Efficacy and safety of human umbilical cord-derived mesenchymal stem cells in the treatment of refractory immune thrombocytopenia: a prospective, single arm, phase I trial.

Patients with refractory immune thrombocytopenia (ITP) frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) present a promising alternative, capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders. This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP. The research design included administering UC-MSCs at escalating doses of 0.5 × 106 cells/kg, 1.0 × 106 cells/kg, and 2.0 × 106 cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase, followed by a dose of 2.0 × 106 cells/kg weekly for the dose-expansion phase. Adverse events, platelet counts, and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period. Ultimately, 12 (with an addition of three patients in the 2.0 × 106 cells/kg group due to dose-limiting toxicity) and six patients were enrolled in the dose-escalation and dose-expansion phase, respectively. Thirteen patients (13/18, 72.2%) experienced one or more treatment emergent adverse events. Serious adverse events occurred in four patients (4/18, 22.2%), including gastrointestinal hemorrhage (2/4), profuse menstruation (1/4), and acute myocardial infarction (1/4). The response rates were 41.7% in the dose-escalation phase (5/12, two received 1.0 × 106 cells/kg per week, and three received 2.0 × 106 cells/kg per week) and 50.0% (3/6) in the dose-expansion phase. The overall response rate was 44.4% (8/18) among all enrolled patients. To sum up, UC-MSCs are effective and well tolerated in treating refractory ITP (ClinicalTrials.gov ID: NCT04014166).

Proteomics landscape and machine learning prediction of long-term response to splenectomy in primary immune thrombocytopenia.

This study aimed to identify key proteomic analytes correlated with response to splenectomy in primary immune thrombocytopenia (ITP). Thirty-four patients were retrospectively collected in the training cohort and 26 were prospectively enrolled as validation cohort. Bone marrow biopsy samples of all participants were collected prior to the splenectomy. A total of 12 modules of proteins were identified by weighted gene co-expression network analysis (WGCNA) method in the developed cohort. The tan module positively correlated with megakaryocyte counts before splenectomy (r = 0.38, p = 0.027), and time to peak platelet level after splenectomy (r = 0.47, p = 0.005). The blue module significantly correlated with response to splenectomy (r = 0.37, p = 0.0031). KEGG pathways analysis found that the PI3K-Akt signalling pathway was predominantly enriched in the tan module, while ribosomal and spliceosome pathways were enriched in the blue module. Machine learning algorithm identified the optimal combination of biomarkers from the blue module in the training cohort, and importantly, cofilin-1 (CFL1) was independently confirmed in the validation cohort. The C-index of CFL1 was >0.7 in both cohorts. Our results highlight the use of bone marrow proteomics analysis for deriving key analytes that predict the response to splenectomy, warranting further exploration of plasma proteomics in this patient population.

Metabolomics profile and machine learning prediction of treatment responses in immune thrombocytopenia: A prospective cohort study.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and impaired platelet production. The mechanisms underlying ITP and biomarkers predicting the response of drug treatments are elusive. We performed a metabolomic profiling of bone marrow biopsy samples collected from ITP patients admission in a prospective study of the National Longitudinal Cohort of Hematological Diseases. Machine learning algorithms were conducted to discover novel biomarkers to predict ITP patient treatment responses. From the bone marrow biopsies of 91 ITP patients, we quantified a total of 4494 metabolites, including 1456 metabolites in the positive mode and 3038 metabolites in the negative mode. Metabolic patterns varied significantly between groups of newly diagnosed and chronic ITP, with a total of 876 differential metabolites involved in 181 unique metabolic pathways. Enrichment factors and p-values revealed the top metabolically enriched pathways to be sphingolipid metabolism, the sphingolipid signalling pathway, ubiquinone and other terpenoid-quinone biosynthesis, thiamine metabolism, tryptophan metabolism and cofactors biosynthesis, the phospholipase D signalling pathway and the phosphatidylinositol signalling system. Based on patient responses to five treatment options, we screened several metabolites using the Boruta algorithm and ranked their importance using the random forest algorithm. Lipids and their metabolism, including long-chain fatty acids, oxidized lipids, glycerophospholipids, phosphatidylcholine and phosphatidylethanolamine biosynthesis, helped differentiate drug treatment responses. In conclusion, this study revealed metabolic alterations associated with ITP in bone marrow supernatants and a potential biomarker predicting the response to ITP.

