Bone Marrow Of Myelodysplastic Syndromes Patients Research Articles

MRNA expression of notch ligand-delta-like-1 and jagged-1 in mesenchymal stem cells of MDS patients

This study was aimed to investigated the mRNA expression levels of Notch ligands- Delta-like-1 and Jagged-1 in bone marrow mesenchymal stem cells of patients with myelodysplastic syndrome (MDS), and to explore their relation with onset of MDS. Bone marrow mesenchymal stem cells of 38 patients with MDS and 16 normal subjects as control were collected to detect mRNA expression of Delta-like-1 and Jagged-1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of Delta-like-1 and Jagged-1 in mesenchymal stem cells of MDS patients were significantly higher than that in normal controls (P < 0.05). According to WHO criteria, the mRNA expression of Delta-like-1 in RA/RAS, RCMD and RAEB groups were significantly higher than that in normal controls (P < 0.05), the mRNA expression of Jagged-1 in RAEB group was also significantly higher than that in normal controls (P < 0.05). The mRNA expression of Delta-like-1 was significantly correlated with the proportion of blasts in the bone marrow of MDS patients (r = 0.502, P < 0.05). The expression levels of Delta-like-1 and Jagged-1 in MDS patients with abnormal karyotypes were significantly higher than those in MDS patients with normal karyotypes (P < 0.05). The mRNA expression of Delta-like-1 in higher risk group according to International Prognostic Scoring System was significantly higher than that in lower risk group (P < 0.05), there was no significant difference in Jagged-1 expression levels between higher risk group and lower risk group (P > 0.05). It is concluded that the changes of Delta-like-1 and Jagged-1 expression level in MSC may play a role in the pathogenesis of myelodysplastic syndrome.

CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes

The myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are initiated and sustained by self-renewing stem cells. We performed transcriptomal profiling of purified MDS hematopoietic stem cells (HSCs) and identified CD99 as a highly expressed cell surface transcript. Flow cytometry of MDS patient bone marrow (BM) samples (n=63) confirmed that CD99 is frequently increased on MDS HSCs (85%). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (81%) and relapse (83%). CD99 is a potential leukemic stem cell (LSC) marker, as CD99 high cells exhibit a CD34+CD38-CD90-CD45RA+ LMPP-like immunophenotype, previously shown to be enriched for LSC activity, whereas CD99 low cells exhibit a CD34+CD38-CD45RA- phenotype resembling normal HSCs and MPPs.Providing additional support that CD99 is a LSC marker in AML, CD99 surface expression is significantly higher in the LSC-enriched CD34+CD38- fraction compared to bulk blasts (p=0.003), as well as in relapsed as compared to diagnostic specimens (p=0.007). To determine if CD99 is preferentially expressed on functional LSCs, we purified the top 10% and bottom 10% of CD99 expressing leukemic blasts from the CD34+CD38-CD90-CD45RA+ fraction of a primary AML specimen. Limiting dilution xenotransplantation experiments demonstrated an estimated LSC frequency of 1 in 24,401 in the top 10% of CD99 expressors, whereas transplantation of up to 360,000 of the bottom 10% of CD99 expressors did not lead to any engraftment. Thus, within the LMPP-like LSC-enriched compartment, LSC activity appears to be restricted to high CD99 expressors.To determine the function of CD99 in AML, we stably transduced MOLM13 AML cells with a CD99 shRNA (8.0-fold knockdown) and xenografted them into NSG mice. Animals transplanted with these cells showed improved survival compared to vector controls (58d vs. 34d, p=0.02). Consistent with its described role in leukocyte trafficking, overexpression of CD99 in AML cell lines promoted transendothelial migration in transwell assays. In primary AML specimens, CD99 expression was higher on PB as compared with BM specimens (p=0.03). Together, these findings suggest that CD99 may promote AML aggressiveness by enhancing transendothelial migration and mobilization.Unexpectedly, CD99 transcript expression in the ECOG 1900 AML patient cohort (n=308) positively correlated with survival (p=0.001). We propose that CD99 may improve survival in the context of chemotherapy by promoting mobilization and thus chemosensitivity of AML cells, and AML cells with low levels of CD99 may exhibit increased retention in chemoprotective BM niches. In line with this hypothesis, the poor prognosis of low CD99 expression appeared to be mitigated by intensification of chemotherapy.To determine whether CD99 may be a relevant therapeutic target, we tested the ability of monoclonal antibodies (mAb) against CD99 to induce direct cytotoxicity in vitro. Anti-CD99 mAbs induced significant cell death in 11 AML and two MDS-derived cell lines, as well as in primary AML blasts (n=7) and MDS HSCs/CD34+ cells (n=3). These mAbs induced cytotoxicity in a CDC and ADCC independent manner, with relative sparing of normal cells expressing low levels of CD99 such as primary human HSCs and endothelial cells, suggesting a mechanism of cytotoxicity unique to AML and MDS. Pre-coating of primary human LSCs with an anti-CD99 mAb prior to transplantation into NSG mice led to impaired engraftment at eight weeks (20% vs. 67%, p=0.009) and improved survival (p=0.05). Immunofluorescence imaging demonstrated that mAb ligation promotes clustering of CD99, and this clustering appears to be critical for cytotoxicity. Accordingly, cross-linking of IgG isotype anti-CD99 mAbs with an anti-IgG secondary antibody enhanced cytotoxicity 3.4-fold. Anti-CD99 mAb treatment was also associated with activation of Src-family kinases, and we propose that anti-CD99 mAbs may promote cytotoxicity by inducing oncogenic stress via dysregulated Src-family kinase activation.Our results establish CD99 as a cell surface marker expressed in AML and MDS stem cells, as a mediator of transendothelial migration, and as a promising therapeutic target for direct targeting by mAbs. Further studies are needed to evaluate the potential of targeting CD99 in AML/MDS in vivo and to further characterize the mechanisms of CD99 mediated cell death. Disclosures:No relevant conflicts of interest to declare.

