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Summary: Rearrangements of T‐ and B‐cell receptor (TCR and BCR) genes are useful markers for clonality assessment as well as for minimal residual disease (MRD) monitoring during the treatment of haematological malignancies. Currently, rearrangements of three out of four TCR and all BCR loci are used for this purpose. The fourth TCR gene, TRA, has not been used so far due to the lack of a method for its rearrangement detection in genomic DNA. Here we propose the first high‐throughput sequencing based method for the identification of clonal TRA gene rearrangements at the DNA level. The method is based on target amplification of the rearranged TRA locus using an advanced multiplex polymerase chain reaction system and high‐throughput sequencing, and has been tested on DNA samples from peripheral blood of healthy donors. Combinations of all functional V‐ and J‐segments were detected, indicating the high sensitivity of the method. Additionally, we identified clonal TRA rearrangements in 57 out of 112 tested DNA samples of patients with various T‐lineage lymphoproliferative disorders. The method fills the existing gap in utilizing the TRA gene for a wide range of studies, including clonality assessment, MRD monitoring and clonal evolution analysis in different lymphoid malignancies. [ABSTRACT FROM AUTHOR]

Both acute GVHD and chronic GVHD remain the leading cause of morbidity and death after allogeneic HSCT. We conducted a retrospective analysis comparing two GVHD‐prophylaxis regimens: 35 patients received "Regimen 1" (horse ATG, tacrolimus, and methotrexate) and 46 "Regimen 2" (rabbit ATG, rituximab, and peritransplant bortezomib). All 81 patients with a median age of 9 (0.6‐23) years with ALL (n = 31) or AML (n = 50) in complete remission received TCRαβ/CD19‐depleted transplants between May 2012 and October 2016, from 40 HLA‐matched unrelated and 41 haploidentical donors. After a median follow‐up of 3.9 years, the CI of acute GVHD II‐IV was 15% (95% CI: 7‐30) in the "Regimen 2" group and 34% (95% CI: −54) in the "Regimen 1" group, P =.05. "Regimen 2" was also more effective in the prevention of chronic GVHD; the CI at 1 year after HSCT was 7% (95% CI: 2‐19) vs 31% (95% CI: 19‐51), P =.005. The CI of relapse at 3 years adjusted for the GVHD‐prophylaxis regimen groups 31% (95% CI: 19‐51) for the "Regimen 1" vs 21% (95% CI: 11‐37) for the "Regimen 2", P =.3. The retrospective observation suggests that the use of the rATG, rituximab, and bortezomib was associated with significantly lower rate of GVHD without the loss of anti‐leukemic activity. [ABSTRACT FROM AUTHOR]

AbstractIn children, therapy-related hematologic neoplasms (t-HN) are uncommon. Many are driven by genetic events independent of clonal hematopoiesis. We sought to understand the clinical and genetic factors of pediatric t-HN in a large independent cohort. Fifty-six t-HN were retrospectively identified. Chromosome microarray, next-generation and/or RNA sequencing were performed. Patients had primary hematologic, solid, or central nervous system tumors. t-HN included myeloid (t-MN) and lymphoblastic (t-ALL) phenotypes. Approximately half of the cases harbored KMTA2A rearrangement (KMT2Ar). Among t-HN without KMT2Ar, genetic drivers were heterogeneous, including diverse fusions or aneuploidy. Approximately 18% harbored 17p deletions and/or TP53 mutations. EFS/OS was not associated with t-HN lineage or KMT2Ar, but HSCT was associated with improved EFS and OS. We detail one of the largest cohorts to date of pediatric t-HN, confirming frequent KMT2Ar and t-ALL. [ABSTRACT FROM AUTHOR]

