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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.

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10−8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10−9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

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.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the ERG gene (rs2836365; P = 3.76 × 10−8; odds ratio [OR] = 1.56), with independent validation (P = .01; OR = 1.43). Imputation analyses pointed to a single causal variant driving the association signal at this locus overlapping with putative regulatory DNA elements. The effect size of the ERG risk variant rose with increasing Native American genetic ancestry. The ERG risk genotype was underrepresented in ALL with the ETV6-RUNX1 fusion (P < .0005) but enriched in the TCF3-PBX1 subtype (P < .05). Interestingly, ALL cases with germline ERG risk alleles were significantly less likely to have somatic ERG deletion (P < .05). Our results provide novel insights into genetic predisposition to ALL and its contribution to racial disparity in this cancer.

To identify the prognostic factors that are useful to improve central nervous system (CNS) control in children with acute lymphoblastic leukemia (ALL), we analyzed the outcome of 7640 consecutive patients treated on Chinese Children’s Cancer Group ALL-2015 protocol between 2015 and 2019. This protocol featured prephase dexamethasone treatment before conventional remission induction and subsequent risk-directed therapy, including 16 to 22 triple intrathecal treatments, without prophylactic cranial irradiation. The 5-year event-free survival was 80.3% (95% confidence interval [CI], 78.9-81.7), and overall survival 91.1% (95% CI, 90.1-92.1). The cumulative risk of isolated CNS relapse was 1.9% (95% CI, 1.5-2.3), and any CNS relapse 2.7% (95% CI, 2.2-3.2). The isolated CNS relapse rate was significantly lower in patients with B-cell ALL (B-ALL) than in those with T-cell ALL (T-ALL) (1.6%; 95% CI, 1.2-2.0 vs 4.6%; 95% CI, 2.9-6.3; P < .001). Independent risk factors for isolated CNS relapse included male sex (hazard ratio [HR], 1.8; 95% CI, 1.1-3.0; P = .03), the presence of BCR-ABL1 fusion (HR, 3.8; 95% CI, 2.0-7.3; P < .001) in B-ALL, and presenting leukocyte count ≥50×109/L (HR, 4.3; 95% CI, 1.5-12.2; P = .007) in T-ALL. Significantly lower isolated CNS relapse was associated with the use of total intravenous anesthesia during intrathecal therapy (HR, 0.2; 95% CI, 0.04-0.7; P = .02) and flow cytometry examination of diagnostic cerebrospinal fluid (CSF) (HR, 0.2; 95% CI, 0.06-0.6; P = .006) among patients with B-ALL. Prephase dexamethasone treatment, delayed intrathecal therapy, use of total intravenous anesthesia during intrathecal therapy, and flow cytometry examination of diagnostic CSF may improve CNS control in childhood ALL. This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IPR-14005706).

