The CRISPR-Cas System and Clinical Applications of CRISPR-Based Gene Editing in Hematology with a Focus on Inherited Germline Predisposition to Hematologic Malignancies.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has begun to transform the treatment landscape of genetic diseases. The history of the discovery of CRISPR/CRISPR-associated (Cas) proteins/single-guide RNA (sgRNA)-based gene editing since the first report of repetitive sequences of unknown significance in 1987 is fascinating, highly instructive, and inspiring for future advances in medicine. The recent approval of CRISPR-Cas9-based gene therapy to treat patients with severe sickle cell anemia and transfusion-dependent ß-thalassemia has renewed hope for treating other hematologic diseases, including patients with a germline predisposition to hematologic malignancies, who would benefit greatly from the development of CRISPR-inspired gene therapies. The purpose of this paper is three-fold: first, a chronological description of the history of CRISPR-Cas9-sgRNA-based gene editing; second, a brief description of the current state of clinical research in hematologic diseases, including selected applications in treating hematologic diseases with CRISPR-based gene therapy, preceded by a brief description of the current tools being used in clinical genome editing; and third, a presentation of the current progress in gene therapies in inherited hematologic diseases and bone marrow failure syndromes, to hopefully stimulate efforts towards developing these therapies for patients with inherited bone marrow failure syndromes and other inherited conditions with a germline predisposition to hematologic malignancies.

[Clinical efficacy of allogeneic hematopoietic stem cell transplantation for myelodysplastic syndrome-evolved acute myeloid leukemia].

Objective: The outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for myelodysplastic syndromes-evolved acute myeloid leukemia (MDS-AML) were explored. Methods: A retrospective review was conducted for 54 patients with MDS-AML treated with allo-HSCT in the Institute of Hematology and Blood Disease Hospital from January 2018 to August 2022. The clinical effects after transplantation were observed, and the related risk factors influencing prognosis were explored. Results: Of the total 54 patients, 26 males, 28 females, and 53 patients achieved hematopoietic reconstruction. After a median follow-up of 597 (15-1 934) days, the 1 year overall survival (OS) rate, disease-free survival (DFS) rate, relapse rate (CIR) and non-relapse mortality (NRM) rate were 75.8%±5.8%, 72.1%±6.1%, 12.7%±4.9%, and 17.1%±5.2%, respectively. The 3 year estimated OS, DFS, CIR, and NRM rates were 57.8%±7.5%, 58.1%±7.2%, 23.2%±6.6%, and 23.7%±6.6%, respectively. The cumulative incidence of acute graft-versus-host disease (aGVHD) was 57.5%±6.9%, and the cumulative incidence of chronic graft-versus-host disease (cGVHD) was 48.4%±7.7%. Hematopoietic cell transplantation comorbidity index (HCT-CI) before transplantation was ≥2, minimal residual disease (MRD) was positive on the day of reconstitution, grade Ⅲ/Ⅳ aGVHD, bacterial or fungal infection and no cGVHD after transplantation were adverse prognostic factors for OS (P<0.05). COX regression model for multivariate analysis showed that HCT-CI score before transplantation, bone marrow MRD on the day of response, grade Ⅲ or Ⅳ aGVHD, and cGVHD after transplantation were the independent adverse factors for OS (P=0.001, HR=6.981, 95%CI 2.186-22.300; P=0.010, HR=6.719, 95%CI 1.572-28.711; P=0.026, HR=3.386, 95%CI 1.158-9.901; P=0.006, HR=0.151, 95%CI 0.039-0.581) . Conclusion: For patients with MDS-AML and high risk of relapse, allogeneic transplantation must be considered as soon as possible. The enhanced management of post-transplantation complications and maintenance treatment should be provided whenever possible after transplantation.

Complete Blood Counts and Research Parameters in the Detection of Myelodysplastic Syndromes.

