Myelodysplastic Syndromes Subgroups Research Articles

Factors Involved in Response to Thalidomide in Myelodysplastic Syndrome: Impace of HLA and Prognostic Factors

Myelodysplastic Syndrome (MDS) is a clonal disease of hematopoiesis characterized by dysplasia in one or more lineages. Since the syndrome is clinically heterogenous, treatment options are complex and hard to define. Thalidomide (T) is the first agent tested in MDS for its immunomodulatory and anti-angiogenic effects. Twenty to 59 % response rates, which is mainly hematological improvement, have been reported. However, because of excessive toxicity and low response rates, thalidomide has not been widely used as an effective treatment option for MDS. Factors invoved in response to T are not well delineated. Better response to immunosupression (IS) in HLA-DRB1*15(+) patients with aplastic anemia and some MDS subgroups have been reported. In this study, the medical records of MDS patients, who received T at least for ≥ 3 months were retrospectively analyzed. Responses could be evaluated in 47 patients with a median age of 59 years (range, 15–84) and M/F: 29/18. According to WHO classification, there were 7 refractory anemia (RA), 9 refractory anemia with ring sideroblasts (RARS), 5 refractory cytopenia with multilineage dysplasia (RCMD), 2 RCMD-RA, 13 RCMD-RS, 6 Hypoplastic MDS, 1 RAEB, 4 MDS/MF. International Prognostic Scoring (IPSS) risk groups consisted of 42 low/intermediate-1 and 5 intermediate-2/high risk patients. Clonal cytogenetic abnormalities were found in 16 patients (5 trisomy 8, 3 monosomy 7, 2 delY, 2 increased chromosomal breaks, 1 del5q, 1 monosomy 15 and 1 del21q). Thalidomide was administered at a dose range of 100–400mg/day and for a range of 3–48 months. Twenty-one patients had hematological response (10 major and 11 minor). Seven patients among responders had received additional erythropoietin (EPO) combined with T. There was no statistically significant difference between responders and non-responders in terms of age (median 53 vs 60.5 years), sex (M/F: 12/9 vs 17/26) and IPSS risk groups (Low risk/high risk: 19/2 vs 23/3). Response rates were 44% in RA; 50% in RCMD-RA; 56% in RARS; 55% in RCMD-RS; 20% in RCMD; 20% in hypoplastic MDS; 75% in MDS/MF. and none in RAEB patient. The EPO levels did not differ significantly between the responders and non-responders (EPO miu/ml <500 in 93% vs 83%). However, compared with non-responders, majority of the responders had ferritin levels <1000 mcg/L (83% vs 50%, p<0.05). A total of 21 patients had their HLA type screened. HLADRB1*15 was detected in 44% of responders (4/9) and 16% of non-responders (2/12) (p<0.05). Interestingly, among 5 patients who had trisomy 8, 4 responded to T. Fourty-five percent of mainly low risk-MDS patients responded to T. Patients with MDS/MF and RARS/RCMD-RS and patients with less iron overload responded better. HLADRB1*15 predominance and the presence of trisomy 8 in T responders should be underlined.

Flow Cytometric Findings Indicative of Myelodysplasia Differ Depending on the Karyotype

