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Targeting Hypomethylation of DNA to Achieve Cellular Differentiation in Myelodysplastic Syndromes (MDS)

Division of Medical Oncology, Mount Sinai School of Medicine, New York, New York, USA

Correspondence: Lewis R. Silverman, M.D., One Gustave L. Levy Place, Box 1129, Mount Sinai School of Medicine, New York, New York 10029, USA. Telephone: 212-241-5520; Fax: 212-348-9233; e-mail: lewis.silverman{at}mssm.edu

	ABSTRACT

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
Considerable progress has been made recently in defining and understanding the diverse members of the group of hematologic disorders now known as the myelodysplastic syndrome (MDS). New systems of classification, based on the latest cytogenetic methodologies, have generated better prognostic data, and basic research has more closely associated molecular mechanisms with clinical subgroups. The mechanisms underlying most cases of myelodysplasia appear to be an array of chromosomal abnormalities leading to suppression of normal myeloid cell differentiation and dominance of abnormal, immature cells. The process is progressive and is mediated by a variety of cytokines, potential loss of tumor suppressor genes, aberrations in signal transduction pathways, and perhaps immune mechanisms. Hypermethylation of specific DNA sequences has been implicated in the pathogenesis of MDS. Until recently, treatment options have been few, high risk, and mostly ineffective. New discoveries, particularly in the area of stimulating remaining normal myeloid cells to resume growth and differentiation, hold promise for safer treatment regimens and improved outcomes. Among the promising new agents are nucleoside analogues, such as 5-azacytidine and decitabine, which reactivate tumor suppressor gene transcription through effects on DNA methylation.

Key Words. Myelodysplastic syndrome • DNA methylation • Gene regulation • 5-azacytidine • Chemotherapy • Quality of life

	INTRODUCTION

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
The myelodysplastic syndrome (MDS) is a hematologic disorder characterized by ineffective hematopoiesis leading to peripheral blood cytopenias and progressive bone marrow failure. Generally 35% to 40% of cases transform to acute myelogenous leukemia (AML), and most patients die from infection or bleeding. [1–4] Although there is no national registry for diagnosis, it is estimated that 15,000 to 20,000 new MDS cases are diagnosed each year, primarily in patients over 60 years of age, for an incidence of 15 to 50 per 100,000 per year. Whether due to better diagnosis, altered classification, an aging population, or a true increase in incidence, the frequency of MDS has risen since 1980 [3]. Statistics are also confounded by changes in classification coincident with advances in the understanding of disease mechanisms that provide new methods of differentiating among similar clinical appearances [3].

Despite these recent events, progress has been slow with regard to treatment. Five-year disease-free survival rates range from <10% to 60% for specific risk groups, as defined by one of the more recent MDS classification systems. Thus, supportive care remains the standard of treatment [5].

	CLINICAL FEATURES

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
MDS initially presents clinically with the subtle and nondiagnostic signs and symptoms of anemia—weakness, fatigue, palpitations, dizziness, headaches, irritability, as well as increases in hemorrhage (petechiae, ecchymosis, frank bleeding) and infection [6]. MDS is characterized morphologically by peripheral cytopenias involving normal or increased cellularity of bone marrow, although there are exceptions in which the typical finding is hypocellular marrow or myelofibrosis [3, 4]. Such exceptions are not rare, again exemplifying the heterogeneous nature of conditions identified as MDS [7].

	PATHOPHYSIOLOGY OF MDS

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
MDS originates from a multilineage hematopoietic progenitor that (in most, but apparently not all cases) is committed to the myeloid, erythroid, and platelet series [2, 5, 8]. MDS can be of primary etiology or can be a consequence of chemotherapy, radiation therapy, or exposure to environmental toxins. Part of the observed increase in its incidence appears to be related to wider use of high-dose chemotherapy regimens with stem cell infusion. This contributes to further stem cell damage beyond that which has accumulated from exposure to prior conventional-dose chemotherapy [9].

A number of chromosomal abnormalities involving critical genes that control hematopoiesis can give rise to MDS [10]. These genetic events affect myeloid cell maturation, resulting in peripheral cytopenia accompanied by the accumulation of immature myeloblasts in the marrow [7]. Another consequence of the underlying genetic aberrations is alteration in cytokine regulation and response to cytokines, leading to increased apoptosis of hematopoietic progenitor cells and aberrant hematopoiesis [11]. The pathophysiology of MDS is represented in Figure 1.

View larger version (21K): [in this window] [in a new window]   Figure 1. Pathophysiology of MDS. This schematic illustrates components that contribute to the development of MDS and their relationships. Mutations in critical growth-regulating genes in the hematopoietic progenitor cells block the cells' normal differentiation and maturation. Cytokine imbalances and aberrant signal transduction that result from these mutations in the affected myeloid cells lead to accelerated apoptosis. Abbreviations: IL-1ß = interleukin-1 ß; TGF-ß = transforming growth factor; TNF- = tumor necrosis factor ; aza C = azacytodine (subcutaneous).

	CLASSIFICATIONS

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
The three major international diagnostic classifications are compared in Table 1.

