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Persistence or recurrence of large B-cell lymphoma after CD19-CAR-T is common, yet data guiding management are limited. We describe outcomes and features following CAR-T treatment failure. Of 305 adults who received CD19-CAR-T, 182 experienced disease recurrence or progression (1-year cumulative incidence 63% [95%CI: 57–69]). Of 52 post-CAR-T biopsies evaluated by flow cytometry, 49 (94%) expressed CD19. Subsequent anti-cancer treatment was administered in 135/182 (74%) patients with CAR-T treatment failure. Median OS from the first post-CAR-T treatment was 8 months (95%CI 5.6–11.0). Polatuzumab-, standard chemotherapy-, and lenalidomide-based treatments were the most common approaches after CAR-T. No complete responses (CRs) were observed with conventional chemotherapy, while CR rates exceeding 30% were seen following polatuzumab- or lenalidomide-based therapies. Factors associated with poor OS among patients treated post-CAR-T were pre-CAR-T bulky disease (HR 2.27 [1.10–4.72]), lack of response to CAR-T (2.33 [1.02–5.29]), age >65 years (HR 2.65 [1.49–4.73]) and elevated LDH at post-CAR-T treatment (HR 2.95 [1.61–5.38]). The presence of ≥2 of these factors was associated with inferior OS compared to ≤1 (56% vs. 19%). In this largest analysis to date of patients who progressed or relapsed after CD19-CAR-T, survival is poor, though novel agents such as polatuzumab and lenalidomide may have hold promise.

Supplementary Table 1. DNA fragments synthesized and inserted into MSCV-CD19-IRES-DsRedFP via EcoRI/BglII or BglII/XhoI digestion, and later moved into pMX-IRES-Blast via EcoRI/XhoI cloning. Supplementary Table 2. Sets of primers used for PCR amplification and Sanger sequencing of CD19 gene and the upstream -1.2Kb enhancer and promoter regions. Supplementary Table 3. Sets of primers for the analysis of CpG-Methylation status after bisulfite modification of genomic DNA. Supplementary Table 4. Sets of primers used for radioactive semiquantitative PCR analysis of CD19 mRNA isoforms. Supplementary Table 5. Sequence of the mini-gene expanding exon2 and 220nt of its flanking introns that was synthesized (Genewiz) and inserted into pBSKii+ via XhoI/NotI cloning. Supplementary Table 6. Antibodies used for pull down assays for the identification of splicing factors that bind to the CD19-int1-exon2-int2 mini-gene. Supplementary Table 7. Primary and secondary antibodies used for immunoblotting and immunoprecipitations after protein separation by SDS-PAGE and transference to PVDF membrane (N/A, not applicable). Supplementary Table 8. Pairs of oligos for semiquantitative PCR amplification of CD19 cDNA followed by visualization in agarose gel. Same primers were used for sequencing after gel purification of specific bands. Supplementary Table 9. Pairs of oligos used for Real Time-qPCR amplification of cDNA.

Supplementary Figure 1. Enhanced skipping of exons 2 and 5-6 in post-CART-19 leukemias. Supplementary Figure 2. Analysis of splicing factors involved in alternative splicing of CD19-exon2.

Anti CD19 chimeric antigen receptor (CAR) T-cell therapy has transformed the care of relapsed and refractory aggressive B-cell lymphoma. However, financial toxicity and manufacturing time represent barriers to its widespread implementation. Study applicability, toxicity, and efficacy of a locally produced autologous CD19-directed CAR T-cell product were studied. We performed a phase 1b/2 clinical trial with a point-of-care (POC) CAR T-cell product that contains a CD28 costimulatory domain. Adult patients with aggressive B-cell lymphoma or transformed low-grade lymphoma who received at least 2 prior regimens were eligible. A total of 73 patients, with a median age of 49 years, met inclusion criteria. CAR T-cell production time from apheresis was 10 days (interquartile range 10-11), negating the need for bridging chemotherapy. Overall and complete response rates were 62.5% and 37.5%. Median progression-free and overall survival were 3.7 and 12.1 months, respectively. Overall and progression-free survival at 12 months were 52.1% (confidence interval [CI]: 40.8%-66.5%) and 40% (CI: 30%-53.7%), respectively. Patients who achieved response had longer progression-free and overall survival. Grade 3-4 cytokine release syndrome was observed in 9.5% of the patients, and immune effector cell-associated neurotoxicity syndrome grade 3-4 in 21.9%. No deaths occurred due to CAR T-cell toxicity. Fifteen patients (20%) underwent allogeneic stem cell transplantation at a median time of 60 days after CAR T-cell therapy; 8 were alive at last follow-up. Of the 6 patients who underwent the transplantation in complete response 2 deceased because of toxicity. POC CAR T-cells are a feasible therapeutic option in aggressive B-cell lymphoma. They provide good efficacy while minimizing production time and the need for bridging therapy.