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) develops from very early cells with the potential for both T-cell and myeloid differentiation. The ambiguous nature of leukemic blasts in ETP-ALL may lead to immunophenotypic alterations at relapse. Here, we address immunophenotypic alterations and related classification issues, as well as genetic features of relapsed pediatric ETP-ALL. Between 2017 and 2022, 7518 patients were diagnosed with acute leukemia (AL). In addition to conventional immunophenotyping, karyotyping, and FISH studies, we performed next-generation sequencing of the T-cell receptor clonal repertoire and reverse transcription PCR and RNA sequencing for patients with ETP-ALL at both initial diagnosis and relapse. Among a total of 534 patients diagnosed with T-cell ALL (7.1%), 60 had ETP-ALL (11.2%). Ten patients with ETP-ALL experienced relapse or progression on therapy (16.7%), with a median time to event of 5 months (ranging from two weeks to 5 years). Most relapses were classified as AL of ambiguous lineage (n = 5) and acute myeloid leukemia (AML) (n = 4). Major genetic markers of leukemic cells remained unchanged at relapse. Of the patients with relapse, four had polyclonal leukemic populations and a relapse with AML or bilineal mixed-phenotype AL (MPAL). Three patients had clonal TRD rearrangements and relapse with AML, undifferentiated AL, or retention of the ETP-ALL phenotype. ETP-ALL relapse requires careful clinical and laboratory diagnosis. Treatment decisions should rely mainly on initial examination data, taking into account both immunophenotypic and molecular/genetic characteristics. [ABSTRACT FROM AUTHOR]

The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells—median for long process [LP] and 1.6 × 109—for short process [SP]), transduction efficiency (43.5%—median for LP and 41.0%—for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays. [ABSTRACT FROM AUTHOR]

Nijmegen breakage syndrome (NBS) is a DNA repair disorder characterized by combined immunodeficiency and a high predisposition to malignancies. HSCT appears to cure immunodeficiency, but remains challenging due to limited experience in long-term risks of transplant-associated toxicity and malignancies. Twenty NBS patients received 22 allogeneic HSCTs with TCRαβ/CD19+ graft depletion with fludarabine 150 mg/m2, cyclophosphamide 20–40 mg/kg and thymoglobulin 5 mg/kg based conditioning regimens (CRs). Twelve patients additionally received low-dose busulfan 4 mg/kg (Bu group) and 10 patients (including 2 recipients of a second HSCT) treosulfan (Treo group) 30 g/m2. Overall and event-free survival were 0.75 vs 1 (p = 0.16) and 0.47 vs 0.89 (p = 0.1) in the Bu and Treo groups, respectively. In the Bu group, four patients developed graft rejection, and three died: two died of de novo and relapsed lymphomas and one died of adenoviral hepatitis. The four living patients exhibited split chimerism with predominantly recipient myeloid cells and predominantly donor T and B lymphocytes. In Treo group, one patient developed rhabdomyosarcoma. There was no difference in the incidence of GVHD, viral reactivation, or early toxicity between either group. Low-dose Bu-containing CR in NBS leads to increased graft failure and low donor myeloid chimerism. Treo-CR followed by TCRαβ/CD19-depleted HSCT demonstrates a low level of early transplant-associated toxicity and enhanced graft function with stable donor chimerism. [ABSTRACT FROM AUTHOR]

Introduction: The live-attenuated BCG vaccine is known to cause disseminated Mycobacterium bovis infection in patients with severe combined immunodeficiency (SCID). However, BCG-related post-hematopoietic stem cell transplantation (HSCT) immune reconstitution inflammatory syndromes, similar to those described in patients with HIV infections, are less-known complications of SCID. Patients and Methods: We reported on 22 BCG-vaccinated SCID patients who had received conditioned allogeneic HSCT with TCRαβ+/CD19+ graft depletion. All BCG-vaccinated patients received anti-mycobacterial therapy pre- and post-HSCT. Post-transplant immunosuppression consisted of tacrolimus in 10 patients and of 8 mg/kg tocilizumab (d-1, + 14, + 28) and 10 mg/kg abatacept (d-1, + 5, + 14, + 28) in 11 patients. Results: Twelve patients, five of whom had BCG infection prior to HSCT, developed BCG-related inflammatory syndromes (BCG-IS). Five developed early BCG-IS with the median time of manifestation 11 days after HSCT, corresponding with a dramatic increase of CD3+TCRγδ+ in at least two patients. Early BCG-IS was noted in only one out of 11 patients who received tocilizumab/abatacept and 4 out of 11 patients who did not. Seven patients developed late BCG-IS which corresponded to T cell immune recovery; at the time of manifestation (median 4.2 months after HSCT), the median number of CD3+ cells was 0.42 × 109/ and CD3+CD4+ cells 0.27 × 109/l. In all patients, late BCG-IS was controlled with IL-1 or IL-6 inhibitors. Conclusion: BCG-vaccinated SCID patients undergoing allogeneic HSCT with TCRαβ+/CD19+ graft depletion are at an increased risk of early and late BCG-IS. Anti-inflammatory therapy with IL-1 and IL-6 blockade is efficient in the prevention of early and treatment of late BCG-IS. [ABSTRACT FROM AUTHOR]

