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Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT), or displaying profound lymphopenia and/or with rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T-cells transduced with a 2nd-generation (4.1BB) CD19-CAR for treatment of patients with BCP-ALL, in a hospital exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR-Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy®-based, manufacturing process (CD19-CAR-Lenti_ALLO). Thirteen children/young adults received ALLO-CAR T-cells between 03/2021 and 10/2022. Doses ranged between 1,0×106 and 3,0×106 CAR T-cells/kg. The toxicity profile was comparable to that of autologous CAR-T cells, characterized mainly by cytopenia, CRS (maximum grade 1) and grade 2 ICANS. One case of acute graft-versus-host disease (GvHD) occurred and was rapidly controlled by steroids and ruxolitinib. None of the other patients, including 3 infused with ALLO-CAR T cells from an HLA-haplo-identical donor, experienced GvHD. Two patients received ALLO-CAR T-cells before HSCT and showed a significant expansion of CAR T cells, without any sign of GvHD. All patients obtained complete remission (CR) with negativity of minimal residual disease in the BM; with a median follow-up of 12 months (range 5-21), 8/13 patients maintain CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly-refractory BCP-ALL relapsing after alloHSCT, without showing increased toxicity as compared to autologous CAR T cells.

We describe a 2 weeks corrected gestational age infant admitted in pediatric intensive care unit (PICU) for severe acute respiratory distress syndrome (ARDS) associated to Bordetella pertussis and Coronavirus infection. He developed leukocytosis as soon as ARDS required intubation and aggressive mechanical ventilation: hence he underwent 3 early therapeutic leukapheresis treatments in order to avoid the worsening of related cardiopulmonary complications, according to recent literature on pertussis infection in infants. The infant was discharged from PICU healthy.

Background: Pediatric myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal disorders, accounting for less than 5% of childhood hematologic malignancies. Usual indications to HSCT are: MDSs with excess of blasts, MDSs secondary to previously administered chemoradiotherapy and RCC associated with monosomy 7, complex karyotype, severe neutropenia, or erythrocyte/platelet transfusion dependence [Locatelli & Strahm, Blood 2018]. We previously demonstrated that TBdepl-haploHSCT is a suitable option for children with acute leukemia, with outcomes comparable to those reported in studies using either an HLA-identical sibling or an unrelated volunteer as donor. Here we present the results of this approach in children with MDSs. Patients and methods: Between February 2013 and February 2021, 23 children with MDSs other than juvenile myelomonocytic leukemia received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù, Rome, Italy or at IRCCS Fondazione Policlinico San Matteo, Pavia, Italy as part of a prospective study (#NCT01810120). All patients were prepared to the allograft using a fully-myeloablative conditioning regimen including a combination of cytotoxic drugs and/or total body irradiation (TBI). Anti-T-lymphocyte globulin (ATLG) was used before transplantation (12 mg/kg total dose, from days -5 to day -3) to modulate bi-directional donor/recipient alloreactivity. Rituximab (200 mg/sqm) was administered on day -1 to prevent post-transplantation EBV-induced lymphoproliferative disorders (PTLD). No patient received any post-transplant pharmacological GvHD prophylaxis. Results: Characteristics of patients enrolled in the study are shown in Table 1 (which reports also donor and graft characteristics). Median follow-up of surviving patients is 4.2 years (range: 0.5 - 8.5 years). Seventeen children were affected by refractory cytopenia of childhood (RCC) (2 cases occurring in the context of inherited bone marrow failure syndromes: one had GATA2 deficiency and the other SAMD9L mutation), while 1 and 5 were affected by MDS with excess of blasts 1 (EB1) and EB2 (one had GATA2 deficiency), respectively. Median time to neutrophil and platelet recovery was 14 (range 10-19) and 11 (range 9-14) days, respectively, with four patients (3 with RCC and 1 with EB2) experiencing primary graft failure, the cumulative incidence of this complication being 17.3% (95% CI 0.3-31.5). All these 4 patients were rescued with a second TBdepl-haploHSCT from the same or the other parent. Cumulative incidence of grade II-III acute GvHD was 11.4% (95% CI 0-25.2). One patient developed skin and gut GvHD after the second TBdepl-haploHSCT, while for all other patients skin was the sole organ involved; no case of grade IV GvHD was observed. One patient developed moderate chronic GvHD [cumulative incidence 5.2% (95% CI 0-14.8)], which completely resolved with low-dose steroids and ruxolitinib. Notably, no patient died for transplant-related complications. Six patients experienced CMV, 2 HHV-6 and 1 adenoviral infection/reactivation; one patient developed lung aspergillosis, which resolved with specific treatment. One patient affected by EB2, not in remission at time of transplant, relapsed 27 months after HSCT, the 5-year cumulative incidence of relapse being 7.1% (95% CI, 0-19.7); she eventually died after failing a second HSCT. The 5-year probability of overall and event-free survival were 92.3% (95% CI 56.6 -98.9) and 76.3% (95% CI 51.3-89.6) (Figure 1A and B), respectively. Five-year disease-free-survival was 90% (95% CI 47.3-98.5). Because of the low number of events, no prognostic factor related to OS and EFS was found. In particular, the MDS variant did not influence the patient's outcome. The median CD3+ cell count on day +30, +90, +180 and +360 were 113, 171, 558 and 1307/mcl, respectively. Conclusions: These data indicate that TBdepl-haploHSCT is a safe and effective transplant option also in children with MDS. Indeed, the low risk of both non-relapse mortality and acute/chronic GvHD makes this approach particularly attractive in the pediatric setting. Moreover, this haplo strategy compares favorably with T-cell replete approaches [Suo et al., 2020]. Figure 1 Figure 1. Disclosures Merli: JAZZ: Consultancy; SOBI: Consultancy. Locatelli: Miltenyl: 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; bluebird bio, Inc.: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αβ T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34+ progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34+ hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony–stimulating factor (G-CSF). Poor CD34+ cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 109 nucleated cells and 494 × 106 CD34+ HSC were manipulated using the CliniMACS device. In comparison to the initial product, αβ T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 106 CD34+ HSC/kg recipient body weight, with median values of residual αβ T cells and B cells of 29 × 103/kg and 33 × 103/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34+ cells was observed after the first year (P = .0005). These data indicate that αβ T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible.