Development Of Myelodysplastic Syndrome Research Articles

The Akt/Mammalian Target of Rapamycin Signal Transduction Pathway Is Activated in High-Risk Myelodysplastic Syndromes and Influences Cell Survival and Proliferation

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is important for both cell growth and survival. In particular, an impaired regulation of the Akt/mTOR axis has been strongly implicated in mechanisms related to neoplastic transformation, through enhancement of cell proliferation and survival. Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by a high risk of evolution into acute myelogenous leukemia (AML). The pathogenesis of the MDS evolution into AML is still unclear, although some recent studies indicate that aberrant activation of survival signaling pathways could be involved. In this investigation, done by means of immunofluorescent staining, we report an activation of the Akt/mTOR pathway in high-risk MDS patients. Interestingly, not only mTOR was activated but also its downstream targets, 4E-binding protein 1 and p70 ribosomal S6 kinase. Treatment with the selective mTOR inhibitor, rapamycin, significantly increased apoptotic cell death of CD33(+) (but not CD33(-)) cells from high-risk MDS patients. Rapamycin was ineffective in cells from healthy donors or low-risk MDS. Moreover, incubation of high-risk MDS patient CD34(+) cells with rapamycin decreased the in vitro clonogenic capability of these cells. In contrast, the phosphoinositide 3-kinase inhibitor, LY294002, did not significantly affect the clonogenic activity of high-risk MDS cells. Taken together, our results indicate that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients. Therefore, this signaling network could become an interesting therapeutic target for treating more advanced MDS cases.

Penumbra Encodes a New Tetraspanin Critical to Effective Erythropoiesis.

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra−/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra−/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra−/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias.

Prognostic Significance of Serial Determinations of LDH Levels in Primary (De Novo) Myelodysplastic Syndromes.

An increased lactate dehydrogenase (LDH) level at diagnosis is associated with a reduced probability of survival and an enhanced risk of AML development in primary (de novo) myelodysplastic syndromes (MDS). However, so far, little is known about the prognostic value of an increase in LDH levels during the follow up in these patients. We have serially determined LDH levels in 221 patients (102 males, 119 females) with de novo MDS (median age 70 years; FAB-types: RA, n=62; RARS, n=46; RAEB, n=48; RAEBT, n=36; CMML, n=29), and examined the prognostic value of LDH as a follow-up parameter. Confirming previous data, an elevated LDH level at diagnosis was found to be associated with a significantly increased probability of AML evolution and a significantly decreased probability of survival (p<0.05). In the follow up, an increase in LDH (from normal to elevated) was found to be associated with progression of MDS and AML evolution in most cases. Moreover, in those patients who progressed to AML, LDH levels were found to be significantly higher in the two three-months-periods preceding progression compared to the two initial three-months-periods examined (p<0.005). In most patients, the increase in LDH was accompanied or followed by other signs of disease progression, such as the occurrence of thrombocytopenia or an increase in blasts. Together, our data show that LDH can be employed as a prognostic follow-up variable in patients with MDS. In those patients in whom an increase in LDH is noted, a thorough re-evaluation of the progression-status of the disease including a bone marrow examination should be considered.

High Density SNP Arrays Reveal That Distinct Clonal Lesions Including Uniparental Disomy Can Be Detected in a Proportion of Patients with Aplastic Anemia with Normal Metaphase Cytogenetics.