Acquired hemophilia A: a single-center study of 165 patients.

Background: Acquired hemophilia A (AHA) is a rare hemorrhagic disorder caused by factor (F)VIII inhibitors. The diagnosis and management of AHA remains challenging because of its rarity and heterogeneity. Objectives: To analyze the characteristics of AHA to enhance our understanding of this disease and identify effective treatment strategies. To analyze the characteristics of AHA to enhance our understanding of this disease and identify effective treatment strategies. Methods: Clinical features of 165 patients with AHA from a single center between July 1997 and December 2021 were retrospectively analyzed. Results: The median age of patients at diagnosis was 45 years. The median time to diagnosis was 30 days. All 165 patients experienced bleeding, with a median bleeding score (BS) of 4 (range, 2-12). Hemostatic therapy was administered to 129 (78.2%) patients. Bleeding control was achieved in 80.0% of patients who received prothrombin complex concentrate and in 92.3% of patients who were treated with recombinant activated FVII. Of the 163 patients who received immunosuppressive therapy, 80 (49.1%) received rituximab-based therapy with a 93.3% complete remission (CR) rate, 50 (30.7%) received steroids plus cyclophosphamide with an 85.0% CR rate, and 22 (13.5%) received steroids alone with an 82.4% CR rate. Six cases relapsed after a median duration of 330 days. Immunosuppressive therapy-related adverse events were reported in 17 patients. Seven deaths were recorded. FVIII inhibitor titer of ≥15 BU/mL and BS of ≥6 were identified as significantly poor prognostic factors for CR. Conclusion: Immunosuppressive therapies yield remarkably high response rates, with a CR rate exceeding 80%; notably, the regimen containing rituximab exhibits a CR rate of approximately 90%. FVIII inhibitor titer of ≥5 BU/mL and BS of ≥6 were poor predictors of CR in patients with AHA.

Single-dose rituximab plus glucocorticoid versus cyclophosphamide plus glucocorticoid in patients with newly diagnosed acquired hemophilia A: A multicenter, open-label, randomized noninferiority trial.

Acquired hemophilia A (AHA) is a rare but serious bleeding disorder. Randomized controlled trial (RCT) comparing the efficacy of immunosuppression therapy for AHA lacks. We conducted the first multicenter RCT aiming to establish whether the single-dose rituximab combination regimen was noninferior to the cyclophosphamide combination regimen. From 2017 to 2022, 63 patients with newly diagnosed AHA from five centers were randomly assigned 1:1 to receive glucocorticoid (methylprednisolone 0.8 mg/kg per day for the first 3 weeks and then tapered) plus single-dose rituximab (375 mg/m2 ; n = 31) or plus cyclophosphamide (2 mg/kg per day until inhibitor becomes negative, for a maximum of 5 weeks; n = 32). The primary outcome was complete remission (CR, defined as FVIII activity ≥50 IU/dL, FVIII inhibitor undetectable, immunosuppression tapered and no bleeding for 24 h without bypassing agents) rate measured within 8 weeks. The noninferiority margin was an absolute difference of 20%. Twenty-four (77.4%) patients in the rituximab group and 22 (68.8%) patients in the cyclophosphamide group achieved CR, which showed the noninferiority of the single-dose rituximab-based regimen (absolute difference = -8.67%, lower limit of the 95% confidence interval = -13.11%; Pnoninferiority = 0.005). No difference was found in the incidence of treatment-related adverse events. Single-dose rituximab plus glucocorticoid regimen showed similar efficacy and safety, without a reported risk of secondary malignancies or reproductive toxicity seen in cyclophosphamide, it might be recommended as a first-line therapy for AHA, especially in China where there is a young age trend in AHA patients. This trial was registered at ClinicalTrials.gov as #NCT03384277.

