Primary MDS Research Articles

Nine-Gene Prognostic Signature for Myelodysplastic Syndrome

Abstract Introduction/Objective Myelodysplastic Syndrome (MDS) is a family of clonal hematopoietic malignancies found frequently in the seventh and eight decades, supporting that aging and acquired somatic mutations drive development of the disease. Without a cure, the prognosis is poor. Patient consideration for bone marrow transplantation remains low (<5%), and enrollment in clinical trials is slightly better (25%). Information regarding MDS-specific whole genome sequencing, a prognostic prediction system, and overall survival is limited and may be beneficial in developing curative therapies. Hence, we aimed to analyze the genomic profile, epidemiology, and overall survival of MDS. Methods/Case Report We utilized cBioPortal cancer genomics [The Cancer Genome Atlas (TCGA) PanCancer Atlas, TCGA Firehose Legacy, and TCGA Nature 2013] to study the mutations associated with MDS. Epidemiological characteristics and genetic profiles available on cBioportal were evaluated. Gene mutations with the most statistically significant, shortest overall survival and survival in days were used for the gene query, and survival rate with the common mutations was calculated. Logrank test and Kaplan–Meier estimators were used in analyzing the survival function. Patients with two or more overlapping mutations were excluded. Results (if a Case Study enter NA) We identified 7,554 patients with MDS out of which 2,852 had a primary MDS. There were 3,323 patients who had gender and age data available. 26.5% were male, and most patients (600) were between the ages of 70-75 years. 1573 (20.8%) were alive and 1600 (21.2%) were deceased. The common mutations associated with reduced survival were ASXL1, NPM1, FLT3, TET2, SF3B1, SRSF2, STAG2, RUNX1, TP53. Median survival of patients with these mutations was 32.02, 27.32, 25.97, 64.21, 80.94, 46.82, 44.28, 16.21, and 10.52 months respectively in comparison with the unaltered mutation group (68.68) (p < 0.0001). Survival was lowest amongst TP53 10.52 months and RUNX1 16.21 months. Leukemia free survival with ASXL1, NPM1, FLT3, TET2, SF3B1, SRSF2, STAG2, RUNX1, and TP53 mutations was 27.88, 13.02, 23.44, 56.52, 79.96, 37.32, 24.89, 14.86, and 9.44 months respectfully in comparison to the unaltered mutation group (68.68) (p < 0.0001). Conclusion Our study found that TP53 and RUNX1 genes were associated with the worst prognosis in MDS with median survival of 10.52 and 16.21 months, respectively. This study may provide insight for genetic predictors and targeted therapies for MDS, as well as provide better understanding of patient prognosis.

Analysis of Clinical Features, Prognostic Characteristics and Gene Mutations of the EB Subtype of Primary Myelodysplastic Syndrome Patients with Myelofibrosis

Objective ：This study aims to analyze the clinical features, prognostic characteristics and gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, to compare the differences and impact of MF on MDS-EB group and MDS-Non-EB group, ultimately to improve the cognition of MF on MDS-EB group. Methods ：From January 1, 2013 to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups (MDS-EB group and MDS-Non-EB group) according to 2016 WHO MDS classification.BM was reassessed on whether associated with fibrosis in grade MF0,1,2,3. (2005 European Myelofibrosis Network criteria). The clinical features, prognosis characteristics and gene alterations were analyzed between the MDS-EB group and MDS-Non-EB group retrospectively. Results ： ① MF was confirmed in 46.3％（193/417）cases of all the MDS patients, of which 66.3％（128/193）were MF-1 , 25.9％（50/193）were MF-2 and 7.8%(15/193) were MF-3 respectively. Compared with MDS-MF0 group, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). ② There was 174 cases of MDS-EB and 243 cases of MDS-Non-EB. The proportion of MF in MDS-EB group was 54.0%(94/174) and significantly higher than in MDS-Non-EB group 40.7%（99/243）(p=0.010).The distribution of MF grade was also differently significant between the two groups(p=0.048). The clinical features analysis showed that there is higher level of C-reactive Protein (CRP), higher level of serum lactate dehydrogenase (LDH) and higher level of β2-microgram in the peripheral blood in the MF(MF1,2,3) group than in the MF0 group, regardless of whether it was in the EB group or the non-EB group. The difference was statistically significant (ALL p value<0.05). ③The impact of MF on MDS-EB patients and MDS-Non-EB patients has different significances concerning OS, PFS and t-AML. As far as the OS prognostic factor, no matter the comparison of MF0, 1,2,3 four groups, the comparison MF1,2,3 three groups, the comparison of MF0-1 with MF2-3, all showed that MF was an adverse prognostic factor in the EB-MF group (All p value <0.05), and it also showed the adverse impact related with the grade of MF, but none of any in the MDS-Non-EB group (All p value>0.05)(Figure1). Comes to the PFS, it showed the MF on MDS-Non-EB was an inferior factor when compared MF+(MF1,2,3) to MF-(MF0)(p=0.0097), but not between the comparison MF1,2,3 groups(p=0.96) and none of the MDS-EB groups（p=0.31 and 0.092 respectively）. The incidence of MF was related with a higher rate of t-AML not only in MDS-EB group but also in MDS-Non-EB group compared with MF0, no matter compared the MF+(MF1,2,3) with MF-(MF0) but also the comparison of MF0,1,2,3) (All P value < 0.001). ④ In the MDS-EB cohort, 66 patients in the MF-(MF-0) group completed Next-generation-sequencing (NGS) test, and 61 patients in the MF+ (MF1,2,3) group completed NGS testing. It seems that the incidence of gene mutation has no difference between the two groups. We conducted univariate COX regression analysis about the 30 genes (which were suggested in the NCCN guideline 2020) with OS, PFS and t-AML in the MDS-EB-MF cohort. The results showed that TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML. Conclusion: MDS-MF has unique laboratory and clinical characteristics, act as an independent risk factor for shorter OS and worse PFS in the MDS-MF patients in our large cohort. In this study, we retrospectively analyzed 417 primary MDS cases in our hospital and analyzed the impact of MF on MDS-EB group and MDS-Non-EB group respectively. Our results showed the MF has a worse OS on the MDS-EB-MF group than the MDS-EB-MF0 group but no impact on MDS-Non-EB cohort. It was also consistent with the 5th edition of the WHO Classification of Haemato-lymphoid Tumors guidelines in which MDS-f was listed as a subtype concerning MDS with fibrosis concomitant with 5-19% blast cells in bone marrow or 2-19% blast cells in peripheral blood. The NGS sequencing showed TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML.As far as we know, this is the first analysis of bone marrow fibrosis in MDS-EB patients. We need more research to help us understand the MDS-f subtype.

Upfront Allogeneic Hematopoietic Stem Cell Transplantation Can Provide Survival Benefit Versus Hypomethylating Agent in Myelodysplastic Syndrome with Increased Blasts I (MDS-IB I)

