Lower-risk Myelodysplastic Syndromes Research Articles

Efficacy and Safety of Luspatercept in Adult Patients with Transfusion-Dependent Anemia Due to Very Low, Low and Intermediate Risk Myelodysplastic Syndromes (MDS) with Ring Sideroblasts, Who Had an Unsatisfactory Response to or Are Ineligible for Erythropoietin-Based Therapy: A Retrospective Multicenter Study By Fondazione Italiana Sindromi Mielodisplastiche (FiSiM ETS)

Background: Patients with lower-risk myelodysplastic syndromes (MDS) who develop red blood cells (RBC) transfusion-dependent anemia, experience worse quality of life and shorter survival. Therapeutic strategies after failure of erythropoiesis stimulating agents (ESAs) are limited. Regulatory approval of luspatercept in lower-risk MDS with ring sideroblasts (MDS-RS) offers a novel approach for the treatment of transfusion-dependent anemia in this subset of patients. However, data about luspatercept effectiveness in a real-world clinical setting are lacking. Here we report the results of a multicenter retrospective study including patients who were treated with luspatercept within the Italian compassionate use program. Methods: Eligible patients were ≥ 18 years old, had MDS-RS (2016 WHO classification), met criteria for very low, low, or intermediate IPSS-R risk; were receiving regular RBC transfusions (≥2 units/8 weeks in the 16 weeks before study entry); were refractory to or unlikely to respond to ESAs. Primary end point was transfusion independence (TI) for 8 weeks or longer during weeks 1 through 24; key secondary end point was TI for 12 weeks or longer, assessed during both weeks 1 through 24 and weeks 1 through 48. Other secondary end points included erythroid response (defined by the IWG 2006 criteria), progression to acute myeloid leukemia (AML) and safety analyses. Exploratory analyses were performed to identify predictive factors for treatment response. Results: 184 patients who were enrolled in the Italian luspatercept compassionate use program between December 2020 and December 2021 were analyzed in this study. Median age at enrollment was 74 years (range, 31-88), median time since diagnosis was 43 months, and median time since onset of transfusion-dependence was 23 months. Overall, 123 (66.8%) patients had at least one comorbidity requiring ongoing treatment, 39 (21.2%) had at least three. Baseline median transfusion burden was 6 RBC units every 8 weeks (range, 2-25). At the time of data analysis (June 30, 2022), 130 (73.4%) patients completed at least 24 weeks of treatment. Median time on treatment was 301.5 days, and median follow-up time was 376 days. During the first 24 weeks of the trial, 59 (32%) patients had TI for 8 weeks or longer. The percentage of patients achieving TI increased to 38.6% in the first 48 weeks of the trial. Median duration of TI was 27.9 weeks. Focusing on secondary endpoints, 36 patients (19.6%) had TI for 12 weeks or longer during the first 24 weeks, 52 (28.3%) had TI for 12 weeks or longer considering the first 48 weeks of the trial. A reduction ≥ 4 RBC units/8 weeks during weeks 1-24 was seen in 66 patients (40.2%). Four (2.2%) patients evolved to AML. Serious adverse events were recorded in 27 (15.2%) patients and led to study discontinuation in 5 (2.8%). Overall, treatment was discontinued in 72 (40.7%) patients, mainly for lack of benefit (table 1) The maximum approved dose (1.75 mg/kg) of luspatercept was administered at least once in 144 (81%) patients. Among patients who achieved TI for 8 weeks or longer in the first 48 weeks, luspatercept dose (mg/kg) at response was 1, 1.33 and 1.75 in 31 (46.3%), 18 (29.9%) and 18 (29.9%%) patients, respectively. Logistic regression analysis showed that higher baseline transfusion burden was significantly associated with a lower probability of achieving TI. We identified 3 group of patients stratified according to baseline transfusion burden (group 1, ≤4 units/8 weeks; group 2, 5 to 7 units/8 weeks, group 3, ≥8 units/8 weeks) with a significantly different probability to achieve both the primary endpoint (51.9%, 35.2% and 16.7%, respectively, P<0.01) and the key secondary endpoint (30.8%, 24.1% and 9%, respectively, P<0.01). Patients with higher transfusion burden had higher luspatercept doses at first response (table 2). In patients with low probability to achieve TI (group 2 and 3), a reduction in transfusions equal or greater than 4 units/8 weeks was achieved in 66 (46.2%). Conclusion: Luspatercept was effective for the treatment of transfusion-dependent anemia in MDS-RS in a real-life setting. The benefit extended beyond the achievement of TI and produced a significant reduction in the number of RBC transfusions. Higher baseline transfusion burden was associated with a reduced probability to achieve TI; in these patients, the reduction of transfused RBC units appears a more reliable treatment target. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Umbrella Trial in Myeloid Malignancies: The Myelomatch National Clinical Trials Network Precision Medicine Initiative

Introduction: To accelerate myeloid cancer therapeutics, the National Clinical Trials Network (NCTN) is launching the National Cancer Institute (NCI) Myeloid Malignancies Molecular Analysis for Therapy Choice (myeloMATCH) precision medicine clinical trial. Sponsored by NCI and conducted across the entire NCTN system with initial trials to be led by SWOG, Alliance, ECOG-ACRIN, and CCTG, the initiative leverages public-private partnerships with pharmaceutical industry and biotech companies to create an efficient regulatory model incorporating cross-company novel-novel combinations in trials for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The goal is to create a portfolio of rationally designed treatment substudies onto which patients sequentially enroll over their treatment journey. As patients transition to higher tiers with increasingly lower remaining tumor burden, the focus will be to target residual disease more precisely. Methods: Newly diagnosed patients are enrolled onto the Master Screening and Reassessment Protocol (MSRP) for baseline clinical and laboratory evaluation. Specimens are sent to the Molecular Diagnostics Network (MDNet) with a 72-hour turn-around for patient assignment to an initial treatment substudy via an integrated informatics system developed by the NCI Precision Medicine Analysis and Coordination Center (PMACC). Assignments will be based on algorithms adjusted for prevalence of co-mutations to enhance accrual of rare molecular subsets to specific targeted treatment trials. As shown in figure 1, there are 4 tiers and 5 clinical baskets. Tier-1 is for initial therapy grouped by MDS, younger AML, and older AML. These are typically randomized phase 2 studies testing sensitivity to novel drug combinations with measurable residual disease (MRD) assessment conducted centrally by MDNet. Subsequent therapy occurs in higher tiers. These assignments are made by MDNet/PMACC based on prior treatment substudy outcome. Flow cytometry and duplex sequencing will be employed in Tier-4 clinical trials that will target residual disease. Statistical designs will evaluate the clinical utility of the assays and biomarkers to determine if targeting residual disease confers clinical benefit. Planned activation is quarter 4 of 2022 with the MSPRP, 2-young adult tier-1 studies and 1 tier-2 study. These are testing combinations of azacitidine, venetoclax, CPX351, 7+3 for ELN defined high risk AML, standard risk AML, and in tier-2 the ability to "erase” residual disease after tier-1 treatment. Additional studies in development include agents for mutant TP53, KIT, FLT-3, NPM1, IDH 1/2, higher and lower-risk MDS and a study for reduced intensity transplant and maintenance to include efforts for diverse populations. Launch for these studies is planned for mid 2023. Tier-4 studies to target KIT, IDH, FLT3 and others are in discussion. Discussion: MyeloMATCH is the largest focused investment of infrastructure and researchers ever coordinated by the US Network Groups and NCI that follows patients from diagnosis thorough all treatment in a single disease area. The charge is to rapidly advance therapeutics in myeloid malignancies. MyeloMATCH is designed to efficiently screen and assign patients to precision treatment trials of promising therapeutic combinations. By using early endpoints to identify large activity signals, myeloMATCH will generate data with promising findings for definitive study. The clinical and laboratory data can be interrogated across the initiative to generate hypotheses for additional focused testing. Participants receiving their treatment journey through myeloMATCH will contribute to a unique clinically annotated database with specimens for "omics” serially collected from pre-treatment and through follow up. This will provide a national resource for understanding drug sensitivity and resistance, as well as clonal evolution. In this manner, myeloMATCH aims to reduce the time and investment in failed phase 3 studies, and instead aims to provide high-quality randomized trial data that will enhance selection of phase 3 priorities. We believe this new paradigm for the collaborative conduct of clinical trials may provide meaningful advances for patients with AML and MDS, mentor investigators, and accelerate drug development. Updates and specific treatment-trial designs will be discussed at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Current Cardiovascular Disease Risk Prediction Models Are Not Applicable in MDS Patients: Preliminary Results of a Prospective Observational Single-Centre Cohort Study

