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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.

The study aimed to compare the efficacy and safety of all-trans retinoic acid (ATRA) plus low-dose rituximab (LD-RTX) with LD-RTX monotherapy in corticosteroid-resistant or relapsed immune thrombocytopenia (ITP) patients. Recruited patients were randomized at a ratio of 2:1 into 2 groups: 112 patients received LD-RTX plus ATRA, and 56 patients received LD-RTX monotherapy. Overall response (OR), defined as achieving a platelet count of ≥30 × 109/L confirmed on ≥2 separate occasions (≥7 days apart), at least a doubling of the baseline platelet count without any other ITP-specific treatment, and the absence of bleeding within 1 year after enrollment, was observed in more patients in the LD-RTX plus ATRA group (80%) than in the LD-RTX monotherapy group (59%) (between-group difference, 0.22; 95% CI, 0.07-0.36). Sustained response (SR), defined as maintenance of a platelet count >30 × 109/L, an absence of bleeding, and no requirement for any other ITP-specific treatment for 6 consecutive months after achievement of OR during 1 year following enrollment, was achieved by 68 (61%) patients in the combination group and 23 (41%) patients in the monotherapy group (between-group difference, 0.20; 95% CI, 0.04-0.35). The 2 most common adverse events (AEs) for the combination group were dry skin and headache or dizziness. Our findings demonstrated that ATRA plus LD-RTX significantly increased the overall and sustained response, indicating a promising treatment option for corticosteroid-resistant or relapsed adult ITP. This study is registered at www.clinicaltrials.gov as #NCT03304288.

INTRODUCTION Steroid-resistant (SR) acute graft-versus-host disease (aGVHD) lacks standard second-line treatment. Mesenchymal stromal cells (MSCs) have potential efficacy in SR aGVHD. To assess efficacy and safety of MSCs combined with anti-CD25 antibody and calcineurin inhibitors as second-line therapy for SR aGVHD. METHORDS A randomized phase 3 trial involved 203 SR aGVHD patients at 10 centers in China (September 2014-March 2019). Final follow-up was on June 30, 2020. Participants were randomized in a 1:1 ratio to receive second-line therapy (anti-CD25 antibody combined with calcineurin inhibitors) with MSCs (n=101) or without MSCs (n=102). The primary endpoint was overall response (OR) at day 28 with a predefined threshold of -20%. Secondary and safety endpoints included durable OR at day 56, failure-free survival, overall survival (OS), chronic GVHD (cGVHD), infection, haematological toxicity and relapse. RESULTS Of 203 patients, 198 (97.5%; mean age, 30.1years; 40.4% women) completed the study. OR at day 28 was higher in the MSCs group than in the control group (82.8% [82 patients] vs 70.7% [70]; odds ratio, 2.00; 95% confidence interval [CI], 1.01-3.94; P=.043). Durable OR at day 56 was also higher in the MSCs group (78.8% [78 patients] vs. 64.6% [64]; odds ratio, 2.02; 95% CI, 1.08-3.83; P=.027). The median failure-free survival was longer in the MSCs group compared with control group (11.3 months vs. 6.0 month; hazard ratio (HR) 0.68; 95% CI, 0.48-0.95, P=.024). The 2-year cumulative incidence of cGVHD was 39.5% (95% CI, 29.3%-49.4%) and 62.7% (51.4%-72.1%) in the MSCs and control groups (HR 0·55, 95% CI, 0·36-0·84; P=0·005). Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections (65 [65.7%] in the MSCs group vs 78 [78.8%] in the control group), haematological toxicity (37 [37.4%] vs 53 [53.5%]). CONCLLUSIONS MSCs plus second-line treatments may increase the efficacy of SR aGVHD, decrease drug toxicity of second-line therapy and cGVHD development, and are well tolerated. Disclosures No relevant conflicts of interest to declare.

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.

Introduction Approximately one-third of immune thrombocytopenia (ITP) patients fail to achieve a response with first-line treatments, and most patients who respond to first-line treatment relapse and require further second-line therapies. Rituximab (RTX) has been frequently used in ITP treatment, and proposed schedules include the standard dose (SD) and low dose (LD). Previous studies on LD-RTX in ITP revealed a comparable overall response (OR) and reduced incidences of short- and long-term complications (e.g., infections) compared with SD-RTX (Blood, 2012; Platelets, 2019). However, LD-RTX does not achieve a rapid and long-lasting response in the treatment of ITP. Our preliminary studies demonstrated the efficacy of all-trans retinoic acid (ATRA) in the ITP model due to its effects on inducing megakaryocyte maturation and its immunomodulatory effects (Stem Cells Dev, 2017; J Thromb Haemost, 2017; Br J Haematol, 2018; Haematologica, 2019). Since RTX and ATRA share disparate mechanisms in treating ITP, a combination of RTX and ATRA may work synergistically based on a "double-hit" mechanism targeting both platelet production and destruction, which may overcome the long TTR and improve the SR rate of LD-RTX. Therefore, our study aimed to determine the efficacy and safety of ATRA plus LD-RTX compared to LD-RTX monotherapy in patients with corticosteroid-resistant or relapsed ITP (NCT03304288). Methods Eligible corticosteroid-resistant or relapsed ITP patients with a platelet count < 30 × 10 9/L or bleeding symptoms at enrolment were randomly allocated in a 2:1 ratio to receive 100 mg of RTX weekly for 6 weeks plus ATRA orally at 20 mg/m 2 daily for 12 weeks or 100 mg of RTX weekly for 6 weeks. Stable doses of concomitant corticosteroids, danazol, and cyclosporin were permitted. Treatment was discontinued if platelet counts in two consecutive tests at least 2 weeks apart were more than 300 × 10 9/L and resumed when the platelet count fell to less than 150 × 10 9/L. Assessments were performed at baseline, weekly during the first 4 weeks, every 2 weeks until week 24, and every 4 weeks thereafter. The primary outcome was OR, defined as a platelet count ≥ 30 × 10 9/L, a doubling of the baseline platelet count and the absence of bleeding. Secondary endpoints included sustained response (SR), initial response, complete response, time to response, duration of response and adverse events. SR was defined as maintenance of a platelet count > 30 × 10 9/L, the absence of bleeding, and no requirement for other ITP-specific treatment for 6 consecutive months. Results One hundred sixty-eight patients from 7 tertiary medical centres in China were included between 2017 and 2020, 112 patients received combination treatment of LD-RTX and ATRA, and 56 patients received LD-RTX monotherapy. All the baseline characteristics were comparable between the two groups. The median ages of patients in the LD-RTX plus ATRA group and LD-RTX monotherapy group were 46.0 years and 44.5 years, respectively. For patients in the combination and monotherapy groups, the median number of previous therapies was 2.0. The median baseline platelet count was 17.0 × 10 9/L in the combination group and 19.0 × 10 9/L in the monotherapy group. All the patients were followed up for more than 11 months. OR was observed in 90 (80.4%) patients in the combination group and 33 (58.9%) in the LD-RTX monotherapy group (between-group difference, 0.22; 95% CI, 0.07 to 0.36), indicating that the combination treatment increased the response rate. SR was achieved by 68 (60.7%) patients in the combination group and 23 (41.1%) patients in the monotherapy group at the 6-month follow-up (between-group difference, 0.20; 95% CI, 0.04 to 0.35). Additionally, 56 (50%) subjects in the combination group and 19 (34%) in the monotherapy group were still in sustained remission at 12 months (between-group difference: 0.16; 95% CI, -0.01 to 0.33). No significant difference was found between the 2 treatment groups in terms of the time to a response. The severity of AEs was primarily of grade 1-2. The 2 most common AEs for the combination group were dry skin and headache or dizziness, with incidences of 40.2% (45/112) and 18.8% (21/112), respectively. Conclusions In conclusion, ATRA plus LD-RTX significantly increased the overall and sustained response, indicating a novel promising treatment option for corticosteroid-resistant or relapsed adult ITP patients. 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 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.

Background: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear. Aims: To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients. Methods: In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes. Results: In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression. Summary/Conclusion: In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients. Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation 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.

Background Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. Immunosuppressive therapy can rescue most patients with AA. However, the pathogenesis of AA is still not well elucidated and the new strategies need to be developed for AA patients. Increasing evidences suggested the dysfunctional BM microenvironment may be involved in the pathogenesis of AA. As important components of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immune. However, whether BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve the hematopoietic and immune status of AA patients remain to be elucidated. Aims To evaluate the quantity and function of BM ECs from AA patients. Moreover, to determine whether the dysfunctional BM ECs are involved in the occurrence of AA by affecting hematopoiesis and regulating immunity in vitro and in vivo. Finally, to uncover the therapeutic potential of repairing dysfunctional BM ECs to alter the hematopoietic and immunological status in AA patients. Methods This study enrolled 30 patients with AA and 30 healthy donors(HD). Flow cytometry and BM in situ immunofluorescence staining were used to analyze the proportion of ECs in BM of the two groups. The level of intracellular reactive oxygen species(ROS) and the proportion of apoptosis were detected by flow cytometry. The functions of BM ECs were evaluated by double-positive staining, migration and tube formation assays. To determine the effect of BM ECs on hematopoiesis and immunity, primary human BM ECs were separately cocultured with CD34 + and CD3 + cells. To further validate the role of BM ECs in the occurrence of AA, a classical AA mice model and VE-cadherin blocking antibody that could antagonize the function of BM ECs were used. Moreover, to explore potential approach of targeting the dysfunctional BM ECs, the exogenous EC infusion or N-acetyl-L-cysteine (NAC, a ROS scavenger) for repairing BM ECs were administrated to the AA mice. To further explore the repairing effect of NAC on BM ECs, the primary BM ECs from AA patients were treated by NAC in vitroand then the functions of BM ECs were evaluated. Results Compared with HD, BM ECs in AA patients were decreased and dysfunctional, which characterized by higher levels of ROS and apoptosis, impaired abilities of migration and angiogenesis. Furthermore, dysfunctional BM ECs from AA patients not only impaired their hematopoiesis-supporting ability but also promoted co-cultured T cells to polarize towards pro-inflammatory T cells in vitro, which resulted in an unbalanced T cell subsets. Consistently, AA mice demonstrated decreased BM ECs with increased level of intracellular ROS. Moreover, hematopoietic failure and immune imbalance in AA mice became more severe when the function of BM ECs was antagonized, whereas the administration of NAC or infusion of exogenous EC improved the hematopoietic and immunological status of AA mice via repairing BM ECs in vivo. In addition, we found the NAC treatment also restored the hematopoiesis-supporting ability and immunity-regulating ability of the primary ECs derived from AA patients in vitro. Summary/Conclusion Our study demonstrates for the first time that dysfunctional BM ECs with impaired hematopoiesis-supporting ability and abnormal immunomodulatory ability are involved in the pathogenesis of AA. Although further validation is required, restoring dysfunctional BM ECs via EC infusion or administration of ROS scavenger NAC might be a potential therapeutic approach for AA patients. Disclosures No relevant conflicts of interest to declare.