Induction of myelodysplasia by myeloid-derived suppressor cells

Myelodysplastic syndromes (MDS) are age-dependent stem cell malignancies that share biological features of activated adaptive immune response and ineffective hematopoiesis. Here we report that myeloid-derived suppressor cells (MDSC), which are classically linked to immunosuppression, inflammation, and cancer, were markedly expanded in the bone marrow of MDS patients and played a pathogenetic role in the development of ineffective hematopoiesis. These clonally distinct MDSC overproduce hematopoietic suppressive cytokines and function as potent apoptotic effectors targeting autologous hematopoietic progenitors. Using multiple transfected cell models, we found that MDSC expansion is driven by the interaction of the proinflammatory molecule S100A9 with CD33. These 2 proteins formed a functional ligand/receptor pair that recruited components to CD33’s immunoreceptor tyrosine-based inhibition motif (ITIM), inducing secretion of the suppressive cytokines IL-10 and TGF-β by immature myeloid cells. S100A9 transgenic mice displayed bone marrow accumulation of MDSC accompanied by development of progressive multilineage cytopenias and cytological dysplasia. Importantly, early forced maturation of MDSC by either all-trans-retinoic acid treatment or active immunoreceptor tyrosine-based activation motif–bearing (ITAM-bearing) adapter protein (DAP12) interruption of CD33 signaling rescued the hematologic phenotype. These findings indicate that primary bone marrow expansion of MDSC driven by the S100A9/CD33 pathway perturbs hematopoiesis and contributes to the development of MDS.

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.

Expression patterns of chemokine receptors on circulating T cells from myelodysplastic syndrome patients

Chemokines and their receptors are involved in the recruitment of leukocytes to sites of inflammation. Recently, chemokine expression signatures have been reported to convey a prognostic value in myelodysplastic syndrome (MDS) patients. In the present study, we investigated the chemokine receptor repertoire on fresh peripheral blood lymphocytes from 31 (22 low-risk and 9 high-risk) patients affected by MDS. Chemokine receptor expression was studied in defined T-cell subsets using eight-color flow cytometry. MDS patients exhibited quantitative differences in peripheral lymphocyte subpopulations. In addition, T cells obtained from MDS patients expressed a chemokine receptor pattern suggesting a dominance of mature and activated T cells. This is illustrated by increased levels of CCR3, CCR5, CX3CR1 and/or by a decreased abundance of CCR7 in defined T-cell subsets. The T-cell subset distribution appears to differ between the peripheral blood and the bone marrow of MDS patients, suggesting a preferential recruitment of specific T-cell subsets to the latter compartment. Alteration in chemokine receptor expression can develop over time even in patients that are considered clinically stable. Elevated expression levels of CXCR4 by CD8+ cells were associated with prolonged patient survival and reduced numbers of bone marrow blasts. We conclude that immunological abnormalities in MDS also involve chemokine receptors on different subsets of T cells, and that these changes may have a prognostic value.

