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Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications–driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Li et al delineate a novel technique for assessing measurable residual disease (MRD) by the assessment of isolated leukemia stem cells (LSCs). They report that assessment of MRD in LSCs provides a better prediction of outcome than standard multiparameter flow cytometry.

Introduction Allogeneic haematopoietic stem cell transplantation (allo-HSCT) has been demonstrated to be the most effective therapy for various malignant as well as nonmalignant haematological diseases. The wide use of allo-HSCT has inevitably led to a variety of complications after transplantation, with bleeding complications such as disseminated intravascular coagulation (DIC). DIC accounts for a significant proportion of life-threatening bleeding cases occurring after allo-HSCT. However, information on markers for early identification remains limited, and no predictive tools for DIC after allo-HSCT are available. This research aimed to identify the risk factors for DIC after allo-HSCT and establish prediction models to predict the occurrence of DIC after allo-HSCT. Methods The definition of DIC was based on the International Society of Thrombosis and Hemostasis (ISTH) scoring system. Overall, 197 patients with DIC after allo-HSCT at Peking University People's Hospital and other 7 centers in China from January 2010 to June 2021 were retrospectively identified. Each patient was randomly matched to 3 controls based on the time of allo-HSCT (±3 months) and length of follow-up (±6 months). A lasso regression model was used for data dimension reduction, feature selection, and risk factor building. Multivariable logistic regression analysis was used to develop the prediction model. We incorporated the clinical risk factors, and this was presented with a nomogram. The performance of the nomogram was assessed with respect to its calibration, discrimination, and clinical usefulness. Internal and external validation was assessed. Various machine learning models were further used to perform machine learning modeling by attempting to complete the data sample classification task, including XGBClassifier, LogisticRegression, MLPClassifier, RandomForestClassifier, and AdaBoostClassifier. Results A total of 7280 patients received allo-HSCT from January 2010 to June 2021, and DIC occurred in 197 of these patients (incidence of 2.7%). The derivation cohort included 120 DIC patients received allo-HSCT and 360 patients received allo-HSCT from Peking University People's Hospital, and the validation cohort included the remaining 77 patients received allo-HSCT and 231 patients received allo-HSCT from the other 7 centers. The median time for DIC events was 99.0 (IQR, 46.8-220) days after allo-HSCT. The overall survival of patients with DIC was significantly reduced (P ＜ 0.0001). By Lasso regression, the 10 variables with the highest importance were found to be prothrombin time activity (PTA), shock, C-reactive protein, internationalization normalized ratio, bacterial infection, oxygenation, fibrinogen, blood creatinine, white blood cell count, and acute respiratory distress syndrome (from highest to lowest). In the multivariate analysis, the independent risk factors for DIC included PTA, bacterial infection and shock (P Conclusions Risk factors for DIC after allo-HSCT were identified, and a nomogram model and various machine learning models were established to predict the occurrence of DIC after allo-HSCT. Combined, these can help recognize high-risk patients and provide timely treatment. In the future, we will further refine the prognostic model utilizing nationwide multicenter data and conduct prospective clinical trials to reduce the incidence of DIC after allo-HSCT and improve the prognosis. Disclosures No relevant conflicts of interest to declare.

Introduction Growing evidence has implicated gut microbiota in the pathogenesis of immune thrombocytopenia (ITP). In a previous research study, we found dysbiosis in the phylogenetic composition and function of gut microbiome in ITP and that corticosteroid treatment may have a strong effect on gut microbiota [Sci China Life Sci, 2020]. Corticosteroids have been widely used in the initial treatment of newly diagnosed ITP patients, but most adult patients relapse upon cessation of steroid treatment. Patients on agents in subsequent therapy may improve at any time, but which patients improve and when is unpredictable. The gut microbiome has been increasingly used in the assessment and prediction of immunomodulatory therapy in autoimmune diseases and cellular immunotherapy in cancers. Here, we provide evidence that gut microbiota and function signatures can be used to predict immune thrombocytopenia patients at high risk of relapse/resistance after corticosteroid treatment and to identify patients that are more likely to benefit from TPO-RAs in subsequent therapy. Methods Seventy-five fecal samples from 60 patients with newly diagnosed ITP (60 specimens before corticosteroid therapy and 15 specimens after corticosteroid therapy) and 41 samples from persistent/chronic ITP before and after treatment with TPO-RAs, including eltrombopag and avatrombopag were collected for deep shotgun metagenomic sequencing. To identify the microbial biomarkers related to relapse/resistance after corticosteroid treatment, we constructed a random forest classifier using machine learning to determine the risk of relapse/resistance of a training cohort of 30 patients from baseline samples and validated the classifier for 30 patients. Patients with persistent/chronic ITP were divided into responders and nonresponders according to their response to TPO-RA treatment in subsequent therapy. After identifying the microbial species and functional biomarkers related to the response to TPO-RA therapy, a random forest classifier was constructed using a training set of 20 patients and validated using a validation set of 21 patients. Results We used a metagenomic sequencing technique to investigate the differences among gut microbiota associated with relapse within 3 months of corticosteroid treatment. We observed that the diversity and composition of the microbial community in ITP patients after corticosteroid therapy (Post-C) changed significantly from the baseline (Pre-C), whereas the gut microbiota of the remission group was similar to that of the HC group, which implies that a shift in the gut microbiome could represent a return to homeostasis. To identify the microbial biomarkers related to early relapse after corticosteroid treatment, the Pre-C samples were divided into a remission group and a resistant/relapse group according to the response to corticosteroid therapy within 3 months. Nine significant associations with the microbial species and function were identified between the remission and resistant/relapse groups. A risk index built from this panel of microbes and functional pathways was used to differentiate remission from resistant/relapsed patients based on the baseline characteristics. The receiver operating characteristic (ROC) curve demonstrated that the risk index was a strong predictor of treatment response, with an area under the curve (AUC) of 0.87. Furthermore, to predict the response to TPO-RAs in subsequent therapy, the baseline gut microbiomes of responders and nonresponders before TPO-RA treatment were compared. Patients who responded to treatment exhibited an increase in Ruminococcaceae, Clostridiaceae and Bacteroides compared to nonresponders, with elevated abundance of the phosphotransferase system, tyrosine metabolism and secondary bile acid biosynthesis pathways according to KEGG analysis. Our prediction model based on the gut microbiome for TPO-RA response was robust across the cohorts and showed 89.5% and 79.2% prediction accuracy for persistent/chronic ITP patients in the training and validation sets, respectively. Conclusions The gut microbiome and function signatures based on machine learning analysis are novel potential biomarkers for predicting resistance/relapse after corticosteroid treatment and response to TPO-RAs, which may have important manifestations in the clinical. Disclosures No relevant conflicts of interest to declare.

The aim of this study is to investigate the prevalence and risk factors of anti-human leukocyte antigen (HLA) antibodies in haploidentical candidates. This study was completed at Peking University People's Hospital, Beijing China. We performed a prospective analysis of patients with hematological diseases concerning the prevalence and risk factors of anti-HLA antibodies. Patients were enrolled between July 2015 - December 2019. Serum was collected for PRAs test within 1 month before haploidentical transplantation. The risk factors, such as age, sex, total transfusion, red blood cell (RBC) transfusion, platelet (PLT) transfusion, pregnancy, disease duration and diagnosis were collected. Univariate and multivariate logistic regression analyses were performed to evaluate the risk factors of anti-HLA antibodies. Six hundred and eighty (17.9%) patients were positive for panel reactive antibodies (PRA)-class I, 360 (9.5%) for class II, 768 (20.2%) class I or II, and 272 (7.1%) positive for class I and II both. Multivariate analysis indicated that female was related to higher risk of having PRAs for class I (P = 0.011), class I or II (P = 0.009), anti-HLA-A (P = 0.015), anti-HLA-DP (P = 0.048) and also for having higher mean fluorescence intensity (MFI) (2000 or more) of PRAs in class I (P = 0.020) and class I or II (P = 0.005). Compared to patients with myelodysplastic syndrome (MDS), patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), aplastic anemia (AA) had a lower incidence for PRAs in class I, class II, class I or II, class I and II, anti-HLA-A. anti-HLA-B, anti-HLA-C, anti-HLA-DQ, anti-HLA-DR, anti-HLA-DP (Table 1). Prior pregnancy was a risk factor for PRAs (P < 0.001), and no previous pregnancy group having lower MFI of PRAs in class I (P = 0.001) and class I or II (P = 0.004). PLT transfusion (more than 4 times) rleted with a higher prevalence of PRAs (P < 0.001), and also had a higher MFI of PRAs in class II (P < 0.001), class I and II (P < 0.001). Patients with RBC transfusion (more than 3 times) had a higher prevalence of PRAs in class I (P = 0.001), class II (P = 0.029), class I or II (P < 0.001), anti-HLA-A (P = 0.001), anti-HLA-B (P < 0.001), anti-HLA-C (P = 0.007), anti-HLA-DQ (P < 0.001) and anti-HLA-DR (P = 0.011). In addition, diseases duration (8 months or more) was also associated with higher MFI of PRAs in class I (P = 0.023) and class I or II (P = 0.004). Subgroup analysis showed that 11.7% of pediatric patients were positive for PRAs in class I; 19.2% of adults, 17.9% of elder patients; 12.4% of males; 26.1% of females; 21.0% of patients with AML; 10.5% of patients with acute lymphoblastic leukemia (ALL); 18.9% of patients with AA; 30.3% of patients with MDS; 16.6% of patients with other hematological diseases. The positive rate of class II PRAs in children was 4.3%; 11.1% for adults; 9.5% for elder patients; 5.5% for males; 15.4% for females; 11.4% for patients with AML; 5.2% for patients with ALL; 10.3% for patients with AA; 17.2% for patients with MDS; 6.6% of patients with other hematological diseases. Multivariate analysis showed that, in children, PLT transfusion and diagnosis were the two main risk factors of PRAs in class I and class II (P < 0.001, P = 0.017). In adults, diagnosis (P = 0.003), transfusion (P < 0.001) and pregnancy (P < 0.001) were the three main factors associated with PRAs in class I and transfusion (P < 0.001) and pregnancy (P < 0.001) were the two main factors associated with PRAs in class II. In males, PLT transfusion (P < 0.001) and diagnosis (P < 0.001) were the two main factors associated with PRAs in class I and class II. In ALL subgroup, gender (P = 0.026, P = 0.048), pregnancy (P < 0.001) and transfusion (P < 0.001) were the three main factors associated with PRAs in class I and II. In AA subgroup, gender (P = 0.004) and PLT transfusion (P < 0.001) were risk factors for class I PRAs, pregnancy (P = 0.008) and PLT transfusion (P = 0.003) were risk factors for class II PRAs. In elder patients, females, AML, MDS and other diseases subgroup, transfusion and pregnancy were the two main factors associated with PRAs in class I and class II. Our results indicated that female sex, diagnosis, pregnancy, transfusion, disease duration were independent risk factors of anti-HLA antibodies in haploidentical allograft candidates, which provided evidence for best haploidentical donor selection. The risk factors of anti-HLA antibodies were different among total patients and those of cases in different subgroups. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Immune thrombocytopenia (ITP) is an acquired, organ-specific, autoimmune disease and one of the most common bleeding disorders seriously endangering human health. Glucocorticoids and intravenous immunoglobulin are first-line treatments recommended by guidelines for patients with ITP. However, approximately 50%-85% of patients relapse during the first year of treatment. In addition, long-term use of glucocorticoids increases the risk for dose- and time-dependent glucocorticoid-related complications and serious side effects. Therefore, in-depth studies investigating new solutions for the first-line treatment of ITP are urgently needed. Tacrolimus is a calcineurin inhibitor, which forms a complex by binding to FK506-binding protein. It is currently widely used in the prevention of graft-versus-host disease for organ transplantation as well as for the treatment of autoimmune diseases. In addition to recent retrospective studies and case reports demonstrating its effectiveness in ITP, tacrolimus has been shown to improve anti-platelet antibody-mediated thrombocytopenia in mice, suggesting it may be a potential treatment for ITP. The aim of this study was to compare two first-line treatment options for ITP-a standard glucocorticoid-only regimen versus tacrolimus in combination with a standard glucocorticoid regimen-to determine which could help patients achieve stable platelet counts faster and experience a longer duration of remission. Methods This open-label, randomized, phase 2 trial, enrolled adult ITP patients from seven different tertiary medical centers in China. Elderly patients had confirmed, newly diagnosed, treatment-naive ITP, platelet counts 50×10 9/L without any additional ITP-modifying therapy at the 6-month follow-up. Key secondary endpoints included initial response by day 14 (OR, platelet count ≥30×10 9/L and at least 2-fold increase in baseline platelet count and absence of bleeding; and CR, platelet count ≥ 100×10 9/L), duration of response, bleeding scores, and adverse events (AEs). This trial was registered with ClinicalTrials.gov (NCT04747080). Results Total 140 patients newly diagnosed with ITP were randomly assigned to either the tacrolimus plus HD-DXM (n=72) or HD-DXM monotherapy (n=68) groups. At the 6-month follow-up, the proportion of patients exhibiting SR was significantly higher in the tacrolimus plus HD-DXM group than in the HD-DXM monotherapy group (65.3% vs 42.6%, p= 0.007). Of the 140 patients with ITP (males accounted for 48.6%), the mean age was 32.8 years, the mean platelet count was 16.7×10 9/L. The combination group exhibited a higher 14-day early remission rate than the monotherapy group (76.4% vs 55.9%, P=0.001). Significantly fewer treatment failures occurred in patients randomly assigned to the combination group(19.4% vs 38.2%, P=0.0014). During the follow-up period, fewer patients in the combination group experienced relapse than in the monotherapy group; the median time to relapse was 77 days (Tacrolimus+HD-DXM) vs 36 days (HD-DXM). The combination group exhibited a lower proportion of bleeding events and a lower bleeding score. The incidence of serious AEs, rescue therapy, and treatment side effects were similar between the two groups, and treatment was well tolerated by all patients, with no grade 4 AEs or treatment-related deaths reported. There was no statistically significant difference in the incidence of treatment-related AEs between the two groups. Conclusions Low-dose tacrolimus plus HD-DXM was an effective and safe treatment for ITP as first-line therapy and elicited a sustained prolonged response in adults. This therapy may be a new treatment option for adult patients with ITP. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: It includes information or discussion of off-label drug use of tacrilimus.