The diagnosis of Myelodysplastic syndromes (MDS) is frequently challenging, especially in terms of the distinction from the other non-neoplastic causes of cytopenia. Currently, it is based on the presence of peripheral blood cytopenias, peripheral blood and bone marrow dysplasia/blasts, and clonal cytogenetic abnormalities, but MDS diagnostic features are polymorphic and non-specific. We investigated the utility of complete blood count (CBC) and research parameters (RUO) from the analyzer BC 6800 Plus (Mindray Diagnostics) to discriminate MDS-related cytopenia. METHODS: 100 samples from healthy individuals were used to establish the values of research parameters in normal subjects. A retrospective study was conducted including 66 patients diagnosed with MDS, 90 cytopenic patients due to other diseases (cancer patients receiving therapy, aplastic anemia, other hematological malignancies) and 50 with macrocytic anemia. The Wilcoxon test was applied to detect statistical differences among the groups of patients, considering p < 0.05 significant. The diagnostic performance of the RUO parameters for discriminating MDS among cytopenias was evaluated using receiver operating characteristic (ROC) curve analysis. Amultivariable logistic regression model was performed to identify the potential predictors for having MDS. The area under curve (AUC) and the Hosmer-Lemeshow test of the model were assessed. The performance of the model was verified in a prospective study including 224 cytopenic patients (validation group). RESULTS: In the MDS group, the mean cell volume (MCV), percentage of macrocytic red cells (MAC), red cell distribution width (RDW) and immature platelets fraction (IPF) had increased values compared to the cytopenic and normal patients, while platelets, red and white cell counts, Neu X (related to the cytoplasmic complexity of neutrophils), Neu Y (related to nucleic acid content) and Neu Z (related to cell size) were lower (p < 0.001). Neu X, Neu Y, and Neu Z showed higher AUC for detecting MDS > 0.80; MAC, RDW and IPF AUC > 0.76. The multivariable model demonstrated that Neu X and Neu Y could be used in the recognition of MDS, AUC 0.88. In the validation group, 89.0% of the MDS patients were well classified. CONCLUSION: MDS are common malignant disorders with a poor prognosis, and early diagnosis is warranted for optimal benefit from treatment. RUO gain insights to detect dysplasia of MDS and could be used in the differential diagnosis of MDS from cytopenias of other etiologies.

Detection of decreased granules in neutrophils by automated hematology analyzers XR-1000 and UniCel DxH 800.

OBJECTIVE: This study aimed to investigate the utility of neutrophil-related cell population data obtained by automated hematology analyzers in assessing myelodysplastic syndrome cases with decreased granules in neutrophils. METHODS: A total of 108 subjects were classified into normal granule (n = 35), hypogranulation (n = 37), or hypergranulation (n = 36) groups. Neutrophil cell area and granule area were measured by ImageJ. All samples were analyzed on the XR-1000 and UniCel DxH 800, and neutrophil-related parameters were compared among the 3 groups. RESULTS: Neutrophil cell area and the ratio of the granular area showed significant differences among the 3 groups; they were the highest in the hypergranulation group and lowest in the hypogranulation group. XR-1000 data showed significant differences in NE-SFL and NE-FSC among the 3 groups (P < .0001). NE-SFL and NE-FSC discriminated most accurately hypogranulation group against other groups. UniCel DxH 800 data showed significant differences in MN-V-NE, MN-MALS-N, MN-UMALS-NE, SD-UMALS-NE (P <.01), MN-LMALS-NE, and SD-LMALS-NE (P <.05) among the 3 groups. The combination of SD-V-NE and SD-LMALS-NE discriminated most accurately the hypogranulation group against the other groups. CONCLUSION: NE-SFL and NE-FSC and the combination of SD-V-NE and SD-LMALS-NE are useful in detecting cases with decreased granules in neutrophils.

Characterization of second primary malignancies post CAR T-cell therapy: real-world insights from the two global pharmacovigilance databases of FAERS and VigiBase.