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 Myeloid sarcoma (MS) is the rare extramedullary proliferation of myeloid blasts that disrupts the normal architecture of various tissues in patients with Acute myeloid leukemia (AML). MS can present concurrently with untreated AML or present as relapsed AML. Based on our clinical observations and emerging immune checkpoint inhibitor (ICI) studies in AML (Davids et al NEJM 2016), MS appears to behave differently based on transplant status and tends to have better ICI sensitivity. One of the prominent mechanisms of relapse post-transplant is immune escape via the loss of leukemia recognition by the graft. This is mediated by down regulation of HLA expression. We hypothesize that the immune signatures in the bone marrow (BM) microenvironment are distinct from those from the local MS microenvironment. Thus, we aimed to comprehensively explore the MS microenvironment at single-cell level, providing clues for more effective therapies including ICI, and insights into the molecular pathogenesis, clinicopathological features and outcomes of MS. Methods This is an investigator-initiated study where we collected paired fresh MS tissues and BM samples from patients with MS as well as retrospective collection of paired MS and BM samples that are already sectioned and paraffin-embedded. Eligible patients were consentable adults > 18 years old and have MS lesions whether at diagnosis or at relapse of their AML disease. We excluded patients who have multiple malignancies or patients who are unable to provide samples. Accepted samples are 10-millimeter tissue or 1 million cells per milliliter. The fresh samples were cryopreserved and used for single cell RNA sequencing and ex vivo immune cellular assays. The sectioned tissues were used for multiplex immunohistochemical staining (mIHC) that tested a panel of 6 antibodies (CD3, CD68, CD4, CD33, PDL-1 and HLA-DR). We sought to further perform spatial transcriptome analysis (Visium) that complements mIHC for in-depth characterization of the MS microenvironment and immunopathogenesis. The clinical and genomic annotations for these samples were abstracted from the patients' charts. We have utilized Next generation sequencing (NGS) panels that interrogated 24- 180 oncogenes, including FLT3, IDH, CALR, NPM1, KRAS, NRAS, and TP53. Survival estimates using K-M curves and multivariate analysis were performed via SPSS. Results Our study recruited 30 patients so far. Mean age at diagnosis was (57.6 ± 14 years); 63.3% of the patients were males (n=19) and 80% of the patients were Caucasian (n=24). In our cohort, 46.6% of the patient had MS diagnosed as the cause of relapse after stem cell transplant (n=14) while the rest had MS at the initial diagnosis of AML. The most common cytogenetic abnormality in our cohort was complex karyotype in 30% of the patients. Molecular landscape of MS at diagnosis and relapse was done in 90% of the patients and shown in figure 1A,B. MS conferred a poor prognosis if diagnosed on initial presentation vs a new presentation at relapse post stem cell transplant with a median OS (4 months vs 25 months p 0.003) (HR 3.9 - 95% CI 1.4-11.1) figure 2. Interestingly, we have noticed that patients who received Venetolcax based therapies have poor prognosis compared to other therapies (IDH/FLT3 inhibitors, conventional chemotherapies, or ICI); median OS (9.3 months vs 24 months p 0.02) (HR 3.9 - 95% CI 1.2-12.6) figure 3. In a prelim analysis using validated mIHC, we have observed abundant HLA-DR expression in both blast cells and nonmalignant stromal cells of the MS microenvironment (n=4) figure-4. By contrast, PD-L1 was expressed mainly on a number of stromal cells rather than blast cells in the MS microenvironment. Further special localization analysis of tumor immune infiltrates (TILs) is being conducted to disclose clinical outcomes and biological significance of PD-L1, HLA-DR and TILs in MS. Spital transcriptomics are in process and will be presented. Conclusion MS diagnosed concurrently with untreated AML appears to confer a poor prognosis and behave differently than MS diagnosed as relapse post-transplant. MS appears to be resistant to Venetoclax based therapies and patients tends to do worse on this therapy. In contrast to BM microenvironment, blast cells within MS appears to have abundant expression of HLA-DR in both neoplastic tumor cells and nonmalignant stromal cells promoting immune recognition which might explain their sensitivities to ICI. Figure 1 Figure 1. Disclosures Abaza: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dinner: Pfizer: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria. Altman: Glycomimetics: Other: data monitoring committee; ALX Oncology Inc.: Research Funding; Loxo: Research Funding; ImmunoGen: Research Funding; Abbvie: Honoraria, Research Funding; Amgen: Research Funding; Aprea: Research Funding; Astellas: Honoraria, Research Funding; Aptos: Research Funding; Boehringer Ingelheim: Research Funding; Celgene: Research Funding; Syros: Membership on an entity's Board of Directors or advisory committees; Fujifilm: Research Funding; Biosight: Membership on an entity's Board of Directors or advisory committees, Other: reimbursement for travel, Research Funding; Kura Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kartos Therapeutics: Research Funding.