Introduction Cysteine and glycine-rich protein 2 (CSRP2) is gaining increasing attention as a therapeutic target due to its high expression in acute leukemias and its involvement in the development of cancer. However, whether it can be used as a reliable marker for minimal residual disease (MRD) remains unknown. Methods A total of 155 adult B-cell acute lymphoblastic leukemia (ALL) patients who received at least two cycles of consolidation chemotherapy were enrolled. Their leukemia-associated aberrant immune phenotypes (LAIPs) and CSRP2 transcript levels at the second consolidation chemotherapy (CON2) were detected by flow cytometry (FCM) and real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR). According to our published work, 1.80% and 0.01% were set as the positive threshold of CSRP2 transcript level and the FCM test for diagnosis, respectively. Pearson correlation coefficient was calculated to describe the relationship between the CSRP2 transcript level and the FCM test. Competing risk model and Cox proportional hazard regression model were conducted to estimated associations between the CSRP2 transcript level at CON2 and the prognosis. Results The median CSRP2 transcript level of all 155 patients was 0.2% (0.02%-108.17%). Among them, 108 patients were negative for both FCM and CSRP2, and 8 patients were positive for both FCM and CSRP2. The coincidence rate was 74.84%. There was a significant positive correlation between FCM and CSRP2 (r=0.73, 95% CI 0.64-0.79; P Patients were divided into a high CSRP2 group (N=17) and a low CSRP2 group (N=138) based on the transcript level of 1.00% settled by the ROC curve. Nine of 17 patients with high transcript level of CSRP2 suffered from leukemia relapse during the follow-up. Moreover, among the nine relapse patients, three patients had positive CSRP2 and negative FCM at CON2. In univariate analysis, patients with high CSRP2 transcript level showed a significantly lower 5-year leukemia-free survival (LFS) (33.0% vs. 48.6%, P =0.014) and 5-year survival (OS) (28.6% vs. 72.5%, P Conclusions Our study suggested that patients with a high CSRP2 transcript level at CON2 had poor survival and was an independent risk factor for relapse. The transcript level of CSPR2 at CON2 may be a valuable marker to complement the MRD assessment system and improve the number of evaluable patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction As a neurological complication following haploidentical haematopoietic stem cell transplantation (haplo-HSCT), immune-mediated demyelinating diseases (IIDDs) of the central nervous system (CNS) are rare, but they seriously affect a patient's quality of life (J Neurooncol, 2012). Although several reports have demonstrated that IIDDs have a high mortality rate and a poor prognosis (J Neurooncol, 2012; Neurology 2013), a method to predict the outcome of CNS IIDDs after haplo-HSCT is not currently available. Here, we reported the largest research on CNS IIDDs post haplo-HSCT, and we developed and validated a prognostic model for predicting the outcome of CNS IIDDs after haplo-HSCT. Methods We retrospectively evaluated 184 consecutive CNS IIDD patients who had undergone haplo-HSCT at a single center between 2008 and 2019. The derivation cohort included 124 patients receiving haplo-HSCT from 2014 to 2019, and the validation cohort included 60 patients receiving haplo-HSCT from 2008 to 2013. The diagnosis of CNS IIDDs was based on the clinical manifestations and exclusion of other aetiologies, including infection, neurotoxicity, metabolic encephalopathy, ischaemic demyelinating disorders, and tumor infiltration. The final prognostic model selection was performed by backward stepwise logistic regression using the Akaike information criterion. The final model was internally and externally validated using the bootstrap method with 1000 repetitions. We assessed the prognostic model performance by evaluating the discrimination [area under the curve (AUC)], calibration (calibration plot), and net benefit [decision curve analysis (DCA)]. Results In total, 184 of 4532 patients (4.1%) were diagnosed with CNS IIDDs after transplantation. Among them, 120 patients had MS, 53 patients had NMO, 7 patients had ADEM, 3 patients had Schilder's disease, and 1 patient had Marburg disease. Grades II to IV acute graft-versus-host disease (aGVHD) (p CNS IIDDs were significantly associated with higher mortality and a poor prognosis (p＜0.001). In a/the multivariate logistic analysis of the derivation cohort, four candidate predictors were entered into the final prognostic model: cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, the cerebrospinal fluid (CSF) IgG synthesis index (IgG-Syn), and spinal cord lesions. The value assignment was completed according to the regression coefficient of each identified independent prognostic factor for CNS IIDDs in the derivation cohort to establish the CELS risk score model. According to the regression coefficient, point values were given to each factor based on the log scale, and 1 point was awarded for each variable. These 4 factors determined the total risk score, ranging from 0 to 4. There was a higher risk of death in IIDD patients with higher CELS scores and we, therefore, defined three levels of risk of death in IIDD patients: a low-risk group for patients with a score of 0, a medium-risk group for patients with a total score of 1 or 2, and a high-risk group for patients with a total score of 3 or 4. The prognostic model had an area under the curve of 0.864 (95% CI: 0.803-0.925) in the internal validation cohort and 0.871 (95% CI: 0.806-0.931) in the external validation cohort. The calibration plots showed a high agreement between the predicted and observed outcomes. Decision curve analysis indicated that IIDD patients could benefit from the clinical application of the prognostic model. Conclusion s We identified the risk factors for IIDD onset after haplo-HSCT, and we also developed and validated a reliable prediction model, namely, the CELS, to accurately assess the outcome of IIDD patients after haplo-HSCT. Identifying IIDD patients who are at a high risk of death can help physicians treat them in advance, which will improve patient survival and prognosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Minimal/measurable residual disease (MRD) determined by multiparameter flow cytometry (MFC) is an important variable for relapse prediction and treatment approach selection in patients with acute myeloid leukemia (AML). We aimed to investigate whether leukemia-stem cell (LSC)-based assay is superior to traditional MFC methods, including LAIP and D-F-N assays, for MRD evaluation in predicting clinical outcomes. Methods: In this cohort study, a total of 360 AML patients who received allogeneic stem cell transplantation (allo-SCT) between July 2018 and November 2019 were prospectively enrolled. The patients were randomized (1:1) and classified into a training set (n=180) and a validation set (n=180). Posttransplantation MRD were according to LSC based assay, mainly including a cocktail of CD7, CD11b, CD22, CD56, Tim-3, and CLL-1 on CD34 +CD38 - cells, and traditional assay determined by MFC, respectively. Findings: In the training set, patients were classified as LSC positive group (group A) and LSC negative group (group B) according to a cutoff value of CD34 +CD38 -cocktail + LSCs as 0.004%. Subjects in group A had a higher cumulative incidence of relapse (CIR, 42.7% vs. 2.6%, P Interpretation: Our data suggest the superiority of LSC-based MRD assays such as higher sensitivity, low false negativity, and longer time for MRD positivity to relapse to traditional MFC MRD methods for outcome prediction in AML patients received allograft. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Transplant-associated thrombotic microangiopathy (TA-TMA) is a severe complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), with mortality over 80%. Effective management of TA-TMA is hampered by obscure pathogenesis and delayed diagnosis. There are no well-acknowledged therapeutic strategies for TA-TMA. TA-TMA-directed therapy includes therapeutic plasma exchange (TPE), eculizumab, rituximab, and defibrotide. The efficacy and outcome of TPE for the treatment of TA-TMA remain controversial. To our knowledge, this is the largest cohort to date of patients treated with TPE for TA-TMA after allo-HSCT. We aimed to identify predictors of response and mortality in patients with TA-TMA who received TPE, and to recognize patients who will benefit from TPE management. Methods A total of 6241 subjects who underwent allo-HSCT were performed at Peking University People's Hospital from January 2010 to December 2019, of whom 538 patients were diagnosed with TA-TMA, with a cumulative incidence of 8.6%. Among them, 82 consecutive critically ill TA-TMA patients received TPE. TA-TMA was diagnosed using the criteria proposed by Cho et al. TPE was not performed in a protocol-defined manner. Patients were classified as achieving complete response (CR) if they showed disappearance of schistocytes, resolution of any changes in mental status, normalization of lactic dehydrogenase, and were not receiving red blood cells and platelet transfusions. Patients were considered to have achieved no response (NR) when they showed no improvement of laboratory features, remained dependent on red blood cell and/or platelet transfusions, or died with active TA-TMA. Subjects were considered to have a partial response (PR) when they did not meet the criteria for either CR or NR (BMT 2010; Blood 2020). Results TA-TMA was diagnosed at a median time of 64.5 [IQR 38.8-158] days post-HSCT. The 42 men (51.2%) and 40 women (48.8%) had an average age of 35.3 years. Renal involvement (59.8%), central nervous system dysfunction (70.7%), gastrointestinal (GI) bleeding (73.2%), and concomitant acute graft-versus-host disease (aGVHD, 78%) were common in our cohort of TA-TMA patients. All 82 patients in our analysis received TPE, and adjunctive TA-TMA-targeted therapy included the use of rituximab (11 patients), rituximab plus eculizumab (1 patient), and defibrotide (1 patient). However, the additional therapy showed no significant difference between the response and nonresponse groups. The median time from TA-TMA to TPE initiation was 8 days [IQR 2.0-16.5], and the cumulative volume of TPE was 6 L [IQR 3.6-8.5]. Our data revealed that the overall response was 52.4% (43/82), comprising 4 CRs and 39 PRs. Early TPE initiation trended towards a better response, but this difference was not statistically significant. The multivariate analysis showed that patients with GI bleeding (OR, 6.26; 95% CI, 1.30-30.12), grade III-IV aGVHD (OR, 5.00; 95% CI, 1.50-16.68), lower cumulative volume of TPE (OR, 8.51; 95% CI, 1.91-38.05), and severe anemia (OR, 7.48; 95% CI, 2.20-25.49) were less likely to respond to TPE. Regarding treatment outcome, 57% (47/82) of cases survived 100 days post HSCT, and 20% (16/82) survived 100 days after the diagnosis of TA-TMA. With a median follow-up of 467 days [IQR 248-1002], the OS at 1 year after TA-TMA was 15%. The leading causes of death were infection, active TA-TMA, and multiple organ dysfunction syndrome (MODS). The Kaplan-Meier analysis showed that GI bleeding, grade III-IV aGVHD, and no response to TPE were associated with poor survival at 1-year post TA-TMA (Figure 1). By multivariate analysis, TA-TMA patients treated with TPE had dismal survival with GI bleeding, lower loading volume of TPE, and elevated total bilirubin. Conclusions The results of this large cohort of real-world practice indicate that TPE may be effective for TA-TMA depending on given clinical circumstances. Our data underscore that GI bleeding is independently associated with poor response to TPE and mortality, while grade III-IV aGVHD is again confirmed as predicting a dismal response to TPE. We hypothesize that higher volume of TPE is warranted to achieve resolution and favorable outcome of TA-TMA. Multicenter prospective studies are required to further verify whether these patients could benefit from TPE and seek a paradigm for TPE in the management of TA-TMA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction SIL-TAL1 rearrangement is relatively common among patients with T-cell acute lymphoblastic leukemia (T-ALL). Pediatric patients with SIL-TL1 positive T-ALL generally have a poor response to early intensive treatment and a lower median relapse-free survival (RFS) and overall survival (OS) rates compared to pediatric T-ALL patients who do not harbor the SIL-TAL1 gene fusion. Therefore, we explored the clinical effect of haploid transplantation among pediatric SIL-TAL1-gene fusion positive T-ALL and aimed to identify potential patient prognostic factors. M ethods We performed a retrospective analysis of 21 children with SIL-TAL1 gene fusion positive T-ALL who underwent haploidentical transplantation at the Lu Daopei Hospital in Langfang, China between July 2014 and June 2020. The median patient age was 9 years (range: 1.6 to 18 years), the male to female ratio was 19:2, and the median white blood cell count at initial onset was 193.65×109/L (range: 3.6-582.17×109/L). Prior to transplantation, 18 patients were bone marrow minimal residual disease (MRD) negative, three patients were bone marrow MRD-positive and the overall median value for MRD positivity by flow cytometry was 0.05% (range: 0.03%-0.35%). Eighteen patients were SIL-TAL1 gene fusion negative and three patients were SIL-TAL1 gene fusion positive prior to transplantation. The quantitative median number is 0.04% (range: 0.033%-0.05%). 13 patients with CR1 and 8 patients with CR2. 11 of 21 patients harbored the SIL-TAL1 gene fusion along with at least one other gene mutation. All patients were given myeloablative pretreatment, of which 17 patients received cyclophosphamide/total body irradiation (TBI/Cy) and 4 patients received Busulfan/cyclophosphamide (Bu/Cy). The median follow-up time was 9.5 months (range: 2 to 51 months). Results The white blood cells (WBCs) of 21 patients were all completely engrafted , two patients did not have platelet engraftment . At one month after transplantation, all patients were bone marrow MRD negative and the quantification of SIL-TAL1 fusion gene was 0% among all the patients. For 20 of these 21 patients, the chimerism rate of peripheral blood CD3+ cells and bone marrow was 100% donor type. The 4-year disease-free survival (DFS) and overall survival (OS) rate were 56.5% and 55.1%, respectively (Figure 1). Causes of death were recurrence (5 patients all of whom had bone marrow recurrence), infection (3 patients) and alveolar hemorrhage after reinfusion (1 patient). Six patients relapsed after transplantation with a median time to relapse of 90 days (range: 54-180 days). The OS rate following transplantation in the CR1 group was higher than in the CR2 groups (69.2% vs 31.3%, respectively) but there was no statistical difference between the two groups (P=0.147) (Figure 2). The OS rate of MRD-negative patients and MRD-positive patients prior to transplantation was 58.4% and 33.4% respectively, (P=0.287) (Figure 3). Prior to transplantation, the OS rate of SIL-TAL1 gene fusion-negative patients was 64.3%, while the OS rate of SIL-TAL1 gene fusion-positive patients was 0.0%--the difference between the two groups was statistically significant (P=0.001) (Figure 4). The OS rate of 11 patients that harbored the SIL-TAL1 gene fusion along with another gene mutation was 47.7% (Figure 5). Among these 11 patients, the OS rate of 9 patients who switched from SIL-TAL1 positive to SIL-TAL1 negative prior to transplantation was 58.3%. In contrast, two patients that remained SIL-TAL1 gene fusion positive had an OS rate of 0% (P=0.027) (Figure 6). Conclusion Pediatric T-ALL patients harboring the SIL-TAL1 gene fusion achieved a higher OS rate through haploid transplantation and were able to avoid extramedullary recurrence. We conclude that if patients with the SIL-TAL1 gene fusion do not switch to SIL-TAL1-negative prior to transplantation, we showed that even if the myeloablative pretreatment program is adopted, the survival rate cannot be improved, and the mortality rate is 100%. Harboring another gene mutation besides SIL-TAL1 at the initial onset of disease did not affect the prognosis in our cohort analysis. We conclude that for pediatric T-ALL patients, intensive chemotherapy should be adopted to make the bone marrow MRD negative and SIL-TAL1 gene fusion negative, and allogeneic hematopoietic stem cell transplantation should be performed as soon as possible to improve OS. Disclosures No relevant conflicts of interest to declare.