Introduction Myelodysplastic syndrome (MDS) is a group of clonal disorders of hematopoietic stem cell. Survival would be significantly shortened if excessive blasts appear in marrow or the periphery. As disease progression accelerating, about 25% of MDS-IB1 and 33% of MDS-IB2 patients were reported to finally transformed into leukemia. Currently, allogeneic stem-cell transplantation (Allo-HSCT) still remains the only curable treatment accompanied with risk of therapy related mortality and decline of life quality. Decitabine (DAC), an of DNA methylation, was also reported to exhibit benefit in treatment of MDS. Thus, comparative studies with allo-HSCT are needed. Objective To retrospectively compare response of upfront allogeneic HSCT with hypomethylating agent (DAC in 87.5% of the group) therapy according to stem-cell donor availability in patients with MDS-IB1. Methods We retrospectively reviewed 103 patients with primary MDS patients who were evaluated and received treatment in our center between August 2011 and August 2020.Overall survival (OS) , event-free survival (EFS), non-relapse mortality (NRM) and cumulative rate of relapse or progression (CIR) at 1 year was calculated according to treatment arm.Cox regression analyses with allo-HSCT as time-dependent covariate (Mantel-Byar approach) were performed. Results According to the International Prognostic Scoring System (IPSS), 55 patients (55%) were stratified into lower-risk and 46 patients (45%) were stratified into higher-risk (IPSS intermediate 2 or high risk). Allogeneic HSCT was performed in 61.2% (n=63) and the rest 38.8% (n=40) received continuous HMA. In the no-donor group, 28 patients (70%) experienced disease progression or relapse after remission and leukemia transformation was noted in 14 patients (35%). The median follow-up time for surviving patients was 30.4months (range: 3.2-122.1 months). In the entire cohort, non-relapse mortality rate at 1 year was significantly higher in the allo-HSCT group compared with HMA group (14.3% vs. 2.5%, P=0.008), and the cumulative incidence of relapse and progression at 1 year was significantly lower (1.6% vs. 45.5%, P<0.001). After matching with propensity-score, the overall survival rate at 3 years was 77.8% in the allo-HSCT group compared with 43.5% in the HMA group with a significant difference by Cox-regression considered allo-HSCT as time-dependent covariate ( P=0.045), and event-free survival rate at 3 years was higher in allo-HSCT patients (73.4%) compared with HMA group (20.6%; P=0.001). The advantage of allo-HSCT was seen in those who aged > 50 years, male, those with ineffective erythropoiesis and without iron overload. Conclusion We concluded that patients with MDS-EB1 significantly benefited from receiving allo-HSCT with prolonged OS and EFS compared with continuous HMA (DAC) therapy. The advantage of allo-HSCT has been seen in subgroups of patients who age >50 years, male, those with ineffective erythropoiesis and without iron overload. Even earlier myeloablative allo-HSCT prevents further progression of MDS-EB1 at risk of higher non-relapse mortality, upfront allo-HSCT should be considered in a donor available scenario.

GDF11/SMAD Regulated Splicing of GATA1 Is Associated with Response to Luspatercept in Lower-Risk Myelodysplastic Syndromes (LR-MDS)

Introduction: Luspatercept is an erythroid maturation agent that promotes late-stage erythropoiesis in patients with LR-MDS. While it is known that luspatercept, a modified activin receptor ligand trap, binds to GDF11 and Activin B, the mechanism by which these ligands trigger a decrease in red blood cell (RBC) formation is unknown. Luspatercept was approved by the FDA following the phase 3 MEDALIST study, but biomarkers of response are currently unknown. Results:In a large cohort of primary MDS CD34+ samples, GDF11 expression was significantly higher in MDS samples than in age-matched healthy controls (183 MDS samples vs 17 controls, P < 0.05). GDF11 was detected in primary MDS sera and was substantially elevated in the refractory anemia (RA) and RA with ring sideroblasts (RARS) MDS subgroups. Activin receptor 2B (ACVR2B), the receptor of GDF11, was also overexpressed in MDS samples, and its expression was confirmed by immunohistochemistry (IHC) in the bone marrows of primary MDS patients. GDF11/ACVR2B activates SMAD2, and we found that the overexpression of SMAD2 in CD34+ cells was associated with more severe anemia in MDS patients. RNA-seq and SMAD2 ChIP-seq analyses were then performed to determine the downstream effects of GDF11 in erythroid progenitors. Human CD34+ primary cells were utilized to generate BFU-E stage erythroid progenitors. We discovered that GDF11 promotes an abnormal erythroid gene expression program characterized by increased expression of pro-apoptotic and early erythroid differentiation-related genes. SMAD2 binding was detected in regulatory regions of early erythroid (including GATA1), proinflammatory, and apoptotic transcripts using ChIP-seq analysis with p-SMAD2. In a zebrafish model, administration of GDF11 caused a decrease in erythroid cells, which was rescued by treatment with luspatercept (Wobus et al. Leukemia 2021 35(10):2936-47). Luspatercept inhibited the phosphorylation and activation of SMAD2 in response to GDF11 stimulation in primary hematopoietic cells and leukemia cell lines. Importantly, luspatercept could reverse the growth-inhibiting effects of MDS-derived sera on primary hematopoietic progenitors. Finally, to identify biomarkers associated with clinical responses, we analyzed phase 3 MEDALIST study samples collected before and after treatment with lupatercept. RNA-seq analysis was performed on bone marrow samples from responders (R; n = 14) and non-responders (NR; n = 9) (Figure). Analysis of pre-treatment/screening bone marrow transcriptomes revealed no significant differences between R and NR in global gene expression. Analysis of splice isoforms between the two cohorts revealed strikingly greater differences. Most differential splicing events were observed in NR (702 in R vs 5232 in NR). The most prevalent splicing modifications were exon skipping events, and most functional pathways affected by differential exon usage were RNA splicing related. GATA1, the master erythroid transcription factor, was spliced differently in R at baseline, which was an intriguing observation. Response to luspatercept was found to be associated with exon 2 skipping, resulting in a shortened isoform of GATA1. R had a greater proportion of the short GATA1 isoform than NR (12% in R versus 6% in NR; P = 0.04). We analyzed ChIP-seq data from primary erythroid cells to determine the effect of GDF11/SMAD2 activation on GATA1 splicing. GDF11 stimulation resulted in SMAD2 binding to the second intron of GATA1. CRISPR-mediated deletion of the SMAD2 binding region in primary erythroid progenitors increased expression of the GATA1 long isoform. Next, we performed a CRISPR-mediated deletion of exon 2 of GATA1, resulting in a predominance of GATA1 short isoform expression and a decrease in erythroid output. In CRISPR-modified primary erythroid cells with a predominant short GATA1 isoform (exon 2-deleted cells), luspatercept was able to reverse the inhibition induced by GDF11. Conclusions: These results demonstrate that MDS cases that respond to luspatercept are associated with a relatively high level of expression of functionally deficient GATA1 short isoform. We further demonstrate that GDF11 stimulates the production of the GATA1 short isoform via SMAD2 binding to intron 2 of the GATA1 gene. The regulation of a GATA1 splicing isoform by GDF11/SMAD2 is associated with clinical responses to luspatercept, according to these novel findings.

Efficacy and Safety of Roxadustat for Treatment of Anemia in Patients with Lower-Risk Myelodysplastic Syndrome (LR-MDS) with Low Red Blood Cell (RBC) Transfusion Burden: Results of Phase III Matterhorn Study

