Cells Of Myelodysplastic Syndromes Patients Research Articles

The Biological Function of DNA Hypermethylation in Murine MDS Model Lacking Tet2 and Ezh2

Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2fl/fl)(Muto T, et al. J Exp Med 2014).In order to determine how DNA methylation contributes to the formation of MDS in Tet2KD/KDEzh2Δ/Δ mice, we transplanted wild type (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Cre-ERT;Tet2KD/KDEzh2fl/fl fetal liver cells in lethally irradiated CD45.1+ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin-Sca1+Kit+ (LSK) cells isolated from Tet2KD/KD and Ezh2Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2KD/KDEzh2Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value<0.01). We found that Ezh2Δ/Δ LSK cells gain significantly larger number of hyper-DMRs at CpG islands (CGIs) over time compared with Tet2KD/KD LSK cells. At 8 months post-deletion, Tet2KD/KD and Ezh2Δ/Δ LSK cells gained hyper-DMRs in 196 and 366 out of 12677 promoters, and 106 and 306 out of 11504 CGIs, respectively. Tet2KD/KDEzh2Δ/Δ-MDS LSK cells at 5 months post-deletion had hyper- and hypo-DMRs in 109 and 51 out of 12677 promoters, and 124 and 27 out of 11504 CGIs, respectively. Hyper-CGI DMRs of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells largely overlapped with those of Tet2KD/KD and Ezh2Δ/Δ LSK cells (p<1.00E-16). Nonetheless, a significant number of hyper-CGI DMRs were newly generated in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells, suggesting unique impact of combined loss of Tet2 and Ezh2 on epigenome. Of note, hyper-DMRs in this mouse MDS model significantly overlapped with those in human CD34+ cells of MDS patients (Maegawa S, et al. Genome Research 2014) (40 genes, p=0.0001). As reported in solid tumor and aging stem cells, polycomb-group (PcG) protein targets were significantly enriched among hyper-CGI DMRs in LSK cells of all three genotypes; Tet2KD/KD, Ezh2Δ/Δ, and Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Indeed, hypermethylated CGIs were enriched for genes encoding DNA binding transcription factors (p=8.96E-05) and cell differentiation (p=3.98E-05) in GO analysis, suggesting that DNA methylation involves the PcG targets such as developmental regulator genes that regulate hematopoietic cell differentiation in the pathogenesis of MDS.To determine an association between levels of differential DNA methylation and transcription of genes, we next performed DNA microarray analysis by using LSK cells. As several studies reported, we observed no significant association between levels of DNA methylation and gene expression (R2=0.003). However, 27 hypermethylated genes showed reduced levels of gene expression in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Those included key transcription factors and epigenetic modifiers for hematopoiesis, such as Gata2, Gata3, Evi1, and Bcor.Finally, we examined whether decitabine, a demethylating agent, reverses the DNA hypermethylation of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells in vivo. Although the administration of decitabine into secondary recipients of MDS BM cells did not improve the survival of mice, platelet counts were significantly increased in decitabine-treated MDS mice, compared to the control MDS mice. After 4 weeks of treatment, a partial restoration of DNA hypermethylation was observed in the decitabine-treated MDS mice (34 out of 124 CGIs; p<1.00-16E). These regions were again enriched in DNA binding transcription factors (p=1.72E-05) and cell differentiation (p=2.25E-07) in GO analysis.Taken together, our work unveiled the biological function of DNA hypermethylation in the pathogenesis of MDS, and will give a clue to understanding how demethylating agents improve the outcome of patients with MDS. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Implications Of PRAME Expression Levels In Myelodysplastic Syndromes

The preferentially expressed antigen of the melanoma (PRAME) gene was first identified in melanoma tissue as a tumour-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. While expression level of PRAME is quite low in most of normal tissues of human, including bone marrow normal CD34+ cells, except for testis, PRAME overexpression is observed in various human cancers, including hematological malignant disorders. Although clinical and biological significance of PRAME expression in human cancers has not been fully elucidated, it has been demonstrated that high PRAME expression is associated poor clinical outcomes in a number of solid cancers. Association between PRAME expression and clinical outcomes in hematological malignancies has not been clarified. In acute myeloid leukemia, high expression of PRAME was shown to be associated with worse progression-free survival or overall survival by some research groups but not by other groups. Our previous study demonstrated that inhibition of PRAME expression in leukemic cells results in cell cycle arrest and induction of apoptosis, suggesting oncogenic function of PRAME expression in leukemogenesis. Clinical significance of PRAME expression in myelodysplastic syndromes (MDS) also has not been fully elucidated. In the present study, we examined PRAME expression of bone marrow cells in MDS patients to clarify clinical significance of PRAME expression.Bone marrow samples of MDS patients were used for analysis. Samples were taken at the time of diagnosis with written informed consent from patients. Total RNA extraction, cDNA synthesis and quantitative real-time RT-PCR by the TaqMan probe method using an ABI 7500 real-time PCR system (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems), were performed. Expression levels were obtained using the standard curve method in each experiment, after normalization with the GAPDH gene for each sample in duplicate wells. The human PRAME primer-probe sets were from Applied Biosystems (assay ID: Hs00196132_m1). Data including patients' demographic, disease status, medical history, clinical and laboratory findings, and outcome, were collected from medical records and laboratory data base.A total of 111 MDS patients, 68 males and 43 females with median age of 69 years (range: 20-91 years) were included in the present study. They were classified as RCUD (n=15), RCMD (n=61), RARS (n=8), RAEB-1 (n=15), and RAEB-2 (n=12) according to WHO classification. Based on the IPSS, they were categorized in four risk groups, low risk (n=29), intermediate-1 risk (n=55), intermediate-2 risk (n=23), and high risk(n=4). Expression level of PRAME was varied among the MDS patients analyzed. Median value of relative PRAME expression level of 111 MDS patients and that of 19 control subjects were 0.073 and 0.07, and there was no significant difference in distribution of expression level of PRAME. However, when we compared expression patterns of PRAME among five WHO-subtypes, statistical difference was observed (P=0.0116). Relative PRAME expression level in WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) was significantly higher than that in WHO-subtypes with less blast counts (RCUD, RCMD, RARS) (median value: 0.44 vs. 0.05, P=0.0012). To investigate prognostic implication of PRAME expression in MDS, we analyzed impact of PRAME expression on overall survival (OS). Based on PRAME expression level, 111 patients were divided into two categories, ‘high expression group' (above median value) and ‘low expression group' (below median value). Kaplan-Meier analysis demonstrated that high expression group showed significantly poorer overall survival than low expression group (P=0.0165). The estimated 5-year OS rates in high expression group and low expression group were 63.5% and 78.4%, respectively.The present study demonstrated that high PRAME expression is associated with poorer clinical outcome, indicating that PRAME expression could be a useful prognostic marker in MDS. Biological significance of PRAME expression in MDS is unclear. Expression level of PRAME was higher in WHO-subtypes with high blasts counts, suggesting that PRAME may play role in disease progression in MDS. Further studies should be necessary to clarify clinicopathological and biological significance of PRAME expression in MDS. Disclosures:No relevant conflicts of interest to declare.