Introduction Current available treatments are limited once patients with B-ALL relapse following allogeneic hematopoietic stem cell transplantation (allo-HSCT). While chimeric antigen receptor (CAR) T-cell therapy offers a chance of remission, long-term outcomes for these patients remain poor. The benefit of bridging into a second transplant after CAR T-cell therapy remains inconclusive and available data are limited. Here, we report the long-term outcomes of 23 B-ALL patients who chose to undergo a second allo-HSCT after achieving complete remission (CR) from CAR T-cell therapy. Methods From April 2017 to April 2020, 23 R/R B-ALL patients (median age of 20 years, ranging from 3 to 58 years) who relapsed after first allo-HSCT received CAR T-cell therapy. The data were aggregated from seven different clinical trials (www.clinicaltrials.gov NCT03173417, NCT02546739 NCT03825718, NCT03825731, NCT03952923, NCT04100187 and www.chictr.org.cn ChiCTR1800016541). Patients' first transplant sources were HLA-identical sibling (n=5), matched-unrelated donor (MUD) (n=1), and haploidentical donors (haplo) (n=17). Eight of the 23 patients had disease relapse within 6 months following the first transplant. The median time from first transplant to CAR T-cell infusion was 261days (range: 117~2181 days). Before CAR T-cell infusion, patients' median bone marrow (BM) blasts by morphology were about 72.5% (1.5%-94.5%) including 12 patients with BM blasts >70% (5 with BM blasts >90%). Three of the 23 patients (13%) had received at least one prior donor lymphocyte infusion. No patients had active graft-versus-host disease (GVHD) prior to CAR T-cell therapy. Second generation CAR T-cells were generated by using purified T-cells from transplant donors (n=15) or patients (n=8). Twenty-two patients received T-cells modified with CD19-targeting CAR T-cells containing either a 4-1BB (n=18) or a CD28 co-stimulatory domain (n=4), and one patient received CD19-CD22 dual specificity CAR T-cells. All patients received a conditioning regimen of IV fludarabine (30mg/m2/d) and cyclophosphamide (250mg/m2/d) for 3 days followed by a single CAR T-cell infusion with a median dose of 3×105 cells/kg (1×105-6×105 cells/kg) in 21 patients. Two patients received a second CAR T-cell infusion in 2-3 months (1/3×105 cells/kg dose). Post CAR-T therapy, all patients bridged into a consolidation second transplantation with conventional myeloablative pre-transplantation conditioning regimens including 15 patients who received total body irradiation-based and 7 patients that received a busulfan-based conditioning regimen. Cyclosporin A, short-term methotrexate, and mycophenolate mofetil were used for GVHD prophylaxis. Results Patients' characteristics are shown in Table 1. On Day 30 post CAR-T-cell infusion, 23/23 (100%) patients achieved minimal residual disease (MRD)-negative CR. A total of 16/23 (69.6%) patients developed cytokine release syndrome (CRS) of which 14/23 (60.9%) had Grade I-II and 2/23 (8.7%) had Grade III CRS. Two patients had Grade III neurotoxicity. All patients with MRD-negative status subsequently bridged into a second transplant (2 from MUD and 21 from haplo donors) with a median interval time of 67 days (39- 329 days) from CAR T-cell therapy to a second transplant. At a median follow-up time of 258 days (84-978 days), no patients relapsed, which was encouraging. Five of 23 patients (21.7%) died from transplant-related mortality (TRM) at a median time of 295 days (103-372 days) (1 from GVHD and 4 from infection). The 1-year overall survival (OS) was 68.0% and 2-year OS was 54.4% (Fig.1). While there was a trend towards a more efficacious OS for patients whose CAR T-cells were derived from donors rather than from patients themselves but the number are too small to reach statistical significance (1-year OS 83.9% vs. 64.3%, 2-year OS 83.9% vs. 42.9%, P=0.739. Fig.2). After the 2nd transplant, four patients developed GVHD. Conclusions Our study demonstrates that even for R/R B-ALL patients who have relapsed following a first allo-HSCT , an MRD-negative CR status can still be achieved through CAR T-cell cell therapy without increasing CRS or neurotoxicity, making consolidation second allo-HSCT feasible for these patients. CAR T-cell therapy combined with a consolidation second HSCT are effective for these heavily pre-treated patients with an encouraging prospect for long-term survival. Disclosures No relevant conflicts of interest to declare.

Introduction: Immune thrombocytopenia (ITP) is a hematological disease associated with thrombocytopenia and bleeding diathesis. Intracranial hemorrhage (ICH) is the most devastating complication in ITP patients. Prophylaxis for ICH is indispensable due to its high mortality. However, the morbidity of ICH is quite low, which makes general prophylaxis unrealistic. A previous study demonstrated risk stratification of ICH in ITP patients before 60 years of age(Platelets, 2020). However, few studies have analyzed the risk factors and outcomes in ICH in elderly ITP patients. Thus, ascertaining the characteristics of ICH in elderly ITP patients is in urgent need to make effective assessments and provide personalized prophylaxis. Here, a retrospective study of a series of ITP patients who developed ICH ≥ 60 years of age was conducted to explore potential risk and prognostic factors, which may contributes to identifying the feasible personalized prophylaxis for ICH in elderly ITP patients. Patients and methods: A total of 44 patients incorporated in the study were over 60 years of age with a diagnosis of primary ITP before the onset of ICH. All measures were chronologically performed before the onset of ICH. Univariate and multivariate analyses were conducted using conditional logistic regression model. Each variables with a p-value < 0.10 in the univariate analysis and well-reported factors were included in the multivariate analysis. A stepwise approach was used to exclude variables with a p-value > 0.10. Results: Platelet counts at ITP diagnosis were significantly lower among patients who developed ICH afterwards (P=0.004). Platelet counts were dichotomized and coded into binary variables. The cut off value was 25,000/μL according to the ROC curve and Youden index. Estimation by the Kaplan-Meier method indicated that the morbidity of ICH was higher among patients with a baseline platelet count less than 25,000/μL (P=0.001). Bleeding severity was also included in the analysis. The control group had a significantly higher probability of no bleeding(P=0.008), while the case group was more likely to suffer life-threatening bleeding(P=0.006). The anatomic sites of bleeding manifestations were then examined. There existed significant difference between cases and controls with respect to skin bleeding (P=0.002), oral cavity bleeding (P < 0.001), gastrointestinal bleeding (P=0.022) and hematuria (P < 0.001). Univariate analysis also revealed that patients with a complication of diabetes mellitus (P=0.007) exhibited a significantly lower incidence of ICH, and fewer patients over 75 years of age developed ICH(P=0.021). Patients who had suffered head trauma before the end point of follow-up had a significantly higher probability of developing ICH (P=0.016). Moreover, patients with a duration of ITP no more than 7 days showed potential relevance (P=0.016). Multivariate analysis was conducted using a conditional logistic regression model. A stepwise approach identified a platelet count ≤ 25,000/μL at ITP diagnosis(OR=3.389, 95% CI 1.290-8.907, P=0.013), head trauma (OR=9.753, 95% CI 1.029-92.414, P=0.047), ITP duration less than 7 days (OR=6.741, 95% CI 1.348-33.712, P=0.020) and life-threatening bleeding(OR=13.077, 95% CI 1.104-154.865, P=0.041) as independent risk factors for ICH in elderly ITP patients. A simple predictive model was established according to the results above. Patients were segregated into a low-risk (Score=0), an intermediate-risk (Score=1-3) and high-risk (Score≥4) groups. Significant differences (P < 0.001) were observed in the frequencies of ICH between the 3 risk groups (Table 1). Kaplan-Meier estimations of ICH incidence were markedly different among the risk groups (Figure 1). As for prognostic factors, skin bleeding (OR=17.400, 95% CI 1.926-157.190, P=0.011) was identified as an independent poor prognostic factor. No significance was found with respect to age, platelet count or ITP duration. Conclusion: Our study revealed that platelet count ≤ 25,000/μL at ITP diagnosis, head trauma, ITP duration < 7 days and life-threatening bleeding are all independent risk factors for ICH in elderly ITP patients. A simple predictive model was established using these factors, and skin bleeding was a poor prognostic factor for ICH in elderly ITP patients. Disclosures No relevant conflicts of interest to declare.

Objective: Established the value of somatic mutations based on next-generation sequencing (NGS) and explore factors associated with prognosis in myelodysplastic syndromes (MDS). Methods: From March 2012 to September 2019, 90 newly diagnosed MDS patients were treated and analysed by a sensitive NGS assay for mutations in 87 candidate genes and target regions. IPSSR higher risk include IPSS-R Intermediate, High, Very High subgroups (risk score >3.5), and IPSSR lower risk include IPSSR risk score ≤3.5. Results: A total of 90 MDS patients were recruited for this study (median age: 51.5 years; range: 16-70 years). Eighty-two (91.1%) patients harbored at least one mutation (median, 3 per patient; range, 0-11), and the most common mutations were found successively in the ASXL1 (28.9%), TP53 (21.1%), TET2 (18.9%), U2AF1 (17.8%), RUNX1 (15.6%), SETBP1 (13.3%), DNMT3A (13.3%), IDH1 (12.2%), NRAS (12.2%), KMT2D (11.1%), CBL (11.1%) and PTPN11 (11.1%) genes. The median follow-up was 32 months (range: 10-96 months). Thirty-seven (92.5%) had at least one point mutation in 40 patients with normal karyotype. ASXL1 and U2AF1 mutations had more frequently platelet levels of Key words: myelodysplastic syndromes; somatic mutations; Prognostic dichotomization based on 3.5 of the revised international prognostic scoring system; overall survival; allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

Introduction Transplant-associated thrombotic microangiopathy (TA-TMA) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which can result in multiorgan injury and increased risk for mortality. Renewed interest has emerged in the prognostication of TA-TMA with the development of novel diagnostic and management algorithms. Our previous study reported an adverse outcome in patients with TA-TMA and concomitant acute graft-versus-host disease (Eur J Haematol, 2018). However, information on markers for the early identification of severe cases remains limited. Therefore, this study is concentrated on the development and validation of a prognostic model for TA-TMA, which might facilitate risk stratification and contribute to individualized management. Methods Patients receiving allo-HSCT in Peking University People's Hospital with 1) a diagnosis of microangiopathic hemolytic anemia (MAHA) or 2) evidence of microangiopathy were retrospectively identified from 2010 to 2018. The diagnosis of TA-TMA was reviewed according to the Overall-TMA criteria (Transplantation, 2010). Patients without fulfillment of the diagnostic criteria or complicated with other causes of MAHA were excluded from analysis. Prognostic factors for TA-TMA were determined among patients receiving HSCT between 2010 and 2014 (derivation cohort). Candidate predictors (univariate P < 0.1) were included in the multivariate analysis using a backward stepwise logistic regression model. A risk score model was then established according to the regression coefficient of each independent prognostic factor. The performance of this predictive model was evaluated through internal validation (bootstrap method with 1000 repetitions) and external temporal validation performed on data from those who received HSCT between 2015 and 2018 (validation cohort). Results 5337 patients underwent allo-HSCT at Peking University Institute of Hematology from 2010 to 2018. A total of 1255 patients with a diagnosis of MAHA and/or evidence of microangiopathy were retrospectively identified, among whom 493 patients met the inclusion criteria for this analysis (269 in the derivation cohort and 224 in the validation cohort). The median age at the time of TA-TMA diagnosis was 28 (IQR: 17-41) years. The median duration from the time of transplantation to the diagnosis of TA-TMA was 63 (IQR: 38-121) days. The 6-month overall survival rate was 42.2% (208/493), and the 1-year overall survival rate was 45.0% (222/493). In the derivation cohort, patient age (≥35 years), anemia (hemoglobin 800 U/L) and elevated total bilirubin (TBIL >1.5*ULN) were identified by multivariate analysis as independent prognostic factors for the 6-month outcome of TA-TMA. A risk score model was constructed according to the regression coefficients (Table 1), and patients were stratified into a low-risk group (0-1 points), an intermediate-risk group (2-4 points) and a high-risk group (5-6 points). The Kaplan-Meier estimations of overall survival separated well between these risk groups (Figure 1). The prognostic model showed significant discriminatory capacity, with a cross-validated c-index of 0.770 (95%CI, 0.714-0.826) in the internal validation and 0.768 (95%CI, 0.707-0.829) in the external validation cohort. The calibration plots also indicated a good correlation between model-predicted and observed probabilities. Conclusions A prognostic model for TA-TMA incorporating several baseline laboratory factors was developed and evaluated, which demonstrated significant predictive capacity through internal and external validation. This predictive model might facilitate prognostication of TA-TMA and contribute to early identification of patients at higher risk for adverse outcomes. Further study may focus on whether these high-risk patients could benefit from early application of specific management. Disclosures No relevant conflicts of interest to declare.

Poor graft function (PGF) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Murine studies suggest that endothelial progenitor cells (EPCs) are preferential supporting cells for hematopoietic stem cells in the bone marrow (BM) microenvironment. Our previous work found that a reduced number of BM EPCs was an independent risk factor for the occurrence of PGF after allo-HSCT. However, little is known about the functional role of BM EPCs and how to improve impaired BM EPCs in PGF. In the current study, we evaluated the function of BM EPCs in subjects with PGF postallotransplant. Moreover, we investigated whether atorvastatin could enhance the number and function of BM EPCs derived from subjects with PGF in vitro. Dysfunctional BM EPCs, which were characterized by impaired proliferation, migration, angiogenesis, and higher levels of reactive oxygen species and apoptosis, were revealed in subjects with PGF. Activation of p38 and its downstream transcription factor cyclic adenosine monophosphate-responsive element-binding protein were detected in BM EPCs from subjects with PGF. Furthermore, the number and function of BM EPCs derived from subjects with PGF were enhanced by atorvastatin treatment in vitro through downregulation of the p38 MAPK pathway. In summary, dysfunctional BM EPCs were observed in subjects with PGF. Atorvastatin treatment in vitro quantitatively and functionally improved BM EPCs derived from subjects with PGF through downregulation of the p38 MAPK pathway. These data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in subjects with PGF postallotransplant.

