High Leukemia Burden Research Articles

Special issue: CAR‐T cell therapy for hematological malignancies

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized the treatment of relapsed/refractory (R/R) B-cell derived hematological malignancies, including acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM). CD19-targeted CAR-T cells yield complete remission (CR) rates of about 90% in R/R ALL and about 50% in R/R NHL, respectively, while BCMA-targeted CAR-T cells yield CR rate of about 50%−80% in R/R MM. Notably, the US Food and Drug Administration have approved six CAR-T cell products (tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, and ciltacabtagene autoleucel) for the treatment of R/R B-NHL, R/R ALL, and R/R MM. However, some patients might not respond to such therapy or quickly relapse after CAR-T cell infusion, which is likely due to the dysfunction and poor persistence of CAR-T cells or the modulation of specific antigen. Thus, alternative treatment strategies must be investigated. In addition, accumulating research have been conducted to discover novel target of antigens with therapeutic efficacy and utility for generating CAR-T cells against acute myeloid leukemia (AML). Safety during CAR-T cell treatment is another important issue, concerning complications such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), pancytopenia, and infection. Despite the fact that the majority of those adverse effects are minimal, the risk of a life-threatening situation still exists. Cautious treatment for severe adverse events requires a multidisciplinary approach with involvement of not only oncologists but also other internal medicine doctors to guarantee diagnosis and treatment in time. Therefore, it is of vital importance to understand the novel advances of CAR-T cell therapy. This special issue of ImmunoMedicine focuses on novel developments of CAR-T cell therapy for hematological malignancies. The review from Xiangmin Wang (https://doi.org/10.1002/imed.1030) summarized the non-BCMA targeted CAR-T cell therapies for MM. Novel targets including CS1, CD38, CD138, NKG2D, CD70, TACI, and etc. might have the potential to prevent the recurrence and enhance treatment efficiency. The review from Elaine Tan Su Yin (https://doi.org/10.1002/imed.1039) summarized the current breakthrough and future perspectives of CD20-targeted CAR-T cell therapy for hematologic malignancies, especially for R/R B-NHL. The review from Yue Huang (https://doi.org/10.1002/imed.1031) summarized the current achievement and potential AML-associated cell markers of CAR-T cell therapy in AML preclinical studies and clinical trials, and discusses the future directions of CAR-T cell therapy in patients with AML. The review from Yuanyuan Hao (https://doi.org/10.1002/imed.1029) summarized the effects of tumor-derived exosomes (TEXs) on the survival and functions of T cell subsets, as well as their clinical applications. This special issue also contains three original research articles reporting clinical trials of CAR-T cell therapy. Linghui Zhou et al. (https://doi.org/10.1002/imed.1036) reported the data of infections post CAR-T cell therapy, and found that it was a common complication in patients with hematological malignancies treated by CD19 CAR-T cells, which means clinicians should pay more attention to such situations. Kaiting Tang et al. (https://doi.org/10.1002/imed.1037) presented three cases of compassionate CAR-T cell therapy in advanced B-ALL, and shared their experiences on treating patients with high disease burden. Yue Huang et al. (https://doi.org/10.1002/imed.1032) reported an R/R ALL patient with high leukemia burden and central nervous system involvement, who responded to donor-derived HLA-matched allogeneic CAR-T treatment and achieved quick CR. We thank all the authors for their insightful contributions. This special issue provides an overview of the current knowledge regarding the most recent advances of CAR-T cell therapy, including CAR-T cell function, diverse applications, and complications with coping strategies. We hope that this issue will appeal to researchers as they explore fundamental and clinical aspects of CAR-T cell therapy.

A real-world study of infectious complications of venetoclax combined with decitabine or azacitidine in adult acute myeloid leukemia.

The purpose of this study was to identify the incidence, sites and main pathogens, and risk factors for infectious complications occurring in patients with adult acute myeloid leukemia (AML) during the first course of venetoclax combined with decitabine or azacitidine. A retrospective cohort analysis was performed of 81 patients with AML older than 14years who received the first cycle of venetoclax combined with a hypomethylating agent (HMA) between March 2018 and March 2021 at our institution. Infectious complications, if any, were documented. Among a total of 81 cases of AML, 59 (72.8%) patients occurred infections, including fever without an identifiable source (28.8%), clinically documented infections (40.7%), and microbiologically documented infections (30.5%). The most commonly isolated organism in culture was Candida albicans, followed by Klebsiella pneumonia, and Pseudomonas aeruginosa. The 4-week and 8-week mortality rates were 3.7% and 7.4%, respectively. In multivariate analysis, a high proportion of blasts in bone marrow, decreased hemoglobin level, and fever with or without a documented infection at baseline were significant independent risk factors for infectious complications. Compared with conventional chemotherapy, the incidence of infectious complications of venetoclax combined with decitabine or azacitidine significantly decreased. Pretreatment high leukemia burden and fever were independent risk factors for infections.

HLA‐matched allogeneic anti‐CD19 CAR‐T therapy in treating a relapsed/refractory acute lymphoblastic leukemia patient with high tumor burden

AbstractThe genetically engineered chimeric antigen receptor T cell (CAR‐T) therapy has shown remarkable clinical efficacy in the treatment of hematological malignancies. Nonetheless, it is difficult to harvest adequate autologous T cells to manufacture potent CAR‐T cell products in patients with high tumor burden and prior tumor‐reductive treatment. Here we reported a relapsed/refractory acute lymphoblastic leukemia patient with high leukemia burden and central nervous system (CNS) involvement. The patient responded to donor‐derived HLA‐matched allogeneic CAR‐T treatment, with the achievement of quick complete remission. And for the first time, we revealed the development of a cerebral CRS in situ after allogeneic CAR‐T therapy.

PREVENTION AND MANAGEMENT OF RELAPSE OF ACUTE LEUKEMIA AND MYELODYSPLASTIC SYNDROME AFTER ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION IN PEDIATRIC PATIENTS

The best way to manage acute leukemia relapse after HCT is to prevent it, buying time for GVL with immunomodulation and, if no GVHD between days +60 and + 90, prophylactic DLI can be indicate for very high or high risk patients. Short-term low dose of cyclosporine or methotrexate can add safety to pro-DLI, particularly after mismatched or unrelated transplantation. Maintenance with imatinib or dastinib, recommended for Ph-positive ALL, with sorafenib, for FLT3-ITD AML, or azacitidine, for myelodysplastic syndrome patients, can be effective in reducing relapse rates. However, target agent maintenance can add toxicity, depends on patient adherence and demands physician experience to know when is safe to start, how adjust the dose according individual tolerance after transplant and to detect undesirable drug interactions. The second step to avoid hematological relapse is preemptive approach guided by measurable residual disease or mixed chimerism. In patients off immunosuppression, chemotherapy followed by DLI is a useful strategy, and if no response, interferon alpha can be associated to enhance GVL. Target-specific agents can be start at this point either. After relapse, antigen-directed therapy with blinatumumab for CD19 ALL, inotuzumab for CD22 ALL are excellent options to induce MRD negativity and facilitate HCT. Disadvantages of new immunotherapies are: high incidence of VOD with inotuzumab and gemtuzumab; lower response in patients with high leukemia burden or concurrent extramedullary relapse; necessity of consolidation with HCT after a bridging therapy with BiTE and probably with CAR-T cell therapy also. It is important to realize that if remission after chemotherapy is associated with the development of GVHD, then there may be limited benefit (and possibly harm) in consolidating with any kind of cellular therapy. However, for patients who achieved remission without GVHD, either DLI or second transplant can be recommend. Further studies are necessary to determine at which point each strategy might yield the best results.

