Primary Myelodysplastic Syndromes Research Articles

Bone Marrow Fibrosis at Diagnosis and during the Course of Disease Is Associated with TP53 Mutations and Adverse Prognosis in Primary Myelodysplastic Syndrome.

Simple SummaryTo understand the role of bone marrow fibrosis and its molecular changes in myelodysplastic syndrome, we retrospectively analyzed data from 814 patients. Older age, lower hemoglobin, unfavorable karyotype and higher BM blast were more often observed in patients with moderate/severe fibrosis. Cases with bone marrow fibrosis had reduced overall survival. TP53, U2AF1 and KMT2D mutations were more frequent in patients with moderate/severe fibrosis. In addition, 15.1% of patients progressed to moderate/severe fibrosis during the follow-up interval. The clinical features, mutation landscape and prognosis of patients with progressed fibrosis were similar to those patients with moderate/severe fibrosis at diagnosis. We concluded that bone marrow fibrosis was associated with reduced overall survival in primary MDS and correlated with TP53 mutations both at the time of initial diagnosis and during the course of the disease.The prognostic significance of bone marrow fibrosis (MF) grade in patients with myelodysplastic syndrome (MDS) is still debated and the molecular changes remain unclear. In our large cohort, a normal reticulum was found in 211 (25.9%) patients, whereas MF1, MF2 and MF3 were detected in 478 (58.7%), 90 (11.1%) and 35 (4.3%) patients at initial diagnosis, respectively. Patients with MF often correlated with some poor prognostic characteristics, including older age, anemia, unfavorable karyotype, higher BM blast and a higher IPSS-R category. For the entire cohort, the median OS was not reached, 30, 16 and 15 months for patients with MF 0, 1, 2 and 3, respectively. After adjusting for IPSS-R, the hazard ratio for mortality was 1.56 (95% CI, 1.18–2.06) for patients with MF1, 2.29 (95% CI, 1.61–3.27) for patients with MF2 and 2.75 (95% CI, 1.69–4.49) for patients with MF3 compared with those with MF0. The mutational landscape of 370 patients showed that TP53, U2AF1 and KMT2D mutations were more frequent in patients with MF2-3. In addition, of the 408 patients with MF0-1, 62 patients (15.1%) progressed to MF2-3 during the follow-up interval. The clinical features, mutation landscape and prognosis of patients with progressed fibrosis were similar to those of patients with MF2-3 at diagnosis. We concluded that BM fibrosis (MF1, 2 and 3) was an adverse prognosis feature in primary MDS and correlated with TP53 mutations both at the time of initial diagnosis and during the course of the disease. Therefore, BM fibrosis should be included in the revised prognostic scoring system and carefully considered in treatment selection.

Characterization of myelodysplastic syndromes hematopoietic stem and progenitor cells using mass cytometry.

Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. Bone marrow samples from healthy donors (n =10), low risk (n =12) and high risk (n =13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n =20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

Patient-specific MDS-RS iPSCs define the mis-spliced transcript repertoire and chromatin landscape of SF3B1-mutant HSPCs

AbstractSF3B1K700E is the most frequent mutation in myelodysplastic syndrome (MDS), but the mechanisms by which it drives MDS pathogenesis remain unclear. We derived a panel of 18 genetically matched SF3B1K700E- and SF3B1WT-induced pluripotent stem cell (iPSC) lines from patients with MDS with ring sideroblasts (MDS-RS) harboring isolated SF3B1K700E mutations and performed RNA and ATAC sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from them. We developed a novel computational framework integrating splicing with transcript usage and gene expression analyses and derived a SF3B1K700E splicing signature consisting of 59 splicing events linked to 34 genes, which associates with the SF3B1 mutational status of primary MDS patient cells. The chromatin landscape of SF3B1K700E HSPCs showed increased priming toward the megakaryocyte- erythroid lineage. Transcription factor motifs enriched in chromatin regions more accessible in SF3B1K700E cells included, unexpectedly, motifs of the TEA domain (TEAD) transcription factor family. TEAD expression and transcriptional activity were upregulated in SF3B1-mutant iPSC-HSPCs, in support of a Hippo pathway-independent role of TEAD as a potential novel transcriptional regulator of SF3B1K700E cells. This study provides a comprehensive characterization of the transcriptional and chromatin landscape of SF3B1K700E HSPCs and nominates novel mis-spliced genes and transcriptional programs with putative roles in MDS-RS disease biology.

