Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment for acute myeloid leukemia (AML). However, most patients experience relapse after allo-HSCT, with a poor prognosis, and treatment options are limited. The lack of an ideal targetable antigen is a major obstacle for treating patients with relapsed AML. CD38 is known to be expressed on most AML and myeloma cells, and its lack of expression on hematopoietic stem cells (HSCs) renders it a potential therapeutic target for relapsed AML. To investigate the clinical therapeutic efficacy and safety of CD38-targeted chimeric antigen receptor T (CAR-T-38) cells, we enrolled 6 AML patients who experienced relapse post-allo-HSCT (clinicaltrials.gov: NCT04351022). Prior to CAR-T-38 treatment, the blasts in the bone marrow of these patients exhibited a median of 95% (92–99%) CD38 positivity. Four weeks after the initial infusion of CAR-T-38 cells, four of six (66.7%) patients achieved complete remission (CR) or CR with incomplete count recovery (CRi); the median CR or CRi time was 191 (range 117–261) days. The cumulative relapse rate at 6 months was 50%. The median overall survival (OS) and leukemia-free survival (LFS) times were 7.9 and 6.4 months, respectively. One case relapsed 117 days after the first CAR-T-38 cell infusion, with remission achieved after the second CAR-T-38 cell infusion. All six patients experienced clinically manageable side effects. In addition, multiparameter flow cytometry (FCM) revealed that CAR-T-38 cells eliminated CD38 positive blasts without off-target effects on monocytes and lymphocytes. Although this prospective study has a limited number of cases and a relatively short follow-up time, our preliminary data highlight the clinical utility and safety of CAR-T-38 cell therapy in treating relapsed AML post-allo-HSCT.
The purpose of the present work was to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo acute myelogenous leukemia by sequencing the entire coding region. EZH2 mutations were identified in 13/714 (1.8%) of AML patients were found to be more common in males (P = 0.033). The presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (P<0.0001) and -7/del(7q) (P = 0.025). There were no differences in the incidence of mutation in 13 genes, ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. No difference in complete remission, event-free survival, or overall survival was observed between patients with and without EZH2 mutation (P>0.05). Overall, these results showed EZH2 mutation in de novo acute myeloid leukemia as a recurrent genetic abnormality to be associated with lower blast percentage in BM and -7/del(7q).
Background
T cells expressing a chimeric antigen receptor (CAR) engineered to target CD19 can treat leukemia effectively but also increase the risk of complications such as cytokine release syndrome (CRS) and CAR T cell related encephalopathy (CRES) driven by interleukin-6 (IL-6). Here, we investigated whether IL-6 knockdown in CART-19 cells can reduce IL-6 secretion from monocytes, which may reduce the risk of adverse events.
Methods
Supernatants from cocultures of regular CART-19 cells and B lymphoma cells were added to monocytes in vitro, and the IL-6 levels in monocyte supernatants were measured 24 h later. IL-6 expression was knocked down in regular CART-19 cells by adding a short hairpin RNA (shRNA) (termed ssCART-19) expression cassette specific for IL-6 to the conventional CAR vector. Transduction efficiency and cell proliferation were measured by flow cytometry, and cytotoxicity was measured by evaluating the release of lactate dehydrogenase into the medium. Gene expression was assessed by qRT-PCR and RNA sequencing. A xenograft leukemia mouse model was established by injecting NOD/SCID/γc-/- mice with luciferase-expressing B lymphoma cells, and then the animals were treated with regular CART-19 cells or ssCART-19. Tumor growth was assessed by bioluminescence imaging.
Results
Both recombinant IL-6 and CART-19 derived IL-6 significantly triggered IL-6 release by monocytes. IL-6 knockdown in ssCART-19 cells dramatically reduced IL-6 release from monocytes in vitro stduy. In vivo study further demonstrated that the mice bearing Raji cells treated with ssCART-19 cells showed significant lower IL-6 levels in serum than those treated with regular CART-19 cells, but comparable anti-tumor efficacy between the animal groups.
Conclusion
CAR T-derived IL-6 is one of the most important initiators to amplify release of IL-6 from monocytes that further drive sCRS development. IL-6 knockdown in ssCART-19 cells by shRNA technology provide a promising strategy to improve the safety of CAR T cell therapy.
Poor graft function (PGF) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Current treatment strategies include the use of growth factors, CD34+-selected stem cell boost, mesenchymal stem cell transfusion, and second allo-HSCT, but these treatments are not effective in all patients. Eltrombopag, an oral thrombopoietin receptor agonist, which showed promising results in severe aplasia anemia, may be an alternative choice for PGF patients. Therefore, we treated 12 patients who responded poorly to standard treatments for secondary PGF after allo-HSCT with eltrombopag. The median duration was 116 (35–1000) days from transplantation to PGF diagnosis and 59 (30–180) days from PGF diagnosis to eltrombopag treatment. Eltrombopag was started at a dose of 25 mg/d for 3 days and then increased to 50 or 75 mg/d. Median treatment duration was 8 (2–23) weeks. Ten patients (83.3%) responded to the treatment: 8 achieved complete response (CR), and the remaining 2 achieved partial response. In the 10 responding subjects, median platelet count was 18 (5–27) × 109/L vs 74 (30–117) × 109/L prior to and after treatment. Neutrophil count was 0.51 (0.28–0.69) × 109/L vs 1.84 (0.78–4.90) × 109/L. Hemoglobin was 88 (63–123) vs 101 (78–134) g/L. In the 8 patients who achieved CR, the time from eltrombopag initiation to achieving CR was 29 (10–49) days; the response lasted until the last follow-up in all 8 CR subjects (10–18 months). The 12-month overall survival rate was 83.3%. There was no treatment-related mortality and no evidence of cataract, thrombosis, or any other grade 3/4 toxicities.
To investigate clinical characteristics and outcomes of transplantation in AML patients with FLT3-ITD/DNMT3A double mutation, we retrospectively analyzed 206 Chinese patients with AML after Sanger sequencing. Our analysis showed that AML patients with FLT3-ITD and DNMT3A R882 mutations had a higher white blood cell count and a lower complete remission (CR) rate after first induction chemotherapy. All 206 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) in status of CR. These results indicate that AML patients with FLT3-ITD and DNMT3A R882 double mutation show a higher 2-year cumulative incidence of relapse (CIR), lower 2-year overall survival (OS) rate, and lower 2-year leukocyte-free survival (LFS) after allo-HSCT. The univariate and multivariate analyses confirmed that disease status prior to transplantation, FLT3-ITD, FLT3-ITD, and DNMT3A R882 double mutation were independent factors for poor prognosis after allo-HSCT. In summary, the present cohort study demonstrated that FLT3-ITD and DNMT3A R882 double mutation predicts poor prognosis in Chinese AML patients receiving chemotherapy or allo-HSCT treatment.
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