Fatigue and health-related quality of life in patients with immune thrombocytopenia: a longitudinal assessment in China.

BACKGROUND: There is lacking studies of longitudinally assessment of fatigue and health-related quality of life (HRQoL) among Chinese immune thrombocytopenia (ITP) adults. We aimed to evaluate changes in fatigue and HRQoL and identify the associated factors. METHODS: Patients' characteristics, Functional Assessment of Chronic Illness Therapy (FACIT-F) and the ITP-specific Patient Assessment Questionnaire (ITP-PAQ) scores at admission (T0), at discharge (T1), and three months after discharge (T2) were collected. Linear mixed effects models were used to examine changes over time. RESULTS: We included 175 patients. The mean score of FACIT-F at T0 was 37.2 and increased at T1 (39.0), while then decreased at T2 (34.7). Patients who were single, retired, had persistent ITP, splenomegaly had more severe fatigue, whereas those who had not received any prior treatment and had a bleeding score of 0 at admission had milder fatigue. The mean score of ITP-PAQ was 57.7 at T0, then gradually increased to 60.3 at T1 and 62.8 at T2. Patients with persistent ITP and those who have never received treatment for ITP have better HRQoL. CONCLUSION: ITP adults' fatigue and HRQoL were impaired. Patients' fatigue improved at discharge but worsened at three months after discharge, while HRQoL gradually improved over time.

Clinical features and current treatment status of essential thrombocythemia in older adults: a multicenter real-world study in China.

Approximately half of patients diagnosed with essential thrombocythemia (ET) are older adults (aged ≥ 60 years), but to date, little is known about the clinical and molecular characteristics of older patients diagnosed according to the 2016 World Health Organization criteria. We retrospectively collected clinical and molecular data from 282 older (≥ 60 years) and 621 younger ET patients (18-59 years) in China from March 1, 2012 to November 1, 2021 and summarized the clinical characteristics and treatment of these older ET patients. Compared to younger patients, older patients had a higher incidence of the JAK2V617F mutation (P = 0.001), a lower incidence of CALR mutations (P = 0.033) and a higher rate of epigenetic mutations (P < 0.001), TP53 mutations (P = 0.005), and RNA splicing mutations (P < 0.001). Older patients had not only a higher incidence of thrombosis but also a higher incidence of bleeding events. Furthermore, older patients had a significantly higher mortality rate after disease progression (P = 0.050) or after thrombotic events (P = 0.013). Risk factors for thrombosis or prognosis were significantly different between older patients and the entire ET cohort. In older patients, non-driver mutations contributed significantly to thrombotic complications and a poor prognosis, while the JAK2V617F mutation was a risk factor for overall survival but not for thrombotic events. The application of interferon in older ET patients was not inferior to that of hydroxyurea in terms of efficacy and safety. Older patients presented unique characteristics different from those of younger patients, which could provide new information for formulating more appropriate treatment and follow-up strategies.

Machine learning analyses constructed a novel model to predict recurrent thrombosis in adults with essential thrombocythemia.

The current study involving 318 essential thrombocythemia (ET) patients with prior thrombosis was designed to identify risk factors that were predictive of recurrent thrombosis. The whole cohort was randomly split into derivation and validation cohorts. The random forest method, support vector machine with built-in recursive feature elimination model, and logistic multivariable analysis were performed in the derivation cohort, and cardiovascular risk factor (CVF) and RBC distribution width with standard deviation (RDW-SD) were finally selected as independent predictors. Subsequently we devise a 3-tiered model (low risk: 0 points; intermediate risk: 1-1.5 points; and high risk: 2.5 points) and it showed good discrimination in all cohorts. Moreover, the model was significantly correlated with rethrombosis-free survival (rTFS) (p = 0.0007 in the derivation cohort; p = 0.0019 in the validation cohort). In the whole cohort, cytoreductive therapy was more effective than antiplatelet agents alone for 10-year rTFS (p = 0.0336). No significant difference in 10-year rTFS was observed among interferon (IFN), hydroxyurea (HU), and IFN + HU therapy (p = 0.444). The present study helps identify individuals who need close monitoring and provides valuable risk signals for recurrence in ET patients with prior thrombosis.

