Myelodysplastic Syndromes Bone Marrow Samples Research Articles

Toll-like receptor and cytokine expression throughout the bone marrow differs between patients with low- and high-risk myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders, the pathogenesis of which involves enhanced immune signaling that promotes or selects for mutant hematopoietic stem and progenitor cells (HSPCs). In particular, toll-like receptor (TLR) expression and signaling are enhanced in MDS, and their inhibition is an attractive therapeutic strategy. Although prior studies have reported increased expression of TLR2 and its binding partners TLR1 and TLR6 in the CD34+ cells of patients with MDS (especially those with low-risk disease), TLR expression in other cell types throughout the bone marrow is largely unknown. To address this, we used mass cytometry to assess the expression of TLR1, TLR2, and TLR6 and cytokines in the bone marrow hematopoietic cells of six low/intermediate-risk and six high-risk unmatched MDS bone marrow samples, as well as healthy controls, both at baseline and in response to TLR agonists. We observed several consistent differences between the groups. Most notably, TLR expression was upregulated in multiple cell populations in the low/intermediate-risk, but not high-risk, patients. In addition, many cytokines, including interleukin-6, interleukin-8, tumor necrosis factor α, transforming growth factor β, macrophage inflammatory protein 1β, and granzyme B, were highly expressed from various cell types in low/intermediate-risk patients. However, these same cytokines, with the exception of transforming growth factor β, were expressed at lower levels in high-risk MDS. Together, these findings highlight the differential role of inflammation, and specifically TLR expression, in low/intermediate- versus high-risk MDS, and suggest that elevated TLR expression and cytokine production in multiple cell types likely influences the pathogenesis of MDS in lower-risk patients.

Human Germline HLTF E259K Mutation Identified in Familial MDS Patients Accumulates DNA Damage through Impaired PCNA Polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been discovered, the genetic landscape and the molecular pathogenesis still remain unclear. To explore novel causal genetic alterations and their pathogenetic significance, we performed whole-exome sequencing analysis using four samples from a Japanese familial MDS pedigree, two affected and two unaffected as controls. In total, 16889 non-synonymous germline substitutions were obtained. Among them, twelve candidate single-nucleotide variations (ANK1, C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, RYK, TTN) were elucidated. Of the candidates, we elucidated HLTF, encoding a member of the SWItch/Sucrose Non-Fermenting (SWI/SNF) family, as a putative associated gene. HLTF works as an E3-ubiquitin ligase and binds with Methyl Methanesulfonate Sensitive 2 (MMS2)/Ubiquitin Conjugating Enzyme E2N (UBC13) complex. This complex is known to promote the Lys-63 linked polyubiquitination of proliferating cell nuclear antigen (PCNA), followed by an error-free postreplication DNA repair by arranging template switching (Mailand et al. Nat Rev Mol Cell Biol 2013). The obtained germline mutation of HLTF was p.Glu259Lys (E259K), heterozygous mutation.To study the functional role of HLTF, we firstly evaluated the effects of Hltf silencing on hematopoiesis by in vitro colony-formation assay with Hltf-knocked down mouse primary bone marrow cells, and found that these cells have enhanced in vitro colony-formation capacity. In agreement with the acquisition of an immature phenotype, the cells expressing c-kit, a hematopoietic stem/ progenitor cell marker, were increased in Hltf shRNA-expressing colonies after the first round. We then focused on DNA damage in HLTF knockdown HEL cells according to the function of HLTF post-replication DNA repair. We examined the phosphorylation of gamma H2AX foci by immunofluorescent staining and found that the number of foci was increased in HLTF knockdown cells compared with control cells, suggesting that DNA damage was accumulated in HLTF knockdown cells.To investigate the effects of exogenous expression of HLTF E259K mutant protein, we evaluated in vitro apoptosis status with HEL cells transduced with empty vector (EV), HLTF wild type (WT), and HLTF E259K mutant (MT), respectively. In apoptosis assay, annexin V positive cells were significantly increased in MT-overexpressed HEL cells compared with WT-overexpressed HEL cells. In addition, we found that the number of gamma H2AX foci was increased in HLTF MT-overexpressed HEL cells, suggesting that DNA double-strand breaks were accumulated in MT-overexpressed cells. To explore the mechanism of accumulated DNA double-strand breaks by mutant HLTF, we evaluated the binding ability of HLTF mutant protein with PCNA, and MMS2/UBC13, all of which play a critical role in the DNA double-strand break repair process. We found that binding of HLTF MT to MMS2 or UBC13 was significantly attenuated compared with HLTF WT although binding to PCNA remained unchanged, suggesting HLTF MT cannot efficiently form a complex that induces polyubiquitination of PCNA. In line with this, PCNA polyubiquitination was impaired in HLTF MT-overexpressed cells compared with HLTF WT-overexpressed cells.Finally, we searched somatic HLTF mutation in sporadic MDS bone marrow samples. Whole HLTF coding regions of 40 samples were sequenced. As a result, a novel HLTF missense heterozygous mutation (p.Thr50Ala, T50A) was identified in one sample (1/40 = 2.5%). These data suggested the possibility that HLTF mutation might contribute to the pathogenesis not only in familial MDS but also in sporadic MDS.In conclusion, our results indicated that HLTF E259K germ line mutation accumulates DNA damage through impaired PCNA polyubiquitination. The aberrant function of HLTF mutant might play a role in the pathogenesis of familial MDS. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Suzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria. Harada:NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding; Celgene: Research Funding. Kurokawa:Bristol-Myers Squibb: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau.

Distinct and convergent consequences of splice factor mutations in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are characterized by recurrent somatic alterations often affecting components of RNA splicing machinery. Mutations of splice factors SF3B1, SRSF2, ZRSR2 and U2AF1 occur in >50% of MDS. To assess the impact of spliceosome mutations on splicing and to identify common pathways/genes affected by distinct mutations, we performed RNA-sequencing of MDS bone marrow samples harboring spliceosome mutations (including hotspot alterations of SF3B1, SRSF2 and U2AF1; small deletions of SRSF2 and truncating mutations of ZRSR2), and devoid of other common co-occurring mutations. We uncover the landscape of splicing alterations in each splice factor mutant MDS and demonstrate that small deletions in SRSF2 cause highest number of splicing alterations compared with other spliceosome mutations. Although the mis-spliced events observed in different splice factor mutations were largely non-overlapping, a subset of genes, including EZH2, were aberrantly spliced in multiple mutant groups. We also verified aberrant splicing of key genes USP9X, USP24 (deubiquitinating enzymes), LUC7L2 (splice factor) and EED (PRC2 component) in MDS harboring small deletions of SRSF2. Pathway analysis revealed that mis-spliced genes in different mutant groups were enriched in RNA splicing and transport as well as several signaling cascades, suggesting converging biological consequences downstream of distinct spliceosome mutations. Our study reveals splicing signatures of each splice factor mutation and identifies shared and distinct sets of mis-spliced genes and affected biological processes in different spliceosome mutant MDS.

Single-Cell RNA Sequencing Reveals Distinct Hematopoietic Stem Cell Hierarchies in MDS

Myelodysplastic Syndromes (MDS) are a group of heterogeneous stem cell disorders that result in inefficient hematopoiesis. Although the genetic and cytogenetic landscapes of MDS have been well characterized (Papaemmanuil 2013, Sperling 2017), little is known about the differentiation abnormalities that underlie the MDS phenotype. Gaining insights on how different hematopoietic stem and progenitor cell (HSPC) types contribute to MDS is essential for the design of new targeted therapies to supplement the currently limited effective therapeutic options. To understand the contribution of different cell types to the pathogenesis of MDS, we analyzed the expression profile of the Lin-CD34+ HSPC compartment at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) analysis of HSPCs isolated from 2 MDS patients and 2 age-matched healthy donor samples revealed distinct cell clusters driven by the sample type and the differentiation potential of the cells. To annotate the specific subsets of HSPCs in each cluster, we scored them on the basis of previously reported population-specific gene signatures (Laurenti 2013, Psaila 2016, Van Galen 2019). Whereas CD34+ cells from the 2 healthy donor bone marrow (BM) samples largely overlapped with each other and displayed 2 distinct erythroid/megakaryocytic (Er/Mk; cluster 3) and lympho/myeloid (clusters 2, 5) differentiation trajectories in line with the current view of hematopoiesis, CD34+ cells from the 2 MDS BM samples clustered separately and showed predominantly myeloid differentiation routes (Fig a). Importantly, differential expression analysis of the HSPCs from the 2 MDS samples (Fig b) showed that cells residing atop of the HSPC hierarchy retained the transcriptional profile of immature HSCs in one of the samples (clusters 2, 4), while they were characterized by the expression of genes involved in the differentiation of myelo/lympho multipotent progenitor cells (clusters 0, 1) in the other. However, pseudotime analysis of the HSPCs' transcriptional dynamics showed that, despite the distinct differentiation state of the early hematopoietic cells in each group, the differentiation trajectories of those cells converged at the late myeloid progenitor state (clusters 3, 5, 6). These results suggest that, although the earlier HSC architecture is heterogeneous across MDS patients, the more differentiated myeloid progenitor compartment is similarly compromised and is responsible for the clinical phenotypes of MDS. To confirm differential cell-type contributions to the MDS hierarchy, we immunophenotyped BM samples from 123 untreated patients using multicolor flow cytometry. We applied principal component analysis and logistic regression to group samples based on their cellular compositions. Our mathematical classifier stratified patients in 2 groups, which had markedly different cellular repertoires consistent with our scRNA-seq results (Fig c). Patients with different MDS stem cell hierarchies did not present with significantly different clinical characteristics at diagnosis. These data confirm that different abnormal hematopoietic trajectories converge in the myeloid bias typically observed in MDS hematopoiesis. Next, we exome-sequenced mononuclear cells and T-cells from 45 untreated MDS patients and identified high-confidence somatic mutations in known oncogenes and/or leukemia driver genes. The median number of mutations (n=3) was not significantly different between MDS groups 1 and 2. We identified 4 genes that were differentially mutated in the 2 MDS architectures (Fig d), which suggested that certain mutations may predispose for a specific HSPC phenotype. However, mutation specificity could not fully account for the origin of the 2 differentiation architectures, which were independent on the genetic background in most patients. In conclusion, we demonstrated that MDS are sustained by distinct and recurrent abnormal HSPC differentiation hierarchies. Diverse cellular compositions suggest that different cell-type specific signaling pathways maintain the disease in each group of patients. Our work shows that the characterization of the cellular diversity in the hematopoietic compartment can be used as a biomarker to stratify MDS patients, and warrants further studies to predict the intrinsic vulnerabilities of the cells involved in the pathogenesis and maintenance of MDS in a patient-specific manner. Figure Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:IONIS: Other: Intellectual property and research material IONIS); Amgen: Research Funding; Abbvie: Research Funding.