Background/aims Cardiovascular disease (CVD) is a leading cause of death in lower-risk Myelodysplastic syndrome (LR-MDS) patients and a shared pathobiology between MDS and CVD has been postulated. However, current evidence emanates only from retrospective cohorts suffering from crucial shortcomings. Prospective annotation can overcome these limitations and delineate the role of MDS as an independent risk factor for CVD. For this reason we conducted a prospective observational single-centre cohort study in LR-MDS patients. Methods Patients underwent thorough evaluation for CVD every 6 months. Cerebrovascular, peripheral and coronary vascular beds were assessed for atherosclerosis by ultrasound and coronary artery calcium (CAC), respectively. Cardiac structure and function was assessed by echocardiography. Multi-Ethnic Study of Atherosclerosis (MESA), Framingham (FRS) risk scores and serum markers of myocardial injury (Serum high-sensitivity troponin T, hsTnT), stress (N-terminal pro-B-type natriuretic peptide, NT-proBNP), and systemic inflammation (high-sensitivity C-reactive protein, hsCRP) were also estimated at each visit. Chi-square, Wilcoxon signed-ranks tests and Pearson's correlation coefficient were used as appropriate. Multiple linear regression was applied for modeling the relationship between a dependent and one or more independent variables. The level of statistical significance was set to P=0.05. Results Table 1 presents baseline characteristics of 26 patients included so far. There were no correlations of CAC, MESA and FRS with either MDS subtype or IPSS-R risk category after adjustment for preexisting CVD, age, sex, and transfusion dependence. In sharp contrast to the general population (Okwuosa, TM et al. JACC 2011), CAC score was not associated with FRS (p=0.7) and was disproportionately increased in low and very low FRS risk patients (Figure 1). Likewise, the number of affected vascular beds (AVB) did not correlate with FRS (p=0,51) (Figure 2), further suggesting that the current CVD risk prediction tools are inaccurate in MDS and arguing for an intrinsic tendency of MDS towards CVD development. A second and a third evaluation was available in 17 and 7 patients, respectively. All patients increased significantly the CAC score both at 6 months (p=0.04) and 1 year (p=0.003). The average increase from baseline was 26.2% at 6 months and 72.6% at 1 year, markedly higher than expected in the general population (McCullough PA et al. Arch Int Med 2009), and was marginally higher in transfusion dependent patients (p=0.049), but not associated with baseline MESA score, FRS, preexisting CVD, exposure to ESAS, statin therapy, sex. Finally, baseline NT-proBNP levels correlated strongly with CAC score P=0.001 (Figure 3) in line with numerous evidence showing that NT-proBNP can potentially serve as a predictor of CVD risk in both MDS and non-MDS individuals. Conclusion To our knowledge this is the first study in LR-MDS patients assessing longitudinally all critical parameters and providing objective measurements of subclinical atherosclerosis in all vascular beds. Our preliminary results indicate that the established CVD prediction models do not operate properly in LR-MDS patients and reinforce the notion of a pro-atherogenic role of MDS. Accurate assessment of CVD risk and prompt detection of subclinical cardiovascular disease in MDS patients will assist clinical decisions on the introduction of intensive monitoring and preventive use of CVD-specific interventions in these individuals. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of PRMT5 Increases Sensitivity to BH3 Mimetics in Aggressive B Cell Malignancies

Introduction: Protein Arginine Methyltransferase 5 (PRMT5) is a type II methyltransferase that catalyzes symmetric dimethylation of arginine. PRMT5 is highly expressed in several types of aggressive B cell malignancies, and increased PRMT5 expression has been associated with poor disease prognosis. Several small molecule inhibitors targeting PRMT5 have been developed and showed promising anti-tumor effect in pre-clinical studies including in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). JNJ-64619178, a potent and specific PRMT5 inhibitor, exhibited broad antiproliferative activity in cancer cell lines from multiple cancer types and cell lines derived xenograft mouse model (Brehmer et al., 2021). In a phase 1 clinical trial of JNJ-64619178 (NCT03573310), patients with advanced solid tumors and non-Hodgkin lymphoma demonstrated manageable toxicity and preliminary evidence of antitumor activity at selected dose levels (Villar et al., 2020). In addition, in patients with lower-risk myelodysplastic syndromes, JNJ-64619178 had manageable hematologic toxicity at the selected expansion dose, but did not have significant clinical activity (Haque et al., 2021). Inhibition of PRMT5 alone could induce cell cycle arrest instead of apoptosis depending on the types of cancer (Zhu et al., 2019), therefore we hypothesized that the combination of PRMT5i and pro-apoptotic BH3 mimetics therapies could be synergistic in aggressive B cell malignancies. Methods: We used dynamic BH3 profiling (DBP) to measure the net pro-apoptotic signaling induced by ex vivo drug treatments (as per Montero et al., 2015). Other standard techniques included shRNA mediated gene knockdown, Western blot, cell viability assays including PI/Annexin V and CellTiter-Glo, and reactive oxygen species (ROS) measurement by flow cytometry with dihydroethidium (DHE) staining. The DLBCL cell lines (HBL1, TMD8, RI-1, OCI-Ly1, Karpas 422, SUDHL4, Toledo), double hit lymphoma (DHL) patient-derived xenograft (PDX) cell lines (DW19), mantle cell lymphoma (MCL) cell lines (Mino, Jeko-1), T cell acute leukemia (T-ALL) cell line (Jurkat) and Burkitt lymphoma (BL) cell line (Raji) were used to investigate the in vitro anti-cancer activity of JNJ-64619178 (Janssen Pharmaceuticals) and BH3 mimetics (venetoclax [BCL2i], S63845 [MCL-1i] and A1331852 [BCL-xLi], Selleckchem). SynergyFinder was used to calculate drug synergism. Results: In all 7 DLBCL cell lines and the DHL-PDX cell line, we found synergistic induction of apoptosis with JNJ-64619178 plus venetoclax (synergy score, 2.82~51.42). The lowest level of synergy was observed in OCI-Ly1 cells, which are intrinsically highly sensitive to venetoclax monotherapy. The combination of JNJ-64619178 with S63845 synergistically induced apoptosis in 5 out of 7 cell lines (synergy score, -1.91~35.64). While the combination of JNJ-64619178 with A1331852 synergistically induced apoptosis in all 7 cell lines (synergy score 1.88~46.73), 4 of them had weak synergy score (1.88~9.4). To confirm the effects we observed with pharmacologic inhibition of PRMT5, we performed shRNA mediated knockdown of PRMT5, which similarly revealed increased sensitivity to venetoclax. In MCL cell lines, combining JNJ-64619178 with venetoclax or A1331852 was more synergistic in inducing cell death than combining with S63845. The T-ALL Jurkat cell line was resistant to JNJ-64619178 and single BH3 mimetic-induced apoptosis. Combining JNJ-64619178 with A1331852 but not venetoclax or S63845 strongly induced apoptosis. The Burkitt cell line Raji was resistant to JNJ-64619178, venetoclax and A1331852, but was sensitive to S63845. The addition of JNJ-64619178 and a single BH3 mimetic in these Raji cells had only a minor synergistic effect. The synergy scores are summarized in Figure 1. Using DBP, JNJ-64619178 was found to increase overall apoptotic priming of DLBCL and MCL cell lines (> 15% increase in cytochrome c loss). By measuring ROS levels, we found increased ROS production (> 2-fold increase in median fluorescent intensity) in response to JNJ-64619178 before the onset of apoptosis, suggesting a possible mechanism by which JNJ-64619178 increases apoptotic priming. Conclusions: The combination of a PRMT5 inhibitor with BH3 mimetics, especially venetoclax, is worthy of further exploration as a potential therapeutic strategy for DLBCL and MCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Imetelstat Achieved Prolonged, Continuous Transfusion Independence (TI) in Patients with Heavily Transfused Non-Del(5q) Lower-Risk Myelodysplastic Syndrome (LR-MDS) Relapsed/Refractory (R/R) to Erythropoiesis Stimulating Agents (ESAs) within the IMerge Phase 2 Study

Imetelstat Achieved Prolonged, Continuous Transfusion Independence (TI) in Patients with Heavily Transfused Non-Del(5q) Lower-Risk Myelodysplastic Syndrome (LR-MDS) Relapsed/Refractory (R/R) to Erythropoiesis Stimulating Agents (ESAs) within the IMerge Phase 2 Study

SF3B1, RUNX1 and TP53 Mutations Significantly Impact the Outcome of Patients with Lower-Risk Myelodysplastic Syndrome