CD19 CAR-T Cell Therapy in Children with Relapsed and Refractory B-ALL

IntroductionCD19 CAR-T is a highly effective therapy among children with relapsed/refractory B-ALL. The optimal approach to delivery of this therapy and the best post-remission strategy remain to be established. We have tested in a prospective academic trial CD19 CAR-T cells manufactured at the point-of-care based on the automatic bioreactor platform. Based on the results of the first part of the trial, a toxicity mitigation strategy with conditional split dosing and refined post-remission therapy based on allogeneic HSCT was implemented. We report here the results of toxicity mitigation strategy approach as well as the long-term outcome with regard to the HSCT consolidation.Patients and methodsA total of 57 pts with relapsed/refractory B-ALL were screened, 54 pts were included in the trial, and additional 3 pts were eligible for compassionate use of CD19 CAR-T cell therapy. The CliniMACS Prodigy T-cell transduction process with lentiviral second generation CD19.4-1BB zeta vector (Lentigen, Miltenyi Biotec) was used for CAR-T manufacturing. All patients received prophylactic tocilizumab before CAR-T cells infusion.In the first part of trial 33 pts received lymphodepleting chemotherapy containing cyclophosphamide (750 mg/m2) and fludarabine (120 mg/m2), CAR-T cells was administered in dose-escalating regimen (0.1, 0.5, 1, and 3х10 6/kg b.w.).After the interim analysis, treatment scheme was modified to adapt the lymphodepletion therapy and the starting CAR-T dose to the leukemia burden. Twenty-four consecutive pts were divided into “low leukemia burden” (n=10) and “highleukemia burden” (n=14) groups, based on the threshold of 15% leukemic cells in the bone marrow.Patients with low leukemia burden received the same lymphodepletion chemotherapy as in the first part of the trial, and a single fixed dose of CAR-T cells at 1x10 6/kg b.w.Patients with high leukemia burden received an escalated lymphodepletion (fludarabine 120 mg/m 2, cyclophosphamide 750 mg/m 2, cytarabine 900 mg/m 2, etoposide 450 mg/m 2, dexamethasone 30 mg/m 2) and a divided dose of CAR-T. Day 0 CAR-T dose was set at 0.1 x10 6/kg. The second dose of 0.9x10 6/kg b.w. was administered between day 7 and day 14 if the following criteria were met: bone marrow leukemia burden by flow cytometry < 15% and CRS and/or ICANS grade within 3 previous days does not exceed grade 2.ResultsThirty patients included in the first part of the trial, were evaluable for response at day 28, and 27 (90%) of them had MRD-negative remission. Interim analysis showed that grade 3-5 CRS and neurotoxicity were associated exclusively with large leukemia burden (>15% blasts in the bone marrow) at the enrollment (p=0,003).With the risk-adapted strategy (part 2 of the trial), 8 patients (80%) with low leukemic burden achieved CR at day 28, and all patients (100%) with high leukemic burden achieved complete remission on day 28. In the high burden cohort 4 patients received the second CAR-T infusion, while the remaining 10 patients did not receive second dose due to either toxicity grade ³2 (4 pts), or persistence of >15% blast cells in bone marrow (6 pts). There were no cases of grade IV-V toxicity among patients with high leukemia burden, Table 1.For all patients the median follow-up for survivors was 490 days (287-1193), the cumulative incidence of relapse after initial response was 69.6%, median time to relapse was 250 days (58-696). HSCT during the CR was performed in 15 patients. The median time between first CAR-T infusion and HSCT was 96 days (41-292). Three patients (20%) relapsed early after HSCT (88, 114 and 155 days). Event-free and overall survival for the total cohort was 19.6% and 56.4%, respectively. Among the 34 pts, who did not receive HSCT in CR after CAR-T therapy, EFS and OS were 14.7% and 55.7%. Among the 15 pts, who received HSCT as consolidation, EFS and OS were 86.1% and 80%, p-value for HSCT vs no HSCT 0.125 (OS) and 0.0001 (EFS).ConclusionLow doses of non-cryopreserved CAR-T cells (0.1*10 6/kg), manufactured at the point-of-care, demonstrated high efficacy in patients with high initial leukemia burden, as well as favorable profile of life-threatening toxicity. The proposed risk-adapted strategy of CAR-T dosing allows to achieve high remission rate in all patients (with high and low leukemic mass). HSCT is likely to be a necessary modality for consolidation and long-term maintenance of remission after CAR-T therapy among a majority of patients with advanced B-ALL. [Display omitted] DisclosuresMaschan: Miltenyi Biotec: Speakers Bureau.

Favorable Outcomes of Single-Unit Cord Blood Transplantation As Salvaged Treatment for Advanced Hematologic Malignancies

Treatments of advanced hematologic malignancies are limited, and the effects are very poor even after allogeneic hematopoietic stem cell transplantation. Recent study showed that cord blood transplantation was benefit for patients with pretransplantation minimal residual disease compared to both HLA-matched and HLA-mismatched unrelated donor transplantation. The relapse rate was lowest in cord blood transplantation group. But the effect of cord blood transplantation in advanced hematologic malignancies as salvaged treatment still remains unknown. We retrospectively analyzed 55 consecutive patients with advanced hematologic malignancies who received single-unit unrelated cord transplantation at our centre between December 2013 and December 2016. Among the patients, 42with acute leukemia, 8 with myelodysplastic syndrome (MDS with refractory anemia with excess blasts [RAEB]), 4 with CML-BC and 1 with plasma cell leukemia. All of them were in non-remission before transplantation. The median age was 10 years (range, 2-43 years), and the median weight was 31 kg (range, 9-82kg).All the patients received intensified conditioning regimen and a combination of CsA and MMF as GVHD prophylaxis. The infused total nucleated cell (TNC) dose was 4.99 (range 2.17-13.9) × 10⁷/kg and CD34⁺ cell dose was 2.53 (range 0.64-7.45) × 10⁵/kg. Neutrophil and platelet engraftment occurred in 49 (89.1%) and 42 (76.4%) patients, respectively. The median times to neutrophil recovery and platelet recovery were 16 days (range, 12-32 days) and 37.5 days (range, 18-83 days), respectively. Forty patients (72.7%) experienced pre-engraftment syndrome (PES). The cumulative incidence rates of II-IV acute GVHD and chronic GVHD were 25.5% (95% CI, 14.8-37.6%) and 18.8% (95% CI, 8.6-32.1%), respectively. The median follow-up period for the surviving patients was 654 days (range, 208 to 720 days). The probability of 2- year OS and LFS were 53.9% and 46.8%, respectively, whereas the cumulative incidence of 2-year NRM and relapse rate were 34.5% (95% CI, 21.6-47.8%) and 20.7% (95% CI, 10.9-32.7%), respectively. To further define the importance of matching at the HLA C locus on our special patient cohort outcomes, we analyzed the effect of HLA C disparity on patient outcomes. Results showed that although OS, LFS and NRM were not significantly different between the two groups, the relapse rate was significantly higher after transplantations with matched at HLA C(n=12) than mismatched at HLA C(n=42)(58.5% VS 27.9%,P=0.0426).Since the favorable outcomes, our results showed that single-unit cord blood transplantation might be a feasible choice as salvaged treatment for advanced hematologic malignancies, even in patients with a high leukemia burden. Choosing the cord blood with mismatch at HLA C locus might reduce the relapse rate without increasing the NRM in our special patient cohort, but this metric still requires further evaluation with a larger cohort. DisclosuresNo relevant conflicts of interest to declare.

