We studied the acute graft-versus-host disease (GVHD) after humanized anti-CD19-CAR T therapy in relapsed B-acute lymphoblastic leukemia (ALL) patients after allogeneic hematopoietic stem cell transplant (allo-HSCT). Fifteen BALL patients were enrolled in our study. Thirteen patients (86.67%) achieved a complete response (CR) or CR with incomplete count recovery. The donor chimerism of the 13 patients reached 99.86 ± 0.21%. The development of aGVHD was observed in 10 patients (66.67%). Six patients developed grade I-II of aGVHD, while the other four patients developed grade III-IV of aGVHD. The notable adverse events were grade 1-2 cytokine release syndrome (CRS) in 10 patients and grade 3-4 CRS in five patients. Two patients died of infection, while another patient died of sudden cardiac arrest. The anti-CD19-CAR T cells were not eliminated in peripheral blood when the patients developed aGVHD. However, we did not observe their expansion peaks again in the process of aGVHD. During the aGVHD, the peaks of IL-6 and TNF-a were correlated with aGVHD levels. By May 31, 2020, the rates of leukemia-free survival (LFS) and overall survival (OS) at 180 days were 53.846 and 61.638%, respectively. All the patients who survived to date experienced aGVHD after humanized anti-CD19-CAR T cell therapy. Trial registration: The patients were enrolled in clinical trials of ChiCTR-ONN-16009862 and ChiCTR1800019622.

The molecular defects which lead to multistep incidences of human T-cell leukemia have yet to be identified. The DNA-binding protein Helios (known as IKZF2), a member of the Ikaros family of Krüppel-like zinc-finger proteins, functions pivotally in T-cell differentiation and activation. In this study, we identify three novel short Helios splice variants which are T-cell leukemic specific, and demonstrate their dominant-negative function. We then test the cellular localization of distinct Helios isoforms, as well as their capability to form heterodimer with Ikaros, and the association with complexes comprising histone deacetylase (HDAC). In addition, the ectopic expression of T-cell leukemic Helios isoforms interferes with T-cell proliferation and apoptosis. The gene expression profiling and pathway analysis indicated the enrichment of signaling pathways essential for gene expression, translation, cell cycle checkpoint, and response to DNA damage stimulus. These data indicate the molecular function of Helios to be involved in the leukemogenesis and phenotype of T-cell leukemia, and also reveal Helios deregulation as a novel marker for T-cell leukemia.