Multiparameter flow cytometry (MFC) is increasingly being used as an adjunct to the diagnostic work-up in myelodysplastic syndromes (MDS). While many antigens have been described to be aberrantly expressed in MDS the findings are generally heterogeneous and there is no consistent finding that would be present in all cases with MDS. The aim of the present study therefore has been to assess the immunophenotype in cases with MDS with clearly defined cytogenetic abnormalities as well as with normal karyotypes. Bone marrow samples of a total of 115 patients with MDS have been analyzed in parallel by MFC and chromosome analysis. They had a complex (≥3) aberrant karyotype (n=7), a normal karyotype (n=74), a deletion of the long arm of chromosome 5 (del(5q), n=19), a deletion of the long arm of chromosome 20 (del(20q), n=8), or a monosomy 7 (−7, n=7), the latter three groups carrying the abnormality as sole chromosome aberration, respectively. A standardized panel of five-color combinations of antibodies has been applied for MFC allowing the detection of previously described aberrantly expressed antigens or antigen expression patterns. As compared to cases with other cytogenetics, in cases with a complex aberrant karyotype a higher bone marrow blast count (median 9.8 vs. 4.1%, p<0.001) as well as a higher frequency of coexpression of CD56 in granulocytes (70.0 vs. 25.9%, p=0.007) were found. Furthermore, the median side-scatter (SSC) signal in granulocytes was significantly lower in these cases (368 vs. 591, p=0.029). When combining the findings in the different cell lineages, significant differences between cases with complex aberrant karyotypes and those with other cytogenetics were found: the presence of more than 4 aberrantly expressed antigens on blasts, granulocytes or monocytes or the presence of >5% blasts were found in 80.0 vs. 29.0% of the cases (p=0.032). Cases with del(5q) as the sole chromosome abnormality more often featured a reduced SSC signal (94.7 vs. 69.3%, p=0.023) while an aberrant coexpression of CD56 on monocytes (21.1 vs. 54.5%, p=0.011) as well as the median number of aberrantly expressed antigens on monocytes (0.32 vs. 0.85, p<0.001) was lower in this group. Accordingly, at least 3 aberrantly expressed antigens on blasts, granulocytes or monocytes or a blast count >5% were found less frequently in cases with del(5q) as the sole chromosome abnormality as compared to other cases (36.8 vs. 69.9%, p=0.009). In cases with del(20q) as sole chromosome abnormality findings hardly differed from other cases with only a lack of CD13 expression in monocytes occurring more often (25.0 vs. 3.8%, p=0.058). Interestingly, in cases with −7 as sole chromosome abnormality a stronger SSC signal in blasts was observed as compared to other cases (median: 147 vs. 129, p=0.041) while the median number of aberrantly expressed antigens in granulocytes was lower (1.33 vs. 1.93, p=0.035). In cases with normal cytogenetics, a stronger SSC signal was found in granulocytes (median: 602 vs. 528, p=0.037) while the ratio of the SSC signal monocytes:lymphocytes was found to be lower (median: 3.0 vs. 3.3, p=0.044). Also the percentage of blasts was lower in these cases (median: 3.9 vs. 5.7%, p=0.032). With regard to the number of aberrantly expressed antigens on granulocytes and monocytes, however, at least 3 aberrantly expressed antigens were present more often in cases with normal cytogenetics (56.8 vs. 31.6%, p=0.016). These findings indicate that aberrantly expressed antigens are present in all of the analyzed cytogenetic subgroups of MDS. Importantly, the frequencies of findings typical for MDS differ between these groups. As a consequence, MFC panels used for the diagnostic work-up of MDS should be designed comprehensively to assure the detection of these diverse findings and should be evaluated prospectively.

Myelodysplastic syndromes. The epidemiological and etiological aspects

From a total of 18,416 bone-marrow biopsy reports entered into the Düsseldorf Bone Marrow Registry between 1975 and 1990 those of patients diagnosed as having preleukaemia, myeloid dysplasia, panmyelopathy with hypercellular bone marrow, refractory anaemia, sideroblastic anaemia or smoldering leukaemia were reanalyzed together with patient-data. If the diagnosis of myelodysplastic syndrome (MDS) was confirmed, the original blood and bone-marrow smear was re-examined and classified, 584 cases in all (3.2%). During the same period acute myeloid leukaemia had been diagnosed in 506 patients (2.8%). The average annual incidence of MDS in the Düsseldorf area was 3.65 per 100,000 inhabitants. Over 80% of cases occurred from the age of 60 years, while 7% were younger than 50 years. The sex ratio was the same in all subgroups of MDS, except chronic myelomonocytic leukaemia (male:female ratio 1:1.57). 31 patients (5.3%) had received ionizing radiation and/or cytostatic or immunosuppressive treatment for various underlying diseases before MDS had been diagnosed (secondary MDS). Preceding occupational contact with organic solvents could not be excluded with certainty in 12 patients. These data suggest that MDS is a relatively frequent disease of hematopoiesis among the older age groups. The proportion of secondary (treatment-induced) forms is small and does not explain the recently observed increased incidence.

Multiparameter Flow Cytometric Analysis Reveals Low Percentage of Bone Marrow Hematogones in Myelodysplastic Syndromes

Diagnosis of myelodysplastic syndromes (MDS) could be difficult. We explored the usefulness of the enumeration of maturing B-lineage precursors (hematogones) by multiparameter flow cytometric analysis in the diagnosis of MDS in bone marrow (BM) specimens. We evaluated 111 MDS, 120 non-MDS (most with cytopenias; control group 1), and 41 noncytopenic lymphoma staging BM (control group 2) specimens. The percentage of total hematogones was significantly lower in MDS (median, 0%; mean, 0.10%) compared with non-MDS (control group 1, median, 0.38%, and mean, 0.91%; control group 2, median, 0.38%, and mean, 0.60%; P < .0001), as was the percentage of the most immature (stage I) hematogones. Thus, hematogone enumeration may serve as a biomarker to aid in the diagnosis of MDS. Interestingly, the percentage of hematogones was not significantly different between MDS subgroups or patients with MDS with and without chromosomal abnormalities, implying that a defect in maturing B-cell precursors may be an early event in the pathogenesis of MDS.