Background: Cytomegalovirus (CMV) infection is a major and potentially life-threatening complication after allogenic hematopoietic stem cell transplantation (allo-SCT). Adoptive transfer of CMV-specific T cells (CTL) from original transplant donor or third-party donor has both emerged as an effective method for CMV infection after allo-SCT. The potential advantages of third party CTL versus transplant donor CTL includes that it is not limited by donor viral immune and can be banked in advance for clinical use. However, as the expansion and persistent of transfused third-party CTL in vivo was considered to be shorter when compared with donor CTL, can third-party CTL provide comparable long-term antiviral efficacy as transplant donor CTL? In fact, the safety and efficacy of these two kinds of CTL have not been compared directly. In addition, the mechanisms driving sustained antiviral immunity induced by these two kinds of CTL remains to be established and compared. Methods: i) We established humanized CMV-infected mouse model and tumor-infiltration mouse model, and compared the antiviral ability of transplant donor and third-party donor derived CTL for CMV infection in mouse models. We comparatively investigated the in vivo recovery of CMV-specific immunity and analyzed the underlying mechanisms driving sustained antiviral immunity induced by these two types of CTL therapy. ii) We collected data from 31 patients who received third-party CTL and selected matched pairs of 62 patients who received donor CTL for refractory CMV infection after allo-SCT, and compared the safety and efficacy of these two kinds of CTL for CMV infection in clinical patients. We also track the infused CTL populations and evaluated the recovery of virus-specific immunity in clinical patients after donor and third-party CTL therapy. Results: i) In mouse models, we observed that adoptively infused donor and third-party CTL could both migrated to the virus or tumor infiltration organs, and contributed to comparable diminishing in CMV pathology and viral burden in target organs. The kinetics of CMV-specific immunity recovery was comparable in donor and third-party CTL group, which further conferred the comparable antiviral response of these two kinds of CTL for CMV infection. When performed a detailed analysis of the recovered source of CTL, we observed a preferential proliferation and expansion of graft-derived endogenous CTL in both donor and third-party CTL therapy group. ii) In clinical patients, adoptive therapy with donor or third-party CTL had comparable clinical response without significant therapy-related toxicity. The cumulative CR rates at 4 th weeks after CTL infusion was 80.6% in donor group and 83.1% in third-party group. We also observed strong expansion of CD8 + tetramer + T cells both after donor and third-party CTL infusion, which were associated with a reduced or cleared viral load. Conclusion:Adoptive therapy with transplant donor or third-party CTL had comparable antiviral response for CMV infection by promoting the restoration of CMV-specific immunity. Our data suggest that both transplant donor or third-party CTL may stimulate the recovery of graft-derived endogenous CMV-specific immunity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Macrophages (MΦs) are an important immune population that are essential for disease pathogenesis. MΦs are now commonly classified as either M1, which produce pro-inflammatory cytokines, or M2, which produce anti-inflammatory cytokines. Our recent study reported that patients who received an allograft with a higher M1/M2 ratio exhibited a higher incidence of grade 2-4 aGVHD(BMT, 2019). Moreover, an aberrant M1/M2 polarization was found in the colon of aGVHD mice, and regulating the polarization of MΦs cultured from aGVHD patients towards M2 phenotype modulated T cells favoring a type 2 response in vitro(Sci China Life Sci, 2020). However, the primary subsets and function of MΦs in aGVHD patients, and the precise roles of different MΦ subsets in the development of aGVHD remain to be elucidated. Aims: To determine the subsets and function of MΦs in primary peripheral blood(PB) of aGVHD patients. Moreover, to investigate whether M1 and M2 had different effects on the development of aGVHD, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 20 patients with aGVHD and 20 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. MΦ subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. M1 and M2 were identified as CD14+CCR2+CD68+ and CD14+CX3CR1+CD163+, respectively. In order to determine the function of MΦs in patients with aGVHD and non-aGVHD, the phagocytosis was analyzed using a DiI-AcLDL assay. The protein expressions for the costimulatory molecules and the cytokine production of MΦs were measured by flow cytometry. To further investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of MΦs. Subsequently, to explore the role of different subsets of MΦs in the development of aGVHD, M1 and M2 were infused into aGVHD mice, respectively. Mice were monitored for survival, weight, and aGVHD score. Histological scores of tissues from aGVHD target organs (liver, intestine, spleen and skin) were evaluated by HE staining. Results: When compared with non-aGVHD patients, MΦs in primary PB of aGVHD patients were polarized towards pro-inflammatory M1, characterized by an elevated proportion of M1 and a reduced proportion of M2. Furthermore, MΦs isolated from aGVHD patients exhibited lower phagocytic function, higher expression of TNF-α and IL-6 and higher expression of costimulatory molecules CD80 and CD86. Consistent with the increased activated MΦs from aGVHD patients, the mRNA levels of genes involved in the pro-inflammatory M1 polarization, antigen presenting and promoting the activation of T cells pathway were substantially elevated in MΦs of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vivo infusion with pro-inflammatory M1 aggravated aGVHD response by modulating immune microenvironment of spleen and liver in mice, characterized by enhanced effector function of T cell, including elevated percentages of Tc1, Th1 and Th17 as well as increased proliferation of T cells from spleen and liver. By contrast, infusion with anti-inflammatory M2 alleviated aGVHD through down-regulating the activity of T cells derived from aGVHD mice, characterized by decreased proliferation and decreased percentages of Tc1, Th1 and Th17. Summary/Conclusion: The current study demonstrated that the primary MΦs in aGVHD patients preferentially polarize into pro-inflammatory M1. Moreover, M1 aggravate aGVHD whereas M2 ameliorate aGVHD by differently modulating immune microenvironment. Although further validation is required, modulating the polarization state of MΦs promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Background Young donors are reported to be associated with better transplant outcomes than old donors in allo-HSCT, but the underlying mechanism is still uncertain. Successful allo-HSCT relies on the rapid reconstitution of donor-derived haematopoietic and immune systems in the recipient. Therefore, characterizing the differences in percentages of HSCs and progenitors and immune cell subtypes between young and old donors may help explain the disparities in transplant outcomes. In humans, HSCs give rise to multipotent progenitors (MPPs) that further segregate into either common myeloid progenitors (CMPs) or multipotent lymphoid progenitors (MLPs), which in turn segregate into either common lymphoid progenitors (CLPs) or granulocyte-macrophage progenitors (GMPs). CMPs further segregate into either megakaryocyte-erythroid progenitor (MEPs), or GMPs. However, differences in the frequencies of HSCs and their progenitors between young and old adults remain uncertain. In addition, aGVHD is generally considered to be associated with increased ratios of donor Th1/Th2, Tc1/Tc2 and M1/M2 macrophage. However, little is known about the cytokine-producing T cell subsets and macrophage subsets in BM between young and old donors. Aims To evaluate the different subsets of HSCs and their progenitors and immune cells among young (aged 45 years). Moreover, to analyze the association between donor characteristics and HSC frequency. M ethods In this prospective study, a total of 60 healthy adult donors, including 20 young donors, 20 middle-aged donors, and 20 old donors were enrolled. The frequencies and ROS levels of BM HSCs(CD34+CD38−CD90+CD45RA−) and progenitors including MPPs(CD34+CD38−CD90−CD45RA−), MLPs(CD34+CD38−CD45RA+), CLPs(CD34+CD38+CD7−CD10+CD45RA+), GMPs(CD34+CD38+CD7−CD10−CD45RA+), CMPs(CD34+CD38+CD7−CD10−CD135+CD45RA+), and MEPs(CD34+CD38+CD7−CD10−CD135−CD45RA−) were quantified by flow cytometry. Furthermore, T cell and macrophage subsets were analyzed in young, middle-aged and old donors by flow cytometry. Effector T cells, naïve T cells, effector memory T cells and central memory T cells were identified as CD45RA+CCR7−, CD45RA+CCR7+, CD45RA−CCR7−, and CD45RA−CCR7+. Th1, Th2, Tc1 and Tc2 were identified as CD3+CD8−IFN-γ+, CD3+CD8−IL-4+, CD3+CD8+IFN-γ+ and CD3+CD8+IL-4+, respectively. In addition, M1 and M2 were identified as CD14+CCR2+CD68+ and CD14+CX3CR1+CD163+. Moreover, the association of donor characteristics with HSC frequency was analysed by univariate and multivariate analysis. Results To determine the differences in HSCs and progenitors in different age donors, HSCs and six subpopulations were compared among young, middle-aged and old donors. The frequencies of HSCs and myeloid progenitors, including CMPs and MEPs in CD34+ cells were significantly lower and the frequencies of lymphoid progenitors including MLPs and CLPs in CD34+ cells were higher in the BM of young donors than in that of old donors. Significantly lower levels of ROS in HSCs and progenitors were observed in young donors than in the other donors. Furthermore, to investigate the differences in the differentiation potential from HSCs to immune cells in different age donors, T cell and macrophage subsets were compared among the three donor age groups. Young donors demonstrated a lower CD4+/CD8+ T cell ratio, lower memory T cell frequency and higher naïve T cell frequency in both CD4+ cells and CD8+ cells. Importantly, BM immune cells from young donors polarized towards less pro-inflammatory T cells characterized by Th1 and Tc1, and more immune suppressor cells, such as M2, than those from old donors. As a result, young donors had lower ratios of Th1/Th2, Tc1/Tc2 and M1/M2 in BM. In addition, multivariate analysis showed that age≥37 was independently correlated with a higher HSC frequency. Conclusion BM HSCs from young donors exhibited a lower frequency, balanced myeloid-lymphoid differentiation potential, lower ROS level and produced more immune suppressors and fewer immune effector cells than those from old donors. Donor age might be a good predictor of HSC frequency. Although further validation is required, the differences in the frequency and immune differentiation potential of HSCs in BM between young and old donors may partly explain the different outcomes of allo-HSCT. Disclosures No relevant conflicts of interest to declare.