International Prognostic Scoring System (IPSS)
Extraordinary progress in cytogenetic analytical methodology identified additional prognostic criteria to supplement the FAB classification. In 1997 a new international group was convened to generate a classification system corresponding to current advances in technology. This group, the International MDS Risk Analysis Workshop, combined cytogenetic, clinical, and outcome data from 816 patients with primary MDS to produce the IPSS [15]. Cytogenetic subgroups, percentage of bone marrow myeloblasts, and number of cytopenias proved to be the major prognostic variables. Multivariate analysis defined four groups with clearly distinctive median rates of survival and of evolution to AML. The cytogenic subgroups that fell into the low-risk category included -Y alone, del(5q) (same as –5q) alone, and del(20q). The high-risk category included any patient whose karyotype showed abnormalities in chromosome 7 and patients with complex abnormalities (3 anomalies). Two intermediate categories emerged during analysis of the data [16].

World Health Organization (WHO) Classification
In 2000 another classification system was published by a working group of the WHO, relying more closely on the FAB system of conventional morphologic criteria but also considering cytogenetic markers [17]. Data from 1,600 patients with primary MDS were evaluated, expanding the FAB system by two categories, for a total of seven, all having a high degree of correlation with prognosis. CMML was eliminated from the WHO categories. RA was split into pure refractory anemia (PRA) and refractory cytopenia with multilineage dysplasia (RCMD). Some conditions previously classified as RARS were placed in the pure sideroblastic anemia group, while those with additional dysplastic features were put into the RCMD category, along with refractory anemias without ringed sideroblasts. RAEB was divided into RAEB I and RAEB II, based on medullary and peripheral blast counts. RAEB-T was included under AML. Additionally, –5q was separated out as a distinct entity, although its relatively benign nature prevails only if the proportion of medullary blasts is below 5% [18].

These new risk-rating systems introduce a certain amount of confusion into studies comparing previous and current therapy, and no system has yet to earn universal acclaim. The classification revisions did, however, set the stage for a more accurate evaluation of the promising new treatment modalities currently being tested [19].

	CURRENT TREATMENT OPTIONS

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
The treatment goals for MDS are to prevent or delay progression to AML, to prolong survival, and to improve the quality of life. Until recently, supportive care has been the mainstay of therapy. Even now, the older age of most patients with MDS argues against employing the newer treatment regimens such as dose-intensive chemotherapy and bone marrow transplantation [8].

To address the problem of hematopoietic dysfunction in myelodysplastic syndromes, various growth factors have been used to stimulate blood cell production. The use of these agents in the treatment of MDS is not without controversy, however, as they have the potential to stimulate proliferation, block apoptosis, and promote survival of both normal hematopoietic cells and preleukemic cells [19]. Among the newer treatments available, erythropoietin is the most commonly used. It is well tolerated, although few ever achieve a transfusion-free state [10, 19]. Patients with low serum erythropoietin levels and minimal red cell transfusion need are most likely to achieve favorable responses after erythropoietin treatment, but other MDS patients respond poorly, exhibiting response rates of 10% or less.

Clinical trials of GM-CSF have shown that it increases circulating neutrophil counts in a majority of MDS patients. These promising results have unfortunately not translated into increased patient survival rates [19]. Clinical trials of G-CSF have also demonstrated significant effects on neutrophil counts in patients with MDS, but not to the extent of treatment with GM-CSF [8]. Although both GM-CSF and G-CSF are effective in improving neutrophil counts in MDS, randomized studies have failed to show a clear benefit from these agents, as both therapies induce thrombocytopenia [8]. In the only randomized trial, treatment with G-CSF was associated with reduced survival compared with supportive care [20]. G-CSF can increase the ineffectiveness of erythropoietin in reducing anemia associated with MDS subtypes RA, RARS, and RAEB when given in combination with erythropoietin, and the demonstrated response rates have approached 40% [21].

Thrombopoietic growth factors are being evaluated in single-agent and combination therapy regimens for MDS, to counteract thrombocytopenia, a leading cause of morbidity and mortality in MDS [22]. Several factors have been shown to stimulate platelet production in humans in a dose-dependent manner, namely, recombinant forms of the ligand for c-mpl receptor present on cells of the megakaryocyte lineage, full-length recombinant human thrombopoietin and a truncated, pegylated form of the same molecule, and recombinant human megakaryocyte growth and development factor [23]. The potential utility of these agents in MDS is under investigation [23]. The biology and clinical development of thrombopoietic growth factors is discussed in the article by Dr. George Demetri in this supplement [24].

Clinical studies have also explored pleiotropic growth factors as a means of hematopoietic support in MDS. Interleukin-3 and interleukin-6 have been evaluated for their effects on enhancing platelet and neutrophil counts in MDS patients, but they have proven to be ineffective because of limited activity and therapy-related toxicities [10, 19]. Interleukin-11 is the only pleiotropic growth factor currently available commercially for the treatment of thrombocytopenia. Studies exploring the value of interleukin-11 as a thrombopoietic support agent in MDS are ongoing.