Aplastic anemia (AA) and myelodysplastic syndrome (MDS) show pathophysiologic and clinical overlap; e.g., MDS can present with hypocellular marrow and a substantial proportion of AA patients develop MDS. A majority of AA patients respond to immunosuppression (IS), also effective in some patients with MDS. Patients with MDS show expansion of single hematopoietic clone frequently characterized by chromosomal aberrations while AA is oligoclonal, likely due to a decrease in available stem cells. Owing to the low resolution of metaphase cytogenetics and its dependence on cell growth in vitro, the ability to detect clonal chromosomal defects is limited in AA and the test is often negative or non-informative in MDS. High-density SNP arrays (SNP-A) can be used for precise identification of unbalanced genomic lesions. We hypothesize that cryptic clonal genomic aberrations do exist in some patients with AA. Their detection may have important clinical implications; it could help to distinguish patients unresponsive to IS, indicate propensity to MDS evolution and point towards pathogenetic genetic lesions. We have applied 50K and 250K SNP-A to analyze bone marrow (N=17), blood (N=49) or both (N=3) of patients with AA (N=26), PNH (N=33) and hypoplastic MDS (N=10). The results were analyzed using CNAG and Exemplar software. Defects identified in marrow of healthy controls (N=36) were used to define the minimal pathologic lesion (only 1 lesion was identified). In AA and PNH, clonal chromosomal abnormalities were found in 5/10 marrow and 15/52 blood samples, respectively, and in 4/10 bone marrows from hypocellular MDS. Of these lesions, known chromosomal defects (following progression to MDS) were confirmed in 3 AA patients and most significantly we also found previously cryptic abnormalities in 17 patients with AA (4 marrow, 13 blood) and normal metaphase cytogenetics. The new lesions involved segmental uniparental disomy (UPD) involving portions of chromosomes 1,2,6,7,8, 9,10,13 and 14. For example, we found LOH due to UPD at 7q (q21.11, q31.33, N=3). The results of 50K SNP-A were validated by 250K scan. LOH due to deletions (monoploid) or UDP (diploid) were confirmed by microsatellite and Taqman PCR copy number analysis, respectively. The lesions were somatic and not present in nonclonal lymphocytes. Overlapping UDP at 2p21.1–p22.1 (2AA, 1PNH) and 2p16.1 (2AA, 1hypoMDS) was detected. Examples of genes linked to SNP in deleted lesions include FANCL, LOC151445 and VRK2. Moreover, LOH due to UDP resulted in homozygosity for very minor alleles of SNPs linked to KIA1607 or NHMT (control population frequency 20%, 19%) and NRX1 or SMEK3 (14%, 27%) in 2q21 and 2p16, respectively. Hematologic response to IS was seen in 7/12 (58%) AA patients in whom lesions were found. However, only 5 of these patients were sampled prior to IS, 2 subsequently responded and 3 remained refractory. In contrast, 16/22 AA patients with normal chromosomes by SNP-A treated with IS showed a hematologic response (72%). Our study, representing the first application of high-density SNP-A for karyotyping of patients with AA, demonstrates that some patients with AA may harbor cryptic clonal defects possibly consistent with the diagnosis of MDS. It is likely that these lesions have clinical and prognostic significance.

Lack of TP53 and FMS gene mutations in children with myelodysplastic syndrome

Myelodysplastic syndromes (MDS) are rare disorders in children. Molecular mechanisms underlying MDS in children are not yet completely understood. Considering the role of FMS and TP53 gene mutations in adult MDS patients, we analyzed mutations of these genes in a cohort of 35 children with MDS. Single-strand conformation polymorphism polymerase chain reaction analysis performed on FMS codon 969 and TP53 exons 5–9 showed no mutations in the analyzed sequences. Our results suggest that molecular mechanisms of MDS evolution in children are different from those in adults.

Frequent Elevation of Akt Kinase Phosphorylation in Mononuclear Cells from High-Risk Myelodysplastic Syndrome Patients.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal stem cell disorders characterized by ineffective and inadequate hematopoesis. Early stages of MDS display an increased apoptosis, while later stages of MDS present an excessive survival and decreased apoptosis of progressing abnormal clonal cells. The identification of the signaling pathways responsible for increased survival of MDS cells is of high importance as they might represent promising targets for novel forms of therapy aimed at preventing MDS evolution in to acute myeloid leukaemia (AML). However, to date, very limited information is available on survival pathways activated in MDS patients. The phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway has been implicated in the aggressiveness of a number of different human cancers, and constitutive activation of the PI3K/Akt pathway has been shown to be necessary for the survival of AML cells. In this study, we have performed an immunocytochemical and flow cytometric staining analysis, using an antibody to Ser473 phosphorylated-At (p-Akt) recognizing the activated form of the Akt kinase, to evaluate the levels of p-Akt in MDS mononuclear cells from either bone marrow or peripheral bllod. We observed a high Ser473 p-Akt staining in 85 % of the patients (n=22) diagnosed as high risk MDS, whereas mononuclear cells from healthy donors or low risk MDS patients showed very low or absent Ser473 p-Akt staining. Furthermore, high levels of the class I PI3K p110d isoform was consistently observed in MDS samples also demonstrating high levels of p-Akt. PI3K p110d was recently shown to have a higher expression in contrast to other class I PI3K isoforms in AML blasts. Finally, the expression of PTEN was correlated to the elevated levels of Ser473 p-AKT and PI3K p110d detected in myeloid cells. PTEN is a lipid phosphatase having the PI3K downstream product phosphatidylinositol 3,4,5 triphosphate (PIP3) as one of its primary targets, thus negatively regulating Akt activity. We have found that PTEN is expressed in mononuclear cells from high risk MDS patients, albeit with a lower staining intensity when compared with peripheral blood mononuclear cells from healthy donors. These findings identify activated Akt and PI3K p110d as potential survival factors in high risk MDS and could be important as therapeutic targets for innovative strategies to treat MDS patients.