CD28-based CD19 chimaeric antigen receptor-modified (CAR-)Tcells were recently FDA-approved for adult acute lymphoblastic leukaemia (ALL). We report long-term outcome of 37 children and young adults treated with autologous CD19 CAR-T cells. The complete remission rate was 86%, of which 71% were polymerase chain reaction (PCR) minimal residual disease (MRD)-negative, 14% were MRD-negative by flow cytometry, and 14% were PCR MRD-positive. 26 patients proceeded to subsequent haematopoietic stem cell transplant (HSCT). 11 patients had a CD19-postive relapse (eight post HSCT and three without) and one had a CD19-negative relapse. All relapse events occurred within two years from cell therapy. With a median follow-up of three years, the median event-free survival (EFS) is 17 months and the median overall survival (OS) is not reached. The three-year EFS is 41% and OS is 56%. Patients with5% blasts in the bone marrow prior to lymphodepletion had an inferior EFS. All patients with a PCR MRD-positive result at day 28 had relapsed after CAR-T-cell therapy. A prior HSCT did not significantly affect outcome, but a consolidative transplant after achieving remission improved long-term results. Overall, prelymphodepletion disease burden and molecular MRD negativity following CAR-T cells are predictors of long-term outcome following CD19 CAR-T-cell therapy for ALL.

Chimeric antigen receptor T cells targeting CD19 (CART-19) have shown remarkable efficacy for relapsed/refractory (R/R) B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated whether prior use of inotuzumab ozogamicin (InO), an anti-CD22 antibody conjugated to calicheamicin, may impact CAR T-cell manufacturing or efficacy via pre-CART-19 depletion of the B-cell compartment. In this international, retrospective analysis, 39 children and young adults receiving InO before (n = 12) and/or after (n = 27) T-cell apheresis as bridging therapy to CART-19 treatment were analyzed. Median age at infusion was 13 years (range 1.4–23 years). Thirty-four out of 39 patients (87.2%) obtained complete remission. With a median follow-up of 18.2 months after CART-19 infusion, 12-month event-free survival (EFS) was 53.3% (95% confidence interval (CI): 38.7–73.4) and overall survival (OS) was 77.8% (95% CI: 64.5–93.9). Seventeen patients (44%) relapsed with a median of 159 days (range 28–655) after CART-19 infusion. No difference in day 28 minimal residual disease negative complete response rate, 12-month OS/EFS, or incidence of CD19-positive or -negative relapses was observed among patients receiving InO before or after apheresis. Compared to published data for patients treated with CART-19 therapy without prior InO exposure, response and OS/EFS for patients treated with InO prior to CART-19 are similar.

Patients with single large-scale mitochondrial DNA (mtDNA) deletion syndromes (SLSMDs) usually present with multisystemic disease, either as Pearson syndrome in early childhood or as Kearns-Sayre syndrome later in life. No disease-modifying therapies exist for SLSMDs. We have developed a method to enrich hematopoietic cells with exogenous mitochondria, and we treated six patients with SLSMDs through a compassionate use program. Autologous CD34 + hematopoietic cells were augmented with maternally derived healthy mitochondria, a technology termed mitochondrial augmentation therapy (MAT). All patients had substantial multisystemic disease involvement at baseline, including neurologic, endocrine, or renal impairment. We first assessed safety, finding that the procedure was well tolerated and that all study-related severe adverse events were either leukapheresis-related or related to the baseline disorder. After MAT, heteroplasmy decreased in the peripheral blood in four of the six patients. An increase in mtDNA content of peripheral blood cells was measured in all six patients 6 to 12 months after MAT as compared baseline. We noted some clinical improvement in aerobic function, measured in patients 2 and 3 by sit-to-stand or 6-min walk testing, and an increase in the body weight of five of the six patients suffering from very low body weight before treatment. Quality-of-life measurements as per caregiver assessment and physical examination showed improvement in some parameters. Together, this work lays the ground for clinical trials of MAT for the treatment of patients with mtDNA disorders.

