Myelodysplastic Syndrome Disease Research Articles

Development of Myelodysplastic Syndrome in a Patient With Pernicious Anemia During the Course of Replacement Treatment.

Megaloblastic anemia (MBA) is a reversible metabolic disorder that responds well to vitamin B12 supplementation. It contrasts with myelodysplastic syndrome (MDS), an irreversible neoplastic condition characterized by hematopoietic stem cell abnormalities. To date, no association has been identified between these two distinct etiologies, and they are considered independent diseases. However, despite their distinct classifications, both conditions present macrocytic anemia, similar bone marrow findings, and sometimes have common chromosomal abnormalities, which can lead to occasional misdiagnoses. Herein, we present a patient initially diagnosed with pernicious anemia (PA)who showed improvement with replacement therapy but subsequently became resistant to treatment and eventually developed MDS. Quantitative assessment of Wilm's tumor-1 (WT1) mRNA has emerged as a valuable tool for gauging MDS disease status and distinguishing it from related disorders, such as aplastic anemia. In our investigation of 30 patients with MBA, we explored WT1 mRNA expression. We observed its presence in 10 patients with PA, which suggests a potential link between PA and hematopoietic tumors.

Establishing a Pathogenic Role for NK Cells As Drivers of Bone Marrow Failure in the Myelodysplastic Syndromes (MDS)

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant bone-marrow (BM) morphology leading to a clonal impairment of hematopoiesis. Worsening BM failure (BMF) and increasing myeloblasts are observed as the predominant consequences of MDS disease progression from very low risk (VLR) to higher risk (HR) to acute myeloid leukemia (AML). Patients are stratified based on the degree of BMF and risk of AML transformation to help guide treatment decisions. Approximately 30% of MDS patients exhibit various autoimmune diseases that can include autoantibodies targeting neutrophils, erythrocytes, and/or platelets. Autoimmune phenomena play an important role in MDS yet are poorly characterized. We hypothesized that early in LR-MDS, dysregulated interactions between stromal cells and developing Natural Killer (NK)/T-cells promote rapid/unregulated development resulting in autoreactivity ( Fig. 1A). We predict dual effector mechanisms: CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) and loss of HLA class I (HLA-I) expression by developing hematopoietic lineage cells making them targets for “missing-self” reactivity ( Fig. 1A). NK cells are important sentinel cells that recognize tumors and dysfunctional cells that lose expression of HLA class I. Such surveillance is guided by interactions between NK inhibitory receptors and HLA class I ligands that ‘train’ NK cells to distinguish diseased cells, exhibiting perturbed HLA expression, from healthy cells with normal expression. The effects of these interactions combined with expression of activating receptors (e.g. CD16, NKG2D, DNAM1, FasL, TRAIL) allow NK cells to engage a target cell through many activating mechanisms, importantly including antibodies eliciting an antibody-dependent cellular cytotoxicity (ADCC) reaction. Autoantibodies are well-established in MDS, yet no one has profiled whether they may trigger ADCC/complement. Further, no one has ever thoroughly characterized BMMCs from LR-MDS patients before. In our study, we observed a clear link between signatures of bone marrow failure at LR-MDS and a significant increase in BM CD57+NK and CD8 T cells expressing a highly activated and dysregulated phenotype. Method: Ex-vivo profiling of 19 MDS patients (N=10 LR/IR-MDS, N=9 HR-MDS) BM mononuclear cells (BMMCs) was performed by mass cytometry (CyTOF)( Fig.1B). Cells were stained with three different antibody panels each consisting of 44 antibodies focused on: specific markers of hematopoiesis and myeloid or lymphoid cell lineages; specific markers of development, effector function, homing, exhaustion, and T-cell memory and proliferation; expression of HLA class I and activating ligands which are critical regulators of NK inhibition. Results: In our study we observed that a major fraction of MDS patients present with clonal-like expansions of BM-derived CD57+CD8+T-cells and natural killer (NK) cells that strongly correlate with autoimmune-cytopenias and BMF-enriched in LR-MDS (Fig.#). We observed that NK-cells and non-classical T-cells were significantly expanded (>2-fold) in LR MDS compared to HR-MDS and healthy-control BM (p<0.01). These non-classical CD57+CD8+T-cells co-expressed HLA-I-reactive and inhibitory NKG2A/KIR receptors as well as activating CD16, NKG2C, NKG2D, NKp46, TRAIL, FasL. In cancers, these cells use innate-like, TCR-independent mechanisms (traditionally ascribed to NK-cells) to target HLA-I-deficient tumor cells. Strikingly, HLA-I was significantly elevated on myeloblasts in LR-MDS compared to HR-MDS (p=0.0007)( Fig.1B), demonstrating NK-evasion strategies, but strongly reduced on dendritic cells, granulocytes, and CD4+T-cells, suggesting susceptible immune targets for destruction by NK-cells (and innate-like CD8+T-cells) (p< 0.0001) ( Fig.1B). BM-derived CD57+NK and CD8+T-cells all co-express very high levels of CD16 making them particularly ADCC reactive. Conclusion: We identified novel innate-like CD8+CD57+ T-cell and NK-cell populations that may play a role in LR-MDS disease pathogenesis. We predict that a strategy to target these cells could help restore hematopoiesis early in the disease state. This may also reduce pressure on myeloblasts to increase HLA-I, permitting improved immune surveillance to control the blast burden and prevention MDS/AML progression. Further experiments are ongoing to validate our observations.