Prognosis in myelodysplastic syndromes: Are the new classifications useful?

Increased understanding of the biologic and clinical parameters that define subgroups of myelodysplastic syndromes has led to continuing refinement of classification strategies for diagnostic and prognostic use. The French-American-British classification, based primarily on morphology, was modified by the World Health Organization system to include the negative impact of multilineage dysplasias and higher blast counts. In addition, this system identifies a distinct clinical subgroup characterized by an isolated chromosome 5 deletion. The International Prognostic Scoring System was created to calculate prognosis, risk of transformation to acute myeloid leukemia, and median survival times. However, therapeutic decisions cannot be solely guided by these systems, and the clinician must decide whether the intent is curative or palliative. Clinical symptoms and degree of transfusion dependency will dictate the degree of therapeutic intervention.

Cutaneous manifestations in relation to immunologic parameters in a cohort of primary myelodysplastic syndrome patients

Cutaneous lesions in myelodysplastic syndrome (MDS) may be specific or not and may reveal bone marrow transformation. Our purpose was to investigate in a cohort of 84 MDS patients the correlation of cutaneous findings with immunologic parameters and prognostic features of MDS in order to clarify their potential clinical significance. We studied a cohort of 84 newly diagnosed MDS patients in order to assess the cutaneous findings present at the time of diagnosis and during 1 to 3 years of follow-up. We described the clinical variety of cutaneous findings ascertained by histology. We also looked for any association between the group of MDS patients with skin manifestations and MDS subtype, immunologic and prognostic features highlighting transformation to acute leukaemia. Twenty-one patients presented cutaneous manifestations: 1 patient developed leukaemia cutis, 6 patients photosensitivity not associated with autoimmune disease, 3 prurigo nodularis, 2 Sweet's syndrome, 6 leucocytoclastic vasculitis, 2 ecchymoses and purpura associated with preexisting relapsing polychondritis, 1 patient subcutaneous nodules associated with Wegener's granulomatosis and 1 patient with malar rash and oral ulcers associated with preexisting systemic lupus erythematosus. Adjusted for age and gender, the presence of skin findings constitutes a significant predictor of the high-risk MDS subgroup (odds ratio, 3.59; 95% confidence interval, 1.18-10.92). Hypergammaglobulinemia was significantly higher in the MDS subgroup with skin manifestations (P = 0.03). Most MDS patients with cutaneous manifestations belong to the high-risk MDS subgroup and present hypergammaglobulinemia. Early biopsy of skin lesions in myelodysplasia is indicated.

SNP Arrays Facilitate Genotyping of Non-Synonymous SNP in MDS To Identify Disease Susceptibility Loci.

Myelodysplastic syndrome (MDS) is a clonal premalignant disease of hematopoietic stem cells characterized by cytopenias and predilection to acute myeloid leukemia (AML). While various exogenous factors (exemplified by chemotherapy-related MDS) constitute known risks for the development of MDS, it is possible that despite long latency, complex genetic traits contribute to MDS susceptibility. Such heritable factors include genes involving DNA repair, apoptosis, senescence, carcinogen catabolism and immune surveillance. Previously, disease association studies were mainly empiric and relied on rational selection of a very limited number of polymorphisms. Recent advances of SNP-array (SNP-A) technology allow for screening of a large number of SNPs. While some SNP-A utilize haplotype tags, custom arrays may specifically target known non-synonymous SNPs. We hypothesized that application of SNP-A genotyping may facilitate identification of potentially pathogenic SNPs. Such a screening approach is a hypothesis forming tool and our study is the first application of this technique to MDS. We have used the 13.9K non-synonymous Genotyping BeadChip (Illumina); DNA from 151 MDS patients (low risk: N=79, advanced: N=51, CMML1/2: N=21) and 99 controls (120 historical controls). In total, ∼2.4 mil genotypes were obtained. Genotype calls were computed and analyzed with Exemplar software. In the initial training Bonferroni correction was not applied. Instead as a hypothesis-forming approach we have ranked all SNPs according to their p value (automated analysis of multiplexed statistical evidence) and case/control ratio. We focused our search on the 100 highest ranking SNPs with a control frequency <5%. Globally, in the whole group 49 SNPs showed a p value of <.001, 75 SNPs present in ≤1% of controls and were found in >5% of cases. For example, the AA (rs3219484) variant of MUTYH, a gene involved in oxidative DNA repair was found in 19% of MDS vs. 2.6% of controls (p=9×10−8). To limit the impact of heterogeneity, subgroups of MDS were also analyzed separately. Among many interesting SNPs found, the AG genotype of (rs8192297) ANPEP was associated with RARS (35% vs. 15% in controls; p=.007). Similarly, the GA form of rs3730947 in DNA repair gene LIG1 was found in 11% of RARS patients (p=.00045), though it was absent in other MDS subtypes and controls. Similar, “enrichment” was observed in patients with CMML1/2, showing e.g., heterozygosity of WDR35 (rs1060742), ALPK2 (rs3809975) at the frequency of 45% and 30% (13% and 4% of controls; p=.0006, p=.0001). In sum, our study constitutes the first application of SNP-A genotyping to study susceptibility loci in MDS.