Characterization Of T Follicular Helper Cells In Patients With Low Risk Myelodysplastic Syndromes

A subpopulation of CD4+helper T cells, T-follicular helper cells (TFH), are characterized by the surface expression of CXCR5, ICOS, and PD1, the transcription factor Bcl-6, and produce mainly IL-21, but also IL-17, IL-4, and IFN-γ. They represent the major population that helps B cells to turn into plasma cells and they are implicated in the pathogenesis of different autoimmune diseases. Peripheral CXCR5+CD4+ T helper cells (p-TFH) are the circulating component of TFH. p-TFH cells have been extensively studied in the context of inflammation and autoimmunity. Patients with systemic lupus erythematosus and rheumatoid arthritis have increased p-TFH. Immune dysregulation characterizes low risk myelodysplastic syndromes (MDS). The expression of p-TFH in patients in low risk MDS has not been previously evaluated. To examine the expression of p-TFH in this disease we isolated peripheral blood mononuclear cells (PBMCs) from patients with low risk MDS (n=20, Refractory Anemia,RA; and Refractory Cytopenias with Multilineage Dysplasia, RCMD) and ten healthy, age- matched controls. Written informed consent was obtained from all study subjects. PBMCs were left untreated or activated with PMA and Ionomycin, in the presence of Brefeldin A, for 5 hours. Subsequently, cells were stained with the surface markers CXCR5, CD4, CD45RO, ICOS and intracellular IL-21, and analyzed by flow cytometry. Patients with MDS show decreased CD4+ICOS+ cells compared to healthy controls (5,34±1,44% vs 8,69±4,08% respectively, p=0.028). The median percentages of CXCR5+CD4+ cells (estimated on CD4+ cells) before stimulation were lower in MDS patients although not statistically significant different from the control subjects. The expansion of the CXCR5+CD4+ population after stimulation was higher in MDS patients compared to healthy individuals (ratio of stimulated to unstimulated CXCR5+CD4+ cells of MDS patients: 1.44±0.47 vs control: 1.23± 0.32, p=0.027). MDS patients at diagnosis showed increased median levels of IL-21 producing CXCR5+CD4+ cells compared to patients previously treated with erythropoietin (EPO), (21,34±10,14% vs 17,55±12,91, respectively). Additionally, patients with RA showed increased CXCR5+CD4+IL-21+ cells compared to the RCMD patients (22,39±11,76% vs 17,84±10,70%, respectively). Collectively, the percentage of IL-21 producing CXCR5+CD4+ cells did not differ significantly between the MDS and control groups (20,57± 10,12% vs 19,86± 11,03%, respectively). The presence of marrow fibrosis did not affect the differences observed and described above. The p-TFH cell subset was identified in every case as a memory component of CD4+ T helper cells, as previously described. Although the number of patients analyzed is limited, our results suggest that low risk MDS patients show a trend of lower CXCR5+CD4+ cells compared to age–matched control subjects. EPO treatment eliminates IL-21 production from CXCR5+CD4+ cells compared to treatment-naïve patients. Further analysis of a larger pool of subjects along with the examination of the specific transcription factor Bcl-6 will reveal the role of p-TFH cells in low risk MDS. Disclosures: No relevant conflicts of interest to declare.

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.

Application of flow cytometry-fluorescence in situ hybridization in the measurement of relative telomere length of bone marrow CD34(+) cells in patients with myelodysplastic syndrome

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.

Centrosome aberrations in bone marrow cells from patients with myelodysplastic syndromes correlate with chromosomal instability

Centrosomes play important roles in the maintenance of genetic stability and centrosomal aberrations are a hallmark of cancer. Deregulation of centriole duplication leads to supernumerary centrosomes, sister chromatid missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 45% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. BM cells of 34 MDS patients were examined cytogenetically. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin to analyze the centrosomal status. Umbilical cord blood specimens and BM cells of healthy persons (n = 11 and n = 4) served as controls. In addition, the protein expression of the protease separase responsible for genetic stability was examined by western blot analysis. Centrosome abnormalities were detected in 10% (range, 4-17%) of cells of MDS samples, but in only 2% (range, 0-4%) of cells of healthy controls. Normal karyotypes were found in control cells and in BM cells of 16/34 MDS patients. The incidence of centrosomal alterations was higher in BM cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). Our results indicate that centrosome alterations are a common and early detectable feature in MDS patients and may contribute to the acquisition of chromosomal aberrations. We assume that centrosome defects could be involved in disease progression and may serve as a future prognostic marker.