Our initial experience with hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD; n = 12) or a haploidentical related donor (n = 6) with T cell receptor (TCR)αβ + /CD19 + graft depletion in patients with Wiskott-Aldrich syndrome (WAS) (n = 18) showed a dramatic decrease in the incidence of graft-versus-host disease (GVHD) and transplantation-related mortality, with an increased overall survival (OS) of 88.9%. Unfortunately, the treatment was associated with mixed myeloid donor chimerism and secondary graft dysfunction (severe thrombocytopenia, n = 2; graft rejection, n = 5). To improve the outcome, we hypothesized that the addition of G-CSF and plerixafor to the conditioning chemotherapy would result in more complete donor stem cell engraftment. This trial was registered at www.clinicaltrials.gov (NCT03019809). A study group of patients with WAS (n = 16) underwent TCRαβ + /CD19 + -depleted HSCT (MUD, n = 6; haploidentical, n = 10). The conditioning regimen was treosulfan-fludarabine-rabbit antithymocyte globulin-melphalan (or thiophosphamide in 1 patient) with G-CSF (10 µg/kg/day for 5 days starting on day −8) and plerixafor (240 µg/kg/day for 3 days starting on day −6). The clinical outcomes in this study were compared to those in a historical dataset (n = 18). No patients had grade III/IV acute GVHD in either the study or the historical control group. Importantly, in the patients with WAS, there was no statistical significance in OS between those who underwent HSCT from haploidentical donors and those who underwent HSCT from MUDs (93.8% versus 88.5%; P = .612). All patients in the study group had full donor chimerism in whole blood and in the CD3 + compartments. The OS was 93.8%, and there were no cases of graft dysfunction. This study demonstrates the efficacy of adding G-CSF/plerixafor to the conditioning regimen before HSCT with TCRαβ + /C D19 + graft depletion in patients with WAS. [ABSTRACT FROM AUTHOR]

T(16;21)(p11;q22)/FUS-ERG is a rare but recurrent translocation in acute leukemias and in some types of solid tumors. Due to multiple types of FUS-ERG transcripts, PCR-based minimal residual disease detection is impeded. In this study, we evaluated a cohort of pediatric patients with t(16;21)(p11;q22)/FUS-ERG and revealed fusion gene breakpoints. We implemented next-generation sequencing (NGS) on long PCR amplicons for the detection of fusion genes with unknown partners or DNA breakpoints. That allowed us to describe different fusion variants of FUS/ERG in different patients and to detect MRD on both RNA and DNA levels. We also found several accompanying mutations in epigenetic regulators (DNMT3A, ASXL1, BCOR) by targeted NGS approach in AML cases. These mutations preceded full transformation by t(16;21)(p11;q22)/FUS-ERG and allowed us to trace clonal evolution on all steps of therapy. As a casual observation, the ASXL1 mutation was found in the unrelated donor hematopoietic cells. [ABSTRACT FROM AUTHOR]