4247 Iron overload is caused by multiple blood transfusion and excess gastrointestinal absorption, leading to most of the mortality and morbidity associated with anemia diseases (i.e. aplastic anemia, myelodysplastic syndromes, thalassemia and myelofibrosis). It can cause tissue damage and ultimately dysfunction of visceral organs (mainly in the heart, liver, and endocrine glands). Nevertheless, it is unknown whether iron overload affects the hematopoiesis of bone marrow (BM). In recent years, a number of papers reported that iron chelation therapy could enhance the erythropoiesis and even reduce the cytopenia in iron overload anemia (i.e. myelodysplastic syndromes and myelofibrosis). As it is well known that iron overload could increase the production of reactive oxygen species (ROS) in the tissue, and also ROS could affect the hematopoiesis of BM, we hypothesized that iron overload could increase the ROS of BM, and then result in the deficient hematopoiesis. To confirm this hypothesis, we studied the relationship between ROS and the hematopoiesis of iron overload BM. The intracellular concentration of ROS in cells were analyzed by a flow cytometer after incubated with 50 μM (final concentration) 2′–7′-dichlorofluorescin diacetate (DCF; Sigma) for 10 min at 37°C in a humidified atmosphere of 5% CO2 in air. We found that the ROS in the hematopoietic cells of BM from 26 patients with iron overload (16 cases with myelodysplastic syndromes and 10 cases with myelofibrosis) was much higher than that of normal control (p<0.05), which in accord with the lower production of hematopoietic colony forming (CFU-E, BFU-E, CFU-GM and CFU-mix) in patients with iron overload. After the patients received iron chelation therapy with deferasirox for more than 6 months, the ROS decreased distinctly and the hematopoietic colony forming recovered subsequently. As a result, 20 cases (76.9%) reduced blood transfusion, 12 cases (46.2%) increased neutrophil counting, 7 cases (26.9%) increased platelet counting, and 4 cases (15.4%) reduced the cytopenia. In vitro, we set up an iron overload model by adding ferric citrate into the mononuclear cells from normal BM and culturing for 24 hours, and confirmed the model by the detecting of labile plasma iron. We found that the concentration of ROS in iron overload BM was increased for 3.5 folds in erythroid cells, 2.1 folds in granulocytes and 1.3 folds in CD34+ cells, respectively. Again, the hematopoietic colony forming in iron overload BM was much lower than that of normal control. Notely, the number of CD34+ cells was decreased about 45% in iron overload BM. When treated the hematopoietic cells from iron overload BM with deferasirox or/and antioxidants (N-acetyl-L-cysteine), the hematopoietic colony forming was recovered in accord with the decreasing of ROS. Furthermore, the apoptosis of hematopoietic cells from iron overload BM was much higher than that of normal control detecting with PI-Annexin V double-stainning method. And also, detecting with western-blot method, we found the ATM kinase was down-regulated and its downstream factor, p38MAPK was notably actived in the hematopoietic cells from iron overload BM, which indicated that ROS related signal pathway was involved in the deficient hematopoiesis of iron overload BM. In sum, our study confirmed that increased intracellular concentration of ROS mediated the deficient hematopoiesis of iron overload BM for the first time. Further study on the mechanism would be helpful to find the target (for example, antoxidant treatment) and improve the efficacy on the treatment of ineffective hematopoiesis in patients with iron overload. Disclosures: No relevant conflicts of interest to declare.

Leukemia is a leading cause of cancer-related mortality in children worldwide, and multidrug-resistance (MDR) is a main reason for tumor chemotherapy failure. The present study investigated the effects of ADR following incubation with cytokine-induced killer (CIK) cells on reversing MDR in K562/ADR cells. Mononuclear cells were isolated from the peripheral blood of healthy individuals and cultured in vitro in the presence of a combination of cytokines to generate CIK for K562/ADR cell treatment. A decreased level of P-glycoprotein expression and glutathione (GSH), an increased intracellular Rh-123 content, decreased mRNA and protein expression levels of MDR gene 1, MDR-associated protein 1, GSH S-transferase-π, B-cell lymphoma 2 and Survivin, and the decreased phosphorylation of AKT and the transcriptional activity of nuclear factor-κB and activator protein 1 were detected following ADR treatment in CIK co-cultured K562/ADR cells. Additionally, the level of ADR sensitivity and the apoptosis rate were increased in the CIK co-cultured K562/ADR cells. These results indicate that pre-treatment with CIK could reverse the MDR of K562/ADR cells, and that patients would be most likely to benefit from the combination of chemotherapy and CIK therapy.

Despite advances in the treatment of acute myeloid leukemia (AML) in recent years, the outcome of elderly AML patients with antecedent hematological disorders remains unsatisfactory. The present study describes a case of complete remission in an elderly patient with AML transformed from chronic myelomonocytic leukemia (CMML) and the treatment of the case with decitabine in combination with cytarabine, aclarubicin and granulocyte colony-stimulating factor (CAG). A 70-year-old male was admitted with fever, pruritus and weakness that had been apparent for two weeks, and a two-year history of monocytosis (22.5–27.0%). Further examinations revealed a hemoglobin level of 106 g/l, a white blood cell count of 39.52×109/l, a platelet count of 81×109/l, Y chromosome loss and uniparental disomy on chromosomes 4q, 2q and 19p. The patient was diagnosed with AML transformed from CMML, with cytogenetic anomalies. A combination regimen of decitabine and CAG was administered. Subsequent to one cycle, the patient achieved complete remission. The patient was then followed up with three courses of the same regimen and achieved clinical remission, with no evidence of AML relapse. The present study suggests that a combination of low-dose decitabine and CAG may offer a novel and potentially effective treatment regimen for elderly AML patients.