Background: Chimeric antigen receptor (CAR) T cells directed against CD19 (CART19) are effective in B-cell malignancies, but little is known about the molecular factors predicting clinical outcome of CART19 therapy. The increasingly recognized relevance of epigenetic changes in cancer immunology prompted us to determine the impact of the DNA methylation profiles of CART19 cells on the clinical course. Methods: We recruited 114 patients with B-cell malignancies, comprising 77 patients with acute lymphoblastic leukemia and 37 patients with non-Hodgkin lymphoma who were treated with CART19 cells. Using a comprehensive DNA methylation microarray, we determined the epigenomic changes that occur in the patient T cells upon transduction of the CAR vector. The effects of the identified DNA methylation sites on clinical response, cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, event-free survival, and overall survival were assessed. All statistical tests were 2-sided. Results: We identified 984 genomic sites with differential DNA methylation between CAR-untransduced and CAR-transduced T cells before infusion into the patient. Eighteen of these distinct epigenetic loci were associated with complete response (CR), adjusting by multiple testing. Using the sites linked to CR, an epigenetic signature, referred to hereafter as the EPICART signature, was established in the initial discovery cohort (n = 79), which was associated with CR (Fisher exact test, P < .001) and enhanced event-free survival (hazard ratio [HR] = 0.36; 95% confidence interval [CI] = 0.19 to 0.70; P = .002; log-rank P = .003) and overall survival (HR = 0.45; 95% CI = 0.20 to 0.99; P = .047; log-rank P = .04;). Most important, the EPICART profile maintained its clinical course predictive value in the validation cohort (n = 35), where it was associated with CR (Fisher exact test, P < .001) and enhanced overall survival (HR = 0.31; 95% CI = 0.11 to 0.84; P = .02; log-rank P = .02). Conclusions: We show that the DNA methylation landscape of patient CART19 cells influences the efficacy of the cellular immunotherapy treatment in patients with B-cell malignancy., Supported by CERCA Programme/Generalitat de Catalunya, Health Department PERIS #SLT/002/16/00374, AGAUR-project #2017SGR1080; MCI/AEI/ERDF project #RTI2018-094049-B-I00; ERC EPIPHARM; Cellex Foundation; “la Caixa” Foundation (LCF/PR/GN18/51140001 and LCF/PR/GN18/50310007), RF-2016–02364388, Accelerator Award—Cancer Research UK/AIRC—INCAR Associazione Italiana Ricerca per la Ricerca sul Cancro (AIRC) Project 5 × 1000 no. 9962, AIRC IG 2018 id. 21724, AIRC MFAG id. 21769 and id. 20450; MIUR (Grant PRIN 2017); and RCR-2019–23669115.

Background Immunotherapy may lead to durable remissions in patients with relapsed and refractory acute lymphoblastic leukemia (R/R ALL). Patients receiving immunotherapy with a lower disease burden tend to have improved long-term outcomes and less toxicity. Thus, an induction protocol to achieve lower disease burden is required. Bortezomib added to a 4-drug induction was shown to lead to high rates of remission in R/R ALL patients. Inclusion of anthracyclines in this protocol may preclude most patients, having maximized the cumulative dose of anthracyclines. Thus, our goal was to evaluate anthracycline-free bortezomib-based induction for patients with R/R ALL. Procedure We conducted a retrospective analysis of patients treated with bortezomib-based protocols for R/R ALL between 2011 and 2019 at our center. Data regarding toxicity and response rate was collected and analyzed. Results Eighteen children with R/R ALL were treated with bortezomib-based induction, 13 of them without anthracyclines. Eleven patients did not complete the induction course: 6 due to toxicity, and 5 due to physician decision to proceed to immunotherapy early. Two events of treatment-related mortality occurred. There was no significant difference in toxicity between patients who treated with anthracycline and those who were not. Ten patients achieved complete remission, with 4 patients having polymerase-chain-reaction minimal residual disease below 10-4. Fifteen patients proceeded directly to immunotherapy: 11 patients received CD19 chimeric-antigen receptor-T-cells, 2 blinatumomab and 2 hematopoietic stem cell transplant. Conclusion Anthracyclines can be safely omitted from bortezomib-based therapies in patients with R/R ALL, when planning to proceed to immunotherapy.

Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

Tisagenlecleucel (tisa-cel) is an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with relapsed/refractory large B-cell lymphoma. Outcomes of patients with out-of-commercial specification (OOS) CAR T products are not well characterized. We therefore assessed 37 adult patients who underwent leukapheresis for tisa-cel therapy in a single center. In nine (24%) patients, manufactured tisa-cel was considered OOS. Three of them (33%) received tisa-cel after institutional review board approval; 2/9 (22%) did not receive tisa-cel due to disease progression; and 4/9 (44%) received academic point-of-care (POC) CAR T-cell as salvage therapy, at a median of 35 days following OOS notification. Three of those four patients achieved a complete response. In univariate analysis, risk factors for OOS were ≥ 4 prior therapies or previous bendamustine exposure. In conclusion, we report high OOS incidence of 24% in real-life setting. Forty-four percent of those patients received POC CAR T-cell as salvage therapy.

Anti-CD19 chimeric antigen receptor T cell (CAR-T) therapy has transformed the care of patients with relapsed/refractory large B cell lymphoma (LBCL). However, approximately 60% of CAR-T recipients ultimately will experience disease recurrence or progression. Salvage therapies after CAR-T treatment failures are of limited efficacy and have a short duration of response. The objective of the present study was to evaluate the role of allogeneic hematopoietic cell transplantation (allo-HCT) after CAR-T therapy in LBCL patients. This was a multicenter observational study reporting the outcome of 39 adult LBCL patients who underwent allo-HCT following anti-CD19 CAR-T therapy. The median patient age was 47 years (range, 20 to 68 years). HLA-matched sibling, HLA-matched unrelated, and alternative donors were used in 36%, 36%, and 28% of transplantations, respectively. Conditioning regimens were primarily of low or intermediate intensity. Disease status at allo-HCT was complete response in 41%, partial response in 38%, and progressive disease in 21%. Allo-HCT was performed at a median of 127 days (range, 82 to 206 days) after CAR-T therapy. A high incidence of hepatic toxicity (28%), including sinusoidal obstruction syndrome (15.4%; 95% confidence interval; [CI], 6.2% to 28.5%), was observed. The 1-year cumulative incidence of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) was 38.5% (95% CI, 23.2% to 53.6%) and 15.4% (95% CI, 6.1% to 28.5%), respectively. The 2-year cumulative incidence of moderate-severe chronic GVHD was 11.1% (95% CI, 3.3% to 24.3%). Overall, 2-year nonrelapse mortality and relapse/progression incidence were 26% (95% CI, 13% to 41%) and 43% (95% CI, 27% to 59%), respectively. With a median follow-up of 32 months, the 2-year overall survival (OS) and progression-free survival (PFS) were 45% (95% CI, 31% to 66%) and 31% (95% CI, 19% to 50%), respectively. In multivariable analyses, pre-HCT elevated lactate dehydrogenase level and transformed lymphoma were predictive of OS and PFS, respectively. Our data suggest that allo-HCT after anti-CD19 CAR-T treatment failure is feasible with a relatively promising efficacy but possibly high toxicity rate.

We report the results of national retrospective study of 45 children with hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) in Israel between the years 2000–2018. Donors were either HLA-matched (n = 26), partially mismatched (n = 7), haploidentical (n = 8), or cord-blood (n = 4). Myeloablative conditioning (MAC) was used in 20 procedures, and reduced-intensity conditioning (RIC) in 25. Forty-two patients engrafted, two had primary graft failure (one successfully retransplanted), one died prior to engraftment, and two developed secondary graft failure. Of the eight patients who had mixed donor chimerism at day 30 (5–95%), five achieved stable mixed or full donor chimerism. The 5-year probabilities of overall survival and event-free survival (EFS) were 86% and 82%, respectively. Five-year EFS was lower for patients receiving RIC compared to MAC (72% vs. 100%, p = 0.018) and following alternative-donor transplant (68% vs. 92% for HLA-matched donors, p = 0.034), mostly due to increased transplant-related mortality (TRM). Thus, both HLA-matched and alternative donor transplant procedures may benefit form a myeloablative conditioning regimen.

CAR T-cells are approved for the treatment of relapsed and refractory leukemia and lymphoma. Here, we studied the infectious complications in 88 patients treated with CD28-based CD19 CAR T-cells. Overall, 36 infections were documented in 24 patients within the first month after CAR T-cell infusion: Six events of bacteremia, sixteen focal bacterial infections, and fourteen systemic or localized viral infections. Seven patients had nine infectious episodes beyond the first 30 days of follow-up, including three events of bacteremia, three focal bacterial, two viral and one fungal infection. The presence of neutropenia, neutropenic fever and lack of response to treatment were associated with a higher rate of infections. Children had less severe infections than adults. In a multivariate analysis lack of response to treatment was the only significant risk factor. Overall, the incidence of bacterial infections following CAR T-cells is modest especially in children and in patients responding to therapy.