Introduction: Prognosis of patients (pts) with myelodysplastic syndrome (MDS), particularly the group with lower-risk disease (LR-MDS) is very heterogeneous. In the last decade, different studies have reported the role of recurrent somatic mutations involved in multiple cellular pathways and incorporated them into clinical practice and current prognostic models, including all risk categories. Recently, the incorporation of genomic data to clinical parameters defined the new Molecular International Prognostic Scoring System (IPSS-M). Materials and Methods: A retrospective study evaluating the impact of molecular profile in a series of 181 pts with LR-MDS and non-proliferative chronic myelomonocytic leukemia (CMML) focused on survival estimates and the probability of leukemic progression. Genomic DNA was isolated from bone marrow mononuclear cells at diagnosis using DNA kit from Qiagen according to the manufacturer´s instructions. A PCR-based targeted was performed sequencing of 40 genes involved in myeloid neoplasms (Oncomine Myeloid Research panel, including hotspots of ABL, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, HRAS, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, U2AF1, WT1 and entire coding regions of ASXL1, BCOR, CALR, CEBPA, ETEV6, EZH2, IKZF1, NF1, PHF6, PRPF8, RB1, RUNX1, SH2B3, STAG2, TET2, TP53, ZRSR2). Sequencing was performed using the Ion Torrent S5 sequencing platform from Life Technologies. Survival (OS) was analyzed from diagnosis until death as a result of any cause or the last follow-up and calculated by the Kaplan-Meier method and log rank test. A Cox proportional hazard regression model was performed to analyze OS as endpoint. Analysis was performed by the IBMÒ SPSSÒ version 26.0.0. The probability of leukemic progression was analyzed using the Gray test in the framework of competing risk and considering death not attributable to AML as a competing event. This study was conducted at Hospital Universitario Virgen del Rocío, approved by the local ethics committee and was performed in accordance with the Declaration of Helsinki. Results: In total, 392 mutations were detected in the 181 pts analyzed with a median of 2 mutations per sample (range: 0-8). Sixty-seven pts (37%) harbored ³3 mutations. Table shows baseline characteristics of pts. Most frequent mutated genes were TET2, SF3B1, RUNX1, ASXL1, TP53, and SRSF2 (all of them present in >10% of pts). Epigenetic regulation was the most recurrently mutated pathway, followed by mutations in splicing factors. After a median follow-up of 47 months (95% CI, 37-56 months), 93 out of 191 pts had died (48%). Median OS for the whole cohort was 41 months (95% CI, 28-53 months). TP53 mutations were detected in 22 pts, with equal distribution of mono-allelic or multi-hit TP53 state. As no significant differences in median OS was observed between groups [23 months (95% CI, 12-53 months) in mono-allelic TP53 state vs. 17 months (95% CI, 11-22 months) in multi-hit cases ( P = 0.1)], TP53 mutation was analyzed as a single parameter. Mutations in SF3B1 were detected in 34 pts and the majority of them (31 out of 34; 91%) were classified as SF3B1a according to the recent segregation of SF3B1-mutated cases by the IPSS-M. In univariate analysis, RUNX1 or TP53mutations correlated with lower median OS (Table 2). In contrast, SF3B1 mutation was associated with prolonged median OS [95 months (95% IC, 32-157) vs. 33 months (95% CI, 19-46) in unmutated pts (P < 0.01)]. In a multivariate Cox regression model, RUNX1 mutations independently associated with shorter OS, while SF3B1 mutation retained its favorable impact on outcome (HR: 0.24, 95% CI, 0.1-0.5; P = 0.001). At last follow-up, 49 patients (25%) progressed to AML. Median time to progression was 16.2 months (range: 2-105 months). Subdistribution hazard ratios (sHRs) for leukemic progression were estimated to identify predictive covariates for progression. TP53 mutation (sHR, 6.3; 95% CI, 3-14); P < 0.001) and RUNX1 mutation (sHR, 6.9; 95% CI, 3.3-14; P < 0.001) were predictive for the probability of leukemic progression (Table 2). Conclusion: Incorporation of molecular testing in LR-MDS identified a subset of pts with expected poorer outcome, either due to lower survival or probability of leukemic progression. Results herein reported may help prognosis stratification in this set of pts with low-risk disease and support the recommendation of molecular testing in pts with MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Risk of Unemployment Among Chronic Myeloproliferative Disorders Patients

Overall survival of chronic myeloproliferative disorders (CMPD) such as Policytemia Vera (PV), Essential Thrombocitemia (ET), Myelofibrosis (MF), Chronic Myeloid Leukemia (CML) and Myelodysplastic Syndrome (MDS) is often long term because of their natural history and therapeutic improvement (ie: TKI, RH-EPO, JAK2 inhibitors). On the other hand, quality of life may be often compromised by persistent symptomatologic burden and chronic therapy (TX) that may worsen work ability. Although the highest incidence falls between 60 and 70 years old, prevalence studies show that nearly 50% of such patients (PTS) are less than 70 YO, hence in their working age. To the best of our knowledge no data have been collected to explore the rate of loss of employment among this group of PTS. Aim of this study is to collect data to estimate among CMPD PTS: rate of long term loss of employment, impact of diagnostic work-up, correlation between loss of employment and diagnosis. METHODS From febbruary 01 until april 30, 2022 all outpatients complying with the following criteria were recruited: CML, PV, TE, MDS, MF diagnosis; life expectancy of more than 5 years based on currently used prognostic scores (CML: ELTS, MF: IPSS, MDS: R-IPSS, PV: IWG-PV score, TE: IPSET); age > 18 years old, ≤ 70 years old. Loss of employment was defined as any modality of abandonement of work after disease diagnosis (retirement, dismissal, abandonment). Demographic and basic disease data were collected. A 29 items questionnaire (QST) mainly based on the "Work Ability Index" was prepared for the purpose of this study. After informed consent acquisition, the QST was administered with psyco-oncology support to PTS planned for routine control. PTS missing control for any reason, were contacted by phone and QST was subsequently administered after informed consent reading. RESULTS 104 PTS have been recruited (M/F: 47/57); mean age at diagnosis was 52,1 for M (range 24-69) and 50,2 for F (range 18-69). PTS diagnosis were: 30 CML, 26 MF, 24 ET, 9 PV, 7 Low-Risk MDS, 4 other CMPD, 4 NA. 68/104 PTS were on active specific TX for their disease (66 on oral TX, 2 on parenteral TX). At diagnosis 58/104 PTS (55,8%) were employed 32 M (55,2%) and 26 F (44,8%). 29 had a permanent employment, 12 had a fixed term employment, 8 flexible employment. Only 8/58 (13.8%) suspended their job during work up (sick leave). 28 of 105 pts (26.7%) (retired, employed, unemployed and housewives) reduced their daily activity and 14 of them haven't recovered yet 1 to 13 YY after diagnosis. 15 of 58 PTS (25,9%) left their employment definitively: 7 could retire because of age; 8 (13,8%), 4M and 4F, left their employment earlier after diagnosis of CMPD (4 LMC, 3 MF, 1 ET). Their jobs were distributed as follows: blue collars (3 F/2 M), driver (1M), white collars (1M/1F). Main reasons for loss of employment were: desease symptoms (10/15), TX side effects (6/15), job characteristics (6/15), anxiety/depression (5/15). Loss of employment caused depression in 9/15 PTS, and in 6/15 PTS echonomic issues and loss of social well being. We could not find any correlation between loss of employment and diagnosis probably due to the low numeber of PTS recruited. CONCLUSIONS In our cohort of 105 retrospectively analyzed good prognosis CMPD PTS, loss of employment rate was quite high soon after diagnosis (25,9%); 13,8% could retire because of age. Surprisingly, also CML PTS left their employment despite the well known prognostic improvement. Most of the early retired PTS were blue collars or drivers (requiring higher phisical commitment). Depression, echonomic issues and loss of social well being were the main consenquences of loss of employment. The high rate of loss of employment may be related to overestimation of disease severity and limited social assistance support. A careful early multidisciplinary approach (hematologist/occupational doctor/psyco-oncologist) could probably help to avoid unnecessary unemployment, improve psycho-social well-being and reduce echonomical anxiety. Further studies are warranted to increase population sample and address methodology biases that limit our study. A specific questionnaire should be validated.

