic arthritis (JIA) in clinical remission have subclinical synovial disease on ultrasound, and whether ultrasound abnormalities predict an early flare of synovitis. Methods Thirty-nine consecutive children who had clinically defined inactive disease (ID) for a minimum of 3 months underwent ultrasound assessment of 52 joints. All joints were scanned for synovial hyperplasia, joint effusion, power Doppler (PD) signal and tenosynovitis. Patients were then followed clinically for up to 2 years until a flare of synovitis occurred in one or more joints, or until the 2-year visit if the disease remained in clinical remission. Results Synovial hyperplasia, joint effusion, PD signal and tenosynovitis in at least one joint were detected in 76.9%, 66.7%, 33.3% and 15.4% of patients, respectively. During the 2-year follow-up, 24 patients (61.5%) experienced sustained ID, whereas 15 patients (38.5%) had a flare of synovitis in a total of 45 joints after a median of 10.6 months (range 6.3–13.7 months). At study entry, the rate of synovial hyperplasia, joint effusion and tenosynovitis was comparable between patients with persistent ID and patients with synovitis flare, whereas patients with persistent ID had a greater frequency of PD signal than patients with synovitis flare. Only 17 of the 45 flared joints had ultrasound abnormalities at study entry. Conclusion The authors found that ultrasound-detected synovial abnormalities are common in children with JIA in clinical remission. However, the presence of ultrasound pathology did not predict an early flare of synovitis in the affected joints.

T-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is a suitable option for patients in need of an allograft who lack a HLA-matched donor. Although it offers the advantage of being immediately applicable to virtually all patients, so far, graft manipulation with removal of all T lymphocyte subsets and of natural killer (NK) cells has been associated with an increased risk of life-threatening infections, as well as, in some studies, of leukemia recurrence. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature NK cells and γδ+ T cells in the graft. We, thus, started a formal study (NCT01810120) in children with acute leukemia aimed at evaluating the safety and efficacy of this approach. As of April 2013, we enrolled 45 patients (pts; 29 males, 16 females). Median age at HSCT was 10.1 years (range 0.9-17.9). Thirty-five pts had acute lymphoblastic leukemia (ALL) and 10 acute myeloid leukemia (AML); all children were transplanted in morphological complete remission (CR). Fifteen pts were transplanted in first CR, 27 in second CR and 3 in more advanced CR. All pts transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease (MRD) at the end of induction therapy. The donor was either the mother (n=25) or the father (n=20); according to the model of KIR/KIR ligand disparity, 22 pts were transplanted from an NK-alloreactive donor. The median number of CD34+ cells, NK cells, γδ+ T cells, B cells and αβ+ T cells were 14.6, 31.7, 7.8, 0.08 and 0.04x106/kg, respectively. A myeloablative regimen, containing Total Body Irradiation in 34 cases, was given to all children, who also received anti-thymocyte globulin (12 mg/kg over 3 days, from -5 to -3). Rituximab (200 mg/m2) was administered on day -1 to further prevent EBV-related lymphoproliferative disorders. No pharmacological graft-versus-host disease (GVHD) prophylaxis was employed after transplantation. Sustained primary engraftment occurred in 44/45 pts, the remaining child being successfully re-transplanted from the other parent. The median time to reach an absolute neutrophil count >0.5x109/L and a platelet count >50x109/L was 13 days (9-18) and 11 days (8-20). No child developed gut or liver acute GVHD. Thirteen pts experienced skin-only grade I-II GVHD, this leading to a cumulative incidence (CI) of 29% (95% confidence interval, CO. IN., 18-45). Only 2 pts developed skin limited chronic GVHD. Two pts died for causes other than disease relapse (both in the first 60 days after HSCT), the CI of transplantation-related mortality (TRM) being 4% (CO. IN. 1-16). Seven pts relapsed, the CI of disease recurrence being 16% (CO. IN. 6-32). With a median follow-up of 11 months (range 2-30), the 2-year Kaplan Meier estimate of leukemia-free survival (LFS) was equal to 75% (CO. IN. 57-86); this value was 73% (CO. IN. 52-85) for pts with ALL. LFS of pts who did or did not experience skin-only acute GVHD was 83% (CO. IN. 48-96) and 72% (CO. IN. 50-86), p=NS. The probability of LFS of the 39 pts with either negative or low ( These data indicate that a selective graft manipulation results into effective prevention of both acute and chronic GVHD, rapid recovery of neutrophil and platelet counts and low TRM. Although the median observation time is still limited, also the CI of disease recurrence is encouraging. Post-transplant add-back of limited numbers of αβ+ T cells transduced with the iCasp9 suicide gene could optimize the recovery of this subset in early weeks after HSCT, maximizing immune-mediated viral and leukemia control, without an unacceptable risk of GVHD. Disclosures: No relevant conflicts of interest to declare.