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: It's well established that conventional chemotherapy doesn't benefit children with refractory/relapsed acute myeloid leukemia (R/R-AML). Though multiple attempts, i.e., novel target agents, and intensive chemotherapy, have been translated into clinics, few have been proved to be successful. Chimeric antigen receptor (CAR) T cell therapy is increasingly becoming a curative candidate for refractory hematological malignancies. Recently, anti-CLL1 CAR T-cells have been reported to effectively treat children with R/R-AML by several groups even though with a small sample size. However, the impact of co-stimulatory domain (CD28/CD27 versus 4-1BB) on the efficacy of CAR T-cells could not be neglected, which is not investigated so far. Thus, we here compare the impact of different costimulatory molecules on the safety and efficacy of anti-CLL1 CAR T-cells in treating children with R/R-AML. Methods: Seven R/R-AML children treated with anti-CLL1-based CAR T-cells were enrolled into this preliminary comparison study. The baseline characteristics, time of CAR T-cells generation, in vivo persistence of CAR T-cells, cytokine release profiling, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), secondary hemophagocytic lymphohistiocytosis (sHLH) or macrophage activation syndrome (MAS), and other adverse events, the length of hospitalization, remission status, and the follow-up data were collected for analysis. Results: Among these seven patients, four received CD28/CD27 based CAR T-cells, and three received 4-1BB based CAR T-cells therapy. The CD28/CD27 group consisted of two boys and two girls, with the median age of 7.5 (range; 5 to 11), while the 4-1BB group consisted of two boys and one girl, with the median age of 12 (range; 8 to 12). Generation of CD28/CD27 equipped CAR T-cells (median, 14 days) were faster than that of 4-1BB ones (median, 21 days). All patients experienced grade 1 to 3 CRS and were well managed with one patient experiencing grade 1-2 ICANS but without any sHLH or MAS. The maximum durations were 156 days for CD28/CD27, ranging from 0 to 156 days and 274 days for 4-1BB, ranging from 16 to 274 days) respect. For hospitalization, CD28/CD27 group experienced subtle longer hospital stays (median, 23 days; range, 14 to 30 days) than 4-1BB group (median, 21 days; range, 18 to 54 days). The overall response rates were 67 % and 75 % in 4-1BB group and CD28/CD27 group respectively. Three of four patients in CD28/CD27 group achieved morphological remission with MRD negativity, while two of three patients in 4-1BB group achieved morphological CR, with one as being MRD negativity. Of all the seven cases, only one patient proceeded to hematopoietic stem cell transplant (HSCT) and died of GVHD seven months after HSCT, however, another four patients who achieved CR could not received HSCT due to poor economic status. Of the remaining six patients, two non-respondent died of disease progression, one patient relapsed and subsequently died, and the other three remained alive and being followed up. Conclusion: Though the enrollment was too small for us to draw a conclusion, we have noticed a similar efficacy/safety index between 4-1BB and CD28/CD27 equipped anti-CLL1-based CAR T-cells in treating children with R/R-AML. In sum, the relatively high efficacy and low grade and manageable side effects were also validated in this study. 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.

Publisher's Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Our understanding of acute lymphoblastic leukemia (ALL) has expanded tremendously in the past few years because of large-scale genomic studies.[1][2],[2][3] ALL is characterized by a relatively

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.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Characterized by high levels of Native American ancestry, Hispanics are disproportionally affected by this cancer with high incidence and inferior survival. However, the genetic basis for this disparity remains poorly understood because of a paucity of genome-wide investigation of ALL in Hispanics. Performing a genome-wide association study (GWAS) in 940 Hispanic children with ALL and 681 ancestry-matched non-ALL controls, we identified a novel susceptibility locus in the

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.

Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin(-)Sca1(+)c-kit(+) cells from mice transplanted with bone marrow cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as in genes involved in myeloid and B-cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia, was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing coexpression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation.

Cyclooxygenases (COXs) and their prostanoid products play important roles in a diverse range of physiological processes, including in the immune system. Here, we provide evidence that COX-1 is an essential regulator in early stages of B-cell development. COX-1-deficient mice displayed systematic reduction in total B cells, which was attributed to the arrest of early B-cell development from pro-B to pre-B stage. We further demonstrated that this defect was mediated through downregulation of the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling and its target genes, including Pax5, in COX-1(-/-) mice. Mechanistic studies revealed that COX-1-derived thromboxane A2 (TxA2) could regulate JAK3/STAT5 signaling through the cyclic adenosine monophosphate-protein kinase A pathway, via binding with its receptor thromboxane A2 receptor (TP). Administration of the TP agonist could rescue the defective B-cell development and JAK/STAT5 signaling activity in COX-1-deficient mice. Moreover, administration of low-dose aspirin caused a significant reduction in total B cells in peripheral blood of healthy human volunteers, coincidentally with reduced TxA2 production and downregulation of JAK/STAT5 signaling. Taken together, our results demonstrate that COX-1-derived TxA2 plays a critical role in the stage transition of early B-cell development through regulation of JAK/STAT5 signaling and indicate a potential immune-suppressive effect of low-dose aspirin in humans.