B ackground: No consensus has been reached on the relationship between kit mutation and prognosis in core banding factor acute myeloid leukemia(CBF-AML). Our preverious analysis of kit mutation subtypes in patients with RUNX1-RUNX1T1 suggested poor prognosis associated with D816/D820 mutation. Objective: To investigate further risk stratification according to different subtypes of kit mutations and minimal residual disease (MRD) in CBF-AML. Methods: A total of 205 patients aged 16-70 years old with CBF-AML, who were diagnosed in our hospital, were analyzed retrospectively from January 2014 to April 2019. The effects of kit mutation subtypes and MRD on the overall survival (OS) and relapse free survival(RFS) were analyzed. Results: Of all, 118 males and 87 females were included. The median age were 38 (15-70) years old. Patients with RUNX1-RUNX1T1 and patients with CBFB-MYH11 accounted for 71.1% (147/205) and 28.3% (58/205) respectively. The ratio of kit mutation was 33.2% (68/205), of which, 52/147(35.4%) patients were RUNX1-RUNX1T1 positive, and 16/58 (27.6%) patients were CBFB-MYH11 positive. Of 68 patients with kit mutation, patients with D816/D820 mutation accounted for 50.0%(34/68) and the rest 34 cases were non D816/D820 mutation. The rate of D816/D820 mutation was 50% (26/52) and 50% (8/16) respectively in Patients with RUNX1-RUNX1T1 and patients with CBFB-MYH11. During the median follow-up time 28 (7-72) months, 10 patients were lost and a total of 195 patients were available. 42.6% (83/195) patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT). 112 patients who did not received allo-HSCT were divided into three group: 85 patients without kit mutation(group A) , 15 patients with D816/D820 nutation(group B), 12 patients with non-D816/D820 nutation(group C). The 3-year overall survival(OS) and 3-year relapse free survival(RFS) between group A and B did not have significance(P=0.603, 0.601). Both the 3-year OS and 3-year RFS of group B were superior to group C(65.5% VS 8.3%; 73.3% VS 14.4%, P=0.002, 0.003), and the 3-year OS and 3-year RFS of group A were superior to group C(68.1% VS 8.3%; 75.2% VS 14.4%, P=0.000; 0.000). So group A and B were defined as low risk group (97 patients), group C defined as high risk group(15 patients). The 3-year OS and 3-year RFS of high risk group were significantly lower than those of low risk group(14.4% VS 75.4%、8.3% VS 68.4%, P=0.000、0.000). After 2 cycles of consolidation, MRD (the transcript of RUNX1-RUNX1T1 or CBFB-MYH11) > 0.1% could predicted disease recurrence(P=0.000). Patients who got MRD < 0.1%(49 patients) after two cycles of consolidation chemotherapy could have higher 3-year OS and 3-year RFS than those who did not (63 patients) (94.1%VS 49.9%; 96.3% VS 36.4%, P=0.000; 0.000). During the follow-up time, patients who achieved MRD < 0.1%(81patients) also had higher 3-year OS and 3-year RFS than those did not(31 patients)(81.8% VS 6.7%; 75.7% VS 12.9%, P=0.000, 0.000). Multivariate analysis showed D816/D820mutation, MRD > 0.1% after two cycles of consolidation chemotherapy, MRD > 0.1% during the follow-up time were poor prognosis on OS and RFS, and kit mutation had not effect on OS and RFS. A total of 91 patients out of 195 patients had D816/D820 mutation (30 patients) or MRD > 0.1% after two cycles of consolidation chemotherapy and MRD > 0.1% (61 patients) during the follow-up time, the 3-year OS and 3-year RFS significantly increased in the group of 57 patients who received allo-HSCT than the group of 34 patients who did not(91.0% VS 22.1%; 91.2% VS 15.9%, P=0.000, 0.000). Conclusion: The real high risk patients must be recognized during the treatment of CBF-AML: D816/D820mutation, MRD > 0.1% after two cycles of consolidation chemotherapy and MRD > 0.1% during the follow-up time. Allo-HSCT can improve the survival of patients who were distinguished under the accurate stratification. 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.