Background: Allogeneic HSCT from either an HLA-identical sibling or an unrelated donor is a potentially curative treatment for patients with hemoglobinopathies and erythroid disorders (ED), such as Thalassemia Major (TM), Sickle Cell Disease (SCD) and Diamond-Blackfan Anemia (DBA). The limited historical experience with HLA-haploidentical HSCT in this setting has reported a disease-free survival probability lower than that reported using HLA-matched donors. In the last few years, we have developed a novel method of graft manipulation, based on the selective depletion of α/β+ T-cells and CD19+ B-cells (ClinicalTrial.gov identifier: NCT01810120), which was shown to be safe and effective in children with multiple types of non-malignant disorders (Bertaina el al, Blood 2014). To further optimize this approach through the acceleration of the recovery of adaptive immunity, we designed an ongoing phase I/II trial aimed to test the safety and efficacy of post-transplant infusion of donor T-cells transduced with the iC9 suicide gene (BPX-501 cells) in children with either malignant or non-malignant disorders (ClinicalTrials.gov identifier: NCT02065869). As the transduced gene contains sequences for the CD19 marker, BPX-501 cells (CD3+/CD19+) can be easily tracked in peripheral blood. We report on 10 children with hemoglobinopathies and ED who were enrolled in the phase II portion of the study. Patients and methods: Five patients were males and 5 were females, and median age at diagnosis and at HSCT was 5.34 and 9.52 years (range 2.33-11.71), respectively. Seven patients had TM (all bo/bo), 2 DBA and 1 SCD. All 10 patients were transfusion-dependent and receiving iron-chelation therapy before haplo-HSCT. Among the thalassemia patients, 4 patients belonged to class I and 3 to class II of the Pesaro classification. All patients were transplanted from a parent. Median number of CD34+ and αβ+ T-cells infused was 22.5 x 106/kg and 0.3 x 105/kg, respectively. In all patients, conditioning regimen included busulfan (16 mg/Kg), thiotepa (10 mg/Kg) and fludarabine (160 mg/m2). Rabbit ATG (12 mg/Kg over 3 days, from day -4 to day -2) was administered to prevent graft-versus-host disease (GvHD) and graft failure and Rituximab (200 mg/ m2 on day -1) was administered to prevent EBV-related lymphoproliferative disorders. No post-transplantation GvHD prophylaxis was given. Median follow-up is 301 days (range 41-420 days). Basic phenotyping of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT. Results: After haplo-HSCT, the median time to reach neutrophil and platelet recovery was 14 days (range 11-28) and 10 days (range 8-12), respectively. After engraftment of the allograft, BPX-501 cells were infused (dose: 1x106 cells/kg) at a median time of 13.5 days after HSCT (range 10-26). Nine of the 10 patients maintained sustained donor engraftment, reaching full chimerism. The patient who experienced secondary graft failure was successfully re-transplanted from the same parent and he is full donor chimeric with transfusion-independence. Grade I/II skin acute GvHD occurred in 2 patients (at 31 and 59 days after HSCT, respectively). There was no occurrence of chronic GVHD. Remarkably, no patient has died and none of the patients have been re-hospitalized after initial discharge. The last erythrocyte transfusion was administered on day +7 post-transplant (range 4-33 days). At last follow-up, the median hemoglobin value of these patients was 11.35 gr/dL (range 10.2-13.4). BPX-501 cells expanded after infusion and still persist in all patients at last follow-up. All children are alive and transfusion-independent. Details on T cell, NK cell and B cell recovery are shown in Figure 1 (Panel A-D). Conclusions: Children with hemoglobinopathies and DBA can benefit from curative haplo-HSCT after depletion of α/β T-cells followed by infusion of BPX-501 cells, which, expanding and persisting over time, contribute to speed immune recovery of adaptive T-cell immunity, thus rendering the procedure safer. Figure 1 Figure 1. Disclosures French: Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Background: Haplo-HSCT after depletion of α/β T and B cells is a suitable and effective option for those children with acute leukemia (AL) who need an allograft and lacking an immediately