Background: The FDA's alerts regarding the T-cell lymphoma risk post CAR-T therapy has garnered global attention, yet a comprehensive profile of second primary malignancies (SPMs) following CAR-T treatment is lacking. Methods: We extracted adverse event reports of hematological malignancies (HMs) patients with clearly definable SPMs from the FAERS and VigiBase databases (2017-2023). Disproportionality analysis using reporting odds ratio (ROR) and adjusted ROR was performed to assess associations between SPMs and CAR-T therapy. Time-to-onset analysis explored factors affecting SPM manifestation. Findings: SPMs post CAR T-cell therapy include HMs and solid tumors. T-cell lymphoma and myelodysplastic syndromes were consistently identified as positive signals across the overall and subgroup analyses. Hematological SPMs showed earlier onset with increasing annual incidence post CAR-T therapy, whereas solid tumors exhibit delayed manifestation. SPMs in CAR-T recipients had significantly earlier onset than non-recipients. Furthermore, age-specific characteristics reveal earlier SPM manifestations in pediatric, adolescent, and young adult populations compared to older populations post CAR-T therapy. Interpretation: The current SPM profile highlights the necessity of long-term safety monitoring for all CAR-T recipients given the observed yearly increase of SPMs. Customizing long-term SPM screening across different age groups may enhance early detection and intervention strategies, ultimately improving patient outcomes in the follow-up of CAR-T recipients. Funding: This work was supported by grants from the Natural Science Foundation of Guangdong Province (2018A030313846 and 2021A1515012593), the Science and Technology Planning Project of Guangdong Province (2019A030317020), the National Natural Science Foundation of China (81802257, 81871859, 81772457, 82172750, 82172811, and 82260546), the Guangdong Basic and Applied Basic Research Foundation (Guangdong-Guangzhou Joint Funds) (2022A1515111212), and the Science and Technology Program of Guangzhou (2023A04J1257).

Molecular monitoring of MDS: Watch this space!

The use of IPSS-M provides a wealth of molecular information in newly diagnosed myelodysplastic syndromes (MDS) patients. Besides the prognostic implications, molecular markers will also help to choose therapeutic options and may also be informative to determine the depth of response. Duployez and Preudhomme provide a comprehensive overview of this area of research, which is particularly complex in MDS. Commentary on: Duployez et al. Monitoring molecular changes in the management of myelodysplastic syndromes. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19614.

Myelodysplasia cutis and VEXAS syndrome initially diagnosed as histiocytoid Sweet syndrome: A diagnostic pitfall.

Histiocytoid Sweet syndrome (H-SS) is a histopathological variant of Sweet syndrome (SS) defined by cutaneous infiltration of immature myeloid cells morphologically resembling histiocytes. The association of H-SS with underlying malignancy, particularly myelodysplastic syndromes, is well-established. Myelodysplasia cutis (MDS-cutis) has been proposed to describe cases historically diagnosed as H-SS but characterized by shared clonality of the myeloid infiltrate in skin and bone marrow. Therefore, identifying patients who might have MDS-cutis is critical for the management of the associated hematologic malignancy. VEXAS syndrome, an adult-onset autoinflammatory disease, should also be included in the histopathologic differential diagnosis of H-SS, as it shares clinical and pathologic features with MDS-cutis. Through the presentation of two cases, we aim to highlight the defining features and key clinical implications of MDS-cutis and VEXAS syndrome.

Phase 1 dose escalation study of the MDM2 inhibitor milademetan as monotherapy and in combination with azacitidine in patients with myeloid malignancies.

BACKGROUND: Mouse double minute-2 homolog (MDM2) plays a key role in downregulating p53 activity in hematologic malignancies, and its overexpression is associated with poor outcomes. METHODS: This phase 1 study assessed the safety and efficacy of different dosing regimens of the MDM2 inhibitor milademetan as monotherapy and in combination with azacitidine (AZA) in patients with relapsed or refractory acute myeloid leukemia or high-risk myelodysplastic syndromes. RESULTS: Seventy-four patients (monotherapy, n = 57; milademetan-AZA combination, n = 17) were treated. The maximum tolerated dose of milademetan was 160 mg once daily given for the first 14-21 days of 28-day cycles as monotherapy and on Days 5-14 in combination with AZA. Dose-limiting toxicities were gastrointestinal, fatigue, or renal/electrolyte abnormalities. Treatment-emergent adverse events related to milademetan occurred in 82.5% and 64.7% of participants in the monotherapy and AZA combination arms, respectively. Two participants (4.2%) in the monotherapy arm achieved complete remission (CR), and 1 (2.1%) achieved CR with incomplete blood count recovery (CRi). Two participants (13.3%) achieved CRi in the combination arm. New TP53 mutations, detected only during milademetan monotherapy, were found pre-existing below standard detection frequency by droplet digital polymerase chain reaction. INTERPRETATION: Milademetan was relatively well tolerated in this population; however, despite signals of activity, clinical efficacy was minimal.