Expansion of CD141int Dendritic Cells in Myelodysplastic Syndrome (MDS)

AbstractAbstract 3856 IntroductionImmunological responses play an important role in the pathogenesis and progression of myelodysplastic syndromes (MDS). Several studies have confirmed that immune dysregulation in MDS may play a critical role in the initiation and progression of the dysplastic clone. We have already shown the importance of expanded Tregs in high risk MDS and its reverse correlation with the number of Th17 cells in low risk disease. However, the potential role of dendritic cells (DCs) in this immune dysregulation and in the expansion of Tregs in MDS is not fully understood. DCs are professional antigen presenting cells (APCs) and potent stimulators of T cells through (cross-)presentation of antigens via MHC class I and II molecules to CD8+ cytotoxic T lymphocytes (CTL) and CD4+ T helper (Th) cells, respectively. DCs are also important in the development of specific anti-tumor T-cell responses. Some subtypes of DCs also play an important role in the expansion of regulatory T cells (Tregs) and induce immune-suppression and editing. There are 3 main subpopulations of DCs in human: plasmacytoid DCs (CD303+ cells) and two types of myeloid DCs (CD1c+ and CD141+ cells).The aim of this study was to investigate the frequency of different DC subsets in the bone marrow and peripheral blood of MDS patients. Patients and methodsTwelve peripheral blood and 26 bone marrow samples from MDS patients and 11 peripheral blood and 4 bone marrow samples from healthy donors (HDs) were studied. Three different DC subsets were investigated by flow-cytometry: plasmacytoid DC (Lineage−, HLA-DR+, CD303+), myeloid DC 1 (Lineage−, HLA-DR+, CD1c+) and myeloid DC 2 (Lineage−, HLA-DR+, CD141hi). ResultsDecreased frequency of DCs in peripheral blood compared to BMFrequencies of all DC subsets were significantly lower in patients’ peripheral blood compared to patients’ bone marrow (CD1c+ DC 2.56×102 v 7.02×102 and 0.06% v 0.33%, p=0.001; CD141+ DC 4.18×103 v 6.04×103 and 0.44% v 2.44%, p=0.005; CD303+ DC 0.06×102 v 1.07×102 and 0.01% v 0.13%, p=0.045). DC subsetsThe percentage of the CD1c+ DC subset was significantly reduced in the peripheral blood of MDS patients compared to HD (1.46×103 v 0.26×103, 0.18% v 0.05%, p=0.001) (figure 1). Interestingly, we have noticed an additional subset of DCs, which was not described before. The definition of this subset was based on intermediate expression of CD141 compared to previously described myeloid DCs. The CD141int subset was the only DC subset that was increased in peripheral blood of MDS patients compared to HD (0.41×102 v 2.30×102, 0.01% v 0.13%, p=0.019). Both CD1c+ and CD141int DC subsets were increased in the bone marrow of MDS patients compared to healthy donors. However, these differences were not statistically significant. DiscussionAlthough the decreased number of circulating DCs is reported in MDS, our data suggest that this decrease is more profound in the peripheral blood compared to BM. We have also shown that this decrease is mainly in CD1c+ myeloid DCs. In this study, we describe two subsets of CD141 DCs, CD141hi and CD141int. The CD141int subpopulation is the only DC subset that is increased in MDS patients compared to HD.Our data suggest that DCs from peripheral blood may migrate to the bone marrow in response to an aberrant BM microenvironment in MDS.Nevertheless, the pattern of decrease in DC numbers is not uniform and CD141int DCs are in fact increased. It is already known that CD141+ DCs, play an important role in the induction of Tregs in an IL-10 dependent manner. We have also reported an increased number of Tregs in high-risk MDS, which correlates, with higher risk of progression toward AML. Our data suggest that CD141+ DCs may play a role in the expansion of Tregs in MDS. However, the exact function of this subset of DCs in MDS and their effect on CD4+ T-cells polarization needs to be investigated in more details. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Localization of the NRAS:BCL-2 complex determines anti-apoptotic features associated with progressive disease in myelodysplastic syndromes