Analysis of Immunogenetic Factors in Myelodysplastic Syndromes (MDS) Reveals Potential Pathogenic Role Cytokine Genotypes Such as TGF-β.

Pathophysiologic and clinical features of MDS are related to the phenotype of the dysplastic hematopoietic clone. The immunogenetic background resulting from complex genetic predisposition traits may influence the quality of the immune response and shape the clinical features of MDS, including the severity of cytopenias and speed of malignant evolution. To test this hypothesis we selected the following immunogenetic factors: KIR and KIR-ligand (KIR-L) genotype, as well as cytokine/receptor single nucleotide polymorphisms (SNP). We genotyped a cohort of 90 patients with MDS/sAML (30 RA/RCMD, 30 RARS/ RCMD-RS, 30 RAEB/sAML); 60 healthy donors matched for ethnicity were analyzed as a control. A group of 66 patients with aplastic anemia (AA) was used as a reference. HLA type, KIR, KIR-L haplotypes, and the following SNPs were analyzed: IL-1α (−889 T/C),IL2 (−330 T/G +166 G/T), IL4 (−1098 T/G -590 T/C -33 T/C, IL-1R (−1970 C/T), IL-1RA (mspa 111100 T/C), IL-4RA (+ 190 G/A), IL-1 β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, -819 C/T), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF- γ (+874 A/T), TNF- α (−308 G/A, -238 G/A), CTLA-4 (exon 1, +49 A/G), FcgIIIR (+559 G/T) as well as SNPs in the CD45 gene (exon 4 +77 C/G, +138 A/G). In the MDS cohort, no difference in the frequency of KIR genotype constellations was identified. However, a higher frequency of 2DS5 (66% vs. 26%, p=.01) and a decreased frequency of 2DL3 (62% vs. 87% p=.02) was found when patients with hypocellular MDS (N=10) were analyzed separately. No significant difference in KIR-L C1/C2 genotype frequency between the group was found. However, an increased incidence of C2/C2 was found in high grade MDS/sAML (RAEB/sAML 44% vs. 13%, p=.02). In MDS, there was a decreased frequency of stimulatory 2DS1/C2 mismatch consistent with potentially enhanced cytotoxicity (17% vs. 44%, p=.01). No significant difference in the MDS cohort compared to control and when MDS subgroup were compared to each other, was found for the SNPs in IL-4RA, IL-12, IL-1α, IFN-γ, IL-2, IL-1 α, IL-1R, and IL4. However, when we examined the frequency of TGF- β genotypes, the MDS population showed a higher rate of TT codon 10 variant (59% vs. 32% in controls, p=.002) and of GG codon 25 variant (71% vs. 35% in controls, p=.0001), consistent with a “high secretor phenotype”. Of note is that, when AA was examined and compared to controls, a higher frequency of TGF-β high secretor genotype was found (GG codon 25 variant; 61% vs 35% in controls, p=.03). We also found a higher incidence of A/A genotype for CTLA-4 in MDS (47 vs 27, p=.001). This relationship was even more pronounced in hypocellular MDS. Moreover, hypocellular MDS was characterized by a higher prevalence of IL10 -819 T/T and -592 A/A phenotypes (40% vs 12% p=.03), which are functionally associated with a lower secretion. In sum, our findings demonstrate that various immunogenetic factors may be demonstrated in MDS patients, which may likely influence the quality of immune response and shape clinical features of MDS. Certain genotypic constellations (e.g., TGF gene variants) resemble, in particular in hypocellular MDS, a constellation seen in AA.

Hemochromatosis‐associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls.