Alpha/beta T cell and CD19 depletion are used to improve the outcomes of hematopoietic stem cell transplantation (HSCT). We evaluated the burden of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in pediatric patients after this HSCT type. A cohort of 182 patients with malignant (n = 114) or nonmalignant (n = 68) disorders was transplanted from either matched unrelated (n = 124) or haploidentical (n = 58) donors. The cumulative incidence of CMV and EBV viremia were 51% and 33%, respectively. Acute graft-versus-host disease (GVHD) grades II to IV, D−/R+ serology, and malignant HSCT indications were associated with increased risk of CMV viremia. CMV disease developed in 10 patients (6%). The occurrence of CMV viremia was not associated with inferior outcomes. Acute GVHD grade ≥ II was the only factor significantly associated with an increased risk of EBV viremia. Rituximab significantly decreased the rate of EBV reactivation in a subgroup that received a higher B cell dose in the graft. The rate of EBV-associated disease was .5%, and EBV viremia did not affect survival. TCR-α/β and CD19 depletion are associated with a significant rate of CMV viremia that does not affect survival. The hazard of EBV post-transplant lymphoproliferative disease (PTLD) is eliminated by the combination of CD19 depletion and rituximab. [ABSTRACT FROM AUTHOR]

The technique of αβ T cell depletion (αβTCD) is a well-established method of hematopoietic stem cell transplantation (HSCT) for children with acute leukemia owing to the low rates of graft-versus-host disease and nonrelapse mortality (NRM). The graft-versus-leukemia effect is generally ascribed to natural killer (NK) cells conserved within the graft. It is not known whether NK-related factors affect the outcome of αβTCD HSCT, however. The aim of this retrospective study was to explore the impact of NK alloreactivity (based on donor-recipient killer immunoglobulin-like receptor [KIR] mismatch), graft NK cell dose, and blood NK cell recovery on day +30 post-HSCT on the incidences of leukemia relapse and NRM. The pediatric acute leukemia cohort comprised 295 patients who underwent their first HSCT from a haploidentical donor in complete remission. During post hoc analysis, the total cohort was divided into subcohorts by diagnosis (acute lymphoblastic leukemia [ALL]/acute myeloid leukemia [AML]), NK alloreactivity prediction (KIR match/KIR mismatch), graft NK cell dose (less than versus greater than the median value), and blood NK cell recovery on day +30 post-HSCT (less than versus greater than the median value). We also investigated the influence of serotherapy (antithymocyte globulin [ATG] group) versus abatacept + tocilizumab combination [aba+toci] group) on relapse risk in the context of KIR mismatch. The risks of relapse and NRM were calculated by the cumulative risk method, and groups were compared using the Gray test. Multivariate analysis revealed no apparent impact of predicted NK alloreactivity or any other studied NK cell-related factors for the entire cohort. For patients with AML, a significantly higher relapse risk associated with high NK cell graft content on the background of no predicted KIR mismatch (P = .002) was shown. Multivariate analysis confirmed this finding (P = .018); on the other hand, for the KIR-mismatched patients, there was a trend toward a lower risk of relapse associated with high NK cell dose. The use of ATG was associated with a trend toward reduced relapse risk (P = .074) in the AML patients. There was no significant impact of NK-related factors in the ALL patients. Overall, the evaluated NK-related factors did not show a clear and straightforward correlation with the key outcomes of HSCT in our cohort of children with acute leukemia. In practice, the data support prioritization of KIR-mismatched donors for patients with AML. Importantly, a potential interaction of KIR ligand mismatch and NK cell content in the graft was identified. Indirect evidence suggests that additional cellular constituents of the graft could influence the function of NK cells after HSCT and affect their role as graft-versus-leukemia effectors., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Mature T/NK-cell lymphomas (MTCLs) are a heterogeneous group of lymphoproliferative disorders, relatively rare in adults and children. Allogeneic hematopoietic stem cell transplantation (HSCT) can be considered in some cases as a consolidation and is the first choice for refractory forms and relapses. We retrospectively analyzed 19 pediatric patients with MTCL who received allogeneic hematopoietic stem cell transplantation from a haploidentical or unrelated donor on the αβ T cell depletion platform. Among the studied patients, cutaneous T-cell lymphoma was diagnosed in 5, hepatosplenic γδT-cell lymphoma in 4, ALK-positive anaplastic large cell lymphoma in 9 patients, and 1 had nasal T/NK cell lymphoma. All patients received myeloablative conditioning based on treosulfan or total body irradiation. Non-relapse mortality was 5%, the cumulative incidence of relapse or progression at 5 years was 27%, 5-year event-free survival was 67%, and 5-year overall survival was 78%. Thus, our data support that allogeneic αβ T-cell-depleted HSCT can provide long-term overall survival of children with high-risk mature T-cell lymphomas., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Langerhans cell histiocytosis (LCH) is a disorder with a variety of clinical signs. The most severe forms affect risk organs (RO). The established role of the BRAF V600E mutation in LCH led to a targeted approach. However, targeted therapy cannot cure the disease, and cessation leads to quick relapses. Here, we combined cytosine-arabinoside (Ara-C) and 2'-chlorodeoxyadenosine (2-CdA) with targeted therapy to achieve stable remission. Nineteen children were enrolled in the study: 13 were RO-positive (RO+) and 6 RO-negative (RO-). Five patients received the therapy upfront, whereas the other 14 received it as a second or third line. The protocol starts with 28 days of vemurafenib (20 mg/kg), which is followed by 3 courses of Ara-C and 2-CdA (100 mg/m2 every 12 h, 6 mg/m2 per day, days 1-5) with concomitant vemurafenib therapy. After that, vemurafenib therapy was stopped, and 3 courses of mono 2-CdA followed. All patients rapidly responded to vemurafenib: the median disease activity score decreased from 13 to 2 points in the RO+ group and from 4.5 to 0 points in the RO- group on day 28. All patients except 1 received complete protocol treatment, and 15 of them did not have disease progression. The 2-year reactivation/progression-free survival (RFS) for RO+ was 76.9% with a median follow-up of 21 months and 83.3% with a median follow-up of 29 months for RO-. Overall survival is 100%. Importantly, 1 patient experienced secondary myelodysplastic syndrome after 14 months from vemurafenib cessation. Our study demonstrates that combined vemurafenib plus 2-CdA and Ara-C is effective in a cohort of children with LCH, and the toxicity is manageable. This trial is registered at www.clinicaltrials.gov as NCT03585686., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

