A validated pediatric disease risk index for allogeneic hematopoietic cell transplantation.

A disease risk index (DRI) that was developed for adults with hematologic malignancy who were undergoing hematopoietic cell transplantation is also being used to stratify children and adolescents by disease risk. Therefore, to develop and validate a DRI that can be used to stratify those with AML and ALL by their disease risk, we analyzed 2569 patients aged <18 years with acute myeloid (AML; n = 1224) or lymphoblastic (ALL; n = 1345) leukemia who underwent hematopoietic cell transplantation. Training and validation subsets for each disease were generated randomly with 1:1 assignment to the subsets, and separate prognostic models were derived for each disease. For AML, 4 risk groups were identified based on age, cytogenetic risk, and disease status, including minimal residual disease status at transplantation. The 5-year leukemia-free survival for low (0 points), intermediate (2, 3, 5), high (7, 8), and very high (>8) risk groups was 78%, 53%, 40%, and 25%, respectively (P < .0001). For ALL, 3 risk groups were identified based on age and disease status, including minimal residual disease status at transplantation. The 5-year leukemia-free survival for low (0 points), intermediate (2-4), and high (≥5) risk groups was 68%, 51%, and 33%, respectively (P < .0001). We confirmed that the risk groups could be applied to overall survival, with 5-year survival ranging from 80% to 33% and 73% to 42% for AML and ALL, respectively (P < .0001). This validated pediatric DRI, which includes age and residual disease status, can be used to facilitate prognostication and stratification of children with AML and ALL for allogeneic transplantation.

Three-year results from phase I of ZUMA-4: KTE-X19 in pediatric relapsed/refractory acute lymphoblastic leukemia.

Here we present the 3-year results of ZUMA-4, a phase I/II multicenter study evaluating the safety and efficacy of KTEX19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia. Phase I explored two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities. Thirty-one patients were enrolled; KTE-X19 was administered to 24 patients (median age 13.5 years, range 3-20; median follow-up 36.1 months). No dose-limiting toxicities were observed. All treated patients had grade ≥3 adverse events, commonly hypotension (50%) and anemia (42%). Grade 3 cytokine release syndrome rates were 33% in all treated patients, 75% in patients given the dose of 2×106 CAR T cells/kg, 27% in patients given the dose of 1×106 cells/kg in the 68 mL formulation, and 22% in patients given the dose of 1×106 cells/kg in the 40 mL formulation; the percentages of patients experiencing grade ≥3 neurologic events were 21%, 25%, 27%, and 11% respectively. Overall complete remission rates (including complete remission with incomplete hematologic recovery) were 67% in all treated patients, 75% in patients given 2×106 CAR T cells/kg, 64% in patients given 1×106 cells/kg in the 68 mL formulation, and 67% in patients given 1×106 cells/kg in the 40 mL formulation. Overall minimal residual diseasenegativity rates were 100% among responders; 88% of responders underwent subsequent allogeneic stem-cell transplantation. In the 1×106 (40 mL) group (recommended phase II dose), the median duration of remission censored at allogeneic stem-cell transplantation and median overall survival were not reached. Pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia achieved high minimal residual disease-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase II of the study is ongoing at the dose of 1×106 CAR T cells/kg in the 40 mL formulation. ClinicalTrials.gov: NCT02625480.

Targeting HLA-DR loss in hematologic malignancies with an inhibitory chimeric antigen receptor.

Chimeric antigen receptor natural killer (CAR-NK) cells have remarkable cytotoxicity against hematologic malignancies; however, they may also attack normal cells sharing the target antigen. Since human leukocyte antigen DR (HLA-DR) is reportedly lost or downregulated in a substantial proportion of hematologic malignancies, presumably a mechanism to escape immune surveillance, we hypothesize that the anti-cancer specificity of CAR-NK cells can be enhanced by activating them against cancer antigens while inhibiting them against HLA-DR. Here, we report the development of an anti-HLA-DR inhibitory CAR (iCAR) that can effectively suppress NK cell activation against HLA-DR-expressing cells. We show that dual CAR-NK cells, which co-express the anti-CD19 or CD33 activating CAR and the anti-HLA-DR iCAR, can preferentially target HLA-DR-negative cells over HLA-DR-positive cells in vitro. We find that the HLA-DR-mediated inhibition is positively correlated with both iCAR and HLA-DR densities. We also find that HLA-DR-expressing surrounding cells do not affect the target selectivity of dual CAR-NK cells. Finally, we confirm that HLA-DR-positive cells are resistant to dual CAR-NK cell-mediated killing in a xenograft mouse model. Our approach holds great promise for enhancing CAR-NK and CAR-T cell specificity against malignancies with HLA-DR loss.