The role of innate immunity in MDS pathogenesis.

The role of innate immunity in MDS pathogenesis.

Data Driven Computational Modeling of Hematopoiesis in Myelodysplastic Syndromes Unveils Differences in Hematopoietic Stem Cell Kinetics Compared to Age-Matched Healthy Controls

Background: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis, peripheral cytopenias and risk of transformation to acute myeloid leukemia (AML). Recently, HSC in MDS have been shown to carry acquired distinct genetic and epigenetic mutations, most frequently in epigenetic regulators or splicing machinery factors, which are considered disease-initiating events and are likely responsible for the dysplastic features observed in this disease. Hierarchies of hematopoiesis have been established for healthy HSC and their progeny but it is unclear whether MDS HSC follow the same patterns. Clonal hierarchies in MDS have been inferred on the basis of mutational data, these only constitute a snapshot of hematopoiesis in the patient at a given time and may not accurately reflect the kinetics of the disease. To understand how clonal dominance of MDS over healthy HSC is achieved, we sought to identify differences in proliferation kinetics and lineage fate decisions of MDS HSC in comparison to healthy hematopoiesis and to fit this real data into a mathematical model of MDS hematopoiesis.Methods: Lower-risk MDS (according to IPSS-R) bone marrow (BM) samples from patients harboring an SF3B1 mutation (n = 5) or higher-risk MDS BM samples from patients with an ASXL1 mutation (n = 5) were chosen for analysis to limit heterogeneity and allow for better comparability. Healthy age-matched control BM samples (n = 5) were obtained from patients undergoing routine hip replacement surgery. Distribution of stem and progenitor cell compartments (HSC, MPP, MLP, CMP, GMP, MEP) in BM on day 0 were determined by multiparameter flow cytometry. Cells were labeled with violet cell trace and HSC (Lin-CD34+CD38-CD90+CD45RA-) were sorted by flow cytometry. A fixed number of HSC as well single HSC cell cultures were set up in vitro in serum-free medium with defined growth factors (FL, KL, TPO, IL-6, G-CSF, GM-CSF and EPO). Cell divisions, proliferation rates, cell death and lineage maturation were determined by flow cytometry on days 1, 3, 5, 7 and 9. Clonogenic capacity was probed by standard CFU assays in methylcellulose on day 0 and day 7. Additional integration of genomic sequencing analyses of bulk BM and HSC was used to assess the contribution of the malignant clone to the population of proliferating cells and to each hematopoietic lineage.Results: While CFU numbers at day 0 were significantly reduced for all MDS samples compared to healthy controls, ASXL1 mutated MDS BM samples contained significantly higher numbers of HSC than SF3B1 MDS or healthy control BM samples. SF3B1 MDS samples had significantly larger CMP and GMP compartments compared to healthy controls or ASXL1 mutated samples. In cultures started with a fixed number of HSC, no apparent differences in proliferation kinetics were observed between MDS and healthy samples over time. However, cell trace labeling and sorting of single HSC into 96 well plates revealed clear differences between MDS and healthy HSC. Overall, MDS HSC were less robust, with fewer wells of the 96 plate containing cells after sorting than for healthy HSC samples. In contrast, the remaining HSC from both both ASXL1 and SF3B1 mutated MDS showed a significantly higher cell division rate than healthy HSC in vitro, suggesting MDS HSC are more active than their normal counterparts. Genotyping analyses to assess single MDS HSC contributions to proliferation are ongoing and data are currently being fitted to mathematical models.Conclusion: Detailed analysis of hematopoietic stem and progenitor cell compartments as well as time-resolved tracking of HSC revealed significant differences between MDS and healthy HSC kinetics in vitro. Integration of this data into a comprehensive mathematical model of MDS hematopoiesis may aid in dissecting the mechanisms of progression of MDS towards acute myeloid leukaemia (AML). DisclosuresGötze:Takeda: Honoraria, Other: Travel aid ASH 2017; Celgene: Honoraria, Research Funding; Novartis: Honoraria; JAZZ Pharmaceuticals: Honoraria.