Cytogenetic characteristics in Vietnamese patients diagnosed with primary myelodysplastic syndromes

BackgroundThe karyotype is the important factor for the diagnosis and prognosis of primary myelodysplastic syndromes (MDS). Some previous studies have suggested that the incidence of chromosomal variations in MDS was related to race. We analyze the chromosomal characteristics in Vietnamese patients with MDS to find differences compared to other races and the association with subtypes by WHO classification. MethodsSixty patients with new primary MDS diagnoses underwent cytogenetic analysis and FISH for del(5q). ResultsTwenty-five patients (41.67%) had an abnormal karyotype at the time of diagnosis, in which 18 patients with a complex karyotype (≥3 chromosomal abnormality) represented the highest percentage (30%). The most frequent chromosomal abnormalities were +8 found in 10/60 patients (16.7%), del (5q) in 9/60 patients (15%), -18 in 5/60 patients (8.3%), only one patient had isolated del(5q) with 1.67%. Patients with abnormal karyotype had higher odds of being MDS-EB (MDS with excess blast) compared to those with normal karyotype (OR = 3.407, 95% CI = 1.164 – 9.976). Patients with complex karyotypes had a higher probability of having MDS-EB compared to those without complex karyotype (OR = 3.25, 95% CI = 1.018 – 10.379). ConclusionsThe complex karyotype was the most frequent chromosomal abnormality. Patients with an abnormal or complex karyotype had a higher probability of having MDS with excess blast. The isolated del (5q) ratio is very low compared to Europe and North Africa, but similar to China and Japan as they are the same countries in East Asia.

Prognostic impact of micromegakaryocytes in primary myelodysplastic syndromes

Micromegakaryocytes (microMKs) are considered a myelodysplastic feature of myeloid neoplasms in adults, with an adverse prognosis connotation. However, this notion in MDS has not been well proved. In our cohort of 287 MDS, patients with microMKs showed lower overall survival (OS) (HR, 2.12; 95% CI, 1.47–3.06; p = 0.000036) and higher risk of acute myeloid leukemia (AML) evolution (HR, 4.8; 95% CI, 2.9–11.01; p = 0.00021). Results were validated with an independent cohort. In multivariate analysis, the presence of microMKs maintained its independent association with OS (HR, 1.54, 95% CI, 1.13–2.1, p = 0.0059) and AML transformation (HR, 2.28, 95% CI, 1.2–4.4, p = 0.014). Moreover, by adding 1 point to the IPSS-R score in patients with microMKs, we improved the IPSS-R accuracy. Interestingly, adding that 1-point, 29% of intermediate IPSS-R risk group patients were upgraded to the high-risk group. In summary, we confirmed that the presence of microMKs implies worse outcomes in MDS and suggested a modification improving IPSS-R.

Humanized three-dimensional scaffold xenotransplantation models for myelodysplastic syndromes

Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34+ hematopoietic stem and progenitor cells (HSPCs). Our study included13 primary MDS patient samples transplanted in parallel according to these five different conditions. Engraftment of MDS samples was assessed by flow cytometry, immunohistological staining, and molecular validation. We determined that three-dimensional ossicle-based methods achieved higher relative rates of engraftment and enabled long-term retrievability of patient-derived MSCs from implanted ossicles. In summary, HSPCs and MSCs derived from MDS BM, which did not significantly engraft in NSG mice after intrafemoral injection, were able to colonize humanized scaffold models. Therefore, these models are promising new xenotransplantation techniques for addressing preclinical and functional questions of the interaction between hematopoiesis and the BM niche in MDS.

Epidemiologic Evaluation of Clinical Outcomes in Ethnic Minorities with Myelodysplastic Syndromes