Presented on behalf of all MATTERHORN (FGCL-4592-082) study investigators Introduction: For patients (pts) with LR-MDS, anemia poses a major clinical challenge, with limited response to first-line erythropoietin (EPO)-stimulating agents (ESAs) and a median duration of response ≤2 years. Further, pts with RBC transfusion dependence (≥2 packed RBC [pRBC] units every 8 weeks [Q8W]) are less likely to respond to ESAs. Anemia treatments with novel mechanisms of action enabling transfusion independence (TI) are needed to reduce frequent RBC transfusion burden. Roxadustat is a first-in-class, hypoxia-inducible factor prolyl hydroxylase inhibitor for treatment of anemia with chronic kidney disease. In the MATTERHORN (NCT03263091) dose-selection stage, roxadustat was well-tolerated, and 37.5% of pts (9/24) with LR-MDS and low RBC transfusion burden ([LTB] 1 pRBC unit Q8W for two consecutive 8-week periods or 2-4 pRBC units Q8W) achieved TI. In the MATTERHORN double-blind stage, TI response rate and safety of roxadustat were further assessed. Methods: MATTERHORN is an ongoing, double-blind, Phase III, randomized, placebo (PBO)-controlled trial. Eligible adult pts (≥18 years of age) had very low-, low-, or intermediate-risk primary MDS per Revised International Prognostic Scoring System (IPSS-R) classification (<5% bone marrow blasts); hemoglobin (Hb) ≤10.0 g/dL at baseline (BL); and LTB. Prior ESA use (>8 weeks before randomization) was permitted. Pts were randomized 3:2 to roxadustat or PBO, then stratified by serum EPO concentration (≤200 or 200-400 mIU/mL), IPSS-R risk, and transfusion burden. Pts received oral roxadustat (starting dosage: 2.5 mg/kg three times weekly based on the dose-selection stage) or PBO with best supportive care (BSC; per institutional criteria, including RBC transfusion) for a 52-week treatment period, followed by a 4-week follow-up period. Primary efficacy endpoint was percentage of pts with TI (the absence of RBC transfusion) for ≥56 consecutive days during the first 28 treatment weeks (TI responder). The percentage of pts with TI and mean Hb increase of ≥1.0 and ≥1.5 g/dL (averaged over 8 weeks) compared with BL pretransfusion Hb was also assessed (to be reported separately). Safety (including treatment-emergent adverse events [TEAEs] and serious TEAEs) was evaluated throughout the study. Results: As of the final 28-week interim analysis of the double-blind stage (data cutoff: April 24, 2023), 140 pts (82 roxadustat, 58 PBO) were randomized and treated. Across arms, median age was 71.5 years (range, 26-96), 59.3% (83/140) were male, and 80.0% (112/140) were white. Most pts (72.1% [101/140]) had IPSS-R low-risk disease and a transfusion burden of 2-4 pRBC units Q8W (92.1% [129/140]). Median (range) BL transfusion burden was 2.5 (1-10) pRBC units. Seventy pts (50.0%) received prior ESAs (98.6% [69/70] were ESA-refractory). Eighty-four pts (41/82 [50.0%] roxadustat, 43/58 [74.1%] PBO) completed 28 weeks of treatment, and 15 pts (6/82 [7.3%] roxadustat, 9/58 [15.5%] PBO) were continuing treatment. Median (range) treatment duration was 24.1 (1.1-28.0) weeks for the roxadustat arm and 28.0 (0.1-28.0) weeks for the PBO arm. A greater percentage of pts in the roxadustat arm compared with the PBO arm were TI responders (47.5% vs. 33.3%). However, this difference did not reach statistical significance ( p=0.22; figure). Percentages of pts with TEAEs of any grade, serious TEAEs, and TEAEs leading to treatment discontinuation were similar across arms (table). Six deaths occurred on study (roxadustat: pneumonia [n=2], acute myocardial infarction and ischemic stroke [n=1], multiorgan failure [n=1]; PBO: urosepsis [n=1], disease progression [n=1]). Three pts (all in roxadustat arm) progressed to acute myeloid leukemia. The study was terminated by the sponsor and is currently being completed. Conclusions: Despite not meeting the primary endpoint, roxadustat plus BSC was well-tolerated, and a high percentage of pts with LR-MDS and LTB were TI responders. The high TI response rate in the PBO arm, historically poor outcomes in pts with ESA-refractory disease, and the inclusion of pts who were not transfusion-dependent (1 pRBC unit Q8W) may have contributed to the lack of a statistically significant difference in TI response rates between arms. MATTERHORN outcomes highlight the continued unmet need for effective and safe therapies that reduce RBC transfusion burden in LR-MDS.

Philadelphia Negative MPNs, MDS/MPNs and MDS in the Context of Recent WHO Changes and Inclusion of Molecular Signatures into Prognostic Tools: A Single Centre Experience

Introduction: The fifth edition of WHO classification of myeloid neoplasms has introduced major changes in the defining criteria and grouping of MDS, MDS/MPNs and MPNs. Recently published literature has also cited the importance of new risk-scoring systems by integrating genomic profiling with hematologic and cytogenetic characteristics, in order to improve the prognostic discrimination of patients and represents a valuable tool for clinical decision-making. Aim: To find out the prevalence and molecular spectrum of Philadelphia-negative MPNs, MDS/MPNs and MDS in our subset of patients and henceforth to evaluate the impact on diagnosis, risk stratification and treatment decision-making of patients. Methods: This retrospective observational study included all newly diagnosed patients of non-Philadelphia positive MPNs, MDS, and MPN/MDS, in whom complete baseline diagnostic work-up was available including complete blood counts, bone marrow morphology and biopsy, cytogenetic and molecular studies. Results: The most frequent entities in our cohort of patients were primary myelofibrosis (32.8%), MDS (32.8%) and CMML (16.4%). In PIMF, 50% patients were JAK2- mutated while 30% were triple negative (JAK-, CALR-, MPL-). The commoner epigenetic modifiers among MPNs were ASXL1, TET2 and IDH2. The predominant CMML molecular signatures in our patients were NRAS, U2AF2, SETBP1, ASXL and SH2B3. There was no significant effect of WHO changes and recently introduced molecular scoring models on the diagnosis and risk stratification of all these MPN and MDS/MPN patients However, in MDS and PIMF patients, recent WHO subtyping plus IPSS-M & GIPSS scoring respectively enabled refining of risk groups. Conclusion: Molecular profiling helps in better risk stratification of patients across all groups as well as in making therapeutic decisions. However, in resource constrained settings, it is not always possible to stratify patients on the basis of molecular signatures and hence, scoring models such as DIPSS and IPSS-R holds their ground strongly even today for offering appropriate therapy to patients without compromising on quality care.

Analysis of Clinical Features, Prognostic Characteristics and Gene Mutations of Primary Myelodysplastic Syndrome Patients with Myelofibrosis

Objective ：This study aims to analyze the clinical features, prognostic characteristics and gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, ultimately to improve the cognition of MDS-MF. Methods ：From January 1, 2013 to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups according to whether BM associated with fibrosis (2005 European Myelofibrosis Network criteria), the MDS-MF group(MF grades 1-3）and the MDS without MF group (MDS-MF0).The clinical features, prognosis characteristics and gene alterations were analyzed between the two groups retrospectively. Results ： ① MF was confirmed in 46.3％（193/417）cases of all the MDS patients, of which 66.3％（128/193）were MF-1 , 25.9％（50/193）were MF-2 and 7.8%(15/193) were MF-3 respectively. The proportion of MDS-EB in MDS-MF group was 48.2%(93/193) and significantly higher than in MDS-MF0 group 35.7%（80/224）,the difference had statistical significance(P=0.008). However, the occurrence of MF does not differ among the MDS subtypes according to the 2016 WHO classification (P=0.342). ② The clinical features of the two groups had statistically significant differences in the higher number of BM blast cells(P=0.006), higher prevalence of death (P=0.031), higher rate of Leukemic transformation (P<0.001), higher level of serum lactate dehydrogenase (LDH) in the peripheral blood (p<0.001), higher level of β2-microgram in the peripheral blood(p=0.003) and higher level of C-reactive Protein (CRP) in the peripheral blood (P=0.004) in the MDS-MF group than in the MDS-MF0 group. The number of WBC, Hb, PLT and Ferritin at the first diagnosed in our hospital were no difference compared the two groups (All P>0.05). The sex, Age and the IPSS-R score(IPSS-R >3.5 v ≤3.5) were no difference in the two groups (ALL P>0.05). ③Compared with MDS-MF0 group, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). The median overall survival was 13.20(0.07-90.70) months in MDS-MF group, and was 23.77(0.53-98.97) months in MDS-MF0 group respectively. Subgroup analysis showed that both OS and PFS of either the MDS-MF1-2 group or the MDS-MF 3 group were also significantly worse than those of the MDS-MF0 group（P＝0.013 and <0.001 respectively）. ④Among all the patients ,337 were finished Next-generation-sequencing (NGS) test, sequencing errors and all known or possible single-nucleotide polymorphisms were excluded. The number of mutations (>2) was more frequently in the MDS-MF, the difference had statical significance(P=0.030). The RNA splicing was more frequently in the MDS-MF than the MDS without MF, the difference also had statical difference(P=0.025). The ratio of U2AF1 mutation occurred in MDS-MF group higher than MDS-MF0 group, the difference had statical significance (P=0.027). There was totally 14.2% (48/337) patients with the mutation of U2AF1, of which 19.7%（29/147) in MDS-MF group and 10.0%（19/190) in MDS-MF0 group. Conclusion: MDS-MF has unique laboratory and clinical characteristics. MF is an independent risk factor for shorter overall survival (OS) and worse progression-free survival (PFS) in primary MDS. Evaluation of MF is very significant for prognosis judgment in primary MDS. The U2AF1 mutation may be a biomarker related to the occurrence and poor prognosis of MDS-MF. However, the mechanism of U2AF1 in MDS-MF patient needs to be further explored. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UBTF tandem Duplications Account for a Third of Advanced Pediatric MDS without Genetic Predisposition to Myeloid Neoplasia

Introduction: Recurrent tandem duplications (TD) in exon 13 of the upstream binding transcription factor gene (UBTF) have recently been identified in 4% of pediatric AML (Umeda et al. 2022). UBTF-TD AMLs were associated with adolescent age, normal karyotype, trisomy 8, as well as FLT3-ITD and WT1 mutations. Response to chemotherapy was poor and probability of overall survival (OS) at 5 years only 44%. Here, we evaluate the role of UBTF-TD in pediatric MDS. Methods: We studied 104 consecutive patients (pts) diagnosed with primary MDS with excess blasts below the age of 18 years enrolled in the prospective studies 98 or 2006 of the European Working Group of MDS in Childhood (EWOG-MDS) in Germany between July 1, 1998 and June 30, 2021. Pts with MDS secondary to chemo- or radiation therapy, acquired aplastic anemia and pts with classical inherited bone marrow failure disorder had been excluded from the analysis. High molecular weight DNA was extracted from stored bone marrow (BM) samples or archived smears. Screening for UBTF-TD was performed by PCR using 6-FAM-labelled primers covering exon 13 of the UBTF gene followed by high resolution fragment analysis. For samples showing aberrant signals indicating UBTF-TDs, Sanger sequencing confirmed the presence of a mutation. All UBTF mutant samples were also analyzed using targeted next generation sequencing (Archer Myeloid panel) for the presence of co-occurring alterations. Results:UBTF-TDs were detected in 25 of the 104 pts (24%; 16 males/9 females), while 79 pts (76%; 45 males, 34 females) showed no UBTF mutation (UBTF-wt). Variant allelic frequencies of UBTF-TD ranged from 11%-79% (median 41%). Median age at diagnosis in UBTF-TD and UBTF-wt pts was not different (11.0 vs. 9.8 years). UBTF-TD pts had higher absolute neutrophil counts (2.7 vs. 0.9 x109/L, p=0.01) and more often normoblasts on peripheral blood smears (92% vs 43%, p<0.01). Blast percentage in BM was similar with <10% blasts in 40% and 38% of UBTF-TD and wt pts, respectively. BM cellularity was normal or increased in 92% of UBTF mutated pts compared to 72% of UBTF-wt pts (p<0.01). Cytogenetic analyses revealed striking differences according to UBTF mutational status with the following karyotypes (TD vs wt): normal (72% vs 33%), -7 (0% vs 30%), del(7q) (0% vs 2%), +8 (24% vs 9%), +8 plus other (0% vs 4%), random (4% vs 7%) and structural complex (0% vs 7%). Probability of OS at 5 years for pts with UBTF-TD was inferior (0.50, 0.28-0.72) to that of pts with UBTF-wt (0.73, 0.62-0.84; p=0.03). Of the 104 pts, 30 pts had a genetic predisposition to myeloid neoplasia, i.e. GATA2 deficiency (n=15), RUNX1 deficiency (n=7), SAMD9/SAMD9L disease (n=6) or neurofibromatosis type 1 (n=2). Of note, none of the pts with predisposition harbored a UBTF-TD. To study the impact of UBTF-TD on outcome of hematopoietic stem cell transplantation (HSCT), pts with germline disease were excluded; of the 72 pts available for HSCT analysis (25 UBTF-TD and 47 UBTF-wt). Therapy prior to HSCT in UBTF-TD and UBTF-wt pts was comparable and consisted of AML-type therapy in 32% vs 21%, less intensive chemotherapy 8% vs 15% and no therapy in 60% vs 64% pts, respectively. Type of donor, stem cell source and conditioning regimen did not differ between the 2 groups, 74% of pts received a busulfan, cyclophosphamide and melphalan conditioning. Event-free survival (EFS) and relapse incidence (CIR) at 5 years of the total cohort were 0.55 (0.39-0.71) and 0.23 (0.15-0.36), respectively. There was no difference in EFS between the 25 UBTF-TD (0.43, 0.22-0.64) and 47 UBTF-wt pts (0.60, 0.45-0.75). However, in pts with normal karyotype, presence of an UBTF-TD was associated with an inferior EFS (0.35 vs. 0.77, p=0.03) and higher CIR compared to wt pts (0.36 vs. 0.06, p=0.03). Co-occurrence of WT1 mutations among UBTF mutated pts was associated with higher CIR (p=0.03); other mutations were less frequent (Figure). Discussion: We identified UBTF-TD in a third of all pediatric pts with advanced primary MDS and absence of germline predisposition. In addition, we noted UBTF-TD at a high frequency in pediatric pts with MDS-related AML (3/15 pts UBTF-TD). In regards to the recent discovery in AML, we conclude that UBTF-TD mutations define a novel subgroup of myeloid neoplasia presenting frequently as pediatric MDS. The high CIR following HSCT particularly in patients with concurrent WT1 mutations outlines an urgent need for new therapeutic strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The sWT1+ - Transcript Confers Cytarabine Resistance Via Upregulation of HOXA3 and GATA2 Axis

The Wilms tumor gene (WT1) encodes for a zinc finger transcription factor which manifests as both tumor suppressive and oncogenic activities. Due to 3 translation start sites and alternative splicing at exon 5 and a lysine, threonine, and serine (KTS) motif, WT1 generates 12 major transcripts. WT1 expression is upregulated in ~90% of leukemia samples and is associated with chemoresistance and disease relapse. However, which WT1 transcripts are associated with chemoresistant has not been defined. We characterized that sWT1+/-, but not other transcripts decreased cell growth, downregulated CD71 expression, and conferred cytarabine resistance. Leukemia blast cells with low CD71 expression are relatively resistant to cytarabine, suggesting that CD71 could be used as a biomarker for cytarabine treatment. Subsequent RNAseq analysis revealed two transcription factors HOXA3 and GATA2 to be uniquely upregulated in sWT1+/-, but not in other transcripts. Consistent with the sWT1 +/- phenotype, HOXA3 or GATA2 overexpression decreased cell growth, reduced CD71 expression, and conferred cytarabine resistance. Real time-PCR analysis showed that overexpression of WT1 and GATA2 upregulates each other and HOXA3 expression; whereas overexpression of HOXA3 upregulates GATA2 expression. Furthermore, relapsed leukemia samples express a relatively higher level of HOXA3 and GATA2 compared to de novo AML samples in the BeatAML cohort. GATA2 and WT1 are also coexpressed in AML samples (TCGA AML and BeatAML). Interestingly, WT1 and GATA2 mutations are mutual exclusive in primary AML and MDS patient samples (TCGA AML, BeatAML, and MDS (MSKCC+ IWG, IPSSM, NEJM)). These data indicates sWT1+-, HOXA3, and GATA2 forms a transcript complex that regulate cytarabine sensitivity. As such, we have identified transcription factors and a biomarker for cytarabine sensitivity in AML.

