Toll-like receptor-4 is up-regulated in hematopoietic progenitor cells and contributes to increased apoptosis in myelodysplastic syndromes.

PURPOSE: To investigate the function and expression of Toll-like receptors (TLR) in bone marrow cells of myelodysplastic syndrome (MDS) patients and to examine their involvement in the apoptotic phenomenon characterizing MDS hematopoiesis. EXPERIMENTAL DESIGN: TLR mRNA and protein expression was investigated in bone marrow cell populations of MDS patients and controls. TLR-4 ability to recognize lipopolysaccharide and up-regulate self mRNA and protein expression was examined. Tumor necrosis factor involvement in the constitutive and lipopolysaccharide (LPS)-induced TLR expression was also evaluated. Possible correlation between TLR-4 overexpression and apoptosis was investigated by simultaneous staining with Annexin V and TLR-4. RESULTS: TLR-2 and TLR-4 are expressed in almost all bone marrow cell lineages including megakaryocytes, erythroid cells, myeloid precursors, monocytes, and B lymphocytes and are up-regulated in MDS patients compared with controls. In hematopoietic CD34(+) cells, TLR-4 is also expressed and significantly up-regulated at both the mRNA and protein levels. Treatment with an anti-tumor necrosis factor antibody reduces both constitutive and LPS-induced TLR-4 levels. Increased TLR-4 expression correlates with increased apoptosis as TLR-4 is almost exclusively found in apoptotic bone marrow mononuclear and CD34(+) cells. The addition of the TLR-4 ligand LPS further enhances the apoptosis of these cells. CONCLUSIONS: TLR-4 and other TLRs are significantly up-regulated in MDS patients whereas TLR-4 is involved in promoting apoptosis, possibly contributing to MDS cytopenia.     

Limited numbers of apoptotic cells in fresh paraffin embedded bone marrow samples of patients with myelodysplastic syndrome

In myelodysplasia (MDS) the precise mechanism of ineffective erythropoiesis is not fully elucidated, but it is suggested that apoptosis may contribute to this process. We performed TdT-mediated dUTP-nick end labelling (TUNEL) staining of paraffin embedded bone marrow specimens to assess the amount of apoptotic cells in 21 MDS patients (7 RA, 3 RARS, 5 RAEB, 3 RAEB-T, 3 CMML) and five normal controls. In 10 MDS patients the TUNEL assay was performed in combination with immunostaining for Glycophorin-A (GpA) to determine apoptosis in the maturing erythroid compartment. To assess the proliferation of the bone marrow cells the expression of Ki-67 antigen was used as a marker. The mean apoptotic index (AI) in MDS patients was not increased (2.3 +/- 3.0% in MDS versus 4.8 +/- 1.2% in normal controls (P < 0.05)). Moreover, no significant difference in mean AI was observed in the GpA+ compartment between MDS and normal controls (0.8 +/- 0.2% versus 0.6 +/- 0.1%). In addition the different FAB-classifications and the different International Prognostic Scoring System (IPSS)-risk groups showed no significant differences between the subgroups. The expression of Ki-67, as marker for proliferative activity, in the GpA+ compartment from MDS did not differ significantly from normal controls (84.0 +/- 12.2% versus 79.9 +/- 20.2%). Our findings suggest that the observed increased apoptosis in in vitro culture assays is related to the detachment of the cells from the microenvironment leading to an increased susceptibility to apoptosis.     