Yoga As Possible Non-Medical Intervention to Improve Quality of Life of Patients with Myelodysplastic Syndromes - a Feasibility Report

Background Despite significant progress in the management of myelodysplastic syndromes (MDS), symptom-burden remains significant and patients report on impaired quality of life (QoL) [Stauder, Leukemia 2018]. There is an ongoing need to explore non-pharmacologic approaches for managing MDS symptom burden. Yoga has proved an effective method to improve various physical and psychosocial outcomes, such as fatigue, anxiety, and sleep disturbances, in patients diagnosed with solid cancers. Aims (1) To examine the practicality of an in-person 8-week yoga class; (2) To explore the feasibility of an 8-week group online yoga within an observational study; (3) Preliminary assessment of QoL parameters. Methods MDS patients from 9 medical centers in Israel were invited to participate in a yoga program, consisting of 8 one hour in-person classes (once weekly). The classes were given by certified Kundalini yoga teachers, who delivered a mild-medium intensity program combining deep breathing, physical movements, and mantras, specifically designed for this patient population. Each medical center hosted its own course. All the exercises were done while the patients were seated on a chair. All MDS patients were invited, regardless of disease risk or performance status. In the second part, patients were offered a further 8-week online program, in which a one-hour weekly session was given via zoom, within an observational study. Symptom-burden was assessed using a modified Edmonton Symptom Self-Assessment Scale (ESAS), a 10-item rating scale (0-10, with 10 being the highest degree), which has been previously recommended by the National Comprehensive Cancer Network to assess QoL in all cancer populations. Patient-reported outcomes included fatigue, sleepiness, depression, nausea, pain and other QoL measures. Questionnaires were filled out by patients before and after each meeting. Results Overall, 120 patients from 9 centers participated in at least one in-person yoga class. Participating patients, including those with poor performance status, were able to engage and complete most exercises throughout the class. Many of the patients were able to return to consequent classes, but some of them encountered barriers, mainly difficulty in commuting. Twenty-three patients signed up for the online yoga live zoom session. Of those, 11 patients were high-risk MDS and 12 were low-risk MDS, 7 were transfusion dependent. During the 8 weeks of study, 3 patients experienced disease progression. Sixteen patients participated in the online program. The main reasons for not participating included video-conferencing installing and enabling issues. Most patients filled out the questionnaires before and after each online lesson. The QoL analysis will be reported separately, but preliminary data suggests a substantial increase in general well-being and decrease in fatigue, after a class. Conclusion This in-person and live online Kundalini yoga program was well-tolerated by many MDS patients of all risk groups. Preliminary analysis suggests improved well-being after completing each class. However, we did encounter some barriers, mainly challenging transportation and software operating difficulties. Despite theses challenges, patients managed to adhere to schedule. We suggest developing a hybrid plan (in-person and live online lessons) for further exploring the benefits of yoga among MDS patients. In the future we will evaluate the effect on QoL, as well as possible effect on disease outcomes.

Targeting S100A9 in the Inflammatory Myelodysplastic Hematopoietic Niche Reprograms the Functional Properties of CD271+ Mesenchymal Stromal Cells

Myelodysplastic syndromes (MDS) belong to the most common hematological malignancies in the elderly population, characterized by ineffective hematopoiesis, peripheral cytopenia and the risk of transformation into acute myeloid leukemia. An involvement of the bone marrow microenvironment (BMME) in disease initiation and progression is indisputable. The profound immune dysregulation and sustained inflammation, essentially mediated by mesenchymal stromal cells (MSCs), are further factors contributing to ineffective hematopoiesis and driving disease progression. The alarmin S100A9 is a TLR4 and CD33 ligand that acts as key player for the inflammasome activation in the MDS BM. The novel small molecular drug Tasquinimod (TASQ, Active Biotech) is a S100A9 inhibitor and has demonstrated antitumor and immunomodulatory properties in a broad range of solid tumors; however, little is known about its effects in myeloid malignancies. Here, we aim to decipher the effects of TASQ on MDS MSC subpopulations in more detail. Therefore, MSCs were isolated from BM aspirates ('asp-MSCs', n=12) or biopsies ('bp-MSCs', n=5) of patients with either low-risk MDS, CMML or age-adjusted healthy donors and were exposed to TASQ (10 µM) in vitro. Flow cytometry analysis of the MSC markers CD44, CD73, CD90, CD105, and CD146 did not show significant differences in neither asp- nor bp-MSCs after treatment. Interestingly, CD271, which is hard to detect in asp-MSCs, was decreased between 40% and 70% in bp-MSCs after TASQ treatment. This was accompanied by an inhibited expression of the checkpoint inhibitor PD-L1. The MDS BM has a higher potency for adipogenic differentiation which further supports the inflammatory conditions and the suppression of normal hematopoiesis. Treatment with TASQ resulted in about 50% inhibition of adipogenesis as demonstrated by significantly lower expression of adipocyte protein 2 in differentiated asp- and bp-MSCs. To investigate possible metabolic modulations of MSCs by TASQ, the oxygen consumption rate was measured as a readout of mitochondrial function by using the Cell Mito Stress Test (Agilent Technologies) in a XF96 analyzer. The basal and the maximal respiratory rate as well as ATP production were increased in TASQ-treated bp-MSC cultures suggesting a positive effect for their bioenergetic capacity. This was supported by detection of higher levels of glutamate dehydrogenase 1, a key enzyme in glutaminolysis converting glutamate to α-ketoglutarate for entry into the TCA cycle, and of the glutamine transporter Slc1a3 in TASQ-treated cells. NMR analyses revealed increased levels of glutamate and citrate as well as of several lipids, such as free cholesterol LDL, phospholipids LDL-6 and Apo-B LDL6 in supernatants of bp-MSCs after TASQ treatment. To investigate the impact of TASQ on CD271 expression in more detail, we sorted CD45-/CD73+/CD90+/CD271+/- bp-MSCs. Gene expression analysis by RNA sequencing of sorted MSCs revealed differential expression of several pathways, e.g. an enrichment of inflammation related molecules in the CD271+ samples, whereby TASQ could abrogate this effect. Moreover, a cytokine array analysis of culture supernatants of sorted cells showed enhanced IFNg, IL1b and IL-6 secretion of CD271+ MSCs compared to CD271- and TASQ-treated cells. To evaluate the impact on the hematopoietic support of MSCs, we analyzed CD271-sorted bp-MSC co-cultures with CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed a significantly decreased number of cobblestone area forming cells (CAF-C) as well as reduced numbers of colonies (CFU) in a subsequent clonogenic assay for CD271+ MSCs, nominating this MSC subpopulation as responsible for the diminished hematopoietic support. Interestingly, the number of CAF-C and CFU-E/CFU-GM could be increased by TASQ pre-treatment of the stromal layer. In conclusion, CD271+ MSCs play a crucial role in the inflammatory MDS BMME. The inhibition by TASQ efficiently blocks adipogenic differentiation and secretion of pro-inflammatory cytokines, thereby modulating the MSC energetic profile and enhancing their potential to support hematopoiesis in vitro.

Characteristics of Germline CHEK2 Mutated Patients in a Large Cohort of 2322 Myeloid Malignancies

Background Checkpoint kinase 2 (CHEK2) is a critical mediator of the DNA damage response and plays a role in DNA double strand break-induced apoptosis and cell cycle arrest (Bartek, 2003). Deleterious germline CHEK2 variants (CHEK2germ) have been associated with an increased risk for breast, colon, prostate, thyroid and kidney cancers (Cybulski, 2004). Early studies suggested that germline CHEK2 variants also predispose to myeloid malignancies, including myelodysplastic syndromes (MDS) (Janiszewska, 2018). Recently, CHEK2 was identified as the most frequent germline mutated gene in a cohort of 391 adult patients with AML (Yang, 2022). However, more data is warranted to clarify the role of germline CHEK2 mutations in the development of myeloid malignancies. Here we analyzed the prevalence of germline CHEK2 variants in a large series of unselected patients diagnosed with myeloid malignancies and describe their clinical features. Methods Between February 2018 and June 2022, 2322 consecutive adult patients diagnosed at Saint-Louis hospital, Paris with a myeloid malignancy were included in the study. DNA obtained from bone marrow or peripheral blood cells was subjected to capture-based next-generation sequencing of genes recurrently mutated in myeloid malignancies, including CHEK2. In selected patients, germline mutation testing was performed using Sanger sequencing of DNA obtained from skin fibroblasts or sorted CD3-positive cells from peripheral blood. Results We identified a CHEK2 variant in 38 out of 2322 (1.6%) unrelated patients. We focused on the 24 patients with a variant allele frequency (VAF) equal or higher than 40%, highly suggestive of a germline origin, which was subsequently confirmed in all patients with available germline DNA. Of these, 16 variants (Fig1 A) were considered as pathogenic/likely pathogenic (P/LP) according to the American College of Medical Genetics as well as 3 missense variants which were semi-functional in an in-vivo, yeast-based, functional assay (Delimitsou, 2019). The 5 remaining variants were considered of unknown significance (VUS). Finally, 19 CHEK2germ variants were considered causal for the myeloid malignancy representing a frequency of 0.8% (19/2322). Noteworthy, two out of them (10.5%) had a second somatic hit in CHEK2. (Fig1 B) The median age of the 19 CHEK2germ patients was 75 years (42-87) at diagnosis of the hematological disease. According to the revised WHO classification, there were 7 (37%) MDS-MLD, 2 (11% ) MDS-RS, 4 (21%) MDS-EB, 3 (16%) MPN, 2 (11%) AML, and 1 (5%) ICUS. Karyotype was normal in 7 patients (39%), 8 patients had an isolated abnormality, 3 patients had a complex karyotype and one had cytogenetic failure. Additional driver mutations (Fig 1 B) were identified in most patients (18/19, 95%), however no particular association was noticed. Seven patients (37%) had a personal history of cancer (prostate n=4, breast n=1, thyroid n=1, colon n=1). Five patients (26%) had a family history of hematologic disorders (MPN, n=2, multiple myeloma n=2, ICUS n=1) and 7 patients (37%) of non-hematological cancer (breast n=2, brain n=2, lung n=1, ovary n=1, intestinal n=1). Eleven patients were classified as low-risk MDS according to the IPSS classification with a median survival of 5.6 years. They mainly received ESA (N=2) or lenalidomide (N=2). Among the 3 high-risk MDS patients 2 received hypomethylating agent with a survival of 5 and 1.7 years, the remaining one is still alive after sequential bone marrow transplant. One AML patient is still receiving hypomethylating agent with BCL2-inhibitor in complete remission with a follow up of 1.1 year, while the other AML patient died of relapse after 2 sequential bone marrow transplants. Regarding the 3 MPN patients, 1 is still alive after 1.3 year and the 2 others died 11.5 and 3.8 years after diagnosis. Conclusion This is the largest study addressing the frequency (0.8%) and characteristics of carriers of germline CHEK2 pathogenic variants within patients with myeloid disorders. Their presentation is rather heterogenous with a majority of low-risk MDS. They did not appear to have a more complex karyotype or a poorer prognosis and seems to be less frequent than previously suggested. Compared to more frequent genetic predisposition to myeloid malignancies like DDX41, CHEK2germ hematological malignancies did not seem to represent a specific entity, in line with recent debate on CHEK2germ solid cancer. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discerning Insights and Perspectives on Treatment for Myelodysplastic Syndrome: Impact of Aligned Patient and Provider Education