Soluble PD-L1 Predicts Poor Outcome and Disease Progression in Patients with De Novo Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a spectrum of myeloid malignancies typified by regular genetic abnormalities, clonal hematopoiesis, dysfunctional myelopoiesis, inefficient blood cell production, peripheral-blood cytopenia, and an elevated risk for progressing to acute myeloid leukemia (AML). Mounting evidence suggests that the dysregulation of immune checkpoints (ICs) plays critical roles in immune evasion of MDS, which thus led to a series of trials using HMAs in combination with immune checkpoints inhibitors (ICIs). Of these, programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) pathway has been studied most extensively. Recently, forms of cell-free soluble PD-1/PD-L1 (sPD-1/sPD-L1) detection in plasm of patients with solid tumors has open a new paradigm for PD-1/PD-L1 investigations. However, within the context of MDS, the potential dysregulation and prognostic impact of sPD-1/sPD-L1 remains uncertain. This study was aimed to assess roles of sPD-1/sPD-L1 in newly diagnosed MDS patients. Method: Between July 2020 and March 2023, 130 MDS/sAML patients matched the inclusion criteria and 55 healthy individuals with no familial or personal history of autoimmune or cancer disorders were included. The criteria for patient inclusion included: (1) a diagnosis of MDS confirmed pathologically according to 2016/2022 World Health Organization (WHO) classification, or sAML patients with a verified prior MDS history; (2) absence of other conditions like autoimmune diseases or different types of tumors; (3) availability of complete clinical information and survival data. MDS patients were categorized by IPSS-R as: lower-risk (very low risk/ low risk; higher-risk (intermediate risk/high risk/very high risk. Follow-up was conducted until June 16th, 2023. The median follow-up was 14.32 months (range 3.2-34.63 months). Profile of sICs (sPD-L1, sPD-1, sCTLA-4, sGITR, sTIM-3, sOX40, sLAG-3, s4-1BB, sICOS) were tested by ELISA, cytokines (IFN-γ, IFN-α2, IL-2, IL-2α, IL-6, IL-7, IL-10, IL-15, IL-17, TNF-α) were detected using Luminex analysis, and the expression levels of PD-1 on T cells (CD3 +, CD4 +, CD8 +, DPT, DNT）were measured using flow cytometry (FCM). Result: In the plasma of 98 newly diagnosed MDS patients, sPD-L1 levels (median 66.80 pg/ml, range 25.00-219.5 pg/ml) were significantly elevated compared to 55 healthy blood donors (median 49.22 pg/ml, range 12.5-218.1 pg/ml). sPD-L1 level was higher in higher-risk IPSS-R groups ( P=0.0380). In addition, higher sPD-L1 levels also showed a correlation with lower hemoglobin concentration, implying a potential link between higher sPD-L1 levels and a more advanced MDS disease state. The AUC for sPD-1 was found to be 0.6333 [95% CI 0.5331-0.7334, P=0.0100] and for sPD-L1, it was 0.6785 [95% CI 0.5836-0.7733, P=0.0002]. The optimal cut-off values were established at 187.5 pg/mL for sPD-1 and 55.08 pg/mL for sPD-L1. Based on these cut-off values, patients with increased sPD-L1 experienced shorter OS ( P=0.0305). By using multivariate Cox model, high expression of sPD-L1 ( P=0.019, HR = 4.172, 95% CI: 1.265-13.755) and high IPSS-R scores ( P=0.030, HR = 2.898, 95% CI: 1.111-7.565) were found to be independent risk factors for newly diagnosed MDS patients. In addition, compared to healthy controls, the PD-1 + T cell subpopulations in MDS patients were significantly reduced, sPD-1 levels were positively correlated with proportion of CD4 + PD-1 + T cells, absolute count of CD4 + PD-1 + T cells and PD-1 + DPT cells, suggesting that CD4 + T cells and DPT cells may be potential sources of sPD-1 in plasma. Subsequently, we analysed the potential roles of cytokines and HMAs in sPD-1/sPD-L1 regulation. It was found that sPD-1 levels had a positive correlation with sPD-L1, IL-2Rα, IP-10, MIG, TNFα, GROα, and TRAIL. sPD-L1 was positively correlated with IL-2Rα, IP-10, MCP-1, MIG, MIP1α, and SCF (Figure 1 A). Notably. We found a reduced sPD-L1 levels in MDS patients who achieved remission after HMA treatment compared to treatment-naïve patients. ( P=0.0302) (Figure 1 B). Conclusions: In conclusion, we found sPD-L1 is an independent risk factors for overall survival in newly diagnosed MDS patients. The elevation of plasma sPD-L1 levels is associated with disease progression in MDS, and may be a potential target for MDS immunotherapy.

Variables fordifferential diagnosis offamilial Mediterranean fever: Multiple correspondence analysis ofa large Japanese cohort.