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Temsirolimus combined with cyclophosphamide and etoposide for pediatric patients with relapsed/refractory acute lymphoblastic leukemia: a Therapeutic Advances in Childhood Leukemia Consortium trial (TACL 2014-001).

Phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is commonly dysregulated in acute lymphoblastic leukemia (ALL). The TACL2014-001 phase I trial of the mTOR inhibitor temsirolimus in combination with cyclophosphamide and etoposide was performed in children and adolescents with relapsed/refractory ALL. Temsirolimus was administered intravenously (IV) on days 1 and 8 with cyclophosphamide 440 mg/m2 and etoposide 100 mg/m2 IV daily on days 1-5. The starting dose of temsirolimus was 7.5 mg/m2 (DL1) with escalation to 10 mg/m2 (DL2), 15 mg/m2 (DL3), and 25 mg/m2 (DL4). PI3K/mTOR pathway inhibition was measured by phosphoflow cytometry analysis of peripheral blood specimens from treated patients. Sixteen heavily-pretreated patients were enrolled with 15 evaluable for toxicity. One dose-limiting toxicity of grade 4 pleural and pericardial effusions occurred in a patient treated at DL3. Additional dose-limiting toxicities were not seen in the DL3 expansion or DL4 cohort. Grade 3/4 non-hematologic toxicities occurring in three or more patients included febrile neutropenia, elevated alanine aminotransferase, hypokalemia, mucositis, and tumor lysis syndrome and occurred across all doses. Response and complete were observed at all dose levels with a 47% overall response rate and 27% complete response rate. Pharmacodynamic correlative studies demonstrated dose-dependent inhibition of PI3K/mTOR pathway phosphoproteins in all studied patients. Temsirolimus at doses up to 25 mg/m2 with cyclophosphamide and etoposide had an acceptable safety profile in children with relapsed/refractory ALL. Pharmacodynamic mTOR target inhibition was achieved and appeared to correlate with temsirolimus dose. Future testing of next-generation PI3K/mTOR pathway inhibitors with chemotherapy may be warranted to increase response rates in children with relapsed/refractory ALL.

Decitabine and vorinostat with FLAG chemotherapy in pediatric relapsed/refractory AML: Report from the therapeutic advances in childhood leukemia and lymphoma (TACL) consortium.

Survival outcomes for relapsed/refractory pediatric acute myeloid leukemia (R/R AML) remain dismal. Epigenetic changes can result in gene expression alterations which are thought to contribute to both leukemogenesis and chemotherapy resistance. We report results from a phase I trial with a dose expansion cohort investigating decitabine and vorinostat in combination with fludarabine, cytarabine, and G-CSF (FLAG) in pediatric patients with R/R AML [NCT02412475]. Thirty-seven patients enrolled with a median age at enrollment of 8.4 (range, 1-20) years. There were no dose limiting toxicities among the enrolled patients, including two patients with Down syndrome. The recommended phase 2 dose of decitabine in combination with vorinostat and FLAG was 10 mg/m2 . The expanded cohort design allowed for an efficacy evaluation and the overall response rate among 35 evaluable patients was 54% (16 complete response (CR) and 3 complete response with incomplete hematologic recovery (CRi)). Ninety percent of responders achieved minimal residual disease (MRD) negativity (<0.1%) by centralized flow cytometry and 84% (n = 16) successfully proceeded to hematopoietic stem cell transplant. Two-year overall survival was 75.6% [95%CI: 47.3%, 90.1%] for MRD-negative patients vs. 17.9% [95%CI: 4.4%, 38.8%] for those with residual disease (p < .001). Twelve subjects (34%) had known epigenetic alterations with 8 (67%) achieving a CR, 7 (88%) of whom were MRD negative. Correlative pharmacodynamics demonstrated the biologic activity of decitabine and vorinostat and identified specific gene enrichment signatures in nonresponding patients. Overall, this therapy was well-tolerated, biologically active, and effective in pediatric patients with R/R AML, particularly those with epigenetic alterations.