available HLA-identical donor. With this approach, recipients can benefit immediately after transplantation from the anti-leukemia effect mediated by donor natural killer (NK) and γd T cells, which can also protect against infections. A further improvement of the results achievable with this platform could achieved with a faster adaptive T-cell immunity recovery, which play a key role to augment the graft-versus-leukemia effect and the capacity to fight infections. In light of these considerations, we designed a phase I/II trial aimed at testing the safety and efficacy of post-transplant infusion of donor-derived T cells transduced with the new iC9 suicide gene (BPX-501) in children with either malignant or non-malignant disorders (NCT02065869). Remarkably, after the activation and transduction with the retroviral iC9 construct, BPX501 cells switch the phenotype towards a preferential CD45RO pattern. Patients and methods: The phase I portion of the trial consisted of a classical 3+3 design with 3 cohorts, receiving escalating doses of BPX-501 cells of 2.5x105, 5x105, and 1x106 cells/kg, respectively. Patients included in the phase II portion were planned to receive the recommended dose identified during the phase I part of the study.Enrollment of patients started in December 2014; so far, 25 patients with AL in morphological complete remission (CR) have been enrolled. Twenty patients had acute lymphoblastic leukemia (ALL) and 5 acute myeloid leukemia (AML). Details on patient, donor and transplant characteristics are reported in table 1. All patients transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease at the end of induction therapy, both factors predicting a high relapse rate. All patients were given a fully myeloablative conditioning regimen (table 1). Before haplo-HSCT, children received rabbit anti-thymocyte globulin (ATG NEOVII, 12 mg/Kg over 3 days, from day -4 to day -2) to prevent both graft-versus-host disease (GvHD) and graft failure, and Rituximab (200 mg/ m2 on day -1) to prevent EBV-related lymphoproliferative disorders. No post-transplantation GvHD prophylaxis was administered. Results: All patients engrafted and no secondary graft failure was recorded. Median time to neutrophil and platelet recovery was 18 days (range 10-22) and 11 days (range 9-13), respectively. Once documented the engraftment of donor cells, BPX-501 T lymphocytes were infused at a median time of 17 days (range 13-52) after the allograft. Two patients were enrolled in the phase I portion of the study; one each received 2.5x105 and 1x106 cells/kg. The remaining 23 children were treated in the phase II, where the recommended dose was 1x106 cells/kg. However, since we did not observe any acute GvHD requiring the infusion of the dimerizing agent (Rimiducid/AP1903) activating iC9 gene in the first 15 children receiving 1x106 cells/kg, we decided to emend the protocol to further increase the BPX501 cell dose infused to 2 and 4x106 cells/kg. Thus, the last 6 patients were enrolled in these 2 last dose levels (3 patients each). Six and 3 patients developed grade II-IV acute and chronic GvHD, respectively. In one child, given 4x106 cells/kg, we infused rimiducid for steroid-resistant grade II skin acute GvHD, with complete resolution of the disease in 24 hours. The cumulative incidence of grade II-III acute and chronic GvHD are shown in figure 1A and B, respectively. Median follow-up of these 25 children is 8 months (range 1-19 months). One of them died due to chronic GvHD-associated bronchiolitis obliterans and one child with ALL transplanted in CR2 relapsed; the cumulative incidence of non-relapse mortality and leukemia recurrence are shown in figure 1C. The probability of disease-free survival at 15 months is 87% (figure 1D). Once infused, BPX501 cells expanded and persisted over time in both peripheral blood and bone marrow. Conclusion: Overall, these data indicate that the infusion of BPX-501 cells in children with AL given selectively manipulated haplo-HSCT results in low non-relapse mortality and chronic GvHD. Although the median observation time is still limited, the cumulative incidence of disease recurrence is promising. Table 1 Table 1. Figure 1 Figure 1. Disclosures Stanson: Bellicum pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees.