We present three patients with aggressive non-Hodgkin's B-cell lymphoma (NHL) who received anti-CD19 chimeric antigen receptor T (CAR T) cells therapy after failure of several lines of chemotherapy that developed pseudo-progression. One-week clinical and radiological findings were consistent with tumor progression. Positron emission tomography-computed tomography (PET-CT) at 1 month post CAR T cells administration was consistent with treatment response. The rapid tumor growth and subsequent resolution are suggestive of tumor pseudo-progression mediated secondary to infiltration and immune activation of CAR T cells. Overall, 56 adult patients with NHL were enrolled in a phase 1b/2 in house clinical study with CD19 CAR T cells. Out of them 22/56 patients progressed as per PET-CT the 1 month post CAR T cells. In 14 patients, signs of progression started 7-10 days after CAR T cells infusion. In 11/14 patients, it was true progression, while in 3 it was pseudo-progression. Additional studies are warranted to describe the extent of this phenomenon and evaluate correlation with the CAR T activity and long-term disease control.

Purpose of the study The prognosis of patients with relapsed/refractory precursor B-acute lymphoblastic leukemia (ALL) is dismal. Antibody-based therapies, such as blinatumomab or inotuzumab ozogamycin (IO) have led to improved outcomes. The impact of prior immunotherapy on chimeric antigen receptor (CAR) T-Cell therapeutic efficacy and toxicity is unknown. Methods We describe a case series of ALL patients with prior exposure to blinatumomab or IO, who were treated with anti-CD19 CAR T cells with CD28 co-stimulatory domain (NCT02772198). We then review the literature on CAR-T post antibody-based therapy with either antibodies. Results Five adult patients with B-ALL were included. Three had active disease, and two were in morphological complete remission (CR) with minimal residual disease (MRD+). Therapy before CAR-T included blinatumomab (3/5 [60 %]) and IO (3/5 [60 %]), with one patient receiving both. One patient experienced severe cytokine release syndrome and central nervous system toxicity and subsequently died. At 28 days following treatment, two patients achieved CR with MRD negativity, and two had an MRD + CR. Two patients received allogeneic hematopoietic stem cell transplantation. At a median of 10 months (range, 5–26, three out of the four patients are still in CR, and one relapsed. The literature review identified a deficiency on data on the influence of blinatumumab and IO on outcomes post CAR-T therapy. Conclusions CD19 CAR T-cell therapy after treatment with blinatumomab and/or IO in patients with relapsed/refractory B-ALL is feasible and results in promising response rates in this case series. Future trails should specifically address outcomes in this population.

ALL is a malignancy of lymphoid progenitor cells, with a bimodal incidence, peaking in early childhood and in older age. In children, ALL tends to have an excellent prognosis, with more than 85% of patients achieving long-term survival. The outcome of younger adults has improved considerably as well. However, overall survival decreases with age (Dores et al. 2012), partially due to the different genetic background of adult ALL, with a higher proportion of Philadelphia chromosome-positive (Ph+) ALL and Ph-like and KMT2A rearrangements in comparison to childhood ALL (Iacobucci and Mullighan 2017). The introduction of paediatric-inspired regimens has improved outcomes in adults, but these regimens are less tolerated in older patients (Curran and Stock 2015).