Real-world experience in treating pediatric relapsed/refractory or therapy-related myeloid malignancies with decitabine, vorinostat, and FLAG therapy based on a phase 1 study run by the TACL consortium.

Current therapies for relapsed/refractory (R/R) pediatric myeloid neoplasms are inadequately effective. Real-world data (RWD) can improve care by augmenting traditional studies and include individuals not eligible for clinical trials. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) consortium recently completed T2016-003, a phase 1 study of decitabine, vorinostat, fludarabine, cytarabine, and granulocyte colony-stimulating factor (G-CSF) in R/R acute myeloid leukemia (AML), which added epigenetic drugs to a cytotoxic backbone. We report results of RWD from six centers that treated 28 pediatric patients (26 with AML, two with other myeloid neoplasms) identically to the TACL study but who were not enrolled. This allowed unique analyses and the ability to compare data with the 35 TACL study patients. The overall response rate (ORR) (complete response [CR] plus CR with incomplete count recovery) among 26 RWD evaluable patients was 65%. The ORR of 13 patients with relapsed AML with epigenetic alterations was 69% (T2016-003 + RWD: 68%, n = 25), of eight patients with refractory AML was 38% (T2016-003 + RWD: 41%, n = 17) and of five patients with therapy-related AML (t-AML) was 80% (T2016-003 + RWD: 75%, n = 8). The mean number of Grade 3/4 toxicities experienced by the T2016-003-eligible RWD population (n = 22) (one per patient-cycle) was not meaningfully different than those (n = 6) who would have been TACL study-ineligible secondary to comorbidities (two per patient-cycle). Overall, this therapy was well tolerated and effective in pediatric patients with R/R myeloid neoplasms, particularly those with epigenetic alterations, t-AML, and refractory disease.

5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with Moxetumomab pasudotox.

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine.

CAR T-cell product performance in haematological malignancies before and after marketing authorisation.

Chimeric antigen receptor (CAR) T cells represent a potent new approach to treat haematological malignancies. Two CAR T-cell therapies, tisagenlecleucel and axicabtagene ciloleucel, have been approved in Europe and the USA, as well as several other countries, for the treatment of leukaemia and lymphoma. These approvals marked a major milestone in the field of cell and gene therapies. However, the clinical development and regulatory evaluation of these innovative therapies faced several challenges that are considered important lessons learned for future similar products. Here, we examine the products' non-clinical and clinical data packages to outline the challenges encountered during the regulatory evaluation process in Europe, and to provide an update on their performance after authorisation.

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

BACKGROUND: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. PROCEDURE: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. RESULTS: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. CONCLUSIONS: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

Results from an international phase 2 study of the anti-CD22 immunotoxin moxetumomab pasudotox in relapsed or refractory childhood B-lineage acute lymphoblastic leukemia.

BACKGROUND: In a multicenter phase 1 study of children with relapsed/refractory acute lymphoblastic leukemia (ALL), moxetumomab pasudotox, an anti-CD22 immunotoxin, demonstrated a manageable safety profile and preliminary evidence of clinical activity. A phase 2 study further evaluated efficacy. PROCEDURE: This international, multicenter, phase 2 study enrolled children with relapsed/refractory B-cell precursor ALL who received moxetumomab pasudotox 40 µg/kg intravenously every other day, for six doses per 21-day cycle. The primary objective was to evaluate the complete response (CR) rate. Secondary objectives included safety, pharmacokinetics, and immunogenicity evaluations. RESULTS: Thirty-two patients (median age, 10 years) were enrolled at 16 sites; 30 received study drug and were evaluable for safety; 28 were evaluable for response. The objective response rate was 28.6%, with three patients (10.7%) achieving morphologic CR, and five patients (17.9%) achieving partial response. Disease progression occurred in 11 patients (39.3%). Ten patients (33.3%) experienced at least one treatment-related serious adverse event, including capillary leak syndrome (CLS; n = 6), hemolytic uremic syndrome (HUS; n = 4), and treatment-related death (n = 1) from pulmonary edema. No differences were observed in inflammatory markers in patients who did or did not develop CLS or HUS. CONCLUSIONS: Despite a signal for clinical activity, this phase 2 study was terminated at interim analysis for a CR rate that did not reach the stage 1 target. Preclinical data suggest enhanced efficacy of moxetumomab pasudotox via continuous infusion or in combination regimens; thus, further studies designed to optimize the efficacy and safety of moxetumomab pasudotox in pediatric ALL may be warranted.

