Abstract: Functional health of transplanted HS patients was significantly diminished. Patients with higher enzyme levels showed favorable results. Psychosocial health appeared unaffected compared with healthy peers.

Abstract: Background Immunotherapy using patient-derived CAR T cells has achieved complete remission and durable response in highly refractory populations. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Allogeneic Cytokine Induced Killer (CIK) cells, a T-cell population characterized by the enrichment of CD3+CD56+ cells, have demonstrated a high profile of safety in acute lymphoblastic leukemia (ALL) patients (Introna M et al. Biol Blood Marrow Transplant. 2017). CIK cells could be easily engineered by the non-viral Sleeping Beauty (SB) transposon for the clinical application (Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017). Methods CIK cells were generated from 50 ml of donor-derived peripheral blood (PB) by electroporation with the GMP-grade CD19.CAR/pTMNDU3 and pCMV-SB11 plasmids according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (300 mg/m2/day) x 2 days, CARCIK-CD19 were infused in pediatric and adult B-cell ALL (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The clinical trial follows a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) using the novel Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and a safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as & lt; 5% bone marrow (BM) blasts, circulating blasts & lt; 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results We manufactured eighteen batches by seeding a median of 126.8x106 allogeneicPBMCs. At the end of expansion, the mean harvesting was 6.46x109 nucleated cells (range 1.39 - 16.00x109). Manufactured cells were mostly CD3+ lymphocytes (mean 98.90% ±SE 0.30%). Of these, 43.57%±3.73% were CAR+, 47.07%±2.74% were CD56+, 80.44%±2.53% were CD8+. The quality requirements for batch release were met in 17 productions. As of the data cut-off date (July 19, 2019), 4 pediatric and 7 adult patients were infused with a single dose of CARCIK-CD19 (n=2 HLA identical sibling, n=4 MUD, n=5 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were a grade I cytokine release syndrome and an infusion-related DMSO-associated seizure, with absence of dose-limiting toxicities, neurotoxicity and graft-versus-host disease (GvHD) in any of the treated patients. Four out of 5 patients, receiving the highest doses, achieved CR and CRi at day 28. The 3 patients in CR were also MRD- (by flow cytometry and RT-PCR) while the CRi was MRD+ and relapsed at day+49. Robust expansion was achieved in the majority of the patients as defined by detectable CAR T-cell detection (vector copy number VCN, range 4645-977992 transgene copies/ug) and flow (range 0.5-30%) in PB. The median time to peak engraftment was 14 days. The magnitude of expansion was correlated with the CD19+ burden in the BM at the time of the infusion (P value = 0.0006, R square 0.7469). CD8+ T cells represented the predominant CARCIK-CD19 T-cell subset (78.88%±5.33% d14 n=6) along with CD3+CD56+ CIK cells and CD4+ T cells to a lesser extent. The majority of CAR T cells had a central and effector memory phenotype. CAR T cells were measurable by VCN up to 6 months with a mean persistence of 70.5 ± 14.85 days (follow up ranging from 28 days to 1 year). No major difference was observed by integration analyses of the patients' PB and the cell products. The vector integration sites reflected the classical random distribution of SB without any tendency for gene dense regions. Conclusions Our ongoing phase I/II trial demonstrates that SB-engineered CARCIK-CD19 cells are able to expand and persist in pediatric and adult B-ALL patients relapsed after HSCT, with important implications for a non-viral technology. These encouraging results prompted us to expand our study. Disclosures Gritti: Autolus Ltd: Honoraria; Roche: Other: Not stated; Abbvie: Other: Not stated; Becton Dickinson: Other: Not stated. Rambaldi:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract: Introduction Iron overload (IOL) is a common complication after HSCT, mainly due to iterative red blood cell (RBC) transfusions with other mechanisms as ineffective erythropoiesis or dysregulation of hepcidin possibly contributing. IOL prevalence is estimated as 30-60% in adults transplanted for hematological malignancies, but few data are available in pediatric malignancies. Patients and Methods All patients (pts) undergoing allogenic HSCT between January 2012 and December 2016 in our institution, who were alive and in continuous complete remission at the last follow-up were included in the study and evaluated for post-HSCT IOL. Follow-up was updated as of 31 May 2018. 