IL10 inversely correlates with the percentage of CD8+ cells in MDS patients

The role of the immune system in myelodysplastic syndrome (MDS) progression has been widely accepted, although mechanisms underlying this immune dysfunction are not clear. CD4+ and CD8+ lymphocyte profiles in the peripheral blood of MDS patients were evaluated and correlated with clinical characteristics, the expression of FOXP3 and the anti-inflammatory cytokines IL10, TGFβ1 and CTLA4. IL10 expression inversely correlated with the percentage of CD8+ cells and was higher in high-risk MDS. Our findings provide further evidence for the role of T cell-mediated IL10 production in MDS and strengthen the idea of distinct cytokine profiles in low and high-risk MDS.

Iron overload impairs proliferation of erythroid progenitors cells (BFU-E) from patients with myelodysplastic syndromes

In patients with myelodysplastic syndromes (MDS) iron overload caused by long-term red blood cell transfusions is an important factor for comorbidity especially in low-risk MDS. In this report we present the results of a comparative study based on colony formation assays of hematopoietic cells in MDS patients with and without iron overload. We demonstrate that iron overload suppresses the proliferation of erythroid progenitors cells (BFU-E), while the myeloid compartment (CFU-GM) was not found to be affected. Even patients with slightly elevated ferritin values show an impaired proliferation capacity in comparison to patients with normal ferritin levels. Furthermore, we show that this negative impact is reversible by sufficient iron chelation therapy.

TET2 Gene Expression in Bone Marrow Cells in Myelodysplastic Syndromes Patients and the Effect of Silencing TET2 by siRNA On Biological Characteristics of Healthy CD34+ Cells

Abstract 4934 ObjectiveTo investigate the expression of TET2 mRNA in bone marrow mononuclear cells(BMMNC), CD3+ T cells and CD34+ cells of patients with myelodysplastic syndrome(MDS)and to study the effect of silencing TET2 by Small interfering RNA (siRNA) on the biological characteristics of CD34+ cells. MethodsCD3+ T cells and CD34+ cells were sorted by magnetic activated cell-sorting system. The expression of TET2 mRNA was detected by qPCR. The silencing effect of RNA interference (RNAi) on TET2 expression was identified by qPCR and Western blot analysis. The cell cycle kinetics and cell apoptosis were detected by flowcytometry. ResultsThe expressions of TET2 mRNA in BMMNC, CD3+ T cells and CD34+ cells were down-regulated in MDS compared with that in controls. The siRNA targeting TET2 suppressed the expression of TET2 in CD34+ cells. Meanwhile, the proliferation activity was significantly enhanced and the apoptosis rate was declined in cells which transfected with TET2 siRNA as compared to those of the cells transfected with scrambled siRNA and control cells. ConclusionsThe mRNA expressions of TET2 in BMMNC, CD3+ T cells and CD34+ cells of MDS patients were decreased. Suppression of TET2 expression renders the CD34+ cells harboring more aggressive phenotype. This preliminary finding suggests that CD34+ cells lowering expression of TET2 may play an oncogenic role on myeloid tumor. Disclosures:No relevant conflicts of interest to declare.

The Proliferation Inhibitor CDKN1C (P57KIP2) Is Over Expressed in CD34+ Cells of Patients with MDS and Determines a Worse Prognosis Independently of IPSS Score Factors

AbstractAbstract 3820 Purpose:Progressive cytopenias are the cause of death for the majority of patients with myelodysplastic syndromes (MDS). In order to investigate if the proliferative activity of CD34+ cells in MDS bone marrow correlates with prognosis, we measured expression levels of the proliferation inhibitor CDKN1C (P57KIP2) in two independent study cohorts. Patients and methods:Gene expression profiling data on bone marrow CD34+ cells were obtained from 183 MDS patients (55 with RA, 48 with RARS, 37 with RAEB1 and 43 with RAEB2). mRNA expression levels of CDKN1C (P57KIP2) were correlated with overall survival (OS) and proliferation markers such as PCNA, cyclins and cyclin dependent kinases.In a second independent patient cohort comprising 93 patients (17 had RA, 13 REAB1, 27 RAEB2, 29 AML arising from MDS and 7 CMML), protein expression levels of CDKN1C (P57KIP2) were evaluated by immune histochemistry (IHC) in trephine biopsies. Protein expression levels of CDKN1C (P57KIP2) were correlated with clinical outcome, OS and the proliferation marker KI67 in CD34+ cells (double staining). Results:In purified CD34+ cells, mRNA expression of CDKN1C (P57KIP2) correlated with higher risk and poorer overall survival of patients with MDS (p=0.0006, n=183, Figure1). Furthermore, increased CDKN1C (P57KIP2) expression was significantly associated with loss of CD38 (p=0.002) as well as loss of proliferating cell nuclear antigen (PCNA, p<0.001), cyclins and cyclin-dependent kinases (p<0.001) underlining the role of CDKN1C (P57KIP2) as a proliferation inhibitor.Similarly, protein expression of CDKN1C (P57KIP2) determined in trephine biopsies predicted a poor prognosis of patients with MDS (p=0.0003, HR=2.2, n=93, Figure 1). No expression of CDKN1C (P57KIP2) could be demonstrated in 10 control patients with normal bone marrow. Separate evaluation of WHO risk categories revealed that in patients with less than 10% blasts (RA, RAEB1 and CMML1), CDKN1C (P57KIP2) was not predictive for OS. In contrast, patients with high CDKN1C (P57KIP2) expression and RAEB2 (p=.0.02, HR 2.4) or AML arising from MDS (p=0.002, HR 2.9) had a significantly worse OS than patients with low CDKN1C (P57KIP2) levels. CDKN1C (P57KIP2) expression analysis within the group of allogeneic stem cell recipients showed no impact on survival after transplant. Multivariate cox regression analysis with the confounding co-variates: age, IPSS score factors (cytopenias, cytogenetic risk profile, blast count) and primary versus secondary MDS confirmed the independent impact of CDKN1C (P57KIP2) expression on OS (p=0.0002, HR 2.9). CDKN1C (P57KIP2) protein expression could also be demonstrated in sorted CD34+ cells of MDS patients by western blot analysis and was significantly higher than in CD34- cells (p=0.03, n=7).KI67 expression was evaluated in CD34+ cells by IHC double staining in 34 trephine biopsies and 10 control patients with normal bone marrow. Percentages of CD34+ and KI67 positive cells were higher in control patients and patients with low risk MDS (RA, RAEB1) than in patients with high risk MDS (RAEB2) (p<0.01). Patients with AML (>20% blasts) had significantly higher levels of KI67 (p<0.05). In patients with MDS (<20% blasts) KI67 percentages correlated inversely with CDKN1C (P57KIP2) expression (p=0.04). Conclusions:High CDKN1C (P57KIP2) expression in CD34+ cells of patients with MDS is associated with a reduced fraction of proliferative CD34+ cells and determines a worse prognosis independently of factors used to calculate the IPSS score. Allogeneic stem cell transplantation could overcome the worse prognosis of high CDKN1C (P57KIP2) expression. Further studies are warranted to determine the impact of CDKN1C (P57KIP2) expression to guide clinical decisions. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Tyrosine Kinase FLT3 and C-KIT Receptor Expression in Patients with Myelodysplastic Syndromes – A Preliminary Study