Background Megakaryopoiesis and platelets production intensely depend on bone marrow(BM) microenvironment. Our previous studies found that impaired BM microenvironment and dysfunctional megakaryopoiesis are responsible for the occurrence of prolonged isolated thrombocytopenia (PT), which is defined as the engraftment of all peripheral blood cell lines other than a platelet count less than 20×109/L or a dependence on platelet transfusions for more than 60 days following allo-HSCT(BBMT 2014; BBMT 2017; Brit J Haematol 2018; Am J Hematol 2018). As an important component of the BM microenvironment, macrophages (MՓs) are heterogeneous and polarized into classically activated (M1) MՓs and alternatively activated (M2) MՓs with distinct phenotypes and function. Although inconsistent effect of BM MՓs was reported on megakaryopoiesis, the functional role of M1 and M2 MՓs and related pathway in regulating megakaryopoiesis and its effect on PT patients post-allotransplant remain to be elucidated. Aims To address the roles of M1 MФs and M2 MФs in regulating megakaryopoiesis as well as PI3K-AKT pathway in the process. Moreover, polarization status and the function of BM MФs in regulating megakaryopoiesis were evaluated in PT patients. Methods This prospective nested case-control study enrolled 12 patients with PT, 24 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 12 healthy donors (HD). BM standard monocyte subsets and M1/M2 MՓs polarization state were analyzed by flow cytometry. To generate M1 and M2 MՓs, both primary BM MՓs and THP1 cell lines were treated with LPS and IFN-γ or with IL-4 and IL-13. The functions of BM MՓs were evaluated by migration, phagocytosis and cytokine secretion assay. The sorted CD34+ cells from HD were co-cultured with BM MՓs from PT and GGF patients or M1 and M2 MՓs respectively for megakaryopoiesis. The quantification of the megakaryocytes(MKs), MKs apoptosis, MKs polyploidy distribution, colony-forming unit MK(CFU-MK) efficiency, and platelet production were analyzed in the coculture system. To understand the underlying mechanism of MՓs polarization in regulating MKs, RNA-seq analyses were performed in BM MՓs from PT and GGF patients. In addition, M1 and M2 MՓs were treated with the chemical inhibitors and lentivirus for PI3K-AKT pathway. Results Elevated intermediate and non-classical monocyte subsets were found in PT patients when compared with those in GGF patients. Moreover, PT patients displayed increased M1 and reduced M2 MՓs, resulting an unbalanced M1/M2 polarization, compared with GGF and HD. BM MՓs from PT patients, with high TNF-α levels and low TGF-β levels, showed decreased megakaryopoiesis-supporting ability. No significant differences in migration and phagocytosis function of MՓs among the three groups. RNA sequencing of BM MՓs showed down-regulated PI3K-AKT pathway in MՓs of PT patients compared with GGF. Consistently, the phosphorylation levels of AKT decreased significantly in MՓs of PT patients, suggesting that PI3K-AKT pathway may functionally regulate megakaryopoiesis-supporting ability of MՓs. Subsequently, BM-M2 and THP1-M2 showed superior effect on megakaryopoiesis-supporting ability compared with BM-M1 and THP1-M1. Specifically, the BM CD34+ cells cocultured with M2 MՓs demonstrated significant increased percentages of MKs and MK polyploidy, CFU-MK efficiency, and platelet count compared with those cocultured with M1 MՓs. Preventing PI3K-AKT pathway by PI3K inhibitor or Akt inhibitor significantly reduced the megakaryopoiesis-supporting ability of M2 MՓs. Moreover, knockdown of AKT1 induced the impairment of megakaryopoiesis-supporting ability via suppressing M2 MՓs polarization, which could be attenuated by AKT1 overexpression complementarily. Summary/Conclusion The current study demonstrated the polarization status of MՓs modulates their ability to support megakaryopoiesis. M2 MՓs, but not M1 MՓs, support megakaryopoiesis via up-regulating PI3K-AKT pathway. Defective M2 MՓs polarization via down-regulating PI3K-AKT pathway may be responsible for the pathogenesis of PT post-allotransplant, which provides a promising therapeutic target for PT patients. 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.