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: In RRMM, the median overall survival (OS) of pts with RRMM who progressed after exposure to ≥3 prior therapies is ~13 mo, indicating a high unmet need. LCAR-B38M is a structurally differentiated CAR-T cell therapy containing a 4-1BB co-stimulatory domain and 2 BCMA-targeting single-domain antibodies designed to confer avidity. Earlier results from LEGEND-2 (NCT03090659), a first-in-human phase 1 study using LCAR-B38M CAR-T cells in 74 pts with RRMM conducted in 4 hospitals in China (Jiangsu Provincial People's Hospital; Ruijin Hospital; Changzheng Hospital; and the Second Affiliated Hospital of Xi'an Jiaotong University), showed encouraging efficacy and manageable safety. Key eligibility criteria included RRMM with ≥3 prior lines of therapy. Here, we present long-term follow-up data on safety and efficacy from the Xi'an site. Methods: In the Xi'an site-specific protocol (n=57), lymphodepletion was performed using cyclophosphamide (Cy; 300 mg/m2)alone for 3 days. LCAR-B38M (median CAR+ T cells, 0.5×106 cells/kg; range, 0.07-2.1 × 106) was infused in 3 split infusions. The primary objective was to evaluate the safety of LCAR-B38M; the secondary objective was to evaluate anti-myeloma response of treatment. Adverse events (AEs) were graded using the NCI-CTCAE v4.03, cytokine release syndrome (CRS) was assessed per Lee et al. 2014, and response was evaluated using IMWG criteria. Results: As of the 12/31/18 cutoff date (median follow-up, 19 mo; 95% confidence interval [CI], 17-22), enrollment at Xi'an is complete, and 57 pts have been infused with LCAR-B38M. AEs were reported by all pts: pyrexia (91%), CRS (90%), thrombocytopenia (49%), and leukopenia (47%). Grade ≥3 AEs were reported by 65% of pts: leukopenia (30%), thrombocytopenia (23%), and increased aspartate aminotransferase (21%). CRS was mostly grade 1 (47%) and 2 (35%); 4 pts (7%) had grade 3 events; no grade 4/5 CRS was observed. Neurotoxicity was observed in 1 pt (grade 1 aphasia, agitation, seizure-like activity). The median time to onset of CRS was 9 days (range, 1-19) with a median duration of 9 days (range, 3-57); all but 1 CRS events resolved. Peak levels of LCAR-B38M (≥1x104 copies/µg genomic DNA) were observed in a majority of pts with blood samples for analysis (n=32). LCAR-B38M was not detectable in peripheral blood in 71% of pts at 4 mo; 5 pts showed CAR-T cell persistence for up to 10 months. The overall response rate (partial response [PR] or better) was 88% (95% CI, 76-95), complete response (CR) was achieved by 42 pts (74%; 60-85), very good partial response (VGPR) by 2 pts (4%; 0.4-12), and PR by 6 pts (11%; 4-22). Of pts with CR, 39/42 were minimal residual disease negative (MRD-neg, 8-color flow cytometry). The median time to first response was 1.2 mo. There was no relationship between best response and baseline BCMA expression level or weight-adjusted CAR+ cells infused (Fig 1a,b). At cutoff, the median follow-up was 19 mo [17-22]. Median OS has not yet been reached. The OS rate at 18 mo was 68% (54─79) with a median duration of response (mDOR) of 22 mo (13-29). Of MRD-neg pts with CR, 91% (75-97) are still alive at data cut, with a 27 mo (16-NE) mDOR. Overall, 26 (46%) of 57 all-treated pts and 25 (64%) of 39 MRD-neg pts with CR remain progression-free. The median progression-free survival (PFS) for all-treated pts was 20 mo (10-28); median PFS for MRD-neg pts with CR was 28 mo (20-31). At 18 months, the PFS rate was 50% (36-63) for all pts and 71% (52-84) for MRD-neg pts with CR. Factors contributing to long-term response are shown in Fig 1c,d. Seventeen pts died during the study and the follow-up period: progressive disease (PD; n=11), disease relapse, PD + lung infection, suicide after PD, esophageal carcinoma, infection, and pulmonary embolism and acute coronary syndrome (n=1 each). Of these, 4 did not achieve PR or better; 1 was not evaluable. Conclusions: This study provides evidence that LCAR-B38M is a highly effective therapy for RRMM, regardless of baseline BCMA expression. LCAR-B38M displayed a manageable safety profile consistent with its known mechanism of action and, with a median follow-up of 19 months, demonstrated deep and durable responses in pts with RRMM. A phase 1b/2 clinical study is ongoing in the United States (CARTITUDE-1, NCT03548207, JNJ-68284528), and a phase 2 confirmatory study has initiated in China (CARTIFAN-1, NCT03758417). Figure 1 Disclosures Zhuang: Nanjing Legend Biotech: Employment. Fan:Legend Biotech: Employment, Equity Ownership.

