ALBERT

Description: Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Background: Chemoresistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). Chimeric antigen receptor T cells (CAR-T) therapy, as a novel immunotherapy modality, demonstrates impressive efficacy and durable remissions in relapsed/refractory(R/R) B-cell malignancies including DLBCL. However, CAR-T therapy is also associated with potentially fatal toxicities, including cytokine release syndrome (CRS) and neurological toxicities which limit the clinical application of CAR-T therapy in R/R DLBCL with high tumor burden. How to improve the prognosis of this population is urgently underway. Here, we conducted a trial testing efficacy and toxicities of CAR-T therapy in R/R lymphoma patients (This study is registered at www.clinicaltrials.gov as NCT03196830). Methods:Human T cells were collected from autologous/allogenous peripheral blood mononuclear cells (PBMC) . CD3+T cells were separated from PBMS of patients or donors by degradable anti-CD3 magnetic microbeads followed by 24h stimulation with CD3 and CD28 monoclonal antibody (5ug/ml). Then CD3+T cells were transduced with lentivirus particles incorporated with a humanized CAR construct targeting CD19, CD20 and CD22 and further expanded for 7 to 10 days in vitro. The transfection efficacy, ratio of CD4+ versus CD8+, antitumor activities, pathogen detection as well as cytokines releasing of CAR-T cells were evaluated before infusion to patients. In this ongoing trial, 14 patients were diagnosed as R/R DLBCL with high tumor burden. Two debulking conditioning regimens including intensive combined chemotherapy or radiation (40 gray in 20 fractions) were carried out before CAR-T therapy followed by sequential FC regimen (cyclophosphamide 300mg/m2 and fludarabine 30mg/m2 X 3d) for lymphodepletion chemotherapy. Two days after FC regimen, autologous/allogenous CAR-T cells targeting CD19 and CD20 or CD22 provided by the unicar-therapy bio-medicine technology co.(Shanghai, China) at average dose of 1-2×107 cells for each target per kilogram(kg) were infused within 3 days. Results: 14 R/R DLBCL patients with high tumor burden were enrolled in the study. Patients ranged from 39 to 66 years of age had received two to four prior lines of therapy. Out of the 14 patients, 7 patients received intensive second-line chemotherapy (median age, 46 years) and 7 received radiotherapy (median age, 63years) to debulking tumor after leukapheresis and during CAR-T cells preparation. All Adverse Events occurring within 30 days of CAR-T cells infusion were graded and reported for the 14(100%) treated patients. All patients experienced CAR-T-related CRS of any grade with severe CRS (grade 3/4/5) reported in all patients of chemotherapy cohort experienced and mild CRS (grade 1/2) reported in all patients of radiation cohort. 4 patients (57.1%) in chemotherapy cohort manifested with neurological toxicity while no patient in radiation cohort manifested with neurological toxicity. Comparable hematological toxicity as well as non-hematological and non-neurological toxicity were observed in two groups. Four of seven (57.1%) patients achieved an objective response after CAR-T infusion, with three of seven (42.9%) achieving a CR in chemotherapy cohort, while seven of seven (100%) achieved an objective response after CAR-T therapy, with four of seven (57.1%) achieving a CR in radiation cohort. Conclusions:Our clinical trial indicates that compared to intensive second-line chemotherapy, radiation is a better approach to enhancing efficacy and decreasing toxicity of CAR-T therapy in R/R DLBCL patients with high tumor burden. Radiotherapy in combination with CAR-T cell infusion may be the optimal alternative treatment model of R/R DLBCL with high tumor burden. Disclosures No relevant conflicts of interest to declare.

Description: Background: Chemo-resistance is a core challenge in successful treatment of diffuse large B cell lymphoma (DLBCL). It has been previously reported that DNA hypermethylation and histone deacetylation are two major epigenetic modifications contributing to chemo-resistance in multiple tumors. Moreover, combination of histone deacetylase inhibitors (HDI) and DNA methyltransferase inhibitors (DNMTI) resulted in synergistic anti-lymphoma effect toward refractory and/or relapsed (R/R) DLBCL cells in vitro and in vivo xenografts. R-GemOx (rituximab, gemcitabine, and oxaliplatin), as a first-line chemotherapy regimen for elderly primary DLBCL patients or a salvage chemotherapy regimen for R/R DLBCL patients not candidates for high-dose therapy, has shown high activity with a relative low toxicity profile. Herein, we therefore aimed to assess the efficacy, safety, and feasibility of the dual epigenetic agents plus R-GemOx regimen (CD-R-GemOx) as a salvage treatment in R/R DLBCL patients. Methods: 13 R/R DLBCL patients including 8 males and 5 females, diagnosed with R/R DLBCL on the basis of the 2008 World Health Organization guidelines, who had failed from previous salvage treatment were exposed to dual epigenetic agents (Chidamide 30mg biw and Decitabine 10mg/m2 qd d1-d5) and sequential R-GemOx(rituximab 375 mg/m² qd d6; gemcitabine 1 g/m² d7,d14; and oxaliplatin 100 mg/m² d8) for salvage chemotherapy. Median age of these patients was 56 (35-67) years old. Most (10/13) patients have advanced Ann Arbor stages. The cycle was repeated every 4 weeks. Clinical efficacy were assessed after two cycles. Results: All thirteen (100%) patients achieved disease control response. Ten (76.9%) patients achieved an overall response at the end of the treatment, with three (23.1%) achieving a complete response. Common grade 3-4 adverse events were haematological toxicities (thrombocytopenia in ten [76.9%] patients, anaemia in six [46.2%], and neutropenia in twelve [92.3%]) and gastrointestinal complications (nausea in four [30.8%] patients, vomiting in one [7.7%], diarrhoea in six [46.2%] and mucositis in six [46.2%]). Besides, 6/13 (46.2%) cases manifested with pyrexia; 3/13(23.8%) cases manifested with increase of ALT and hyperbilirubinemia. All above toxicities were reversible and recovered within 1 month after chemotherapy. No neurological toxicities and treatment-related deaths were observed. Conclusions: Our study indicates that combination of dual epigenetic agents and R-GemOx is a safe and promising salvage approach to managing R/R DLBCL patients and may serve as an optimal salvage treatment for bridging autologous transplantation. Disclosures No relevant conflicts of interest to declare.