AML-317: Outcomes of Second Allogeneic Hematopoietic Cell Transplantation for Patients with Acute Myeloid Leukemia

Context Relapse after allogeneic hematopoietic cell transplantation (allo-HCT1) leads to poor survival in patients with acute myeloid leukemia (AML). A second allo-HCT can achieve durable remission in a subset of patients. Objective To assess outcomes of patients who underwent allo-HCT2 for relapsed AML and identify risk factors associated with survival. Design We reviewed consecutive medical records of 91 adult patients (age >18 years) with AML who underwent allo-HCT2 for relapsed disease at our institution from January 2000 until August 2019. Results At allo-HCT2, median age was 43 (range, 18–67), donor types were HLA-identical sibling 41%, HLA-matched-unrelated 35%, haploidentical 21%, and cord-blood 1%. Donors were different at allo-HCT2 in 53% patients. The majority of patients received reduced intensity conditioning (78%) and were in remission (58%) at allo-HCT2. The median remission duration after allo-HCT1 was 8.4 months (range, 1–70); median time between transplants 13.6 months (range, 2.6–73). The median follow-up of surviving patients after allo-HCT2 was 25 months (range, 4–70), with 32% alive at time of analysis; the most common cause of death was disease recurrence (87%). At 2-years, progression-free-survival, overall survival, and cumulative incidence of relapse were 30% (95% CI, 18–37%), 36% (95% CI, 26–47%) and 42% (95% CI, 32–54%), respectively. The cumulative incidence of grades II–IV acute and chronic GVHD were 35% and 18%, respectively. In multivariate analysis, persistent disease at time of allo-HCT1 and allo-HCT2, along with high leukemia burden at allo-HCT2 (defined as BM blast >10%) [HR,2.4; p=0.006], development of cGVHD after allo-HCT1 [HR,3.1; p 2) [HR, 2.1; p=0.01] were associated with inferior PFS after allo-HCT2. These factors also predicted for OS. A total of 19, 55, and 17 patients had neither, one, or more than one adverse factors, respectively. Two-year OS was 60%, 40%, and 0%, and PFS was 47%, 28%, and 0% in these 3 respective groups. Conclusions Long-term survival benefit is possible after allo-HCT2 in patients with AML who relapsed after allo-HCT1. This can be achieved in patients without cGVHD after allo-HCT1 who are fit and in remission at time of allo-HCT1, along with

Clinical Experience of CAR T Cell Immunotherapy for Relapsed and Refractory Infant ALL Demonstrates Feasibility and Favorable Responses

Background: The youngest patients referred for CAR T cell therapy are those with relapsed or refractory (R/R) KMT2A-rearranged infant B-ALL. Infants with relapsed ALL following Interfant-99 therapy have a dismal reported 3-yr OS of 20.9%, indicating the need for novel therapies. Smaller patient size, heavily pre-treated disease and high leukemia burden are often characteristics of this subgroup of patients that pose unique challenges to apheresis and manufacture of a T cell product. Additionally, reports of KMT2A-rearranged leukemia undergoing lineage switch following CD19-targeting pressure raises concern for an increased risk of myeloid leukemia relapses after B-lineage targeted CAR T cell therapy in this population. Here we report our experience using CAR T cell immunotherapy for patients with R/R infant ALL enrolled on clinical trials PLAT-02 (NCT02028455) and PLAT-05 (NCT03330691). Methods: PLAT-02 is a phase 1/2 trial of CD19-specific (FMC63scFv:IgG4hinge:CD28tm:4-1BB:ζ) CAR T cells. PLAT-05 is a phase 1 trial of CD19xCD22 dual specific CAR T cells, transduced with two separate lentiviral vectors to direct the co-expression of the CD19-specific CAR above and a CD22-specific CAR (m971scFv:IgG4hinge-CH2(L235D)-CH3-CD28tm:4-1BB:ζ). Eligible subjects on both studies have R/R B-ALL, an absolute lymphocyte count ≥100 cells/µL, and were at least 1 year of age. In addition, subjects on PLAT-02 were ≥ 10kg, and ≥ 8kg on PLAT-05. For cell manufacture, apheresis products were immuno-magnetically selected for CD4 and CD8 cells. Selected T cells were activated with anti-CD3/CD28 beads, transduced, and grown in culture with homeostatic cytokines to numbers suitable for clinical use. Infant ALL subjects received a range of 5x105 to 10x106 CAR+ T cells/kg following lymphodepleting chemotherapy. Disease response assessments were required at Day 21 and Day 63 following CAR T cell infusion. Adverse events were graded according to CTCAEv4, except CRS which was graded according to 2014 Lee criteria. Results: Eighteen subjects with R/R infant ALL have enrolled on PLAT-02 (n=14) or PLAT-05 (n=4), with a median age of 22.5 months at enrollment (range: 14.5 - 40.1 months). Of these, 2 (11.1%) had primary refractory disease, 8 (44.4%) were in 1st relapse, 7 (38.9%) were in 2nd relapse and 1 (5.6%) was in 3rd or greater relapse. Ten subjects (55.6%) had an M2 marrow or greater at enrollment prior to apheresis, and 9/18 had a history of hematopoietic cell transplant (HCT). The mean ALC was 1309 cells/µL (range 253-6944). Successful CAR T cell products were manufactured in 17/18 subjects, including in 9/9 subjects with no prior history of HCT. Of these, 16/17 subjects with available products were infused, with a median follow up of 26.9 months. One subject died of disease complications prior to CAR T cell infusion. Of the 16 treated subjects, 1 is pending disease and toxicity assessments. The maximum grade of CRS was 3 and occurred in two of 15 evaluable subjects (13%) and neurotoxicity was limited to a maximum grade of 2. Fourteen of 15 (93.3%) achieved an MRD negative complete remission (MRD-CR) by Day 21. Of the 14 subjects with an MRD-CR, 6 went on to HCT with 1 subsequent CD19 negative relapse. Of the 8 subjects who did not proceed to HCT, 1 developed lineage switch at one month following CAR T cells, and 1 died of infectious complications with aplasia. A "wait and watch" approach was taken for the remaining 6 subjects, and 2 developed CD19+ relapse. The incidence of lineage switch among the infant ALL group was 1/15 (6.7%). The estimated 1-year LFS was 66.7% and 1-year OS was 71.4%. Conclusion: This is the largest reported cohort to date of R/R infant B-ALL subjects treated with CAR T cell therapy. We report successful manufacture and administration of a CAR T cell product in the significant majority of infant subjects. Toxicity and MRD-CR rates are comparable to that of non-infant ALL subjects. In our experience, subjects with R/R infant ALL are not at increased risk for lineage switch relapse compared with the entire study populations following B-antigen targeting CAR T cell immunotherapy. Numbers in this report are too small to make definitive conclusions about the value of consolidative HCT. However, the LFS of this cohort is remarkably higher when compared with historical controls. Future work is focused on overcoming feasibility issues for the smallest of subjects, to enable a larger number of these cases to access CAR T cell therapy. Disclosures Pulsipher: Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wayne:AbbVie: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding; Servier: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. Jensen:Bluebird Bio: Research Funding; Juno Therapeutics, a Celgene Company: Research Funding. Gardner:Novartis: Honoraria.

