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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.

High genetic heterogeneity in the human leukocyte antigen (HLA) increases the likelihood of efficient immune response to pathogens and tumours. As measure of HLA diversity, HLA evolutionary divergence (HED) has been shown to predict the response of tumours to immunotherapy and haematopoietic stem cell transplantation (HSCT) in adults. We retrospectively investigated the association of HED with outcomes of 153 paediatric/young adults patients, treated for malignant disorders with HSCT from 9–10/10 HLA-matched unrelated donors. HED was calculated as pairwise genetic distance between alleles in patient HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, using the locus median to stratify patients with ‘high’ or ‘low’ HED. Patients with high HED-B and -DRB1 showed significantly improved disease-free survival (DFS), especially when combined (70.8% vs 53.7% p = 0.008). High HED-B + -DRB1 was also associated with improved overall survival (OS) (82.1 vs 66.4% p = 0.014), and concomitant reduction of non-relapse-mortality (5.1% vs 21.1% p = 0.006). The impact on OS and DFS of combined HED-B + -DRB1 was confirmed in multivariate analysis [hazard ratio (HR) 0.39, p = 0.009; and HR 0.45, p = 0.007 respectively]. Only high HED scores for HLA-DPB1 were associated, in univariate analysis, with reduced incidence of relapse (15.9% vs 31.1%, p = 0.03). These results support HED as prognostic marker in allogeneic HSCT and, if confirmed in larger cohorts, would allow its use to inform clinical risk and potentially influence clinical practice.

Langerhans cell histiocytosis (LCH) is due to aberrant monoclonal proliferation and accumulation of dendritic cells, ranging from a self-limiting local condition to a rapidly progressive multisystem disease with poor prognosis. Pathogenic cells originate from a myeloid-derived precursor characterized by an activation of the MAPK/ERK signaling pathway in about 70% of cases. In particular, BRAF V600E mutation is usually associated with a more severe clinical course and poor response to chemotherapy. We report on a newborn with multisystem LCH in life-threatening medical conditions. At diagnosis, the patient was successfully treated with the early association of BRAF inhibitor Vemurafenib to standard chemotherapy representing a new approach in first-line treatment. A rapid clinical improvement with a prompt fever regression from day 2 and complete resolution of skin lesions by week 2 were observed; laboratory data normalized as well. Vemurafenib was discontinued after 12 months of treatment. No signs of relapse occurred after 12 months of discontinuation. This case indicates that early combination of target therapy with standard treatment may induce rapid response and prolonged disease remission without significant toxicities in infants. This approach represents a valid and safe option as first-line treatment in multisystem disease, especially in high-risk patients.

The wide variety of IPEX symptoms leads to diagnosis and treatment delay with fatal outcomes if left untreated before two first years of life. Cow's milk allergy non‐responsive to amino acid‐based formula must raise suspicion of this syndrome.

LCH is an aberrant monoclonal proliferation of dendritic cells, ranging from a self-limiting local condition to a rapidly progressive multisystemic disease. Pathogenic cells expressed, in almost 70% of cases, an activation of the MAPK/ERK signaling pathway, in particular BRAF V600E mutation. We report on a newborn with multisystemic disease diagnosed in life-threatening medical conditions, who was successfully treated with the early association of BRAF inhibitor Vemurafenib to chemotherapy. After 12 months, Vemurafenib was discontinued, without any signs of relapse. This case indicates that early combination of target therapy with standard treatment may induce rapid response and prolonged disease remission.

Cow’s milk allergy non-responsive to amino acid-based formula must raise suspicion of Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. This is a potentially fatal disease associated with food allergy, sometimes leading to diagnostic delay. In our case, early diagnosis and management provided life-saving therapy.