CD4+CD25high Foxp3+ regulatory T cells in myelodysplastic syndrome (MDS)

AbstractFoxp3+ regulatory T cells (Tregs) play a central role in maintaining immune tolerance. A reduction in the function of Tregs is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to the suppression of host antitumor responses. We analyzed the absolute number of CD4+ and CD8+ Tregs in the peripheral blood of 52 patients with myelodysplastic syndrome (MDS) and show a significant correlation between increased number of CD4+ Tregs and MDS subgroups with 5% or more bone marrow blasts (P < .001), high International Prognostic Scoring System (IPSS) score (P < .001), and disease progression (P < .001), whereas no correlation between CD8+ Tregs and prognostic variables was observed. The CD4+ Tregs showed a polyclonal spectratype, and the percentage of the naive subset was significantly higher in the high-risk patients compared with low-risk or healthy age-matched donors (P = .032). Our data suggest that CD4+ Treg expansion is a feature of high-risk MDS and progression to aggressive subtypes of the disease.

Flow cytometry in myelodysplastic syndromes: Report from a working conference

Since new therapeutic strategies are emerging in myelodysplastic syndromes (MDS), a refined diagnostic procedure of the several subgroups of MDS is of increased importance. Multidimensional flow cytometry may add significantly to a more detailed analysis of the hematopoietic lineages with respect to qualification and quantification of bone marrow cells and is described in detail. Clearly defined aberrancies on myeloid immature and maturing cells are now identified with possible impact on diagnosis, classification and prognostication in the near feature.

Bone marrow morphology and classification systems in myelodysplastic syndromes

Summary One of the hallmarks of myelodysplastic syndromes (MDS) is their morphological heterogeneity which complicates the diagnosis and classification of these bone marrow disorders. The French–American–British (FAB) classification system which is based on cytological findings and the blast cell percentage in the bone marrow (BM) and peripheral blood (PB) was the universally accepted reference standard for categorization of MDS samples from 1982 to 1999 when it was modified by an expert panel of the World Health Organization (WHO). The WHO classification system distinguishes eight subgroups of MDS and is based on three principles: distinction between unilineage and multilineage dysplasia of haematopoietic cells, definition of refined blast cell percentages in BM and PB for diagnosing patients with RAEB and AML, as well as incorporation of genetic data into the classification scheme. When compared to the FAB proposals, the WHO classification appears to define more homogeneous subgroups of patients and to provide greater prognostic power. However, prospective studies of large patient populations will be required to decide whether the WHO classification allows accurate risk assessment and facilitates clinical decision-making for individual MDS patients.

Iron body stores in patients treated with red-cell transfusions

Background: Consequence of chronic transfusion therapy is a secondary iron overload, which adversely affects the heart, liver and endocrine glands. This can be minimized by iron chelating therapy. The assessment of iron overload is essential to assign and adjust this treatment. Serum ferritin is a widely used marker for the estimation of iron stores. However, a variety of conditions alter the relationship between serum ferritin levels and body iron stores. Soluble transferrin receptors in serum (sTfR) reflect the degree of erythropoiesis and the status of body iron. The data on usage of serum transferrin receptors for the assessment of iron overload are sparse. The purpose of our work was to estimate iron overload in transfusion dependent patients and to assess the combinative value of serum transferrin receptors and ferritin in prediction of iron load. Methods: Serum levels of iron, transferrin saturation, ferritin and sTfR were evaluated for 36 transfusion dependent patients. Twenty of them had myelodysplastic syndrome (MDS). Body iron (mg/kg) was calculated from the sTfR/ferritin ratio with the use of regression formula developed by Cook et al. Levels of serum parameters and calculated body iron were compared with estimated iron overload. We used absolute number of transfused units of blood as a measure of iron overload. Results: Serum ferritin above 1000 ug/L and above 2000 ug/L was found in 69 % and in 47 % of transfusion dependent patients. For MDS patients these levels were found in 85 % and 65 %. Concentration of sTfR was lower in the group of patients than it was in control group of healthy people (p = 0.001). We found a significant inverse correlation between sTfR and transferrin saturation. The estimated iron overload correlates well with serum ferritin (n = 0.66, p 1000 ug/L and good prognosis (subgroup of MDS) should need intensification of chelating treatment. In this study we didn’t find any advantages of calculating body iron in comparison to measuring serum ferritin levels for the estimation of iron overload.

The JAK2 V617F Mutation Identifies Specific Subgroups of Myelodysplastic Syndrome (MDS).

The myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal stem cell disorders characterised by ineffective erythropoiesis and peripheral blood cytopenias. However, specific subgroups of MDS with both myeloproliferative (MPD) and dysplastic features are recognized, which include chronic myelomonocytic leukaemia (CMML), refractory anaemia with ringed sideroblasts and thrombocytosis (RARS-T) and MDS/MPD unclassified. In addition cases of 5q- syndrome may present with a marked elevation in platelet count and a hypercellular bone marrow. The molecular events which determine the predominant morphological features however remain unclear. The JAK2 somatic point mutation (V617F) results in constitutive activation of the tyrosine kinase and culminates in both proliferation and differentiation signals to the cell and thus may contribute to the proliferative features identified in MDS as reported in the chronic MPDs. We analysed 194 patients from six centres with a diagnosis of MDS or MDS/MPD for the presence of the JAK2 mutation. 11 cases of RA; 70, RARS; 60, 5q- syndrome; 28, refractory anaemia with excess blasts (RAEB); 9, acute myeloid leukaemia with multi-lineage dysplasia (AML-MLD); 4, MDS/MPD-U; 1, CMML; and 1, hypoplastic MDS were analysed. DNA samples were obtained from bone marrow aspirate or peripheral blood. Ethical approval was obtained. Genomic DNA was prepared using standard methods (Qiagen). A modified allele specific PCR (AS PCR) was used to screen DNA samples. JAK2 mutated DNA samples were further subjected to pyrosequencing for confirmatory testing. The median age of the JAK2 mutant versus wild type (WT) cases was 66years (46–80years) v 67years (30–92years). The JAK2 mutation was detected in 13/194 (6.7%) cases overall. 4/70 (5.7%) RARS, 5/60 (8%) 5q- syndrome, 1/28 RAEB (associated with del 5q), 1/4 CMML, 1 MDS/MPD-U and 1/8 AML-MLD demonstrated the mutation. Within the RARS cohort, the prevalence of JAK2 mutation increased to 4/6 (67%) of cases with thrombocytosis (platelets >500x109/l). The presence of the mutation was confirmed in 8/13 cases. In cases analysed, the mutation was detected in CD34+, CD14+, CD15+ and CD61+ cell fractions but not CD3+ or CD19+ cell lineages. On analysis of the haemoglobin (Hb), platelet count and total white cell count (WCC) in the JAK2 mutant versus wild-type groups, there was no difference in median Hb (10.8 v 9.2 g/dl, p=0.275) but a significantly higher median platelet count (526 v 209 x109/l, p<0.001) and WCC (6.43 v 4.84x109/l, P<0.001) was observed in the JAK2 mutant cases. In addition, all JAK2 mutant cases were associated with marked increase in bone marrow cellularity. The JAK2 mutation identifies a subset of MDS patients with proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis. The presence of JAK2 mutation helps to identify a subgroup of MDS patients who may benefit from therapies that specifically target the mutant JAK2.

Expansion of Polyclonal CD4+CD25high Foxp3+ Regulatory T-Cells in High Risk Myelodysplastic Syndromes (MDS).