Human Acute Leukemia Is Addicted to Branched-Chain Amino Acid Metabolism to Maintain Leukemia Stemness

Hematopoietic stem cells (HSCs) have capabilities to self-renew, maintaining an undifferentiated status, as well as to proliferate and mature into blood cells. Similarly, cancer stem cells (CSCs) self-renew and propagate to form cancer tissues. In human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), CSC-like populations that can reconstitute human leukemia in immuno-deficient mice have been found, and are called leukemic stem cells or leukemia-initiating cells (LICs). In order to obtain "cure" by eradicating LICs, it should be critical to understand molecular machineries as to how LICs self-renew and expand to exert their cancer stemness properties. Recent studies revealed that several specific metabolism pathways actively contribute to the maintenance of stemness in several types of stem cells including ES/iPS cells. However, little is known about the LICs-specific metabolic activity. To clarify metabolic features of human LICs, we comprehensively analyzed 116 cellular metabolites of human CD34+ normal hematopoietic stem progenitor cells (HSPCs) (n=10), CD34+ AML (n=30) and CD34+ ALL cells (n=18). The metabolome analysis revealed that CD34+ AML and ALL cells commonly contain extremely high levels of branched chain amino acids (BCAA) as compared to normal CD34+ HSPCs. AML and ALL cells, but not normal HSCsexpressed BCAA-metabolism related enzymes as well as BCAA-transporters at high levels, and actively transport BCAAs into cytoplasm. Enzymatic inhibition of BCAA metabolism induced apoptosis in LICs but not in normal HSCs. Furthermore, deprivation of BCAA from daily diet in mice xeno-transplanted with human LICs caused significant inhibition of their reconstitution activities in vivo. Serial transplantation experiments revealed that hCD34+AML/ALL cells from BCAA-free-dieted mice exhibited extremely impaired reconstitution of human AML/ALL in secondary recipients fed with conventional diet, but those from mice with control diet developed AML/ALL with a high leukemia burden. To clarify how BCAA metabolism pathway regulates the leukemia-initiating activity, the global gene expression of primary CD34+AML /ALL cells and cell lines including THP-1(AML), Kasumi-9(B-ALL), Jurkat cells (T-ALL) were compared before and after pharmacological BCAA-metabolism inhibition and BCAA-deprivation. Gene set enrichment analysis (GSEA) revealed BCAA metabolism inhibition induced the expression of ES-related PRC2 target genes, which should be suppressed in ES cells via H3K27me3 histone modification, and ChIP-Seq analysis confirmed the significantly decreased H3K27me3 level around thetranscription start site ofPRC2 target genes both in CD34+ AML and ALL cells. Furthermore, BCAA metabolism inhibition or BCAA-deprivation resulted in the down regulation of EZH2 and EED, critical components of PRC2, at mRNA and protein level in human AML/ALL cells. Thus, BCAA-metabolism activity is a common metabolic machinery to maintain PRC2 function through potentiating EZH2 and EED expression in AML/ALL. We also found that human AML/ALL cells, but not normal HSPCs are dependent on BCAA metabolism for maintaining energy production through TCA cycle activity, leading to the adequate supply of alpha ketoglutarate (α-KG), an intermediate metabolite of TCA cycle. Since BCAT1, a catalytic enzyme for BCAA, requires α-KGas a substrate, the active energy production driven by BCAA metabolism contributes to prevent α-KG exhaustion to maintain the high BCAA catabolism activity in human AML/ALL. Consistent with persistent α-KG supply through enhanced BCAA metabolism, primary CD34+AML/ALL cells, which highly express BCAT1 did not exhibit decreased cellular α-KG level as compared to normal HSPCs in our metabolome analysis. Thus, the energy production and regulation of PRC2 function driven by enhanced BCAA metabolism constitute a vicious molecular cycle to maintain the leukemia stemness through the maintaining α-KG level in human acute leukemia. In summary, the current study demonstrates that human LICs are addicted to the activated BCAA metabolism to maintain their leukemia stemness irrespective of their lineage origin, and that the BCAA metabolic pathway is a generic therapeutic target in human acute leukemias. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Local Manufacture of CD19 CAR-T Cells Using an Automated Closed-System: Robust Manufacturing and High Clinical Efficacy with Low Toxicities