Introduction:Myelodysplastic syndromes (MDS) are a spectrum of disorders with variable degrees of cytopenias, morphological dysplasia and risk of progression to acute myeloid leukemia (AML) (Hellström-Lindberg, Tobiasson, Greenberg, Haematologica 2020). The Surveillance, Epidemiology, and End Results (SEER) cancer registry reports incidence and survival data for MDS, and since 2001 also for clinical subgroups. Since socioeconomic status (SES), including economic and educational disadvantage (EED), may impact overall survival (OS), the extent to which EED and race/ethnicity impact OS of MDS require improved analysis. Understanding the impact of EED and race/ethnicity on OS could help quantify health disparities and provide insights that inform treatment decisions. This study analyzed the impact of race/ethnicity and EED on OS in Non-Hispanic White (NHW), Non-Hispanic Black (NHB) and Hispanic patients with MDS by clinical risk category.Methods:A retrospective review was performed to identify all adult patients (aged ≥ 50 y) diagnosed with primary MDS in the SEER US population (~30% of US population) between January 1, 2001 and December 31, 2016. Following the World Health Organization morphologic classification, primary MDS clinical risk categories were higher risk (RAEB-1 & -2), lower risk (other defined categories), and MDS not otherwise specified (NOS). Factor analysis was used for 23 county-level SES indicators obtained from the American Community Survey via SEER for features of EED (concordance was noted between economic and educational factors). Univariable and multivariable Cox proportional hazards models were used to determine the association between prognostic factors, including age, sex, clinical risk category, race/ethnicity and EED by tertile with MDS OS. Survival was documented from the time of diagnosis to death or the last follow-up day available in the SEER registry. All analyses were two-tailed with the level of significance of ≤0.05. The statistical software used was SAS 9.3 (SAS Institute, Cary, NC) for data analyses.Results:There were 52,739 patients with primary MDS identified. The most prominent SES factor extracted by factor analysis was related to EED (explaining 38% of the variance). Table 1 summarizes patient characteristics, hazard ratios (HR), 95% confidence intervals (CI) and p-values to illustrate the association between prognostic factors and MDS OS. Increased age at diagnosis, male sex, MDS higher risk category and EED were all associated with reduced OS. Multivariable results showed that patients with higher risk MDS had lower OS (HR 2.62 [2.54-2.71], p <0.001) compared to lower risk MDS (Figure 1A). NHB patients had the highest OS (HR 0.89 [0.86-0.93], p <0.001) compared to NHW (Figure1B). There was no statistically significant difference in OS for Hispanics compared to NHW. EED by tertile showed lower OS for most compared to least disadvantaged for all patients (HR 1.14 [1.11-1.17], p <0.001). More NHB and Hispanics (51 and 49%) were in the most disadvantaged category compared with NHW (30%) (p <0.001). Figure 1C demonstrates EED reduced the median OS in all patients with lower risk MDS from 41 [39-43] to 35 months [33-36] in patients with least and most disadvantage, respectively (HR 1.14 [1.08-1.19], p <0.001), although without such OS decrement for higher risk patients. Figure 1D indicates EED reduced median OS for Hispanic patients with MDS from 29 [25-33] to 20 months [18-23] for least and most disadvantage, respectively (HR 1.24 [1.13-1.36], p <0.001). Patients with higher risk MDS were more likely to expire from hematologic causes, including 25% from AML transformation and 35% from MDS complications. Patients with lower risk MDS were more likely to expire from bone marrow failure or non-hematologic causes, such as 21% from cardiovascular disease. No significant differences were noted for causes of death by race/ethnicity.Conclusion:For primary MDS patients, higher OS was noted in NHB compared to NHW and Hispanics. This association persisted after adjustment for age, sex, and clinical risk category. There was no statistically significant difference in OS for Hispanics compared to NHW. However, greater EED was associated with lower OS, which was most striking in patients with lower clinical risk MDS and those of Hispanic origin. These findings demonstrate the added importance of EED and racial/ethnic factors being associated with OS in MDS. [Display omitted] DisclosuresGreenberg: Notable Labs: Research Funding.

Updated Efficacy of Eltanexor Monotherapy in Patients with Higher Risk Hypomethylating Myelodysplastic Syndrome Primary Refractory to Hypomethylating Agents

Updated Efficacy of Eltanexor Monotherapy in Patients with Higher Risk Hypomethylating Myelodysplastic Syndrome Primary Refractory to Hypomethylating Agents

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.

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.

Frequency of Chromosomal Translocations Are Significantly Higher in Therapy Related Myeloid Neoplasm As Compared to Primary MDS