Prediction of thrombosis in polycythemia vera: Development and validation of a multiple factor-based prognostic score system.

Background: Thrombosis is an important cause of death in patients with polycythemia vera (PV). The conventional stratification of thrombosis may ignore some potential risk factors. Objectives: This study aimed to develop and validate a multiple factor-based prediction model of thrombosis for the 2016 World Health Organization-dened PV. Methods: Clinical and next-generation sequencing data from 2 cohorts of patients with PV were analyzed. Multivariable Cox regression analyses were conducted for the identification of thrombotic risk factors and model development. Results: The study involved 372 patients in the training cohort and another 195 patients in the external validation cohort. Multivariable analyses indicated that age ≥60 years (hazard ratio [HR] 2.56, 95% CI 1.51-4.35, P < .001), cardiovascular risk factors (HR 4.22, 95% CI 2.00-8.92, P < .001), at least 1 high-risk mutation for thrombosis (mutations in DNMT3A, ASXL1, or BCOR/BCORL1) (HR 4.35, 95% CI 2.62-7.21, P < .001), and previous thrombosis (HR 5.93, 95% CI 3.29-10.68, P < .001) were independent risk factors of thrombosis. After assigning coefficient-weighted scores to each risk factor mentioned above, a multiple factor-based prognostic score system of thrombosis (MFPS-PV) was developed, classifying patients into low-risk, intermediate-risk, and high-risk groups. Patients in the 3 groups had notably different thrombosis-free survival rates (P < .001). The MFPS-PV outperformed the conventional model in discrimination power (C-statistic: 0.87 [95% CI 0.83-0.91] vs 0.80 [95% CI 0.74-0.86]). The MFPS-PV was well calibrated and remained consistent during external validation. Conclusion: The MFPS-PV, integrating genetic and clinical characteristics for the first time, shows excellent accuracy and utility for thrombosis prediction in WHO-defined PV.

Increased reactive oxygen species lead to overactivation of platelets in essential thrombocythemia.

INTRODUCTION: Platelet function, rather than platelet count, plays a crucial role in thrombosis in essential thrombocythemia (ET). However, little is known about the abnormal function of platelets in ET. Here, we investigated the functional characteristics of platelets in ET hemostasis to explore the causes of ET platelet dysfunction and new therapeutic strategies for ET. MATERIALS AND METHODS: We analyzed platelet aggregation, activation, apoptosis, and reactive oxygen species (ROS) in ET patients and JAK2V617F-positive ET-like mice. The effects of ROS on platelet function and the underlying mechanism were investigated by inhibiting ROS using N-acetylcysteine (NAC). RESULTS: Platelet aggregation, activation, apoptosis, ROS, and clot retraction were elevated in ET. No significant differences were observed between ET patients with JAK2V617F or CALR mutations. Increased ROS activated the JAK-STAT pathway, which may further influence platelet function. Inhibition of platelet ROS by NAC reduced platelet aggregation, activation, and apoptosis, and prolonged bleeding time. Furthermore, NAC treatment reduced platelet count in ET-like mice by inhibiting platelet production from megakaryocytes. CONCLUSIONS: Elevated ROS in ET platelets resulted in enhanced platelet activation, function and increased risk of thrombosis. NAC offers a potential therapeutic strategy for reducing platelet count.

Preclinical studies of a factor X activator and a phase 1 trial for hemophilia patients with inhibitors.