Increase of DNA damage and alteration of the DNA damage response in myelodysplastic syndromes and acute myeloid leukemias

Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We used immunofluorescence staining of γH2AX and 53BP1 for analyzing DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of the DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53. As compared to γH2AX foci levels in normal bone marrow samples (0.2 focus per CD34+ cell±0.0; mean±standard error of mean), increased levels of γH2AX foci were detected in 16/16 MDS bone marrow samples (2.8 foci per CD34+ cell±0.5), 18/18 AML bone marrow samples (5.5 foci per blast±0.5), 1/1 MDS cell line (6.4 foci per cell) and 6/6 AML cell lines (12.0 foci per cell±0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. Further, DDR proteins were expressed heterogeneously suggesting impairment of the DDR. In summary, our results provide evidence for a continuous increase of DSB across the spectrum from MDS to AML in conjunction with an impaired DDR. Co-localization of γH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, γH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML.

γH2AX Foci Increase Across the Spectrum from Myelodysplastic Syndromes to Acute Myeloid Leukemias and Co-Localize with 53BP1 in Specific Patterns in the Context of an Impaired DNA Damage Response

Purpose: Increased DNA damage and alteration of the DNA damage response (DDR) are critical features of genetic instability presumably implicated in pathogenesis of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML).Materials and methods: We performed combined γH2AX/53BP1 immunofluorescent focus staining of DNA double-strand breaks (DSB) in MDS and AML cell lines, in CD34+ selected cells of normal and MDS bone marrow (including three cases of chronic myelomonocytic leukemias) and in blasts of AML bone marrow. In addition, we screened for activation of DDR by immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53.Results: Compared to γH2AX foci levels in normal bone marrow samples (0.18 focus per CD34+ ± 0.6), increased levels of γH2AX foci were detected in 1/1 MDS cell line (6.4 foci per cell ± 0.0), 6/6 AML cell lines (12.0 foci per cell ± 0.6), 12/12 MDS bone marrow samples (2.8 foci per CD34+ ± 0.7) as well as 12/12 AML bone marrow samples (6.0 foci per blast ± 0.6). γH2AX and 53BP1 co-localized in all tested samples forming diffuse, clustered and marginal patterns. DDR proteins were expressed heterogeneously suggesting impairment of DDR.Conclusions: Our results provide evidence for a continuous increase of constitutive DSB across the spectrum from MDS to AML in the context of an impaired DDR. γH2AX and 53BP1 co-localize in unique patterns in nuclei of MDS and AML presumably owing to a non-random spatial organization of the genome and foci formation implies promotion of ineffective nonhomologous end-joining repair mechanisms at sites of constitutive DSB. DisclosuresNo relevant conflicts of interest to declare.

Interlaboratory Variation in CD34+ Cell Quantification in Low Risk Myelodysplastic Syndromes

Background: Accurate bone marrow (BM) blast count is essential for establishing prognosis in lower-risk myelodysplastic syndromes (MDS). Flow cytometry immunophenotyping (FCI) helps to evaluate the most immature BM cell compartment, but it is not known whether the CD34+ myeloid cell count in MDS are comparable among different observers. We investigated the FCI approaches used for enumeration of CD34 + myeloid cells in MDS in the routine practice in 6 clinical Spanish centers, and then explored if data were reproducible among participants.Methods: In this prospective study, participants exchanged 47 FCI files from MDS BM samples with <5% blasts, and quantified the CD34+ myeloid cells in a blind manner. No agreement regarding cytometers, number, reagents, fluorochromes and events recorded were previously established.Results: The mAb combinations selected to identify BM myeloid CD34+ cells was highly variable: CD45 was included in 45/47 (95.7%) files, CD34/CD45/CD117 in 37 (78.7%), and HLA-DR in 19 files (40.4%). The FCI assays used in every centre were quite different, but a very good concordance among the 6 FCI observers for CD34+ cell enumeration was observed with the intraclass correlation coefficient (0.720). Considering the specific analysis of each sample, 2 groups were distinguished. Twenty-five cases (53.2%) exhibited the highest concordance, with an interlaboratory coefficient of variation (CV) <30%, or a difference among observers <0.5%. In the remaining 22 cases, the concordance among participants was low. Comparison of the characteristics of high and low agreement assays showed no statistical differences. In order to determine whether the lower concordance obtained among observers after the analysis of these 22 samples could be improved, we designed a protocol with specific guidelines with gating strategies for identification of the CD34+ myeloid cells in several consecutive steps. The strategy was designed for samples with the CD34/CD45/CD117 mAb combination (n=15), with or without HLADR. Using this strategy, the interlaboratory CV improved in 7/15 cases (46.66%), but no improvement was obtained in the remaining 8 cases. Finally, the percentage of CD34+ myeloid cells was reproducible in 32/47 cases (68%) diagnosed with low risk MDS. Conclusions: Despite different methodology, accuracy among observers in CD34 cell quantification is feasible. Selection of appropriate protocols and targeted training would help to enhance the reproducibility of the technique among different observers. However, the intrinsic heterogeneity of these cells in MDS might justify the difficulty for their identification in some cases. DisclosuresNo relevant conflicts of interest to declare.

Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

The Challenge Of Quantifying CD34+ Myeloid Cells In Myelodysplastic Syndromes With Less Than 5% Bone Marrow Blasts. Reproducibility Among 6 Flow Cytometry Observers

IntroductionIn low-risk myelodysplastic syndromes (MDS), the morphological bone marrow (BM) blast cell count between 0 and 2%, and >2 - <5%, has demonstrated prognostic value and is critical for R-IPSS. Flow cytometry immunophenotyping (FCI) provides an accurate way for quantification of the immature BM cell compartment through the identification of CD34+ cells and may contribute to a better characterization of these MDS low-risk categories. However, there is a wide variety of FCI strategies to study the CD34+ cells, without a universal consensus on how many and which markers should be used to reach their best identification. There are some studies evaluating the correlation between FCI and the morphological blast cells count, but it is not clear if FCI allows a good concordance among several observers when the morphological blast count is <5%. Objectives1-To explore the concordance among 6 FCI observers to quantify the CD34+ myeloid BM cells from patients diagnosed with MDS with <5% BM blasts 2- To study the correlation between FCI and morphology for detecting <5% BM blast cells. 3- To determine if the mophological threshold of 2% is reproducible by FCI. MethodsFCI data files from 48 MDS BM samples with <5% blasts were simultaneously and independently evaluated by 6 FCI observers from 6 different Spanish hospitals. According to the WHO criteria patients were distributed as follows: 3 refractory cytopenia with unilineage dysplasia; 13 refractory anemia with ring sideroblasts; 25 refractory cytopenia with multilineage dysplasia; 1 unclassifiable MDS; 2 chronic myelomonocytic leukemia and 4 therapy-related myeloid neoplasms. Each participant contributed with 8 samples and all files were exchanged among them. All of them used the INFINICYTTM software program for analysis according to their usual strategies. The morphological quantification of BM blast cells was provided by each centre and was blinded to the others. Each centre processed the samples for FCI according to their usual strategies in their clinical practice, without previous agreement on standardization of the protocols used. All centres used the stain-lyse-wash protocol but panels of monoclonal antibodies were different: combinations of 4, 6 and 8 fluorochrome–conjugated monoclonal antibodies were included in 28, 8 and 12 files respectively. Median number of events recorded per file was 157,200 (range 10,000-500,000). The combination CD34/CD45/CD117 was included in 38 files and 20 also associated HLA-DR. The fluorochrome attached to CD34 was PerCP-Cy5 in 26 samples. 8G12 was the clone used in 40 samples.The degree of agreement among the 6 observers for quantification of CD34+ myeloid cells was evaluated using the intraclass correlation coefficient (ICC). The generalized kappa statistic for multiple rates (κ) calculated the concordance among observers after categorization of quantitative variables. Both the ICC and the generalized κ statistic were interpreted as follows: 0-0.2 poor; 0.3-0.4: fair; 0.5-0.6 moderate; 0.7-0.8 strong; >0.8 almost perfect agreement. ResultsFinally, 47 samples could be evaluated by the FCI observers. The ICC showed a strong agreement among observers (0.720), and also a good concordance on the quantification of CD34+ cells at the critical level of 2% (k=0.587). Regarding the comparison between FCI and morphology, only one participant counted >5% CD34+ cells in a sample. However, the absolute quantification of BM blasts <5% by FCI showed poor agreement with morphology (ICC ranged from 0.106 to 0.458). Indeed, none of the FCI observers could reproduce the new morphological categories using the threshold of 2% BM blasts (k= 0.320). ConclusionsIn our study, FCI seems a reproducible tool to quantifying CD34+ cells in MDS patients with <5% BM blasts, despite of the great heterogeneity of the protocols used. A FCI threshold of 2% CD34+ cells was also reproducible among observers. However, the lack of a precise correlation between the morphological blast cell count and the number of CD34+cells by FCI, illustrates the importance of considering each value independently. Probably, homogenization of the FCI protocols will contribute to improve the correlation between the 2 techniques. Disclosures:No relevant conflicts of interest to declare.