109 pts who fulfilled the inclusion criteria were included in the study. Pts characteristics are shown in Table 1. Overall pts affected with malignancies were 77 (71%) and with non malignancies were 32 (29%). IOL was initially assessed using serum ferritin pre and post HSCT, considered as maximum and minimum stable ferritin, defined respectively as the highest value of ferritin detected anytime after HSCT and the last ferritin value detected after HSCT and before starting IOL therapy in pts treated or the minimum ferritin value closer to 12 months after HSCT in pts never treated for IOL. In pts with a minimum stable ferritin 〉 500 ng/mL, LIC was assessed by MRI-T2* or SQUID in younger pts who would have needed sedation to underwent MRI. Liver biopsy was performed in case of liver abnormalities. Imaging to detect IOL was not planned in pts with expected low compliance or when serum ferritin was lowest. These pts were classified according to their minimum stable ferritin values 〈 or ≥ 1000 ng/mL. IOL was defined for all pts with a T2* value ≤3.8 msec or a LIC by SQUID 〉 1000 μgFe/g liver ww or, in pts without imaging for IOL, with a minimum stable ferritin value ≥1000 ng/mL. For liver biopsy, IOL was assessed by Deugnier's score. A phlebotomy program was proposed to all pts with any grade of IOL. In pts with venous access difficulties, anemia or early severe IOL treatment with iron chelator (deferasirox or deferoxamine) was performed. Results Statistically significant differences between malignancies and non malignancies were found in terms of median number of pre-HSCT RBC transfusions (11 vs 7, p 0.002), median pre-HSCT ferritin (1490 vs 643 ng/mL, p 〈 0.001), median maximum ferritin (2419 vs 1131 ng/mL, p 〈 0.001) and median minimum stable ferritin (1286 vs 575 ng/mL, p 0.002). Overall 71 pts have been investigated for IOL (65%) by MRI (64), SQUID (3) or liver biopsy (4). Prevalence of moderate-severe IOL was 37% (42% in malignancies and 25 % in non malignancies). Among the 38 pts who did not performed IOL assessment with instrumental evaluation, 25% of the malignancies group and 47% of the non malignancies group had a minimum stable ferritin 〈 1000 ng/mL. The proportion of pts undergoing IOL assessment (by MRI, SQUID or liver biopsy) was significantly higher in pts with malignancies (72%) compared with non malignancies (47%, p 0.02). 81% of the pts with any grade of IOL have been treated. 51 pts (43 malignancies and 8 non malignancies) underwent phlebotomies, 10 (8 malignancies and 2 non malignancies) were treated with iron chelators and 5 (all malignancies) received both treatments after HSCT. Main side effects of phlebotomies were hypotension and difficult venous accesses. Few pts treated with iron chelator had drug-related side effects, mainly related with renal tubular function impairment, which was transient and rapidly reverted after drug discontinuation. Furthermore, factors significantly associated with IOL were: 1) in univariate analysis: age at HSCT 〉 10 years (odds ratio (OR) 3.63, 95% confidence intervaI (CI) 1.61-8.5, p 0.002), total number of RBC transfusion 〉 20 (OR 10.47, 95%CI 4.09-29.19, p 〈 0.001) and radiation-based preparative regimen (OR 3.02, 95% CI 1.0-7.15, p 0.011); 2) in multivariate analysis: age at HSCT 〉 10 compared with younger age (OR 4.02, 95% CI 1.49-11.8, p= 0.007) and RBC transfusion 〉 20 (OR 10.83, 95% CI 4-32.7, p 〈 0.001). Conclusion IOL is a frequent complication post HSCT, even among children affected with malignancies. In our series the iron burden was worse for older pts and highly transfused. IOL is assessed by MRI-T2*. The minimum stable serum ferritin, approximately 1 year after HSCT and after, can be used to monitor IOL treatment. Phlebotomy is a well tolerated therapy but also pharmacological approaches could be safely used. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 2306 In the last few years the usage of third party mesenchymal stem cells (MSC) as therapy for steroid-refractory Graft versus Host Disease (GvHD) is constantly increasing and holds big promises. Nevertheless, at our knowledge, studies on MSC efficacy have been scarcely corroborated by biological analysis of patient response to cell infusion. Here, we report the immunological monitoring of 8 patients (7 male, 1 female; aged 4 to 33 years), with steroid-refractory GvHD (grade II to III), who received MSCs, between August 2009 and June 2010. GvHD presented as acute in 6 cases and chronic in 2 cases. In 5 cases GvHD occurred as a single organ pathology (2 skin, 2 gut, 1 liver), while in 3 cases GvHD had multi-organ involvement (1 liver and oral mucosa, 1 skin and oral/ocular mucosa, 1 skin, gut and liver). All patients received 2 to 3 MSC infusions from third party donors aiming at 1 × 106/kg recipient body weight MSCs for each infusion. After MSC therapy, 2 patients showed complete response, 3 patients showed partial response, whereas 3 patients did not respond to MSC infusion. To better comprehend the immunomodulatory effects of MSC infusions, we studied GvHD plasmatic markers, inflammatory cytokines and CD4+ T-cell subsets circulating in the peripheral blood (PB) of enrolled patients before MSC infusion and at day 7, 14 and 28 after cell therapy. In accordance with clinical observations, in patients responding to MSC infusions, we observed a dramatic decrease of three validated GvHD plasmatic markers TNFRI, IL2Rα and elafin (Paczesny S et al. Blood 2009) to the mean levels of Healthy Donors (HD). In particular, at day 28 after therapy, TNFRI and IL2Rα levels decreased of 2 times (range=1.9-2.4 and range=1.4-2.8, respectively) and elafin levels decreased of 2.5 times (range=1.7-3.6). Partially responding patients showed a transient decrease of TNFRI, IL2Rα and elafin levels, while non responding patients showed stable or even increasing levels of all analysed markers. Moreover, we investigated the effect of MSC infusion on lymphocyte counts. We demonstrated that patients responding to MSC infusion, oppositely to non responders, strongly decreased total and CD4+ lymphocyte counts in the PB (mean total T-cell Fold Decrease (FD)=11.85, range=1.3-116; mean CD4+ T-cell FD=12, range=1.5-116). Interestingly, after MSC infusion, CD4+ T-cell subsets changed significantly: Tregs increased and Th1 and Th17 populations decreased, and a new CD4+ cell subset balance was observed starting from day 7 after therapy. In particular, the mean FD of Th1/Treg ratio was 4.1 (range=4-4.2) and the mean FD of Th17/Treg ratio was 4.7 (range=3.3-6). Correspondingly, patient symptoms also gradually improved, suggesting an association between GvHD clinical course and CD4+ T-cell imbalance, reverted by MSCs in responding patients. In partially responding patients Th1/Treg and Th17/Treg showed a transient decreased and even slightly increased in the case of non responding patients. In accordance with the decrease of Th1 CD4+ T cells in the PB of patients responding to MSC infusion, we observed a valuable decrease of IFNγ plasma concentrations (mean FD=48, range=30-65 in complete responders), which reached the levels typical of HD. In summary, despite its limited size, the present study suggests that MSCs, upon infusion, are able to convert an inflammatory environment to a more physiological one, both at a cellular level, promoting the expansion of circulating Tregs, and at a molecular level, diminishing inflammatory cytokines. Further studies on a larger group of patients, clarifying the mechanisms of action used in vivo by MSC to tune ongoing allo-reactions, will be fundamental to provide the rationale for improving current clinical trials. Disclosures: No relevant conflicts of interest to declare.