Introduction Mesenchymal stem cells (MSCs) are being recognized as one of the treatment options for acute graft versus host disease (aGVHD), but their therapeutic mechanisms have not been fully elucidated. Pyroptosis, a novel form of inflammation related programmed cell death, often occurs in myeloid cells. Many studies have found that macrophage pyroptosis plays an important role in multiple inflammatory and autoimmune diseases (Journal of Autoimmunity, 2018). As an immune disease with involvement of various inflammatory factors, aGVHD exhibits macrophage dysfunction according to our previous study (Sci China Life Sci, 2020). However, whether macrophages undergo pyroptosis and their role in aGVHD remain unknown. MSCs have been reported to inhibit pyroptosis, and some cytokines that suppress pyroptosis can also be secreted by MSCs (Nature Immunology, 2016). Whether inhibition of macrophage pyroptosis represents a therapeutic mechanism for MSCs to alleviate aGVHD needs further exploration. Methods Twenty patients with aGVHD and 20 patients without aGVHD after hematopoietic stem cell transplantation were enrolled in our study. Macrophages were derived from CD14+ monocytes of patients and the THP-1 cell line. CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. MSCs were obtained from fresh umbilical cord of healthy puerpera. Morphological analysis of macrophages was performed by scanning electron microscopy. Expression of GSDMD and NLRP3 inflammasome associated components was assessed by real-time transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. The subgroup of CD4+T cells was analyzed by flow cytometry. RT-PCR, ELISA and RNA interference were used to evaluate relevant immunomodulatory factors which were involved in the inhibitory effect of MSCs on macrophage pyroptosis. Additionally, an aGVHD mouse model was established to observe the therapeutic effect and mechanism of MSCs on macrophage pyroptosis. Results Scanning electron microscopy images showed the formation of membrane pores in macrophages of aGVHD patients. Meanwhile, expression of the pyroptosis executioner GSDMD, NLRP3 inflammasome associated components, IL-1β, IL-18, and LDH release were elevated in macrophages from aGVHD patients, indicating that macrophages in aGVHD underwent NLRP3 inflammasome activation and pyroptosis. Furthermore, NLRP3 inhibition reduced macrophages pyroptosis, suggesting that macrophages pyroptosis in aGVHD are mediated by NLRP3 inflammasome activation. Since CD4+T cells play a critical role in the pathogenesis of aGVHD, we investigated the effect of macrophage pyroptosis on CD4+T cells. In vitro, macrophage pyroptosis increased the proportion of CD69+, Th1 and Th17 cells among CD4+T cells, which was partially reversed by blocking IL-1β/IL-1R and IL-18/IL-18R signaling. We also observed that the proportion of macrophage pyroptosis was more increased in patients with III-IV aGVHD than in those with I-II aGVHD. In addition, administration of a pyroptosis inhibitor into aGVHD model mice greatly attenuated clinical and histopathological scores. Taken together, these results indicate that macrophage pyroptosis might be involved the development of aGVHD. Expression of GSDMD, NLRP3 inflammasome associated components, IL-1β, IL-18, and LDH release in aGVHD macrophages were reduced when cells were cocultured with MSCs, indicating that MSCs inhibit aGVHD macrophage pyroptosis by suppressing NLRP3 inflammasome activation. Furthermore, secretion of prostaglandin E2 (PGE2) was increased in MSCs cocultured with aGVHD macrophages, blocking which by small interfering RNA (siRNA) or inhibition of PGE2 induced CAMP-PKA signaling with antagonists both largely abrogated MSC effects. Consistently, the effect of MSCs on macrophage pyroptosis and the NLRP3 inflammasome in vivo was also dampened after transfection with prostaglandin E synthase (PTGES) siRNA, and the therapeutic effect in the aGVHD mouse model was impaired. Conclusions Our results demonstrate that macrophage pyroptosis plays a crucial role in the pathogenesis of aGVHD by promoting activation and differentiation of CD4+ T cells. MSCs suppress macrophage pyroptosis in aGVHD via PGE2/cAMP/PKA signaling, which might represent a therapeutic mechanism of MSCs for aGVHD. Disclosures No relevant conflicts of interest to declare.

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

Background The optimal dose of antithymocyte globulin (ATG) with respect to the prevention of graft-versus-host disease (GVHD) following haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is under investigation. In our previous single-center randomized study, as compared with 6 mg/kg ATG, 10 mg/kg ATG was found to be associated with better GVHD prevention and superior GRFS, but an increase in infection-related deaths. Later on, in our multi-center randomized trial, 7.5 mg/kg ATG for GVHD prophylaxis was associated with reduced EBV and CMV infections without increased incidence of GVHD in comparison with 10.0 mg/kg ATG in haplo-HSCT. Methods We reanalyzed and updated the prospective, randomized trial (clinicaltrials.gov, NCT01883180) identifying the influence of 7.5mg/kg versus 10.0 mg/kg of ATG on clinical outcomes in haplo-HSCT with extended follow-up (N=145. Seventy-six patients received 7.5 mg/kg ATG (ATG-7.5), whereas the remaining patients received 10 mg/kg ATG (ATG-10). Results The median follow-up period was 1702 days (range, 23-2036 days). The rate of infection-related deaths in ATG-10 arm was double that of the ATG-7.5 arm (20.0% vs 11.8%; P=0.024). The 5 year cumulative incidence of relapse was not significantly different between the ATG-7.5 and ATG-10 groups (16.8% vs. 5.7%, P = 0.053). The 5 year cumulative incidence of non-relapse mortality was comparable between the ATG-7.5 and ATG-10 groups (27.6% vs. 28.7%, P = 0.938). The 5 year cumulative incidence of chronic GVHD (46.7% vs. 48.3%, P = 0.913), moderate-to-severe chronic GVHD (32.8% vs. 25.3%, P = 0.248), and severe chronic GVHD (17.1% vs. 13.3%, P = 0.505) were comparable between the ATG-7.5 and ATG-10 groups. The 5 year probabilities of disease-free survival (DFS) in the ATG-7.5 and ATG-10 groups were 55.6% and 65.7%, respectively (P = 0.281). The 5 year probability of GVHD-free/relapse-free survival (GRFS) in the ATG-10 group was significantly higher than that in the ATG-7.5 group (48.1% vs. 29.5%, P = 0.020). The 5 year cumulative incidence of late effects of grades 1-5 (67.2% vs. 71.2%, P = 0.695) and multiple late effects (26.2% vs. 25.4%, P = 0.920) were comparable between the ATG-7.5 and ATG-10 groups. In multivariate analysis, ATG-7.5 was associated with a significantly lower GRFS compared to ATG-10 (hazard ratio, 1.819; 95% confidence interval, 1.106-2.994; p=0.019). Conclusion it appears that 10 mg/kg ATG was found to be associated with superior GRFS and comparable GVHD and late effects, but an increase in infection-related deaths as compared with 7.5 mg/kg ATG for haplo-HSCT. Figure Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) often relapses following chemotherapy-induced remission and is generally chemo-resistant. Given the potential role for cancer stem cells in relapse, targeting of the leukemia-initiating cell (LIC) in AML may provide improved outcome following remission induction. However, due to overlap in their self-renewal program with normal hematopoietic stem cells (HSCs), therapeutic targeting of the LIC may have an adverse effect on long-term hematopoietic recovery. Here we used a mouse model of relapsed AML to explore whether the hypoxia-inducible factor (HIF)1α inhibitor echinomycin can be used to treat relapsed AML without affecting host HSCs. We show that echinomycin cured 40% to 60% of mice transplanted with relapsed AML. Bone marrow cells from the cured mice displayed normal composition of HSCs and their progenitors and were as competent as those isolated from nonleukemic mice in competitive repopulation assays. Importantly, in mice with complete remission, echinomycin appeared to completely eliminate LICs because no leukemia could be propagated in vivo following serial transplantation. Taken together, our data demonstrate that in a mouse model of relapsed AML, low-dose echinomycin selectively targets LICs and spares normal hematopoiesis.

The best donor for a related donor for a human leukocyte antigen (HLA) haplotype-mismatched transplant for hematological neoplasms is controversial. We studied outcomes in 1210 consecutive transplant recipients treated on a uniform protocol. Younger donors and male donors were associated with less nonrelapse mortality (NRM; hazard ratio [HR] = 0.30; 95% confidence interval [CI] = 0.01-0.39; P = .008 and HR = 0.65; 95% CI = 0.49-0.85; P = .002) and better survival (HR = 0.73; 95% CI = 0.54-0.97; P = .033 and HR = 0.73; 95% CI = 0.59-0.91; P = .005). Father donors were associated with less NRM (HR = 0.65; 95% CI = 0.45-0.95; P = .02), acute graft-versus-host disease (GVHD) (HR = 0.69; 95% CI = 0.55-0.86; P = .001), and better survival (HR = 0.66; 95% CI = 0.50-0.87; P = .003) compared with mother donors. Children donors were associated with less acute GVHD than sibling donors (HR = 0.57; 95% CI = 0.31-0.91; P = .01). Older sister donors were inferior to father donors with regard to NRM (HR = 1.87; 95% CI = 1.10-3.20; P = .02) and survival (HR = 1.59; 95% CI = 1.05-2.40; P = .03). Noninherited maternal antigen-mismatched sibling donors were associated with the lowest incidence of acute GVHD compared with parental donors and noninherited paternal antigen-mismatched sibling donors. Specific HLA disparities were not significantly correlated with transplant outcomes. Our data indicate which HLA haplotype-mismatched related donors are associated with the best transplant outcomes in persons with hematological neoplasms.

Purpose or Background: We aimed to develop a disease risk comorbidity index (DRCI) based on Disease Risk Index (DRI) and Hematopoietic Cell Transplantation-Specific Comorbidity Index (HCT-CI) in patients receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Method or Case: We examined 889 patients undergoing haplo-HSCT from 2015 to 2016. We used a Cox multivariable model to identify factors prognostic of disease-free survival (DFS) in a training subset (n = 593). A weighted score using these factors was assigned to the remaining patients (validation cohort; n = 296). This work was supported by the Capital's Funds for Health Improvement and Research (grant number 2018-4-4089). Results or Progress: The multivariable model identified two independent predictors of DFS: DRI and HCT-CI before HSCT. A weighted score of 2 was assigned to very high risk DRI, and a weighted score of 1 was assigned to high-risk DRI and intermediate- and high-risk HCT-CI in the scoring system (i.e., haplo-HSCT). In the validation cohort, the 3-year DFS was 65.2% (95% CI, 58.2-72.2%), 55.8% (95% CI, 44.9-66.7%), and 32.0% (95% CI, 5.8-58.2%) for the low-, intermediate- and high-risk group, respectively (P=0.005). Haplo-DRCI can predict relapse (P Conclusion or Discussion: These data confirmed that haplo-DRCI can effectively risk stratifies haplo-HSCT recipients and provide the tool to better predict who will best benefit from haplo-HSCT. Disclosures No relevant conflicts of interest to declare.

Introduction Late-onset hemorrhagic cystitis (LOHC) is a common complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with an incidence ranging from 6.5% to 70% and leads to prolonged hospitalization and even death (Silva Lde P et al, Haematologica, 2010; LAM et al, Transpl Infect Dis, 2017). The pathogenesis of LOHC remains obscure, but in our previous study, viral infections and acute graft-versus-host disease (aGVHD) have frequently been shown to be associated with its development (Han et al., Am. J. Hematol, 2014). Therapeutic strategies for LOHC are still not standardized due to the complicated clinical background and given the paradoxical phenomenon that immunosuppressive agents, such as steroids, are necessary for aGVHD but cause an immunosuppressive state. We aimed to investigate the incidence, risk factors and outcomes of LOHC after allogeneic stem cell transplantation. Additionally, for the first time, we propose four therapeutic patterns and their impact on prognosis of LOHC to aid in clinical decision making. Methods This retrospective, nested, case-control study reviewed data from 2158 patients who received allo-HSCT from January 2014 to December 2018. In total, 364 (16.87%) patients were diagnosed with LOHC. Individual matching was performed by randomly selecting 3 controls from the same cohort for each identified LOHC patient according to the time of allo-HSCT. Standard basic measures included hyperhydration, forced diuresis, insertion of a vesical catheter for intermittent or continuous bladder irrigation and evacuation of clots. Patients with grade III-IV LOHC were divided into four groups according to immunosuppressant use (steroid, MMF and Basiliximab) 1 week before and after the onset of LOHC: an intensified-intensified (I-I) group, intensified-not intensified (I-N) group, not intensified-intensified (N-I) group and not intensified-not intensified (N-N) group. "Intensified" was defined as an increase in any immunosuppressant, and "not intensified" was defined as maintenance or tapering of all immunosuppressants. Data concerning the baseline characteristics, incidence, treatment patterns and outcomes were recorded. Results In total, 364 patients developed LOHC at a median of 29 days (range, 23-37.75 days), with a cumulative incidence of 16.87%. AML (HR =0.493 95% CI, 0.244-0.997; p=0.049), AA (HR =0.444, 95% CI,0.18-1.096; p=0.078), HLA match(HR =0.149, 95% CI, 0.036-0.616; p=0.009), days to platelet engraftment(HR =1.023, 95% CI, 0.998-1.049;p=0.069)and CMV viremia (HR =2.365, 95% CI, 1.179,4.733;p=0.015)were associated with LOHC. Only HLA match (HR =0.111, 95% CI, 0.014-0.878; p=0.037)remained significant in the multivariate analysis. 6.5%, 12.9%, 48.39% and 32.26% patients with III-IV LOHC were divided into I-I, I-N, N-I and N-N groups, respectively. There were no significant differences in overall survival (p=0.05) and the incidence of CMV viremia (p=0.187) among the four groups. The incidence of relapse (p=0.007) and the incidence of aGVHD (p=0.01) was highest in the I-I group, followed by the N-I, I-N and N-N groups. 50% of the patients in N-I group showed the improvement of LOHC, which is significantly highest among the four groups(p=0.000). However, no statistical significance was found regarding the CMV viremia turning shade or the improvement of aGVHD. Non-relapse mortality (NRM) was observed in 25% of patients without resolution of LOHC. NRM was 0% among patients without intensified immunosuppression but was 25%, 0% and 50% among those with intensified immunosuppression before LOHC, after LOHC and before and after LOHC, respectively. Conclusion The independent risk factors for LOHC after allo-HSCT were HLA mismatch. Avoiding intensified immunosuppression that damages endothelium or reactivates the related virus may improve transplant outcome. The N-N pattern could reduce the incidence of CMV viremia or aGVHD in the LOHC patients, and the N-I pattern might be more promising as a therapeutic strategy for LOHC. Disclosures No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

Background: B-acute lymphoblastic leukemia (B-ALL) with t(v;11q23)/MLL-rearrangement (MLL-r) in children (1 year or older) is rare, and its outcome and optimal treatment options remain controversial. This study aimed to analyze the clinical characteristics and outcomes of these patients, and to explore the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially haploidentical HSCT (haplo-HSCT) in the treatment of these patients. Methods: At the time of the last follow-up (July 1, 2019), we retrospectively analyzed clinical data of 42 non-infant children with t(v;11q23)/MLL-r B-ALL. Comparison of outcomes was made between patients received allo-HSCT in the first complete remission (CR1) and chemotherapy only. Results: The median follow-up was 41 (1-106) months. The median age at diagnosis was 4.5(1-14) years and the median leukocyte count was 56.0 (2.2-735.2)×109/L. One was excluded for death during induction. For the remaining 41 patients, the complete remission rate after induction therapy was 40/41 (97.6%), the estimated 4-year probabilities of overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 59.7%, 51.6% and 43.4%, respectively. 19 patients received allo-HSCT in CR1 (HSCT in CR1 cohort), notably, 18/19 cases in this cohort received haplo-HSCT, and the remaining 22 patients continued the consolidation therapy (Non-HSCT in CR1 cohort). The estimated 4-year probabilities of OS, EFS and CIR in the HSCT in CR1 cohort were 86.6%, 89.2% and 5.3%, respectively. Meanwhile, the estimated 4-year probabilities of OS, EFS and CIR in the Non-HSCT in CR1 cohort were 37.5%, 19.9% and 75.6%, respectively. They were considered to be statistically significant. Of the 17 patients who relapsed during consolidation chemotherapy, 9 patients who underwent chemotherapy only (Non-HSCT after relapse cohort) all died within 44 months. For the remaining 8 patients who chose allo-HSCT (HSCT in CR2 cohort) when they achieved the second complete remission (CR2), the estimated 4-year probability of OS was 47.6% (P=0.002). Multivariate analysis showed that HSCT in CR1 was the only independent protective factor for OS, EFS and CIR, and age at diagnosis (≥10 years) was an independent risk factor of OS. Conclusions: Allo-HSCT (especially haplo-HSCT) in CR1 may reduce the risk of relapse and improve prognosis in non-infant children with MLL-r B-ALL. In addition, allo-HSCT also seemed to be an effective approach to improve the prognosis of relapsed patients. Thus, haplo-HSCT could be an alternative approach for non-infant children with MLL-r B-ALL. Disclosures No relevant conflicts of interest to declare.