AbstractAbstract 4620Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by dyshematopoiesis and high susceptibility to acute myeloid leukemia.Deregulated epigenetic mechanisms are likely involved in the pathogenesis of MDS. Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Aberrant methylation of gene promoter region is responsible for inappropriate gene silencing, and it has been associated to initiation and progression of cancer. However, in the MDS disease process, more and more gene dysfunction has been related with the pathogenesis. FLT3 and c-KIT are important members of the receptor tyrosine kinase family that are overexpress or dysexpress in many malignant hematologic diseases. However, little is known about the distribution and the role of these proteins in MDS.The study is to investigate the role of receptor tyrosine kinase FLT3 and c-KIT expression in patients with myelodysplastic syndromes (MDS) and their clinical implication.We have at moment examined c-kit protein (CD117) expression by flow cytometry, in CD34 bone marrow cells collected at diagnosis of 12 patients with de novo MDS and 5 non-neoplastic patients (controls). FLT3 mutations, in particular Internal Tandem Duplications (ITD) and the D835 mutation were analysed by PCR-RFLP. The median age was 72 years (22–89), gender M/F=5/7, WHO subtypes: RCMD (n=6), RA (n=3), RARS (n=1), AREB-2 (n=1), CMML (n=1) and IPSS: low (n=6), intermediate-1 (n=5) and intermediate-2 (n=1). None of the patients evolved to acute leukemia, with a median follow up of 24 months (7–74).Our preliminary results show an increase in c-KIT expression in CD34 positive cells in MDS patients as compared with controls. However, the percentage of c-KIT protein expressing cells was also higher than in the controls in particular in CD34 negative cells. There was a correlation of the c-kIT protein expression with the CD34 antigen of the cells. Expression is correlated with the WHO MDS classification and with IPSS, being highest in RAEB-2 and INT2 MDS prognostic group. These results suggest that the elevated c-KIT expression could maintain the affected clone in MDS. Besides that we didn't find any FLT3 mutations in our population However further data and refinement of data analysis are needed to confirm our results and to predict clinical outcomes. The preliminary results suggest that c-KIT expression could be helpful to the pathogenesis and prognosis prediction of MDS patients. Disclosures:No relevant conflicts of interest to declare.

Interphase Chromosome Flow-FISH

AbstractA 2-day method using flow cytometry and FISH for interphase cells was developed to detect monosomy 7 cells in myelodysplastic syndrome patients. The method, Interphase Chromosome Flow-FISH (IC Flow-FISH), involves fixation of leukocytes from blood, membrane permeabilization, hybridization of cellular DNA with peptide nucleic acid probes with cells intact, and analysis by flow cytometry. Hundreds to thousands of monosomy 7 cells were consistently detected from 10-20 mL of blood in patients with monosomy 7. Proportions of monosomy 7 cells detected in IC Flow-FISH were compared with results from conventional cytogenetics; identification of monosomy 7 populations was verified with FACS; and patient and donor cells were mixed to test for sensitivity. IC Flow-FISH allows for detecting monosomy 7 without requiring bone marrow procurement or the necessity of metaphase spreads, and wider applications to other chromosomal abnormalities are in development.

Let-7a, Mir-17 and Mir-20a Expression Levels in CD34+ Bone Marrow Cells of Patients with Myelodysplastic Syndromes (MDS) Are Associated with Established Prognostic Factors, Supporting Their Implication in the Pathogenesis of the Disease,

Abstract 3792 Introduction:MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose:In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods:We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results:In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion:To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures:No relevant conflicts of interest to declare.

Detection of Highly Clonal CD34+, Mononuclear Bone Marrow and Peripheral Blood Cells From Patients with Myelodysplastic Syndrome and Age Related Increase of Clonal Hematopoiesis In Healthy Subjects Using a Novel Transcriptional Pyrosequencing Based Clonality Assay