Development Of a Preclinical Human Xenograft Model For Myelodysplastic Syndrome In Immunodeficient Mice Expressing Human Growth Factors

Myelodysplastic syndromes (MDS) are a diverse class of hematopoietic disorders, defined by progressive bone marrow failure, myeloid dysplasia, and an increased risk of acute myeloid leukemia (AML). The development of treatments for MDS has been hampered by the lack of an adequate in vivo model of disease. Mouse xenograft models for MDS have been challenging, as samples from patients with MDS have exhibited poor engraftment when transplanted into immunodeficient mice, with little evidence of persistent disease in recipient mice. Here, we tested a novel xenograft mouse model of MDS using an immunodeficient mouse strain that constitutively expresses human hematopoietic growth factors, SCF, GM-SCF and IL-3 (SGM3). These mice demonstrate improved engraftment of human hematopoietic cells from MDS patient samples. We have used bone marrow from patients diagnosed with MDS with blast counts ranging from 2-15% and differing cytogenetic profiles. Unfractionated bone marrow cells we injected into both NOD/LtSz-scid IL2RG-/- (NSG) and NOD/LtSz-scid IL2RG-/- SGM3 (NSGS) mice. These studies demonstrate significantly improved engraftment in the NSGS mice compared to NSG mice. Specifically, 1 out of 4 MDS patient samples demonstrated engraftment in NSG mice, but 3 out of 4 MDS samples demonstrated engraftment in NSGS mice. In NSG mice, we noted approximately 5-6% of human CD45+ cells in peripheral blood at 6 weeks after injection in one MDS sample, but subsequent to that, there was a drop to less than 1 percent of human CD45+ cells detected at 9 and 12 weeks post injection. The NSGS mice demonstrated more robust engraftment up to 15% human CD45+ cells in the peripheral blood at the 6 week time point in some samples, but subsequently fall to 1-2 % at 12 weeks post injection. This demonstrates that the NSGS strain of mice demonstrates improved xenograft efficiency of MDS bone marrow samples. Surprisingly, higher levels of blasts in the patient samples did not necessarily correspond to increased human CD45+ cell engraftment in the NSGS mice. This suggests the NSGS mice do model the MDS patient characteristics rather than overt leukemia. These results support our hypothesis that constitutive expression of human growth factors can promote engraftment of hematopoietic cells from patients with MDS. These studies will allow us to now use of this NSGS mouse model to test novel drug and/or immunotherapy approaches against human MDS in an in vivo mouse model system. Disclosures:No relevant conflicts of interest to declare.

Biology of Myelodysplasia Clonality: Clinical Implications of Whole-Genome Sequencing

Abstract SCI-32The discovery of acquired genetic mutations associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has rapidly expanded with the use of next-generation sequencing technologies. The distinction between MDS and de novo or secondary AML (arising after MDS) and therapeutic recommendations are largely based on the presence of morphologic dysplasia and the percentage of bone marrow myeloblasts. Using whole genome sequencing to define the allele burden of hundreds of mutations, our group has shown that nearly all the bone marrow cells in seven patients with MDS and secondary AML were clonally derived (∼85%), regardless of the myeloblast count. We now have experimental evidence suggesting that the vast majority of mutations in these samples are randomly acquired during aging (passenger mutations) and carried forward in a cell when it is transformed. By using all the validated mutations in a sample (passengers and drivers) we found that five of the seven MDS samples harbored two or more malignant clones and that all seven contained a founding clone that gave rise to subsequent daughter clones. Thus, the MDS bone marrow samples are a mosaic of several tumors with unique sets of mutations each shaped by the acquisition of a series of mutations and clonal diversification. Each clone contained at least one coding gene mutation, and we identified 11 recurrently mutated genes in the seven samples, including recently described mutations in U2AF1, a spliceosome gene that is typically mutated in the MDS founding clone. In addition, the MDS founding clone always gave rise to the dominant secondary AML clone, suggesting that therapies targeting mutations in the founding clone may have benefit in treating both MDS and secondary AML. It is possible that therapeutic response and disease progression is driven not only by the presence of recurrent mutations, which have prognostic value, but also by the clone (i.e., founding and daughter) in which they arise. We are currently testing this hypothesis by monitoring the dynamic change in clone size, including driver mutation allele burdens, during treatment of patients with AML and MDS. Disclosures:No relevant conflicts of interest to declare.

Multiparameter Flow Cytometry in Patients with Suspected Myelodysplastic Syndromes Adds Significant Prognostic Information: A Study on 1,013 Patients