Apoptosis susceptibility and cell-cycle distribution in cells from myelodysplastic syndrome patients: modulatory in-vitro effects of G-CSF and interferon-alpha

Susceptibility to apoptosis varies in different forms of myelodysplastic syndromes (MDS). Our in vitro study aimed at better defining the cell kinetic profile by investigating whether G-CSF and interferon-alpha (IFNalpha) were capable of controling apoptotic/proliferative mechanisms in RAEB as well as in RAEB-t forms. Apoptosis and cell-cycle distribution were measured in mononuclear and in CD34+ cells from bone marrow samples of 27 MDS patients with RAEB (n = 15) and RAEB-t (n = 12). In selected samples, the in vitro influence of G-CSF and lymphoblastoid (Ly)-IFNalpha on the apoptotic susceptibility and on the cell kinetics of the above MDS populations was evaluated. RAEB samples showed a significantly greater apoptosis than RAEB-t ones, both in mononuclear cells (14.76%+/-8.73 vs. 5.95%+/-3.88, P= 0.0058) and in CD34+ cells (24.66%+/-16.08 vs. 3.96%+/-2.57, P = 0.0007). Short-term cell culture in the presence of G-CSF reduced apoptosis in CD34+ cells in all 4 RAEB samples tested (39.1%+/-40.7 vs. 21.0%+/-23.5, P = n.s.); the percentage of cells in S-phase significantly increased in 3/4 samples (19.90%+/-4.40 vs. 32.40%+/-7.85, P = 0.03). Ly-IFNalpha protected CD34+ cells from apoptosis in 3/4 RAEB samples (25.7%+/-8.06 vs. 10.9%+/-8.8, P = n.s.), but did not modulate cell-cycle distribution. G-CSF and Ly-IFNalpha failed to affect apoptosis and proliferation in RAEB-t. These observations indicate that in RAEB forms increased apoptosis can be efficiently counteracted in most of the samples by both G-CSF and Ly-IFNalpha, suggesting that only in these forms a retained regulatory mechanism on the apoptotic/ proliferative balance may allow therapeutic intervention with apoptotic regulators.     

Significance of fetal hemoglobin-containing erythroblasts (F blasts) and the F blast/F cell ratio in myelodysplastic syndromes.

To investigate the relationship between the fetal hemoglobin-containing erythroblasts (F blasts) and apoptosis in myelodysplastic syndromes (MDS), we immunohistochemically assessed F blasts, F cells, and apoptosis in 137 patients with MDS. A marked increase in the number of F blasts in the bone marrow was identified in 116 of 137 patients (84.7%), and the number of F cells was elevated in 54 patients (39.4%). Among the erythroblasts stained by anti-glycophorin C antibody, the mean percentage of F blasts was 14.63 +/- 9.17% in MDS, which was significantly higher than that in non-MDS patients with stress erythropoiesis (4.82 +/- 3.35%, P < 0.01), although there were no significant differences in the number of F cells between these groups. In particular, 62 of the 137 MDS patients (45.3%) had an apparent increase in F blasts but no elevation of F cells. The apoptotic rate was significantly higher in the patients with a F blast/F cell (Fb/Fc) ratio >or=5.0 than in those with a Fb/Fc ratio <1.0 (P < 0.01). The results indicate that F cell precursors are incapable of maturing into functioning end-stage F cells, presumably owing to apoptotic cell death. The measurement of F blasts in the bone marrow is needed for the precise evaluation of fetal-type erythropoiesis in MDS.     

Expression of FLIP(Long) and FLIP(Short) in bone marrow mononuclear and CD34+ cells in patients with myelodysplastic syndrome: correlation with apoptosis.

Several apoptosis-inducing systems, including Fas/Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) and its receptors, are upregulated in myelodysplastic syndrome (MDS). FLIP (FLICE (FAS-associated death-domain-like IL-1beta-converting enzyme)-inhibitory protein)) was identified as an inhibitor of FAS and TRAIL signals. Here, we characterized FLIP(Long) (FLIP(L)) and FLIP(Short) (FLIP(S)) expression in bone marrow mononuclear cells (BMMNCs) and in CD34+ cells of 29 MDS patients, and in 17 normal volunteers. The expression was correlated with apoptotic indices. In CD34+ cells, FLIP(L) levels were higher among normal individuals than in MDS patients (P=0.04). Among total BMMNC, FLIP(L) levels also tended to be higher in normal subjects than in MDS patients, although this difference was not significant (P=0.71). FLIP(L) levels in CD34+ cells were negatively correlated with apoptosis in both normal and MDS marrows (P=0.03). FLIP(Short) RNA expression was higher in MDS patients than in normal controls in both BMMNC (P=0.03) and CD34+ cells (P=0.08). In contrast to FLIP(L), FLIP(St) levels were positively correlated with apoptosis. At the protein level FLIP was most readily detectable in patients with high blast counts. The data suggest that FLIP(L) and FLIP(S) are differentially regulated, and that the relative levels of both isoforms play a role in the regulation of apoptosis in MDS.     