Background: The outcomes of alternative donor haematopoietic stem cell Transplantation(HSCT)for Inherited bone marrow failure syndromes(IBMFS) have not been well described,especially the conditioning regimens are major challenges ,each disease type has different characteristics, whether engraftment can be achieved with less toxicity. Method : With this background,we retrospectively analyzed alternative donor HSCT for 42 patients with IBMFS in our single center from November 2012 to August 2018. Results: 27 cases were Fanconia anemia(FA),7 cases were dyskeratosis congenital(DC), 8 cases were severe congenital neutropenias(SCN). The median age at diagnosis and transplantation were 4(1 to 25 ) years and 10(1.9 to 26)years respectively. Male to female was 28 :14. All patients were confirmed to have BMF and disease-specific pathogenesis-related gene mutations. 16 cases had disease specific congenital anomalies, 10 patients had family history. Chromosomal fragility test was positive in 8 cases of FA group. Indication of HSCT for FA and DC patients which were 30 patients had BMF or transfusion dependency at transplantation;4 cases had clonal disease (2 cases myelodysplasia, 2 cases acute myeloid leukaemia). Indication of HSCT for SCN patients were uncontrollable severe infection .FA received low dose Busulfan (Bu;total dose of 6.4 mg/kg, IV), Fludarabine (Flu; total dose of 120 mg/m2, IV) , Cyclophosphamide (Cy; total dose of 2.0 g/m2, IV) based-reduced intensity conditioning(RIC) ; DC patients received low dose TBI (total 300cGy, Special position, supine) , Flu(total dose of 120 mg/m2, IV) , Cy( total dose of 3.0 g/m2, IV) based-RIC, while SCN patients had Bu(total dose of 12.8 mg/kg, IV),Cy( total dose of 3.6 g/m2, IV) or Flu(total dose of 160 mg/m2, IV) based -myeloablative conditioning(MA); and all patients combinated either of 2 different rabbit ATG ,ATG-T , rabbit anti-human thymocyte immunoglobulin, total dose 5.0 -10 mg/kg in 26 cases or ATG-F ,rabbit anti-human lymphocyte immunoglobulin, total dose 20 mg/kg in 14 cases. Campath-1, Anti-CD52 mAb was accepted with total dose 1mg/kg in 2 cases. Donor types were matched unrelated donor(MUD) in 22 patients ,Haploidentical donor (HID) in 17 patients,unrelated cord blood (UCB) in three cases. Unmanipulated stem cells were used for all patients. The Haplo-HSCT cohort received granulocyte colony-stimulating factor (G-CSF)-primed BM combined with peripheral blood stem cells (PBSCs) , The MUD HSCT cohort only received G-CSF PBSCs. The UCB HSCT cohort received one unit CB . No primary graft failure was observed. The median myeloid engraftment time was 14 (range, 10 to 21) days.Survivor median follow-up time was 38 months (range, 9-63 months), the overall survival in all patients was 76.1% ,in FA,DC,SCN were 72.4% ,100%,53.0% respectively. Cumulative incidence of 100 days acute graft-versus-host disease(GVHD) was 48.1%,Cumulative incidence 1 year and 3 years of chronic GVHD were 35.0% and 69.3% respectively. The positive chromosomal fragility test was the only independent adverse prognostic factor in multivariate analysis for FA patients rather than age ,donor type and graft source. Main causes of death were GVHD (50%) and infection (20%).No secondary malignancies occurred after HSCT till the last follow up time. Conclusion: In our study, alternative donor and disease-specific conditioning regimen HSCT for IBMFS showed promising prognosis especially for DC patients. Chromosomal fragility test positive was the only independent adverse prognostic factor in HSCT for FA patients. Disclosures No relevant conflicts of interest to declare.

A prospective, clinical trial in non-manipulated haploidentical allografts was initiated to define the efficacy of a single dose of 375 mg/m2 rituximab as a desensitization regimen for donor-specific anti-HLA antibody (DSA)-positive patients with 2,000 ≤ mean fluorescence intensity (MFI) < 10,000. In the clinical cohort, compared to pretransplantation [median, 4791, n=28], the median MFI levels at day 7 [0], 14 [0], 21 [0], 30 [0], and 45 [0] following transplantation were significantly decreased (P This study is registered at http://www.chictr.org.cn/ChiCTR-OPC-15006672. Disclosures No relevant conflicts of interest to declare.

Introduction Cytoreduction either with hypomethylating agents (HMA) or induction chemotherapy (IC) before hematopoietic cell transplantation (HCT) for patients with advanced myelodysplastic syndromes (MDS) has been used in an attempt to decrease post-HCT relapse, but the benefit for post-HCT long-term survival remains a debatable issue. We investigated the impact of previous therapy, and depth of response, as well as donor source on the outcome of allo-HCT for patients with advanced MDS. Method After excluding patients who had chronic myelomonocytic leukemia, we analyzed 303 advanced MDS patients (96 (32%) with RAEB-I, 148 (49%) with RAEB-2, and 59 (19%) with tAML) who underwent transplantation from a matched related/unrelated (MSD/URD, n=75/12) or haploidentical (n= 216) donor (HID) after preparation with myeloablative conditioning regimens (identical with BUCY for MSD and BUCY+ATG for HID) between year 2015 and 2018. Results Baseline characteristics Median age was 42 years old. Of the 303, 142 (47%) received only best supportive care (BSC) before HCT; 18 (6%) received IC, 54 (18%) received HMA, and 89 (29%) received both (IC+HMA). Before therapy, 45 (15%), 141 (46%) and 117 (39%) patients had intermediate 1,2 or high-risk scores per the International Prognostic Scoring System (IPSS). Thirty-six (12%) achieved complete remission (CR), 81 (27%) had marrow CR, 160 (53%) had stable disease, and 26 (9%) had progressive disease before HCT defined by International Working Group (IWG). Patients in CR or marrow CR were grouped together as responders (OR) while patients not in CR were grouped together as non-responders (NR) at HCT. Patients had more high-risk disease by WHO and IPSS in the IC and IC+HMA groups. Other patient and donor characteristics known to affect outcomes such as age, time from diagnosis to HCT, IPSS-R, donor source are comparable between groups. Outcome for the entire cohort CR rate at HCT was 6%, 33%, and 30% for HMA, IC, and IC+HMA groups, respectively (P =.001) while OR rate was 37%, 56%, and 70%, respectively (P =.001). With a median follow-up of 727 days, 2-year disease-free survival (DFS) was 77%, 69%,60%, and 62% for BSC, HMA, IC, and IC+HMA groups, respectively (P = .075); 2-y DFS was 77%, 72%,and 56% for untreated, OR, and NR groups, respectively (P = .026); 2-y DFS was 77%, 78%,and 60% for untreated, CR, and non-CR groups, respectively (P = .008); 2-y DFS was 80% and 57% for MSD/URD and HID groups, respectively (P = .051). Multivariate analyses revealed that older age (hazard ratio [HR], 1.03; P< .0001); higher-risk histologic subtypes (HR, 2.3; P =.003), time from diagnosis to HCT (HR,1.005; P =.027), haploidentical donor (HR, 1.9; P =.012) and NR at HCT (HR, 1.6; P =.05) were poor prognostic factors for DFS. Outcome for patients with EB2 and t-AML Focusing on the 207 patients who had BM blasts of 10% or higher before therapy, CR rate at HCT was 6%, 35%, and 33% for HMA, IC, and IC+HMA groups, respectively (P =.007) while OR rate was 44%, 59%, and 74%, respectively (P =.001); 2-year DFS was 77%, 68%,58%, and 62% for BSC, HMA, IC, and IC+HMA groups, respectively (P = .20); 2-y DFS was 77%, 71%,and 50% for untreated, OR, and NR groups, respectively (P = .025); 2-y DFS was 77%, 77%,and 58% for untreated, CR, and non-CR groups, respectively (P = .015); 2-y DFS was 75% and 64% for MSD/URD and HID groups, respectively (P = .43). Propensity score analysis To allow for potential confounding factors between treatments that could influence outcome, a 1:1 ratio propensity score matching was also performed. Included in the propensity score model were: WHO stage, IPSS score, age, time from diagnosis to HCT, and donor source. We were able to pair-match 104 untreated with 104 cytoreductive and 2-y DFS were 80% vs 68% (p= .14); 2-y DFS was 80%, 76%,and 63% for untreated, OR, and NR groups, respectively (P = .19); 2-y DFS was 80%, 94%,and 64% for untreated, CR, and non-CR groups, respectively (P = .015); 2-y DFS was 85% and 68% for MSD/URD and HID groups (P = .044) and the rate was 82% vs 70% after excluding EB-1 (p= .30). After excluding IC group (n=8), 2-year DFS was 80%, 67%, and 75% for BSC, HMA, and IC+HMA groups, respectively (P = .28). Conclusion In this analysis, various therapy approaches before HCT did not lead to different transplantation outcomes. There was no evidence of a benefit in post-HCT outcome associated with prior cytoreductive therapy for advanced MDS. Further randomized studies need to delineate the role of debulking strategy. Disclosures No relevant conflicts of interest to declare.

Background About one-half of persons with acute myeloid leukemia have normal cytogenetics but have diverse outcomes which might be explained, at least in part, by specific mutations. We focused on DDX11, the budding yeast ortholog of ChlR1, which encodes an ATP-dependent RNA- and DNA-helicase involved in diverse cell processes such as sister chromatid exchange cohesions and implicated in other cancers and which is associated with telomere shortening. Methods DNA from 359 consecutive, newly-diagnosed adults with AML and normal cytogenetics were interrogated by deep target regional sequencing (TRS). Average effective sequencing depth was 1678.5X (1000.0-1800.0X) and median coverage, 99.9% (98.0, 100.0%). Outcomes studied included cumulative incidence of relapse (CIR) and event-free survival (EFS). DDX11 mutants were transfected into a human AML cell line which was studied in vitro and in immune deficient mice. Results We identified 2909 non-synonymous somatic variants. 284 subjects (79%; 95% confidence interval [CI], 75, 83%) had ≥5 mutations with a median of 8 mutations per subject (range, 1-20). We identified mutations at 4 new loci including DDX11 in 29 subjects (8%), NPIPA5 in 15 (4%), CYP2F1 in 13 (4%), and MED12 in 9 (3%). In multi-variable analyses DDX11 mutation was independently associated with CIR (Hazard Ratio [HR]= 2.276 [1.318, 3.931]; P=0.003) and EFS (HR=2.319 [1.391, 3.867], P=0.001). Two missense DDX11-mutant alleles (DDX11P316L/WT or DDX11Q363K/WT) were generated and introduced to the kasumi-1 leukemia cells by lentivirus transfection. Both mutants showed proliferative and anti-apoptotic activities in functional analyses and increased resistance to killing by Cytarabine and Daunorubicin in vitro and in transplanted immune deficient mice. Conclusions Subjects with AML with normal cytogenetics who have a DDX11 mutation have a higher CIR and worse EFS compared with subjects with wild-type DDX11. Functional studies of a transfected DDX11 human AML cell line provide a functional explanation of our clinical findings. * Correspondence Profs. Guo-Rui Ruan and Xiao-Jun Huang Peking University Peoples Hospital and Institute of Hematology No.11 Xi-Zhi-Men South Street, Beijing 100044, China T 86-10-88324672 F 86-10-88324672 E ruanguorui@pkuph.edu.cn OR huangxiaojun@bjmu.edu.cn Disclosures No relevant conflicts of interest to declare.

Introduction Sphingosine-1-phosphate (S1P) is now emerging as a vital lipid mediator. Activation of sphingosine kinase (SphK) produces intracellular S1P, which in turn can be secreted out of the cell and act extracellularly by binding to S1P receptors (S1PR). Recent studies suggest that the "inside-out" signaling by S1P in megakaryocytes (MKs) plays a critical role in proplatelet formation (PPF) (Blood, 2013; J EXP MED, 2012). PPF requires a profound reorganization of the MK actin and tubulin cytoskeleton. Rho GTPases which can be activated by S1P, including Rac1 and Cdc42, have been shown to be master regulators of cytoskeletal rearrangements. The pathogenesis mechanisms of immune thrombocytopenia (ITP) are not entirely understood. Our previous data indicated that impaired PPF contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that S1P-mediated microtubule reorganization is defective in PPF of ITP. All-trans retinoic acid (ATRA), which has demonstrated to be a promising option for ITP patients in our previous study (Lancet Haematology, 2017), could correct the altered microtubule reorganization and promote PPF. Methods Thirty consecutive patients with primary ITP and 20 healthy donors were enrolled in our study. MKs were isolated from bone marrow samples, and they were collected again after ITP patients received ATRA therapy. MK mRNA sequencing by microarray was used to assess the difference of gene expression between ITP and controls. Microtubule regrowth assay was performed to observe microtubule dynamic behavior. In this assay nocodazole was first used to induce complete depolymerization of microtubule network, followed by drug washout to allow microtubule regrowth over time. ATRA was added to the culture medium of MKs to determine the mechanism of ATRA in correcting impaired PPF. Additionally, ITP mice model was established to observe the therapeutic effects of ATRA in PPF. Pf4-Cre/loxP system was used to specifically knock down gene of MKs. Results S1P concentration in bone marrow from ITP patients was lower compared to healthy donors. MKs mRNA sequencing demonstrated that S1P synthetase SphK2 and S1P receptor S1PR1 gene were downregulated while S1P lyase (SPL) gene was upregulated in ITP patients, which caused abnormal S1P signaling. Furthermore, we observed that PPF capacity of MKs in patients with ITP was reduced. Pharmacological disruption of S1PR1 blocked PPF, exogenous S1P corrected impaired PPF. Collectively, deregulation of S1P signaling was associated with impaired PPF in ITP. To verify the downstream role of S1P in regulating PPF, the Rho GTPases detection of MKs revealed a decrease in Cdc42 and Rac1 levels from ITP patients. Immunofluorescence of the differentiated MKs showed that the expression and distribution of β1 tubulin were abnormal from ITP patients. Early PPs from MKs of healthy donors displayed a well-organized tubulin bundles resembling bunches of grapes. In contrast, in MKs from ITP patients, tubulin was disorganized in thick bundles. In addition, TEM analysis of the MKs showed an irregular distribution of granules, tortuous membranes and impaired proplatelet structure. In microtubule regrowth assay, MKs from ITP patients had significantly lower microtubule regrowth at 10 min post-nocodazole washout compared with controls. Together, microtubule alteration resulted in impaired PPF in ITP. We tested whether S1P pathway were required for microtubule reorganization, both SphK2-/- and S1PR1-/- mice displayed significantly reduced S1P, Cdc42 and Rac1, altered microtubule architecture and defective PPF. Taken together, abnormal S1P pathway accounted for impaired microtubule reorganization in ITP. Next, we explored the effect of ATRA on microtubules reorganization in ITP patients, our data showed that in vitro treatment with ATRA restored microtubules structure by upregulating S1P and activating Rho GTPases. In vivo studies showed that ARTA could rescue the impaired PPF in both patients and mice model with ITP. Conclusions The MKs of ITP patients displayed defective cytoskeletal reorganization regulated by S1P pathway. ATRA restored cytoskeletal structure and corrected impaired PPF by upregulating S1P and activating Rho GTPases. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of ARTA in ITP patients. Disclosures No relevant conflicts of interest to declare.