[Background]: Children aged younger than 3 years were excluded from the ELIANA phase 2 trial of tisagenlecleucel in children with acute lymphoblastic leukaemia. The feasibility, safety, and activity of tisagenlecleucel have not been defined in this group, the majority of whom have high-risk (KMT2A-rearranged) infant acute lymphoblastic leukaemia and historically poor outcomes despite intensification of chemotherapy, and for whom novel therapies are urgently needed. We aimed to provide real-world outcome analysis of the feasibility, activity, and safety of tisagenlecleucel in younger children and infants with acute lymphoblastic leukaemia., [Methods]. We did an international, multicentre, retrospective cohort study at 15 hospitals across ten countries in Europe. Eligible patients were children aged younger than 3 years at screening between Sept 1, 2018, and Sept 1, 2021, who were screened for tisagenlecleucel therapy for relapsed or refractory B-cell precursor acute lymphoblastic leukaemia according to licensed indications. Patients received a single intravenous infusion of tisagenlecleucel. We tracked chimeric antigen receptor T-cell therapy outcomes using a standardised data reporting form. Overall survival, event-free survival, stringent event-free survival, B-cell aplasia, and toxicity were assessed in all patients who received a tisagenlecleucel infusion., [Findings]: 38 eligible patients were screened, of whom 35 (92%) received a tisagenlecleucel infusion. 29 (76%) of 38 patients had KMT2A-rearranged acute lymphoblastic leukaemia, and 25 (66%) had relapsed after previous allogeneic haematopoietic stem-cell transplantation (HSCT). Patients had previously received a median of 2 lines (IQR 2–3) of (non-HSCT) therapy. Seven (18%) of 38 patients had received inotuzumab and 14 (37%) had received blinatumomab. After a median of 14 months (IQR 9–21) of follow-up, overall survival at 12 months after tisagenlecleucel infusion was 84% (64–93; five patients had died), event-free survival was 69% (47–83; nine events), and stringent event-free survival was 41% (23–58; 18 events). The probability of ongoing B-cell aplasia was 70% (95% CI 46–84; seven events) at 12 months. Adverse events included cytokine release syndrome, which occurred at any grade in 21 (60%) of 35 patients and at grade 3 or worse in five (14%), and neurotoxicity at any grade in nine (26%), none of which were severe. Measurable residual disease-negative complete response with or without haematological recovery occurred in 24 (86%) of 28 patients who had measurable disease., [Interpretation]: These data suggest that tisagenlecleucel has antitumour activity and has an acceptable safety profile for young children and infants with B-cell precursor acute lymphoblastic leukaemia.

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.

Axicabtagene ciloleucel (YESCARTA; Kite Pharma, a Gilead Company, Los Angeles CA) and tisagenlecleucel (KYMRIAH; Novartis Pharmaceuticals Corp., Basel, Switzerland) are two CD19-directed chimeric antigen receptor (CAR) T cell products currently approved by the US Food and Drug Administration; the European Medicines Agency; Health Canada; Ministry of Health, Labor and Welfare (Japan); and Therapeutic Goods Administration (Australia) for treatment of specific subtypes of relapsed/refractory aggressive B cell non-Hodgkin lymphoma (NHL). Although this approval has been transformative in the use of cellular immunotherapy in lymphoma, there are concerns regarding appropriate use of this novel therapy and of short- and long-term toxicities. To address these issues, representatives of the American Society of Transplantation and Cellular Therapy convened to recognize and address key issues surrounding the clinical application of CD19 CAR T cell therapy in B cell lymphomas, in collaboration with worldwide experts. The aim of this article is to provide consensus opinion from experts in the fields of hematopoietic cell transplantation, cellular immunotherapy, and lymphoma regarding key clinical questions pertinent to the use of CD19 CAR T cell products for the treatment of NHL. As the clinical practice using CAR T cells grows worldwide, we anticipate that this guidance will be relevant for hematology/oncology physicians who care for patients with lymphomas.

Klotho, a single-pass transmembrane protein associated with premature aging, acts as a tumor suppressor gene by inhibiting insulin/insulin-like growth factor-1 and fibroblast growth factor pathways. Downregulated Klotho expression is reported in melanoma, mesothelioma, bladder, breast, gastric, cervix, lung, and kidney cancers and is associated with a poor prognosis. Klotho expression and Klotho promoter hypermethylation are predictive factors for patient prognosis.To investigate the potential role of Klotho in glioblastoma-multiforme (GBM), 22 GBM samples were collected from the Sheba Tumor Bank and examined.We found that increased Klotho messenger ribonucleic acid (RNA) expression predicted longer survival (P = 0.03) of GBM patients. Methylation analysis was performed on bisulfite-treated deoxyribonucleic acid from the GBM patient samples using ionization time-of-flight mass spectrometry according to the Sequenom EpiTYPER protocols. Klotho promoter hypermethylation was detected in 65% of the GBM samples and correlated significantly with improved survival (P0.04). We found 3 major Klotho promotor hypermethylation sites located 585-579 bp, 540-533 bp, and 537-534 bp upstream of the transcription start site. Methylated deoxyribonucleic acid immunoprecipitation studies confirmed these results. Notably, the messenger RNA expression in these GBM samples revealed an unexpected linear correlation with methylation of these 3 hypermethylation sites identified in the Klotho promotor. Thus Klotho expression and methylation could predict prognosis in patients with GBM.Epigenetic regulation in GBM appears to be complicated. Specific CpG islands affect genes or micro RNAs that interact to control Klotho expression. The diverse effects of these islands may be due to unique factors of GBM.