Introduction CD19 CAR-T cell products were recently approved as therapy for B-lineage malignancies. We initiated an IIT trial where manufacture of CAR-T cells was performed locally using a unique CD19 CAR with potent anti-leukemic effects. Patients and methods A total of 37 pts with relapsed/refractory B-acute lymphoblastic leukemia (12 female, 25 male, median age 10 y) were screened, 27 pts were enrolled for a trial, 10 were eligible for compassionate use of CD19 CAR-T cell therapy. Sixteen patients had relapsed B-ALL after haploidentical HSCT, 19 pts refractory relapse, 2 induction failure, 13 patients had previous blinatumomab infusion. Eighteen patients had &gt;20% blast cells, median bone marrow leukemia burden for patients with full blown disease was 89%, 19 pts had minimal residual disease (MRD) &gt;0.1% in BM, 3 had skeletal involvement with multiple mass lesions, one had CNS involvement. The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells. The automated production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct and transduced with lentiviral vector expressing the CD19CAR gene (second generation CD19.4-1BB zeta with alternate transmembrane domain derived from the TNF superfamily) (Lentigen, Miltenyi Biotec company). T cells were expansion over 10 days in the presence of serum-free TexMACS GMP Medium supplemented with MACS GMP IL-7 and IL-15. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab at 8mg/kg before CAR-T cell infusion. Patients did not receive HSCT as consolidation after CAR-T therapy. Results Thirty-five manufacturing cycles were successful. Median transduction efficacy was 60% (20-80). Median expansion of T cells was x 46 (18-51). CD4:CD8 ratio in the final product was 0.73. The cell products were administered at a dose of 3*106/kg of CAR-T cells in 4 pts, 1*106/kg in 9 pts, 0.5*106/kg in 14 pts, 0.1*106/kg in 8 pts. Two patients received 0.1*106/kg of CAR-T cells produced from haploidentical donors. The cytokine release syndrome (CRS) occurred in 22 (59%) pts and was mostly mild and moderate: grade I - 15 pts, grade II- 4 pts, grade III - 2 pt, grade IV - 1 pt. CAR-T cell related encephalopathy occurred in 15 (40%). Grade I-II neurotoxicity developed in 10 pts, grade III - in 2 pt, grade IV - 1 pt, grade V - 2 pt. In one patient with grade V neurotoxicity concomitant K. pneumonia encephalitis was documented. Severe (grade 3-5) CRS and neurotoxicity were associated exclusively with large leukemia burden (&gt;20% in the bone marrow) at enrollment, p=0,002. Thirty-one patient was evaluable for response at day 28. Four pts had persistent leukemia. In 27 (87%) cases Flow MRD-negative remission was achieved. Disease relapse after initial response was registered in 9 (33%) cases (7 patients had CD19 negative, 2 had CD19 positive relapse). At the moment of reporting, 10 patients have died (3 due to sepsis, 1 due to brain edema, 1 due to brain edema and K. pneumonia encephalitis, 5 due to progression of disease or relapse). Twenty-seven pts are alive, 19 in complete remission with a median follow up of 223 days (41-516 days). Conclusion CliniMACS Prodigy TCT process is a robust CAR-T cell manufacturing platform that enables rapid and flexible provision of CAR-T cells to patients in need. Significant toxicity of CD19 CAR-T cells was associated exclusively with high leukemia burden at enrollment. In the absence of HSCT consolidation relapse rate exceeds 30%. Disclosures Schneider: Lentigen Technology, A Miltenyi Biotec Company: Employment. Preussner:Miltenyi Biotec: Employment. Rauser:Miltenyi Biotec: Employment. Orentas:Lentigen Technology Inc., a Miltenyi Biotec Company: . Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Maschan:Miltenyi Biotec: Other: lecture fee.

Chemotherapy plus DLI for relapse after haploidentical HSCT: the biological characteristics of relapse influences clinical outcomes of acute leukemia patients.

This study investigated the prognostic factors in patients (n = 89) who experienced relapse and received chemotherapy plus donor leukocyte infusion (Chemo-DLI) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Patients with early relapse (< 6 vs. > 6 months after haplo-HSCT), higher bone marrow blast count before chemo-DLI (> 20% vs. 5-19%), and without chronic graft-versus-host disease (cGVHD) after chemo-DLI had a higher rate of progressive disease (PD) and worse progression-free survival (PFS) and overall survival (OS). In multivariate analysis, non-cGVHD after Chemo-DLI and high blast count predicted a higher risk of PD and poorer PFS, and non-cGVHD after Chemo-DLI and early relapse predicted poorer OS. The patients were stratified into three groups according to these three risk factors. Patients with all three risk factors (n = 14) had the highest PD rate and poorest survival compared with those with one or two risk factors (n = 63) or no risk factors (n = 12). Thus, early relapse, high leukemia burden before Chemo-DLI, and non-cGVHD after Chemo-DLI can predict outcomes in patients who have experienced relapse and received Chemo-DLI after haplo-HSCT. New therapeutic strategies should be identified for these patients.

Efficacy and Safety of CD19 Chimeric Antigen Receptor (CAR) T Cell Therapy for B-Cell Acute Lymphocytic Leukemia (B-cell ALL) in a Large Cohort Including Patients with Extramedullary Disease(EMD), High Leukemia Burden, BCR-ABL (+) Mutation,TP53 Mutation, and Post-Transplant Relapse

Introduction: CD19 CAR-T therapy is effective for B-cell ALL. However, whether patients (pts) with EMD, High Leukemia Burden (HLB), TP53 mutation, BCR-ABL (+) mutation, or post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) relapse can benefit from the treatment remains to be investigated. Here we show the efficacy and safety of CD 19 CAR-T therapy for these subgroups.Patients and Methods: From April 2017 to April 2018 with a median follow-up of 172 (27-325) days, 83 pts with refractory or relapsed (R/R) B-cell ALL were enrolled (US NIH Clinical#:NCT03173417). The cohort included 17 with EMD, of whom 11 had central nervous system leukemia (CNSL), 13 with HLB (≥70% blast in bone marrow), 10 with BCR-ABL+, 8 with TP53 mutation, and 11 with relapsed ALL after allo-HSCT. The median age of the cohort was 10 (2-61) years. Patients' peripheral blood mononuclear cells were collected, and CD3+ T cells were separated and stimulated with CD3/CD28 dynabeads and cultured in X-VIVO 15 media with interleukin-2. T cells were transduced with a lentivirus vector encoding anti-CD19-CD3ζ either with a CD28 (21 pts) or a 4-1BB (62 pts) co-stimulatory domain, and cultured (8-10 days) until there were sufficient cells. All pts received a conditioning regimen of IV fludarabine (25 mg/m2/day) and cyclophosphamide (250 mg/m2/day) for 3 days before a single infusion of CAR-T cells with a median dose of 1x105 (0.1-10x105) cells/kg.Results: On day 30 after CAR-T, 76/83 (91.6%) pts achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 70/76 (92.1%) achieved minimal residual disease (MRD)-negative CR. One-year overall survival (OS) was 76.5% (Fig. 1) and relapse-free survival (RFS) was 62.6%. Grade 0-II cytokine release syndrome (CRS) incidence was 82%, and grade III-IV, 16%. There was one CAR-T-related death. Severe CNS reaction occurred in 10 pts (12%). CR was achieved in 82.4% (14/17) of pts with EMD, including 11 pts with CNSL, who had a CR rate of 82% (9/11). Incidence of severe CRS (esp. CNS toxicity) in CNSL group was not significantly increased. No significant difference in RFS was seen between pts with or without EMD (54.9% vs. 66.3%, p=0.6). However, OS (45.1% vs 88.3%, p=0.0003) favored those without EMD. 13 pts with HLB achieved 76.9% CR/CRi. Severe CRS occurred more frequently in HLB pts vs. low leukemia burden (LLB) pts (46.2% vs. 7.1%, p = 0.001). The risk was controllable.No significant difference in CR/CRi (76.9% vs. 90.4, p=0.07) or RFS (61.1% vs 70%, p=0.55) was seen between the HLB and LLB pts, but the OS in HLB pts was inferior (54.9% vs. 80.8%, p=0.03). There was no difference in CR/OS/RFS between pts with or without the BCR-ABL mutation, including pts with 3 cases of chronic myeloid leukemia (CML) transformed into B-ALL. 88% (7/8) pts with TP53 mutation achieved CR/CRi, but only 3/7 remained leukemia free. The OS (45.7% vs. 79.5%, p=0.026) and RFS (0% vs. 67.9%, p=0.0023) were much lower for pts with TP53 mutation. 11 pts with relapsed ALL after allo-HSCT achieved 91% CR/CRi after CAR-T. The OS was similar to those treated with CAR-T before allo-HSCT (77.8% vs. 77.1%, p=0.85), with 3 pts remaining in CR by 8-12 months after CAR-T. No significant GVHD developed in these pts. Of 76 pts with D30 CR, 57 underwent subsequent allo-HSCT in a median time of 2 months post CR. The remaining 19 (including 10 post-HSCT CAR-T-treated pts) did not undergo transplant. The 1-year OS/RFS (Fig. 2) of the CAR-T bridge to allo-HSCT group was better than that of the non-transplant group (OS 87.5% vs. 63.4%, p=0.013; RFS 87.4% vs. 7.5%, p=0.001). There was a trend towards improved in OS (83.7% vs. 50%, p=0.68) and RFS (64.9% vs. 30%, p=0.18) in the MRD(-)CR group vs. the MRD(+)CR group. While the OS of CR pts was significantly better than that of CRi pts (100% vs. 73.4%, p=0.038), the RFS was not yet statistically significant (75% vs. 56.4%, p=0.25). The median recurrence time in the group without additional allo-HCST was 100 days.Conclusion: High CR rate was achieved from CAR-T for R/R B-ALL including pts with high-risk features such as EMD, HLB, BCR-ABL+, TP53+,and relapse after allo-HSCT. CRS was manageable. CNSL is not a contraindication for CAR-T therapy. An early and high relapse rate was observed in TP53+ group. Overall, RFS was superior for pts bridging to allo-HSCT after CAR-T treatment than for those receiving CAR-T- treatment only. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Therapy of 4s Chimeric Antigen Receptor T Cells Achieves Long-Term Disease-Free Survival with No Severe CRS or Cres in Patients with Relapsed and Refractory Acute Lymphoblastic Leukemia