Dynamics of Mortality and Transformation Risk within Different Risk Groups of Patients with Myelodysplastic Syndromes Stratified According to the IPSS-R - Comparison of Treated and Untreated Patients and Consequences for the Description of Risk Categories

Background In MDS, most risk assessment tools such as the IPSS-R estimate risk at time of diagnosis. Risk assigned by the IPSS-R categories, however, is not constant over time. Changes in hazard (= time specific risk in a short time interval) during course of the disease affect the use of prognostic tools and may influence clinical decisions. We have previously shown time related hazard changes in untreated patients leading to declining risk in higher risk groups and diminution of differences between risk groups over time. Aim of this study was to investigate hazard changes in patients undergoing disease-modifying treatment. Patients and methods 16 international partners of the IWG-PM contributed data from 9179 primary MDS patients of which 2015 had disease modifying treatment: median age 70.0 yrs, 60% male, median follow up 4.1 yrs, median OS 3.8 yrs, IPSS-R categories: very low 16%, low 35%, intermediate 22%, high 15%, very high 12%. Treatment modalities included hypomethylating agents, lenalidomide, cytotoxic therapy, 17% had stem cell transplantation. Time variations were described by smoothed hazard graphs and by the Cox zph test including rho, a measure for a linear time dependent impact of a feature on hazard. Semiparametric accelerated failure time models were estimated by direct optimization procedures. Results and discussion Using hazard graphs and the Cox zph-test we confirmed changes in hazard relations over time in the untreated subsample (rho -0.20 for OS). In treated patients a similar diminution was observed (rho -0.26 for OS). Results from treated patients revealed temporary increased hazard after some months especially in higher risk patients. Possible reasons are decision to start treatment when hazard increases or transient risk by therapy-associated toxicity, as well as a "guarantee time bias", induced by delayed treatment start, which in treated patients implicitly guarantees survival until start of treatment. To account for the latter, analyses were repeated excluding "guarantee time” in treated patients, again showing hazard reduction in higher risk patients and diminution between risk categories: untreated: rho -0.20 for OS, treated: rho -0.08 (fig A-B). Differently developing changes in hazards over time contradict the proportional hazards assumption of the Cox model. Therefore, as an alternative we investigated accelerated failure time models - fitting semi parametric models for IPSS-R risk categories: results show that patients in different risk categories share similar course of hazards - only at different "speeds” (speed-factors excluding guarantee time to achieve average survival: VERY LOW 0.34, LOW 0.63, INT 1.15, HIGH 2.23, VERY HIGH 5.11.) Transforming time accordingly makes common patterns visible, as in figure C, where most risk categories show hazard peaks before reaching the time of their median survival. Conclusions In MDS, hazards are not constant over time, possibly slightly rising until around the category specific median survival times and later clearly falling, except for the very high risk category. Longitudinal changes of hazards are not proportional between IPSS-R categories, instead diminution of hazard proportions between risk categories is observed. To describe the development of hazard for different IPSS-R categories the concept of different "speeds” of the disease development may be appropriate. Clinicians need to be aware of the dynamics of hazards in MDS when counselling patients both treated or those receiving supportive care only. Risk assessment at time of diagnosis by the IPSS-R assigns average group differences but changes over time need to be taken into account. Our data suggest that median survival times for IPSS-R categories approximately express different speeds and also indicate the time, when survival hazards are declining. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone marrow mesenchymal stem cells regulate the dysfunction of NK cells via the T cell immunoglobulin and ITIM domain in patients with myelodysplastic syndromes

BackgroundMyelodysplastic syndrome (MDS) is a clonal disease of hematopoietic cells, characterized by hematopoietic cell hematopoiesis and a high risk of transformation into acute myeloid leukemia (AML). Although the underlying mechanism is unclear, MDS is often associated with immune system disorders, especially cellular immune abnormalities. We analyzed the number of lymphocyte subsets by flow cytometry assay and explored the alteration of lymphocyte subsets in MDS.MethodsHealthy controls, inpatients with primary MDS and patients with AML diagnosed from January 2017 to July 2021 were included. Flow cytometry assays were used to study lymphocyte subsets obtained from the bone marrow of the participants as well as changes in natural killer (NK) cell function. One-way analysis of variance and Student’s t-test were used to analyze the data.ResultsWe found a reduction in the number and function of NK cells in patients with MDS. By further measuring the activating and inhibitory receptors on the surface of NK cells, we found that the T cell immunoglobulin and ITIM domain (TIGIT) was the highest expressed marker on NK cells. Additionally, the expression of CD155, which is the ligand of TIGIT, was significantly higher than expressions of CD112 and CD113 on bone marrow mesenchymal stem cells (BMSCs).ConclusionsThe co-culture results of BMSCs and NK cells demonstrated that BMSCs regulate NK cells through the TIGIT/CD155 interaction, indicating that NK cells play a vital role in MDS progression. BMSCs regulate the function of NK cells via TIGIT/CD155.EvM_ycHj6U8mjawjepZzLzVideo

BMSCs Regulate the Function of NK Cell By CD155/Tigit/CD226 Pathway in MDS Patients

Objective To analyze the immune status of MDS patients and evaluate its impact on prognosis.To investigate the specific mechanism of how mesenchymal stromal cells (MSCs) affecting immunity.Methods The study included 84 healthy controls and 84 inpatients with primary MDS, 22 patients with AML who were initially diagnosed in the Department of Hematology, General Hospital of Tianjin Medical University from January 2017 to July 2021.We collected flow cytometry(FCM) reports and clinical data. All MDS patients were stratified by IPSS-R for disease risk. According to IPSS-R classification, Very Low/Low and Int/High/Very high were divided into lower-risk MDS group and higher-risk MDS group, respectively.1. Statistical analysis of lymphocyte subset counts and cytokines in peripheral blood of different types of patients and healthy controls. 2. Correlation analysis between hemoglobin , platelets,neutrophil, blast cells, cytogenetics and lymphocyte counts was performed.3.We generated MSCs in BM samples from MDS patients and HCs.The expression of CD155 in MSCs and TIGIT/CD226 in lymphocytes were detected by FCM.Results 1.Percentage counts of NK cells in the HC group(19.94,95%CI 17.86-22.02，P<0.001)were significantly higher than other groups.The higher-risk group of MDS(11.51,95%CI 9.34-13.68) was obviously lower than the lower-risk group(16.91,95%CI 13.85-19,97)(P=0.006),close to the AML group(9.77,95%CI 6.33-13.20,Figure1).Meanwhile, the level of IFN-Ƴ in serum which is secreted mainly by NK cells was also the highest in the HC group(5.50,95%CI 3.74-7.26), followed by the MDS group and the lowest in the AML group(2.64,95%CI 1.91-3.37,Figure1).In addition, CD8+ T cells and CD19+ cells had similar results as NK cells, but there was no significant difference with CD3+ cells and CD4+ T cells.2.Significantly higher level of NK cells percentage counts were found in patients with hemoglobin ≥ 100 ×10 12/L compared to patients with lower hemoglobin(23.23,95%CI 17.26-29.21 and 15.77,95%CI 13.00-18.55,P=0.010). While the levels of NK cells were higher in patients with blast cells≤5% than in those with higher blast cells.(19.51,95%CI 16.09-22.93 and 14.65,95%CI 11.55-17.74,P=0.027). No significant difference was found between NK cell levels with regard to platelet and neutrophil counts.CD8+ T cells behaved similarly to NK cells. 3.The expression of CD155 on the surface of BMSCs was significantly higher in the MDS group than that in the HC group(P=0.001，Figure3).Moreover,MDS patients had higher expression of TIGIT(P=0.012) and lower expression of CD226(P=0.001,Figure4B) on the surface of NK cells in bone marrow than that in HCs.And CD8+ T cells also have similar results as NK cells.We analyzed the correlation between TIGIT of NK cells and CD155 of BMSCs, and found that they were negatively correlated（P=0.0297）, while CD226 was positively correlated with CD155（P=0.0706，Figure 4C）.Conclusions The decreased immune function in MDS patients is associated with prognosis.BMSCs are responsible for inducing an immune-suppressive microenvironment in MDS through CD155/TIGIT/CD226 pathway, the underlying mechanism remains to be further study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Hypomethylating Therapy Does Not Improve Outcome of Therapy-Related Myeloid Neoplasm Including TP53 Mutated and Complex Karyotype Subgroups