We compared the efficacy and safety of eltrombopag (ELTR) combined with immunosuppressive therapy (IST) and IST alone in treatment-naïve children with severe (SAA) and very severe (vSAA) aplastic anemia. Ninety-eight pediatric patients were randomized to receive horse antithymocyte globulin (hATG) and cyclosporin A (CsA) with (n = 49) or without (n = 49) ELTR. The primary endpoint was the overall response rate (ORR) at 4 months. After 4 months, nonresponders were crossed over to the alternative group. In all patients, the ORR in ELTR + IST and IST groups was similar (65% vs 53%; P = .218); however, the complete response (CR) rate was significantly higher in the ELTR + IST group (31% vs 12%; P = .027). In severity subgroups, the ORR was 89% vs 57% (P = .028) in favor of IST + ELTR in SAA, but it did not differ in patients with vSAA (52% vs 50%; P = .902). At 6 months after the crossover, 61% of initial ELTR(-) patients achieved a response compared with 17% of initial ELTR(+) patients (P = .016). No significant difference in ELTR + IST and IST groups was observed in the 3-year overall survival (OS) (89% vs 91%; P = .673) or the 3-year event-free survival (EFS) (53% vs 41%; P = .326). There was no unexpected toxicity related to ELTR. Adding ELTR to standard IST was well tolerated and increased the CR rate. The greatest benefit from ELTR combined with IST was observed in patients with SAA but not in those with vSAA. The second course of IST resulted in a high ORR in initial ELTR(-) patients who added ELTR and had limited efficacy among patients who received ELTR upfront. This trial was registered at Clinicaltrials.gov as #NCT03413306., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Graft failure (GF) remains a serious issue of hematopoietic stem cell transplantation (HSCT) in inborn errors of immunity (IEI). Second HSCT is the only salvage therapy for GF. There are no uniform strategies for the second HSCTs and limited data are available on the second HSCT outcomes. 48 patients with various IEI received second allogeneic HSCT from 2013 to 2020. Different conditioning regimens were used, divided into two main groups: containing myeloablative doses of busulfan/treosulfan (n = 19) and lymphoid irradiation 2-6 Gy (n = 22). Irradiation-containing conditioning was predominantly used in suspected immune-mediated rejection of the first graft. Matched unrelated donor was used in 28 patients, mismatched related in 18, and matched related in 1. 35 patients received TCRαβ/CD19 graft depletion. The median follow-up time was 2.4 years post-HSCT. One patient died at conditioning. The OS was 0.63 (95% CI: 0.41-0.85) after busulfan/treosulfan and 0.68 (95% CI: 0.48-0.88) after irradiation-based conditioning, p = 0.66. Active infection at HSCT significantly influenced OS: 0.43 (95% CI: 0.17-0.69) versus 0.73 (95% CI: 0.58-0.88) without infection, p = 0.004. The cumulative incidence of GF was 0.15 (95% CI: 0.08-0.29). To conclude, an individualized approach is required for the second HSCT in IEI. Low-dose lymphoid irradiation in suspected immune-mediated GF may be a feasible option., (© 2022. The Author(s).)