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders with variable outcomes. Acquired chromosome abnormalities in patients with myelodysplastic syndromes (MDS) are among the most valuable determinants of prognosis. However, chromosomal translocations are rare in MDS, and their prognostic implication is not well known. To elucidate factors driving chromosomal translocations in MDS, we compared the frequency of MDS patients with and without prior exposure to DNA damaging cytotoxic therapies.Methods: We evaluated diagnostic bone marrow metaphase cytogenetic data of MDS patients enrolled in South Australian MDS/AML registry data. Appropriate Institutional Review Board approval was obtained. Diagnosis and subclassification of MDS were done according to World Health Organization (WHO) 2016 criteria and risk stratified according to the revised International Prognostic Scoring System (R-IPSS) (Greenberg et al., 2012). Standard G-banding method was used for cytogenetic analysis.To decipher changes induced by cytotoxic therapy (CT) from that of myeloid neoplasm in general, we compared cytogenetic profile of three carefully selected cohorts: (i) tMN, in which a myeloid neoplasm occurred in a cancer survivor following exposure to cytotoxic (n = 206, 15.5%); (ii) myeloid neoplasm developing in unrelated cancer survivors with NO prior exposure to CT (a diagnosis of primary MN and another cancer; pMN+Ca) (n =148, 12%). This would include myelodysplastic syndrome or acute myeloid leukemia diagnosed in patients managed with surgical resection of breast cancer and NO exposure to CT; (iii) primary MDS without preceding independent cancer and/or exposure to CT (pMDS) (n = 893,72.5%)Results: The median age of 1247 patients analyzed was 72.50 (IQR 64.9 to 79) and 64% patients were male. Majority of patients were diagnosed with pMDS (n=893; 72.5%), while 206 (15.5%) and 148 (12%) were classified as t-MN and pMN+Ca respectively.Ten percent (114/1247) patients harbored translocations at the time of diagnosis. The most frequent chromosomes involved in translocation are chromosome 1 (37/114, 34.5%), 3 (15/114, 13.8%), 5 (6/114= 5.3%) (Fig.1A). Most common chromosome partners for translocation with chromosome 1 were chromosome 3, 7 and 21.The frequency of chromosomal translocations in patients with monsomal (55/114, 48%) and complex (70/114, 61%) karyotype were higher than patients without. There is a clear relationship in incidence of chromosomal translocations with increasing IPSS-R scores. The frequency of translocations in the Very high risk IPSS-R risk category was significantly higher compared to patients with Very low risk category (27% vs.1.1%).Importantly, frequency of chromosomal translocations was significantly higher in t-MN as compared pMN+Ca and P-MN patients who were not exposed to DNA damaging cytotoxic therapies prior to their diagnosis (17% vs. 4% vs. 8%, P=0.0004; Fig 1B). Notably, the survival of patients with chromosomal translocation was significantly poorer as compared to patients without translocation (Fig.1C). Poor OS of patients with translocation is most probably due to association with complex karyotype (Fig.1D).Conclusion: This comprehensive analysis demonstrates that chromosomal translocations are observed in 10% of MDS patients. Chromosomal translocations are significantly higher in patients who had prior exposure to cytotoxic therapy as compared to patients without. [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.

RVU120 (SEL120) CDK8/19 Inhibitor - a Drug Candidate for the Treatment of MDS Can Induce Erythroid Differentiation

Background: Myelodysplastic syndromes (MDS) are a group of malignant blood disorders characterized by ineffective hematopoiesis and cytopenias and frequent evolution to acute myeloid leukemia (AML). MDS results from the expansion of genetically and epigenetically changed clones with impaired differentiation and maturation. Primary clinical goals in MDS are to achieve remissions, alleviate symptoms associated with cytopenias, and minimize the transfusion burden. While supportive red blood cell transfusions, erythropoiesis-stimulating agents and novel targeted agents may lead to clinical improvement, an allogeneic bone marrow transplant (BMT) remains the only potential curative option for patients with MDS. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human Phase Ib clinical trial with RVU120 in patients with AML or high risk (HR)-MDS is currently ongoing. Preclinical studies indicated the strong antileukemic potential of RVU120 that was often associated with the multilineage commitment of CD34+ AML cells. Moreover, RVU120 could improve proliferation and induce erythroid differentiation of CD34+ cells derived from Diamond-Blackfan anemia (DBA) patients.Aims: Primary aim was to evaluate the erythroid differentiation potential of RVU120 in primary MDS and transformed cord blood CD34+ blood cells characterized with an early block in erythroid differentiation.Methods: Efficacy and mechanism of action of RVU120 and other CDK8 inhibitors were investigated in cord blood (CB) cells transduced with TLS-ERG, a fusion gene generated from t(16;21)(p11;q22). Transformed cells displayed an increased capacity for self-renewal, proliferation, and altered erythroid differentiation. Efficacy was further tested in CD34+ cells isolated from BM of MDS patients and established MDS cell lines. Cells were treated with RVU120 and global transcriptional changes and chromatin status were analyzed by RNA-seq, ATAC-seq, and ChIP-seq. Cell cycle, proliferation, and lineage-specific markers were studied by flow cytometry in liquid and semi-solid methylcellulose-based media.Results: RVU120 treatment leads to transcriptional reprogramming of transformed CD34+ CB cells. The most profound changes included decreased CDK8 occupancy followed by increased STAT5 and RNA Polymerase II loading at transcription start site and gene bodies. RVU120 treatment transcriptionally represses multiple genes associated with hematopoietic and leukemia stem cells such as CD34, FLI1, ENG and RGS18 and importantly induce the expression of genes involved in erythroid commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1, INHBA, GATA1/2 and hemoglobin genes. Detailed analysis of transformed CB and MDS CD34+ cells by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, leading to erythroid differentiation.Conclusions: Presented results indicate strong erythroid differentiation potential of RVU120 in CD34+ cells that acquired genetic abnormalities leading to arrested erythroid commitment, characteristics of many MDS and AML subtypes. Observed differentiation phenotype strikingly resembles effects of RVU120 in DBA cells caused by disruption of genes encoding ribosomal proteins. Detailed transcriptomic profiling strongly links the differentiation with enrichment of genes representing regulators of erythroid commitment and hemoglobin metabolism. Further studies are warranted to investigate the efficacy of RVU120 in chronic anemias associated with bone marrow failures in AML and MDS patients. [Display omitted] DisclosuresPakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Dziedzic: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company.