Abstract: Introduction. Although rabbit anti-thymocyte globulin (ATG) is largely used for preventing immune-mediated complications in patients given allogeneic hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD), the optimal dosage of this drug in children is still undefined. We conducted an academic open-label, multicenter, randomized trial comparing two different dosages of rATG in children with hematological malignancies transplanted with either bone marrow (BM) or peripheral blood stem cell (PBSC) from a MUD selected using high-resolution typing for HLA class I/II (i.e. A, B, C, DRB1) loci. The study aimed at testing whether a higher dose of rATG (30 mg/Kg over 3 days, from day -4 to day -2) was superior to a lower dose (15 mg/Kg over 3 days, from day -4 to day -2) in terms of grade II-IV acute graft-versus-host disease (GvHD) prevention. Secondary end-points included cumulative incidence (CI) of chronic GvHD, non-relapse mortality (NRM), disease recurrence and event-free survival (EFS). The drug was kindly provided by Fresenius/Neovii. Patients and methods. Inclusion criteria were: diagnosis of acute or chronic leukemia in remission/chronic phase, non-Hodgkin lymphoma or myelodysplasia; age at HSCT 0-19 years; availability of an unrelated donor selected using high-resolution molecular typing of HLA-A, B, C and DRB1 loci, completely matched or with a single allelic disparity at one of the HLA loci or with 2 allelic disparities, or with one antigenic disparity; use of G-CSF-mobilized PBSC or BM-derived hematopoietic stem cells. From January 2008 to September 2012, 180 patients were enrolled in the study. Eight patients, 5 randomized to the rATG 30 mg/kg group and 3 to the 15 mg/kg group, did not proceed to transplant due to further relapse before HSCT. The remaining 172 patients, 84 belonging to the 30 mg/kg group and 88 belonging to the 15 mg/kg group, were transplanted and included in this analysis; 94 children had acute lymphoblastic and 42 acute myeloid leukemia. The 2 randomization groups were comparable for all demographic and transplant-related variables evaluated (see Table 1). Patients were stratified according to the stem cell source (BM vs. PBSC), degree of HLA compatibility with the donor (unrelated donor perfectly matched or with 1 allelic disparity vs. unrelated donor with 2 allelic disparities or with 1 antigenic disparity) and disease risk group (standard- vs. high-risk, SR and HR). All patients received a fully myeloablative preparative regimen. All patients received cyclosporine-A and short-term methotrexate as post-transplant GvHD prophylaxis. The trial was registered at ClinicalTrials.gov (NCT00934557). Data were analyzed as of January 31st, 2016. Results. With a median follow-up of 4.5 years (range 3.3-7.6 years), the 100-day CI of grade II-IV acute GvHD in the high and low rATG group was 29% and 36%, respectively (P=NS, Figure 1A); the CI of extensive chronic GvHD in the two groups was 6% and 10%, respectively (P=NS, Figure 1B). The CI of NRM was 19% and 9% in the high and low rATG group, respectively (P=0.09). Children receiving the higher rATG dosage had a greater incidence of viral reactivations as compared to those who received the lower dosage. The difference was statistically significant for EBV reactivation (37% vs. 23%; P = 0.038) and for Adenovirus reactivation (12% vs. 1%; P = 0.004). The overall CI of disease recurrence was 17% and did not differ between high and low dose rATG. The 5-year EFS for the whole cohort of patients was 69%; it was 61% and 77% for children given high and low dose rATG, respectively (P=0.028, Figure 1C). EFS was 78% and 55% in the SR and HR groups, respectively (P=0.001, Figure 1D). EFS of the 136 children with acute leukemia given either high or low dose rATG was 60% and 77%, respectively (P=0.049). In multivariate analysis on EFS, the following variables were associated with an unfavorable outcome: rATG dose of 30 mg/kg (relative risk 1.90; P=0.026), HLA mismatch 〉 1 allele (relative risk 2.08; P=0.01) and HR disease (relative risk 2.46; P=0.0015). Conclusions. Our data indicate that, in children with hematological malignancies transplanted from a MUD selected through high-resolution HLA typing, the use of low dose rATG results into decreased incidence of NRM and better EFS. Low dose rATG is able to spare life-threatening viral infections, without significantly increasing the incidence of acute and chronic GvHD. Table 1 Table 1. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract: Abstract 2288 Poster Board II-265 Background. More than 80% of children with de novo acute myeloid leukemia (AML) achieve complete remission (CR) after aggressive induction chemotherapy. However, post-remission treatment plays a crucial role in the final outcome of these patients. In this regards, possible post-remission treatments include high dose chemotherapy, as well as autologous or allogeneic hematopoietic stem cell transplantation (HSCT). In the last years, several groups have compared allogeneic HSCT with chemotherapy or autologous HSCT in adults as well and children with AML in first remission. These studies have demonstrated that allogeneic HSCT is superior to the other forms of post-remission therapy in children with AML not carrying favourable cytogenetic characteristics. We herein report the long-term results of a prospective multicenter study aimed at evaluating the safety and efficacy of an original combination of 3 alkylating agents, busulfan (BU), cyclophosphamide (CY) and melphalan (L-PAM) as conditioning regimen for patients affected by de novo AML other than the FAB M3 subtype and given allogeneic HSCT from a matched family donor in first haematological CR. Patients and methods. Seventy consecutive patients (40 males and 30 females), affected by de novo AML in first CR, received an allogeneic HSCT from a HLA-matched family donor after a conditioning regimen combining BU (4 mg/Kg/day for 4 days), CY (60 mg/Kg/day for 2 days) and L-PAM (140 mg/m2). BU dosage was adjusted after the pharmacokinetic study performed following the first administration, in order to maintain a concentration to the steady state (Css) comprised between 600 and 900 ng/mL. Transplants were performed in one of the transplant centers of the Italian Association for Pediatric Hematology / Oncology (AIEOP) between 1992 and 2002. Median patient age at HSCT was 7 years (0.5 – 17). Two patients had FAB M0 AML, 25 M1, 17 M2, 12 M4, 21 M5, 1 M6 and 2 M7 AML. Children with M3 AML were excluded from the study. Five patients with CBF anomalies, either isolated or associated with loss of sex chromosome, were considered to have favourable cytogenetics. The median interval between diagnosis and HSCT was 3.9 months (2.9 - 9). All patients were given bone marrow stem cells. GVHD prophylaxis consisted of cyclosporine-A (Cs-A) alone in 89% of patients, the remaining children receiving a combination of Cs-A with short-term methotrexate. Results. Median follow-up for surviving patients is 9 years (range, 6 – 16 years). The conditioning regimen was well tolerated, and no patient died for causes directly attributable to the myeloablative treatment. All patient engrafted, the median time to reach neutrophil and platelet recovery being 13 (7 - 28) and 21 (13 – 115) days, respectively. The cumulative incidence (CI) of grade II-IV acute GVHD was 58% (48 – 71), while that of grade III-IV acute GVHD was 14% (8 – 25). Chronic GVHD incidence was 27% (18 – 39). Ten-years survival probability was 77% (67 - 87), while 10-years disease-free survival (DFS) was 76% (65 - 86). The cumulative incidence of relapse was 17% (10 – 29), while the cumulative incidence of transplant-related mortality was 7% (3 – 17). Results improved over time, DFS being 59% (41 – 78) for patients transplanted before 1997 and 86% (75 – 96) for those transplanted after 1997. No other variables influenced the probability of both survival and DFS. Conclusions. Our study, conducted on a significant number of children affected by AML in first CR and with a very long follow-up demonstrate that allogeneic HSCT from a matched family donor can cure a large proportion of patients. The combination of dose-adjusted BU, CY and L-PAM was safe and well tolerated, with an incidence of TRM of only 7% and no death directly attributable to the conditioning regimen. Furthermore, the combination of 3 alkylating agents showed a good anti-leukemic activity, the probability of leukaemia recurrence being only 17%. Disclosures: No relevant conflicts of interest to declare.