BACKGROUND: Current therapies for myelodysplastic syndrome (MDS), such as allogeneic bone-marrow transplantation, erythropoiesis stimulating agents, lenalidomide, and hypomethylating agents, are not adequate for many patients. Recently approved and emerging investigational therapies with different mechanisms of action are providing additional treatment options for patients with low-, intermediate-, or high-risk MDS. Clinical trials offer treatment options for those patients with newly diagnosed or untreated MDS, which remains an unmet need in the treatment paradigm. The substantial accumulation of new data has created knowledge and practice gaps among clinicians that can impact management. To meet these needs, a patient-provider education initiative was developed to bring awareness to new therapies, including diversifying clinical trials, and assist clinicians in integrating advances in the field. METHODS: In collaboration with The Aplastic Anemia & MDS International Foundation (AAMDS) two, 1-hour online video-based programs were hosted on CancerCoachLive.com and OMedLive.com in 2021 for patients/families and HCPs, respectively. In 2022, an HCP activity which included the 'patient voice' was hosted on OMedLive.com and remains on-demand through June 2023. The programs were interactive featuring an expert physician panel, patient interviews, polling, and question/answer sessions. Knowledge and competence questions were administered pre-, immediate post-, and 2 months post-activity. Repeated knowledge and competence questions were tested for statistical significance using Chi-Square. RESULTS: As of August 2nd, the 1,758 HCPs attended the HCP activities with 15.309 patients/caregivers engaging in the patient/caregiver activity. Of the HCPs in attendance, 75% sought CME certificates for participation, and 83% practiced in the community setting. Of the patients/caregivers participating, 59% were female, 89% were older than 55 years of age, and 91% identified as White, non-Hispanic. Behavioral impact was assessed at 2 months following the program with write-in responses (>90) from patients and HCPs of what behaviors changed or were implemented as a result of participation in the education. Thematic analyses of these responses reveal greatest impact on treatment practices for HCPs and engagement in shared decision-making for patients/caregivers. Clinician gaps in care were also revealed, including 67% of participants being unable to identify which classification system categorized MDS with greater weights on cytogenetic abnormalities and severity of cytopenias. Analyzing the baseline practice pattern data showed that 42% of HCPs would offer allogeneic stem cell transplant for patients with low-risk MDS; 73% were unaware of novel agents and clinical trials results for intermediate-, high-, and very-high-risk MDS; and 60% of HCPs unable to discern which patient profiles would benefit the most with newer therapies. Initial outcomes from the 2022 program highlight barriers to patient enrollment and HCP recommendation of clinical trials for newly diagnosed MDS. CONCLUSIONS: The multi-year initiative provided insights into both HCP and patient persistent questions and behavioral changes regarding MDS management. Patients/caregivers reported behavioral improvements in engagement with their treatment teams while providers reported an impact on clinical practice. Additional gaps revealed provide an opportunity for continued education across the risk-stratification of MDS.

Inter-Modality Variance of Blast Quantification in Patients with Myelodysplastic Syndrome (MDS) and Its Impact on Risk Stratification and Overall Survival

Introduction: Risk stratification is an essential step after diagnosing a myelodysplastic syndrome (MDS). It defines patient prognosis and guides therapeutic decisions (ranging from "watch and wait", best supportive care to hypomethylating agents and allogeneic stem cell transplantation). In the Revised International Prognostic Scoring System (IPSS-R), the percentage of marrow blasts (inferred via cytology) plays a pivotal role in addition to karyotype, hemoglobin level, platelet count and absolute neutrophil count. However, blast quantification in MDS is subject to inter-observer and inter-modality variance. In the recently published Molecular IPSS (IPSS-M), the variable blasts had a small weight compared to some molecular aberrations. Blast count can be performed with three different methods: bone marrow cytology (BM-c), BM flow cytometry (BM-f) and BM histology (BM-h). The aims of this retrospective study were to analyze the differences of blast evaluation among the three methods BM-c, BM-f and BM-h and what impact these differences have on categorizing patients according to IPSS-R and overall survival (OS). Methods/Results: Since 2012, we prospectively annotated comprehensive clinical, laboratory and bone marrow data of newly diagnosed MDS. In this study, we included 214 cases of MDS (median age 71 years, male/female: 142/72) with blast enumeration performed via BM-c, BM-f and/or BM-h. In 178/214 (83%) cases, precise blast counts were available in at least two methods (mostly BM-c and BM-f). Overall, discordance (defined as a shift within the IPSS-R blast categories) regarding blast counts were detected in 79/178 (44%). In 57/88 (65%) of cases, blast counts were concordant within all three methods. In cases with blast enumeration available in two methods (BM-c and BM-f, only), blast counts were similar in 26/48 (54%) cases, only. Interestingly, within the cases with blast count discordance (and availability of BM-c), patients had either a) no change (19/58, 33%), b) a down-grade (25/58, 43%) or c), an upgrade (14/58, 24%) of IPSS-R category when BM-f or BM-h were applied instead of BM-c. Furthermore, a possible shift of the IPSS-R category in individual cases led to re-stratification from low-risk MDS (LR-MDS, IPSS-R very low, low, intermediate) to high-risk MDS (HR-MDS, IPSS-R high, very high) in 3/39 (8%) and viceversa in 8/39 (21%), respectively. In the cohort of patients with blast count discordance (n=58), the IPSS-R failed to stratify between LR- and HR-MDS regarding OS (Figure 1b) in contrast to the cohort of all patients (Figure 1a). Conclusion: In this study, we could demonstrate that, a) significant discordance regarding blast count was detected in about 50% of patients, b) discordant blast count led to significant shifts (down or upgrade) within the IPSS-R categories (which may have high impact on treatment decisions), c) in the cohort with blast discordance the IPSS-R failed to stratify between LR- and HR-MDS, and d) blast enumeration is clearly highly vulnerable and its value on risk-stratification should be down-graded which is the case in the new IPSS-M. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes in Lower-Risk, Non-Del(5q) Myelodysplastic Syndromes: Findings from a Global Real-World Medical Record Review Study