PLAGL2 Independently Drives Aberrant Erythropoiesis and Initiation of Preleukemic State

BackgroundThe identification and understanding of early drivers in malignancy is crucial to prevent or revert preleukemic events. Del(20q) is one of the most common primary cytogenetic abnormalities found in preleukemic malignancies from myeloproliferative neoplasms to myelodysplastic syndrome (MDS). Previous studies have identified a “common retained region” within 20q11.21 that is often amplified in a subset of MDS patients. PLAGL2 is one of the 4 genes identified to be within the minimally conserved amplified region. (MacKinnon et al. 2010) Indeed, in previously published datasets of MDS hematopoietic stem and progenitor cells (HSPCs) transcriptome, PLAGL2 is significantly elevated in del(20q) patients compared to healthy controls. However, we have found that its level is also higher in HSPCs of cytogenetically normal MDS patients with low blasts. Given these findings, we sought to define the role of PLAGL2 as a potential early driver of myeloid malignancies.ResultsIn healthy cord blood (CB) HSPCs, PLAGL2 overexpression enhanced proliferation ex vivo, better maintained stemness and decreased apoptosis. Colony formation assays also identified increased output of the erythroid lineage. Xenotransplanted CB CD34+ HSPCs overexpressing PLAGL2 exhibited increased engraftment competitiveness and led to splenomegaly with signs of hypercellularity after 20 weeks, features consistent with clinical observations of hematological malignancy. Grafts derived from PLAGL2 overexpressing cells reproducibly maintained a significantly larger CD34+ HSPC compartment. Intriguingly we also identified that ~50% of PLAGL2-overexpressing grafts exhibited a significant erythroid (CD71+/CD235a+) component where none was observed in the control group. This unique finding of aberrant erythropoiesis is reminiscent of clinical observations in patients with 20q11.21 amplification, where a high proportion of erythroblasts in the marrow and in some cases progression to erythroleukemia was noted. To evaluate the progression of PLAGL2-overexpressing grafts, further secondary transplantations were carried out and showed the persistence of only immature erythroid progenitors (CD71+/CD235a-) coupled with a near complete absence of lymphopoiesis in the same grafts. Together, our data strongly suggests ectopic levels of PLAGL2 can independently drive the expansion of human HSPCs and enforce features of myeloid malignancy.To uncover the molecular mechanism underlying PLAGL2 function, we performed RNA-seq and Cut&Run in human CB CD34+ HSPCs overexpressing PLAGL2. Geneset enrichment analysis of the transcriptome and over-representation analysis of bound genes both identified signatures consistent with LSCs. We compared these findings with identically-derived omics profiles of HSPCs overexpressing PLAG1, a closely related family member that our lab has identified as a potent expander of HSCs ex vivo but not capable of promoting malignant features. We found a strong common feature in the downregulation of ribosomal components and translation machinery, then functionally validated reduced protein synthesis in PLAGL2 overexpressing HSPCs through OP-Puro assays. We have shown dampened mRNA translation to be one of the mechanisms by which PLAG1 enhances stemness and survival of HSCs, one that potentially extends to PLAGL2 as well. However, we also identified discordant signatures, notably PLAGL2's unique capacity to reduce mitochondrial translation, a pathway associated with ineffective erythropoiesis and MDS and one that we are currently exploring as a means by which PLAGL2 can enforce malignant phenotypes.Finally, to investigate the potential of PLAGL2 as a therapeutic target in MDS, we performed shRNA knockdown in MDSL, a human MDS cell line. In vitro competitive assays with mixed wildtype cells showed steady dropout of PLAGL2 depleted cells. Currently, we are continuing to purse the dependence of primary MDS cells on PLAGL2 through in vivo xenograft models.ConclusionWe have identified PLAGL2's potential as an early independent driver of myeloid malignancy and aberrant erythroid differentiation. An understanding of PLAGL2 and its downstream mechanisms will not only further our understanding on the development of early myeloid malignancies but also potentially provide another avenue to treat or prevent leukemia before it manifests. DisclosuresDick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