AbstractAbstract 977X-chromosome inactivation occurs in female cells leading to stochastically random silencing of one X-chromosome. Accordingly, investigation of X-chromosome inactivation patterns (XCIP) in healthy female tissues revealed similar ratios of cells carrying either an active maternal or paternal X-chromosome. Genomic XCIP markers like the HUMARA locus have been used to elucidate clonality of myelodysplastic syndromes (MDS). However, analysis of healthy female peripheral blood (PB) cells has shown a strong age-related increase in skewing of XCIP suggesting the frequent manifestation of clonal hematopoiesis in the elderly. Since the occurrence of clonality in blood cells from healthy individuals remains controversially discussed (Swierczek et al, Blood 2008), we developed a novel highly accurate transcription based approach using Pyrosequencing to detect clonal hematopoiesis. In addition, we present the first comprehensive XCIP analysis of CD34 purified (CD34+) and unfractioned bone marrow (BM) cells of MDS patients and healthy females.BM, CD34+ and PB cells were obtained from patients with MDS (IPSS-low/int-1-risk BM: n=25, CD34+: n=13, PB: n=21, IPSS-int-2/high-risk BM: n=16, CD34+: n=9, PB: n=12) and age related healthy donors (BM: n=19, CD34+: n=15, PB: n=105) after informed consent. Genomic DNA SNP genotyping was carried out in order to screen for heterozygous clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS, MPP1 and G6PD. After PCR-amplification of informative marker loci from cDNA transcripts, SNP allele ratios, representative for XCIP, were quantified using the PyroMark ID system (Qiagen, Hilden, Germany). Clonality was assumed for samples exhibiting skewed XCIP ratios >80% (allelic ratio of >4:1).Standard curves from pyrosequencing reactions with predefined allelic ratios revealed strong correlations for assessment of XCIP ratios in all markers (R2>0.99). Furthermore, excellent inter-marker correlations of XCIP ratios from individuals with multiple informative markers were achieved ranging from R2>0.83 to R2<0.98. These findings indicate a balanced inactivation of the whole X-chromosome and corroborate the suitability of our method. Of note, distinctively increased numbers of clonal PB samples from healthy elderly donors (age: 65–93 years) with skewed XCIP ratios in 55% of all cases were detected as compared to young donors with skewed XCIP ratios in 13% and 25% for age groups ranging from 18–40 and 18–65 years, respectively. Analysis of XCIP ratios in MDS samples revealed clonality in 96% of BM, 100% of CD34+ and 90% of PB specimen from MDS low/int-1-risk and 100% of BM, 100% of CD34+ and 92% of PB specimen from MDS int-2/high-risk patients. In contrast, only 44% of BM and 43% of CD34+ samples from healthy elderly (age range 65–93 years) and 20% of BM and 25% of CD34+ samples from healthy young females (age range 18–65 years) were graded as clonal. Furthermore, we observed extremely skewed XCIP ratios of >95% in 56% of BM, 62% of CD34+ and 57% of PB cells from MDS low/int-1-risk and 50% of BM, 67% of CD34+ and 67% of PB cells from MDS int-2/high-risk patients but only in 22% of BM, 29% of CD34+ and 3% of PB cells from healthy elderly subjects.Our detection of highly clonal cells in MDS patients in 3 hematopoietic cell compartments and frequent detection of extreme XCIP skewing provides strong evidence for the manifestation of a malignant MDS clone even in early progenitor cells. Moreover, detection of non-clonal PB cells in patients with clonal CD34+ cells indicates the presence of residual healthy hematopoietic cells in the peripheral blood. Notably, in our hands, age related increases of XCIP skewing have been detected in PB cells and, for the first time, in CD34+ and BM cells of healthy females suggesting the clonal outgrowth of hematopoietic cells in the early progenitor compartment of healthy elderly. In conclusion, pyrosequencing based clonality assessment has proven to be suitable for quantitative monitoring of clonal and potentially malignant cell populations in hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Detection of Karyotype Evolution From Peripheral Blood by Sequential FISH Analyses of Circulating CD34+ Cells In MDS Patients: Results of the Ongoing German Multicenter Prospective Diagnostic Study

AbstractAbstract 2937 Introduction:Chromosomal aberrations in myelodysplastic syndromes (MDS) play a major role in diagnostics, pathogenesis, prognosis, and, more recently, in treatment allocations. The acquisition of clonal abnormalities in pts with initially normal karyotype, the expansion of an aberrant cell clone with a given anomaly or the occurrence of new abnormalities are called karyotype evolution (KE). A model of stepwise cytogenetic changes is proposed, but only a few systematic studies had focused on this phenomenon as yet. Chromosomal anomalies can be detected by conventional chromosome banding analyses of bone marrow (bm) metaphases and most of them are provable by Fluorescence in Situ Hybridization (FISH) of circulating CD34+ cells from peripheral blood (pb). Aims:To closely follow chromosomal aberrations from pb, to assess karyotype evolution and rare anomalies and to correlate the molecular-cytogenetic results with pb blood counts, bm morphology and treatment modalities. For this purpose, we initiated a German multicenter prospective, non-interventional diagnostic study in October 2008. Methods:Sequential FISH analyses were performed on immunomagnetically enriched circulating CD34+ cells from pb as follows: a “super-panel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, p53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R) was used for initial screening, every 12 months during follow-up and in every case of suspected progression. A “standard-panel” (EGR1, D7/CEP7, CEP8, p53, D20, CEP X/Y, TEL/AML1) was performed for analyses at short intervals every 2 months in the 1st year and every 3 months during the 2nd year. Results were also correlated with those of conventional banding and FISH analyses performed on bm samples, which typically were collected every 6 months. Results:As yet, 205 pts have been included in the study. Concerning median age, gender distribution, and MDS subtypes (according to WHO and IPSS) the study cohort was representative for the disease. Up to date, chromosomal aberrations were detectable in 126 pts. (62%) by FISH from pb. Most of them had 1 or 2 anomalies, but there were even pts with 4 or more aberrations. The most common aberrations- as known from other studies based on bm analyses- were del(5q) (64%), aberrations of chromosome 7 (del(7q)/-7) (25%), allelic loss of p53 (12%), +8 (11%), del(20q) (10%) and aberrations of chromosome 21 (del(21q)/-21/+21) (7%). Interestingly, a 12p-deletion was detectable as often as a trisomy 8 (11%), and in most cases as part of complex aberrations. A loss of p53 was significantly associated with aberrations of chromosome 7: 67% of pts with aberrations of chromosome 7 showed an allelic loss of p53 at the same time. In 4 cases, a coexistence of del(7q) and -7 in the same clone was observed.Within a median observation time of 7 months (2-21), a KE was detectable from pb by FISH in 17 pts (13%) out of 135 with at least 2 sequential analyses. In most cases del(7q)/-7 developed as a new anomaly, and was followed in frequency by aberrations of chromosome 21. Conclusions:FISH analyses of enriched circulating CD34+ cells are a feasible, less-invasive method to detect chromosomal aberrations in MDS pts and to follow the clone size from peripheral blood. By that method we are able to observe karyotype evolution as a step wise process in detail and to follow even rare abnormalities to learn more about their clinical and prognostic impact. Disclosures:No relevant conflicts of interest to declare.

CD38 Expression On CD34+ Cells as a Single Flow Cytometric Parameter to Distinguish Myelodysplastic Syndromes (MDS) From Non-Clonal Cytopenias.

Abstract 2783Poster Board II-759Myelodysplastic syndromes are a heterogeneous group of bone marrow (BM) disorders characterised by clonal proliferation. The diagnosis of MDS can be challenging, especially in cases with minimal dysplastic changes by morphology and without characteristic karyotypic abnormalities. Recent investigations show that multi parameter analysis of BM samples of MDS patients by flow cytometry (FCM) might be of value in the diagnosis of MDS and for discriminating MDS from other disease entities. Detection of immunophenotypic abnormalities in the immature and the maturing myelomonocytic compartment is even included as a co-criterion in the minimal diagnostic criteria for MDS (Valent et al., Leuk Res 2009,31:727–36). Integration of FCM as a diagnostic tool in MDS requires a simple method with high sensitivity and specificity with low inter laboratory variation. Recently, reduced CD38 expression was demonstrated to discriminate between MDS and other disease entities (Goardon et al., Haematologica 2009, 94:1160–63).To validate the value of CD38 in the diagnosis of MDS, we analysed BM samples of 27 MDS patients (refractory anemia n=4; refractory anemia with multi lineage dysplasia n=15; refractory anemia with excess blasts [RAEB]-1 n=4 and RAEB-2 n=4), age-matched healthy volunteers (n=16) and pathologic controls (n=39). The pathologic control group consisted of aplastic anemia (n=3), iron deficiency (n=6), Multiple Myeloma (n=8), B-cell non-Hodgkin lymphoma (n=6) and autoimmune thrombocytopenia (n=3) patients. We found that CD38 RMFI on CD34+ cells was significantly decreased in MDS with less than 5% blasts (mean±SD: 216.5±95.4) as compared to pathologic controls (mean±SD: 377.2±246.1, P=0.01) and healthy volunteers (mean±SD: 322.8±138.1, P=0.02). In RAEB-1/-2, CD38 RMFI (mean±SD: 111.3±74.9, P=0.001) was even more decreased in comparison with both pathologic controls and healthy volunteers. Expression of CD38 on CD34+ cells is heterogeneous and the most immature cells or hematopoietic stem cells are considered to be negative for CD38 expression. By using the median fluorescence intensity (RMedFI), the skewed distribution of CD38 expression on CD34+ cells is taken into account. In our cohort, RMedFI is significantly decreased in MDS with <5% blasts (mean±SD: 368.1±210.3, P=0.03) and MDS RAEB -1/-2 (mean±SD: 139.7±108.5, P<0.001) as compared to pathologic controls (mean±SD: 521.6±314.2). However, no significant differences were observed between RMedFI of CD38 of MDS with less than 5% blasts and healthy volunteers. In conclusion, we show that CD38 expression is reduced in CD34+ cells of MDS patients and might be of value in the diagnosis of MDS. However, there is considerable overlap in CD38 expression between the MDS with less than 5% blasts group and pathologic controls group and healthy volunteers group. Differentiation of MDS from other disease entities by FCM is based on an accumulation of aberrancies rather than a single aberrancy which might reflect the degree of deviation from normal. The number of aberrancies in the myelomonocytic and blast compartment can be translated by a flow cytometric scoring system into a flow score (Wells et al., Blood, 2003,102:394–403, van de Loosdrecht et al., Blood, 2008,111:1067–77). A flow score above 3 is suggestive for MDS. The clinical impact of CD38 expression as a single flow cytometric parameter or as part of a multi parameter analysis requires validation with emphasis on inconclusive cases such as idiopathic cytopenia of unknown significance and idiopathic dysplasia of unknown significance. Flow cytometry in MDS is highly promising and prospective studies are warranted. Disclosures:No relevant conflicts of interest to declare.

Circulating Malignant Clonal Cells in Peripheral Blood of Patients with Myelodysplastic Syndrome: Verification by Interphase Fluorescent in Situ Hybridization Study and Its Correlation with Bone Marrow Clonal Cells.

Abstract 4840 IntroductionBone marrow (BM) cells are believed as the source neoplastic cells in patients with myelodysplastic syndrome (MDS). We examined whether the clonal proliferating pattern in peripheral blood truly reflects clonal haemopoiesis in the bone marrow. Materials and MethodsWe performed interphase fluorescence in situ hybridization (iFISH) with peripheral blood mononuclear cells in MDS patients with clonal abnormalities detected using iFISH and conventional G-banding. Nine patients were included in the study: 3 at initial diagnosis (patient 4, 6 and 9), 2 at follow up without stem cell transplantation (SCT) (patient 1 and 3) and 4 at follow-up after SCT (patient 2, 5, 7 and 8). We used 5 types of probes (Vysis Inc.) to detect 5q, 7q, 8, 20q and 1q abnormalities. The results were compared with iFISH results of bone marrow at the initial diagnosis and at the follow up (if any). ResultsSame clonal abnormalities were detected in similar quantity in the initial bone marrow, the follow-up bone marrow (if any) and the peripheral blood mononuclear cells of 3 patients at initial diagnosis and 2 follow up patients who did not received SCT. The follow up bone marrow biopsies of those 2 without SCT showed disease progression to advanced disease (RAEB-1 to RAEB-2, patient 1) or persistent disease (RCUD-RA, patient 3). One patient who received SCT (patient 2) had the same clonal abnormality (trisomy 8) in peripheral blood mononuclear cells at the follow up as in the bone marrow cells at the initial diagnosis in a very reduced dose (69.0% vs. 2.5%). Another patient with SCT (patient 5) did not showed clonal abnormality in the bone marrow and the peripheral blood at the follow up, with bone marrow biopsy findng of poor engraftment. The other 2 patients with SCT (patient 7 and 8) did not showed the clonal abnormality in the peripheral blood mononuclear cells, which were observed in the initial bone marrow. Their bone marrow biopsies showed complete remission of the disease. The results are specified in Table 1. Conclusion and DiscussionUsing interphase FISH technique, we observed the same clonal abnormalities in the peripheral blood mononuclear cells as in the bone marrow mononuclear cells in the cases of initial diagnosis or the cases with disease progression or persistent disease. The quantity of clonal abnormality was also similar in between. The clonal abnormalities were not detected in the peripheral blood in the cases with stem cell transplantation and improved bone marrow findings. We concluded that the type and the quantity of clonal abnormalities in peripheral blood mononuclear cells well reflect those of bone marrow and bone marrow morphology. Our results demonstrate the potential role of disease monitoring with peripheral blood in the MDS patients, even when not accompanied by invasive bone marrow exam. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interferon-γ and Tumor Necrosis Factor-α Induce An Immunoinhibitory Molecule, B7-H1, Via NfκB Activation in Blasts of Myelodysplastic Syndromes.