We have previously demonstrated that two prognostic features of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML), mutant NRAS and over-expressing BCL-2, cooperate physically and functionally in vivo. Screening of MDS patient bone marrow (BM) identified NRAS:BCL-2 co-localization in 64% cases, correlating with percentage BM blasts, apoptotic features and disease status (p<0.0001). Localization of the complex at the plasma membrane or the mitochondria correlated with disease and apoptosis features in MDS patients, whilst caspase-9 mediated mechanism was elucidated in vivo and in vitro. The intensity and localization of the RAS:BCL-2 complex merits further evaluation as a novel biomarker of MDS.

Alteration of lipids and the transcription of lipid-related genes in myelodysplastic syndromes via a TP53-related pathway

Myelodysplastic syndromes (MDS) are clonal stem cell diseases of the bone marrow characterized by abnormalities in maturation of hematopoietic cells of all lineages. MDS patients frequently have lower lipids and high rates of apoptosis and p53 (TP53) expression. An association between the reduced lipids in MDS and the expression of lipid-related genes was sought. We further evaluated whether 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGcoAR) and low-density lipoprotein receptor (LDL-R) are regulated by TP53 invivo and invitro. Gene expression was measured using real-time reverse transcription polymerase chain reaction on RNA extracted from bone marrow and peripheral blood from eight newly diagnosed MDS patients and eight controls and from mice livers. Serum lipid profile was measured using colorimetric enzymatic procedures. Total- and LDL cholesterol were lower in MDS patients in comparison to controls (p = 0.04 and p = 0.01, respectively). HMGcoAR messenger RNA increased in peripheral blood and bone marrow of MDS patients compared to controls (p = 0.04 and p = 0.01, respectively). LDL-R messenger RNA was higher only in the peripheral blood of MDS patients (p = 0.05). Comparable results were obtained invivo. The transcription of these genes correlates with TP53 activation as documented by p21 messenger RNA elevation, a surrogate for TP53 activation and by using TP53 temperature-sensitive cells treated with adriamycin. To conclude, an association between reduced lipids in MDS and expression of HMGcoAR and LDL-R genes was documented. The transcription of these genes can be regulated by TP53.

Abstract 190: p53 target gene expression, DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy

Abstract The myelodysplastic syndromes (MDS) are a heterogeneous group of blood disorders characterized by peripheral cytopenias, a cellular bone marrow with dysplasia and increased intramedullary apoptosis, and are associated with risk of progression to acute myelogenous leukemia (AML). MDS are often fatal due to complications from cytopenias and/or progression to AML. DNA methyltransferase inhibitors (DNMTi), including 5-azacytidine and 5-aza-2’-deoxycytidine (decitabine) have recently assumed a major role in the clinical management of MDS, and are two of only three FDA-approved treatments for these disorders. The mechanistic basis for the activity of DNMTi in MDS is unclear. We recently reported that decitabine treatment induces the expression of p53-inducible ribonucleotide reductase p53R2/RRM2B in tumor cell lines and MDS patient bone marrow (Link et al., Cancer Research 68; 9358-9366, 2008). Importantly, p53R2 induction was independent of DNA hypomethylation at its promoter region, and the level of p53R2 induction correlated with clinical response in a cohort of 13 patients. These data suggest that important pharmacodynamic responses to DNMTi may be associated with DNA damage and/or p53 activation. To better understand the overall nature of the molecular responses occurring in the bone marrow of MDS patients treated with decitabine, in the current study we examined a variety of pharmacodynamic endpoints including: p53 target gene expression, global DNA methylation, tumor antigen gene expression and methylation, and tumor suppressor gene expression. Specifically, we measured the expression of three classical p53 target genes (p21, GADD45-α, NOXA), LINE-1 repetitive element methylation, the expression and methylation of cancer-germline (CG) antigens including MAGE-A1, NY-ESO-1, XAGE-1, and CTCFL/BORIS, and the expression of the tumor suppressor genes (TSGs) p15INK4b and PDLIM4. Our data reveal that decitabine treatment causes robust induction of p53 target genes in MDS patient bone marrow, which coincides with LINE-1 hypomethylation and clinical responses. In contrast, while CG antigen genes showed some evidence for DNA hypomethylation post-therapy, this did not coincide with CG antigen gene induction. Surprisingly, TSGs were also not induced by decitabine therapy in MDS patient bone marrow. Ongoing studies seek to define p53 mutational status in MDS patients and to determine how this relates to p53 target gene induction and/or clinical response. In summary, our data reveal a connection between p53 target gene activation, global DNA hypomethylation, and clinical response in MDS patients undergoing decitabine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 190.