Foxp3+ regulatory T-cells(Tregs) play a central role in maintaining tolerance. A reduction in Tregs activity is a key feature of autoimmune diseases, whereas their expansion in malignant diseases leads to suppression of the host anti-tumour responses. Expansion of oligoclonal cytotoxic T cells, inhibition of MDS CD34+ progenitors proliferation and response to immunosuppressive therapy suggest that autoimmunity contributes to the pathophysiology of low risk MDS. However, this is not generally a feature of high risk MDS. We hypothesise that in high risk MDS patients an increased number of Tregs may suppress immune responses against the dysplastic clone. In early MDS, these cells may be reduced and or be associated with coexistent autoimmune diseases, an uncommon but recognised association of low risk MDS. We therefore studied CD4+ CD25high Foxp3+ and CD8+ CD25+Foxp3+ Tregs in peripheral blood of MDS patients and examined correlation with bone marrow blasts, cytogenetic Status, IPSS score and progression to AML. Clonality of CD4+CD25+ Tregs was assessed by TCR spectratype analysis of CDR3 size distribution and by CDR3 sequence analysis.52 patients with MDS (30 male, 22 female) with a mean age of 62 years (range 40 to 82 years) were studied. According to WHO classification, 5/52 (10%) had a diagnosis of 5q− syndrome, 9/52(17%) refractory anaemia (RA), 18/52 (35%) refractory cytopenia with multilineage dysplasia (RCMD), 16/52 (31%) refractory anaemia with excess blasts (RAEB) and 4/52 (7%) patients MDS/MPD (one with JAK2 V617F Mutation). Cytogenetic study shows normal pattern in 57%, 5q- in 22%, stable cytogenetic in 10% and complex findings in 11%. All samples were taken at diagnosis prior to any treatment. In addition 5 samples were analyzed pre and post 5-Azacytidine therapy. The absolute number of CD4+CD25highFoxp3+ regulatory T cells in 5q- syndrome was 0.5±0.28×107/l, RA0.6±0.56×107/l, RCMD1.42±0.97×107/l, RAEB2.8±2.2×107/l and MDS/MPD 2.9±2×107/l. In cases with <5% bone marrow blasts (RA, 5q− & RCMD)absolute number and percentage of Tregs was significantly lower than those with ≥5% BM blasts (p=0.001). The mean number was also significantly lower at 0.73±0.57×107/l in low risk cases(IPSS 0) compared with 2±1.5×107/l in intermediate and high risk groups (p=0.008). CD8+ Tregs were not significantly different between the subtypes of MDS and between low and high risk IPSS subgroups. Tregs number did not differ significantly between various cytogenetic subgroups. The spectratype of CD4+CD25+ TCR amplicons, showed a polyclonal pattern and the overall complexity of Vβ subfamilies was not different between low risk and high risk MDS, suggesting that the expanded Tregs in high risk MDS are not clonal and likely to arise by peripheral expansion rather than an antigen-driven response. CD4+ CD25high Foxp3+ Tregs in five patients were studied pre and post 5-Azacytidine. The numbers were significantly decreased after treatment in the one patient who responded to treatment (p=0.001), whereas Treg numbers were unchanged or increased in non responsive cases. In 5 RCMD cases with concomitant autoimmune diseases the percentage of Tregs was lower than other patients within the same subgroup of MDS, however, this did not achieve statistical significance.The findings indicate Tregs are altered in MDS and may be important in the pathophysiology of MDS. Monitoring of Tregs numbers can be a useful indicator for disease progression and response to immunosuppressive therapy.

Immunosuppressive Therapy in Children with Refractory Anemia: Results of Japanese Childhood MDS Study Group Trial (MDS99).

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder and uncommon in childhood. Especially, refractory anemia (RA), which is a subgroup of MDS without an increased number of blasts, is quite rare in this age group. Although hematopoietic stem cell transplantation (HSCT) is thought to be a curative therapy for pediatric MDS, it may cause severe complications and mortality. Several reports have shown encouraging results with immunosuppressive therapy (IST) in adult patients with RA. We report the outcome of 12 children with RA enrolled on a prospective multicenter trial conducted by Japanese childhood MDS study group. In this study, a child who was suspected of having RA required repetitive bone marrow aspiration at 6–8 weeks intervals. If the disease was stable and blood transfusion was not urgent, the patient could be monitored closely without any therapy. If physicians decided to start therapy due to progression of cytopenia, the patient received IST consisting antithymocyte globulin (ATG), cyclosporine (CyA) and methylprednisolone (mPSL). Of the 12 children, 9 received IST (IST group), 2 were followed without treatment (observation group) and one underwent HSCT without IST. Seven children showed hematological response in the IST group, and a response rate was 77.8%. Of note, 1 patient with monosomy 7 showed complete cytogenetic response after IST and remained in remission. One patient became refractory to IST after relapse and underwent bone marrow transplantation (BMT) from a human leukocyte antigen (HLA) 1-antigen mismatch relative, and she is alive without disease on 351 days after HSCT. One patient received BMT from an HLA-matched unrelated donor without IST because he had monosomy 7, but he relapsed and died from disease progression. Neither of 2 patients in the observation group experienced disease progression. There were 8 children who showed chromosomal abnormalities, including monosomy 7 and trisomy 8, 7 of whom received IST and 6 children showed hematological response. No severe adverse events related to IST were reported in this study. Eleven of the 12 patients are alive after a median follow-up of 1,319 days. The probability of survival at 5 years was 88.9%, which was superior to our previous retrospective analysis of children with RA. We conclude that IST for children with RA seems an effective modality and warrants an international clinical trial.

Can Molecular Profiling of Cytogenetic Subgroups Draw A Roadmap For Individualizing Therapy In Myelodysplastic Syndromes?