Cytogenetic aspects of adult primary myelodysplastic syndromes: Clinical implications

Myelodysplastic syndrome (MDS) is a heterogeneous disease from the clinical, biological and morphological point of view. The pathogenesis of MDS is not well established and it appears to occur complex changes in the stem cell biology. Clonal chromosomal aberrations are found in 30–50% of primary MDS and no specific cytogenetic abnormality has as yet been defined. The chromosomal abnormalities are predominantly characterized by partial/total chromosomal losses or chromosomal gains. These chromosomal abnormalities include mainly −5/del(5q), −7/del(7q), del(11q), del(12p), del(20q), −Y, and +8. The role of cytogenetic analysis in the diagnosis, prognosis, taking treatment decisions and follow up of patients with MDS has been clearly defined. Despite its difficulties in obtaining for analysis high quality metaphases conventional cytogenetics continues to be the basic technique for cytogenetic evaluation of a MDS patient. Other molecular cytogenetic methods have been shown to be complementary, without replacing the information obtained with this technique. Further investigations with both conventional and molecular cytogenetics in relation to clinical features as well as other molecular methods will undoubtedly contribute to improve understanding of the underlying genetic events responsible for the development and evolution of MDS leading to more accurate classification and management of MDS patients.

Expression of the proliferation-associated nuclear protein MIB-1 and its relationship with microvascular density in bone marrow biopsies of patients with myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a group of disorders characterized by dysplastic hemopoiesis and an increased risk of leukemic transformation. The process of angiogenesis has been implicated in the pathogenesis and evolution of MDS. In this study the proliferative activity and extent of angiogenesis was examined in bone marrow samples from 54 patients with MDS in relation to clinicopathologic features. Cellular proliferation and microvascular density (MVD) were examined immunohistochemically, using the monoclonal antibody MIB-1 (Ki-67) and an anti-CD34 monoclonal antibody respectively. Serum concentrations of interleukin-6 (IL-6) were measured by ELISA. The results showed that the MIB-1 Labeling Index (MIB-1 LI), MVD and IL-6 increased significantly with advancing severity of disease. Among the MDS-FAB subtypes, MIB-1 LI, MVD and IL-6 were significantly higher in RAEB-t, RAEB and CMML in comparison to RA and RARS (p < 0.0001 in all cases). Similarly, MIB-1 and MVD were increased in patients with score 3 in comparison to scores 0 and 1 in the IPSS system (p < 0.05). All parameters studied were significantly higher in patients versus controls. We conclude that cellular proliferative activity and angiogenesis are associated with disease progression in MDS patients.

Evolution Of Clonal Cytogenetic Abnormalities in Aplastic Anemia

Prior to the introduction of effective therapies, the high mortality rates of severe aplastic anemia (AA) precluded recognition of late complications of this disease. Once the survival of AA improved, observation of clonal evolution raised questions as to whether the development of secondary myelodysplastic syndrome (MDS) is a part of the extended natural history of the disease or is related to the therapies applied. Clinical features of myelodysplasia and AA can overlap, and typical MDS may evolve as a complication of AA. Common pathophysiologic elements operate in these diseases and are subject to many studies and theories as to what mechanisms in AA may lead to the late evolution of MDS. Similarly, AA has been hypothesized to be a reflection of an over-reactive immune response triggered by the appearance of genetically altered and/or phenotypically abnormal dysplastic clones. Hypocellular variants of myelodysplasia and responsiveness of certain forms of MDS to immunosuppressive regimens serve as the most appealing examples of the intricate and close pathophysiologic relationship of this disease with AA. The diagnosis of clonal evolution in the course of AA can be obvious if secondary cytopenia involves hypercellularity and a high percentage of blasts. In addition, the occurrence of a new karyotypic defect objectively heralds the progression of disease to MDS. However, the diagnostic imprecision of dysplasia recognition in the context of marrow hypocellularity, inability to obtain informative cytogenetics, and a high proportion of MDS cases with normal karyoptype have hampered studies designed to determine the frequency and timing of MDS evolution in AA. In addition, the diagnostic criteria and definitions used are not unified. While some centers recognize that the abnormal karyotype does not preclude the diagnosis of AA; in others, the diagnosis of AA includes the presence of normal karyoptype. Many typical features of dysplastic evolution in AA have been clarified. For example, karyotypes most frequently encountered in MDS secondary to AA involve chromosomes 6, 7 and 8. The evolution rates seem to be in the range of 10 – 15% in 10 years, but there are no predictive clues as to which patients are at greatest risk for this complication. Study of the mechanisms of clonal evolution in AA may help understand the pathophysiology of other forms of MDS and leukemia and also the mechanisms of antileukemic surveillance. Clinically, identification of patients at increased risk for clonal complications may influence the choice of therapies applied.

Normal bone marrow function over 6 years in a patient with dysplastic hematopoiesis and a complex karyotype

Myelodysplasia associated with a complex karyotype is usually associated with advanced stage myelodysplastic syndrome (MDS) and an enhanced risk to develop secondary leukemia. We report on a 36-year-old female patient who was first presented in 1997 because of ‘Pseudo Pelger–Huet’ neutrophils. The remaining blood and differential counts were normal. Bone marrow examination revealed dysplasia in the erythroid and granulocytic series, no increase in blasts, and a karyotype with complex aberrations involving chromosomes 7, 13, 20 and 22. Almost all metaphases examined appeared to be affected. During the next few months, the patient was closely monitored and considered as candidate for bone marrow transplantation. However, blood counts remained stable without occurrence of significant cytopenias or an increase in blasts. Re-examinations of the bone marrow in 1998 and 1999 disclosed identical results compared to that obtained in 1997. After a total follow up of 6 years, the patient is still in good health with normal blood counts and persisting ‘Pseudo Pelger–Huet’ neutrophils. This exceptional case supports the notion that complex chromosomes are not invariably associated with rapid disease evolution in MDS.

Interstitial Deletion of the Short Arm of Chromosome 12 during Clonal Evolution in Myelodysplastic Syndrome with t(5;12)(q13;p13) Involving the ETV6 Gene

We report here a 65-year-old man with a myelodysplastic syndrome (MDS), refractory anemia with excess of blasts. He had received chemotherapy with tegafur for renal carcinoma. Chromosome analysis of bone marrow cells revealed complex karyotypes; del(5)(q13) was observed in all 20 metaphase spreads, and two related aberrations, add(12)(p11) and add(12)(p13), were detected in 13 and 7 cells, respectively. Fluorescence in situ hybridization (FISH) analysis with chromosome-specific DNAs revealed that these alterations originated from a reciprocal translocation (5;12)(q13;p13). Therefore, del(5)(q13), add(12)(p11), and add(12)(p13) were revised as der(5)t(5;12)(q13;p13), der(12)del(12)(p11p13)t(5;12)(q13;p13), and der(12)t(5;12)(q13;p13), respectively. Fluorescence in situ hybridization with a series of cosmid probes spanning the ETV6 gene showed that the 12p13 breakpoint on the der(12)t(5;12)(q13;p13) was located in intron 1, but the exon 1 signal was deleted. Our results suggest that a fusion gene was generated between the 5′-end of an unidentified partner at 5q13 and the 3′-end of ETV6 by t(5;12)(q13;p13), and that the interstitial deletion (12)(p11p13) occurred following t(5;12) during clonal evolution. del(12)(p11p13), including the rearranged ETV6 gene, may be implicated in the progression of MDS.