Fatal posterior reversible encephalopathy syndrome after blood transfusion in a patient with myelodysplastic syndromes.

BACKGROUND: Posterior reversible encephalopathy syndrome (PRES) is known as a transfusion-related complication with typically favorable prognosis and no report fatalities. Pathological evaluation of PRES is also scarce. CASE REPORT: An 88-year-old female with myelodysplastic syndromes (MDS) attended our hospital because of a compression fracture and chronic heart failure with chronic anemia. While her hemoglobin levels improved from 4.6 to 8.0 g/dL and the pleural effusions substantially decreased following six units of red blood cell transfusion and diuretic therapy, a gradual decline in cognitive function and speech reduction was noted. PRES was diagnosed by magnetic resonance imaging of the head. Despite treatment of intensive supportive care, the patient fell into a coma by the 20th day and passed away on the 22nd day. Although the pathophysiological link between blood-transfusion-related PRES and its impact on survival is not fully understood, autopsy findings confirmed the diagnosis of PRES and revealed multiple cerebral hemorrhages that were not detected in earlier imaging studies. CONCLUSION: This case highlights the importance of vigilant monitoring and management of PRES, especially in high-risk populations such as elderly patients with multiple comorbidities or those with thrombocytopenia. Further studies are needed to elucidate the mechanisms of PRES in patients with hematologic diseases.

Reclassification of Myelodysplastic Neoplasms According to the 2022 World Health Organization Classification and the 2022 International Consensus Classification Using Open-Source Data: Focus on SF3B1- and TP53-mutated Myelodysplastic Neoplasms.

Background: In 2022, the WHO and International Consensus Classification (ICC) published diagnostic criteria for myelodysplastic neoplasms (MDSs). We examined the influence of the revised diagnostic criteria on classifying MDSs in a large population. Methods: We retrieved an open-source pre-existing dataset from cBioPortal and included 2,454 patients with MDS in this study. Patients were reclassified based on the new diagnostic 2022 WHO and ICC criteria. Survival analysis was performed using Cox regression to validate the new criteria and to assess risk factors. Results: Based on the 2022 WHO criteria, 1.4% of patients were reclassified as having AML. The 2022 WHO criteria provide a superior prognostic/diagnostic model to the 2017 WHO criteria (Akaike information criterion, 14,152 vs. 14,516; concordance index, 0.705 vs. 0.681). For classifying MDS with low blast counts and SF3B1 mutation, a variant allele frequency cut-off of 5% (2022 WHO criteria) and the absence of RUNX1 co-mutation (2022 ICC criteria) are diagnostically relevant. For classifying MDSs with mutated TP53, a blast count cut-off of 10% (2022 ICC criteria) and multi-hit TP53 (2022 WHO criteria) are independent risk factors in cases with ≥10% blasts. Conclusions: Our findings support the refinements of the new WHO criteria. We recommend the complementary use of the new WHO and ICC criteria in classifying SF3B1- and TP53-mutated MDSs for better survival prediction.

NPM1-mutated myeloid neoplasms are a unique entity not defined by bone marrow blast percentage.