The pathogenesis of corticosteroid-resistant immune thrombocytopenia (ITP), a clinically challenging condition in which patients exhibit either no response to corticosteroids or are corticosteroid-dependent, remains poorly understood. Murine studies suggest that bone marrow (BM) endothelial progenitor cells (EPCs) play a crucial role in regulating megakaryocytopoiesis. However, little is known regarding the number and function of BM EPCs or how to improve impaired BM EPCs in corticosteroid-resistant ITP patients. In the current case-control study, we evaluated whether the BM EPCs in corticosteroid-resistant ITP differed from those in corticosteroid-sensitive ITP. Moreover, whether atorvastatin could enhance the number and function of BM EPCs derived from corticosteroid-resistant ITP patients was investigated in vitro and in vivo. Reduced and dysfunctional BM EPCs, characterized by decreased capacities of migration and angiogenesis as well as higher levels of reactive oxygen species and apoptosis, were observed in corticosteroid-resistant ITP patients. In vitro treatment with atorvastatin quantitatively and functionally improved BM EPCs derived from corticosteroid-resistant ITP patients by downregulating the p38 MAPK pathway and upregulating the Akt pathway, and rescued the impaired BM EPCs to support megakaryocytopoiesis. Subsequently, a pilot cohort study showed that atorvastatin was safe and effective in corticosteroid-resistant ITP patients. Taken together, these results indicate that reduced and dysfunctional BM EPCs play a role in the pathogenesis of corticosteroid-resistant ITP, and the impaired BM EPCs could be improved by atorvastatin both in vitro and in vivo. Although requiring further validation, our data indicate that atorvastatin represents a promising therapeutic approach for repairing impaired BM EPCs in corticosteroid-resistant ITP patients.