Introduction-About 25% of persons with new-diagnosed acute myeloid leukemia (AML) have normal cytogenetics and no NPM1 or FLT3-ITD mutation. The prognosis and best therapy of these persons is controversial. Methods-We evaluated 809 consecutive newly diagnosed adult with normal cytogenetics and 231 of whom had no NPM1 or FLT3-ITD mutation identified by targeted regional sequencing. 158 achieved a complete remission within 2 cycles of induction therapy and were assigned to 2 different post-remission strategies: (1) 6 courses of consolidation chemotherapy (N=95); or (2) 2-4 courses of consolidation chemotherapy and an allotransplant (N=63). Results-In multi-variable analyses a WBC ≥13·6×10E+9/L, mutated IDH2, not having a bi-allelic CEBPA mutation at diagnosis, a positive measurable residual disease (MRD)-test during consolidation and not receiving an allotransplant were independently associated with a higher cumulative incidence of relapse (CIR) and worse event-free survival (EFS). Amongst subjects with IDH2 mutations, non-bi-allelic CEBPA mutations or a positive MRD-test, subjects receiving an allotransplant had a lower 5-year CIR (16% [95% confidence interval, 6, 26%]; vs. 83% [72, 95%]; hazard ratio, HR=8·77 [4·05, 13·49]; P < 0·001) and better 5-year EFS (74% [60, 88%] vs. 15% [5, 25%]; HR=0·16 [0·09, 0·29]; P < 0·001). In contrast, in subjects with none of these adverse predictive variables there was no difference in CIR and EFS between those receiving an allotransplant and those who did not. Conclusions-Our data suggest a strategy to identify which persons with AML with normal cytogenetics and no NPM1 or FLT3-ITD mutation benefit from an allotransplant. Trial Registration: Registered in the www.clinicaltrials.gov, NCT01455272 and NCT02185261. Keywords: Acute myeloid leukemia, mutations, prognosis, targeted regional sequencing, measurable residual disease, risk stratification. *Correspondence Profs. Guo-Rui Ruan and Xiao-Jun Huang Peking University Peoples Hospital and Institute of Hematology No.11 Xi-Zhi-Men South Street, Beijing 100044, China T 86-10-88324672 F 86-10-88324672 Disclosures No relevant conflicts of interest to declare.

Aim: Graft-versus-host disease (GVHD), particularly the multidrug-resistant (MDR) GVHD, is one of the most important complications for allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. The purpose of our study is to identify the efficacy of ruxolitinib in allo-HSCT recipients with MDR-GVHD. Methods: A total of 44 patients who received ruxolitinib treatment for MDR-GVHD after allo-HSCT between 2017 and 2019 were enrolled in this study (HLA-identical sibling donors: n=10; HLA-haploidentical related donors: n=34). MDR-GVHD was defined as GVHD showing no improvement after at least 3 types of treatments. Patients were treated with ruxolitinib as an add-on immunosuppression therapy at a dose of 5 mg orally twice daily, and it could be increased to 10mg twice daily if hematologic parameters were stable and no treatment-related toxicities were observed after the first 7 days of treatment. This work was supported by the Capital's Funds for Health Improvement and Research (grant number 2018-4-4089). Results: The median number of previous GVHD-therapies was 4 for both MDR-aGVHD and MDR-cGVHD. For MDR-aGVHD (n=16), the median time to response was 10 days (range, 2 to 65), and the overall response rate (ORR) was 62.5% (10/16) in, including 37.5% (6/16) complete response (CR) and 25.0% (4/16) partial response (PR). Patients with 3 organs involvement had a lower ORR compared to that of those with 1-2 organs involvements (88.9% vs 28.6%, P=0.035). The 1-year probability of overall survival after ruxolitinib was 62.1%. The rates of hematologic and infectious toxicities were 75.0% and 50.0% after ruxolitinib treatment. For MDR-cGVHD (n=28), the median time to response was 30 days (range, 6 to 270), and the ORR was 78.6% (22/28), including 28.6% (8/28) CR and 50% (14/28) PR. The 1-year probability of overall survival after ruxolitinib was 90.0%. The rates of hematologic and infectious toxicities were 46.4% and 42.9% after ruxolitinib treatment. Conclusions: Ruxolitinib is an effective and safe salvage treatment for both MDR-aGVHD and MDR-cGVHD in allo-HSCT recipients. Disclosures No relevant conflicts of interest to declare.

Background:B cell acute lymphoblastic leukemia (B-ALL) is a clonal hematopoietic stem cell neoplasm. Despite advances in its therapeutic strategies, including induction chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT), relapse remains the major barrier to the improvement of survival. Unfortunately, the underlying mechanism of relapse in B-ALL is largely unknown. Myeloid-derived suppressor cell (MDSC), a relatively novel population of immunomodulatory cells, has been identified to be associated with the onset of solid tumor and poor prognosis. However, the role of MDSCs played in prompting B-ALL relapse is vague. Aims:The present study was performed to explore the association between the percentage of MDSCs in bone marrow (BM) and B-ALL relapse and to investigate the effects of MDSCs on relapse of B-ALL. Methods:Firstly, twenty relapsed B-ALL patients and 10 non-relapsed patients after allo-HSCT as well as 14 healthy donors were enrolled. The percentage of CD33+HLA-DR-CD11b+ MDSCs in BM was analyzed by flow cytometry (FCM). Secondly, we co-cultured GFP+ Nalm-6 cells (a B cell precursor leukemia cell line initiated from an adolescent male) with MDSCs depleted bone marrow mononuclear cells (BMMCs) or replete of MDSCs from relapsed B-ALL patients post-transplantation. Then, we tested the direct effects of MDSCs on leukemia cells proliferation by co-culturing Nalm-6 cells with MDSCs. In addition, the effects of MDSCs on the functions of T cells and natural killer (NK) cells were also investigated. Briefly, CD3+ T cells or CD3-CD56+ NK cells purified from BMMCs via FCM were co-cultured with the same donor's CD11b+HLA-DR-CD33+ MDSCs using anti-CD3/CD28 beads, PMA or K562 cells as activators. The functional indexes of T cells or NK cells, including proliferation, IFN-γ, CD107a, Granzyme B, and perforin were detected by FCM. Furthermore, we analyzed the expression levels of immune response regulating proteins, including Foxp3 and PD-1 in T cells co-cultured with MDSCs. Finally, we detected the cytokines regulating MDSCs proliferation and activation in the sera of BM from relapsed B-ALL patients after allo-HSCT . Results:We demonstrated an increased percentage of MDSCs in BM of relapsed patients compared with paired non-relapsed cases (n=10 vs. 10, 28.76% vs. 5.18%, P=0.0068) and healthy donors (n=20 vs. 14, 22.76% vs. 6.57%, P=0.0007). In vitro experiments showed that the MDSCs sorted from relapsed patients' BM enhanced the proliferation of tumor cells (n=3, the mean number of GFP+Nalm-6 cells in co-culture with MDSCs group 2.2×104, vs. without MDSCs group 1.2×104, P=0.0447) in a BMMCs condition rather than a co-culture of tumor cells with MDSCs directly. MDSCs obtained from relapsed B-ALL patients' BM could suppress T cell proliferation and IFN-γ production in a cell dose-dependent manner. The percentages of proliferated CD4+T cells were 89.5%, 61.2%, 49.7%, 7.1% at a ratio of T: MDSC 1:0, 1:0.5, 1:1, 1:2 respectively (n=3, P Conclusions:Our results suggest the increased MDSCs might contribute to the relapse of B-ALL via promoting leukemia proliferation, suppressing the function of T cells and NK cells. Our findings suggest that MDSC could be served as a potential therapeutic target for this disease. Disclosures No relevant conflicts of interest to declare.

We report the results of a prospective, patient self-selected study evaluating whether haploidentical related donor stem cell transplantation (HRD-HSCT) is superior to chemotherapy alone as postremission treatment for patients with intermediate- or high-risk acute myeloid leukemia (AML) in first complete remission (CR1). Among totally 419 newly diagnosed AML patients, 132 patients with intermediate- and high-risk cytogenetics achieved CR1 and received chemotherapy alone (n = 74) or HSCT (n = 58) as postremission treatment. The cumulative incidence of relapse at 4 years was 37.5% ± 4.5%. Overall survival (OS) and disease-free survival (DFS) at 4 years were 64.5% ± 5.1% and 55.6% ± 5.0%, respectively. The cumulative incident of relapse for the HRD-HSCT group was significantly lower than that for the chemotherapy-alone group (12.0% ± 4.6% vs 57.8% ± 6.2%, respectively; P < .0001). HRD-HSCT resulted in superior survival compared with chemotherapy alone (4-year DFS, 73.1% ± 7.1% vs 44.2% ± 6.2%, respectively; P < .0001; 4-year OS, 77.5% ± 7.1% vs 54.7% ± 6.3%, respectively; P = .001). Multivariate analysis revealed postremission treatment (HRD-HSCT vs chemotherapy) and high WBC counts at diagnosis as independent risk factors affecting relapse, DFS, and OS. Our results suggest that HRD-HSCT is superior to chemotherapy alone as postremission treatment for AML.

Previous authors have suggested that tumor suppressor expression promotes aging while preventing cancer, but direct experimental support for this cancer-aging hypothesis has been elusive. Here, by using somatic, tissue-specific inactivation of the p16INK4a tumor suppressor in murine T- or B-lymphoid progenitors, we report that ablation of p16INK4a can either rescue aging or promote cancer in a lineage-specific manner. Deletion of p16INK4a in the T lineage ameliorated several aging phenotypes, including thymic involution, decreased production of naive T cells, reduction in homeostatic T-cell proliferation, and attenuation of antigen-specific immune responses. Increased T-cell neoplasia was not observed with somatic p16INK4a inactivation in T cells. In contrast, B lineage–specific ablation of p16INK4a was associated with a markedly increased incidence of systemic, high-grade B-cell neoplasms, which limited studies of the effects of somatic p16INK4a ablation on B-cell aging. Together, these data show that expression of p16INK4a can promote aging and prevent cancer in related lymphoid progeny of a common stem cell.

The level of minimal residual disease (MRD) is one of the most important prognostic indicators for acute lymphoblastic leukemia (ALL). In this study, the data about 390 cases of ALL patients who obtained MRD negative after chemotherapy or CART therapy underwent allogenic hematopoietic stem cell transplantation (HSCT) in our center were retrospectively analyzed. The MRD was detected by flow cytometry or molecular methods such as fusion genes or gene mutations. 235 were males and 155 were females. Median age was 15 years old (range 2-64). According to 2016 WHO classification, the diagnosis were Pro-B-ALL(n=24), Common B-ALL(n=113), Pre-B-ALL(n=17), Hyperdiploid (n=8), BCR-ABL positive B-ALL(n=51), MLL rearranged B-ALL (n=19), TEL-AML positive B-ALL (n=13), E2A-PBX B-ALL (n=16), BCR-ABL1-like B-All (n=4), Pro-T-ALL(n=8), Pre-T-AL(n=21), Cortical T-ALL(n=17), Medullary T-ALL (n=6) and ETP T-ALL(n=1). Other 5 patients have complex karyotypes and 67 patients cannot be grouped because of absence complete information about immunophenotypes or genetic profiles. Total number of B-ALL was 295 (54 of which received CART therapy before transplant.) and T-ALL was 92. Another one was T-B mixed lineage and remaining two's lineages was unknown. 14% of the patients had sibling identical donors (n=54), 66% of the patients had haplo-identical relative donors (n=258) and others are unrelated donor (n=77) or cord blood (n=1) transplantation. Disease status before transplant are CR1 (n=228), CR2(n=134) and ≥CR3(n=28). MNC dose was 8.30(2.50-22.60)×108/kg, CD34+cells dose was 4.51(0.89-19.61) ×106/kg and CD3+cells was 1.58(0.01-37.63) ×108/kg. Preparative regimens were based on TBI (n=352) or Bu (n=38). The median time to neutrophil and platelets engraftment was 14 and 12 days. Five-years OS and disease-free survival (DFS) for all patients were 71.7% and 71.1% . Univariate analysis showed difference of impact on overall survival about the patient' gender, age, T or B immunotype, WHO classification, whether or not receiving CART therapy before transplant, conditioning regimen based on TBI or Bu, years of transplant, and time from diagnosis to transplant (≤1year, >1year, ≤2year, >2year, ≤3year, >3year) was not statistically significant. 5-ys OS for CR1, CR2, >CR2 were 77.4%, 60.9%, 67.8% (p=0.0018). Five-years OS for sibling-identical , unrelated donor and haplo-identical transplant were 74.8%, 68.8% and 79.6% (p=0.026). The incidence of gradeⅠ-Ⅱ aGVHD and grade Ⅲ-ⅣaGVHD within 100 days were 1.5% and 2.1%. Limited cGVHD was 10.3% and extensive cGVHD was 9.2%. 5 years cumulative relapse rate was 12.2%. In summary, when MRD were negative before transplantation, the result of allo-HSCT for ALL is good. The factors affecting the survival rate are disease status and interval between diagnosis and transplant time rather than gender, age, WHO classification, immunotype, whether or not receiving CART therapy and conditioning regimen. Disclosures No relevant conflicts of interest to declare.