We investigated differential diagnoses that should be noted with familial Mediterranean fever (FMF) and useful variables for differentiation in a large Japanese cohort. Patients aged ≥13 years who were clinically suspected of having FMF by Livneh criteria were studied 1 year after MEFV genetic testing. Patients ultimately diagnosed with other diseases were studied, and the association among each disease, patient characteristics, and clinical variables were analysed using multiple correspondence analysis. In total, 504 patients were included in this study; 34 (6.7%) were diagnosed with a disease other than FMF. The most common diagnosis was Behçet's disease, followed by periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome, inflammatory bowel disease, myelodysplastic syndromes (MDS), and infectious diseases. Although none of the non-FMF patients had exon 10 variants, some responded to colchicine treatment. Multiple correspondence analysis suggested that atypical symptoms such as stomatitis were associated with Behçet's disease and PFAPA syndrome, whereas characteristic situations such as disease onset ≥40 years were associated with MDS and infectious diseases. Careful follow-ups and reanalysis of the diagnosis should be performed for patients with atypical findings and no exon 10 variants, even if their symptoms meet the clinical criteria for FMF.

Reduced murine double minute 2 and 4 protein, but not messenger RNA, expression is associated with more severe disease in myelodysplastic syndromes and acute myeloblastic leukaemia.

The human homologues of murine double minute 2 (MDM2) and 4 (MDM4) negatively regulate p53 tumour suppressor activity and are reported to be frequently overexpressed in human malignancies, prompting clinical trials with drugs that prevent interactions between MDM2/MDM4 and p53. Bone marrow samples from 111 patients with acute myeloblastic leukaemia, myelodysplastic syndrome or chronic myelomonocytic leukaemia were examined for protein (fluorescence-activated cell sorting) and messenger RNA (mRNA) expression (quantitative polymerase chain reaction) of MDM2, MDM4 and tumour protein p53 (TP53). Low protein expression of MDM2 and MDM4 was observed in immature cells from patients with excess of marrow blasts (>5%) compared with CD34+ /CD45low cells from healthy donors and patients without excess of marrow blasts (<5%). The mRNA levels were indistinguishable in all samples examined regardless of disease status or blast levels. Low MDM2 and MDM4 protein expression were correlated with poor survival. These data show a poor correlation between mRNA and protein expression levels, suggesting that quantitative flow cytometry analysis of protein expression levels should be used to predict and validate the efficacy of MDM2 and MDM4 inhibitors. These findings show that advanced disease is associated with reduced MDM2 and MDM4 protein expression and indicate that the utility of MDM2 and MDM4 inhibitors may have to be reconsidered in the treatment of advanced myeloid malignancies.

Rker-050, a Novel Activin Receptor Type II Ligand Trap, Rescued Anemia and Reduced Bone Loss in a Mouse Model of Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell diseases characterized by ineffective hematopoiesis, peripheral blood cytopenias and dysplasias resulting from molecular defects and dysregulated signaling that govern the regulation of hematopoietic stem and progenitor cells (HSPCs). Crosstalk between cells in the osteo-hematopoietic (O-H) niche are necessary for the maintenance and self-renewal of HSPCs. Additionally, MDS patients commonly present with osteopenia and osteoporosis further demonstrating the critical interplay between the O-H cells in the bone marrow (BM). Alterations in the interaction between bone precursors and HSPCs within the BM microenvironment have been linked to the pathophysiology of MDS. Transforming growth factor-beta (TGF-β) superfamily signaling is instrumental in regulating both normal hematopoiesis and bone homeostasis. KER-050 is a modified investigational activin receptor type II ligand trap designed to bind and inhibit select TGF-β superfamily ligands, including activin A, activin B, GDF8 and GDF11, to promote hematopoiesis and bone growth. Preclinical studies have demonstrated that treatment with a research form of KER-050 (RKER-050) increased bone mass and multi-lineage hematopoiesis in healthy mice. To further investigate the effect of RKER-050 on hematopoietic and bone systems within the MDS disease setting, the NUP98/HOX13 MDS mouse model was utilized. Five-month-old male MDS mice were treated with vehicle (VEH, n=10), or RKER-050 (050, 7.5mg/kg, n=9) once weekly for 6 weeks along with a wildtype vehicle (WT, n=7) control group. To assess changes in hematological parameters, CBCs were measured, bone marrow (BM) and splenocytes were characterized via flow cytometry. To investigate alterations of bone microarchitecture, femurs were assessed using microCT imaging. MDS-VEH mice showed a reduction in red blood cells (RBC; 30%), hemoglobin (HGB; 19%), hematocrit (HCT; 13%) and reticulocytes (Retic; 16%) compared to WT; all hallmarks of human MDS. Relative to MDS-VEH, MDS-050 showed increased RBC, HGB, HCT and Retic by 25%, 13%, 10% and 39%, respectively. The erythroid Ter119 positive (Ter119+) cell population was slightly reduced in BM and spleen of MDS-VEH mice compared to WT. The BM Ter119+ cells were unchanged in the MDS-050 cohort relative to MDS-VEH; however, the spleen Ter119+ cells were increased similar to the levels in WT mice. Specific erythroid precursor populations were increased in BM and spleen of MDS-VEH mice versus WT, consistent with the ineffective erythropoiesis in this model. Interestingly, erythroid precursor populations were similar between MDS-VEH and MDS-050, yet MDS-050 exhibited recovered RBC production, suggesting an increase in maturation of erythroid precursors with RKER-050 treatment. Given the importance of the O-H niche in regulating hematopoiesis along with osteoporosis and osteopenia being comorbidities of MDS, bone microarchitecture was also analyzed. In the distal femoral trabecular bone, relative to WT mice, MDS-VEH mice had significant increases in trabecular (Tb) separation (33%) and decreases in Tb number (28%), changes that are consistent with reduced bone strength. Bone volume (34%) and bone volume fraction (26%) decreased but were not statistically significant. MDS-050 treated mice had a significant increase in bone volume (71%), higher bone volume fraction (88%), increased Tb number (44%) and decreased Tb spacing (20%) compared to the MDS-VEH, supporting RKER-050's role in improving bone mass. Taken together, these results suggest that RKER-050 can mitigate anemia and bone loss in an MDS mouse model, potentially by rebalancing hematopoiesis and bone turnover. In particular, the ability of RKER-050 to improve bone microarchitecture within the trabecular region, where the O-H niche resides, could be an important step in reestablishing healthy blood cell production. Furthermore, exploratory PD data from an ongoing phase 2 clinical trial demonstrate improved blood parameters with KER-050 treatment, including platelet and RBC in MDS patients (Tan et al. 2022 EHA #P776), and changes in bone biomarkers are currently being evaluated in the study. KER-050, thus, represents a potentially promising approach for patients with MDS and other hematological diseases, including myelofibrosis, where ineffective hematopoiesis and defects in bone homeostasis occur.