INTRODUCTION: NPM1-mutated (NPM1mut) myeloid neoplasms (MNs) with <20% bone marrow (BM) blasts (NPM1mut MNs<20) are uncommon, and their classification remains inconsistent. METHODS: The clinicopathologic features of 54 patients with NPM1mut MNs <20 were evaluated and compared with wild-type NPM1 MNs <20 and NPM1mut MNs≥20, respectively. RESULTS: NPM1mut MNs had similar features regardless of blast percentage, except for higher IDH2 (29% vs 7%, p = .023) and FLT3 (70% vs 11%, p < .001) frequency in patients with ≥20% BM blasts. Thirty-three (61%) patients with NPM1mut MNs <20 received low-intensity chemotherapy (LIC) and 12 (22%) received intensive chemotherapy (IC). Higher complete remission rates (75% vs 27%, p = .006) and median overall survival (mOS) (not reached vs 30.4 months, p = .06) were observed with IC compared to LIC. Young patients (age <60 years) did not reach mOS either when treated with LIC or IC. Stem cell transplant was associated with increased survival only in patients treated with LIC (HR, 0.24; p = .025). No differences in mOS were observed by BM blast strata (32.2 months, not reached and 46.9 months for <10%, 10%-19%, and ≥20% blasts, p = .700) regardless of treatment modality (LIC: p = .900; IC: p = .360). Twenty-three patients (43%) with NPM1mut MNs <20 had marrow blast progression to ≥20%. CONCLUSIONS: Overall, NPM1mut MNs define a unique entity independent of BM blast percentage.

Real-world study of the use of azacitidine in myelodysplasia in Australia.

Hypomethylating agents are the most widely used upfront therapy for patients with myelodysplastic syndrome (MDS) who are not suitable for hematopoietic stem cell transplantation. In Australia, azacitidine was, until recently, the only approved and subsidized treatment for patients with intermediate-2 and high-risk MDS, chronic myelomonocytic leukemia, and low blast acute myeloid leukemia. We analyzed prescription data to evaluate the real-world persistence and overall survival (OS) of patients prescribed azacitidine for the first time in Australia. A retrospective cohort analysis of patients who had been prescribed Pharmaceutical Benefits Scheme (PBS)-listed azacitidine for the first time, between January 2016 and April 2021, was conducted using the PBS 10% dataset. Treatment persistence and OS were estimated using Kaplan-Meier methods. The impact of the number of treatment cycles and treatment adherence on OS was also estimated. There were 351 patients in the PBS 10% dataset who initiated treatment with azacitidine. The average age (standard deviation [SD]) at azacitidine initiation was 71.9 (11.1) years and the average number (SD) of azacitidine prescriptions was 5.6 (0.2). The median persistence on azacitidine was 15.6 months, and the OS was 13.4 months. The median OS for patients who had six or more cycles of azacitidine treatment was greater compared to patients who had five or less cycles of treatment. The data from this real-world study illustrate the unmet medical needs of patients with MDS treated with azacitidine in Australia. The majority of patients are not treated with the optimal number of cycles of azacitidine, which is negatively correlated with patient outcomes.

Development of the treatment preference in myelodysplasia questionnaire for clinicians, carers, and patients.

This study reports the development activities for the Treatment Preference Myelodysplasia Questionnaires (TPMQ) for clinicians (mTPMQ), carers (cTPMQ), and patients (pTPMQ). These tools are intended to evaluate treatment preferences for patients with myelodysplastic syndromes (MDS). This was a non-interventional, cross-sectional qualitative interview study consisting of interviews with clinicians, patients, and those caring for patients with MDS. All participants were located in Australia and data were collected from qualitative mixed-method interviews composed of concept elicitation and cognitive debriefing related to initial drafts of the questionnaires. Fifteen individuals participated in interviews (five from each group). Based on the concept elicitation portion of interviews, concepts of importance were classified and reasons for treatment preference were documented. From cognitive debriefing, the questionnaires were generally deemed to be clear and easy to understand. Participant input from both concept elicitation and cognitive debriefing portions was used to revise initial drafts of the questionnaires. The mTPMQ, cTPMQ, and pTPMQ were developed with direct input from clinicians, patients, and caregivers to assess the key concepts of interest related to the preference for the treatment of MDS and are ready to be used and evaluated further in clinical trials.

Analyzing Differences in Hematological and Immunological Characteristics Related to Common Gene Mutations in Myelodysplastic Syndromes.