Phase 1 study of the anti-CD22 immunotoxin moxetumomab pasudotox for childhood acute lymphoblastic leukemia.

Novel therapies are needed to overcome chemotherapy resistance for children with relapsed/refractory acute lymphoblastic leukemia (ALL). Moxetumomab pasudotox is a recombinant anti-CD22 immunotoxin. A multicenter phase 1 study was conducted to determine the maximum-tolerated cumulative dose (MTCD) and evaluate safety, activity, pharmacokinetics, and immunogenicity of moxetumomab pasudotox in children, adolescents, and young adults with ALL (N = 55). Moxetumomab pasudotox was administered as a 30-minute IV infusion at doses of 5 to 50 µg/kg every other day for 6 (cohorts A and B) or 10 (cohort C) doses in 21-day cycles. Cohorts B and C received dexamethasone prophylaxis against capillary leak syndrome (CLS). The most common treatment-related adverse events were reversible weight gain, hepatic transaminase elevation, and hypoalbuminemia. Dose-limiting CLS occurred in 2 of 4 patients receiving 30 µg/kg of moxetumomab pasudotox every other day for 6 doses. Incorporation of dexamethasone prevented further dose-limiting CLS. Six of 14 patients receiving 50 µg/kg of moxetumomab pasudotox for 10 doses developed hemolytic uremic syndrome (HUS), thrombotic microangiopathy (TMA), or HUS-like events, exceeding the MTCD. Treatment expansion at 40 µg/kg for 10 doses (n = 11) exceeded the MTCD because of 2 HUS/TMA/HUS-like events. Dose level 6B (ie, 50 µg/kg × 6 doses) was the MTCD, selected as the recommended phase 2 dose. Among 47 evaluable patients, an objective response rate of 32% was observed, including 11 (23%) composite complete responses, 5 of which were minimal residual disease negative by flow cytometry. Moxetumomab pasudotox showed a manageable safety profile and evidence of activity in relapsed or refractory childhood ALL. This trial was registered at www.clinicaltrials.gov as #NCT00659425.

The American Society of Pediatric Hematology/Oncology workforce assessment: Part 1-Current state of the workforce.

The American Society of Pediatric Hematology/Oncology (ASPHO) recognized recent changes in medical practice and the potential impact on pediatric hematology-oncology (PHO) workforce. ASPHO surveyed society members and PHO Division Directors between 2010 and 2016 and studied PHO workforce data collected by the American Board of Pediatrics and the American Medical Association to characterize the current state of the PHO workforce. The analysis of this information has led to a comprehensive description of PHO physicians, professional activities, and workplace. It is important to continue to collect data to identify changes in composition and needs of the PHO workforce.

Myeloid lineage switch following chimeric antigen receptor T-cell therapy in a patient with TCF3-ZNF384 fusion-positive B-lymphoblastic leukemia.

A pediatric patient diagnosed initially with B-lymphoblastic leukemia (B-ALL) relapsed with lineage switch to acute myeloid leukemia (AML) after chimeric antigen receptor T-cell (CAR-T) therapy and hematopoietic stem cell transplant. A TCF3-ZNF384 fusion was identified at diagnosis, persisted through B-ALL relapse, and was also present in the AML relapse cell population. ZNF384-rearrangements define a molecular subtype of B-ALL characterized by a pro-B-cell immunophenotype; furthermore, ZNF384-rearrangements are prevalent in mixed-phenotype acute leukemias. Lineage switch following CAR-T therapy has been described in patients with KMT2A (mixed lineage leukemia) rearrangements, but not previously in any patient with ZNF384 fusion.

Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation.

Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.

Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL): Overview and introduction to the proceedings of the 2016 TACL investigator meeting.