S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE

Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model (Zrsr2m/mTet2−/−) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated. Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS. Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin-Sca-1+c-kit+ (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR. Results: RNA-seq analysis of Zrsr2m/mTet2−/−cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2m/m and 1203 in Tet2−/−. From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2m/mTet2−/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2m/mTet2−/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2m/mTet2−/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study. Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2m/mTet2−/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2m/mTet2−/− mice.

Miyelodisplastik sendrom hastalığında P53 polimorfizmlerinin önemi

Amaç: Miyelodisplastik sendrom (MDS) akut miyeloid lösemiye dönüşüm riski yüksek olan, artmış apoptozis ve azalmış hematopoez oranlarıyla karakterize klonal bir hastalıktır. MDS patogenezi tam olarak açıklanabilmiş değildir. Olguların ~%50' si anormal karyotiptedir ve bu oran ikincil MDS' de %80 civarındadır. P53 proteini kök hücre homeostazının önemli düzenleyicisidir ve hücre döngüsünün düzenlenmesi, apoptotik ile enflamatuar yanıt gibi bir dizi hücresel olayda yer alır. Genomik bütünlüğün korunmasında da önemli görevleri bulunan TP53 geni kanserlerde sıklıkla mutasyona uğramaktadır; ancak, mutasyonlarının yanında bazı gen polimorfizmlerinin de kanserle ilişkili oldukları bilinmektedir. Çalışmadaki amacımız, dört yaygın p53 tek nükleotid polimorfizminin MDS’ deki yaygınlıklarını ve hastalık gelişimi üzerine etkilerini belirlemektir. Bu amaçla, Ege Üniversitesi Tıp Fakültesi Dahiliye Anabilim Dalı Hematoloji Bilim Dalı' nda MDS tanısı ile takip edilen ya da yeni tanı almış 100 olgu çalışmaya dahil edildi. Gereç ve Yöntem: MDS’li olguların periferik kan lökositlerinden izole edilen DNA’lar gerçek-zamanlı PCR yöntemiyle çalışılarak, p53 polimorfizmleri (rs35163653, rs35993958, rs1800371, rs1042522) uygun probların kullanımıyla ve erime eğrisi analizleriyle belirlendi. Bulgular: İncelenen dört yaygın p53 polimorfizmin arasında özellikle rs1042522 polimorfizmindeki atasal olmayan G alelinin MDS’li olgularda artmış olduğu gözlenmiştir (C:%30.3; G:%69.7). Fonksiyonel olduğu, yani sentez edilen proteinin fonksiyonunu etkilediği bilinen bu polimorfizmde 417. pozisyonundaki C nukleotidinin G’ye transisyonu (C&amp;gt;G), proteinin 72. pozisyonundaki prolin amino asidinin arjinine (P72R) kodlanmasına yol açmaktadır. Sonuç: Çalışmamız, MDS hastalık grubunda rs35163653, rs35993958, rs1800371 ve rs1042522 p53 polimorfizmlerinin araştırıldığı ilk çalışmadır. Bunlardan, rs1042522 polimorfizminin kansere yatkınlık ve duyarlılıkla ilişkili olduğu yapılan diğer bazı çalışmalarla gösterilmiş olması nedeniyle, MDS hastalığı için de yüksek risk oluşturabileceği düşünülmektedir. Sonuç olarak, MDS hastalığı için gerçekleştirilen bu çalışmanın daha geniş bir olgu grubuyla tekrarlanmasıyla rs1042522 polimorfizmi ileride MDS teşhisinde belirteç olarak kullanılabilecektir.

Machine learning application to find patients with lower-risk myelodysplastic syndrome from real-world data.