Comparative multi-color flow cytometric analysis of cell surface antigens in bone marrow hematopoietic progenitors between refractory anemia and aplastic anemia

Refractory anemia (RA) in myelodysplastic syndrome (MDS) without prominent dysplasia closely resemble the mild type of aplastic anemia (AA) in their hematological features. This sometimes makes it difficult to distinguish clearly between the two diseases. Using the multi-color flow cytometric technique, we compared cell surface antigen expression patterns on bone marrow hematopoietic progenitor cells which were isolated as a CD34 positive- CD45 dull positive with low side scatter intensity (CD34 +CD45 dull+SSC low) population in flow cytogram between RA ( n=12) and AA ( n=11). The antigens analyzed in CD34 +CD45 dull+SSC low mononuclear cells were: CD38 and CD71 for cell growth-related antigens, CD 33 and CD13 for myeloid and monocytoid lineage-associated antigens, CD7 and CD19 for lymphoid lineage, and CD14 for a monocytic lineage specific antigen. The percentages of CD34 +CD45 dull+SSC low cells in bone marrow non-erythroid mononuclear cells, and the expression frequencies of CD38, CD71, CD33 and CD13 antigens in CD34 +CD45 dull+SSC low progenitors were all significantly decreased in AA compared to normal bone marrows ( n=7) ( P<0.005). In contrast, in RA bone marrows the percentages of CD34 +CD45 dull+SSC low cells showed wide distribution and the cell surface antigen expression patterns varied among each case: some cases showed low frequencies of CD38 and CD71 expression as well as AA, whereas the others showed high expression frequency of specific antigen(s) which may reflect the clonal expansion of an abnormal clone in bone marrow. An MDS patient who had progressed from RA to RAEB showed further projecting pattern of expression of CD38 and CD33 in CD34 +CD45 dull+SSC low population in accordance with the disease progression. These data suggest that analysis of cell surface antigen expression patterns of CD34 +CD45 dull+SSC low progenitor cells by multi-color flow cytometry appears to be a useful method for qualitative and quantitative assessment of marrow progenitor states in AA and RA, therefore this method could be helpful for early detection of clonal evolution in MDS.

Molecular Mechanisms in Myelodysplastic Syndromes and Implications for Evolution to Acute Leukemias

This article reviews the molecular lesions that occur in the clonal hematopoietic disorders classified as myelodysplastic syndromes (MDS). A systematization of these molecular lesions is attempted based on the types of molecular abnormalities. Characteristically, these molecular lesions affect pluripotent hematopoietic progenitors and therefore affect myeloid, monocytic, erythroid, and megakaryocytic lineages. Progression of MDS to acute myelogenous leukemia is common; transformation is considered the final stage in the multi-step process of accumulation of molecular lesions over a prolonged latency period of MDS evolution. Although no molecular lesion is MDS specific, multiple combinations of molecular lesions are common and a systematic approach based on the type of molecular abnormality may offer a better understanding of MDS pathogenesis and the basis for new therapeutic strategies.

Apoptosis, bcl-2 Expression and p53 Accumulation in Myelodysplastic Syndrome, Myelodysplastic-Syndrome-Derived Acute Myelogenous Leukemia and de novo Acute Myelogenous Leukemia

Apoptosis and its dysregulation have been implicated in dysplastic and ineffective hematopoiesis and the neoplastic transformation of bone marrow in myelodysplastic syndrome (MDS). To explore the role of apoptosis in hematological disorders, we examined the frequency of apoptotic cells by the in situ end labeling method in bone marrow specimens from 37 patients with MDS [refractory anemia (RA) 10 cases, RA with excess of blasts (RAEB) 27 cases including 12 cases with leukemic transformation], 12 patients with MDS-derived acute myelogenous leukemia (AML) and 13 patients with de novo AML. In addition, we investigated the relationship of apoptosis to the immunohistochemical expression of bcl-2 and p53 in these cases, and the association of apoptosis, bcl-2, and p53 with the leukemic evolution of MDS by examining sequential bone marrow samples of the same patient from the time of initial diagnosis to the time of overt leukemia. The percentage frequency of apoptotic cells was significantly greater in MDS (RA: 9.46 ± 2.99%, m ± SD; RAEB: 5.60 ± 3.09) as compared with those in MDS-derived AML (0.62 ± 0.37), de novo AML (0.28 ± 0.11) and controls (1.00 ± 0.59). On the other hand, the cases of RAEB with leukemic transformation exhibited a lower frequency of apoptotic cells and a higher frequency of bcl-2- and p53-positive cells than those without transformation. When the RAEB cases transformed to AML, the frequency of apoptotic cells was significantly reduced (2.96 ± 1.54 → 0.62 ± 0.37), while the frequencies of bcl-2-positive cells and p53-positive cells were greater (10.88 ± 3.66 → 30.54 ± 7.14, and 20.21 ± 6.21 → 32.34 ± 14.71, respectively). In contrast to MDS-derived AML, over a half of de novo AML cases showed few p53-positive cells. These findings corroborate the earlier notion that apoptosis may play a substantial role in dysplastic and ineffective hematopoiesis in MDS. It is also suggested that the suppression of apoptosis associated with enhanced bcl-2 expression and p53 accumulation increases the probability of developing leukemia in MDS, and that oncogenetic development might be different between MDS-derived AML and de novo AML.