RUNX1 Expression Can be Complementary to RUNX1 Mutation in MDS Prognostication

▪RUNX1 is a member of the core-binding factor family of transcription factors and is imperative for establishing definitive haematopoiesis. Mutated RUNX1 is an adverse risk factor for myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Meanwhile, high expression of RUNX1 is correlated with dismal prognosis in cytogenetically normal AML patients and critical for maintaining leukemic stem cells across AML genetic subgroups. However, the clinical relevancy of RUNX1 expression in MDS patients remains elusive. This study aimed to investigate the prognostic and biologic impacts of RUNX1 expression in MDS patients. We recruited 341 primary MDS patients who had enough bone marrow (BM) samples for RNA and next-generation sequencing. We first examined the difference in RUNX1 expression among patients with wild RUNX1 and N-terminal or C-terminal RUNX1 mutation. Among the 341 patients, 54 (15.8%) harboured RUNX1 mutation, 15 (27.8%) in N-terminal and 39 (72.2%), C-terminal. Patients with C-terminal RUNX1 mutant had higher RUNX1 expression than those with N-terminal RUNX1 mutant or wild RUNX1 (p<0.001 ). Patients were then divided into two groups with higher- and lower-RUNX1 expression (median as cut-off). Higher RUNX1 expression was closely associated with lower platelet counts, higher blast percentages in the BM and peripheral blood and complex karyotypes at diagnosis. Patients with higher-RUNX1 expression were more frequently categorized into higher-risk groups based on the revised international prognosis scoring system (IPSS-R). Higher RUNX1 expression was intimately associated with ASXL1, NPM1, RUNX1, SRSF2, STAG2, TET2, TP53, and ZRSR2 mutations, whereas lower RUNX1 was associated with SF3B1 mutation.Regarding survival, we first examined the impact of RUNX1 mutation on survival in this cohort. As expected, patients with mutated RUNX1 had significantly inferior leukaemia-free survival (LFS) and overall survival (OS) than those with unmutated RUNX1 (p=0.007, and p=0.008, respectively). We then explored the effects of RUNX1 expression on patients' survival. Patients with higher RUNX1 expression had significantly inferior LFS and OS than those with lower expression (both p<0.001, Figure 1a and 1b). We further interrogated RUNX1 expression and mutation statuses for risk stratification. The higher-RUNX1 group consistently had shorter LFS and OS than the lower-RUNX1 group no matter RUNX1 was mutated or not (Figure 1c-1f). Subgroups analysis revealed the same findings in IPSS-R lower-risk (very low, low, and intermediate-risk) and IPSS-R higher-risk (high and very high risk) subgroups (all p<0.05). Moreover, time-dependent ROC curves indicated that RUNX1 expression had better predictive power for LFS and OS than RUNX1 mutation. In multivariate analysis, higher RUNX1 expression appeared as an independent adverse risk factor for LFS and OS irrespective of age, IPSS-R, and mutations in ASXL1, EZH2, RUNX1, SF3B1, SRSF2, STAG2, TET2, and TP53 (Table). The prognostic significance of RUNX1 expression was further validated in two external public cohorts, GSE 114922 and GSE15061. Patients with higher-RUNX1 expression consistently had significantly inferior survival than those with lower-RUNX1 expression (Figure 2). Bioinformatic analysis revealed that higher-RUNX1 patients had more robust IL-17 and MAPK signallings but exhausted antioxidant activities and antimicrobial humoral responses. In summary, we present the characteristics and prognosis of MDS patients with various RUNX1 expressions and propose that RUNX1 expression can complement RUNX1 mutation in MDS prognostication, wherein patients with wild RUNX1 but high expression may need more proactive treatment. [Display omitted] DisclosuresTien: AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding.