Despite great success in the development of curative therapies for pediatric hematologic malignancies, new approaches are needed to overcome resistance to treatment and to reduce associated side effects. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium is an early phase clinical trial group dedicated to developing innovative therapies for currently incurable pediatric leukemias and lymphomas ( https://tacl.chla.usc.edu/tacl/ ). In November of 2016, a TACL Investigator Meeting was held, the proceedings of which appear in this edition of Pediatric Hematology Oncology. This introductory article provides an overview of TACL and introduces the five-part proceedings.

Investigating the biology of relapsed acute leukemia: Proceedings of the Therapeutic Advances for Childhood Leukemia & Lymphoma (TACL) Consortium Biology Working Group.

During the 2016 Therapeutic Advances for Childhood Leukemia & Lymphoma (TACL) Consortium investigators' meeting (Los Angeles, CA), a Biology Working Group was established to support the consortium's mission of developing innovative therapies for currently incurable childhood leukemias and lymphomas. The charge of the Biology Working Group was to address how TACL could advance biological investigations of pediatric relapsed/refractory hematologic malignancies while undertaking forward-looking therapeutic trials. To this end, the TACL Biology Committee was established to provide the scientific platform needed to further develop preclinical and translational studies that will advance the understanding and treatment of relapsed and refractory disease. The Biology Committee will focus on ensuring state-of-the-art studies that address biological components of early phase clinical trials, and developing a central biology bank of materials from these early phase trials for interrogations into the mechanisms of disease resistance.

Induction of Immune Response after Allogeneic Wilms' Tumor 1 Dendritic Cell Vaccination and Donor Lymphocyte Infusion in Patients with Hematologic Malignancies and Post-Transplantation Relapse.

Relapse of hematologic malignancies is the primary cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). Treatment for post-HCT relapse using donor lymphocyte infusion (DLI) has limited utility, particularly in the setting of acute leukemia, and can result in the development of graft-versus-host disease (GVHD). The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies, with limited expression in normal tissues. In this pilot trial, we assessed safety and feasibility of a WT1 peptide-loaded donor-derived dendritic cell (DC) vaccine given with DLI designed to enhance and direct the graft-versus-leukemia effect. Secondary objectives were to evaluate immunologic and clinical responses. A total of 5 subjects, median age 17 years (range, 9 to 19 years), with post-HCT relapse were enrolled. Disease subtypes included acute lymphoblastic leukemia (n = 3), acute myelogenous leukemia (n = 1), and Hodgkin lymphoma (n = 1). Successful vaccine production was feasible from all donors. DC vaccination and DLI were well tolerated. One recipient developed grade 1 skin GVHD not requiring systemic therapy. The most common adverse events included grade 1 reversible pain and pruritus at the vaccine injection and delayed-type hypersensitivity (DTH) skin testing sites. There were no grade 3 or higher adverse events related to the research. Immune responses consisted of ELISpot response in 3 recipients and positive DTH tests to WT1 peptide cocktail in 2 subjects. Our study provides 1 of the first attempts to apply tumor-specific vaccine therapy to the allogeneic setting. Preliminary results show the DC-based vaccination is safe and feasible after allogeneic HCT, with a suggestion that this approach can be used to sensitize the repopulated allogeneic-donor immune system to WT1. Future directions may include testing of vaccination strategies in the early post-transplantation setting for relapse prevention.

T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.

BACKGROUND: Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells. METHODS: This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696. FINDINGS: Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients). INTERPRETATION: CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur. FUNDING: National Institutes of Health Intramural funds and St Baldrick's Foundation.

Immunotoxins for leukemia.

Unconjugated monoclonal antibodies that target hematopoietic differentiation antigens have been developed to treat hematologic malignancies. Although some of these have activity against chronic lymphocytic leukemia and hairy cell leukemia, in general, monoclonal antibodies have limited efficacy as single agents in the treatment of leukemia. To increase their potency, the binding domains of monoclonal antibodies can be attached to protein toxins. Such compounds, termed immunotoxins, are delivered to the interior of leukemia cells based on antibody specificity for cell surface target antigens. Recombinant immunotoxins have been shown to be highly cytotoxic to leukemic blasts in vitro, in xenograft model systems, and in early-phase clinical trials in humans. These agents will likely play an increasing role in the treatment of leukemia.