Gene Expression Changes in Bone Marrow of MDS Patients with Del(5q) During Lenalidomide Treatment.

Abstract 2622Poster Board II-598 Introduction:Genetic alterations reported in myelodysplastic syndromes (MDS) are not disease-specific and the underlying molecular causes of the disease remain poorly understood. It has been suggested that one or more of the genes mapping within the commonly deleted region of the 5q syndrome, together with other distant genes, may be critical to the development of the 5q syndrome. Potential candidate genes have been identified including the tumor suppressor gene SPARC, and the ribosomial protein gene, RPS14. Haploinsufficiency of RPS14 has been demonstrated and recent evidence indicates RPS14 as a causal gene for the 5q syndrome. Lenalidomide has proven efficacy in MDS patients with del(5q). Rapid and durable responses include transfusion-independence, with a rise in Hb, suppression of the 5q-deletion clone and improvement in bone marrow morphologic features. Methods:In a multicenter Italian phase II trial to evaluate safety, changes in quality of life and efficacy of lenalidomide in primary MDS patients with del(5q) and low or Int-1 risk IPSS, we investigate changes in bone marrow cytogenetics and gene expression patterns during treatment. The starting dose of lenalidomide is 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Dosing is based upon clinical and laboratory findings. Bone marrow cytogenetics and gene expression profiling are performed on study entry and every 12 weeks up to end of study (week 52). Gene expression assays of 51 candidate genes from the published literature and genomic databases have been selected and are carried out with TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative dd CT method, according to manufacturer's instructions. Using an 18S mRNA gene pre-designed assay from Applied Biosystems to detect the expression of the housekeeping gene 18S in each sample, target gene expression is normalized with 18S gene expression derived from a bone marrow pool of normal healthy subjects and for each sample the ratio between the target and 18S are expressed. Results:Baseline values for 23 patients (mean age 73 ± 10 years) are available and 16 have been re-evaluated after 12 weeks. Mean Hb was 8.6 ± 0.9 g/dL and 20 patients were transfusion-dependent. Seven patients had additional cytogenetic abnormalities. At baseline, RPS14 was under-expressed in 19 out of 21 patients evaluated. After 12 weeks RPS14 was re-evaluated in 13 patients: all had erythroid responses and RPS14 increased significantly from 0.07 (IQ Range 0.03–0.13) to 76.1 (0.73– 304.0, p=0.002). SPARC expression was under-expressed in 15/23 patients and variations during treatment were not significant. Baseline FAS gene was under-expressed in all patients and increased above reference values (p=0,006) after 12 weeks in 7/14 cases. IL7R was over-expressed in all patients at baseline (median 3263.3, IQ range 1998.3–5027.1) and was significantly reduced after 12 weeks (median 0.17, IQ range 0.05–2.20, p<0.0001). TINAGL1 gene expression was very high at baseline (mean 5214 ± 3661) and significantly reduced below normal values during treatment (0.63±0.87, p<0.0001). WTI was over-expressed in almost all patients (median 3246, IQ range 1590–7785) and, though reduced during treatment, still remained highly over reference values at 12 weeks (p=0.148). The expression of the other genes did not vary significantly during treatment. Conclusions:A preliminary study evaluating short-term effects of lenalidomide in low and Int-1 IPSS risk MDS and del(5q) demonstrates that gene expression is abnormal and varies during treatment. RPS14 is down-expressed and a significant increase in its expression is observed during treatment with lenalidomide in responsive patients. There is also an increase in FAS expression; the protein encoded by FAS gene is a member of the TNF-receptor superfamily and its interaction with its ligand leads to apoptosis.The IL7R gene on chromosome 5 (5p13) codifies for the IL7 receptor, which blocks apoptosis during differentiation and activation of T lymphocytes. TINAGL1 gene on chromosome 1 codifies for a Cathepsin B-like protein implicated in a number of human diseases such as cancer. During lenalidomide treatment, the expression of IL7R and TINAGL1 are reduced. Further results and repeated testing during this trial may develop new insights in the pathogenesis of MDS with del(5q) and in long-term effects of lenalidomide on bone marrow changes. Disclosures:Oliva:Celgene: Consultancy. Balleari:Celgene: Consultancy. Finelli:Celgene: Consultancy.