Abstract 1722 Backgroundn:Multiparameter flow cytometry (MFC) has been demonstrated capable of identifying aberrant antigen expression related to myelodysplastic syndromes (MDS). The exact role and place of MFC in the diagnostic work-up of patients with suspected MDS, however, remains to be defined. Aim: Evaluation of the diagnostic impact of MFC in relation to cytomorphology (CM) and cytogenetic (CG) by determining the association of MFC results to overall survival (OS). Patients and Methods: In 1,013 patients with suspected MDS bone marrow samples had been analyzed in parallel by MFC, CM, and CG. CM confirmed MDS in 511 patients, excluded MDS in 277 patients, and showed dysplastic features but not sufficient to unequivocally diagnose MDS by CM in 225. The MFC diagnostic result was in agreement with MDS (“MDS by MFC”) in 446 patients including 382/511 patients with MDS proven by CM. CG revealed an aberrant karyotype in 245/1,013 patients. The median follow-up time amounted to 14.8 months, a total of 156 deaths was recorded. Results: The first set of analyses was performed on the cohort of 511 patients with MDS confirmed by CM. The median total number of aberrantly expressed antigens amounted to 3 (range, 0–11) and included expression of mature antigens in myeloid progenitors; abnormal CD13-CD16- and CD11b-CD16-expression patterns, aberrant expression of myeloid markers and reduced side scatter signal (SSC) in granulocytes; reduced expression of myelomonocytic markers in monocytes; aberrant expression of CD71 in erythroid cells; as well as expression of lymphoid markers in all myeloid cell lines. A higher total number of aberrantly expressed antigens as a continuous variable correlated with a shorter OS (Cox analysis, p=0.008). Next, patients were categorized based on the three parameters i) at least 3 aberrantly expressed antigens, ii) significantly reduced SSC in granulocytes, and iii) >5% myeloid progenitor cells in MFC. Patients with at least one of these criteria had a significantly shorter OS than those without (median 48.5 months vs. not reached (n.r.), p<0.001). Overall, the global diagnostic rating of “MDS by MFC” was the strongest MFC parameter: Patients with “MDS by MFC” had a shorter OS as compared to patients without (median 56.8 months vs. n.r., p=0.001). Non-MFC parameters related to OS in univariable Cox analysis included WBC count, thrombocyte count, CG (grouped according to IPSS), % blasts by CM (p<0.001 each), Hb level (p=0.001), and age (p=0.002). In order to determine the clinical relevance of “MDS by MFC” a multivariable analysis for OS was performed on this parameter together with non-MFC parameters (blood counts excluded due to incomplete data sets). It revealed an independent relation between “MDS by MFC” and OS (p=0.045). This was also true for relation of OS to the other parameters (CG, p<0.001; age, p=0.001, % blasts by CM p=0.014). Given this strong prognostic value of “MDS by MFC” in cases with MDS proven by CM a second set of analyses on the relation between MFC findings and OS were performed for the complete cohort of 1,013 patients, i.e. additionally including all cases with a diagnostic result by CM of “no MDS” or “dysplastic features not sufficient to diagnose MDS”. Again, significant relations to OS was found for the total number of aberrantly expressed antigens as a continuous variable (Cox analysis, p<0.001), for at least one of the above mentioned criteria i), ii) or iii) (median 75.6 months vs. n.r., p<0.001), as well as for “MDS by MFC” (median 60.5 months vs. n.r., p<0.001). Again, “MDS by MFC” proved to be the most relevant MFC parameter. Multivariable Cox analysis for OS including “MDS by MFC” and non-MFC parameters revealed a trend only for “MDS by MFC” (p=0.135) and significance for the other parameters (age, p<0.001; CG, p<0.001; blasts by CM, p=0.045). Conclusions: 1) The present data indicates the diagnostic use of MFC for MDS results in independent prognostic information for cases with MDS as proven by CM. 2) Furthermore, the diagnosis of MDS by MFC has a strong prognostic impact even without prove of MDS by CM which strengthens the diagnostic value of MFC even more. 3) This analysis therefore argues in favour of diagnosing MDS not only based on a combination of CM and CG but of adding also MFC for better classification and even prognostication in the future. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: Report from the Dutch Working Party on Flow Cytometry in MDS

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005–2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Myelodysplastic Syndromes Are Characterized by Gene Expression Changes in Hematopoietic Stem Cells and Alterations in Hematopoietic Stem Cell and Myeloid Progenitor Composition.

Abstract 1762Poster Board I-788The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders characterized by peripheral cytopenias due to impaired hematopoietic differentiation. To date, most data characterizing the immature hematopoietic compartment in MDS have relied on evaluation of CD34+ bone marrow cells, which are a heterogeneous population containing a predominance of oligo- and unilineage-potent progenitors and few hematopoietic stem cells (HSC). In this study we show that MDS are disorders of HSC, evidenced by the presence of recurrent cytogenetic alterations, including -5q, -7, and -20q, in highly purified HSC (Lin-CD34+CD38-CD90+CD45RA-) by FISH. Because MDS HSC harbor cytogenetic changes, we sought to better characterize the molecular basis of MDS HSC function by performing whole transcriptome analysis of highly purified HSC and committed myeloid progenitor populations from low-risk (n=8) and high-risk (n=2) MDS patients. When compared to control HSC from healthy patients (n=10), MDS HSC showed broad transcriptional changes. Using the significance analysis of microarrays (SAM) algorithm and Ingenuity Pathways Analysis software, we identified 3,258 differentially expressed genes (FDR < 0.1) with increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). Interestingly, while MDS common myeloid progenitors (CMP, Lin-CD34+CD38+CD123+CD45RA-) showed increased expression of cell death-related genes when compared to normal CMP (p < 0.001), neither MDS HSC nor multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-) showed significant differential expression of these genes when compared to their normal counterparts. To assess the cellular and developmental correlates of HSC/committed progenitor transcriptional changes, we evaluated by flow cytometry the frequency of HSC and committed myeloid progenitors in bone marrow aspirates from 35 low-risk MDS, 6 high-risk MDS and 32 healthy patient samples (range 4-84 yo). Low-risk MDS bone marrow samples showed significantly increased numbers of HSC compared to normal bone marrow samples (+3-fold change, p < 0.03). In addition, myeloid progenitor composition was frequently altered in low-risk MDS patients, with decreased percentages of granulocyte-macrophage progenitors (GMP, Lin-CD34+CD38+CD123+CD45RA+) when expressed as a percentage of total myeloid progenitors [including GMP, CMP and megakaryocyte-erythroid progenitors (MEP, Lin-CD34+CD38+CD123loCD45RA-)] (-2.3-fold change, p < 1e-6). This altered myeloid progenitor profile was highly specific to MDS, even when MDS patient samples were compared to a group of control bone marrow samples from non-MDS patients exhibiting at least one cytopenia (n=34, p < 1e-5), allowing for the distinction of MDS samples from non-MDS cytopenias with 0.89 sensitivity and 0.89 specificity. Together, these data indicate that MDS HSC exhibit significantly altered gene expression profiles and suggest that gene expression changes in MDS HSC induce the altered developmental fate decisions and transcriptional changes observed in MDS committed myeloid progenitors. These data also demonstrate that the changes in MDS myeloid progenitor composition may provide a novel, flow cytometric method for distinguishing MDS from other hematologic conditions that mimic MDS. Finally, these studies indicate that molecular characterization of MDS phenotypes may require evaluation of purified hematopoietic progenitors in order to account for the differential effect of MDS-associated changes on specific hematopoietic progenitor populations. DisclosuresWeissman:Amgen: Equity Ownership; Cellerant Inc.: Founder; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Comprehensive Genomic Copy Number and Sequence Analysis of 28 Chromosome 5q31.2 Candidate Genes in De Novo MDS.