Background: allogeneic HSCT is the only potentially curative treatment for many non-malignant diseases (NMD), either inherited or acquired. However, many patients lack an HLA-matched donor (familiar (MFD) or unrelated (MUD)) and the outcome of children transplanted from an HLA-haploidentical relative (haplo) was historically inferior to that of transplants from a MFD or a MUD. We previously published promising results in a cohort of 23 children with NMD given this type of allograft (Bertaina et al., Blood 2014), demonstrating a low transplant-related mortality (TRM) and high cure rates. Here, we report the outcome of a large cohort of children affected by NMD who received a TBdepl-haploHSCT at our Center (NCT01810120). Patients and methods: Between February 2011 and June 2020, 80 consecutive patients affected by NMD received TBdepl-haploHSCT from an HLA-partially matched relative at Ospedale Pediatrico Bambino Gesù in Rome, Italy. Patients had many different disorders (see Table for details on patient- and transplant-related characteristics). Median time from diagnosis to transplant for the whole cohort was 12 months (range 1-177), while it was 2.5 months (range 1.3-11.2) for SCID patients. All patients, including children with SCID, received a conditioning regimen, which varied according to the original disease. Pre-transplant anti-thymocyte globulins (from day -4 to day -2) were given to modulate bi-directional donor/recipient alloreactivity, while rituximab (on day -1) was administered to prevent PTLD. Moreover, no post-transplant pharmacological GvHD prophylaxis was given. Results: fifty-eight patients (72.5%) achieved primary donor cell engraftment, while 3 patients experienced secondary graft failure (GF); the cumulative incidence of either primary or secondary GF was 27.8% (95% CI 17.2-37.0). Median time to neutrophil and platelet recovery was 13.5 (range 9-33) and 10 days (range 7-51), respectively. As expected, GF occurred more frequently in children with disorders known to be associated with an increased GF risk (i.e., HLH, thalassemia, SAA or osteopetrosis) (see also Figure 1A). Three children (4%) experiencing GF died because of infectious complications before retransplant. Sixteen of the 22 patients with either primary or secondary GF were successfully retransplanted (2 with a mismatched unrelated cord blood unit, the other having received a second TBdepl-haploHSCT from either the same donor or the other parent). Since 3 other patients died [all because of infectious complications, 2 due to disseminated adenovirus infection and 1 to CMV pneumonia)], TRM is 7.8% (95% CI 1.6-13.7). Eighteen patients experienced acute GVHD of any grade, the cumulative incidence of this complication being 22% (95% CI 13.5-31.8); 10/18 patients developed grade II acute GVHD (no patient developed grade III or IV aGVHD), this resulting into a cumulative incidence of 12.9% (95% CI 6.6-21.4). Only one patient at risk developed mild chronic GVHD. Twenty-two and 7 patients developed clinically-relevant (i.e., with a viral load > 1000 copies/ml and/or requiring specific antiviral-treatment) CMV and adenovirus infection, respectively, at a median time of 4 (range 0-16) and 1 (range 1-4) weeks from HSCT. Time averaged area under the curve (i.e., viral burden under the curve/weeks at risk for infection) for CMV and ADV are reported in Figure 1B. With a median follow-up of 36 months (range 2 - 110), the 5-year probability of overall survival and event-free survival for the entire cohort of patients is 92.1% (95% CI 83.3-96.4) (Figure 1C) and 68.1% (95% CI 56.4-77.2), respectively. Considering the 16/22 given a successful 2nd allograft, the 5-year disease-free survival is 88.4% (95% CI 78.9-93.8). Details on reconstitution of CD3+, CD4+ and CD8+ lymphocytes are reported in Figure 1D. Conclusions: TBdepl-haploHSCT is an effective option for children with different NMD. GF (either primary or secondary) is a challenging problem in a sub-group of patients at risk (i.e., those with HLH, thalassemia, SAA or osteopetrosis): thus, new strategies to overcome this problem are desirable. However, a second transplant is able to rescue most of these patients. Prompt availability of this type of transplant, limiting infectious risk, low incidence of both acute and chronic GvHD preserving a good quality of life in patients makes this strategy an attractive choice in patients with NMD. Figure 1 Disclosures Merli: Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; SOBI: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria; Sanofi-Genzyme: Honoraria; Atara Therapeutics: Honoraria. Algeri:BlueBird Bio: Membership on an entity's Board of Directors or advisory committees; Atara Therapeutics: Membership on an entity's Board of Directors or advisory committees. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

TcRαβ/CD19-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) represents a promising new platform for children affected by acute leukemia in need of an allograft and lacking a matched donor, disease recurrence being the main cause of treatment failure. The use of zoledronic acid to enhance TcRγδ+ lymphocyte function after TcRαβ/CD19-cell depleted haplo-HSCT was tested in an open-label, feasibility, proof-of-principle study. Forty-six children affected by high-risk acute leukemia underwent haplo-HSCT after removal of TcRαβ+ and CD19+ B lymphocytes. No post-transplant pharmacological graft-versus-host disease (GvHD) prophylaxis was given. Zoledronic acid was administered monthly at a dose of 0.05 mg/kg/dose (maximum dose 4 mg), starting from day +20 after transplantation. A total of 139 infusions were administered, with a mean of 3 infusions per patient. No severe adverse event was observed. Common side effects were represented by asymptomatic hypocalcemia and acute phase reactions (including fever, chills, malaise, and/or arthralgia) within 24–48 h from zoledronic acid infusion. The cumulative incidence of acute and chronic GvHD was 17.3% (all grade I-II) and 4.8% (all limited), respectively. Patients given 3 or more infusions of zoledronic acid had a lower incidence of both acute GvHD (8.8 vs. 41.6%, p = 0.015) and chronic GvHD (0 vs. 22.2%, p = 0.006). Transplant-related mortality (TRM) and relapse incidence at 3 years were 4.3 and 30.4%, respectively. Patients receiving repeated infusions of zoledronic acid had a lower TRM as compared to those receiving 1 or 2 administration of the drug (0 vs. 16.7%, p = 0.01). Five-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 67.2 and 65.2%, respectively, with a trend toward a better OS for patients receiving 3 or more infusions (73.1 vs. 50.0%, p = 0.05). The probability of GvHD/relapse-free survival was significantly worse in patients receiving 1–2 infusions of zoledonic acid than in those given ≥3 infusions (33.3 vs. 70.6%, respectively, p = 0.006). Multivariable analysis showed an independent positive effect on outcome given by repeated infusions of zoledronic acid (HR 0.27, p = 0.03). These data indicate that the use of zoledronic acid after TcRαβ/CD19-cell depleted haploHSCT is safe and may result in a lower incidence of acute GvHD, chronic GvHD, and TRM.

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.

In solid tumors and lymphomas, the neutrophil/lymphocyte (N/L) ratio at diagnosis has been shown to be a prognostic factor. The aim of our study was to validate the originally reported N/L ratio cut-point of 3.5 in patients with diffuse large B-cell lymphoma (DLBCL) registered in an Italian real-life database. The prognostic role of the N/L ratio at diagnosis on event-free survival (EFS) and overall survival (OS) was assessed in 505 patients with DLBCL. Patients with an N/L ratio3.5 (