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.

Transforming growth factor beta is an important regulator of hematopoiesis and its overactivation has been shown to occur in myelodysplastic syndromes. Even though TGF-beta leads to activation of the transcription factor SMAD2, the exact gene expression program and chromatin changes stimulated in human hematopoiesis are not well studied. We generated primary erythroid progenitors from human CD34+ stem cells and assessed genome occupancy of SMAD2 upon TGF-beta stimulation by Chip-seq analysis. We observed widespread occupancy of important stem cell and differentiation regulators with approximately 7000 binding regions. Parallel RNA-seq identified gene expression changes that are associated with smad2 binding at respective promoters and enhancers. Genes involved in stem cell maintenance as well as erythroid differentiation were prominently altered by TGF-beta and contained prominent SMAD2 peaks in hematopoietic progenitors. Next, to determine the relative contribution of various TGF-beta ligands in bone marrow failure, we analyzed a large cohort of MDS serum samples and found TGF-beta 1 (TGF-b1) to be the predominantly elevated ligand in 30% samples. Exposure to MDS patient serum led to erythroid differentiation blocks in primary hematopoietic stem cells in clonogenic cultures. Finally, we analyzed the effect of constitutive secretion of TGF-b1 in a transgenic mouse containing TGF-b1 gene driven by an albumin promoter. These mice constitutively secrete TGF-b1 and become cytopenic and show marrow histological changes that mimic human MDS. We observed stem cell expansion and block at the stage of common myeloid progenitor (CMP) in these mice; findings that are similar to stem and progenitors alterations seen in human MDS patients. To inhibit TGF-b1 signaling we next used a novel ALK5 (TGF-beta receptor I kinase) inhibitor that has been shown to be safe for human use. Vactosertib is specific small molecule inhibitor of ALK5 with a IC50 between 1 x 10-8 M and 1 x 10-9 M and has been shown to have an acceptable safety profile in phase I trial in solid tumors (NCT02160106). Vactosertib was able to inhibit smad2 phosphorylation and activation in a variety of hematopoietic cell lines. Vactosertib treatment led to reversal of hematopoietic alterations upon exposure to MDS serum in clonogenic assays. Vactosertib dosing in vivo also led to increased blood counts in TGF/alb transgenic mice and resulted in normalization of stem and progenitor changes. In conclusion, we demonstrate increased TGF-b1 levels in subset of MDS samples. We also determined TGF-b1 stimulated smad2 binding in important regulatory regions of the genome in primary human erythroid progenitors. We also demonstrate the preclinical efficacy of novel TGF-beta receptor 1 kinase inhibitor in vitro and in vivo. These studies provide preclinical rationale for using this ALK5 inhibitor in trials. Disclosures Huh: MedPacto Inc: Employment. Hwang:MedPacto Inc: Employment. Seong-Jin:MedPacto Inc: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