Introduction: Therapy related myeloid neoplasm (t-MN) is a rare but devastating complication of cytotoxic therapy used for management of unrelated malignancy or autoimmune diseases. Therapeutic options for t-MN are limited and although hypomethylating agents (HMA) are frequently used, the benefit of it in t-MN is unclear. There are no prospective trials of HMA in t-MN and published experience is based on retrospective analysis of small number of cases (n=42 to 54). The aim of this study was to assess the efficacy of HMA therapy in t-MN patients and evaluate predictors for it.Methods: t-MN patients managed with first line HMA therapy were analyzed. This study was approved by the participating institutional review board. Criteria for inclusion were a pathologically confirmed diagnosis of t-MN based on WHO 2016 criteria, age >18 years, and ≥1 cycle of decitabine or azacitidine.Patient characteristics were summarized by frequency (percentage). Overall survival was calculated from the time of starting azacitidine to last follow up or death date using Kaplan-Meier analysis. Multivariable analysis was performed using Cox proportional hazard model. Statistical analysis was performed using R program and significance was defined as P<0.05.Results: Of the 554 t-MN patients analyzed, 184 (33%) patients were treated with HMA as the first line therapy and were included in the study. Median age at t-MN diagnosis was 70 (IQR 64-75) years and 112 (60%) patients were male. At the time of diagnosis, 84% (n=154) and 16% (n=30) patients were classified as t-MDS and t-AML and 47% (87/184) and 46% (49/107) of the evaluable patients had complex karyotype and TP53 somatic mutations (TP53 Mut) respectively. Azacitidine (n=145, 79%) was most frequently used HMA followed by decitabine (n=39, 21%). Median survival of the whole cohort since t-MN diagnosis was 13.2 months.At the time of analysis further treatment details were available for 78 patients and outcome of these patients were compared with primary MDS (p-MDS) and oligoblastic AML managed at the same institute. Completion of six-cycles of HMA (50% vs. 62%; P=0.07) and overall response rate (47 vs. 50%, P=0.22) was not significantly different between t-MN and p-MDS. However, median OS of t-MN was significantly poor compared to p-MDS (10 vs. 20 months, P=0.0004; Figure 1A).In t-MN patients treated with HMA as the first-line agent, median OS was significantly shorter in t-AML compared to t-MDS (7 vs.10 months, P=0.04; Figure 1B). Median OS was significantly shorter in patients with TP53 Mut and complex karyotype (Figure 1C-D). In Cox proportional hazard analysis, t-AML and complex karyotype were independently associated with poor outcome.Conclusion: This study demonstrates significantly poor outcome of t-MN patients treated with HMA therapy as first line. The outcome is particularly poor in t-AML and patients with complex karyotype and TP53 Mut. Hence there is urgent need for clinical trials of novel therapies for t-MN. [Display omitted] DisclosuresSharplin: Kite Gilead: Honoraria; Novartis: Other: Travel Support. Ross: Keros Therapeutics: Consultancy, Honoraria; Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Unexpected High Frequency of Pathogenic Germline Variants in Older Adults with Primary Myelodysplastic Syndrome

Background: Germline predisposition is increasingly being recognised in myeloid neoplasms (MN) including primary myelodysplastic syndrome. An unequivocal diagnosis of germline predisposition carries actionable considerations for patient management including donor stem cell source for allogeneic transplantation, dose-reduction of conditioning regimes and screening for extra-hematological disease (such as pulmonary abnormalities in patients with telomere biology disorders). In addition, the identification of MDS predisposition syndromes can avoid misdiagnosis (for example, distinguishing idiopathic thrombocytopenic purpura from thrombocytopenia due to RUNX1 germline variant). However, the prevalence of pathogenic germline variants (PGVs) in unselected pMDS patients presenting at older age remains unknown.Aim: This study assesses frequency and type of pathogenic germline variants in MDS patients and compares with age matched healthy controls and patients with other cancers.Method: We analysed 68 known cancer predisposition genes in germline samples of 146 samples from myeloid neoplasms. Study included primary MDS (n=51) and MDS diagnosed in cancer survivors with (n=77) or without prior exposure to cytotoxic therapy (n=18). Using uniform American College of Medical Genetics and Genomics (ACMG) guidelines for annotating germline mutation, we also compared the frequency of pathogenic germline variants in the same genes with patients with single cancer and age-match healthy controls (>70 years).Results: Pathogenic germline variants (PGVs) were identified in 19% (28/146) patients compared to 4% and 3% patients with single cancer and age-matched controls respectively (P<0.0001) (Fig. 1A). Median age at diagnosis was similar between MN patients with or without PGVs [66 years (19-81) vs. 70 years (33-87); P=0.06]. PGVs were most frequent in DDX41 (n=7, 33%) followed by BRCA1 (n=2, 10%), GATA2 (n=2; 10%) and TP53 (n=2; 10%) (Fig.1B). We also identified pathogenic copy number variations (CNV) in 4 patients. The distribution of PGVs was also different, with DDX41 PGVs absent in single cancers and more prevalent in MN than age-matched controls (35% vs. 4%, P<0.001). The frequency of PGV was not significantly different between P-MN and T-MN/ MC-MN (17% versus 10%, P = 0.32 (Fig.1C). The frequency of PGV was 30%, 6%, 19%, 15% and 18% in patients ≤50, 51-59, 60-69, 70-79 and >80 years of age (Fig. 1D).Phenotypic features such as monocytopenia and mycobacterium infections (MonoMAC; SA460) and personal and family history of pulmonary fibrosis (SA918) were present in only two cases with PGVs. Family history of MDS/AML was present in only in four cases with PGVs, in which PGVs were found in typical myeloid malignancy genes (DDX41, GATA2). Importantly, some patients with family history of solid cancers carried PGVs in genes traditionally associated with solid cancers (e.g. MSH6, NF1, TP53 and BRCA1). SA927 had a PGV in MSH6 and multiple first-degree relatives with solid cancers including colon, renal and brain cancers. Moreover, 41% of adults with hematological disorders and a personal and/or family history of interstitial lung disease had PGVs in telomere biology disorder genes. Hence, family history should not be restricted to hematological disorders, but also solid cancers and non-malignant phenotypes (e.g. hepatic and pulmonary fibrosis). The frequency of PGVs was not different in patients with and without family history of cancer (23% vs. 13%, P=0.32).Conclusion: The frequency of PGVs is significantly high in MN compared to age matched healthy control and other cancer patients. Our observation of a high frequency of PGVs in the older MDS population warrants standardization of germline testing at diagnosis to guide optimal management of patients and their families. [Display omitted] DisclosuresHiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Isogenic MDS-RS Patient-Derived iPSCs Define the Mis-Spliced Transcript Repertoire and Chromatin Landscape of SF3B1-Mutant Hematopoietic Stem/Progenitor Cells

▪The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WT cells, recapitulating hallmark phenotypes of MDS cells.To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot.By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WT iPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells.In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700E mutant HSPCs.G. Asimomitis, A.G. Deslauriers: shared 1 st authorshipE. Papaemmanuil, E.P. Papapetrou: shared senior authorship DisclosuresDeslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.