1555 Background: It is a challenge to identify patients with myelodysplastic syndrome (MDS) using structured data from electronic health records (EHRs). Current claims-based algorithms incorporating diagnosis codes, clinical labs, and procedures have not been validated against an expert reference standard. A machine learning-based approach was investigated to identify erythropoietin-stimulating agent (ESA)-treated, lower-risk (LR)-MDS patients from structured EHR data. Methods: A sample of 1,549 patients from the Syapse Learning Health Network (SLHN) was identified as potential ESA-treated LR-MDS patients by a team of clinicians and epidemiologists based on diagnosis and medication data from multiple health systems’ EHRs and cancer registries. Of these, 404 (25%) were confirmed as ESA-treated LR-MDS patients through a review of patient records by certified cancer registrars (CTRs). The sample was divided into training and validation sets at a ratio of 80/20, stratified by the outcome. Age, sex, diagnosis codes corresponding to MDS and chronic kidney disease, medication (ESA, luspatercept, lenalidomide), clinical lab tests (hemoglobin, absolute neutrophils, platelet, blast percentage), and evidence of bone marrow biopsy were included as the predictive variables for the models. Gradient boosting machines with a nested cross-validation scheme were adopted to build the optimal model on the training set. Model acceptance was evaluated based on precision and recall on the validation set. The optimal model was then applied to the remaining unscreened SLHN patient population. Results: The optimal model identified an additional cohort of 157 patients based on the predicted likelihood. Among these, 69 (44%) were CTR-confirmed ESA-treated LR-MDS patients, all of whom were previously missed by the initial expert-determined selection criteria, as shown in the table. Conclusions: The application of machine learning methods increased the rate of ESA-treated MDS patient identification even after the expertly-determined population was depleted. This suggests the application of machine learning models using EHR data may improve the efficiency of MDS patient identification and screening efforts for research, quality improvement, and clinical care. [Table: see text]

389 - Elevated Pre-Transplant Ferritin Is Associated with Development of Liver Graft-Vs-Host Disease in Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation

389 - Elevated Pre-Transplant Ferritin Is Associated with Development of Liver Graft-Vs-Host Disease in Myelodysplastic Syndromes Following Allogeneic Stem Cell Transplantation

How low risk are low risk myelodysplastic syndromes?

ABSTRACT Introduction Risk stratification is crucial to the appropriate management of many diseases, but in patients with myelodysplastic syndromes (MDS), for whom expected survival can vary greatly, accurate disease prognostication is especially important. This is further supported by a relative lack of therapies in MDS, and thus we must prognosticate carefully and accurately. Currently, patients with MDS are often grouped into higher-risk (HR) versus lower-risk (LR) disease using clinical prognostic scoring systems, but these systems have limitations. Areas covered The authors reviewed the literature on diagnostics, prognostics, therapeutics and outcomes in MDS. Factors such as disease etiology, specific clinical characteristics, or molecular genetic information not captured in the international prognostic scoring system revised IPSS-R can alter risk stratification, and identify a subset of LR-MDS patients who actually behave more like HR-MDS. Expert opinion This review will describe the current identification and management of patients with LR MDS disease whose condition is likely to behave in a less favorable manner than predicted by the IPSS-R. The authors comment on clinical and molecular features which are believe to upstage a patient from lower to higher risk disease.

A Focus on Phenotype and Genotype: Racial /Ethnic Disparities in Myelodysplastic Syndromes

Background: There is a paucity of research on racial and ethnic disparities in myelodysplastic syndromes (MDS). Research focused on racial and ethnic disparities for MDS is essential to improve knowledge and understanding to deliver racial and ethnic sensitive care. There are limited studies that delineate the incidence of cytogenetic and molecular features of MDS by race and ethnicity (Yan, et al. 2021, Ramadan, et al.,2016). The aim of this abstract is to report the difference in clinical phenotype, genotype and outcomes of White, Black and Hispanics from a large MDS data base.Methods: Adult patients were identified through the Moffitt Cancer Center MDS data base and divided into racial/ethnic categories. Fisher exact test and chi-square tests were used to compare categorical variables. Univariate survival analysis was estimated using the Kaplan-Meier method.Results: From analysis of 4414 patients with MDS, 4131 (93%) were White, 116 (3%) Black and 167 (4%) Hispanic. Table-1 summarizes baseline characteristics. There were more Black and Hispanic women diagnosed with MDS (p < .001). Black and Hispanic patients were younger at diagnosis (p<0.01), Hispanic patients had a lower platelet count at baseline (p=0.02). There were no racial differences in WHO 2016 MDS classification or disease risk according to R-IPSS. Black MDS patients had less complex karyotype (p<0.05) while abnormalities in chromosome 5 were more common in White patients 785(19.6%, p<0.05).Table-2 summarizes somatic mutations (SM) landscape among the different racial groups, IDH2 SM (p=0. 01) and NPM-1 SM (p=.004) were more common in Black MDS patients. MPL (p < 0.005) was observed more frequently among Hispanic patients.There was no difference in response to any modality of treatment based on race. Hispanic patients were more likely to undergo allogeneic hematopoietic stem cell transplant 50 (29.9%, p<0.01). Clinical trial participation rates among the groups were similar.The median overall survival (mOS) was 35 months (mo), 31 mo, and 52.5 mo for White, Black and Hispanic patients respectively, p= .01 shown in Figure 1. For very low/low R-IPSS the mOS was 67.6, 52 and 93 mo respectively, p= .028, Figure 2a. For intermediate risk R-IPSS the mOS was 33, 30 and 51 mo respectively, p=0.2, and for high/very high-risk R-IPSS the mOS was 16.8, 21.7, and 25 mo respectively, p=.025. See Figures 2b and 2c. There was no difference in rate of AML transformationConclusions: This large retrospective study revealed racial/ethnic differences in clinical and molecular features of MDS. Hispanic patients had better overall survival. Continued research in this area is recommended to better understand the phenotype and genotype of patients from diverse ethnic/racial backgrounds.Acknowledgement of Funding: NINR Grant # 1K23NR018488-01A [Display omitted] DisclosuresTinsley-Vance: Novartis: Consultancy; Celgene/BMS: Consultancy, Speakers Bureau; Taiho: Consultancy; Fresenius Kabi: Consultancy; Incyte: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Abbvie: Honoraria; Jazz: Consultancy, Speakers Bureau. Padron: Stemline: Honoraria; Blueprint: Honoraria; Kura: Research Funding; Taiho: Honoraria; Incyte: Research Funding; BMS: Research Funding. Sweet: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Lancet: Celgene/BMS: Consultancy; BerGenBio: Consultancy; AbbVie: Consultancy; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Millenium Pharma/Takeda: Consultancy; Astellas: Consultancy; Agios: Consultancy; Jazz: Consultancy. Kuykendall: PharmaEssentia: Honoraria; Novartis: Honoraria, Speakers Bureau; Incyte: Consultancy; Prelude: Research Funding; CTI Biopharma: Honoraria; Celgene/BMS: Honoraria, Speakers Bureau; BluePrint Medicines: Honoraria, Speakers Bureau; Abbvie: Honoraria; Protagonist: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sallman: Incyte: Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding; Syndax: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Intellia: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees. Komrokji: AbbVie: Consultancy; Geron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Acceleron: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Speakers Bureau.