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.

Background: Bone marrow(BM) endothelial cells(ECs), a key component of BM microenvironment, is essential for the physiology and regeneration of hematopoietic stem cells (HSCs). The damage of ECs is recognized by us and other researchers as a mainstay in the pathophysiology of a serious of life-threatening complications after chemoradiotherapy and myeloablative hematopoietic cell transplantation(HSCT), including poor graft function (PGF) (2013BBMT, 2015BMT, 2016Blood, 2019Blood Advances). Despite numerous researches focused on the BM ECs contributing to HSC regeneration following myelotoxicity, the mechanisms underlying the injured BM ECs itself remain to be elucidated. Under physiological conditions, energy metabolism plays an instrumental role in maintaining EC function, and markedly perturbed of EC metabolism is linked to many pathologies, like cancer and diabetes. However, little is known about the metabolism state and its role in impaired BM ECs. Aims: The current study was performed to investigate the metabolism status in BM ECs after chemotherapy-induced injury. Moreover, we evaluated the metabolic state and its role in BM ECs of PGF patients post-allotransplant. Finally, we evaluated the therapeutical potential of anti-metabolic drugs to the dysfunctional BM ECs derived from PGF patients. Methods: Two EC injury models in vitro were established with the cultivated human BM ECs treated by 5-Fluorouracil (5-FU) and hydrogen peroxide. The findings from the above models were further validated by a prospective case-control study enrolled 15 patients with PGF, 30 matched patients with good graft function (GGF) and 15 healthy donors (HD). To determine the metabolic status of BM ECs, the expression of metabolism regulating genes was analyzed by qRT-PCR (mRNA level) and flow cytometry (protein level). Glucose metabolism levels were measured by glucose consumption and lactate production assays. To evaluate the functions of BM ECs, apoptosis, migration and tube formation assays were performed. To investigate the effect of anti-metabolic drugs to injured BM ECs, the glycolysis inhibitor 3PO and PPARd agonist GW501516 were administrated to the cultivated BM ECs treated by 5-FU , hydrogen peroxide or derived from PGF. Results: We demonstrated that the glycolysis in BM ECs could be induced by the treatment with either 5-FU or hydrogen peroxide in vitro, consistent with the dysfunction(impaired migration, angiogenesis, and higher level of apoptosis) of BM ECs, which could be attenuated by glycolysis restoration. Mechanistically, we revealed that the aberrant glycolysis and dysfunction of BM ECs could be triggered by PPARd knockdown in vitro, while the PPARd were down-regulated by either 5-FU or hydrogen peroxide treatment in vitro, Furthermore, PPARd agonist GW501516 treatment attenuated the perturbed function and number of injured BM ECs treated by either 5-FU or hydrogen peroxide. Subsequently, the prospective case-control study demonstrated elevated expressions of the glycolytic activator PFKFB3 and decreased PPARd were observed in BM ECs of PGF patients, when compared with those of GGF patients and HD, indicating that BM ECs of PGF patients have a hyper-glycolytic metabolism. Moreover, either glycolysis (PFKFB3) inhibitor 3PO or PPARd agonist GW501516 treatment reduced the aberrant glycolysis and improved the number and function of BM ECs derived from patients with PGF in vitro, revealing the critical role of defective glycolysis in the impaired BM ECs of PGF. Summary / Conclusions: These findings reveal that hyper-glycolysis mediated by PPARd inhibition is involved in the dysfunction of BM ECs after injury. Defective glycolysis may contribute to the pathobiology of BM ECs of PGF patients, which could be attenuated by glycolysis inhibitor 3PO or PPARd agonist GW501516 in vitro. Our findings might merit further consideration of targeting BM ECs glycolysis or PPARd as a promising therapeutic approach for PGF patients post-allotransplant in the future. Disclosures No relevant conflicts of interest to declare.