BACKGROUND: Bleeding episodes in hemophiliacs with inhibitors are difficult to control. Staidson protein-0601 (STSP-0601), a specific factor (F)X activator purified from the venom of Daboia russelii siamensis, has been developed. OBJECTIVES: We aimed to investigate the efficacy and safety of STSP-0601 in preclinical and clinical studies. METHODS: In vitro and in vivo preclinical studies were performed. A phase 1, first-in-human, multicenter, and open-label trial was conducted. The clinical study was divided into parts A and B. Hemophiliacs with inhibitors were eligible for this study. Patients received a single intravenous injection of STSP-0601 (0.01 U/kg, 0.04 U/kg, 0.08 U/kg, 0.16 U/kg, 0.32 U/kg, or 0.48 U/kg) in part A or a maximum of 6 4-hourly injections (0.16 U/kg) in part B. The primary endpoint for each part was the number of adverse events (AEs) from baseline to 168 hours after administration. This study was registered at clinicaltrials.gov (NCT-04747964 and NCT-05027230). RESULTS: Preclinical studies showed that STSP-0601 could specifically activate FX in a dose-dependent manner. In the clinical study, 16 patients in part A and 7 patients in part B were enrolled. Eight (22.2%) AEs in part A and 18 (75.0%) AEs in part B were reported to be related to STSP-0601. Neither severe AEs nor dose-limiting toxicity events were reported. There were no thromboembolic event. The antidrug antibody of STSP-0601 was not detected. CONCLUSION: Preclinical and clinical studies showed that STSP-0601 had a good ability to activate FX and had a good safety profile. STSP-0601 could be used as a hemostatic treatment in hemophiliacs with inhibitors.

Molecular analysis of phenotypic heterogeneity in JAK2V617F-positive myeloproliferative neoplasms reveals a potential target for therapy.

JAK2V617F is the most frequent mutation in BCR-ABL-negative myeloproliferative neoplasms (MPNs). It is an important but not the only determinant of MPN phenotype. We performed high-throughput sequencing on JAK2V617F+ essential thrombocythaemia (ET) and polycythaemia vera (PV) patient samples to unveil factors involved in phenotypic heterogeneity and to identify novel therapeutic targets for MPN. Two concurrent mutations that may affect phenotype were identified, including mutations in SH2B3, which is primarily prevalent in PV, and SF3B1, which is more commonly mutated in ET. Next, we conducted transcriptomic analysis at the haematopoietic stem cell (HSC) and megakaryocyte (MK)-erythroid progenitor (MEP) levels. Inflammatory signalling pathways were elevated in both ET HSCs and MEPs, unlike in PV HSCs and MEPs. Notably, Wnt/ß-catenin signalling was uniquely upregulated during ET haematopoietic differentiation from HSC to MEP, and inhibiting Wnt/ß-catenin signalling blocked MK differentiation in vitro. Consistently, Wnt/ß-catenin inhibitor administration decreased platelet counts in JAK2V617F+ MPN mice by blocking MEPs and MK progenitors and by inhibiting maturation of MKs, while in wild-type mice, Wnt/ß-catenin inhibitor did not significantly reduce platelet counts. In conclusion, our findings provide new insights into the mechanisms underlying phenotypic differentiation of JAK2V617F+ PV and ET and indicate Wnt/ß-catenin signalling as a potential therapeutic target for MPN.

Hematopoietic Stem Cell Heterogeneity Is Linked to the Initiation and Therapeutic Response of Myeloproliferative Neoplasms.