BACKGROUND: Genetic mutations play a crucial role in the development and progression of myelodysplastic syndromes (MDS), impacting the immune microenvironment and influencing the choice of treatment regimen, as well as the efficacy and prognosis of patients. The objective of this study was to examine variations in hematological and immunological characteristics associated with common gene mutations in MDS patients and establish a foundation for the precise treatment of MDS. METHODS: The hematological, immunological, and other clinical features of 71 recently diagnosed MDS patients from January 1, 2019, to July 31, 2023, were retrospectively analyzed. These patients were categorized based on their gene mutations, and the variances in hematological and immunological characteristics among distinct groups were compared. RESULTS: Hematological variances were observed among different gene mutation groups. Specifically, platelet counts in the splicing factor 3B subunit 1 (SF3B1) mutation group were notably higher compared to the wild-type group (p = 0.009). Conversely, in the additional sex combs like 1 (ASXL1) mutation groups, monocyte ratios were significantly elevated in comparison to the wild-type group (p = 0.046), and in the ten-eleven translocation 2 (TET2) mutation group, lymphocyte ratios were significantly lower (p = 0.022). Additionally, the leukocyte (p = 0.005), neutrophil ratio (p = 0.002), and lymphocyte ratio (p = 0.001) were significantly higher in the Runt-related transcription factor 1 (RUNX1) mutation group. Regarding immunological distinctions, the Natural Killer (NK) cell ratio demonstrated a significant increase in the SF3B1 mutation group (p = 0.005). Moreover, the TET2 mutation group exhibited a significantly higher Interleukin-8 (IL-8) level (p = 0.017). In contrast, the U2 small nuclear RNA auxiliary factor 1 (U2AF1) group displayed significantly lower levels of IL-1ß (p = 0.033), IL-10 (p = 0.033), and Tumour Necrosis Factor-α (TNF-α) (p = 0.009). CONCLUSION: Distinct variations exist in the immune microenvironment of MDS associated with different genetic mutations. Further studies are imperative to delve into the underlying mechanisms that drive these differences.

Monitoring molecular changes in the management of myelodysplastic syndromes.

The ongoing or anticipated therapeutic advances as well as previous experience in other malignancies, including acute myeloid leukaemia, have made molecular monitoring a potential interesting tool for predicting outcomes and demonstrating treatment efficacy in patients with myelodysplastic syndromes (MDS). The important genetic heterogeneity in MDS has made challenging the establishment of recommendations. In this context, high-throughput/next-generation sequencing (NGS) has emerged as an attractive tool, especially in patients with high-risk diseases. However, its implementation in clinical practice still suffers from a lack of standardization in terms of sensitivity, bioinformatics and result interpretation. Data from literature, mostly gleaned from retrospective cohorts, show NGS monitoring when used appropriately could help clinicians to guide therapy, detect early relapse and predict disease evolution. Translating these observations into personalized patient management requires a prospective evaluation in clinical research and remains a major challenge for the next years.

No advantage of antimicrobial prophylaxis in AML/MDS/CMML patients treated with azacitidine-a prospective multicenter study by the Polish Adult Leukemia Group.

Introduction: Infections represent one of the most frequent causes of death of higher-risk MDS patients, as reported previously also by our group. Azacitidine Infection Risk Model (AIR), based on red blood cell (RBC) transfusion dependency, neutropenia <0.8 × 109/L, platelet count <50 × 109/L, albumin <35g/L, and ECOG performance status ≥2 has been proposed based on the retrospective data to estimate the risk of infection in azacitidine treated patients. Methods: The prospective non-intervention study aimed to identify factors predisposing to infection, validate the AIR score, and assess the impact of antimicrobial prophylaxis on the outcome of azacitidine-treated MDS/AML and CMML patients. Results: We collected data on 307 patients, 57.6 % males, treated with azacitidine: AML (37.8%), MDS (55.0%), and CMML (7.1%). The median age at azacitidine treatment commencement was 71 (range, 18-95) years. 200 (65%) patients were assigned to higher risk AIR group. Antibacterial, antifungal, and antiviral prophylaxis was used in 66.0%, 29.3%, and 25.7% of patients, respectively. In total, 169 infectious episodes (IE) were recorded in 118 (38.4%) patients within the first three azacitidine cycles. In a multivariate analysis ECOG status, RBC transfusion dependency, IPSS-R score, and CRP concentration were statistically significant for infection development (p < 0.05). The occurrence of infection within the first three azacitidine cycles was significantly higher in the higher risk AIR group - 47.0% than in lower risk 22.4% (odds ratio (OR) 3.06; 95% CI 1.82-5.30, p < 0.05). Administration of antimicrobial prophylaxis did not have a significant impact on all-infection occurrence in multivariate analysis: antibacterial prophylaxis (OR 0.93; 0.41-2.05, p = 0.87), antifungal OR 1.24 (0.54-2.85) (p = 0.59), antiviral OR 1.24 (0.53-2.82) (p = 0.60). Discussion: The AIR Model effectively discriminates infection-risk patients during azacitidine treatment. Antimicrobial prophylaxis does not decrease the infection rate.