Therapeutic options for the heterogeneous hematopoietic disorders grouped under the myelodysplastic syndromes (MDS) have been difficult to develop, even though the incidence of this disease is increasing because of the ageing population. Several drugs have now been shown to have therapeutic efficacy in subgroups of patients, but the main challenge is still the preselection of the patient for a given strategy. To state the problem simply, effective therapies may already exist for a substantial number of MDS patients, but we do not know how to match the right drug to the right patient. Cytogenetic abnormalities have provided some treatment guidance, however these are generally restricted to patients known to have a better prognosis. To develop reliable predictive assays in patients with complex or more advanced diseases, we will have to delve deeper than cytogenetics. This review summarizes what is known about the clinical and biological characteristics of various karyotypic subgroups of MDS, and proposes a roadmap for combining the bedside-to-bench approach with the use of DNA microarray analysis in developing expression profiles that can serve as a guide in the preselection of treatment options for individual MDS patients.

Trisomy 19 as the sole chromosomal abnormality in proliferative chronic myelomonocytic leukemia

Distinct morphologic and clinical features associated with specific chromosomal abnormalities have been described in subgroups of myelodysplastic syndromes (MDS), which often are losses or gains and only rarely translocations. Among 103 consecutive MDS patients diagnosed and karyotyped at the Albert-Ludwigs University of Freiburg (ALU) between 1993 and 1999, two chronic myelomonocytic leukemias (CMMoL) displayed trisomy 19 (+19) as the sole chromosomal abnormality. Three further CMMoL cases with +19 as the single abnormality, two of which previously reported, were collected from other centers. Four of the five patients presented with leukocytosis and splenomegaly, and an increased number of ringed sideroblasts was observed in two cases. Treatment was low-dose Decitabine (cases 1 and 2), oral steroids (case 3), hydroxyurea (case 4), and daunorubicin/Ara-C (case 5). Transformation to acute myeloid leukemias (AML) occurred in three/five patients (cases 1, 2, and 4) 26, 12, and 22 months after diagnosis of CMMoL, respectively. We conclude that +19 as the sole anomaly is a rare but recurrent change in CMMoL, in particular of the proliferative type. It is at present unclear which gene(s) located on chromosome 19 might have a functional role for the development of this phenotype.

Promoter hypermethylation of p15INK4B, HIC1, CDH1, and ER is frequent in myelodysplastic syndrome and predicts poor prognosis in early‐stage patients

The propensity of myelodysplastic syndrome (MDS) to transform into acute myeloid leukemia (AML) suggests the existence of common pathogenic components for these malignancies. Here, four genes implicated in the development of AML were examined for promoter CpG island hypermethylation in cells from 37 patients with different stages of MDS. Aberrant methylation was detected by polymerase chain reaction amplification of bisulfite-treated DNA followed by denaturing gradient gel electrophoresis. The highest rate of methylation was found for p15INK4B (51%), followed by HIC1 (32%), CDH1 (27%), and ER (19%). Concurrent hypermethylation of > or = 3 genes was more frequent in advanced compared with early-stage MDS (P < or = 0.05), and hypermethylation of p15INK4B was associated with leukemic transformation in early MDS (P < or = 0.05). The median overall survival was 17 months for cases showing hypermethylation of > or = 1 genes vs. 67 months for cases without hypermethylation (P = 0.002). Specifically, promoter hypermethylation identified a subgroup of early MDS with a particularly poor prognosis (median overall survival 20 months vs. 102 months; P = 0.004). In multivariate analysis including stage and thrombocyte count, hypermethylation of > or = 1 genes was an independent negative prognostic factor (P < 0.05). These data suggest that hypermethylation of p15INK4B, HIC1, CDH1, and ER contribute to the development and outcome of MDS.

Usefulness and prognostic impact on survival of WHO reclassification in FAB low risk myelodyplastic syndromes

In 1999, WHO proposed a revised classification for myelodysplastic syndromes (MDS). According to this system, FAB low risk MDS (RA and RARS) were defined as such when the presence of dysplastic features was only restricted to the erythroid lineage, and new categories, refractory cytopenia with multilineage dysplasia (RCMD) and refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), were added. In a retrospective analysis of 240 consecutive patients diagnosed at our institution as having FAB RA and RARS, we reclassified the disease following the WHO criteria and we found that 179/214 patients (84%) still remained in the RA category, while 35/214 (16%) moved to RCMD. Moreover, 17/26 patients (65%) maintained the RARS diagnosis, whereas 9/26 (35%) were re-classified as RCMD-RS. We detected differences among the WHO subgroups as to age and sex distribution as well as to median survival observed by stratifying patients according to different prognostic scoring systems. Furthermore we confirmed the usefulness of WHO segregation with regard to its predictive value for evolution into acute leukaemia. Our study provides evidence that WHO classification may have prognostic impact on MDS subgroups which are usually categorized by FAB as having a favourable outcome.