Anti-CD123 CAR T-Cell Therapy for the Treatment of Myelodysplastic Syndrome

INTRODUCTIONMyelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis characterized by bone marrow dysplasia resulting in cytopenias. Currently, treatment options for MDS are limited to supportive care and hypomethylating agents with most patients eventually succumbing to the disease (anemia, infection or hemorrhage) or progressing to leukemia. Previously it have been demonstrated that CD123 is an important target for the treatment of MDS due to its aberrantly high expression in high-risk MDS cells compared to low-risk MDS cells and normal cells. As such, CD123-directed therapy may be useful in treating patients with high-risk MDS. METHODSChimeric antigen receptor (CAR) vector containing a CD123-specific single-chain variable fragment in combination with a CD28 costimulatory domain, CD3ζ signaling domain and tEGFR was expressed on healthy donor and patient derived T lymphocytes utilizing lentiviral vector delivery to target high-risk MDS cells. An anti-CD19 CAR was used as a negative control. CAR expressing or control T-cells were cocultured with MDS cell line (MDS-L) or primary MDS cells from patient bone marrow to examine in vitro anti-tumor function. Eradication of MDS cells was analyzed by flow cytometry 48hr after coculture. Intracellular TNF-α and cell surface CD107A were examined in effector cells 5 hours after cocultured with MDS cells. For in vivo analysis of CD123 CAR T cells, NSG-S mice were engrafted with 1.5x106 primary high-risk MDS cells for eight weeks, and were infused with autologous anti-CD123 CAR T-cells or control. Tumor burden was then analyzed on day 28. RESULTSHealthy donor derived anti-CD123 CAR T-cells effectively eliminated MDS cell line (MDS-L) (>99%), and primary bone marrow derived MDS cells (>70%) in vitro . The killing was associated with increased cytokine release and degranulation by anti-CD123 CAR T-cells, which did not occur with control anti-CD19 CAR T-cells. In addition, anti-CD123 CAR T-cells generated from patients with high-risk MDS showed 50~70% transduction efficiency. These cells successfully eradicated autologous CD123+ MDS cells at >99.9% in vitro compared to untransduced control T-cells (Figure 1A). The killing was also confirmed by increased intracellular TNF-α expression (Figure 1B). Furthermore, a patient derived xenograft system (PDX) was utilized to examine the killing ability of autologous anti-CD123 CAR T-cells in vivo . High-risk MDS bone marrow cells were engrafted in NSG-S mice. Eight weeks following engraftment, 7x104 autologous EGFRt+ anti-CD123 CAR T-cells or mock T-cells were infused via tail vein. On day 28 after autologous anti-CD123 CAR T-cell infusion, we observed significant decreases in both CD34+/CD38-/CD123+ MDS cells and MDS bulk (hCD45+/EGFRt-) while mice infused with mock CAR T-cells were unable to reduce tumor burden (Figure 1C and 1D). To extend the utility of our approach, we performed drug screening to identify the agents that increase expression of CD123 on MDS cells. We hypothesize that drugs inducing increased expression of CD123 will increase efficacy of anti-CD123 CAR T cell and avoid resistance arising from CD123 low/dim MDS progenitor cells. 134 various oncologic drugs were dosed at multiple concentrations (1nM, 100nM, 1uM, 0uM) and incubated with MDS-L cell line for 6hr, 16hr or 48hr. MDS-L cells were then analyzed for CD123 expression (Mean fluorescence) and cell viability (%live out of total) by flow cytometry. Eight drugs with low cytotoxicity significantly increased CD123 expression, including trifluridine (1uM), sunitinib (100nM), metoxantrone (1nM), dasatinib (10uM), imatinib (1uM), regorafenib (10uM), dabrafenib (100nM) and DEAB (100nM). These drug candidates will be examined further in primary MDS bone marrow samples and in combination with anti-CD123 CAR T-cells. CONCLUSIONThese results demonstrate that anti-CD123 CAR T-cells exhibit activity against high-risk MDS, and represent an effective therapeutic option for patients with high-risk MDS. [Display omitted] DisclosuresBudde:Mustang Therapeutics: Research Funding; Merck: Research Funding, Speakers Bureau; Amgen: Research Funding; KITE Pharmaceutical: Speakers Bureau; Precision Biosciences: Consultancy.

PPM1D Mutations in MDS Patients with Deletion of Chromosome 5q

Background: Patients with myelodysplastic syndrome with deletion of chromosome 5q (MDS del(5q)) have a favorable prognosis, but TP53 mutations occur in 13-18% of these patients and are known as unfavorable prognostic marker. Recently, Protein Phosphatase Magnesium-Dependent 1 (PPM1D) mutations have been described in clonal hematopoiesis and were found more commonly in patients with therapy-related MDS than in patients with primary MDS (15% vs. 3%) (Lindsley et al. 2017). PPM1D encodes a serine-threonine protein phosphatase that inhibits the p53 tumor suppressor pathway. It is therefore of great interest to evaluate the frequency and potential co-occurrence of PPM1D mutations in patients with MDS del(5q).Aim: To determine the frequency and potential clinical impact of PPM1D mutations in a large cohort of MDS del(5q) patients.Methods: 219 patients with MDS or AML following MDS (sAML) and deletion of chromosome 5q were included in the analysis. Patients were classified following the 2008 WHO classification. Patients with MDS (n=196) or sAML (n=23) were cytogenetically characterized by chromosome banding analysis and molecularly analyzed for mutations in all exons (1 to 6) of PPM1D by Sanger or next-generation sequencing (NGS). Patients were also analyzed for mutations in TP53 and CSNK1A1 .Results: A total of 11 PPM1D mutations were found in 10 (4.6%) MDS patients with del(5q). The mutation frequency was similar in patients with MDS with isolated del5q (8 of 167, 4.8%) compared to patients with more advanced MDS or secondary AML (2 of 52, 3.8%, P=.78). Four patients had nonsense mutations, 3 patients had frameshift mutations (one patient with 2 frameshift mutations), and 3 patients had missense mutations. Nine mutations were found in the previously described hotspot region of PPM1D between amino acids 427 and 542, which is located downstream of the phosphatase domain of PPM1D. Three of the 10 PPM1D mutated patients had additional cytogenetic aberrations besides del(5q) (trisomy 8; complex karyotype in two patients). TP53 was also mutated in the two patients with complex karyotype and in one additional patient with a nonsense mutation of PPM1D . None of the PPM1D mutated patients had a mutation in CSNK1A1 .Patients with mutated PPM1D were slightly older than PPM1D wildtype patients (median age 77.7 vs 71 years, not significant). Eight of 10 PPM1D mutated patients had a diagnosis of MDS del(5q) and 2 patients had MDS RAEB2 with del(5q) accompanied by a complex karyotype. There were no significant differences in the distribution of sex, WHO subtypes, cytogenetic risk according to the international prognostic scoring system (IPSS), WBC count, hemoglobin, platelet count and IPSS risk score between PPM1D wildtype and mutated patients. Two of the 10 patients were transfusion dependent and none of 7 patients with available information transformed to AML.Survival information was available for 156 patients with MDS del(5q) (excluding other WHO subtypes of MDS/sAML) including 8 patients with mutated PPM1D and 15 patients with mutated TP53 . Median follow-up from the time of sample harvest was 2.5 years. The probability of survival at 2 years was 100% for PPM1D mutated and TP53 mutated patients, respectively, and 84% for PPM1D/TP53 wildtype patients (P=.18, log-rank test, Figure 1A). None of 7 PPM1D mutated patients and 1 of 13 TP53 mutated patients with available information underwent allogeneic hematopoietic cell transplantation.Survival information was available for 45 patients with del(5q) and more advanced MDS or secondary AML. The 14 patients with TP53 mutation (including 2 patients with additional PPM1D mutation) had a significantly shorter OS than TP53 wildtype patients (P<.001, 2-year OS 0% vs 51%, Figure 1B). One PPM1D mutated patient responded well to lenalidomide treatment for the duration of 3.2 years.Conclusion:PPM1D mutations are recurrently mutated in MDS patients with del(5q) with a frequency of 4.6% and may co-occur with TP53 mutations. PPM1D mutated patients were not specifically associated with patient characteristics or outcome. Interestingly, in our cohort of patients with MDS with isolated del(5q), TP53 mutations were not associated with unfavorable outcome.VP and MM contributed equally as first authors. TH and MH contributed equally as last authors. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Platzbecker:Novartis: Consultancy, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Thiede:Novartis: Consultancy, Speakers Bureau; Roche: Consultancy; Agendix: Employment; Bayer: Consultancy, Speakers Bureau. Germing:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Schroeder:Celgene: Consultancy, Honoraria, Other: travel support. Kobbe:Celgene: Other: Advisory Board, Research Funding. Haase:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Dohner:Novartis: Honoraria, Research Funding. Sperr:Novartis: Other: Register; Phadia: Research Funding; Meda: Research Funding; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria. Valent:Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Incyte: Honoraria; Blueprint: Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Pfizer: Honoraria; Deciphera: Honoraria, Research Funding; Teva: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Activation of Autophagy in Models of SF3B1 Mutant MDS