Background: CD19 targeting chimeric antigen receptor T cells(CART19) therapy have shown great therapeutic potential in relapsed and refractory B cell acute lymphoblastic leukemia (ALL), but associated with risk of life-threatening adverse effects as severe cytokine release syndrome (sCRS) and CAR-T-cell-related encephalopathy syndrome (CRES).It's been reported that high leukemia burden before CART therapy, and high does infused CART cells are associated with severity of CRS and CRES. To decrease the risk of severe adverse effect, we applied integrated therapeutic strategy of using fourth generation CART cells , reducing leukemia blasts burden in bone marrow, and decreasing the dose of infused CART cells (www.clinicaltrials.gov; #NCT03407859 and #NCT03125577 ).Methods: Between May 23, 2016 and July 2, 2018,the trial enrolled 20 patients (pts) who were exhausted with all available treatment options, life expectancy >2 months,CD19-positive and diagnosis of B lineage ALL.T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentivector CAR with the following intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR). In vitro amplication of CART was not performed. In pts with bone marrow blasts exceeding 50.0%, VDCP or similar chemotherapy was given to reduce the tumor burden, and then received FC conditioning regimen (FLU 30mg/m2, d1-3; CTX 300mg/m2, d1-3), while FC regimen was directly carried out in pts with bone marrow blasts less than 50.0%.In this trial, Pts received single CD19-targeted CARTs or multi-CARTs targeting CD19 and an additional antigen of CD22 or CD123.The level of CAR-T cells and cytokines in peripheral blood, as well as tumor burden was measured after infusion.Results: 20 pts were enrolled and infused with CAR-T cells. The median age is 37.5 (16-67) years old. Of these pts, 5 had prior HSCT and 14 had adverse genetic abnormalities, including 4 pts (20%) who were Philadelphia chromosome-positive(Ph+). All pts were previously treated with 2-22 courses of cytotoxic chemotherapy regimens. The overall objective response rate was 85%(17/20), and the complete response(CR) rate was 80%(16/20). The complete remission rate of 12 pts receiving single CD19-targeted CART therapy was 83 %(10/12), while 33%(4/12) of them had disease relapse at 6 months after infusion. Of the 7 people who received multi-CARTs infusion, 71%(5/7) achieved complete remission, with relapse rate of 29%(2/7) at 6 months after infusion. 3 pts who relapsed post transplantation received combination therapy of anti-CD19 CART and anti-CD123 CART, and all achieved minimal residual disease-negative CR within 1 month after CART infusion, 2 of whom maintained disease-free survival for 7 months and 11 months to date, respectively. Among the 7 people who underwent HSCT after achieving CR, 6 of them maintained disease-free survival for 3 months to 9 months. At a median follow-up of 115.5 days (ranging from14 to 384), the median overall survival was 269 days and the median event-free survival was 232 days. During treatment and follow-up, the most common adverse events were grade 1-2 cytokine release syndrome (CRS), with an incidence of 55%.No grade 3 or higher CRS was observed.12 pts were infused with a dose equal to or exceeding 5*10^5/kg, 10 of whom had CRS response. While only 2 of the 8 pts who received the infusion dose of less than 5*10^5/kg had CRS reaction and both of them were grade 1 CRS, suggesting low CAR-T cell doses decrease the risk of CRS (P=0.009). Interestingly, the objective response rate did not differ significantly between the low dose and high dose group.Conclusions: Based on fourth generation CART system, a therapeutic strategy of low tumor burden and low CART infusion dose shows a safer profiling while remaining potent efficacy against leukemia. DisclosuresNo relevant conflicts of interest to declare.

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.

In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell-mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.

CD19 CAR-T Cells Administered after High-Dose Cyclophosphamide Chemotherapy Induce Rapid Remissions of Chemotherapy-Refractory Acute B-Cell Leukemia with High Tumor Burden