Antibody Response to Vaccination with BNT162b2, mRNA-1273, and ChADOx1 in Patients with Myeloid and Lymphoid Neoplasms

Background:In general, patients with hematological diseases are predisposed to develop infections. Severe COVID-19 infection associated with high mortality is more likely in these patient cohorts compared to the general population. Due to immune defects related to the primary disease and/or to immunosuppressive treatment regimes, vaccination efficacy may be reduced in patients with hematological diseases. So far, data on this area are limited.Aim:To evaluate vaccination-related antibody response to BNT162b2, mRNA-1273, and ChADOx1 in patients with hematological disorders.Patients and methods:In this interim analysis of a prospective, observational single-center study, we report antibody levels at least 2 weeks after COVID-19 vaccination. A FDA/CE approved electrochemiluminescent assay (ECLIA) (Elecsys®, Roche, Mannheim, Germany) was used to quantify antibodies, pan Ig (including IgG) against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The assay has a measurement range of 0.4 to 250 U/mL, with a concentration ≥0.8 U/ml considered as positive. Data were analyzed for patients without detection of anti-N (nucleocapsid) SARS-CoV-2 antibody (i.e., without having passed SARS-CoV-2 infection). All tests were performed according to the manufacturer's instructions in an accredited laboratory at the University Hospital Mannheim.Results:Between February 2021 and July 2021, a total of 175 patients with hematological diseases were included in this study. The median age was 66 years (range 21-90 years), and 81 (46.3%) were female. The antibody levels were measured at least 14 days (median, 58 days) after the 2 nd vaccination. The patients were vaccinated with BNT162b2 (BioNTech, n=134), mRNA-1273 (Moderna, n=19), ChADOx1 (AstraZeneca, n=12), or got the first vaccination with BNT162b2 and the second with ChADOx1 (n=10). Overall, 145/175 (82.9%) were diagnosed with a malignant hematological disease (myeloid neoplasms, n=108; lymphoid neoplasms, n=37) and 30/175 with a non-malignant hematological disease (autoimmune disease, n=24; benign, n=6). 124 patients (70.1%) were on active therapy, and 51 patients (29.1%) were previously treated or treatment naïve. Correlation to specific therapies is ongoing and will be presented.In general, vaccination-related antibody response was positive (≥0.8 U/mL) in 148/175 (84.6%) patients with a median level of 208.6 U/mL (range 0.8-250.00) and negative (<0.8 U/mL) in 27/175 (15.4%) patients. The distribution of the negative cohort regarding the disease subgroups were as followed: myeloid neoplasms 7/27 (25.9%), lymphoid neoplasms 16/27 (59.3%), non-malignant hematological disease 4/27 (14.8%). Within the negative cohort, 21/27 (77.8%) were treated on active therapy, 6/27 (22.2%) were previously treated or treatment naïve.In myeloid neoplasms, patients with classical myeloproliferative neoplasm (MPN) had the highest negative result for antibodies with 4/7 (57.1%) followed by myelodysplastic syndrome (MDS) 2/7 (28.6%). Interestingly, all patients with chronic myeloid leukemia (CML) had a measurable immune response.In lymphoid neoplasms, patients with low-grade non-hodgkin lymphoma (NHL) (predominately chronic lymphocytic leukemia, CLL) had the highest negative antibody result 13/16 (81.3%) followed by high-grade NHL 4/8 (50%; predominately diffuse large b-cell lymphoma, DLBCL).In non-malignant hematological diseases, only patients with autoimmune diseases had a negative result.Conclusion:A remarkable group of patients with hematological disease were measured with no or low immune response after 2 nd COVID-vaccination, especially those with low-grade NHL, MDS and autoimmune disease. It seems that the percentage of patients with MPN and low response is less critical. No problems appeared in CML patients.Further explorations are needed with focus on potential risk of COVID infections despite full vaccination: The potential of 3 rd booster vaccination should be explored within clinical trials. DisclosuresReiter: AOP Orphan Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel support; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Blueprint Medicines: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Abbvie: Membership on an entity's Board of Directors or advisory committees; Deciphera: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses. Kreil: Novartis: Research Funding. Hofmann: Amgen: Honoraria; BMS: Honoraria; Novartis: Honoraria. Jawhar: Takeda: Honoraria, Other: Travel support; Blueprint Medicines: Honoraria; Stemline: Consultancy, Honoraria; Celgene: Other: Travel support; Novartis: Consultancy, Honoraria, Other: Travel support, Speakers Bureau. Saussele: Roche: Honoraria; Pfizer: Honoraria; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Mesenchymal Stromal Cells (MSCs) from Myelodysplastic Syndromes (MDS) Are Not Clonally Mutated I n Vivo