The long-term outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in chemorefractory acute myeloid leukemia (AML) remains suboptimal because of a high relapse rate. Enhancement of conditioning regimens by the incorporation of targeted anti-leukemia agents is a potential approach to improve the efficacy of HSCT. In a pilot trial and extended access cohort, we evaluated the safety and potential value of adding combinations of venetoclax and daratumumab to a preparative regimen among children with chemorefractory acute myeloid leukemia grafted with αβ T-cell-depleted peripheral blood stem cells. All 20 patients had active disease status of AML at the time of transplantation. The preparative regimen included myeloablative conditioning based on either total body irradiation or treosulfan. A haploidentical related donor was used as a graft source for all patients. Engraftment was not compromised, and no excess toxicity was noted. Minimal residual disease-negative complete remission was achieved in 17 patients (85%). The cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 17%, and the cumulative incidence of chronic GVHD was 7%. At 2 years, nonrelapse mortality was 10%, relapse incidence was 46%, event-free survival was 44%, and overall survival was 65%. Our data show the possibility of safely adding targeted agents to conditioning regimens; however, no evidence of a significant improvement in long-term transplantation outcomes in this cohort of patients was observed., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Relapse of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) may occur in the central nervous system (CNS). Most clinical trials of CAR T-cell therapy excluded patients with active CNS leukemia, partially for concerns of neurotoxicity. Here, we report an international study of fifty-five children and adolescents who received CAR T-cell therapy for relapsed BCP-ALL with CNS involvement at the time of referral. All patients received bridging therapy, 16 still having active CNS disease at the time of lymphodepletion. Twelve patients received CD28-based CAR T-cells, 9 being subsequently treated with allogeneic hematopoietic stem-cell transplantation (allo-HSCT). Forty-three patients received 4-1BB-based CAR T-cells. Cytokine-release syndrome (CRS) and neurotoxicity occurred in 65% and 38% of patients, respectively, more frequently following treatment with CD28-based CARs. Fifty-one of 54 evaluable patients (94%) achieved complete response following this therapy. Relapse occurred in 22 patients: 19/43 following 4-1BB-based CARs (12 CNS relapses), and 3/12 after CD28-based CARs with subsequent HSCT (no CNS relapse). Patients treated with tisagenlecleucel for an isolated CNS relapse had a high incidence of a subsequent CNS relapse (6 of 8). CAR T-cells were found to be effective in this cohort, though the risk of CNS relapse was not completely mitigated by this approach., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5‒97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies., (© 2021. The Author(s).)