Analysing Circulating CD34+ Cells by FISH Is Representative for the Clone Size in Bone Marrow in MDS Patients Under Different Therapy Modalities

Introduction: In myelodysplastic syndromes (MDS) the knowledge about chromosomal aberrations is important for diagnosis, pathogenesis, prognosis and treatment of cytogenetic subgroups of MDS patients. Usually, chromosomal anomalies in MDS patients are detected in bone marrow (bm) cells by chromosome banding analyses of metaphases. Alternatively or additionally they can be diagnosed by Fluorescence-in-Situ-Hybridization (FISH). We here present a novel method for cytogenetic monitoring of MDS patients from peripheral blood (pb) which is representative for the clone size in bm cells.Patients and methods: After initial diagnosis by chromosome banding analyses of bm cells, CD34+ cells from pb were enriched by immunomagnetic activated cell sorting (MACS®) and then analysed by FISH. Sixteen MDS patients (7 AML, 4 RAEB-2, 1 RAEB- 1, 3 RCMD, 1 RA) treated with 5-Azacytidine (5-Aza) and 3 patients (all 5q-syndromes) treated with Lenalidomide (Len) were included in this study. FISH analyses of circulating CD34+ cells were performed once a month in every patient during follow-up.Results: Our data show that, irrespective of FAB-/WHO subtypes or peripheral blast counts, the successful collection of enough CD34+ cells for FISH analyses from pb is possible: in average 80 000 to 400 000 CD34+ cells per sample (20ml pb) were enriched. Morphologic features of CD34+ cells by microscopic analyses were the same in all patients and did not depend on WHO subtypes or on the percentage of peripheral blasts: homogeneous, pure populations of cells with an immature appearance. Additionally, we compared the results of FISH analyses of non-enriched pb, enriched circulating CD34+ cells, non-enriched bm cells and enriched CD34+ bm cells with the results of classical cytogenetic analyses of bm using conventional banding techniques: Constantly, the smallest clone sizes were measured in non-enriched pb, followed by non-enriched bm cells, enriched CD34+ bm cells and enriched circulating CD34+ cells. In all cases the clone sizes of enriched circulating CD34+ cells measured by FISH were close to the results of classical cytogenetic analyses of bm metaphases representing the gold standard of cytogenetic examinations. In our patient cohort 10/16 patients treated with 5-Aza and 3/3 patients treated with Len developed a cytogenetic remission (cyR) detected by FISH analyses of circulating CD34+ cells. CyR was defined according to modified IWG criteria. Because there are no standardized response criteria for FISH analyses available, especially not for FISH from pb, as yet, we definded cyR as a reduction of more than 50% of circulating CD34+cells showing the respective aberration. Remarkably, in all cases with cyR under 5-Aza the hematologic improvement (HI) followed 1–3 cycles later. Under therapy with Len, cyR and HI were measured simultaneously. During follow up HI was still onlasting while there was a cytogenetic relapse detectable by a step-wise increase of aberrant circulating CD34+ cells.Conclusions: Analysing circulating CD34+ cells by FISH is a reliable method to survey the size of an aberrant clone in pb in high-risk as well as in low-risk MDS patients. It is representative for the clonal situation in the bm and allows a very frequent monitoring during therapy with obviously predictive value. It may reduce the need for repeated bm biopsies. In all cases where a bm biopsy is not possible or unsuccessful or metaphases for conventional banding analyses are not available, our method still allows the measurement of the clone size from pb.

The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.

It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.

Identification and Hematopoietic Potential of CD45-Negative Clonal Cells with Very Immature Phenotype (CD45−CD34−CD38−Lin−) in Patients with Myelodysplastic Syndromes.