Haploidntical hematopoietic stem cell transplantation has been increasingly used in recent years for patients without a matched donor. The αβTCR+/CD19+ depletion technique provide a graft that is enriched with CD34 cells, γδ-T-cells and natural killer. The current experience with αβTCR+/CD19+ depleted grafts in pediatric patients with malignant and non-malignant disorders, demonstrated rapid engraftment, improved immune reconstitution and low risk of GVHD. Future studies will need to define the optimal conditioning regimen in order to improve transplant outcome.

By increasing disease-free survival and offering the potential for long-term cure, chimeric antigen receptor (CAR) T-cell therapy has dramatically expanded therapeutic options among those with high-risk B-cell malignancies. As CAR T-cell utilization evolves however, novel challenges are generated. These include determining how to optimally integrate CAR T cells into standard of care and overcoming mechanisms of resistance to CAR T-cell therapy, such as evolutionary stress induced on cancer cells leading to immunophenotypic changes that allow leukemia to evade this targeted therapy. Compounding these challenges are the limited ability to determine differences between various CAR T-cell constructs, understanding the generalizability of trial outcomes from multiple sites utilizing unique CAR manufacturing strategies, and comparing distinct criteria for toxicity grading while defining optimal management. Additionally, as understanding of CAR behavior in humans has developed, strategies have appropriately evolved to proactively mitigate toxicities. These challenges offer complimentary insights and guide next steps to enhance the efficacy of this novel therapeutic modality. With a focus on B-cell malignancies as the paradigm for effective CAR T-cell therapy, this review describes advances in the field as well as current challenges and future directions.

On August 30, 2017 the US Food and Drug Administration approved tisagenlecleucel (Kymriah; Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present, ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring, and disease assessments. To address these issues experts representing the American Society for Blood and Marrow Transplant, the European Society for Blood and Marrow Transplantation, the International Society of Cell and Gene Therapy, and the Foundation for the Accreditation of Cellular Therapy formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

Autologous T cells transduced with CD19-directed chimeric antigen receptors have recently been approved by several regulatory agencies for the treatment of relapsed and refractory leukemia and lymphoma, after demonstrating remarkable remission rate in advanced patients. The most common adverse events reported are cytokine-release syndrome (CRS), neurotoxicity, and hematologic toxicity. Here, we focus on early and late cytopenia occurring after CD19 CAR-T cells in 38 patients treated with CD19 CAR-T cells. Neutropenia, thrombocytopenia, and anemia occur frequently (94, 80, and 51%, respectively) after CAR-T cell infusion, and are associated with a biphasic nature, as in 93% of patients hematologic toxicity occurs after 21 days from cell infusion. Late hematologic toxicity was more common in patients with high grade CRS and in patients treated after a recent stem cell transplantation. Interestingly, since these events occur late after the lymphodepleting chemotherapy and after resolution of CRS, we found perturbations in SDF-1 levels to correlate with events of late neutropenia, likely associated with B-cell recovery.

BackgroundAdoptive cell therapy with T cells genetically engineered to express a chimeric antigen receptor (CAR-T) or tumor-infiltrating T lymphocytes (TIL) demonstrates impressive clinical results in patients with cancer. Lymphodepleting preconditioning prior to cell infusion is an integral part of all adoptive T cell therapies. However, to date, there is no standardization and no data comparing different non-myeloablative (NMA) regimens.MethodsIn this study, we compared NMA therapies with different doses of cyclophosphamide or total body irradiation (TBI) in combination with fludarabine and evaluated bone marrow suppression and recovery, cytokine serum levels, clinical response and adverse events.ResultsWe demonstrate that a cumulative dose of 120 mg/kg cyclophosphamide and 125 mg/m2 fludarabine (120Cy/125Flu) and 60Cy/125Flu preconditioning were equally efficient in achieving deep lymphopenia and neutropenia in patients with metastatic melanoma, whereas absolute lymphocyte counts (ALCs) and absolute neutrophil counts were significantly higher following 200 cGyTBI/75Flu-induced NMA. Thrombocytopenia was most profound in 120Cy/125Flu patients. 30Cy/75Flu-induced preconditioning in patients with acute lymphoblastic leukemia resulted in a minor ALC decrease, had no impact on platelet counts and did not yield deep neutropenia. Following cell infusion, 120Cy/125Flu patients with objective tumor response had significantly higher ALC and significant lower inflammatory indexes, such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). Receiver-operating characteristics curve analysis 7 days after cell infusion was performed to determine the cut-offs, which distinguish between responding and non-responding patients in the 120Cy/125Flu cohort. NLR≤1.79 and PLR≤32.7 were associated with clinical response and overall survival. Cytokine serum levels did not associate with clinical response in patients with TIL. Patients in the 120Cy/125Flu cohort developed significantly more acute NMA-related adverse events, including thrombocytopenia, febrile neutropenia and cardiotoxicity, and stayed significantly longer in hospital compared with the 60Cy/125Flu and TBI/75Flu cohorts.ConclusionsBone marrow depletion and recovery were equally affected by 120Cy/125Flu and 60Cy/125Flu preconditioning; however, toxicity and consequently duration of hospitalization were significantly lower in the 60Cy/125Flu cohort. Patients in the 30Cy/75Flu and TBI/75Flu groups rarely developed NMA-induced adverse events; however, both regimens were not efficient in achieving deep bone marrow suppression. Among the regimens, 60Cy/125Flu preconditioning seems to achieve maximum effect with minimum toxicity.