We aimed to identify the impact of stool cultures on clinical outcomes among patients undergoing haploidentical hematopoietic stem cell transplantation (haplo-HSCT) (n=332). Positive stool cultures (PSC group) were observed in 61 patients (18.4%). Enterobacteriaceae in stool specimens was associated with a higher risk of bloodstream infection, and Candida in stool specimens was associated with a higher risk of platelet engraftment failure. The cumulative incidence of non-relapse mortality at 1 year after haplo-HSCT in the PSC group was higher than that of the patients who showed persistently negative stool cultures (NSC group; 22.2% vs. 12.7%, P=0.059). The probabilities of overall survival (71.4% vs. 83.8%, P=0.031) and disease-free survival (69.6% vs. 81.0%, P=0.048) at 1 year after haplo-HSCT were significantly lower for the PSC group than those of the NSC group, particularly for patients who had Candida in their stool specimens. In multivariate analysis, Candida in stool specimens significantly increased the risk of non-relapse mortality and was associated with poorer survival. Our results showed that PSC impacted the clinical outcomes after haplo-HSCT, particularly for those who had Candida in their stool specimens. Disclosures No relevant conflicts of interest to declare.

Introduction Myeloid-derived suppressor cells (MDSCs) are proposed to control graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the definition of human MDSCs has not yet reached consensus. Granulocyte colony-stimulating factor (G-CSF) has been routinely used to mobilize stem cells to peripheral blood in healthy donors. It was also recognized as a novel mediator of T-cell tolerance. However, the effects of G-CSF administration on donor-derived MDSCs and the further regulatory effects of these MDSCs on GVHD remained unclear. Amis The aim of this study is to evaluate the in vitro and in vivo effects of G-CSF expanded, donor-derived MDSCs (HLA-DR-/lowCD16-CD33+) in preventing acute GVHD after allo-HSCT. Methods The frequency and cell numbers of different kinds of MDSCs in peripheral blood before and after G-CSF administration from 10 healthy donors were analyzed by flow cytometry. Cells morphological features were detected by May-Grünwald-Giemsa cytospin. Secondly, the suppressive and regulatory functions of HLA-DR-/lowCD16-CD33+ population on CD3+ T cells were assessed via in vitro experiments. A humanized xenogeneic acute GVHD model was established to determine whether this population could prevent acute GVHD in vivo. Furthermore, a clinical prospective cohort study enrolled one hundred consecutive transplant recipients was performed to assess the effects of HLA-DR-/lowCD16-CD33+ contained in HSC grafts on the occurrence of acute GVHD. Results The findings of this study include: First, a novel phenotype of HLA-DR-/lowCD16-CD33+ MDSCs with suppressive function and morphological features similar to those of immature monocyte was identified. The median of percentages of this subset were significantly increased both in peripheral blood (PB, 6.5% vs. 4.6%, P=0.0122) and peripheral blood stem cells harvest (PBSCs, 15.5% vs. 4.6%, P Conclusion Our results suggest that MSDCs with HLA-DR-/lowCD16-CD33+ phenotype in G-CSF-mobilized PBSCs have monocytic features and immune-regulatory properties, which could alleviate acute GVHD in the allo-HSCT settings. Key words: Myeloid-derived suppressor cells; granulocyte colony-stimulating factor; graft-versus-host disease Figure Figure. Disclosures No relevant conflicts of interest to declare.

Myeloid malignancies including AML, CMML, MPN and MDS are considered clonal blood disorders. Hematopoietic stem and progenitor cells (HSPCs) with mutation(s) in AML-related genes such as TET2 or DNMT3A represent what are commonly defined as preleukemic HSPCs. The selection and expansion of preleukemic-HSPC clones precedes the development of AML. Additionally, preleukemic-HSPCs can transform through serial acquisition of additional somatic mutations over time and contribute to the development of full-blown AML. What is unclear is the nature of environmental signals that might contribute to the "switch" from a preleukemic state to a leukemic state in these cells. In this context, inflammation has been hypothesized to play an essential role, but precisely how inflammatory signals influence the growth, survival, differentiation and the overall engraftment potential of preleukemic-HSPCs is yet to be determined. Mouse models carrying loss of function alleles in Tet2 or Dnmt3a manifest an expanded HSPC pool, including a hematopoietic stem cell (HSC)-enriched fraction defined by cell surface markers Lineage-/Sca-1+/c-Kit+ (LSK). Some of these genetically modified mice go on to develop CMML or MPN with modest penetration when aged. Majority of pre-leukemic mutations on their own are insufficient to cause AML in mice, suggesting that a single mutation among the above-described mutations define a pre-leukemic state and perhaps additional cooperating mutations are necessary to provide a more effective selection advantage for preleukemic-HSPC leading to the development of full-blown leukemia. Inflammation has been linked to tumor induction and transformation in solid tissues. Inflammation caused by environmental exposure, infection, autoimmunity, or ageing may result in mutations and genomic instability in somatic cells as well as in reprogramming of the tumor microenvironment (i.e. through regulating angiogenesis and expression of cytokines and chemokines). Considering that both innate and adaptive immune cells are generated from HSPCs and are involved in regulating local as well as whole-body inflammatory processes, the relationship between inflammation and hematopoietic malignancies is likely to be complex. While the influence of inflammatory stress on normal HSPCs has recently gained significant attention, little is known about how preleukemic HSPCs respond to inflammation. Because HSPCs reside in the bone marrow and are surrounded by mature immune cells, the inflammatory microenvironment is likely to influence the growth and self-renewal of these cells in part by producing pro-inflammatory cytokines and chemokines. In support of this hypothesis are epidemiologic findings demonstrating that infection may act as a trigger for AML development in humans. In the present study, we asked whether Tet2-deficient HSPCs maintain growth and survival advantage during pathological stress by examining how Tet2-KO preleukemic-HSPCs respond to acute and chronic inflammation. We show a rapid increase in the frequency and absolute number of Tet2-deficient mature myeloid cells and HSPCs in response to acute inflammatory stress, which results in enhanced production of inflammatory cytokines, including IL-6, and resistance to apoptosis. Functionally, Tet2-deficient preleukemic-HSPCs showed resistance to inflammation-induced damage and apoptosis relative to controls. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic lncRNA Morrbid in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss, with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2-deficient mice rescues inflammatory stress-induced abnormalities in HSPCs and mature myeloid cells including clonal hematopoiesis. Our results suggest that the anti-inflammatory drugs E3330 and SHP099, which suppress the hyper-activation of Morrbid, could be of clinical benefit for TET2 mutations-induced clonal hematopoiesis and leukemogenesis. Our results provide insight into the selection advantage that might render Tet2-deficient HSPCs susceptible to transformation and suggest that anti-inflammation therapy could be of clinical benefit for individuals carrying TET2 mutations. Disclosures Kelley: Apexian Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Mohseni:Novartis Institutes of Biomedical Research: Employment.

Introduction: Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation. Mesenchymal stem cells (MSCs) can modulate immune response and have been used as a treatment for aGVHD. The immune-modulating factors of MSCs are secreted and reside in supernatant fractions that are enriched for extracellular vesicles (EVs). MSC-derived EVs (MSC-EVs) also exhibit immunosuppressive activity, providing many advantages compared to MSCs and have been proven therapeutic in aGVHD. Arsenic trioxide (ATO) exhibits potent antitumor effects and increasing studies indicate its immunosuppressive effects. However, ATO at high concentrations can cause severe adverse effects. If encapsulated in some kind of drug vehicles, ATO can be made less toxic. Therefore, we believed that the combination of MSC-EVs with a low dose of ATO would be an effective therapy for aGVHD. Methods: We used a classical GVHD model (BALB/c→B6) and developed 4 groups: the control group (TCD-BM), the GVHD control group (TCD-BM + spleen T cells), the MSC-EVs treatment group and the MSC-ATO-EVs (MSC-derived ATO-encapsulating EVs) treatment group. OS, GVHD clinical and histological scores were evaluated. A20-luc lymphoma cells were injected to generate the GVL model. Using flow cytometry analysis, we analyzed Th cell subsets, cytokines and transcription factors (Th1*IFN-γ/TNF-α*T-bet, Th2*IL-4*GATA3, Th17*IL-17*RORγt, Treg*IL-10*Foxp3) and sorted CD8+ SLECs, and CD8+ MPECs in BM and spleen of recipients. Dll4 expression was analyzed on DCs. B6 cells were incubated with or without BALB/c spleen cells and complete medium alone, with 10 mM ATO alone. T cell apoptosis was determined with Yopro-1 staining. We used MLR assays to examine Th subsets, cytokines and notch targeted genes with or without ATO or neutralizing Ab specific to Dll4 (anti-Dll4). Results: BALB/c mice receiving B6 TCD-BM alone developed no sign of GVHD, whereas all BALB/c mice receiving B6 donor TCD-BM + spleen T cells died of GVHD. In contrast, injection of MSC-EVs and MSC-ATO-EVs inhibited GVHD in T cell recipients, with 20% and 29% of them surviving without severe GVHD, respectively. These survival rates were accompanied by significantly lower clinical and histological scores. GVL effects mediated by MSC-EVs and MSC-ATO-EVs were comparable to those obtained in the GVHD control group. Compared to the control group, CD4+T and CD8+T cells increased substantially in T cell recipients, resulting in severe GVHD. In contrast, treatment with MSC-ATO-EVs significantly reduced the number of CD4+T and CD8+T cells, while MSC-EVs recipients retained approximately the same number of T cells as the GVHD group. Compared to the GVHD control group, Th2 and Treg cells derived from the spleen increased, while Th1 and Th17 cells were reduced significantly in both the MSC-EVs and MSC-ATO-EVs groups. We also detected lower serum levels of TNF-α and IFNγ as well as lower expression of RORγt and T-bet in blood and BM CD4+ T cells in these two groups, while the expression of GATA3 and Foxp3 increased significantly. Treatment with MSC-ATO-EVs markedly raised the MPEC/SLEC ratio compared to the MSC-EVs and GVHD control groups. We also examined Dll4high DCs in different organs and different groups and found that only MSC-ATO-EVs significantly reduced the Dll4high DCs, especially in the spleen and intestine. Treatment of stimulated B6 CD4+ T and CD8+ T cells with ATO increased production of H2O2. Yopro-1 staining of activated B6 CD4+ T and CD8+ T cells indicated that ATO dramatically triggered apoptosis in those cells. DCs were isolated and cultured with B6 mouse-derived CD4+ T or CD8+ T cells, with or without addition of ATO or anti-Dll4. ATO and anti-Dll4 both led to significant reduction of IFN-γ and TNF-α, while IL-4 and IL-10 increased slightly. We next assessed the notch pathway targeted genes in T cells and found there were significantly increased GATA3 and reduced Dtx expression levels. Conclusion: Altogether, our findings demonstrate that MSC-ATO-EVs might be a highly promising therapy for aGVHD through reducing T cell amounts and modulating Th subsets and CD8+ T cell differentiation. These effects can be explained with the inhibition of the Dll4-notch pathway by ATO. Therefore, further exploitation of the potential application of ATO in aGVHD and the mechanisms of action of ATO may improve outcomes after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Background: Human cytomegalovirus (HCMV) infection, especially persistent HCMV infection, is an important cause of morbidity and mortality after allogenic stem cell transplantation (allo-SCT). Antiviral agents remain the mainstay of treatment and are recommend as the first-line therapy for HCMV. However, drugs are associated with significant toxicity, and their efficacy is limited in the absence of cell-mediated immunity. In recent years, adoptive immunotherapy with HCMV-specific T cells (CTLs) has been developed as an alternative option for HCMV, and data from previous studies have indicated that infusion of CTLs at early-stage of HCMV infection may have better benefits compared to salavage therapy. However, because CTLs remains time consuming and cost-intensive, so far there have no reports of first-line therapy with CTLs for HCMV infection, and the mechanisms driving the sustained antiviral immunity induced by adoptive T cells transfer remain undetermined. Our previous study had demonstrated that patients with acute graft-versus-host disease (aGVHD) were at high risk to develop persistent HCMV infection. Therefore, in the current study, we selected patients who developed aGVHD before HCMV reactivated and started CTLs generation in advance. This risk-stratified measure successfully selected patients who had high risk resisting to conventional anti-HCMV therapy , and spared low risk patients as well, making it feasible and financially viable to use CTLs as a first-line therapy. Aims: To provide robust support for the safety and efficacy of CTLs given as a first-line therapy for HCMV infection after allo-SCT, and gain some insight into the underlying mechanisms. Methods: Firstly, using humanized HCMV infected mice model, we explored where the adoptive transferred CTLs cells trafficked, evaluated the antiviral efficacy of CTLs and investigated the recovery of HCMV-specific immunity after T cell transfer. Secondly, we conducted a prospective clinical trial enrolled 35 allo-SCT patients who diagnosed with acute GVHD and had high risk developing persistent HCMV infection, intervened with antiviral agents combined with CTLs as first-line therapy and evaluated the long-term safety and durability of antiviral responses. As controls, we selected a cohort of 70 high-risk patients as well as another cohort of 70 low-risk patients who only received antiviral agents as first-line therapy without CTLs. We also evaluated the immune response after infusion and analyzed the association between immune recovery and HCMV clearance. Results: i) In humanized HCMV infection mice, adoptive infused CTLs had the ability to homing to organs, and effectively combated systemic HCMV infection by promoting the restoring of stem cell derived endogenous HCMV-specific immunity. ii) In clinical trial, first-line therapy with CTLs significantly reduced the rate (2.86% vs. 20.00%, P=0.018) and the cumulative incidence (HR=7.60, 95%CI=1.22-10.15, P=0.020) of persistent HCMV infection, and showed a lower one-year treatment related mortality (TRM) (HR=6.83, 95%CI=1.16-8.90, P=0.030) and a better one-year overall survival (OS) (HR=6.35, 95%CI=1.05-9.00, P=0.040) compared to high-risk control cohort. The cumulative incidence of persistent HCMV infection, one-year TRM and OS in CTL cohort were comparable to those in low-risk control cohort. Moreover, first-line therapy with CTLs promoted the quantitative and functional recovery of CTLs in patients, which was associated with HCMV clearance. Conclusion: In this study, we firstly demonstrated the safety and efficacy of CTLs administration as a first-line therapy for HCMV infection in humanized HCMV infection mice, and in a large clinical cohort study. The data provided robust support for the benefits of donor derived CTLs in treating HCMV infection as a first-line therapy, and suggested that infused CTLs might probably stimulate the recovery of donor derived HCMV-specific immunity. This trial was registered at www.clinicaltrials.gov as #NCT02985775. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