The implications of stem cell heterogeneity for disease pathogenesis and therapy are poorly defined. JAK2V617F+ myeloproliferative neoplasms (MPNs), harboring the same mutation in hematopoietic stem cells (HSCs), display diverse phenotypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These chronic malignant disorders are ideal models to analyze the pathological consequences of stem cell heterogeneity. Single-cell gene expression profiling with parallel mutation detection demonstrated that the megakaryocyte (Mk)-primed HSC subpopulation expanded significantly with enhanced potential in untreated individuals with JAK2V617F+ ET, driven primarily by the JAK2 mutation and elevated interferon signaling. During treatment, mutant HSCs were targeted preferentially in the Mk-primed HSC subpopulation. Interestingly, homozygous mutant HSCs were forced to re-enter quiescence, whereas their heterozygous counterparts underwent apoptosis. This study provides important evidence for the association of stem cell heterogeneity with the pathogenesis and therapeutic response of a malignant disease.

Multilevel defects in the hematopoietic niche in essential thrombocythemia.

The role of the bone marrow niche in essential thrombocythemia (ET) remains unclear. Here, we observed multilevel defects in the hematopoietic niche of patients with JAK2V617F-positive ET, including functional deficiency in mesenchymal stromal cells (MSC), immune imbalance, and sympathetic-nerve damage. Mesenchymal stromal cells from patients with JAK2V617F-positive essential thrombocythemia had a transformed transcriptome. In parallel, they showed enhanced proliferation, decreased apoptosis and senescence, attenuated ability to differentiate into adipocytes and osteocytes, and insufficient support for normal hematopoiesis. Additionally, they were inefficient in suppressing immune responses. For instance, they poorly inhibited proliferation and activation of CD4-positive T cells and the secretion of the inflammatory factor soluble CD40-ligand. They also poorly induced formation of mostly immunosuppressive T-helper 2 cells (Th2) and the secretion of the anti-inflammatory factor interleukin-4 (IL-4). Furthermore, we identified WDR4 as a potent protein with low expression and which was correlated with increased proliferation, reduced senescence and differentiation, and insufficient support for normal hematopoiesis in MSC from patients with JAK2V617F-positive ET. We also observed that loss of WDR4 in MSC cells downregulated the interleukin-6 (IL-6) level through the ERK-GSK3ß-CREB signaling based on our in vitro studies. Altogether, our results show that multilevel changes occur in the bone marrow niche of patients with JAK2V617F-positive ET, and low expression of WDR4 in MSC may be critical for inducing hematopoietic related changes.

[Clinical Characteristic of "triple-negative" Essential Thrombocythaemia Patients and Mutation Analysis by Targeted Sequencing].

BACKGROUND: Essential thrombocythemia is a subgroup of myeloproliferative neoplasms. Previous studies identified mutations of JAK2, CALR, and MPL that are closely related with the pathogenesis of myeloproliferative neoplasms. All these mutations contribute to the hyperactivation of JAK2/STAT pathway. However, a small proportion of essential thrombocythemia patients does not display such mutations. The pathogenesis of "triple-negative" form of essential thrombocythemia remains unknown. OBJECTIVE: To investigate the clinical characteristics of triple-negative essential thrombocythemia and related mutation genes. METHODS: To identify the mutations associated with triple-negative essential thrombocythemia, next-generation sequencing was used to conduct targeted sequencing of 360 genes in samples from 68 patients. RESULTS: At least one missense mutation was detected in all the patients and all the detected genes. After screening the data, it was observed that 10 genes with the 10 highest mutation were follows: FLT3, SH2B3, ASXL1, ADAMTS1, TET2, TP53, EGFR, CUX1, GATA2, and MPL.When only rare genes (i.e., with a frequency in Asian populations lower than 5%, as estimated by the 1000 Genomes Project) were analyzed, the most frequently mutated genes in the patients were TET2 (33.82%), SH2B3(29.41%), and ASXL1 (23.53%). Our study identified some mutations that did not previously reported. Although all these mutations need further validation, high incidence rates may indicate relevance of the respective mutations to essential thrombocythemia pathogenesis. Some of the detected mutations have been previously reported; these mutations were also found in a large proportion of our subjects. CONCLUSION: whole-exon sequencing can provide a higher level of accuracy for gene mutation analysis and assist in identifying mutations that contribute to illustrate the pathogenesis of essential thrombocythemia.