Abstract 2766Poster Board II-742 (Introduction)B7-H1 (CD274) is a costimulatory/coinhibitory molecule that plays a crucial role in T cell regulation in various immune responses. B7-H1 expression is detected not only on professional antigen-presenting cells but also on some tumor cells. It has been reported that B7-H1 molecules on tumor cells inhibit the proliferation of tumor-specific cytotoxic T-lymphocytes and are associated with poor prognosis in patients with some tumors such as renal and breast cancers. In this study, we investigated whether functional B7-H1 molecules are expressed on blasts from myelodysplastic syndromes (MDS) and, if so, are associated with pathophysiology in specific immune evasion in MDS. (Materials and methods)(1) First, we analyzed the expression of B7-H1 mRNA and protein in MDS cell lines (OIH-1, SKM-1, and F36P) using reverse transcription-PCR and flow cytometry (FCM), respectively. Although F36P cells alone expressed B7-H1 molecules at mRNA and protein levels, interferon (IFN)-γ and more efficiently a combination of IFN-γ and tumor necrosis factor (TNF)-α enhanced or induced B7-H1 expression in all of these cell lines. Notably, the combination of IFN-γ and TNF-α induced nuclear factor (NF) γB activation of MDS cell lines, and the blocking of NFκB activation by pyrrolidine dithiocarbamate (PDTC) or NFκB inhibitor peptide turned off the B7-H1 induction. (2) To investigate whether B7-H1+ MDS blasts have a proliferative advantage, we compared the cell cycle and colony formation between B7-H1+ and B7-H1– cell fractions in F36P cells. B7-H1+ cells had fewer G0/G1-phase cells and more G2/M-phase cells compared with B7-H1– cells. Furthermore, purified B7-H1+ cells formed more colonies than B7-H1– cells in the colony-forming assay using the methylcellulose. (3) We investigated the immunomodulatory effects of B7-H1+ blasts on T cells. When normal CD3+ T cells were cultured with or without irradiated B7-H1+ F36P cells, the B7-H1+ F36P cells had markedly increased percentages of T cells showing apoptosis. The blocking antibody for B7-H1 or PD-1 inhibited the T-cell apoptosis induced by B7-H1+ cells, decreased the percentage of caspase-3+ cells in T cells, and increased T-cell proliferation. (4) We then examined B7-H1 expression on blasts from 41 MDS patients, 20 patients with acute myeloid leukemia transformed from MDS (AML-MDS), and 10 hematologically normal individuals using FCM. B7-H1+ cases, in whom more than 5% of blasts expressed B7-H1 molecules, were observed in 6 of 14 (43%) patients with refractory anemia with excess blasts (RAEB), 6 of 12 (50%) with RAEB in transformation (RAEB-t), and 4 of 20 (20%) AML-MDS patients, but in none of 15 RA patients and normal individuals. Similar to the data obtained using MDS cell lines, B7-H1 expression on blasts purified from MDS patients was induced by stimulation with IFN-γ or TNF-α, and more strikingly by their combination. PDTC inhibited the induction of B7-H1 expression. The percentages of G0/G1-phase cells were lower and those of G2/M-phase cells higher in B7-H1+ blasts than in B7-H1– blasts from patients. Furthermore, in allogeneic coculture, in which B7-H1+ blasts from an MDS patient and CD3+ T cells from healthy volunteers were cultured, T-cell proliferation was significantly augmented by blocking B7-H1 molecules. (5) Finally, we investigated the expression of PD-1, a counterreceptor of B7-H1, on circulating T cells in 34 MDS patients. PD-1 expression on CD3+, CD4+, and CD8+ T cells in MDS patients was significantly higher than that in healthy volunteers. (CONCLUSIONS)Taking the results together, in MDS: 1) B7-H1 expression on blasts occurs more often in the advanced disease stages; 2) B7-H1 expression on blasts may be induced by cytokines derived from the bone marrow environment via NFκB activation; 3) cell cycle and proliferation were more active in B7-H1+ blasts than in B7-H1– blasts; and 4) B7-H1 molecules on blasts suppress T-cell immunity. These findings indicate that B7-H1 molecules on blasts may be involved in the pathophysiology of MDS. Disclosures:No relevant conflicts of interest to declare.

Is It Relevant to Determine Routinely Levels of Circulating NK and NK Like Cells in Myelodyplastic Syndroms?.