Background: Mantle cell lymphoma (MCL), which accounts for around 7% of non-Hodgkin lymphomas, is currently incurable. MCL's key survival pathway is the B-cell receptor pathway, and Bruton's tyrosine kinase (BTK), the critical component of this pathway, is fundamentally activated in MCL. Ibrutinib, an agent that inhibits BTK, produced a 68% clinical response rate in patients with relapsed/refractory MCL, leading to its FDA approval. However, if MCL patients experience relapse following ibrutinib therapy or do not respond to ibrutinib, the 1-year survival rate is only 22%. Therefore, determining the mechanisms underlying ibrutinib resistance and identifying alternative therapeutic options are urgent unmet needs. Our group and others have shown that the BTK mutation associated with ibrutinib resistance is rare in MCL, and targeting alternative survival pathways to overcome ibrutinib resistance has not shown promising results. Methods: To explore novel biomarkers or therapeutic targets, we performed whole exome and transcriptome sequencing and RNA-seq of ibrutinib-treated clinical specimens. Patient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with western blots. Functional studies targeting this molecular pathway were conducted, and in vivo efficacy was investigated in the ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model. Results: Co-deletion of CDKN2A and MTAP was frequently detected in ibrutinib-resistant tumors but not in ibrutinib-sensitive tumors. We also found that metabolic reprogramming towards oxidative phosphorylation (OXPHOS) potentially drives ibrutinib resistance. IACS-010759, a small molecule developed by MD Anderson Cancer Center, inhibits OXPHOS and overcomes ibrutinib resistance in vitro and in PDX models. Nevertheless, the potential drivers of this observed metabolic reprogramming towards OXPHOS remain unclear. MTAP encodes 5-methylthioadenosine phosphorylase, a critical enzyme in the methionine salvage pathway, and is frequently lost in cancer cells, resulting in accumulation of metabolite methylthioadenosine and overexpression of protein arginine methyltransferase 5 (PRMT5). Thus, PRMT5 is a potential druggable therapeutic target to overcome ibrutinib resistance in MCL. We found that PRMT5 is highly expressed in ibrutinib-resistant MCL but not ibrutinib-sensitive MCL (7 ibrutinib-resistant vs. 15 ibrutinib-sensitive patient samples, p = 0.0051), especially in MTAP-deleted MCL specimens. Furthermore, patients with high PRMT5 levels had poor clinical outcomes on ibrutinib treatment. PRMT5 regulates tumor survival pathways by the methylation of histones H3R8, H4R3, and post-translational methylation of non-histone proteins, including p53, E2F1, BCL6, p65, and others. A novel PRMT5 inhibitor, GSK3226595, significantly inhibited tumor growth in an ibrutinib-resistant MCL PDX model (n = 5; vehicle vs. GSK3226595, p = 0.000048) and had no significant mouse body weight change (GSK3226595 vs. vehicle, p = 0.08). Conclusion: This study may pave the way to develop a PRMT5/OXPHOS-based therapeutic strategy to overcome ibrutinib resistance in MCL. Disclosures Wang: Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; MoreHealth: Consultancy, Equity Ownership; Acerta Pharma: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding.

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.

OBJECTIVE To determine whether dasatinib given at 80 mg/m2 is more effective than imatinib at 300 mg/m2 to improve event-free survival of children with Philadelphia chromosome-positive ALL, in the context of intensive chemotherapy without prophylactic cranial irradiation. DESIGN, SETTING, AND PARTICIPANTS This open-label phase III randomized study was conducted at 20 hospitals in China. Enrollment began in January 2015 and randomization was stopped in October 2018 when the early stopping criterion of the trial was met. Patients aged between 0 and 18 years were recruited. Of the 225 patients with the diagnosis, 35 declined and 1 died before treatment. INTERVENTIONS Patients were randomized to receive daily dasatinib (n=92) or imatinib (n=97) continuously for the entire duration of ALL therapy from the time of diagnosis made during remission induction to the end of continuation therapy. MAIN OUTCOMES AND MEASURES The primary outcome was event-free survival, analyzed by intent-to-treat. The secondary outcomes were relapse, toxic death, and overall survival. RESULTS With a median follow-up of 26.1 months (IQR 16.3-34.1), the 4-year event-free survival rate was 71.0% (95% CI 56.2-89.6) in the dasatinib group and 48.9% (95% CI 32.0-74.5, p=0.005) in the imatinib group (hazard ratio 2.36, 95% CI 1.27-4.40, p=0.007). The 4-year cumulative risk of any relapse was 19.8% (95% CI 4.2-35.4) in the dasatinib group and 34.4% (95% CI 15.6-53.2) in the imatinib group (p=0.01), while the 4-year cumulative risk of an isolated central-nervous-system relapse was 2.7% (95% CI 0.0-8.1) in the dasatinib group and 8.4% (95% CI -1.2-15.6) in the imatinib group (p=0.06). There were no significant differences in the frequency of severe toxicities between the two treatment groups. CONCLUSION AND RELEVANCE Intensive chemotherapy including dasatinib at 80 mg/m2 per day yielded superior results in the treatment of Philadelphia chromosome-positive ALL compared to imatinib at 300 mg/m2 per day and provided excellent control of central-nervous-system leukemia without the use of prophylactic cranial irradiation. Disclosures No relevant conflicts of interest to declare.