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell malignancies defined by ineffective hematopoiesis and peripheral blood cytopenias. Patients often present with anemia and frequently require red blood cell transfusions (RBCTs), which may lead them to become transfusion-dependent (TD). This results in poor quality of life and reduced survival. In patients with non-del(5q) lower-risk (LR)-MDS, the standard of care includes erythropoiesis-stimulating agents (ESAs) ± granulocyte colony-stimulating factor as first-line (1L) therapy; however, although clinical trials have demonstrated efficacy, there is little evidence from real-world settings. This study aimed to assess real-world data to investigate treatment patterns and prevailing hematologic outcomes in patients with non-del(5q) LR-MDS. Methods: In this retrospective medical record review, eligible patients ≥ 18 years of age diagnosed with non-del(5q) LR-MDS between July 1, 2013 and September 30, 2018 were identified by participating hematologist-oncologists in the US, Canada (CAN), UK, and EU (France, Germany, Spain). Demographics, clinical characteristics, treatment patterns, and hematologic outcomes data were abstracted (June-November 2021) and reported descriptively. Aggregated analyses were performed using pooled US/CAN and UK/EU data sets for the overall cohort and for patient subgroups treated with clinician-defined 1L and second-line (2L) ESA-containing regimens. Results: Medical record data were abstracted for 119 patients with non-del(5q) LR-MDS in US/CAN (median age, 61.5 years; 71.4% male) and 245 patients in UK/EU (median age, 67.3 years; 71.4% male). Median follow-up duration from MDS diagnosis was 50.5 months (US/CAN) and 67.8 months (UK/EU). At initial diagnosis, 50.4% (US/CAN) and 53.5% (UK/EU) of patients had Low-risk MDS (per Revised International Prognostic Scoring System). Of the 71 patients in US/CAN and 123 patients in UK/EU who were tested successfully for the SF3B1 mutation, 16.9% (US/CAN) and 15.5% (UK/EU) were positive. Most of the patients initiated 1L therapy (US/CAN: 109 [91.6%]; UK/EU: 225 [91.8%]) and more than one-third initiated a 2L therapy (US/CAN: 45 [37.8%]; UK/EU: 89 [36.3%]). In 1L, 2.8% patients in US/CAN and 2.2% in UK/EU received treatment with lenalidomide. Most patients were treated with ESA-containing regimens in 1L, with a median duration of therapy of ≥ 2 years (US/CAN: 97 [89.0%], of which 86.6% was ESA monotherapy; UK/EU: 203 [90.2%], of which 85.7% was ESA monotherapy). Treatment characteristics and hematologic outcomes for patients treated with ESAs are presented in the Table. Among patients who were TD before 1L ESA treatment (36 [37.1%] US/CAN; 104 [51.2%] UK/EU), a small proportion (2.8% US/CAN; 14.4% UK/EU) became transfusion independent (TI) during 1L; RBCT reduction threshold (≥ 50%) was met by 41.7% in US/CAN (median duration, 23 months) and 52.9% in UK/EU (median duration, 13 months). Among patients treated with ESA-containing regimens in 2L (12 [26.7%] US/CAN; 26 [29.2%] UK/EU), most had been treated with ESA in 1L (US/CAN: 10 [83.3%]; UK/EU: 22 [84.6%]). The most frequently reported reasons for reinitiating treatment with ESAs in 2L were overall health status (54.6% US/CAN; 40.9% UK/EU), treatment efficacy (45.5% US/CAN; 63.6% UK/EU), and disease characteristics (36.4% US/CAN; 45.5% UK/EU). In the subgroup of patients who reinitiated ESA in 2L, the most frequently reported reasons for discontinuation of 1L ESA were patient decision (45.5% US/CAN; 22.7% UK/EU), completion of planned course of therapy (45.5% US/CAN; 18.2% UK/EU), and progressive disease (36.4% US/CAN; 68.2% UK/EU). Conclusions: In this real-world study, the most frequently used treatments for the management of anemia in patients with non-del(5q) LR-MDS were ESA-containing regimens. A considerable proportion of patients were TD before initiating 1L ESA therapy. For those who were re-treated with ESAs in 2L, the most common reasons for treatment reinitiation were health status, disease characteristics, and reported treatment efficacy, although only a small fraction of patients who were TD at 1L ESA initiation became TI. These findings highlight an unmet need in anemia management among patients with non-del(5q) LR-MDS, and a requirement for new and more effective treatment options. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First Single-Center Experience with Luspatercept Therapy in Low-Risk Myelodysplastic Syndrome (LR-MDS) Patients with Transfusion Dependence Refractory to Erythropoietin Therapy

Introduction: In the treatment of low-risk myelodysplastic syndromes (MDS) patients, anemia is the predominant problem. A recent breakthrough in the therapy of anemia in low-risk MDS patients are drugs involved in TGFβ pathway blockade. Luspatercept is a recombinant fusion protein derived from human activin receptor type IIB. It binds TGFβ superfamily ligands and inhibits the SMAD2/3 pathway leading to potentiation of red cell line differentiation and proliferation. Luspatercept was approved last year by the FDA and EMEA in the treatment of anemic low-risk MDS patients with ring sideroblasts (MDS-RS-MLD, MDS-RS-SLD) and/or SF3B1 mutated patients who are not eligible for erythropoiesis-stimulating agents (ESA) or for patients who failed on this therapy. Luspatercept showed high clinical activity for the treatment of anemia in the phase 2 trial (PACE-MDS) and subsequently in the phase 3 trial (MEDALIST). It has led to transfusion independence (TI) in about 40 % of patients. Methods: From January 2021 to July 2022, 27 patients were treated with luspatercept at the hematology department of Charles University Hospital in Prague, Czech Republic. The median age of the patients was 77 years (range 57-89) with a male to female ratio 17:10. WHO 2016 diagnosis were: MDS-RS-MLD (N = 23) and MDS/MPN-RS-T (N = 4). Median of IPSS-R was 2.5 (range 1-4). Median of IPSS-R cytogenetic score scaled from 0-2 with the median of 1. Median follow-up was 7.8 months (range 1-38). Luspatercept was administered in subcutaneous injections once every 3 weeks with an initial dose of 1 mg/kg. Dose escalation was done according to the level of response to 1.33 and 1.75 mg/kg. The median number of cycles administered was 7 (2-17). Transfusion dependency before luspatercept initiation ranged from 2 transfusion units (TU)/8 weeks to 12 TU/8 weeks, 11 patients had low transfusion burden (LTB), 16 high transfusion burden (HTB). Baseline erythropoietin (EPO) level was 374 IU/l in average (range 150-770) and ferritin level was 1307 μg/l in average (range 234-3832). 9 patients received chelation therapy. 22 patients were tested for SF3B1 mutation with 21 positive results (95.5 %) using Sanger sequencing (N = 3) or NGS approach (N = 18). Isolated mutation of SF3B1 was present in 8 patients (44 %). The most frequent mutations besides SF3B1 were TET2 (N = 8; 44 %), ASXL1 (N = 3; 17 %), DNMT3A and RUNX1 (N = 2; 11 %). Average mutations count per one patient was 1.7 (range 0-5). We did not observe any TP53 mutation. Results: There were 24 evaluable patients in terms of response. Thus far 11 (45 %) patients have responded by achieving TI, 5 (20 %) patients have had a reduction in transfusion dependence (HI, according to IWG criteria 2006), 8 (33 %) patients have not responded. One patient progressed to the higher risk MDS, and one transformed to acute myeloid leukemia. Median duration of response was 7.4 months (range: 0.7-31.8). Median number of cycles to achieve response was 4. Eleven patients responded to the basic dose 1 mg/kg, 2 additional patients on the dose 1.33 mg/kg and 2 more on the dose 1.75 mg/kg. There were differences in response according to transfusion burden, in LTB group achieved TI 8 patients and HI 1 patient, in HTB group we observed TI in 3 patients and HI in 4 patients. In 9 patients, we added ESA to luspatercept therapy, as they act both at different levels of erythropoiesis, which led to the improvement of response in 5 cases. In general, patients with lower initial baseline EPO level were better responders. We did not observe decrease in ferritin level during therapy. There was no significant effect of mutation burden on patient's OS, although patients with single mutated SF3B1 achieved better ORR (75 %), than patients bearing mutations in multiple genes (ORR = 40 %). There were no adverse effects of Grade II or more. Conclusion: In conclusion, luspatercept is certainly the next major step in the treatment of anemia in MDS patients. The advantage of the drug is very good tolerance and easy administration. We observed better responses in patients bearing single mutation in SF3B1, patients with LTB and lower initial baseline EPO levels. The higher response rate in our follow-up may be influenced by the combination with ESA and rapid dose escalation. This work was supported by the following grants: grant AZV 2021-2024 NU21-08-00312, Grant AZV 2019-2023 NV19-08-00144, UNCE/MED/016

Clinical Significance of a Miniscule GPI(-) Granulocyte Population in Patients with Bone Marrow Failure