Graft failure requires urgent salvage HSCT, but there is no universally accepted approach for this situation. We investigated T-cell replete haploidentical HSCT with post-transplantation cyclophosphamide following serotherapy-based, radiation-free, reduced intensity conditioning in children with non-malignant disorders who had rejected their primary graft. Twelve patients with primary or secondary graft failure received T-cell replete bone marrow grafts from haploidentical donors and post-transplantation cyclophosphamide. The recommended conditioning regimen comprised rituximab 375 mg/m2, alemtuzumab 0.4 mg/kg, fludarabine 150 mg/m2, treosulfan 20–24 g/m2 and cyclophosphamide 29 mg/kg. After a median follow-up of 26 months (7–95), eleven of twelve patients (92%) are alive and well with complete donor chimerism in ten. Neutrophil and platelet engraftment were observed in all patients after a median of 18 days (15–61) and 39 days (15–191), respectively. Acute GVHD grade I was observed in 1/12 patients (8%) and mild chronic GVHD in 1/12 patients (8%). Viral reactivations and disease were frequent complications at 75% and 42%, respectively, but no death from infectious causes occurred. In summary, this retrospective analysis demonstrates that a post-transplantation cyclophosphamide-based HLA-haploidentical salvage HSCT after irradiation-free conditioning results in excellent engraftment and overall survival in children with non-malignant diseases.

BackgroundCD19 chimeric antigen receptor T (CAR-T) cells demonstrate remarkable remission rates in pediatric and adult patients with refractory or relapsed (r/r) acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). In 2016, we initiated a clinical trial with in-house produced CD19 CAR-T cells with a CD28 co-stimulatory domain. We analyzed, for the first time, differences in production features and phenotype between ALL and NHL patients.MethodsNon-cryopreserved CAR-T cells were produced from patients’ peripheral blood mononuclear cells within 9 to 10 days. 93 patients with r/r ALL and NHL were enrolled under the same study. CAR-T cells of ALL and NHL patients were produced simultaneously, allowing the head-to-head comparison.ResultsAll patients were heavily pretreated. Three patients dropped out from the study due to clinical deterioration (n=2) or production failure (n=1). Cells of ALL patients (n=37) expanded significantly better and contained more CAR-T cells than of NHL patients (n=53). Young age had a positive impact on the proliferation capacity. The infusion products from ALL patients contained significantly more naïve CAR-T cells and a significantly higher expression of the chemokine receptor CXCR3. PD-1, LAG-3, TIM-3, and CD28 were equally expressed. 100% of ALL patients and 94% of NHL patients received the target dose of 1×10e6 CAR-T/kg. The overall response rate was 84% (30/36) in ALL and 62% (32/52) in NHL. We further compared CAR-T cell infusion products to tumor infiltrating lymphocytes (TIL), another common type of T cell therapy, mainly clinically effective in solid tumors. CAR-T cells contained significantly more naïve T cells and central memory T cells and significantly less CCR5 compared to TIL infusion products.ConclusionsThe in-house production of CAR-T cells is highly efficient and fast. Clinical response rate is high. CAR-T cells can be successfully produced for 99% of patients in just 9 to 10 days. Cells derived from ALL patients demonstrate a higher proliferation rate and contain higher frequencies of CAR-T cells and naïve T cells than of NHL patients. In addition, understanding the differences between CAR-T and TIL infusion products, may provide an angle to develop CAR-T cells for the treatment of solid tumors in the future.Trial registration numberClinicalTrials.gov; CAR-T:NCT02772198, First posted: May 13, 2016; TIL:NCT00287131, First posted: February 6, 2006.