Modeling Clonal Progression in SF3B1-Mutant Myelodysplastic Syndrome

SF3B1-mutant myelodysplastic syndrome (MDS) has recently been proposed as a distinct disorder characterized by ring sideroblasts, ineffective erythropoiesis and good prognosis. Selected co-occurring genetic abnormalities were reported associated with significantly worse outcome and suggested as exclusion criteria for the proposed entity. However, it remains unclear how a limited spectrum of co-occurring drivers affects SF3B1-mutant MDS biology to determine evolution from a relatively indolent condition to high risk malignancy.To gain a better insight into the clonal progression of SF3B1-mutant MDS, we analyzed SF3B1 co-mutations in a cohort of 176 SF3B1-mutated patients diagnosed with a myeloid neoplasm. RUNX1 and STAG2 were the only co-mutated genes found significantly associated with advanced disease phenotype (i.e. MDS with excess blasts and secondary acute myeloid leukemia) (OR=18.36 (2.18-862.91), P=0.001, and OR= Inf (3.57-Inf), P<.001, respectively), and reduced overall survival (P<.001). Based on these data, we hypothesized that acquisition of RUNX1 or STAG2 co-mutations in patients with SF3B1-mutant MDS drives progression to high-risk neoplasms.RUNX1 and STAG2 control hematopoietic stem and progenitor cell self-renewal and differentiation by regulation of gene expression. To explore the biological impact of RUNX1 or STAG2 loss in the context of SF3B1-mutant MDS, we disrupted RUNX1 or STAG2 using CRISPR/Cas9 in an induced pluripotent stem cell (iPSC) model of MDS-RS that we previously established by reprogramming of bone marrow cells from SF3B1-mutant individuals. This patient-derived system allows hematopoietic progenitor expansion through doxycycline-mediated expression of 5 transcription factors (5F-HPC) and multilineage differentiation upon doxycycline withdrawal. We asked how RUNX1 or STAG2 disruption affected SF3B1-mutant HPCs self-renewal and differentiation in our model of MDS-RS, and sought to define the underlying transcriptional changes.RUNX1-edited cells maintained significantly higher proportion of CD34 + HPCs, suggesting that RUNX1 mutation increases self-renewal capacity of SF3B1-mutant progenitors. Consistently, SF3B1/RUNX1 double-mutant 5F-HPCs showed positive enrichment of HSC-specific gene signatures. This was accompanied by broad upregulation of inflammatory programs, recapitulating the activated gene signatures we identified in SF3B1/RUNX1 co-mutated patients in our cohort. RUNX1 disruption promoted myeloid skewing at the expense of erythroid differentiation in SF3B1-mutant cells. Consistent with this, granulocyte-monocyte progenitors (GMP) and myelo-erythroid transcriptional programs were positively and negatively enriched, respectively. By contrast, in SF3B1/STAG2 double-mutant 5F-HPCs, both erythroid and myeloid populations were reduced compared to SF3B1 single mutant controls, suggesting a block in both myeloid and erythroid differentiation, despite the presence of pro-differentiation signals. This was supported by a profound down-regulation of genes involved in the response to external stimulus and suppression of GMP specific transcriptional signature.In summary, we identified RUNX1 and STAG2 mutations as main drivers of disease progression in SF3B1-mutant patients, and generated extensive cell line panels to interrogate their functional interaction with mutant SF3B1. By applying CRISPR/Cas9 editing to our MDS-RS model, we could overcome limited availability of primary MDS samples to show that RUNX1 or STAG2 co-mutations drive progression through distinct biological mechanisms in SF3B1-mutant HPCs. DisclosuresNo relevant conflicts of interest to declare.

Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition

Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1 mutant HSPCs using a multi-model approach of in vivo zebrafish and mouse systems, and in vitro assays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1 point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivo STATTIC treatment selectively depleted Sf3b1 mutant cells over wildtype in vivo. RNAseq of sf3b1 homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1 heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1 heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivo while sparing wildtype cells. We sought to rescue HSPCs in sf3b1 homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1 and SRSF2 heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666N cells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1 homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment. DisclosuresShastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.

Myelodysplasia: A report of 6 cases

Introduction: Myelodysplasia is a typical feature of Myelodysplastic Syndrome (MDS), but not restricted to MDS, it may be found in non-clonal hematological as well as non-hematological diseases. The essential component of the WHO definition of MDS is the clonal nature of myelodysplastic hematopoiesis. MDS needs to be separated from the numerous neoplastic and non-clonal hematologic disorders mimicing MDS, because, while MDS is potentially preleukemic, disorders with secondary myelodysplasia are not neoplastic or preleukemic and are reversible when the underlying factor is removed. Methods: We carried out an analysis of 6 cases presenting with marrow dysplasia between Feb2018 to Apr2019 and present their diagnostic approach with clinical history, complete blood count, biochemical tests, peripheral smear, bone marrow aspirate and biopsy, flowcytometry and cytogenetics. Results: We hereby present a series of 6 cases of myelodysplasia. Two cases are primary MDS with clonality established. One case is associated with acute leukemia, and one therapy-related, and two cases secondary to an autoimmune disorder. Conclusion: MDS is diagnosed by exclusion, because numerous signs of dysplasia might also occur in the context of other, including non-hematological diseases. It is therefore important to exclude other hematological and non-hematological diseases by a combination of clinical features, blood tests and molecular studies.

Immune signatures of bone marrow cells can independently predict prognosis in patients with myelodysplastic syndrome.