[Background] Small (0.003-1.0%) populations of glycosylphosphatidylinositol (GPI)-deficient granulocytes (Grs) (sPNH-GPs) are often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndromes, and are thought to represent a marker of immune-mediated bone marrow (BM) failure. On the other hand, our recent studies revealed that miniscule (<0.003%) GPI(-) Gr populations (mPNH-GPs), which are derived from mono- or oligo-clonal PIGA-mutated hematopoietic stem cells (HSCs), are detectable in the peripheral blood (PB) of 80% of healthy individuals (HIs) and some cord blood (CB) samples (ASH 2021, #2181). The high prevalence of mPNH-GPs in HIs and the similarly high (60%) prevalence of PNH-phenotype Grs in AA patients suggest that the proliferation of PIGA-mutated HSCs may inevitably occur in immune-mediated AA. However, our earlier study failed to detect mPNH-GPs in some AA patients in long-term remission who possessed HLA-class I allele-lacking (HLA[-]) Grs after immunosuppressive therapy (IST) (unpublished data). In patients with long-standing AA, the prevalence of mPNH-GPs may be lower than that in HIs due to the replacement of mPNH-GPs by other aberrant Grs. [Methods] To test our hypothesis, we examined PB samples from 40 patients who developed AA 1-43 (median 11) years previously and were all negative for sPNH-GPs for the presence of mPNH-GPs. At the time of sampling, 28 including 6 patients possessing HLA(-) Grs were in remission after IST (n=8), thrombopoietin-receptor agonists (TPO-RAs, n=14), anabolic steroids (AS) (n=3) after failing IST, or spontaneous resolution (n=3). Eight patients were refractory to all therapies, 3 of which were transfusion dependent. The remaining 4 with non-severe AA had not been treated. Four patients with pure red cell aplasia (PRCA) who had a 12-32 year history of anemia responding to cyclosporine (CsA) were included in this analysis. PB samples from 93 HIs and 22 CB served as controls, and samples from a patient with chronic myeloid leukemia (CML) were used as a positive control for clonal hematopoiesis. mPNH-GPs were deemed positive when 4 or more CD11bhigh dots that formed tight clusters were present in the FLAER- fraction of Grs that were enriched using microbeads coated with PE-labeled anti-CD55 and anti-CD59 antibodies. [Results] We first determined the prevalence of mPNH-GPs in HIs (n=93) at different ages. The prevalence was approximately 80% in all different age groups with the exception of the CB, which showed a prevalence of 40.9% (Figure 1). In contrast, mPNH-GPs (0.101-1.998x10-3%) were detected only in 7 (17.5%) of the 40 patients with a long history of AA (Figure 1). These 7 patients were refractory to IST but were in remission at the time of sampling after responding to TPO-RAs (n=3, Figure 2) and AS (n=1); while 3 experienced spontaneous resolution of AA after cessation of treatment. Of the 33 AA patients who were negative for mPNH-GPs, only 8 were in remission, and 5 of the 8 possessed HLA(-) Grs that accounted for 75%-99% (median 96%) of the total Grs (Figure 2). All 4 patients with PRCA who were negative for sPNH-GPs possessed mPNH-GPs, despite the fact that they responded to CsA. The lack of mPNH-GPs in AA patients with complete clonal hematopoiesis with HLA(-) Grs prompted us to examine a CML patient whose Grs were 100% BCR-ABL-positive. mPNH-GPs were undetectable at the diagnosis, but became detectable 2 weeks after the initiation of nilotinib therapy, and increased after 6 months when the patient achieved cytogenetic remission (Figure 2). [Discussion] The persistence of mPNH-GPs in AA patients who failed to respond to IST suggests that immune mechanisms may have been not involved in the patients in whom GPI-deficient Grs remained miniscule. Given that mPNH-GPs were undetectable in AA patients possessing HLA(-) Grs and in many patients with long-standing AA refractory to IST, the presence of mPNH-GPs may be a marker of oligo- or poly-clonal hematopoiesis, which can be eliminated by the clonal expansion of HLA(-) HSCs or some other HSCs with intrinsic abnormalities in patients with long-standing AA, findings that are compatible with the absence of mPNH-GPs at the diagnosis in a CML patient. Lastly, the presence of mPNH-GPs without expansion in PRCA patients responding to CsA suggests that immunopathogenesis of PRCA is distinct from that of CsA-responsive AA, which turns mPNH-GPs into sPNH-GPs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular Patterns Identify Distinct Subclasses of Myeloid Neoplasia

The diverse molecular pathogenesis of MDS underpins its pathomorphologic and clinical heterogeneity, ultimately converging in a stepwise progression to leukemia. The identification of hallmarks morphological features, hampered by inter-observer variability, limited a precise resolution of pathogenic differences, partially mitigated by the inclusion of cytogenetic abnormalities, the two pillars of MDS classification. The advent of NGS led to the discovery of a multitude of mutations, and their combinations unveiled a tremendous molecular diversity in MDS. Intuitively, molecular patterning has been used in supervised fashion to fit morphologic classification, but this approach ensured only a limited success. Machine learning (ML) analytic methods, as demonstrated in acute myeloid leukemia (AML), provide new opportunities to integrate molecular data and identify functionally convergent patterns manifesting with similar clinical outcomes. Particularly, ML approaches can identify cryptic molecular interactions and pathogenic mechanisms irrespective of their often comparable impact on outcomes. Here, we took advantage of a large, well-annotated multicenter cohort of MDS and secondary AML (sAML) cases (N=3588) to test the hypothesis that related molecular patterns can be analyzed in an unbiased/unsupervised fashion to characterize molecularly-defined configurations. Our aim was not meant to compete with or replace current prognostication tools (including the new molecular prognostic score)2 but rather to illuminate the genetic sub-classification of MDS and related conditions in an operator-independent fashion, according to molecular correlations and mutual functional proximity, irrespective of their impact on clinical outcomes. Molecular signatures driving each cluster were identified and used for genomic subclassification that separated functionally related subtypes in an outcome independent fashion. We then validated the model on 412 patients from two external institutions. Among the 3588 patients included in this cohort, 735 (20%) had sAML, 774 (22%) had higher-risk MDS (HR-MDS), and 2079 (58%) had lower-risk MDS (LR-MDS). Abnormal karyotype was found in 43% of the cases, and 77% had at least one somatic mutation, with 8% harboring >4 mutations. ML analysis identified 14 distinctive molecular clusters (MCs) with unique pathobiological associations, treatment responses, and prognosis (Fig.1A). Normal karyotype (NK) was enriched in MC2, MC4, MC6, MC9, MC10, and MC12 with different distributions of ASXL1, DNMT3A,JAK2, RAS, SF3B1, and TET2 mutations. Complex karyotype and trisomy 8 were enriched in MC13 and MC1, respectively. Remarkably, our ML approach identified functionally relevant genomic clusters (Fig.1A). For instance, MC2, MC6, and MC8 demonstrated distinct genomic signatures: MC2 included cases with NK only (100%) and some DNMT3A (11%), JAK2 (11%), and RAS pathway (10%) mutations; MC6 cases had similar features to MC2 but were also enriched in SRSF2 (49%) and RAS mutations (23%); MC8 was instead characterized by the presence of del5q/-5 (100%), DNMT3A (17%), and TP53 (17%) mutations. MC13 contained cases with complex karyotype (100%) and TP53 (44%) mutations (Fig1.B). We then identified five risk groups to reflect the biological differences between clusters. Our clustering model highlighted the significant survival differences among patients assigned within similar IPSS-R risk groups but displaying heterogeneous molecular configurations. Different response rates to hypomethylating agents (e.g., MC9 and MC13 [OR: 2.2 and 0.6, respectively]) paralleled the biological differences across the clusters. Interestingly, our MCs retained significant survival differences regardless of the bone marrow blast percentage. Our model is available via web-based access resource (https://drmz.shinyapps.io/mds_latent). Despite the complexity and the diversity of molecular alterations in MDS/sAML, by deploying ML we were able to identify and distinguish functional and pathologically related MCs irrespective of the anamnestic clinico-morphological features. In recognition that similar outcomes do not warrant similar molecular features, our model provides genomic signatures for a better understanding of the pathobiological mechanisms of disease, progression to higher stages, and identification of future therapeutic vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase 1 Results of the First-in-Class CXCR1/2 Inhibitor SX-682 in Patients with Hypomethylating Agent Failure Myelodysplastic Syndromes