Gut Microbiota Composition Correlates with Disease Severity in Myelodysplastic Syndrome.

The myelodysplastic syndrome (MDS) is a heterogeneous group of clonal disorders of hematopoietic progenitor cells related to ineffective hematopoiesis and an increased risk of transformation to acute myelogenous leukemia. MDS is divided into categories, namely lineage dysplasia (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS with multilineage dysplasia (MDS-MLD), MDS with excess blasts (MDS-EB). The International Prognostic Classification System (IPSS) ranks the patients as very low, low, intermediate, high, and very high based on disease evolution and survival rates. Evidence points to toll-like receptor (TLR) abnormal signaling as an underlying mechanism of this disease, providing a link between MDS and immune dysfunction. Microbial signals, such as lipopolysaccharides from gram-negative bacteria, can activate or suppress TLRs. Therefore, we hypothesized that MDS patients present gut microbiota alterations associated with disease subtypes and prognosis. To test this hypothesis, we sequenced the 16S rRNA gene from fecal samples of 30 MDS patients and 16 healthy elderly controls. We observed a negative correlation between Prevotella spp. and Akkermansia spp. in MDS patients compared with the control group. High-risk patients presented a significant increase in the genus Prevotella spp. compared to the other risk categories. There was a significant reduction in the abundance of the genus Akkermansia spp. in high-risk patients compared with low- and intermediate-risk. There was a significant decrease in the genus Ruminococcus spp. in MDS-EB patients compared with controls. Our findings show a new association between gut dysbiosis and higher-risk MDS, with a predominance of gram-negative bacteria.

Potential Diagnostic Value of Abnormal Pyroptosis Genes Expression in Myelodysplastic Syndromes (MDS): A Primary Observational Cohort Study.

Background: Myelodysplastic syndromes (MDS) are determined by ineffective hematopoiesis and bone marrow cytological dysplasia with somatic gene mutations and chromosomal abnormalities. Accumulating evidence has revealed the pivotal role of NLRP3 inflammasome activation and pyroptotic cell death in the pathogenesis of MDS. Although MDS can be diagnosed with a variety of morphologic and cytogenetic tests, most of these tests have limitations or problems in practice. Materials and Methods: In the present study, we evaluated the expression of genes that form the inflammasome (NLRP3, ASC, and CASP1) in bone marrow specimens of MDS patients and compared the results with those of other leukemias to evaluate their diagnostic value for MDS. Primary samples of this observational cohort study were collected from aspiration samples of patients with myelodysplastic syndromes (27 cases) and patients with non-myelodysplastic syndrome hematological cancers (45 cases). After RNA extraction and c.DNA synthesis, candidate transcripts and housekeeping transcripts were measured by real-time PCR method (SYBER Green assay). Using Kruskal-Wallis the relative gene expressions were compared and differences with p value less than 0.05 were considered as significant. Discrimination capability, cut-off, and area under curve (AUC) of all markers were analyzed with recessive operation curve (ROC) analysis. Results: We found that Caspase-1 and ASC genes expressed at more levels in MDS specimens compared to non-MDS hematological malignancies. A relative average expression of 10.22 with a p-value of 0.001 and 1.86 with p=0.019 was detected for Caspase-1 and ASC, respectively. ROC curve analysis shows an AUC of 0.739 with p=0.0001 for Caspase-1 and an AUC of 0.665 with p=0.0139 for ASC to MDS discrimination. Conclusion: Our results show that Caspase-1 and ASC gene expression levels can be used as potential biomarkers for MDS diagnosis. Prospective studies with large sample numbers are suggested.

