ALBERT

Description: Introduction. Allogeneic stem cell transplantation remains the only curative therapy for patients with myelofibrosis, but is associated with relevant morbidity and mortality, possiby due to the specific nature of the disease. The outcome is significantly worse when alternative donor transplants are compared to matched siblings (MSD): in a recent cooperative study, TRM was 22% in MSD vs 59% in patients receiving unrelated donor grafts (UD) (Blood 2014 124:1183). Aim of the study. This is a retrospective analysis of 95 patients with myelofibrosis (MF) allografted in our Unit between 2001 and 2014. The aim of the study was to assess whether (a) the outcome of alternative donor grafts has improved with time and (b) how this compares with the outcome of identical sibling grafts. Patients were studied in two time intervals-2000-2010 (n=58) and 2011-2014 (n=37). The DIPSS score was comparable in the two time periods, but differences in the most recent group included older age (58 vs 53 years, p=0.004), more family haploidentical donors (54% vs 5%, p

Description: Leukemia relapse remains a significant problem in patients with AML undergoing an allogeneic stem cell transplant(HSCT). Wilms Tumour 1 (WT1) expression has been shown to be a sensitive marker of minimal residual disease (MRD), both in patients after induction chemotherapy, as well as in patients undergoing an allogeneic HSCT. Hypotheses. The present study had 2 hypotheses: (1) WT1 expression in marrow cells of AML patients post-HSCT, will predict leukemia relapse and (2) WT1 based pre-emptive immunotherapy (IT) such as abrupt cyclosporin discontinuation and/or donor lymphocyte infusion (DLI), will prevent leukemia relapse. Patients. Bone marrow WT1 expression, was monitored in 207 patients with acute myeloid leukemia (AML) before and monthly after an allogeneic HSCT, until day +150, and then at every other outpatient access. Eligible for IT were patients without acute or chronic GvHD, with increased WT1 expression and a a marrow in hematologic remission. The trigger for IT was 180 WT1 copies in a first group of 122 patients (group A): this was based on the fact that WT1 expression in normal bone marrow is up to 180 copies . In a subsequent group of 85 patients (group B) the cut off for IT, was 100 copies, due to the fact that a first analysis of group A had shown 100 copies to be an earlier predictor of relapse (BJH 2013; 160: 503). DLI were given in escalating doses, starting at 1x105 CD3+ cells/kg in alternative donor grafts and at 1x106/kg in HLA identical grafts. DLI were escalated ½ log every month, in the absence of GvHD, to a maximum dose of 1x107/kg. Sixtyfour patients were eligible for IT, but only 35 received IT: reasons for non intervention were ongoing GHD, unavailable donor and delay in WT1 results. Results-Hypothesis N.1. Following transplantation, WT1 expression, was highly predictive of leukemia relapse: 12 relapses in 99 patients with WT1 〈 100 copies /104 abl (12%); 19 relapses in 55 patients with WT1 between 101 and 180 copies (35%) and 37 relapses in 53 patients with WT1 〉180 copies (70%) (p

Description: BACKGROUND AND AIMS Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for patients with high-risk acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are the most common tools to evaluate MRD. We recently reported that combining WT1 expression and MFC for MRD detection after induction therapy strongly impacts on relapse risk in AML. The aim of this study was to analyze the role of pre-BMT MRD assessment as predictor for the post-transplant relapse risk. MATERIALS AND METHODS We retrospectively analyzed the outcome of 253 consecutive AML patients receiving allo-BMT. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation . Median age at transplant was 45 years. Disease phase was CR1 in 161, CR2 in 63, and CR3 in 29 patients. One hundred eighty-two received myeloablative conditioning, whereas 71 patients received reduced intensity conditioning. Median follow-up was 59 months (95% CI 46.2 - 71.8 months). Relapse-free survival (RFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall Survival (OS) was calculated from the time of transplantation until death by any cause or last follow-up. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression. RESULTS Relapse occurred in 81 patients (32%). Three-year estimate of RFS was 63.7% (median not reached). The probability of relapse was significantly affected by disease status (first or subsequent CR, p

Description: Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients. Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse. Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression. Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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

Description: Abstract 2200 Poster Board II-177 Cardiotoxicity is a limiting factor of successful anthracycline-based anticancer regimens. Recently, cardiac side effects have been also reported for Tyrosine Kinase Inhibitors, among which Imatinib Mesylate (IM) has been able to change the natural history of Chronic Myeloid Leukemia (CML) and Gastrointestinal Stromal Tumors. The mechanism of IM-related cardiac dysfunction has not been fully elucidated. Since we have demonstrated the presence of resident cardiac stem cells controlling homeostasis of the human and murine heart, we advanced the hypothesis that IM-induced cardiotoxicity involves myocardial cell turnover resulting in an impairment of the adaptive response of the heart to the toxic insult. To this purpose, we studied the in vitro genotoxic effect of 5mM, 25mM and 50 mM concentrations of IM on human and rat Cardiac Progenitor Cells (CPCs). Freshly isolated human BM CML cells carrying Bcr/Abl translocation served as control. In addition, to ascertain whether IM affects cardiac structure and function, young rats were treated by three weekly i.p. injections of 50, 100 and 200 mg/kg IM for 4 weeks when hemodynamic parameters and LV remodelling were analyzed. A dose and time dependent DNA damage was observed in both CPCs and CML after in vitro exposure to IM. As early as 6 hours after 50 mM IM, DNA double strand breaks formation (dSBs), as shown by immunostaining of Histone A2X, involved 50% and 70% of CPCs and CML, respectively. At later time points more than 80% of both cells died. However, low concentrations (5mM), corresponding to therapeutic doses, exerted a permissive reparative response on CPCs that persisted with time. As a result of this divergent effect, CPCs proliferation and survival increased from 12 to 24 h in spite of the presence of IM. In vivo studies showed that, following 200 mg/kg IM, all animals died within the first 2 weeks. In the presence of moderate anemia, diastolic dysfunction was present even at 50 mg/kg IM and a dose dependent impairment in Left Ventricular End Diastolic Pressure (LVEDP) and +/- dP/dT was observed. Anatomical parameters indicated that only higher doses IM resulted in a smaller LV with a concentric type of remodelling. In the absence of diffuse myocardial fibrosis and/or high levels of apoptosis, small foci of myocardial damage were observed only with 200 mg/kg IM. Preliminary data on myocardial cell turnover indicate that high doses of IM blunted the proliferative response.of cardiomyocytes to the cytotoxic injury. Conversely, in agreement with in vitro data, low IM doses were associated with a reparative response. In conclusion, IM, by inhibiting the multiple pathways modulated by tyrosine kinases, affects in a dose dependent manner CPCs and cardiac function suggesting possible alternative mechanisms of its effects on the cardiovascular system. Disclosures: No relevant conflicts of interest to declare.