Increasing evidence supports the role of the immune microenvironment and associated signalling in the pathogenesis of myelodysplastic syndromes (MDS). Nevertheless, the clinical relevancy of immune signals in patients with MDS remains elusive. To address this, we used single-sample gene-set enrichment analysis to score immune signatures of bone marrow (BM) samples from 176 patients with primary MDS. Enhanced signatures of 'immature dendritic cells' and 'natural killer cells with cluster of differentiation (CD)56bright' were correlated with better overall survival (OS), whilst higher 'CD103+ signature' was associated with reduced survival. An MDS-Immune-Risk (MIR) scoring system was constructed based on the weighted sums derived from Cox regression analysis. High MIR scores were correlated with higher revised International Prognostic Scoring System (IPSS-R) scores and mutations in ASXL transcriptional regulator 1 (ASXL1), Runt-related transcription factor 1 (RUNX1), and tumour protein p53 (TP53). High-score patients had significantly inferior leukaemia-free survival (LFS) and OS than low-score patients. The prognostic significance of MIR scores for survival remained valid across IPSS-R subgroups and was validated in two independent cohorts. Multivariable analysis revealed that a higher MIR score was an independent adverse risk factor for LFS and OS. We further proposed a model with the combination of MIR score and gene mutations to be complementary to IPSS-R for the prognostication of LFS and OS of patients with MDS.

Overview of myelodysplastic syndrome in Baghdad, Iraq- clinical picture and outcome

Background: Myelodysplastic syndromes (MDS) can represent a challenge in diagnosis and treatment because of heterogeneous presentation, unpredictable response to variable measures with propensity for leukemic transformation. Aim of study: To review MDS Iraqi patients in term of presentation, risk stratification and treatment options and to assess outcome of different treatment options in term of response criteria, quality of life and progression. Patients and method: a hospital based study conducted over a period of 21 months from January 2018 till October 2019 in different hematology centers in Baghdad. Any patient with primary or therapy related MDS diagnosed were enrolled in this study. Each patient stratified into different risk groups when applying cytogenetic versus non cytogenetic scoring systems, the therapeutic options for every patient. They observed for their response to different therapeutic modalities and any evidence of progression. Results: Mean age at diagnosis for MDS was 58.7± Anemia was the most common presenting symptom representing 68.4% of patients. MDS with excess blasts-II is forming 28.8%. Majority of patients categorized as intermediate risk group. Hypomethylating agents used for 50% of MDS patients while 32.1% received best available therapy. Those on HMA achieve partial remission in 7.1%, 25.2% reached to stable disease, hematological improvement in 25.2%. Death outcome in 21.6 with a mean survival is 7.7 months. Disease progression in 10.8%, 7.2% showed leukemic transformation. Conclusion: Iraqi MDS patients present at younger age with advanced manifestation that needs standard risk stratification. The non-cytogenetic score systems can't be of equivalent application even low resources circumstances.

Quantitative analysis of myelofibrosis and its prognostic significance in patients with myelodysplastic syndrome

Objective: To quantitatively analyze the reticulin fiber intensity density (RFD) in patients with myelodysplastic syndrome (MDS) by using the computer-aided grid point method, and preliminarily explore its correlation with the prognosis of MDS patients. Methods: Bone marrow (BM) slices from 32 primary MDS patients treated in Tongren Hospital Shanghai Jiao Tong University School of Medicine from February 2017 to December 2019 were observed. Images were taken by the optical microscope imaging system after the Gomori staining. The computer grid marking software was developed according to the principle of the mesh micrometer to assess RFD, meanwhile, the artificial semi-quantitative were used to assess the fibrosis of bone marrow. The co-relation between the above two methods was evaluated, and the relationship between RFD and prognosis of MDS patients were further investigated with Cox regression analysis. Results: Of the patients, there were 17 males and 15 females with a median age of 69 years (32-91 years). The RFD quantitatively analyzed by the computer-based method was positively correlated with the myelofibrosis grade by the artificial semi-quantitative analysis (r=0.497, P=0.004). The RFD in patients diagnosed with MDS complicated with excess blasts (MDS-EB) was significantly higher than that in the non-MDS-EB group((9.55%±0.75%) vs (1.71%±0.23%), P<0.001). Cox regression model analysis showed that the RFD of MDS patients had better prognostic value when compared with the artificial semi-quantitative analysis, which was also a poor prognostic factor (RR=1.337, 95%CI: 1.085-1.648, P=0.006). The overall survival (OS) of patients with RFD>5.54% was significantly shorter than that with RFD≤5.54% (P=0.001). The OS of MDS-EB patients with RFD>9.81% was significantly shorter than that in patients with RFD≤9.81% (P=0.003). Conclusion: Abnormal proliferative fibrosis of bone marrow is a potential high-risk factor for poor prognosis of MDS patients.