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.     

Impaired clonogenic growth of myelodysplastic bone marrow progenitors in vitro is irrelevant to their apoptotic state

Excessive intramedullary apoptosis has been considered to account for the paradox of hypercellular marrow and refractory cytopenias in myelodysplastic syndrome (MDS). However, a causative relationship of apoptosis to the progenitor's defective clonogenic growth has not been sufficiently demonstrated. We investigated the degree of apoptosis and its contribution to ineffective hematopoiesis in MDS, by assessing the differential clonogenic capacity of purified "apoptotic" and "non-apoptotic" bone marrow progenitors in a short-term semisolid culture system. Although increased apoptosis was indeed detected in MDS bone marrow progenitors, there was no correlation between the existence of apoptosis and culture performance. Non-apoptotic as well as apoptotic CD34+ cells gave similar patterns of growth, both defective compared to normal. The ability of "apoptotic" CD34+ cells to proceed in colony formation as well as the abnormal growth of "non-apoptotic" progenitors are probably pointing towards the need to reconsider the role of apoptosis in the defective clonogenicity of MDS.     

Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome

Clonal expansion of leukemic cells is thought to be due to proliferation in excess of apoptosis. To define and compare proliferation and apoptosis between various leukemias and myelodysplastic syndrome (MDS), we measured proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation as surrogate markers for proliferation and caspase 3 activity and annexin V surface binding as surrogate markers for activation of the apoptotic cascade in patients with MDS, chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). We found high proliferation in bone marrow cells from MDS and CMML as measured by PCNA and BrdU incorporation. The lowest level of proliferation was found in CLL. Apoptosis was also highest in MDS and CMML as measured by annexin V and caspase 3 activity. Unexpectedly, we found no significant difference in proliferation in bone marrow CD34+ cells from various leukemias or MDS. Apoptosis was significantly higher in bone marrow CD34+ cells from MDS and CML in chronic phase as compared to CD34+ cells from AML patients. Our results illustrate differences in proliferation and apoptosis between acute and chronic leukemias and MDS. These differences may have diagnostic and therapeutic implications.     

Over-Expression of Cancerous Inhibitor of PP2A (CIP2A) in Bone Marrow Cells from Patients with a Group of High-Risk Myelodysplastic Syndromes

Cancerous inhibitor of PP2A (protein phosphatase 2A) (CIP2A) is an inhibitor of PP2A, a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. The aim of this study was to investigate whether CIP2A plays a role in the progression of myelodysplastic syndromes (MDS). Immunohistochemical analysis revealed that a fraction patients having refractory anemia with excess blasts (RAEB)-1 (4 out of 12) and RAEB-2 (10 out of 14) exhibited significant expression of CIP2A in bone marrow hematopoietic cells, while all patients with refractory cytopenia with unilineage or multilineage dysplasia (RCUD/RCMD) (0 out of 18) and the control group (0 out of 17) were negative. CIP2A was mainly expressed by the MPO-positive myeloid series of cells and partly by the CD34-positive cells in association with the expression of phosphorylated c-MYC (p-c-MYC) protein and the cell cycle-related proteins Ki-67, MCM2, and geminin. The percentage of p-c-MYC-positive cells in the bone marrow of CIP2A-positive MDS cases was significantly higher than that in CIP2A-negative MDS cases (P?<?0.01). The expression levels of mRNA for CIP2A and PP2A exhibited positive correlation in MDS/control bone marrow. These results suggest that up-regulated expression of CIP2A might play a role in the proliferation of blasts in the MDS bone marrow and in disease progression in at least some cases.     

High-level CD34 expression on megakaryocytes independently predicts an adverse outcome in patients with myelodysplastic syndromes

Expression of CD34 on mature-appearing megakaryocytes can be seen in various intrinsic bone marrow (BM) disorders as well as reactive bone marrows. In this study we investigate the clinical significance of CD34+ megakaryocytes in myelodysplastic syndromes (MDSs). Expression of CD34 on megakaryocytes was assessed on BM biopsies obtained from 202 patients with MDS. High-level (≥20%) CD34 expression on megakaryocytes was found in BM of 29 patients (14%). The expression of CD34 on megakaryocytes is correlated with severe cytopenia, higher numbers of myeloblasts, more frequent and higher risk cytogenetic abnormalities, and a shorter overall survival. Multivariate analysis indicated that the expression of CD34 on megakaryocytes could be a strong and an independent poor prognostic factor in MDS, with a hazard ratio of 2.53.     

Standards and impact of hematopathology in myelodysplastic syndromes (MDS)

The diagnosis, classification, and prognostication of patients with myelodysplastic syndromes (MDS) are usually based on clinical parameters, analysis of peripheral blood and bone marrow smears, and cytogenetic determinants. However, a thorough histologic and immunohistochemical examination of the bone marrow is often required for a final diagnosis and exact classification in these patients. Notably, histology and immunohistology may reveal dysplasia in megakaryocytes or other bone marrow lineages and/or the presence of clusters of CD34-positive precursor cells. In other cases, histology may reveal an unrelated or co-existing hematopoietic neoplasm, or may support the conclusion the patient is suffering from acute myeloid leukemia rather than MDS. Moreover, histologic investigations and immunohistology may reveal an increase in tryptase-positive cells, a coexisting systemic mastocytosis, or bone marrow fibrosis, which is of prognostic significance. To discuss diagnostic algorithms, terminologies, parameters, and specific issues in the hematopathologic evaluation of MDS, a Working Conference involving a consortium of US and EU experts, was organized in June 2010. The outcomes of the conference and resulting recommendations provided by the faculty, are reported in this article. These guidelines should assist in the diagnosis, classification, and prognostication in MDS in daily practice as well as in clinical trials.     

Apoptosis and prognostic factors in myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hyper-cellular bone marrow. This is thought to be due to apoptosis of hematopoietic bone marrow cells, resulting in ineffective hematopoiesis. Several studies have confirmed the presence of a high apoptotic rate and proliferative state in the bone marrow of MDS. However, MDS is a heterogeneous disease from the point of view of prognosis. Some patients develop only anemia and show long survival with or without maintenance therapy, while others develop fatal pancytopenia or leukemic changes and therefore show a poor prognosis. This review focuses on the relationship between prognosis and apoptotic or proliferative processes affecting hematopoietic cells in the bone marrow of patients with MDS.     

Diagnostic value of histology and immunohistochemistry in myelodysplastic syndromes

The histologic and immunohistochemical analysis of the bone marrow yields important information for the diagnosis of myelodysplastic syndromes (MDS), thereby often exceeding the information obtained by cytological analysis of smears. Notably, tissue-fibrosis, angiogenesis, or the abnormal localization of megakaryocytes and CD34+ progenitor cells can only be assessed histologically. Many of these parameters are also of prognostic significance. Moreover, evaluation of bone marrow histology is of crucial importance in cases with dry-tap or blood-contaminated marrow-smears, especially in hypoplastic states. Histologic/immunohistochemical investigation of the bone marrow therefore is strongly recommended for patients with (suspected) MDS, the minimum marker-panel suggested being CD31, CD34, and tryptase.     

Correlation of tumor necrosis factor alpha (TNF alpha) with high Caspase 3-like activity in myelodysplastic syndromes.

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor alpha (TNF alpha) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNF alpha signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNF alpha in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8 +/- 13.9 at 0 h vs. 177.8 +/- 55.2 units/mg protein at 4 h, n = 14, P = 0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76% +/- 0.25% vs. 3.99% +/- 1.1%, n = 14, P = 0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNF alpha levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (p = 0.517, n = 13, P = 0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (p = 0.593, n = 13, P = 0.033). Additionally when TNF alpha-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNF alpha-positive marrows (189.6 +/- 66.2 vs. 25.0 +/- 14.6 units/mg protein, respectively, P = 0.043). The increase in AI%, though not statistically significant, was also higher in the TNF alpha-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNF alpha-induced apoptosis were evaluated. TNF alpha treatment significantly increased AI% (P < 0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 microM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 microM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 microM/l) as well as PTX (250 microM/l) significantly curtailed the AI% induced by TNF alpha. The present studies thus identify the downstream effectors of TNF alpha-inducible apoptosis in MDS and so also the suppressors of TNF alpha apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.     

Apoptosis in patients with myelodysplastic syndromes: differential involvement of marrow cells in 'good' versus 'poor' prognosis patients and correlation with apoptosis-related genes.

Apoptosis has been implicated in the pathogenesis of marrow failure in MDS and the coexistence of marrow hypercellularity along with blood cytopenias was seen as evidence of extreme cell death of mainly mature cells in the marrow (ineffective hematopoiesis). We investigated apoptosis in 53 patients with MDS, by using single-step DNA extraction and gel electrophoresis and then by separating fresh marrow mononuclear cells in CD34+ and CD34- populations and in situ single cell evaluation of the process. We also studied the expression of apoptosis-related genes, in total and separated mononuclear marrow cells and correlated the findings with clinical and laboratory characteristics. Patients with apoptosis had increased marrow cellularity, longer overall survival and a longer period for transformation to AML. In 'good' prognosis MDS patients, total mononuclear marrow cells, as well as isolated populations of CD34+ and CD34- cells showed significant degrees of apoptosis; in 'poor' prognosis cases, however, apoptosis was evident only in a large percentage of CD34+ marrow cells and not in total or CD34- cells. Absence of expression of both c-myc and p53 in total marrow cells was associated with significant degrees of apoptosis and in isolated CD34+ and CD34- marrow cells the phenomenon was inversely correlated with the level of bcl-2 expression. In conclusion, marrow apoptosis is detected in both CD34+ and CD34- cells in early MDS and seems to be restricted to CD34+ cells in advanced MDS cases.     

Telomerase activity in myelodysplastic syndromes

Myelodysplastic syndromes are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral cytopenias. Telomeres are thought to be critical in maintaining normal hematopoiesis. In this study, we assessed telomere dynamics in order to obtain further insight into the pathogenesis of MDS. We studied telomerase activity (TA) in mononuclear cells from peripheral blood (PB) and bone marrow (BM) from patients with myelodysplastic syndrome (MDS; n=24), acute myeloid leukemia (AML; n=14), chronic myeloid leukemia (CML; n=12) and 11 normal controls using a polymerase chain reaction-based telomeric repeat amplification assay. Telomerase activities (mean+/-S.D.) were found as 0.199+/-0.09, 0.414+/-0.55, 0.253+/-0.26 and 0.181+/-0.05 pg/ml in PB mononuclear cells, respectively (P>0.05). Comparison of TA of BM mononuclear cells from 19 MDS patients versus 10 BM samples from normal controls revealed no significant difference (P=0.3). There was no correlation between the levels of TA and clinical and prognostic parameters of the patients with MDS, such as degree of anemia, platelet counts on presentation, gender, presence of organomegaly, bone marrow fibrosis and BM blast percentages. Patients who had higher TA had significantly inferior survival compared with patients who had lower TA (P=0.005). Consistent with previous data, our results suggest that in patients with MDS, telomerase activity might be insufficient to compensate for the telomere shortening. Furthermore, TA might be prognostically important in patients with MDS. Measurements of enzymatic activity in association with telomere length studies may help to understand the prognostic role of telomere dynamics in patients with myelodysplastic syndromes more reliably.     

CD10 is a marker for cycling cells with propensity to apoptosis in childhood ALL

CD10 constitutes a favourable prognostic marker for childhood acute lymphoblastic leukaemia. Since correlations between CD10, cell cycle and apoptotic abilities were demonstrated in various cell types, we investigated whether differences existed in the cycling/apoptotic abilities of CD10-positive and CD10-negative B acute lymphoblastic leukaemia cells. Twenty-eight cases of childhood acute lymphoblastic leukaemia (mean age of 6.8 years) were subdivided into two groups according to high (17 cases, 93.2+/-4.5%, MRFI 211+/-82 CD10-positive cells) or low (11 cases, 11.5+/-6.2%, MRFI 10+/-7 CD10-negative cells) expression of CD10. CD10-positive acute lymphoblastic leukaemia cells were cycling cells with elevated c-myc levels and propensity to apoptosis, whereas CD10-negative acute lymphoblastic leukaemia cells had lower cycling capacities and c-myc levels, and were resistant to apoptosis in vitro. A close correlation between all these properties was demonstrated by the observations that the few CD10-positive cells found in the CD10-negative acute lymphoblastic leukaemia group displayed elevated c-myc and cycling capacities and were apoptosis prone. Moreover, exposure of CD10-positive acute lymphoblastic leukaemia B cells to a peptide nucleic acid anti-gene specific for the second exon of c-myc caused inhibition of c-myc expression and reduced cell cycling and apoptotic abilities as well as decreased CD10 expression.     

Angiogenesis in myelodysplastic syndromes

It is now well established that solid tumour growth depends on angiogenesis. However, less is known about the generation of new vessels in haematological malignancies and, in particular, in preleukaemic-myelodysplastic syndromes (MDS). In this study, bone marrow microvessel density (MVD) was assessed by immunohistochemistry and compared in trephine biopsies from 14 controls, five infectious disease (ID), 82 MDS, 15 acute myeloid leukaemia (AML) and 14 myeloproliferative disorder (MPD) patients. Statistical analysis (P < 0.001) demonstrated that MDS MVD was higher than in controls and ID (21 +/- 9 vs 6 +/- 2 and 10 +/- 8 respectively) but lower than AML (30 +/- 12) and MPD (40 +/- 12). Among MDS-FAB subtypes, MVD was significantly higher in RAEB-t, CMML and fibrosis subsets compared to RA, RARS and RAEB subsets (P= 0.008). To further investigate angiogenesis machinery, the expression of vascular endothelial growth factor (VEGF) was evaluated by means of immunohistochemistry in control, MDS, AML and MPD biopsies. Even though VEGF mRNA expression was reported in the past in AML cell cultures and cell lines, in our samples VEGF expression was found to be particularly strong in most of the megakaryocytes but significantly less prominent in other cell populations including blasts. Since our findings suggest a correlation between angiogenesis and progression to leukaemia, additional work is now warranted to determine what regulates the generation of new vessels in MDS and leukaemia.     

Increased CD133 expression in bone marrow of myelodysplastic syndromes

Fresh frozen bone marrow biopsies were evaluated immunohistochemically, applying monoclonal antibodies against CD31, CD34, VEGFR-2 and CD133, a novel marker identifying human endothelial progenitor cells (EPCs). Specimens of 51 patients diagnosed with MDS were compared with 16 AML and 18 controls. The percentage of CD34 expressing cells was increased and CD31 expression was decreased in advanced stages of MDS compared with normal BM. VEGFR-2 expression was also raised in MDS. Here we show for the first time that increased numbers of CD133 positive cells are present in the majority of MDS patients. Additionally, those cells occasionally seem to contribute to capillary forming units in bone marrow.