Introduction Primary disease relapse remains the leading causeof death inadult acute lymphocytic leukemia (ALL) patients despite the availability of high-dose chemotherapy, targeted drugs and allogeneic hematopoietic stem cell transplantation. Relapsed or refractoryALL especially with high leukemia burden has very poor prognosis. Chimeric antigen receptor-modified T cells (CAR-T) targeting CD19 may overcome many limitations of conventional therapies and induce remission in patients with refractory disease.Patients and Methods Refractory or relapsed CD19+ ALL adult patients with more than 60% blast cells in the bone marrow at the time of evaluation were enrolled. Eligible patients underwent leukapheresis, and T cells were transduced with a Lenti-virus encoding a CAR construct composed of anti-CD19 scFV linked to 4-1BB and CD3¦Æ signaling domains. To enhance the activity of the transferred CAR-T cells, all patients received lymphodepleting chemotherapy with fludarabine (30mg/m2/day Days -4, -3, -2) and cyclophosphamide (1000mg/m2/day Days -3, -2) followed with 1x10E6 to 10x10E6 CD19CAR-T cells/kg 2 days later. The primary objective of the study was to evaluate the safety and anti-tumor activity of CD19CAR-T cells in ALL. Peripheral blood and bone marrow samples were collected for immunophenotypic, cytokine, and molecular studies at pre-specified times after CD19CART cell infusion.Results 2 patients were enrolled. The patient characteristics are listed in Table 1. All the 2 patients had once achieved complete remission, with MRD<0.01%. Patient 1 suffered from high-risk ALL and achieved CR after 3 courses of induction chemotherapy. Because of lung infection and liver disfunction, he could not receive the 4th chemotherapy. At the time of enrollment on our protocol, he relapsed with 66% blast cells in bone marrow. When the lymphodepleting chemotherapy ended, percentage of his marrow blast cells was 45%. 5x10E6/Kg CAR-T cells were infused. Except for B-cell depletion (Figure 1A, B), the most prominent toxicities included fevers, fatigue and chills (Table 1). The peak blood and bone marrow levels of CAR-T cells were 41.8% and 28.3% of CD3+ lymphocytes in day 8 respectively (Figure 1A). Detailed cytokine analysis showed marked increases of IL-6 and IFN-¦Ã (Figure 1C). Bone marrow blasts were 0.047% and <0.01% in day 8 and day 12 respectively. Patient 2 exhibited persistent chemotherapy refractory disease with 74% blast cells in the bone marrow following salvage chemotherapy. When the lymphodepleting chemotherapy ended, percentage of his marrow blast cells was 77%. 10x10E6/Kg CAR-T cells were infused. This patient suffered severe CRS with marked increases of IL-6 and IFN-¦Ã (Figure 1C). Administered with the IL6-receptor antagonist Tocilizumab she recovered from CRS soon. The peak blood and bone marrow levels of CAR-T cells were 83.65% and 75.34% of CD3+ lymphocytes in day 8 respectively (Figure 1B). Bone marrow blasts were <0.01% on day 8.Conclusion Our study revealed that CD19CAR-T cells could express robust expansion and anti-leukemia activity in relapsed and refractory ALL patients with high tumor burden. All the 2 patients achieved CR with negative MRD within 12 days after CART cell infusion. No uncontrollable clinical toxicities were manifested. The robust anti-leukemia activity of CD19CAR-T and high-dose CTX chemotherapy might contribute to the good therapeutic efficacy in our clinical trial. More patients will be enrolled in this trial and results will be reported in ASH meeting this year.Table 1Patient characteristicsPatientAge/sexNumber of prior therapiesLymphodepleting chemotherapyMarrow blastToxicitiesPre-CARPost-CAR151y/M3Flu(30mg/m2/d -4 to -2) and CTX(1g/m2/d -3, -2)66%<0.01%Fever Fatigue Chills Anorexia229y/F22Flu(30mg/m2/day Days -4 to -2) and CTX(1g/m2/day Days -3, -2)73%<0.01%Fever Fatigue Vomit Hypoxemia Hypotension [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Halpo-Identical Hematopoietic Stem Cell Transplantation for High-Risk Leukemia

Objective To retrospectively evaluate the results of allogeneic halpo-identical hematopoietic stem cell transplantation for high-risk leukemia.Methods From June 2012 to January 2015, total 60 patients with high-risk leukemia were enrolled, including 18 cases of ALL, 37cases of AML and 5 case of CML-BP. Including criterions: 1) ≥CR1; 2) relapse within 6 months after remisson. The average leukemia burden was 53% in bone marrow. All patients received HLA haplo-identical stem cells transplantation from parent or sibling donors. Myeloablative conditioning regimens consist of 7cases of BuCy, 26 cases of TBI/FLAG, 15 cases of TBI/Cy, and 12 cases of FLAG that followed by reduced-intensified BUCY. All patients received cyclosporine A, MMF and methotrexate for GVHD prophylaxis. Analyzed outcomes were hematological engraftment, incidence of acute and chronic GVHD, incidence of relapse, and nonrelapse mortality (NRM), Overall survival (OS) and Disease-free survival (DFS).Results The median mononuclear cells and CD34+ for transfusion were 9.08(7.02-24.4)*108/Kg and 3.42(0.8-12.1)*106/Kg. All 60 patients achieved stable engraftment. The median time of ANC≥0.5*10^9/L was 16 (8-23) days. And for platelet ≥20*10^9/L, the median was 22 (8-150) days. 38 patients developed acute GVHD, the accumulative incidence of aGVHD was 66.4%, the accumulative incidence of II-IV grade aGVHD was 35%, and the accumulative incidence of III-IV grade aGVHD was 15%. 26 patients developed cGVHD (12 patients extensive, 14 patients limited), the accumulative incidence of cGVHD was 88.2% and for extensive type, the accumulative incidence was 67.4%. The accumulative incidence of CMV infection was 54.1%, and the accumulative incidence of EBV infection was 16.3%. 10 patients developed virus cystitis. The number of Bacterial and fungal infected patients were 51 and 27, respectively. The median follow-up time post transplantation was 11(1-36) months, 14 patients relapsed and the accumulative incidence of relapse was 27%. For AML, ALL and CML-BP patients, the accumulative incidence of relapse were 26.6%, 34.8% and 0%, respectively. The median follow-up time post transplantation was 11months, 21 patients died and the main causes were relapse (11 cases), infection (5 cases), cGVHD (2 cases) and diffuse alveolar hemorrhage (3 cases). Among 60 patients, 39 patients survived. The one-year and two-year accumulative incidences of OS were 61.8% and 49.5%, respectively. The one-year and two-year accumulative incidences of DFS were 53.8% and 47.8%, respectively. For AML, ALL and CML-BP patients, the two-year accumulative incidence were 52.6%, 34.4% and 66.7%, respectively. The non-relapse mortality was 10. The one-year and two-year accumulative incidences of NRM were 19.4% and 28.4%, respectively.Conclusion Our clinical results have shown that the salvaged HSCT is a promising modality for treatment of high-risk AL with high leukemia burden. DisclosuresNo relevant conflicts of interest to declare.

Antileukemic potency of CD19-specific T cells against chemoresistant pediatric acute lymphoblastic leukemia

Adoptive therapy with chimeric antigen receptor (CAR) T cells (CART cells) has exhibited great promise in clinical trials, with efficient response correlated with CART-cell expansion and persistence. Despite extensive clinical use, the mechanisms regulating CART-cell expansion and persistence have not been completely elucidated. We have examined the antileukemia potency of CART cells targeting CD19 antigen using second-generation CAR containing a CD28 co-stimulatory domain cloned into piggyBac-transposon vector and patient-derived chemoresistant pediatric acute lymphoblastic leukemia samples. In the presence of large numbers of target cells characteristic of patients with high leukemia burden, excessive proliferation of CART cells leads to differentiation into short-lived effector cells. Transient leukemia growth delay was induced by CART-cell infusion in mice xenografted with rapidly growing CD19+ acute lymphoblastic leukemia cells and was followed by rapid CART-cell extinction. Conditioning with the hypomethylating agent 5-aza-2'-deoxycytidine-activating caspase 3 and promotion of apoptosis in leukemia cells maximized the effect of CART cells and improved CART-cell persistence. These data suggest that the clinical use of 5-aza-2'-deoxycytidine before CART cells could be considered. Coculture of leukemia cells with bone marrow stroma cells reduced target cell loss, suggesting that leukemia cell mobilization into circulation may help to remove the protective effect of bone marrow stroma and increase the efficacy of CART-cell therapy.