IntroductionThere is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures.Materials and MethodsWe performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients.ResultsIn the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases.To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo.DiscussionTogether with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. DisclosuresSchmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

The Impact of BMI on Next Generation Sequencing Panels for Patients with Myelodysplastic Syndromes

Introduction:The usage of cytogenetics and next generation sequencing (NGS) has become more prominent in recent years for risk stratification of patients (pts) with myelodysplastic syndromes (MDS). Management of these patients is a multi-faceted endeavor which requires understanding of how MDS genetics interacts with a patient's lifestyle and co-morbidities. Obesity rates in the United States continue to rise; the current rate in Virginia is 30% (Centers for Disease Control and Prevention, CDC). This study analyzed whether body mass index (BMI) at diagnosis was correlated with the number or type of MDS mutations seen on initial NGS and whether obesity influenced outcomes stratified by individual mutations.Methods:Adult pts diagnosed with MDS at the University of Virginia from August 2012 to December 2019 who had cytogenetics and NGS panels done prior to initiation of MDS therapy were included. Any patient without the aforementioned initial diagnostic information was excluded. MDS disease characteristics, treatment type, comorbidities, and patient demographics were assessed including weight, height, and BMI (categories defined per CDC) at time of diagnosis. The primary aim of this analysis was to evaluate the association of BMI with the number and type of mutations on NGS panel at diagnosis. Wilcoxon-Mann-Whitney tests were used to explore the difference in BMI by presence of 13 mutations of interest: ASXL1, DNMT3A, FLT3, IDH1, IDH2, NPM1, NRAS, SETBP1, SF3B1, SRSF2, TET2, TP53, and U2AF1. The Spearman correlation coefficient was used to assess the relationship between BMI and total number of mutations. Kaplan-Meier estimates, log rank tests, and Cox proportional hazard models were used for time-to-event analyses. All analyses were performed using SAS 9.2 (SAS Institute, Cary, NC).Results:143 MDS pts met inclusion criteria with a median age at diagnosis of 69 (range 39-87) and 60.7% (n=88) were male. The median weight was 81.3 kg and the median BMI at diagnosis was 27.7 kg (range 16.04-52.66). There were 29 pts in the normal BMI class (20%), 4 pts in the underweight class (3%), 38 pts in the obese class (26%), and 72 pts in the overweight class (50%). By IPPS-R risk scoring system, 64 pts were low or very low risk (44%), 39 pts intermediate risk (27%), and 41 pts high or very high risk (29%).There was no difference in the median number of mutations across BMI groups with a spearman correlation coefficient of 0.05 (Figure 1). However, the median BMI was significantly lower in patients with the NPM1 mutation versus those without (p=0.04), diagnosed prior to the WHO re-classification of these patients to acute myeloid leukemia (AML). Presence of the NPM1 mutation in MDS is also associated with worse PFS and an increased hazard ratio of 9.4 (p=0.0003), likely behaving similarly to AML. In comparison, presence of the SF3B1 mutation was found to be protective wherein patients who lacked the mutation had an increased hazard ratio of 6.0 (p=0.0133). BMI alone did not predict for progression free survival (PFS) or overall survival (OS); this was also true regardless of patient residency based on zip code. However, obese patients exhibiting the NPM1 and SF3B1 had better PFS (Figure 2). Moreover, the landmark analysis indicates among patients who are alive and free of progression at 20 months after MDS diagnosis, overweight patients exhibit better PFS than normal/underweight or obese patients (p=0.0192) (Figure 3).Conclusion:While BMI in pts with MDS does not appear to predict the gross number of NGS mutations, NPM1 mutations occurred more often in pts with low BMIs. Overweight pts who did not progress by 20 months have a better PFS. Prospective and larger retrospective studies are needed to expand on the findings that BMI may have an unexplored potential in stratifying pts with newly diagnosed MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gender Disparities in Myelodysplastic Syndromes: Phenotype, Genotype, and Outcomes

Gender Disparities in Myelodysplastic Syndromes: Phenotype, Genotype, and Outcomes

Looking beyond VEXAS: Coexistence of undifferentiated systemic autoinflammatory disease and myelodysplastic syndrome

It has been established that individuals with myelodysplastic syndromes (MDS) have a higher frequency of systemic inflammatory disorders. On the other hand, patients with autoimmune diseases are at increased risk of MDS development. Both diseases can be associated with various genetic lesions and share diverse pathogenetic mechanisms. Recently identified VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, associated with somatic mutations in UBA1, encompasses a range of inflammatory conditions involving multiple organs along with hematological pathologies, including MDS, as well as characteristic bone marrow vacuolization of myeloid and erythroid precursors. This novel syndrome drove further attention to complex associations between MDS and adult-onset inflammatory conditions. The present narrative literature review discusses the clinical presentation, pathophysiology, management of concurrent MDS and systemic inflammatory diseases in parallel to the clinical picture of VEXAS syndrome.