For AML patients with positive pre-transplant minimal residual disease (MRD), determined by multicolor flow cytometry (MFC), haploidentical stem cell transplantation (haplo-SCT) could achieve lower CIR rates than human leukocyte antigen (HLA)-matched sibling donor transplantation (MSDT) (J Hematol Oncol. 2017;10(1):134).However, these data only provide indirect evidence supporting the idea that haplo-SCT might have a strong graft-versus-leukemia (GVL) effect compared with MSDT. Therefore, direct evidence is needed to determine whether treating AML with haplo-SCT has a stronger GVL effect. The real time-polymerase chain reaction (RT-PCR) has greater sensitivity and specificity than MFC for detecting leukemia-specific genes, such as RUNX1/RUNX1T1, and provides a better estimation of the leukemia burden. Based on the levels of RUNX1/RUNX1T1 transcripts determined by RT-PCR, our group found that treating high risk cases with t(8;21) AML, who did not achieve major molecular remission (MMR, defined as a >3-log reduction in RUNX1/RUNX1T1 transcripts), with allogeneic stem cell transplantation (allo-SCT) could reduce the CIR compared with chemotherapy alone (22.1% vs 78.9%, P < 0.0001). These findings suggest that allo-SCT is necessary for high-risk t(8;21) AML patients, which provides an opportunity to investigate whether haplo-SCT has a superior GVL effect compared with MSDT in treating AML. Therefore, we focused on t(8;21) AML cases with positive MRD pre-transplant who underwent allogeneic SCT. The purpose of this study was to investigate and provide direct evidence that a haploidentical allograft has superior anti-leukemia effects compared with a HLA-matched sibling allograft. In the present study, One hundred and thirty-five t(8;21) acute myeloid leukemia (AML) patients with positive pre-transplantation minimal residual disease who underwent a haplo-SCT or MSDT were enrolled in this study. The levels of RUNX1/RUNX1T1 transcripts were monitored and quantified by real-time quantitative PCR. The 3-year cumulative incidence of relapse (CIR), non-relapse mortality (NRM), leukemia-free survival (LFS), and overall survival (OS) were 19%, 21%, 60%, and 61%, respectively. The levels of RUNX1/RUNX1T1 were significantly lower in Haplo-SCT patients compared with MSDT patients at 60 (P=0.039) and 90 (P=0.004) days after transplant (Figure 1). Compared with pre-transplant, the leukemia burden in the haplo-SCT group was significantly more reduced than in the MSDT group at 30 (P=0.068), 60 (P=0.005), 90 (P Figure 1. Kinetics of log-transformed leukemia burden reduction (evaluated by RT-PCR for RUNX1/RUNX1T1) in all patients and subgroup cases who either received haplo-SCT or MSDT, using a repeated measures ANOVA. Disclosures No relevant conflicts of interest to declare.

Introduction: CD19-CAR-T cells induce high rates of initial response among patients with refractory/relapsed and MRD positive high risk B-cell acute lymphoblastic leukemia (B-ALL). Afterwards allogeneic hematopoietic stem cell transplantation (HSCT) can further reduce relapse rate. Our previous results had shown that CR obtained by CD19 CAR-T had comparable significance to CR by chemotherapy before Allogeneic HSCT in B-ALL. Patients and Methods: Between July 2015 and Mar 2018, consecutive 135 patients with refractory/relapsed or high risk B-ALL obtained CR with CD19-CART therapy followed by allogeneic HSCT were retrospective analyzed. Median follow-up of survivors was 13 months (range, 3-32 months). Results: The median age was 11 (2-49) years. The median disease course before transplant was 21(4-143) months. The median time from CART therapy to HSCT was 69 (35-312) days. Disease status was 108 cases relapsed diseases, 11 cases refractory, and 16 persistent/recurrent measurable residual diseases (MRD). MRD pre-conditioning measured by flow cytometry and QT-PCR was positive in 20(14.8%) subjects. Donor source was haploidentical donors in 107(79.3%), identical sibling in 7(5.1%), and unrelated in 21(15.6%). Most subjects (87.4%) received conventional myeloablative pretransplant conditioning with total body radiation (TBI), the rest with busulfan (Bu). Antithymocyte globulin was used in haploidentical and unrelated transplants. Cyclosporine, short-term methotrexate, and mycophenolate mofetil were employed for GVHD prophylaxis. There were no cases of graft-failure except one early death on Day 0 for septic shock. The median time to neutrophil engraftment was 14 days (10, 26 days), and median time to platelet engraftment 14 days (5, 70 days). The incidences of non-relapse mortality within 100 days were 4.4% (0.8, 7.9%) The incidence of grades II-IV acute graft-versus-host disease (GvHD) were 32.1% (24.3, 39.9%) and grades III-IV GvHD 10.5% (5.4, 15.6%). Chronic GvHD and extensive chronic GvHD were 69.7% (60.7, 78.7%) and17.6% (10.7, 24.5%). Cumulative incidence of relapses (CIRs) at 2-year was 11.1% (5.4, 16.8%). There were totally 14 subjects relapsed after HSCT, among which 8 were CD19 negative relapse, 5 CD19 positive and 1 partial CD19 positive. And among the 8 CD19 negative relapse after transplant, 4 subjects had CD19 negative MRD before conditioning. Leukemia-free survival (LFS) was 76.5% (64.2, 88.8%) and overall survival (OS) was 80.8% (72.6, 89.0%) at two years after transplant. In multivariate analysis subjects who were MRD- positive pre-transplant had a higher 2-year CIR (43.5% [18.4, 68.6%] vs. 5.9% [1.2, 10.6%]; p=0.000) and worse 2-year OS (61.5% [35.6, 87.4%] vs. 83.6% [75, 92.2%]; p=0.034). Conclusions: Our clinical results showed that CART therapy followed by allogeneic HSCT was a promising modality for refractory/relapsed B-ALL. CD19 negative relapse accounted for most relapse after allogeneic HSCT. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background Poor hematopoietic reconstitution including poor graft function (PGF), characterized by pancytopenia, and prolonged isolated thrombocytopenia (PT), defined as the engraftment of all peripheral blood cells other than platelets, remains a life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and the clinical management is challenging. Endothelial cells (ECs) play a crucial role in regulating hematopoiesis in bone marrow (BM) microenvironment. N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, is clinical used as a mucolytic drug. In this regard, we reported that PGF and PT patients had impaired BM ECs post-HSCT (BBMT 2013; BMT2016;Blood 2016; AJH2018). Recently, we reported in vitro treatment with NAC could improve the defective HSCs through repairing the impaired BM ECs of PGF and PT patients (Blood 2016; AJH2018). However, our previous studies evaluated BM ECs in PGF and PT patients at +3 month(M) post-HSCT. Therefore, whether BM ECs dysfunction in PGF and PT patients is responsible for the defective hematopoiesis, or vice versa, requires to be further clarified. Moreover, prophylactic intervention to improve the impaired BM ECs and HSCs remains unidentified. Aims In order to investigate whether the defective BM ECs pre-HSCT is the risk factor for the occurrence of PGF and PT post-HSCT. Moreover, to evaluate whether prophylactic NAC intervention could repair the impaired BM ECs and reduce the incidence of PGF and PT. Methods Two registered prospective clinical trials were included. The first trial compared the dynamic reconstitution of the BM ECs pre- and post-HSCT among PGF, PT, and good graft function (GGF) patients at Peking University People's Hospital. Multivariate analyses were performed to identify the risk factors for the occurrence of PGF and PT. Receiver operating characteristic (ROC) curves were used to identify the cut-off percentage of BM ECs pre-HSCT to predict high risk patients for PGF and PT. Subsequently, the second trial was performed to investigate whether prophylactic NAC intervention could reduce the incidence of PGF and PT and its underlying mechanism. The quantity and function of BM ECs were evaluated at -14 day (D), 0D pre-HSCT, and +1M, +2M post-HSCT in the patients who were willing to provide BM samples after the written consent. Results In the first trial, 15 patients of the enrolled 68 patients developed PGF or PT, whereas the remaining 53 patients were GGF at +2M post-HSCT. PGF and PT patients demonstrated impaired BM ECs at -14D pre-HSCT and defective dynamic reconstitution at +1M, +2M post-HSCT. Moreover, the BM ECs impairment positively correlated with their ROS levels. Multivariate analysis identified BM EC The second trial enrolled EC Summary / Conclusion: BM EC Disclosures No relevant conflicts of interest to declare.

Introduction Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by abnormal immune response. Though many therapies have been used, corticosteroid-resistance remains to be a challenge clinically. Extensive research has improved our understanding of ITP, showing that environmental factors affect the disease profile, such as Helicobacter pylori being proven to be associated with thrombocytopenia. Though evidence that the gut microbiota contributes to the development of auto-immune disorders is accumulating, there is no information available on relationship between gut microbiome and ITP. Berberine(BBR), a traditional compound isolated from a Chinese herb, has been widely used as a nonprescription drug to treat diarrhea. Recently, BBR has been reported to modulate microbiota structures, which contributes to improving metabolic disorders. Here, we hypothesized that BBR might modulate gut microbiota to treat ITP. Methods In order to investigate the relationship between gut microbiome and ITP, we performed deep shotgun sequencing on 253 fecal samples totally from consecutive ITP patients and healthy controls. Metagenome-wide association study (MWAS) was conducted, and clinical characteristics of patients were collected to analyze the correlation with gut microbiome (Nan Qin, et al. Nature. 2012). Certainly, a clinical cohort study was performed to assess the efficacy of BBR in corticosteroid-resistant ITP patients. To better characterize the role of gut microbiota in the development of ITP and to verify the modulating effect of BBR on gut microbiota structure, we performed colonization of mice with specific bacterium and established active murine models (immunized-splenocyte engraftment) of BBR treatment. Result We integrated the sequencing data into an existing gut microbial reference-gene catalog and identified 35275 genes that differ in abundance between ITP patients and healthy controls. We then clustered the genes into metagenomic species (MGS) and finally identified 15 MGS which were significantly different in both discovery cohort and validation cohort. Dysbiosis was detected in the gut microbiome of ITP patients, as both phylogenetic analysis and MGS annotation indicated that Lachnospiraceae bacterium, Clostridium asparagiforme were over-represented while Bacteroides spp was depleted in ITP patients comparing with healthy controls. Functionally, KEGG annotation showed that the most enriched orthologs in ITP patients were related to membrane transport. Moreover, the biosynthesis of microbe-associated molecular patterns (MAMPs) such as lipopolysaccharides (LPS), peptidoglycan biosynthesis and flagellin were highly abundant in patients. Gene biomarkers and cluster markers based on gut microbiome were established to identify ITP patients and were validated in an independent cohort. The alterations of gut microbiome in ITP patients are partly reversed after treatment. Furthermore, L. bacterium shows more abundant in corticosteroid-resistant ITP comparing with newly diagnosed ITP. Specifically, BBR treatment could improve the microbial dysbiosis of corticosteroid-resistant ITP patients, the complete response (CR), response(R), and overall response (OR) rates being 26.3%, 47.4% and 73.7%, respectively. Targeted QPCR assay determined that L. bacterium accumulated in corticosteroid-resistant ITP, consistent with the result of shotgun sequencing data. Gavage with L. bacterium results in significantly alterations of gut microbiota structure in mice comparing with mice without bacterial administration or those receiving Clostridium asparagiforme administration. Moreover, colonization of L. bacterium caused more severe thrombocytopenia and impaired the response to corticosteroid therapy in active ITP model. Intriguingly, BBR treatment, but not any other antibiotics, could reverse the effect of L. bacterium colonization on gut microbiota structure and enhance the response to corticosteroid therapy. Conclusion Our findings demonstrate that the gut microbiome alters in ITP and is partly normalized after treatment. Gut microbiota dysbiosis may contribute to the development of corticosteroid-resistant ITP. BBR may correct corticosteroid-resistance by modulating the gut microbiota structure, thus being a novel potential second-line candidate to treat ITP. Disclosures No relevant conflicts of interest to declare.

Background Bone disease is an important determinant of quality-of-life and survival of persons with plasma cell myeloma (PCM). Biomarkers of bone disease could be useful to predict risk and monitor therapy. In our prior analyses OCSTAMP (osteoclast stimulatory trans-membrane protein) mRNA levels were identified as increased above normals in persons with PCM. OCSTAMP encodes a membrane-anchored cell surface receptor promoting nucleation of osteoclasts involved in bone resorption and osteoclast differentiation. Aims Measure levels of OCSTAMP mRNA in subjects with PCM and interrogate clinical associations. Methods OCSTAMP mRNA levels were quantified by quantitative real-time polymerase chain reaction (RT-qPCR) in 224 bone marrow samples from 160 subjects with PCM including 160 newly-diagnosed; 55 in remission and 9 with recurrent PCM. Results were compared with 42 normals and data expressed as ratio of OSSTAMP mRNA/ABL mRNA. Associations with clinical variables were interrogated and comparisons analyzed using the Chi-square test. Results OCSTAMP mRNA levels were significantly greater than normals in 111 subjects (69%, [95% confidence interval l[CI], 62-77%], P Conclusions OCSTAMP is highly transcribed in persons with newly-diagnosed and recurrent PCM compared with normals whereas persons in remission have similar levels to normals. OCSTAMP mRNA levels correlate with several clinical variables including Durie/Salmon stage, serum albumin C-reactive protein levels and likelihood of pathological bone fractures. OCSTAMP mRNA levels are potentially useful as a biomarker if our data are validated. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Leukemia is the most common malignant tumor in children under 15 years old, which is divided into several subtypes, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL) and chronic myeloid leukemia (CML), based on the disease phases and effected cells. Each subtype has its specific molecular feature and key regulation factors. In our previous studies, we reported that β-arrestin1 (ARRB1), the pivotal scaffold protein to transduce various cellular signals, could bind with EZH2 to increase Bcr/Abl H4 acetylation level and thus promote CML progression (Brit J Cancer 2014, 111(3): 568-76). ARRB1 could enhance DNMT1 activity and PTEN methylation, decrease PTEN expression and promote self-renew of B-ALL leukemia initiating cells (LICs) (Cancer Lett 2015, 357(1): 170-8.). ARRB1 could increase P300 to bind with SP1 to hTERT promoter, and thus increase hTERT transcription/expression, telomerase activity, telomere length and cell senescence in B-ALL LICs (Cell Death Diff 2017, 8(4): e2756.). However, little is known in the T-ALL, which about 70% have the mutations of NOTCH1 gene. Here, we unveil ARRB1 could curb the progression of T-ALL cells in vitro and in vivo, while the expression of ARRB1 was suppressed by the aberrant increased miR-223. Mechanistically, ARRB1 could recruit DTX1, the E3 ubiquitin ligase, to promote the ubiquitination and degradation of NOTCH1 protein in T-ALL. Furthermore, Overexpression of ARRB1-derived miR-223 sponge BUTR was incompatible with cell proliferation and induces apoptosis in T-ALL cells. Collectively, our results for the first time revealed that ARRB1 acted as a tumor suppressor by promoting NOTCH1 degradation in T-ALL cells where miR-223 effectively antagonized ARRB1 functions. This provides that miR-223 may serve as a valid drug target for developing novel and efficacious T-ALL therapeutics. Disclosures No relevant conflicts of interest to declare.