Reversal of in situ T-cell exhaustion during effective human antileukemia responses to donor lymphocyte infusion

AbstractIncreasing evidence across malignancies suggests that infiltrating T cells at the site of disease are crucial to tumor control. We hypothesized that marrow-infiltrating immune populations play a critical role in response to donor lymphocyte infusion (DLI), an established and potentially curative immune therapy whose precise mechanism remains unknown. We therefore analyzed marrow-infiltrating immune populations in 29 patients (22 responders, 7 nonresponders) with relapsed chronic myelogenous leukemia who received CD4+ DLI in the pre–tyrosine kinase inhibitor era. Immunohistochemical analysis of pretreatment marrow revealed that the presence of >4% marrow-infiltrating CD8+ (but not CD4+) T cells predicted DLI response, even in the setting of high leukemia burden. Furthermore, mRNA expression profiling of marrow-infiltrating T cells of a subset of responders compared with nonresponders revealed enrichment of T-cell exhaustion–specific genes in pretreatment T cells of DLI responders and significant downregulation of gene components in the same pathway in responders in conjunction with clinical response. Our data demonstrate that response to DLI is associated with quantity of preexisting marrow CD8+ T cells and local reversal of T-cell exhaustion. Our studies implicate T-cell exhaustion as a therapeutic target of DLI and support the potential use of novel anti-PD1/PDL1 agents in lieu of DLI.

Salvaged Cord Blood Transplantation For 19 Patients With Progressive Hematologic Malignancies

Few clinical studies have investigated the role of salvaged unrelated cord blood transplantation (CBT) for progressive hematologic malignancies. The aim of this report is to identify the potential benefits of unrelated CBT in progressive hematologic malignancies.19 consecutive patients with progressive myeloid and lymphoid malignancies who received salvaged CBT following myeloablative conditioning regimens (12 TBI/CY/Ara-C, 6 Bu / CY / Ara-C, and 1 Bu /CY) from July 2005 to December 2012 were analyzed retrospectively. Of the 19 patients, 6 suffered from acute myeloid leukemia(AML), 5 acute lymphoid leukemia(ALL),：1 acute mixed lineage leukemia (AMLL), 1 myelodysplastic syndrome-refractory anemia with excess blasts(MDS-RAEB), 2 acute myeloid leukemia transformed from myelodysplastic syndrome, and 4 lymphoma, all of them in non-remission (NR) before transplantation. Median age of them were 13（range 6-32）years and median body weight were 45 (range 18–73 ) kg. All patients received 1 CBT unit ≤2 loci HLA-mismatched with the recipient. Infused TNC was 4.07（range 2.76-6.02）×107/kg and CD34+ stem cell 2.08（range 0.99-8.65）×105/kg. All patients were engrafted with neutrophil exceeded 0.5×109/L on median day +17（range 14-37d）and plt counts of＞20×109/L on median day +35 (range 17-70d). 10 patients (52.6%) experienced pre-engraftment syndrome (PES) and 3 (15.8%) patients progressed to acute GVHD. The incidence of Ⅱ-Ⅳ aGVHD and cGVHD were 10.5% and 21.1%. With a median follow up of 10(range 1-58) months，10 cases survived and 2 relapsed．The estimated 2 year overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM) rate was 45.6%, 34.2% and 39.8%. Salvaged CBT might be a promising modality for treatment of progressive hematologic malignancies, even with high leukemia burden. Disclosures:No relevant conflicts of interest to declare.

Potentiation of the Effects of Nilotinib by Combination with Plerixafor in a Mouse Model of BCR-ABL-Positive Residual Disease

Abstract 2737 Background:It has been shown that a small, residual pool of leukemic CD34+ progenitor cells can survive in the marrow microenvironment of chronic myeloid leukemia (CML) patients after years of kinase inhibitor treatment. Bone marrow stroma contributes to the expansion and proliferation of both transformed as well as normal hematopoietic cells, and has been implicated in the long-term survival of leukemic cells. We previously demonstrated the stromal protection of leukemic cells from the anti-proliferative effects of nilotinib, and identified stromal-derived viability factors, including IL-6 and GM-CSF, as possibly mediating stromal protection of tyrosine kinase inhibitor-treated leukemic cells (Weisberg et al. Mol Cancer Ther 2008;7:1121). Additionally, we have found high leukemia burden in the tissues of nilotinib-treated mice that have significant sources of hematopoiesis-promoting stroma, suggesting that significant reservoirs for tumor growth may be tissues that are able to support normal and malignant hematopoietic stem cell development. These studies revealed a leukemia distribution pattern consistent with that observed in imatinib or nilotinib-treated patients.One strategy for overriding stromal-mediated chemoresistance is the use of inhibitors of the stroma-derived factor (SDF-1a) receptor, CXCR4, which mediates the migration of hematopoietic cells to the bone marrow and plays a key role in leukemic cell-stromal cell interactions. Studies have shown small-molecule CXCR4 inhibitors, such as plerixafor, to be effective in mobilizing hematopoietic cells from bone marrow and enhancing chemotherapy- and tyrosine kinase inhibitor-induced apoptosis of bone marrow stroma-protected leukemic cells in vitro and in vivo. Thus, imatinib has been shown to up-regulate CXCR4, which induces CML migration to the bone marrow microenvironment and leads to stroma-mediated chemoresistance of quiescent CML progenitor cells. Methodology:Utilizing a functional in vivo assay system that allows monitoring of the growth of progressive disease, as well as baseline level (“residual” disease) resulting from treatment with a moderate-to-high dose of nilotinib, we investigated the ability of stem cell mobilization to enhance the efficacy of nilotinib by suppressing leukemia recurrence following nilotinib treatment. Specifically, 32D.p210-luc+ cells were injected into the tail-vein of mice, which were then imaged 10 days later to determine baseline bioluminescence and quantify tumor-burden. Mice were subsequently treated by oral gavage for 10 days with nilotinib (75 mg/kg qd) and reimaged (“Induction Phase” of treatment); mice were at this stage considered to have minimal residual disease, with reduced tumor burden > 2 logs. Mice were then divided into four treatment groups (“Consolidation Phase” of treatment addressing minimum residual disease) with similar mean bioluminescence (n=8 or 9 per group): Group 1: Vehicle (PEG300 po), Group 2: plerixafor (5 mg/kg sq qd), Group 3: nilotinib (75mg/kg po qd), and Group 4: Combination (plerixafor+nilotinib). Results:Nilotinib was highly efficacious in reducing disease burden in leukemia-engrafted mice. However, with continued treatment, animals developed resistance to nilotinib with increasing disease burden despite continued therapy. Although plerixafor had no single-agent activity, combination with nilotinib significantly delayed time to relapse, and significantly prolonged survival when compared to nilotinib monotherapy (p<0.0001). Since plerixafor had no monotherapeutic efficacy, there results demonstrate that plerixafor, at a well-tolerated dose, acts synergistically with nilotinib to suppress the growth of 32D.p210 leukemia. Conclusion:We demonstrated the ability of plerixafor to delay the onset of recurring BCR-ABL-positive disease in mice carrying an extremely low tumor burden following treatment with a moderate-to-high dose of nilotinib. These results support the idea of using stem cell mobilization in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease. Disclosures:Manley:Novartis Pharma AG: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Griffin:Novartis Pharmaceuticals: Consultancy, Research Funding.