Background:SF3B1 is a key pre-mRNA splicing factor that is abundantly expressed in normal erythroid cells and whose mutation has been linked with high frequency to myelodysplastic syndromes with ringed sideroblasts (MDS with RS) implying that this gene may be an important driver of the pathogenesis of this subtype. Given that MDS with RS is characterized by ineffective erythropoiesis, we previously investigated the link between SF3B1 aberrations and the regulation of pathways that control erythroid maturation and iron homeostasis. Indeed, knockdown of SF3B1 led to significant impairment of the growth of erythroid cells and delayed erythroid differentiation. MDS erythroid cells were unable to properly process iron, resulting in its accumulation in mitochondria. Iron build-up inhibited the proliferation of erythroid progenitors and induced mitochondrial DNA-damage. During erythroid maturation, autophagy plays critical roles in the clearance of damaged mitochondria and maintaining iron homeostasis. Knockout of the essential autophagy gene, Atg7 leads to anemia due to a delayed clearance of mitochondria with a consequent increase in iron levels, ultimately resulting in increased reactive oxygen species formation. As such, we set out to determine if autophagy upregulation alleviates oxidative stress and thereby increases erythropoiesis in MDS with RS.Methods: Transmission electron microscopy (TEM) was performed to examine the cytoplasmic vacuolization, autophagosome content, and morphology of vacuoles and mitochondria in SF3B1 mutant (SF3B1MUT) vs. wild-type (SF3B1WT) cells. Immunoblotting was used to detect microtubule-associated protein 1 light chain 3 (LC3B), p62, and cathepsin D (marker of autophagic degradation). Whole exome sequencing (WES) was used to detect somatic alterations in autophagy genes and RNA-Sequencing and qPCR were conducted to evaluate differences in mRNA expression levels of autophagy genes. A missense mutation in SF3B1 resulting in K700E amino-acid substitution was introduced using EGE-CRISPR/Cas9-stimulated homology-mediated repair to generate heterozygous K562 cells expressing K700E.Transgenic heterozygous SF3B1 mice were used as an in vivo model. Temsirolimus (CCI-779), an mTOR inhibitor with autophagy-inducing properties, was tested in vitro and in vivo.Results: We identified numerous autophagosomes, double-membraned autophagic vacuoles, and single-membrane cytoplasmic structures in SF3B1MUTvs. SF3B1WT cells. LC3B protein was higher in primary SF3B1MUTvs. SF3B1WT cells. WES did not detect any somatic mutations in autophagy genes in the patients analyzed by TEM. RNA-Sequencing and qPCR showed that primary SF3B1MUT MDS with RS cells displayed increased expression levels of key genes of the ATG-autophagy pathway: [ATG2A/B, ATG3, ATG5 (FC=2), ATG9, (FC=5); P=.05], serine/threonine kinases [ULK1 (FC=2), ULK3 (FC=4); P=.05], and cathepsins [CTSB/D (FC=2), CTSE (FC=6); P=.01]. CRISPR/Cas9 K700E cells showed high autophagosome counts, increased basal expression of LC3B protein, and a 2-fold increase in autophagy genes (ATG5, ATG7, ULK1) compared to parental cells. Treatment with temsirolimus (10-100 nM) for 24 hours induced growth inhibition (IC50, 50 nM), enhanced the amount of autophagosomes containing degraded organelles, increased the percentage of dividing CD71+ cells and induced a 2-fold increase in LC3B in CRISPR/Cas9 K700E cells compared to parental cells. Temsirolimus (10 mg/kg i.p. 5d/wk) administered for two weeks to 6-8-month-old transgenic SF3B1 heterozygous mice (N=20) induced autophagy by increasing the number of autophagosomes and autophagolysosomes containing entrapped mitochondria, increased hemoglobin (7.5 vs. 6.6 g/dL; P=.05), and doubled the percentage of CD71+ erythroid cells (5-10%) in temsirolimus-treated vs. untreated SF3B1 mice.Conclusions: Basal autophagy is significantly upregulated in SF3B1MUT cells. Further stimulation of autophagic activity using FDA approved pathway-activating drugs may improve rates of erythrocyte production by SF3B1MUT clones and thus improve control of anemia in MDS with RS cases. DisclosuresAdvani:Takeda/ Millenium: Research Funding; Pfizer: Consultancy. Kelly:Pharmacyclics: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Jannsen: Honoraria. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees. Carew:Millenium/ Takeda: Research Funding.

Additional Prognostic Impact of the Percentage of Erythroid Cells in the Bone Marrow of MDS Patients

Introduction:There is no direct correlation between the dysplastic features in the bone marrow as well as bone marrow cellularity with the degree of cytopenias in the peripheral blood in patients with MDS. Furthermore, the impact of the percentage of erythroid precursors in the bone marrow has been the subject of considerable debate over the past several decades. We therefore were interested in the prognostic impact of the percentage of erythroid cells in the bone marrow (bmery) for MDS patients. We analyzed the prognostic impact of the amount of erythroid cells in the bone marrow with regard to overall survival and AML evolution, both as single feature and in addition to the IPSS-R.Methods:Data from 2453 primary untreated MDS patients from Germany, France, Italy, and Austria with available percentage of erythroid cells in the bone marrow at the time of diagnosis were analyzed. Excluding CMML, the WHO2008 subgroups were RCUD 12%, RARS 13%, RCMD 41%, RAEB-1 15%, RAEB-2 14%, others 5%.Results:The median age of the entire patient cohort was 73 years (range: 14-100 years). After a median follow up of 72 months, 1502 patients have died. At the time of data analysis, 282 patients showed a progression to AML. Baseline characteristics are shown in table 1. To evaluate the influence of the percentage of erythroid bone marrow cells on prognosis, we grouped the percentage of cells in <=10%, 11-25%, 26-45% and >45% and found in univariate analysis different median survival rates. OS for bmery <=10% was 23.0 months, for 11-25% 39.9 months, for 26-45% 49.4 months, and for >45% 41.5 months, respectively. Therefore the following three risk categories were defined: “<=10%” (poor), “11-25 or >45%” (intermediate), and “26-45%” (good). Categorized that way, bmery shows prognostic impact on survival (Dxy=0.08, p<0.001) and time to AML progression (Dxy=0.15, p-value <0.001). Taking into account the IPSS-R (by stratified analyses), the Dxy was 0.09 for survival, and 0.18 for transformation, both with p<0.001. Added to the IPSS-R, bmery slightly enhances the prognostic power for both survival (Dxy=0.39) and time to AML (Dxy=0.59). Bmery seems to be more important for time to AML progression, in particular in IPSS-R lower risk categories, but this interaction is not significant. On the other hand, subtracting the bmery from total nucleated cells when calculating the bone marrow blast percentage does not enhance the prognostic power of the IPSS-R. Dxy fall from survival: 0.37 and time to AML: 0.58 to 0.36, and 0.52, respectively. Looking at the peripheral blood count, we could not find a correlation between the percentage of bmery and hemoglobin levels, ANC or platelet count.Conclusion:Survival and time to AML progression differ according to the amount of erythroid cells in the bone marrow although we could not demonstrate a correlation between bmery and peripheral blood counts, especially hemoglobin levels. Longest survival and time to AML is found at approximately the normal range of bmery between 26% and 45%. Lower, as well as higher percentages are unfavorable. This u-shaped influence has to be taken into account, when studying prognostic power. Adding bmery as differentiating feature to the IPSS-R may enhance especially the prognostic power for time to AML in lower risk categories. [Display omitted] DisclosuresFenaux:Janssen: Honoraria, Research Funding; Astex: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astex: Honoraria, Research Funding. Germing:Janssen: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Nachtkamp:Celgene: Other: Travel Support. Sperr:Amgen: Consultancy, Honoraria, Research Funding; Phadia: Research Funding; Meda: Research Funding; Celgene: Consultancy, Honoraria; Teva: Honoraria; Novartis: Other: Register. Stamatoullas:Takeda: Consultancy; Celgene Corporation: Honoraria. Valent:Celgene: Honoraria, Research Funding; Incyte: Honoraria; Teva: Honoraria; Blueprint: Research Funding; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Ariad: Honoraria, Research Funding; BMS: Honoraria; Novartis: Honoraria, Research Funding.