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.

Objective: To investigate the long-term prognostic factors of CBFB-MYH11-positive acute myeloid leukemia in low、 intermediate risk patients. Methods:58 CBFB-MYH11-positive patients were analyzed retrospectively in 1525 acute myeloid leukemia patients who were diagnosed in Peking University Institute of Hematology from October 2013 to August 2018, and the clinical feature、induction remission rate and survival were compared. Results: 58 (8%) patients with CBFB-MYH11-positive were found in 1525 newly diagnosed actue myeloid leukemia, 41 males and 17 females were included. The median age was 38(17-66) years , and median follow-up time was 29.8(4.8-74.4)months, 25(43.1%) patients received allogenic hematopoietic stem cell transplantation, in which there were 10 patients with c-kit mutation. According to the NCCN guideline, there were 40 patients in low risk group and 18 patients in intermediate risk group (16 patients with c-kit mutation). The rate of complete remission (CR) in the first induction chemotherapy was 98.3%(57/58), the cumulative remission rate of two cycles was 100%, the relapse rate was 25.9%(15/58), and the mortality rate was 19.0% (11 ≤ 58). After 1 and 2 cycles of consolidation, the level of CBFB-MYH11/ABL decreased to less than 0.18% and 0.094% respectively, which were related to the low recurrence rate. Among the 33 patients who did not undergo allogenic hematopoietic stem cell transplantation after two courses of consolidation, the recurrence rate of patients with CBFB-MYH11/ABL level > 0.1% (n =21) was significantly higher than that of patients with < 0.1% (n = 12) (61.9% VS 0%, P =0. 001), and the relapse-free survival(RFS) (3-year RFS rate 37.6% VS 100%, P =0.005 ) 、event-free survival (EFS)(3-year EFS rate 33. 3% VS 100%, P = 0.004) were significantly decreased. The RFS and EFS were lower than those of patients received allogenic hematopoietic stem cell transplantation (n=25) (3-year RFS rate 31.4% VS 84.6%, P = 0.000; 3-year EFS rate 31.4% VS 76.0%, P =0.004). Conclusions: To the CBFB-MYH11-positive acute myeloid leukemia patients, if the level of CBFB-MYH11/ABL gene presented more than 0.1% at any time after 2 cycles of consolidation, poor prognosis was related to, and allogenic hematopoietic stem cell transplantation could improve the survival. 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.

Background: DNAM-1 (DNAX accessory molecule-1, CD226) is a costimulatory molecule that is constitutively expressed by NK cells and CD8+ T cells. Interaction between DNAM-1 on NK cells and CD8+ T cells with its ligands on target cells triggers cell-mediated cytotoxicity and cytokine production. Previous mouse model had showed that the expression of DNAM-1 is associated with NK education. Moreover, Monika's group found that DNAM-1 serves as an intrinsic, readout-independent marker for NK cell education in health donor. Our previous study had demonstrated that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells would achieve better functional education and contribute to least relapse for the patients post allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the roles of DNAM-1 in NK education post allo-HSCT were unknown. Aims: In this study, we have investigated the contribution of DNAM-1 expression to NK education post transplantation. Methods :We prospectively enrolled 114 patients undergoing haplo-SCT between May 2016 and April 2017 to explore the NK cell dynamic education at days 30, 90 and 180 post-transplant. Peripheral blood mononuclear cells of each sample were analyzed by 15-colors flow cytometry to evaluate of the phenotype as well as functional recovery of NK cells with different maturation expression levels of NKG2A, KIR, and CD57, as well as of activating receptors (NKp30, NKp46, NKG2D, DNAM-1) and CD25, CD122 as well as CD107a and IFN-gamma on NK cells. To study the correlation between the expression of DNAM-1 and effect of donor and host HLA on NK cell education, the expression of DNAM-1 on single-KIR+ NK cells (exhibiting NKG2A, KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) was compared based on the combination between donor/host HLA and donor inhibitory KIR. Results: The DNAM-1 expression on single-KIR+ NK cell ligated by sole donor HLA, or sole host HLA, or both donor and host HLA is higher compared to those single-KIR+ NK cells lacking ligands from donor or host or both. From the donor point of view, when donor only presented C1C1 ligand for KIR2DL2/L3, the MFI of DNAM-1 on single KIR2DL2/L3+ NK cells was significantly higher than KIR2DL1+ NK cells and KIR3DL1+ NK cells at 90day (P Summary: This study demonstrated that no matter from donor or/and host point of view, DNAM-1 expression contributes to optimal NK cells education post allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. 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.

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.

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.