Clinical Outcomes and Healthcare Resource Utilization for Patients With Lower-Risk Myelodysplastic Syndromes Treated With Erythropoiesis-Stimulating Agents.

INTRODUCTION: Real-world studies of lower-risk myelodysplastic syndromes (LR-MDS) are limited. We evaluated treatment patterns, clinical outcomes, and healthcare resource utilization (HCRU) among patients with LR-MDS treated with erythropoiesis-stimulating agents (ESAs) in the United States. PATIENTS AND METHODS: This retrospective study included patients with LR-MDS who initiated treatment with ESAs between January 1, 2016 and June 30, 2019. The primary analysis assessed patient demographic and clinical characteristics, treatment patterns, clinical outcomes (hematologic response, transfusion requirements, disease progression), and HCRU (medical encounters, laboratory tests, and medication use). Subgroup analyses of patients repeatedly treated with ESA therapy evaluated selected clinical outcomes and primary ESA failure by SF3B1 mutational status, per recently updated NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines©). RESULTS: A total of 142 patients were included with a median follow-up time of 17 months (interquartile range [IQR], 7-33). Median age at ESA initiation was 79 years (IQR, 73-85). Patients were predominantly male (54%), overweight or obese (32% and 23%, respectively), of White race (96%) and non-Hispanic ethnicity (89%). Overall, 57% patients were initially treated with darbepoetin alfa and 43% with epoetin alfa. Clinical outcomes were poor, and there was a significant burden on both the health system and individual patients treated with ESA therapies. Hematologic improvement- erythroid was only seen in 26% of 142 patients treated with ESAs, and 65% of 82 retreated patients experienced primary ESA failure. CONCLUSION: Our results indicate that primary ESA failure is largely unrecognized and that many patients should be considered for alternative treatments.

GSH exhaustion via inhibition of xCT-GSH-GPX4 pathway synergistically enhanced DSF/Cu-induced cuproptosis in myelodysplastic syndromes.

The clinical application of the therapeutic approach in myelodysplastic syndromes (MDS) remains an insurmountable challenge for the high propensity for progressing to acute myeloid leukemia and predominantly affecting elderly individuals. Thus, the discovery of molecular mechanisms underlying the regulatory network of different programmed cell death holds great promise for the identification of therapeutic targets and provides insights into new therapeutic avenues. Herein, we found that disulfiram/copper (DSF/Cu) significantly repressed the cell viability, increased reactive oxygen species (ROS) accumulation, destroyed mitochondrial morphology, and altered oxygen consumption rate. Further studies verified that DSF/Cu induces cuproptosis, as evidenced by the depletion of glutathione (GSH), aggregation of lipoylated DLAT, and induced loss of Fe-S cluster-containing proteins, which could be rescued by tetrathiomolybdate and knockdown of ferredoxin 1 (FDX1). Additionally, GSH contributed to the tolerance of DSF/Cu-mediated cuproptosis, while pharmacological chelation of GSH triggered ROS accumulation and sensitized cell death. The xCT-GSH-GPX4 axis is the ideal downstream component of ferroptosis that exerts a powerful protective mechanism. Notably, classical xCT inhibitors were capable of leading to the catastrophic accumulation of ROS and exerting synergistic cell death, while xCT overexpression restored these phenomena. Simvastatin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase, has beneficial effects in repurposing for inhibiting GPX4. Similarly, the combination treatment of DSF/Cu and simvastatin dramatically decreased the expression of GPX4 and Fe-S proteins, ultimately accelerating cell death. Moreover, we identified that the combination treatment of DSF/Cu and simvastatin also had a synergistic antitumor effect in the MDS mouse model, with the reduced GPX4, increased COX-2 and accumulated lipid peroxides. Overall, our study provided insight into developing a novel synergistic strategy to sensitize MDS therapy by targeting ferroptosis and cuproptosis.