Long-term administration of G-CSF for aplastic anaemia is closely related to the early evolution of monosomy 7 MDS in adults.

There is an increasing incidence of the evolution of myelodysplastic syndrome (MDS) from aplastic anaemia (AA) with immunosuppressive treatment. In paediatric patients G-CSF is also reported to increase MDS evolution, but this process is not precisely understood in children or in adults. Therefore risk factors of MDS evolution in adults are evaluated here. Of 72 patients, five developed MDS. In 47 patients without cyclosporine (CyA) or antithymocyte globulin (ATG) therapy, only one developed MDS with trisomy 8, 242 months after diagnosis. But of 25 patients treated with either CyA or ATG, four developed monosomy 7 MDS within 3 years. Of these 25 patients, 18 were treated with G-CSF and the four patients (22.2%) who developed MDS were found in this group. The cumulative dose and the duration of G-CSF administration were significantly elevated in patients who developed MDS when compared with those who did not, 822.3 +/- 185.0 v 205.4 +/- 25.5 microg/kg (P<0.05) and 187.5 +/- 52.5 v 72.0 +/- 24.6 d (P<0.002), respectively. However these two values for CyA did not differ significantly. Statistically, treatment with CyA, G-CSF and combined G-CSF and CyA were significantly related to MDS evolution. The administration of G-CSF for more than a year was the most important factor (P=0.00). These results suggested that a close relationship exists between G-CSF and subsequent monosomy 7 MDS from AA in adults who receive immunosuppressive therapy. Long-term administration of G-CSF should be prohibited in order to prevent MDS evolution.

Apparent expansion of CD34+ cells during the evolution of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

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.

76 The absolute number of CD34+ cells and CD34+ cells in S-phase increase during the evolution of myelodysplastic syndromes (MDS)

76 The absolute number of CD34+ cells and CD34+ cells in S-phase increase during the evolution of myelodysplastic syndromes (MDS)

Infrequent microsatellite instability during the evolution of the myelodysplastic syndrome to acute myelocytic leukemia

Microsatellites are highly polymorphic, short-tandem repeat sequences dispersed throughout the genome. Instability of these repeat sequences at multiple genetic loci may result from mismatch repair errors and occur in hereditary nonpolyposis colorectal cancer and several other sporadic cancers, including chronic myelocytic leukemia as it progresses to blastic crisis. We investigated whether genetic instability occurred as myelodysplasia progressed to acute myelocytic leukemia. To this end, we studied microsatellite instability in 20 patients with myelodysplastic syndrome (MDS). These included five patients with refractory anemia (RA), three with refractory anemia with ringed sideroblast (RARS), nine with refractory anemia with excess blasts (RAEB) and three with chronic myelomonocytic leukemia (CMML). All of these patients transformed to acute myelocytic leukemia (AMD of various subtypes: three patients with M1, 11 with M2 and six patients with M4 (according to FAB classification). The DNA from both the MDS and AML phases of their disease was analyzed at 16 loci, and only four microsatellite instabilities were found in the 240 paired samples (1.6%) analyzed. These results indicate that mismatch repair errors such as microsatellite instability are not important in the evolution of MDS to AML.

Clinical aspects, cytogenetics and disease evolution in myelodysplastic syndromes.

Myelodysplastic syndrome (MDS) is a morphologically characterized hematologic entity that is one of the clonal myeloproliferative disorders. Approximately 50 approximately 70% of MDS patients have cytogenetic abnormalities; these are usually chromosomal deletions, but some involve translocations such as t(1;7) (q10;p10). Translocations involving chromosomal regions 3q26 or 22q11 are often therapy-related. Recent studies have demonstrated that cytogenetic changes in MDS patients have clinical relevance. Accordingly, there are now scoring systems for predicting the prognoses of MDS patients. In this review, we describe the clinical significance of cytogenetic changes in MDS. We include MDS with some atypical forms, such as MDS with hypocellular bone marrow, MDS with minimal dysplasia, and MDS with myelofibrosis.