Introduction. Allogeneic HSCT currently represents the only consolidated curative approach for Fanconi anemia (FA) patients, with best results observed in the HLA-identical sibling setting. For patients lacking an HLA-matched related or unrelated donor, haploidentical HSCT virtually assures the opportunity for nearly all patients to benefit from HSCT, offering the advantage of immediate accessibility to the transplant procedure. In order to overcome the limitation of delayed immune recovery, historically associated with this type of allograft, in the last few years we developed a novel method of ex vivo graft manipulation, consisting of the negative depletion of T-cell receptor (TCR) αβ+ T-lymphocytes and CD19 B-cells from peripheral blood stem cells (PBSC) grafts (ClinicalTrial.gov identifier: NCT01810120) (Bertaina el al., Blood 2014). Here we report our analysis in a subgroup of FA patients given TCRαβ/CD19-depleted haploidentical HSCT at our Institution. Patients and methods. Ten consecutive FA patients (6 girls and 4 boys) underwent a TCRαβ/CD19-depleted HSCT from an HLA-haploidentical relative between September 2011 and July 2015. Median age at diagnosis was 6.6 (range 2.7-22.0) years and median age at time of transplantation was 8.1 (range 4.4-22.2) years. The conditioning regimen included Cyclophosphamide 300 mg/m2/day and Fludarabine 30 mg/m2/day for 4 consecutive days (days -6 to -3), with 200 cGy single-dose TBI. Pretransplantation Fresenius® ATG was administered at a dose of 4 mg/kg/day for 3 consecutive days (days -5 to -3) in order to prevent both graft failure and graft-versus-host disease (GVHD). All patients received Rituximab 200 mg/m2 to reduce the risk of Epstein-Barr virus-related post-transplant lymphoproliferative disorders. Selective removal of TCRαβ+ and B-cells was performed on G-CSF-mobilized donor PBSC through labeling with biotinylated anti-TCRαβ antibodies and anti-CD19 antibodies, followed by incubation with anti-biotin antibodies conjugated to paramagnetic beads (CliniMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). No immunosuppressive therapy was administered as post-transplantation prophylaxis against GVHD. Results. The TCRαβ/CD19-depletedgrafts contained a median of 20.40 x106/kg (range 15.80-33.40) CD34+ cells, 5.60 x106/kg (range 1.78-69.60) CD3+ lymphocytes, 0.021 x106/kg (range 0.002-0.043) TCRαβ+ lymphocytes, 5.60 x106/kg (range 1.78-69.60) TCRγδ+ lymphocytes, 0.036 x106/kg (range 0.013-0.079) CD20+ lymphocytes, and 45.30 x106/kg (range 16.2-177.0) NK cells. Engraftment with sustained full donor chimerism was achieved in 9 out of 10 patients, the cumulative incidence of graft rejection being 10% (95% CI, 0-26.8). The patient who rejected his first allograft achieved a complete engraftment after a second HSCT from one-antigen mismatched unrelated donor. No secondary graft failures were observed. The median time for neutrophil and platelet engraftment was 12 days (range, 9-15) and 9 days (range, 8-12), respectively. No patient experienced acute or chronic GVHD in the follow-up period. No transplant-related deaths occurred in our cohort. With a median follow-up of 28 months (range 13.2-39.1), the Kaplan-Meier estimates of OS and DFS were both 100%, while the EFS probability was 90% (95% CI, 47.3-98.5). Discussion. These data suggest that haploidentical HSCT after removal of TCRαβ+ and CD19+ lymphocytes is able to guarantee engraftment with excellent OS and DFS in patients affected by FA. Moreover, given the very low incidence of both acute and chronic GVHD, which has been shown to contribute to the increased risk of developing late post-transplantation malignancies in FA patients, this approach can be considered a very attractive option for FA patients in need of an allograft and lacking an HLA-identical family donor. Disclosures No relevant conflicts of interest to declare.