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38−Lin− cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34−CD38−Lin− cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site was reported to be committed myeloid progenitors and, more recently, the CD45+CD34+CD38−Lin− HSCs. In this study, we detected CD45−CD34−CD38−Lin− cells in the peripheral blood and bone marrow of MDS patients. Fluorescence in situ hybridization showed that CD45−CD34−CD38−Lin− cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-negativity, they lacked CD117 and CD133 expressions. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some cases. Freshly-isolated CD45−CD34−CD38−Lin− cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When these cells were co-cultured with stroma cells, CD45−CD34−CD38−Lin− cells showed only weak potential for proliferation/differentiation, yet differentiated to CD34+ cells and then mature myeloid cells. This newly-identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.

Immunophenotyping of acute leukemias and myelodysplastic syndromes.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

Assessment of growth and differentiation processes in myelodysplastic syndromes by PCR analysis of Galpha16 and 5'-lipoxygenase.

Development of further diagnostic and prognostic tools in myelodysplastic syndromes (MDS) is warranted. In this study we tested two molecular markers in bone marrow of MDS patients: Galpha16, a hematopoiesis-specific G protein alpha subunit serving as an intracellular marker of hematopoietic activity, and 5'-lipoxygenase (5-LO), a putative differentiation marker. The results were correlated with clinical and laboratory features and outcome. Bone marrow mononuclear cells were evaluated by block cycler PCR in 32 patients for Galpha16 and in 25 patients for 5-LO. In 12 patients cDNA analyzed by the block cycler method was quantified by real-time quantitative (RTQ) PCR for both Galpha16 and 5-LO. The results confirmed concordance of the two methods. All FAB and WHO subtypes were positive for at least one of the two genes. Strikingly, only 1 of 11 patients with refractory anemia with ringed sideroblasts was positive for Galpha16. No correlation between Galpha16 or 5-LO and bone marrow cellularity or cytogenetic risk factors (IPSS) was detected. Only combined evaluation of Galpha16 and 5-LO expression showed a correlation with extent of anemia and thrombocytopenia. A relationship between the two markers and preleukemic duration was found. Our findings show that both Galpha16 and 5-LO can be expressed in MDS, reflecting proliferation and differentiation processes in this disease. Different expression patterns of the two molecules indicate that proliferation and differentiation of hematopoietic cells may occur independently. These parameters could constitute a new class of risk factors in MDS. Determination of Galpha16 and 5-LO expression may provide a tool for observing growth and maturation in these diseases.

Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes.

To clarify whether regulatory cytokines inhibit hematopoiesis in patients with myelodysplastic syndromes (MDS), malignancies characterized by the formation of cytopenias despite the presence of cellular bone marrow, expression of TNF-alpha and IFN-gamma by bone marrow cells was investigated using specific reverse transcriptase-polymerase chain reaction assays. An enhanced expression of the mRNA for TNF-alpha was observed in most of the samples from MDS patients (11/14, 79%), whereas no enhancement was observed in bone marrow samples from AML (0/6), CML (0/2) or control cases (0/8). The expression of IFN-gamma was also enhanced in some of MDS cases (5/12, 42%) while AML (0/5), CML (0/2) and control cases (0/6) showed very low levels of IFN-gamma mRNA expression. Immunohistochemical examination confirmed the scattered presence of TNF-alpha or IFN-gamma producing cells in the bone marrow of MDS patients. The majority of these cells were CD68-positive macrophage lineage cells. These results suggested that disruption of hematopoiesis in MDS might be caused by enhanced production of inhibitory regulatory cytokines especially TNF-alpha and occasionally IFN-gamma by bone marrow macrophages.

Histopathology and clinical aspects of primary myelodysplastic syndrome

The histopathology of bone marrow in primary myelodysplastic syndromes (MDS) is described, with reference to the FAB classification. Variants such as hypoplastic, thrombocythemic and fibrotic MDS are recognized from their histopathology and must be incorporated in the FAB classification. The clinical significance of hypoplastic, thrombocythemic and fibrotic variants is illustrated by the survival rates and leukemic transformation in these patients. Histopathological classification according to the FAB system corresponds with cytological classification, as proven by the distribution of the subtypes, blood values, leukemic transformation rates and survival times. Finally it is even possible to elaborate a prognostic score for survival based on histological features of diagnostic biopsies, which emphasizes the importance of histopathological examination of bone marrow in MDS patients.

Quantitative and Qualitative Characteristics of Colony-Forming Unit-Erythroid Colonies in Myelodysplastic Syndrome Patients

The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (lambda = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.