Background: SX-682 is an oral dual allosteric inhibitor of the CXCR1 and CXCR2 (CXCR1/2) chemokine receptors, a signaling pathway involved in the recruitment of immunosuppressive MDSCs and autocrine growth of disease-initiating leukemic stem cells (LSCs) in AML and myelodysplastic syndromes (MDS). CXCR2 overexpression is predictive of transfusion dependence in MDS. CXCR2 inhibition reduces proliferation of leukemic cell lines and primary MDS/AML patient (pt) samples in vitro and impairs leukemic growth in an in vivo AML animal model. CXCR2 is also involved in normal neutrophil egress from the bone marrow and thus absolute neutrophil count (ANC) serves as a pharmacodynamic (PD) marker for CXCR1/2 inhibition by SX-682. In pts with normal bone marrow, 25-400 mg SX-682 twice-daily (BID) dose-dependently reduces ANC, with maximum ANC reduction (-66% of baseline) obtained at >150 mg BID, consistent with maximum CXCR1/2 inhibition above this dose. ANC rapidly (24 hrs) normalizes with dose-holding. This NHLBI funded trial (HL142389, NCT04245397) explored the safety and efficacy of SX-682 monotherapy in pts with hypomethylating agent (HMA) failure MDS. Methods: This Phase 1 dose-escalation with expansion study treated five cohorts of HMA failure MDS pts with escalating 25-400 mg BID doses of SX-682 in six continuous 28-day cycles with responding pts continuing treatment. Steady-state pharmacokinetics (PK) were obtained. Response assessment was at the end of cycles 1, 3 and 6 and every third cycle thereafter using IWG criteria (Cheson 2006). This interim analysis after dose-escalation to select the expansion dose was pre-specified. Results:17 MDS pts received SX-682 (median 1 prior therapies; range 1-4) with 6 pts in the 200 mg cohort, 3 pts in each of the 25, 50 and 100 cohorts and 2 pts in the 400 mg cohort (Table). Median age was 76, and pts were low (n=1), intermediate-1 (n=10), intermediate-2 (n=5) and high (n=1) risk by the International Prognostic Scoring System (IPSS). All pts were transfusion-dependent and all had failed prior HMA and 24% lenalidomide. Steady-state PK exhibits increasing drug plasma concentration with dose comparable to pts with solid tumors. The ANC PD marker exhibited a dose-dependent decrease from baseline (ΔANC) that plateaued in the 200 and 400 mg dose cohorts consistent with maximum receptor inhibition (Table). Marrow MDSCs and LSCs were reduced after initiation of SX-682. SX-682 was well-tolerated with no maximally tolerated dose and no pt discontinued treatment for adverse events. Treatment-emergent adverse events > grade 3 were most common in the 200 and 400 mg dose cohorts, and those related to neutrophils are consistent with being related to SX-682. Effects on neutrophils reversed on cessation of drug dosing. There was a dose-dependent increase in overall response rate (ORR) from 0% at 25 mg BID to 50% at 200 mg BID consistent with the observed clinical benefit being related to CXCR1/2 inhibition by SX-682 (Table). Four of the five responders had mutant genes (MGs) involving splicing factors (60% of responders; SF3B1, SRSF2) and/or epigenetic regulation (60% of responders; TET2, ASXL1, IDH2). Across all dose cohorts, 8 of 17 (47%) pts had a reduction in marrow blasts after initiating SX-682 (Figure). The 200 mg dose yielded the most rapid and deep reduction in blasts with 2 of 6 pts (33%) achieving a marrow complete remission (mCR) in keeping with this dose providing maximum receptor inhibition. There was hematologic improvement (HI) in 3 pts with one having a durable near-CR response (on SX-682 >500 days) consisting of trilineage HI (hemoglobin > 10 g/dL) and transfusion independence for over 4 months (pt required 16 transfusions in the 7 weeks before starting SX-682 therapy). Based on ANC PD, ORR and marrow blast responses, the 200 mg BID dose was selected for the expansion phase of the trial. Conclusions: SX-682 is a novel therapy in HMA failure MDS with dual activity against LSCs and MDSCs. SX-682 was well tolerated with encouraging efficacy with a biologically effective oral dose selected at 200 mg BID. An expansion cohort in both lower and higher risk MDS pts is ongoing and will additionally include HMA naïve patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Aberrant Proportion of Monocyte Subsets in Bone Marrow of High-Risk Myelodysplastic Syndrome Patients

Background: Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic stem cells characterized by peripheral blood (PB) cytopenias due to ineffective hematopoiesis in bone marrow (BM). Patients face increased morbidity from infection and/or bleeding, while having a high propensity for leukemic transformation. It is evident that aberrant chromosomal and genetic lesions are the key drivers of MDS pathogenesis, which also contributes towards chronic activation of the innate immune response and a hyperinflammatory BM microenvironment (Barreyro et al, Blood, 2018). Various types of immune cells like monocytes, dendritic cells, myeloid-derived suppressor cells (MDSCs), T cells, and NK cells also add to the aberrant cytokines within the BM microenvironment. In hematological malignancy, altered frequencies of monocytes, granulocytes, and MDSCs are well documented. Thus, in-depth understanding of this alteration is critical in order to provide clues about its role in MDS progression and develop targeted immunotherapies. Method: BM myeloid cell compartment of MDS patients (n=11) was profiled by mass cytometry (CyTOF) and compared with age-matched healthy controls (n=4). Patients were further categorized into Low-risk (LR) MDS (n=8) and High-risk (HR) MDS patients (n=3) based on the IPSS-R score. All the patients included in this study were treatment naïve. Result: Immunophenotypically, we were able to differentiate three subsets of monocytes: classical (CD14+/brightCD16+), intermediate (CD14+/brightCD16+) and non-classical monocytes (CD14dimCD16+). HR-MDS patients showed a significantly reduced proportion of classical monocytes (p<0.05) and a higher proportion of intermediate monocytes (p<0.05) compared to LR-MDS patients and healthy individuals (Figure A and B). However, the frequency of classical and intermediate monocyte subsets was comparable in healthy individuals and LR-MDS patients (Figure A and B). The proportion of non-classical monocyte was not statistically significant in LR-MDS and HR-MDS (data not shown). Furthermore, LR-MDS patients demonstrates reduced granulocyte frequency as compared to healthy individuals, HR-MDS patients shows lower frequency compared to LR-MDS patients, but the difference was not statistically significant (Figure C). We also observed a significant increase in the expansion of Mo-MDSCs from healthy individuals to LR-and HR-MDS patients (p<0.05) (Figure D). Conclusion: Increased frequency of intermediate monocyte and Mo-MDSCs in HR-MDS patients points towards a hyperinflammatory phenotype and immunosuppressive microenvironment in HR-MDS patients which may facilitate the immune escape of blast cells and MDS progression. Additional studies are underway to further characterize the immune landscape in treatment-naive and treated MDS patients and to study the interaction of different immune components with blast cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal Trajectories and Therapeutic Targeting of High-Risk SF3B1-Mutant Myelodysplastic Syndromes

Myeloid neoplasms are driven by acquisition of clonal genetic lesions, yet it remains unclear how these lesions cooperate to transform hematopoietic stem and progenitor cells (HSPCs) programs and modify drug responses. The integration of genomic profiling with clinical and cytogenetic data in the recent IPSS-M has significantly improved risk stratification of patients with myelodysplastic syndromes (MDS). Spliceosome mutations in SF3B1 are common initiating events in MDS and confer favorable prognosis, while secondary mutations in RUNX1 and STAG2 transform this low-risk condition into high-risk MDS or secondary acute myeloid leukemia. To understand how these events cooperate with SF3B1 mutations, we modeled these clonal trajectories in primary human HSPCs. SF3B1 K700E mutation was introduced in CD34+ cells isolated from cord blood or peripheral blood of healthy donors, using CRISPR/Cas9 editing and AAV6-mediated homology directed repair. By introducing an intronic fluorescent reporter, this system allows for the tracking and isolation of SF3B1-mutant cells for molecular and functional analysis. Edited cells were heterozygous for the K700E mutation and recapitulated alternative 3' splice site selection of genes previously identified in SF3B1-mutant patients. To model disease progression, we introduced secondary RUNX1 (S-R), STAG2 (S-S), or control AAVS1 (S-A) mutations into SF3B1 K700E knock-in CD34+ HSPCs, leading to a reduction of RUNX1 or STAG2 expression in SF3B1-mutant cells (50% and 90%, respectively). The S-R and S-S trajectories induced divergent alterations in lineage specification of SF3B1-mutant HSPCs. S-R promoted myeloid skewing at the expense of the erythroid lineage, while S-S induced a block in differentiation, impairing both myeloid and erythroid differentiation. Consistently, introduction of secondary RUNX1 or STAG2 mutations in an induced pluripotent stem cell (iPSC-HPCs) model of SF3B1-mutant MDS recapitulated S-R myeloid expansion and S-S maturation arrest identified in primary HSPCs. S-R and S-S genotypes resulted in divergent transcriptional programs including inflammation, immune response, and myeloid differentiation. However, both high-risk clonal trajectories expanded the immature CD34+CD38- HSCs/multipotent progenitors (MPPs). S-R selectively expanded CD34+CD38-CD133+ HSCs, whereas S-S expanded the more mature CD34+CD38-CD133- population. These data suggest that, despite divergent molecular pathways, high-risk mutations converge on expansion of stem cell potential of SF3B1-mutant HSPCs. To understand how genetic (co-mutation patterns) and epigenetic (HSC state) heterogeneity can influence drug responses, we profiled a panel of spliceosome inhibitors and 166 FDA approved and investigational compounds. Both S-R and S-S genotypes conferred decreased response to single and combinatorial agents conventionally used for the treatment of myeloid neoplasms. High-risk genotypes maintained elevated sensitivity to SF3B inhibition, but conferred differential response to novel classes of spliceosome modulators, with STAG2 but not RUNX1 loss selectively promoting response to type I PRMTs inhibitor MS023. By contrast, CHK1 inhibitor Prexasertib was highly selective for SF3B1-mutant cells irrespective of co-mutations, inhibiting growth and cell cycle progression. Taken together these data indicate that the presence of SF3B1 mutation offers therapeutic vulnerabilities that can be leveraged to target high-risk genotypes, and different SF3B1 co-mutated genes can further modulate drug response of SF3B1-mutant cells. In conclusion, progression from low-risk SF3B1-mutant MDS to high-risk disease is mediated by molecularly distinct trajectories driven by RUNX1 and STAG2 mutations that converge on expansion of the HSC compartment. Moreover, clonal progression is associated with genotype-specific drug responses and increased resistance to standard agents, and ongoing studies are elucidating how genetic and epigenetic states affect therapeutic responses.