U2AF1 mutation promotes tumorigenicity through facilitating autophagy flux mediated by FOXO3a activation in myelodysplastic syndromes

Mutations in the U2 small nuclear RNA auxiliary factor 1 (U2AF1) gene are the common feature of a major subset in myelodysplastic syndromes (MDS). However, the genetic landscape and molecular pathogenesis of oncogenic U2AF1S34F mutation in MDS are not totally understood. We performed comprehensive analysis for prognostic significance of U2AF1 mutations in acute myeloid leukemia (AML) cohort based on The Cancer Genome Atlas (TCGA) database. Functional analysis of U2AF1S34F mutation was performed in vitro. Differentially expressed genes (DEGs) and significantly enriched pathways were identified by RNA sequencing. The forkhead box protein O3a (FOXO3a) was investigated to mediate the function of U2AF1S34F mutation in cell models using lentivirus. Chromatin immunoprecipitation, immunoblotting analyses, and immunofluorescence assays were also conducted. U2AF1 mutations were associated with poor prognosis in MDS and AML samples, which significantly inhibited cell proliferation and induced cellular apoptosis in cell models. Our data identified that U2AF1-mutant cell lines undergo FOXO3a-dependent apoptosis and NLRP3 inflammasome activation, which induces pyroptotic cell death. Particularly, an increase in the level of FOXO3a promoted the progression of MDS in association with restored autophagy program leading to NLRP3 inflammasome activation in response to U2AF1S34F mutation. Based on the result that U2AF1S34F mutation promoted the transcriptional activity of Bim through upregulating FOXO3a with transactivation of cell cycle regulators p21Cip1 and p27Kip1, FOXO3a, a potentially cancer-associated transcription factor, was identified as the key molecule on which these pathways converge. Overall, our studies provide new insights that U2AF1S34F mutation functions the crucial roles in mediating MDS disease progression via FOXO3a activation, and demonstrate novel targets of U2AF1 mutations to the pathogenesis of MDS.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3′ splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12–13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+ Tet2−/− mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+ Tet2−/− mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Activation Levels of b-Catenin Signaling in Osteoblasts Define a Threshold for the Development of Myeloid Neoplasia of Variable Severity

A growing body of evidence indicates that osteoblasts have a direct pathogenetic role in the development of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. Models of osteoblast-induced MDS or AML (OIAML) include b-catenin activation in osteoblasts inducing AML in mice and seen in more than 30% of MDS and AML patients; deletion of Dicer1 in osteoblast progenitors leading to MDS in mice; Noonan syndrome mutation (PTPN11) in stromal cells driving MPN in mice; and Schwachman-Diamond syndrome mutation in stromal cells driving MDS in mice and predicting AML progression in patients. To further understand the mechanism of OIAML we examined whether a defined threshold of AML-inducing signal by osteoblasts is required for disease development. For this purpose we constitutively activated b-catenin in osteoblasts using three different lines of 2.3Col1 a 1-Cre transgenic mice characterized by different recombination efficiencies in bone. Line 1, in which recombination efficiency was 80%. Line 2 was the same original line in which after several inter-breedings we detected a reduction in recombination efficiency to 20%. Line3, which has 75% recombination efficiency. Activation of b-catenin in osteoblasts using Line 1, led to the development of MDS which rapidly transformed to full-blown AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. In contrast, activation of b-catenin in osteoblasts using Line 2 did not lead to AML development. Mice remained healthy and survived until at least 10 months of age, the entire time they were observed. Activation of b-catenin in osteoblasts using Line 3 led to anemia, with the appearance of myeloid blasts in the blood, marrow and spleen and segmented neutrophils in blood. In addition to what was observed with Line 1, Pelger Huet neutrophils and Howell-Jolly bodies were detected in the blood. The latter are RBCs with inclusions of nuclear chromatin remnants (damaged chromosome fragments) indicating genomic instability and a dysfunctional spleen, unable to clear them from the circulation, due to dense infiltration with blasts and megakaryocytes. A spectrum of atypical immature monocytes with abundant pale blue agranular cytoplasm with vacuoles were observed in the blood. Taken together these features resemble characteristics of human MDS/CMML. Mice die between 4 and 14 weeks of age, when they undergo “blast crisis” indicating transformation to AML. To examine whether and how these observations relate to the human disease, we compared the levels of b-catenin activation in osteoblasts (calculated as a function % osteoblasts (Lin-/CD34-/RUNX2+ cells) expressing activated b-catenin) with the different MDS diseases subtypes as those are defined by the 2008 World Health Organization (WHO) classification system. We found that increased levels of b-catenin activation in osteoblasts correlate with the severity of the MDS type and with conversion to AML. Collectively, these observations suggest that a minimal activation level of a key leukemogenic signal from the stroma, is required to initiate cancer development, and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML and the transformation of MDS to AML. DisclosuresRaza:Onconova Therapeutics: Research Funding, Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Janssen R&D: Research Funding; Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding.