Disease Progression In Myelodysplastic Syndromes Research Articles

The prognostic value of maturation-associated phenotypic abnormalities in myelodysplastic syndromes

Several phenotypic abnormalities of bone marrow (BM) hemopoietic precursors have been associated with disease progression in myelodysplastic syndromes (MDS). We analyzed the influence on overall survival of the expression of lineage and maturation-associated antigens of BM hemopoietic cells quantified in a previous study. In the univariate Cox regression the peripheral platelet count was a significant favourable factor for overall survival. Unfavorable prognostic factors were: WPSS, increase in BM CD34+ cells, increased mean fluorescence intensity (MFI) of CD13 on myelocytes, metamyelocytes and mature neutrophils as well as increased CD45 of myelocytes and mature neutrophils. In a model containing platelet count, WPSS and MFI of CD45 and CD13 on mature neutrophils, only hyperexpression of CD13 and degree of thrombocytopenia were independent risk factors. Therefore, phenotypic features that can also be obtained from PB might be useful for predicting survival in MDS.

Lenalidomide in Myelodysplastic Syndrome and Multiple Myeloma

The use of thalidomide is limited by adverse effects of sedation, constipation, neuropathy and thromboembolism. In order to discover more potent and less toxic immunomodulators than thalidomide, its chemical structure was modified and lenalidomide was formed. Lenalidomide is approved by the US FDA for the treatment of patients with low-risk myelodysplastic syndrome (MDS) with deletion 5q cytogenetic abnormality. Two studies and a case report have evaluated lenalidomide in these MDS patients and showed significantly higher cytogenetic responses and durable red blood cell transfusion independence. Lenalidomide should be the drug of choice for patients with low and intermediate-1 risk MDS (based on the International Prognostic Scoring System) with chromosome 5q31 deletion with or without other karyotype abnormalities. Lenalidomide, in combination with dexamethasone, has been compared with dexamethasone alone in patients with relapsed or refractory multiple myeloma (MM) in two studies (MM-009 in North America and MM-010 in Europe, Israel and Australia). In these two phase III trials, lenalidomide demonstrated impressive (58-59%) response rates with dexamethasone. Lenalidomide has also been shown to overcome thalidomide resistance in MM patients. Therefore, the lenalidomide plus dexamethasone regimen provides another treatment option, in addition to first line MM treatment regimens of bortezomib, thalidomide or high-dose dexamethasone, for the treatment of relapsed or refractory MM. Lenalidomide does not produce significant sedation, constipation or neuropathy, but does lead to significant myelosuppression, unlike thalidomide. The prescribing information has a black box warning for risk of myelosuppression, deep vein thrombosis/pulmonary embolism and teratogenicity. Administration of lenalidomide is recommended at a starting dose of 10 mg/day orally for deletion 5q in MDS patients. Significant risk of myelosuppression may lead to dose reduction in the majority of these patients. Clinical trials of relapsed and refractory MM have shown that lenalidomide is clinically efficacious at a dosage of 25 mg/day when administered in combination with dexamethasone. Lenalidomide should be continued until disease progression in both MDS and MM patients.

Reduced Natural Killer Function Accompanies MDS Disease Progression and Is Restored by Lenalidomide (CC5013, Revlimid®) Via a Mechanism Divergent from Interleukin-2 (IL-2).

Background: Patients with myelodysplastic syndromes (MDS) display several abnormalities of T cell and NK cellular immunity. Tumor lysis by NK cells occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs. Lenalidomide is an immunomodulatory (IMiD) drug structurally related to thalidomide, which has proven clinical efficacy for the treatment of low-risk MDS (List et al, NEJM 2005; 352:549). Investigations in patients with multiple myeloma (MM) reported that thalidomide was associated with enhanced cytolytic function and increased NK cells in responding patients. We investigated the action of lenalidomide on NK function and activating NK receptor expression in patients with MDS.Methods: MDS patients were categorized according to WHO category, age, sex, IPSS, and cytogenetics. Peripheral blood cells (PBMCs) were isolated from patients and normal donors and NK receptor expression determined in paired healthy and patient PBMCs by 3-color flow cytometry. NK receptor expression was analyzed for CD158a (KIR2DL1, KIR2DS1), CD158b (KIR2DL2, KIR2DL3, KIR2DL3), KIR3DL1, KIR2DS4, NKG2A, NKG2D, NKp30, NKp44, and NKp46. Cytotoxicity assays were performed using 5-hour 51Cr-release assays.Results: Forty-eight MDS patients and 37 normal donors were analyzed, demonstrating that NK cytolytic function was reduced in the patient population (19% ± 21 S.D. vs. 44% ± 21) (p<0.001). Reduced NK function in MDS was significantly associated with higher IPSS (p=0.01), abnormal karyotype (p=0.05), presence of excess blasts (p=0.01), and age-adjusted bone marrow hypercellularity (p=0.04). MDS patients had reduced display of the activating receptor NKp30, accompanied by NKG2D (an activating C lectin-like (NKG2)-family receptor) downregulation that closely correlated with impaired NK function (p=0.001). NK-mediated cytotoxicity and NKR expression were examined after treatment with lenalidomide 5μM for 72 hours or IL-2 in normal donors and MDS patients. Lenalidomide augmented NK lytic function in normal donors (vehicle, 42% ± 15 vs. lenalidomide, 71% ± 17; P≤0.01 by t test) and in 10 out of 17 (59%) of the MDS patients (vehicle, 25% ± 25 vs. lenalidomide 42% ± 33). IL-2 increased NK cytolytic function in all MDS patients (vehicle, 16% ± 16 vs. lenalidomide, 47% ± 33) and the percentage of NK cells expressing CD69 was increased from both MDS patients and normal controls. Similarly, the percentage of NK cells expressing NKp30, NKG2D, NKp44 but not NKp46 was significantly enhanced by IL-2 culture. In contrast, lenalidomide treatment failed to induce NKR expression in MDS patients and normal controls.Conclusions: These findings suggest that impairment of NK cytolytic function in MDS derives in part from reduced display of the activating NK receptors such as NKG2D, which accompanies disease progression. Lenalidomide augments NK function in some patients; however, the mechanisms of functional augmentation by IL-2 and lenalidomide are divergent. Evasion of NK immunosurveillance may be an important factor associated with MDS disease progression.

Bone Marrow Patterns of Expression of p53, Caspase-3, Ki-67, P21waf and WT-1 Proteins May Provide a Tool To Differentiate Inherited from Acquired Cytopenias: A Study of 199 Patients.

Cell cycle regulation plays a major role in controlling hematopoiesis. Loss of appropriate mechanisms regulating cell cycle may lead to increased bone marrow apoptosis with subsequent cytopenias or to the accumulation of DNA-damaged cells, with an associated increased risk of leukemogenesis. p53 protein, a regulator of cell cycle, induces maturation arrest and apoptosis through its downstream mediator p21waf or other pathways. Cleaved caspase-3 is a common pathway for most apoptotic mechanisms. Wilms' tumor 1 (WT-1) protein, which is normally not detected in the bone marrow, has been correlated with disease progression in myelodysplastic syndrome. Ki-67 is a marker of cell proliferation. To study bone marrow (BM) cell cycle patterns in inherited and acquired cytopenias, we used immunohistochemical methods against p53 (DO-7, Dako, Carpinteria, CA), WT-1 (6F-H2, Dako), cleaved caspase-3 (Cell Signaling, Beverly, MA), Ki-67 (Dako), and p21waf (Dako) in BM biopsies from patients with various inherited bone marrow failure syndromes (IBMFS, 68), low blast refractory cytopenias (RC, 29), acquired aplastic anemia (AA, 10) and acquired cytopenias (AC, 92 of infectious, endocrine, hepatic, and renal origins). The IBMFS included: 16 Shwachman-Diamond syndrome, 18 Diamond Blackfan anemia, 15 Fanconi's anemia, 4 dyskeratosis congenita, 1 Kostman's syndrome, and 14 unclassified inherited neutropenias, as well as 21 control subjects. p53 protein was significantly overexpressed (≥1% positive marrow cells) in 68% of IBMFS, 52% of RC, and none of the AA, AC, or control subjects. Intense p53 protein overexpression (≥5%) was detected in 83% of the p53-positive IBMFS but in only 26% of the p53-positive RC patients (P<0.0001). None of the subjects expressed WT-1 protein while the overexpression of p21waf was rare. Detectable caspase-3 (any positive cells) was seen in 40% of IBMFS, 55% of RC, 20% of AA, 33% of AC, and 52% of the controls with no statistical significance among the groups. Ki-67 staining was detected in 64% of IBMFS (p<0.00001 compared to controls), 28% of RC, 0% of AA, 7% of AC, and 9% of the controls. Most IBMFS marrows with p53 protein overexpression had concomitant Ki-67 expression (80%) compared to 40% of RC suggesting deregulation of cell cycle control pathways. p53 protein overexpression and caspase-3 expression were not correlated indicating a defective p53 apoptotic pathway in IBMFS and RC. The following immunohistochemical patterns are observed: IBMFS p53+ intense, Ki67+; RC p53+ weak, Ki67±; AA p53-, Ki-67-; AC p53-, Ki-67+. WT-1 protein plays no role in the development of RC or the RC stage of IBMFS. The p53 pathway may play a role in leukemogenesis in RC and IBMFS. The absence of a positive correlation between p53 protein overexpression and increased caspase-mediated apoptosis in RC and IBMFS suggests that a dysfunctional p53 apoptotic pathway may play a role in the pathogenesis of these disorders. Disease-specific patterns of bone marrow cell cycle characteristics may provide an additional tool for discrimination of bone marrow failure disorders.

Expression and methylation status of the FHIT gene in acute myeloid leukemia and myelodysplastic syndrome

To clarify the role of fragile histidine triad (FHIT) in hematological malignancies, we examined the methylation status and the expression level of the FHIT gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cells in comparison with the methylation of the p15(INK4B) gene. The FHIT methylation was found in 13 of 94 (13.8%) AML and 22 of 40 (55.0%) MDS cases, but not in normal mononuclear cells (MNCs). Both the frequency and density of methylation increased in the advanced-stages MDS and the relapsed AML cases. Although FHIT and p15(INK4B) methylations were not correlated in MDS and AML, increased FHIT methylation at the relapse in AML was associated with p15(INK4B) methylation. The median expression level in AML was significantly higher than in normal MNCs, although the median expression level in those with methylation was significantly lower than in those without methylation. Furthermore, the methylation level at relapse was significantly higher than at diagnosis in AML. These results suggested that FHIT methylation was accumulated through the disease progression of MDS and AML, and the role of the FHIT gene as a tumor suppressor seemed different in AML and MDS.

High IGFBP-3 levels in marrow plasma in early-stage MDS: effects on apoptosis and hemopoiesis

The pathophysiology of the myelodysplastic syndromes (MDS) is incompletely understood. Tumor necrosis factor (TNF)alpha levels are elevated, particularly in early-stage MDS, and apoptosis in marrow cells is upregulated. Observations in other models have shown a role for insulin-like growth factor binding protein 3 (IGFBP-3) in TNFalpha-mediated apoptosis. We observed increased levels of IGFBP-3 in the marrow plasma of patients with MDS (P = 0.005) and hypothesized that altered IGFBP-3 levels contribute to the dysregulation of hemopoiesis in MDS by affecting proliferation and apoptosis. Western analysis of marrow plasma from MDS patients revealed an increase in the ratio of intact vs fragmented IGFBP-3 in early-stage MDS (relative to controls) that decreased with MDS disease progression, suggesting increased proteolysis with more advanced disease. Thus, these results provide evidence for dysregulation of IGFBP-3 in patients with MDS. While the data are complex, they are consistent with a modulatory effect of IGFBP-3 on hemopoiesis in MDS. Conceivably, understanding these mechanisms may allow for the development of novel therapeutic strategies.

Bmi-1 Expression Predicts the Progression of Malignant Hematopoietic Stem Cells in Myelodysplastic Syndrome.

Bmi-1 is a member of the Polycomb group (PcG) of transcriptional repressor genes, which could have an essential role in embryogenesis and be expressed restrictedly in the stem cells and proliferating cells. Some studies have recently reported that Bmi-1 is required to regulate the adult self-renewing hematopoietic stem cells. Others have shown that overexpressed Bmi-1 gene could cause the neoplastic proliferation of cells. A series of evidence on Bmi-1 suggests that it might be closely associated with the progression of the hematopoietic malignancies. Myelodysplastic syndrome (MDS) is a clonal bone marrow disorder characterized by aberrant hematopoiesis susceptible to acute leukemia. This category is quite heterogeneous with a variable clinical course and prognosis. Therefore, it is difficult to decide when to initiate therapies because some patients survive for years without treatment whereas others come to die in rapid progress in overt leukemia. Thus, we investigated whether the expression of Bmi-1 protein in CD34+ cells could be correlated with disease progression of MDS. Primary cells from patients with RA, RAEB, and MDS-AML, were stained with anti-CD34 antibody-PE and then fixed in PFA followed by staining with anti-Bmi-1-antibody-FITC. These cells were subjected to flow cytometric analysis. Bmi-1 expression in CD34+ cells was preferentially seen in RAEB and MDS-AML compared with RA (13.86%±6.40% in RA(n=4), 63.05%±8.19% in RAEB(n=6), 73.99%±33.40% in MDS-AML(n=6)). Patients were dead with >70% at Bmi-1 expression level in CD34+. Moreover, two patients with RA at >10% of Bmi-1 positivity made disease progression to RAEB. One patient with RAEB at <10% at the Bmi-1 expression in CD34+ is still alive. Furthermore, we are dissecting gene profiling of cells from different types of MDS. These results show that the expression of Bmi-1 can distinguish the proliferating malignant stem cells from resting cells by using flow cytometry. Alternatively, IPSS in MDS patients is closely correlated with Bmi-1 expression of CD34+ cells. It may lead to the prediction of prognosis in patients with MDS in quality. Taken together, Bmi-1 expression could predict the disease progression and prognosis of MDS. Finally, Bmi-1 gene might be a target to control leukemogenesis by immunotherapy or gene therapy.

Delineation of Promoter Methylation in Patients with Myelodysplastic Syndromes: Widespread and Concurrent Hypermethylation of Multiple Genes and Increased mRNA Expression of the DNA Methyltransferases DNMT1, 3A and 3B.

Transcriptional silencing of tumor suppressor genes by promoter hypermethylation is associated with hematological malignancy, including myelodysplastic syndromes (MDS). Specifically, hypermethylation of the p15 gene has previously been associated with MDS and seems to be acquired with disease progression. The methylation status of other genes associated with the development of myeloid malignancies is, however, largely unknown. We have elucidated this in total bone marrow mononuclear cells (BM-MNC) as well as CD34 enriched cells from 17 patients with different stages of MDS by determining the promoter methylation status in four genes believed to be associated with the development of acute myeloid leukemia (AML), namely those encoding for the p15, HIC, E-cadherin (ECAD), and the Estrogen receptor (ER). Employing Bisulfite-Denaturing Gradient Gel Electrophoresis (Bisulfite-DGGE) we found all four genes to be hypermethylated in MDS, albeit with varying frequencies (19/37, 12/37, 10/37, and 7/37, respectively). Since the Bisulfite-DGGE allows for the detection of heterogeneous methylation patterns we were able to compare these patterns. Similar Bisulfite-DGGE band patterns were observed in total BM-MNC and CD34 cells in all positive patients suggesting that methylation is a process encompassing both immature and end-stage hematopoietic cells. In a total of 37 patients (14 RA, 3 RAS, 11 RAEB, 4 RAEB-t and 5 with AML secondary to MDS) analyzed, we found promoter hypermethylation of the HIC gene to be significantly increased in advanced MDS (p<0.05). To determine the kinetics of promoter methylation in the single patient, bone marrow from eleven MDS patients, where two or more samples were available, were analyzed (observation time between sampling ranged from 43 to 1132 days, median 284 days). These analyses showed that the patterns of methylation are surprisingly stable over time. When two or more samples were analyzed we found that the number of methylated genes only changed in three of the eleven patients. To delineate the process of hypermethylation we determined the mRNA levels of the DNA methyltransferases (DNMT) 1, 3A, and 3B. Interestingly, all three DNMTs were up-regulated in MDS compared to normal bone marrow (DNMT1 and DNMT3A: p<0.01; DNMT3B: p<0.0001) and DNMT3A and 3B were up-regulated in advanced MDS compared to low-risk MDS (p<0.01). Taken together, these results imply an involvement of promoter hypermethylation in MDS disease progression, and DNMT overexpression as a contributing factor for the aberrant methylation in these patients.Figure: Promoter hypermethylation status over time. A) Development in methylation status in 11 patients. Arrows indicate patients where methylation status changes over time. a) RA, b) RAS, c) RAEB, d) RAEB-t, e) sAML. Black quarters: methylated; white quarters: unmethylated. B) Bisulfite-DGGE experiments for methylation in the p15, HIC , and ER promoters in UPN 2. [Display omitted]

Th1-biased humoral immune responses against Wilms tumor gene WT1 product in the patients with hematopoietic malignancies

The Wilms' tumor gene WT1 is highly expressed in leukemias and myelodysplastic syndrome (MDS), and WT1 expression levels increase along with the disease progression in chronic myeloid leukemia and MDS. We previously reported that IgM and IgG WT1 antibodies were detected with significantly higher detection rate and antibody titers in leukemias and MDS compared to those in healthy volunteers. In this study, whether IgG humoral immune responses against WT1 protein were Th1- or Th2-type were determined by measurement of four subclasses of IgG WT1 antibody, IgG1, IgG2, IgG3, and IgG4. In leukemias and MDS, Th1-type WT1 antibodies such as IgG1, IgG2, and IgG3 were significantly increased in both detection rate and antibody titers compared to those in healthy volunteers, whereas Th2-type WT1 antibody such as IgG4 did not increase. These results showed that Th1-biased humoral immune responses against WT1 protein were generated in leukemias and MDS. These results should allow us to consider that Th1-biased cellular immune responses against WT1 protein, which was essentially needed for cancer immunotherapy targeting WT1, should be elicited in patients with hematopoietic malignancies.

Survivin Expression in Acute Leukemias and Myelodysplastic Syndromes

We analyzed by immunocytochemistry the expression of survivin in bone marrow cells from 36 acute myeloid leukemia (AML) cases, from 98 patients with myelodysplastic syndrome (MDS), and from 41 non hemopathic subjects. Our aim was to evaluate whether abnormalites in survivin expression were associated with peculiar laboratory and clinical findings, altered apoptosis levels or altered proliferative rate. In normal samples survivin was never detectable. It was detected in almost all AML and MDS cases. In AML and in MDS with more than 5% bone marrow blasts survivin levels higher than in RA and RARS were observed (P = 0.04). In MDS a tendential inverse correlation between survivin and TUNEL positivity was identified (P = 0.08), whereas survivin expression was independent of the proliferative rate. Survivin levels did not predict disease progression in MDS; among AML patients treated with intensive polichemotherapy, survivin expression was significantly higher in resistant cases (P = 0.01). Our findings confirm the high incidence of survivin expression in AML. Its abnormal expression also in MDS may play a role in promoting aberrantly increased cell viability and contribute to the altered homeostatic balance between cell growth and cell death.

Understanding the pathogenesis of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal disorders of an immature hematopoietic progenitor cell. MDS hematopoiesis is characterized by abnormal progenitor proliferation and impaired cellular differentiation and maturation. As a consequence, the majority of MDS patients have a gradual evolution towards progressive bone marrow failure and/or leukemic transformation. The efforts made by several research groups during the last few years have helped to unravel the complex underlying pathogenesis of MDS. In this review, we will focus on the principal cytogenetic and molecular abnormalities involved in MDS initiation and disease progression. Additionally, we will discuss the influence of the marrow microenvironment on survival and proliferation of hematopoietic progenitors in MDS.

Blood levels of angiogenin and vascular endothelial growth factor are elevated in myelodysplastic syndromes and in acute myeloid leukemia.

Angiogenesis is of prognostic importance not only in solid tumors but also in malignant blood diseases. We measured levels of vascular endothelial growth factor (VEGF), angiogenin (ANG), and basic fibroblast growth factor (bFGF) in peripheral blood samples from 65 patients with myelodysplastic syndrome (MDS), from 25 patients with de novo acute myeloid leukemia (AML), and from 50 healthy donors. In matched samples, VEGF levels in serum were substantially higher than VEGF levels in plasma (380.7 +/- 56 pg/ml vs. 45.3 +/- 4.5 pg/ml, mean +/- SEM, p < 0.001), whereas serum and plasma levels of ANG were comparable and significantly correlated (r = 0.8; p < 0.01). Compared to normal controls (1.3 +/- 0.09 pg), serum levels of VEGF corrected for the peripheral blood platelet count (VEGF/10(6) platelets, VEGF(PLT)) were elevated in patients with refractory anemia (RA; 3.1 +/- 0.8 pg, p < 0.01), and reached maximal values in patients with advanced stage MDS (RAEB, RAEB-t) (3.5 +/- 0.6 pg, p < 0.001), de novo AML (3.6 +/- 1.1 pg, p < 0.05), and chronic myelomonocytic leukemia (CMML; 3.7 +/- 0.9 pg; p < 0.001). Levels of soluble ANG were elevated in RA (351 +/- 25.7 ng/ml, p < 0.001), in RAEB/RAEB-t (402 +/- 17.9 ng/ml; p < 0.001), in CMML (413.8 +/- 29.5 ng/ml; p < 0.001), and in patients with AML (305.1 +/- 17.1 ng/ml; p < 0.01, controls 255.4 +/- 8.1 ng/ml). Serum bFGF was neither elevated in MDS nor in AML patients. These results suggest that VEGF(PLT) is a marker of disease progression in MDS. Moreover, we show for the first time that elevated blood levels of ANG can be found in patients with myeloid malignancies, suggesting a role of ANG in the pathogenesis of these diseases.

Wilms' tumor gene WT1: its oncogenic function and clinical application.

The Wilms' tumor gene WT1 is a gene responsible for the childhood renal tumor. Wilms' tumor, and is defined as a tumor suppressor gene. However, the wild-type WT1 gene is highly expressed in leukemic blast cells of myeloid and lymphoid origin, and thus, WT1 messenger RNA provides a novel tumor marker for detection of minimal residual disease of leukemias and for monitoring disease progression of myelodysplastic syndromes. The WT1 gene exerts an oncogenic function rather than a tumor-suppressor gene function in solid tumors as well as leukemias, and the WT1 gene product is an attractive tumor antigen capable of eliciting cytotoxic T lymphocytes against WT1-expressing tumors.

Utility of blasts with a clear halo around the nucleolus as a predictive indicator for disease progression in patients with myelodysplastic syndromes and aplastic anemia.

This study was designed to evaluate the utility of blasts with a clear halo around their nucleoli (BCHN) as a predictive indicator of disease progression in myelodysplastic syndromes (MDSs) and aplastic anemia (AA). Bone marrow aspirates from 75 patients with MDSs and 18 with AA were fixed in 95% ethanol solution or 10% neutral formalin and stained with the Papanicolaou method. BCHNs were detected in 57 of 75 patients with MDSs and in 10 of 18 AA patients. Disease progression was restrictedly observed in 17 patients with MDSs who had BCHNs at onset and in 1 patient with AA. The proportion of BCHNs increased with disease progression in these 16 of 17 patients with MDSs. The presence of BCHNs at onset and the increase in proportion of BCHNs during the clinical course of MDSs were significant indications for predicting disease progression.

The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.

RAS, FMS and p53 mutations and poor clinical outcome in myelodysplasias: a 10-year follow-up.

The molecular mechanisms underlying the development and evolution of myelodysplastic syndrome (MDS) are largely unknown. The increasing number of blast cells in the bone marrow correlate with poor prognosis and risk of developing acute leukemia. Such progression is frequently associated with increasing chromosomal abnormalities and genetic mutations. A cohort of 75 MDS patients were investigated for RAS, FMS and p53 mutations, and these molecular findings were related to cytogenetics, clinical status, transformation to acute leukemia, prognostic scores and survival. A mutation incidence of 57% (43/75) was found, with 48% (36/75) RAS mutations, 12% (9/75) FMS mutations and 8% (4/50) p53 mutations. The mutation status for RAS and FMS was related to MDS subgroup, increasing with poor-risk disease. The highest incidence was in the chronic myelomonocytic leukemia (CMML) subgroup. The most frequent RAS mutations were of codon 12 and a predominance of FMS codon 969 mutations was observed. A statistically significant increased frequency of transformation to AML was observed in MDS patients harboring RAS or FMS mutations (P < 0.02). Patients with oncogene mutations had a significantly poorer survival compared with those without mutations at 2 years and at the end of the period of follow-up (P < 0.02). Multivariate analysis including mutation, age, gender, diagnosis (FAB), cytogenetics and International score shows that the International score and mutation and age is the best predictive model of a poor outcome, (P < 0.0001). When the analysis was undertaken without the International score, mutation and gender was the best predictor of poor survival (P = 0.005). This study shows that oncogene mutation, indicative of genetic instability, is associated with disease progression and poor survival in MDS.

Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome

A cell line designated SKM-1 was newly established from leukaemic cells of a 76-year-old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis, the cloned cells were positive for butyrate esterase, but negative for the Epstein-Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA-DR. Cells from the primary peripheral blood and those from 50 passages of the SKM-1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46,XY, del(9)(q13;q22), der(17) t(17;?)(p13;?). The SKM-1 cells have two mutations in p53 gene and overexpress the p53 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.

Pathogenetic implications of internuclear bridging in myelodysplastic syndrome. An Eastern Cooperative Oncology Group/Southwest Oncology Group Cooperative Study.

Numerous morphologic features have been described in bone marrow and peripheral blood in myelodysplastic syndrome (MDS). We draw attention to a high incidence of a subtle morphologic feature, internuclear bridging (INB), not previously recognized as a morphologic feature in MDS. The occurrence of INB in MDS suggests an underlying abnormality of mitotic division that could explain the impaired production of hematopoietic cells, the addition and deletion cytogenetic changes, and the stepwise disease progression and cytogenetic progression characteristic of MDS. Lack of awareness that INB occurs in MDS may cause confusion of MDS and congenital dyserythropoietic anemia type I, a congenital process also characterized by INB.