Deletions involving the long arm of chromosome 5 are among the most common recurring cytogenetic abnormalities detectable in human myelodysplastic syndromes (MDS). The minimally deleted segment has been mapped by several groups to a 2.3 megabase interval at 5q31.2. This region contains 21 annotated genes, one pseudogene, 4 small RNAs, and 2 predicted genes (SPOCK1, KLHL3, HNRPA0, NPY6R, MYOT, PKD2L2, C5orf5, WNT8A, NME5, BRD8, KIF20A, CDC23, GFRA3, CDC25C, FAM53C, JMJD1B, REEP2, EGR1, ETF1, HSPA9B, SNORD63, LOC391836, LOC729429, CTNNA1, LRRTM2, SIL1, MATR3, SNORA74A). Extensive study of many of these genes by other investigators has raised the possibility that gene(s) in this interval contribute to MDS pathogenesis by haploinsufficiency. To address this hypothesis, we selected samples from 46 de novo MDS patients that had adequate amounts of paired tumor (bone marrow) and germline (skin) DNA available. The 46 patients include all FAB subtypes, with a range of IPSS scores from 0–3 (median = 1), and a median blast count of 5%. The majority of these samples (n=37) do not have cytogenetically apparent 5q31.2 deletions. We first asked whether these samples might contain deletions in this interval below the limit of detection by cytogenetics. We used a custom oligomer based array comparative genomic hybridization (aCGH) platform developed by NimbleGen Systems, Inc. that has ∼385,000 probes spanning human chromosome 5, providing an average probe spacing of 500 base pairs. Analysis of aCGH data for 20 patients demonstrated that cytogenetically apparent 5q31.2 deletions could be detected by this platform, but no cytogenetically “silent” interstitial deletions were seen. We next asked whether genes in this interval might instead be targeted by point mutations. To define the sensitivity of our sequence-based studies, we used standard agarose gel electrophoresis and the Agilent LabChip to detect FLT3 internal tandem duplications (ITD) in 3 of 90 MDS samples. We chose one sample with an ITD and performed 6 serial dilutions of MDS DNA with wild-type genomic DNA and performed PCR amplification followed by DNA sequencing of the PCR products. We could detect the FLT3 ITD when 12% of the alleles harbored the mutation, or when ∼20–25% of cells contain a heterozygous mutation. From these results, we are confident that our strategy can detect mutant alleles in unpurified MDS bone marrow samples, but may miss mutations occurring in a rare cell. We then designed and validated primers for 415 amplicons covering the coding region and proximal introns of all 28 genes in the 5q31.2 interval and produced 7.2 megabases of sequence for these genes in the 46 patient samples. No somatic changes were identified. Twelve novel non-synonymous SNPs were discovered. The allele and genotype frequencies of 49 known SNPs in these genes were similar to the frequencies in race-matched HapMap individuals, apart from an enrichment in a non-synonymous CDC25C SNP in the MDS cohort (rs3734166, Odds Ratio=1.87, 95% confidence interval=1.06–3.32, p=0.036). The over-representation of this SNP and the other rare novel SNPs in the MDS population suggest that they may mark susceptibility alleles for MDS. Taken together, these data suggest that small deletions and/or point mutations of genes in 5q31.2 are not common events in the pathogenesis of MDS, and that larger deletions leading to haploinsufficiency of several genes in the interval appear to be the dominant mechanism.