Abstract 2786Poster Board II-762 Purpose:Myelodysplastic syndromes (MDS) are characterized by an ineffective haematopoiesis due to different pathways: Intrinsic factors as cytogenetic and molecular abnormalities and extrinsic factors as disturbance in cellular immunity. An excess or abnormal natural killer (NK) cells cytolytic function have been previously described in MDS. A sub-group of MDS characterized by bone marrow hypoplasia, an excess of NK, HLA-DR15 and trisomy 8 may respond to an immune-suppressive therapy by antithymocyte globuline (ATG) or Cyclosporin. Recent advances in immunophenotyping of hematopoietic dysplastic cells in blood and bone marrow pointed to a useful role for multiparameter flow cytometry (FCM) in the diagnosis of MDS. This prospective study reports the rates of NK and NK like circulating cells in MDS patients (pts) assessed by routine FCM. Methods:This study concern a cohort of pts with MDS, in a university hospital, proved by morphological criteria on blood samples and bone marrow aspirate or trephine biopsy. Pts were classified according to 2001 WHO classification. IPSS score has been determined for pts according to cytopenia, blasts percent and cytogenetic results. We performed between 02/2005 and 09/2008 a prospective analysis of immunophenotyping data using a double platform method that allowed the enumeration of NK cells according to two different antibodies combinations (CD45+, CD3-, CD16 or CD56+, so called “total” NK cells vs CD45+ CD2+ CD3- so called 2+3- NK cells) and NK like cells (CD3+, CD16+ or CD56+). The statistical analysis was made with STATVIEW software. Results:The cohort counts 58 pts, median age 75 years (50-86), sex ratio 0,93. MDS subtype were distributed as 34.5% RAEB-1 (n=20), 22.4% RAEB-2 (n=13), 15.5 RA (n=9), 13.7% CMML (n=8), 8.6% RARS (n=5), 1.7% RA/RCMD (n=1), 1.7% 5q- syndrome (n=1), 1.7% inclassifiable (n=1). Cytogenetic analysis could be performed in 96.5% of cases (56/58) and revealed 78.5% of favourable prognosis (44/56) with 60.7% normal, 7.1% del (5q), 7.1% -Y, 3.6% del (20q), intermediate prognosis 12.5% (7/56) with 10.7% trisomy 8 and 1.8% inv(9), 5.4% of unfavourable prognosis (3/56) with 3.6% complex and 1.8% del(7q) and 3.6% failure (2/56). IPSS was calculated for 93% of the files (54/58) with 68.5% of low risk (18.5% low IPSS (n=10), 50% int-1 IPSS (n=27)) and 31.5% of high risk (25.9% int-2 IPSS (n=14), 5.6% high IPSS (n=3). Median rate of 2+3- NK cells, obtained on 96.5% of pts, was 79 cells/μl (6.7-798) (normal values : 130-430). This rate is decreased for 69.6% of pts (n=39), normal for 25% (n=14) and increased for 3.4% (n=3). The classification of these 3 pts was RARS, RAEB-2 and CMML. All had a normal cytogenetic profile. Median rate of “total” NK cells (determined on 60% of pts) was 119.7 cells/μl (17.6-473.1) (normal values 130-430). This rate is decreased for 57.1% (n=20), normal 40% (n=14) and increased 2.9% (n=1). This pts was suffering CMML with absence of cytopenia, 11% blast excess, normal cytogenetic and IPSS Int-2. Individually, the two approaches for NK numeration gives, as expected, close values with superior values for “total” NK cells that comprised the subset of CD2- NK cells. Median rate of NK like cells (60% of pts) was 58.5 cell/μl (7-1264) (normal values: 5-95). This rate is normal for 71.4% of pts (n=25) and increased in 28.6% (n=10). The classification of these 10 pts was 10% of RARS (n=1), 40% RAEB-1 (n=4), 20% RAEB-2 (n=2) and 30% CMML (n=3). Cytogenetic was normal 60% (n=6), trisomy 8 20% (n=2), inv 9 10% (n=1) and –Y 10% (n=1). IPSS was low 10% (n=1), Int-1 50% (n=5), Int-2 40% (n=4). Conclusion:European Leukemia Net recommendation proposes to use FCM for diagnosis and prognostication of MDS, based abnormal phenotype of myeloid lineages. The interest of lymphocytes immunophenotyping is discussed. Our study shows that cases with elevated circulating NK cells are rare, in addition without correlation with IPSS score and cytogenetic data. By contrast, an increase in circulating NK like cells is more frequent, and seems correlated with a blast excess. Such data could be of interest regarding to the potential therapeutic issues using immunosupressors. Disclosures:No relevant conflicts of interest to declare.

Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome

An activated immune system has been observed in patients with myelodysplastic syndrome but its exact contribution to disease development and control is not fully clarified. On the one hand an activated and skewed T-cell repertoire has been reported, but on the other hand, decreased natural killer cell function has been found. Immune activation could reflect undesired autoimmune reactions against normal hematopoietic precursor cells as well as effective immune-surveillance against dysplastic clones. We have investigated immune effector cells (lymphocyte subsets, lymphocyte activation markers, and natural killer cells) of 40 low and intermediate risk myelodysplastic syndrome patients and compared them to those of 10 age-matched healthy donors. Furthermore, we have analyzed the cytotoxic capacity of effector cells against autologous bone marrow hematopoietic precursor cells of 8 myelodysplastic syndrome patients and 2 healthy donors. In myelodysplastic syndrome patients, we have found an activated state of lymphocytes, determined by increased percentages of effector T cells with cytotoxic profile, more skewing of the T-cell receptor Vbeta (TCR-Vbeta) repertoire, and decreased frequencies of regulatory T cells, when compared to healthy donors. The percentage of natural killer cells did not differ between myelodysplastic syndrome patients and healthy donors, but natural killer cells of myelodysplastic syndrome patients expressed increased levels of granzyme B. Finally, we have demonstrated non-MHC restricted autologous cytotoxicity up to 90% against aberrant hematopoietic precursor cells, presumably mediated by natural killer cells. Our data point to a role for active immune-surveillance in myelodysplastic syndrome, as demonstrated by activated T cells and TCR-Vss skewing. Autologous cytotoxicity against hematopoietic precursor cells was natural killer cell dependent, which points to an additional role for the innate immune system in immune-surveillance of myelodysplastic syndrome patients.