For younger patients, allogeneic stem cell transplantation has produced cure rates between 30% and 50%, albeit with a high treatment-associated mortality (20%). Patients with RAEB and RAEB-T have high rates of relapse [25]. Multiple improvements in transplant technology, including the use of peripheral blood stem cells and minitransplants, are in investigational stages. Transplants carry the additional advantage of exerting a graft-versus-tumor (or graft-versus-leukemia) effect [26].

Chemotherapy is designed to eliminate abnormal cells in MDS. Standard induction therapy with an anthracycline and cytarabine produces response in approximately 50% to 60% of patients, but the relapse rate is 90% within a median duration of less than one year [2, 8, 15, 27]. These regimens are also accompanied by significant toxicities and risk of mortality with considerable decrease in quality of life.

In a strategy similar to the cytokine therapy, several agents expected to stimulate a return to normal growth and differentiation are under evaluation. These include anti-tumor necrosis factor- agents such as etanercept, infliximab, and pentoxifylline. Other agents such as ciprofloxacin, dexamethasone, vitamin D₃ analogues, dimethyl sulfoxide, lipopolysaccharide, glucocorticoids, lectins, nucleoside analogues, hemins, and short-chain fatty acids, are being tested as differentiating agents [28]. Retinoic acids both inhibit proliferation of malignant cells and promote differentiation of normal cells [29].

Finally, experience with aplastic anemia in children has identified an immune component in certain cases of marrow failure. Treatment with antithymocyte globulin (ATG) and cyclosporin has restored normal hematopoiesis in 55% to 77% of patients [30]. Immune suppression has also improved the outcome of bone marrow transplantation in patients with MDS and a variety of leukemias—high-dose ATG reduced the incidence of acute graft-versus-host disease in 55 adults without adverse effects or compromise of donor chimerism [31]. These results led one investigator to test ATG in 25 patients with hypoplastic MDS. Patients with this uncommon variant include those with RA, RARS, or RAEB, as classified according to the FAB system. A single course of ATG produced a response in two-thirds of RA patients, one-third of RAEB patients, and no RARS patients, reflecting the disparate nature of the conditions classified as MDS [32].

	DNA METHYLTRANSFERASE INHIBITION

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
DNA methylation recently has been shown to play a key role in myelodysplasia. DNA methyltransferase enzymatically methylates cytosine residues in newly synthesized DNA, thus maintaining the parental pattern. Methylation usually suppresses gene transcription as effectively as it suppresses gene deletion [33]. Two nucleosides that affect this process have produced promising clinical results in inducing remissions: 5-azacytidine, a ring analogue of cytidine, and decitabine (5-aza-2'-deoxycytidine). Azacytidine is incorporated into DNA, where it produces a dose- and time-dependent inhibition of DNA methyltransferase activity [29]. Newly synthesized DNA is consequently hypomethylated, resulting in expression of previously quiescent genes (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Figure 2. 5-Azacytidine-induced DNA hypomethylation and gene activation. 5-Azacytidine inhibits DNA methyltranferase (DMT), causing hypomethylation and transcription of previously quiescent genes.

On a slightly larger scale, chromatin structure, which is dependent upon histone chemistry, is a crucial factor in regulating transcription. Alterations in histones, specifically hypoacetylation, and the consequent chromatin remodeling, have been shown also to be responsible for gene silencing. This implies that histone deacetylase inhibitors may be useful agents in cancer treatment [33].

Clinical Studies With Azacytidine and Decitabine
In preliminary studies, azacytidine and decitabine inhibition of DNA methyltransferase has shown promise in treating MDS. In a study of 29 elderly patients with high-risk MDS, decitabine produced complete remission in 29% of patients and partial remissions in 18% of patients, median duration of remission is 7.75 months. Median survival in these patients was 28 months [37]. In a single-arm phase I/II trial in which patients with RAEB and RAEB-T received a continuous infusion of azacytidine, 75 mg/m²/day for 7 days repeated every month, 49% of patients responded, with 37% having trilineage responses, either complete or partial. Complete remission occurred in 5 of the 43 patients and partial remission in 11. Median survival for all patients was 13.3 months, and median duration of remission was 14.7 months (Table 2) [1]. (Patients who did not respond after 4 months were discontinued from the study.) A subsequent study using subcutaneous bolus injection of azacytidine produced a response in 50% of patients, with 27% having trilineage responses (Table 2) [38].

View larger version (19K): [in this window] [in a new window]   Figure 3. CALGB 9221 trial design.

	SUMMARY

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

Future studies will be necessary to define the optimal timing and dosing regimens for inhibiting DNA methyltransferase and to select the most effective concomitant therapies, such as histone deacetylase inhibitors, cytokines, and standard chemotherapy agents.

	ACKNOWLEDGMENT

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 
Supported in part by grants from the T.J. Martell Foundation for Leukemia, Cancer, and AIDS Research.

	REFERENCES

Top Abstract Introduction Clinical Features Pathophysiology of MDS Classifications Current Treatment Options DNA Methyltransferase Inhibition Summary References

 

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This Article Abstract Full Text (PDF) eLetters: Submit a response to this article Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services E-mail this article link to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Silverman, L. R. Search for Related Content PubMed PubMed Citation Articles by Silverman, L. R.