Correspondence *Department of Haematology, King's College London, Denmark Hill Campus, London SE5 9RS, UK

Email ghulam.mufti@kcl.ac.uk

Introduction

Myelodysplastic syndrome (MDS) comprises a group of clonal stem cell disorders characterized by ineffective hemopoiesis due to exaggerated apoptosis and abnormal bone marrow blast proliferation. Deaths result from peripheral blood cytopenias and myeloid leukemic transformation. Anemia is the most frequent presenting feature, and infections due to neutropenia and bleeding secondary to thrombocytopenia are common.¹ Although the disease's prognostic criteria are diverse, description of morphological classification by the French–American–British (FAB)^{2, 3, 4} and World Health Organization⁵ groups and by the International Prognostic Scoring System (IPSS) (Tables 1 and 2)⁶ allow for the identification of prognostic subgroups and patients who require early therapeutic intervention.

Table 1 International Prognostic Scoring System^a for survival and acute myeloid leukemia evolution in myelodysplastic syndrome

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Table 2 Survival related to score on the International Prognostic Scoring System

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MDS primarily affects individuals aged 65 years and older.¹ Considering the increasing life expectancy within the Western world, it is anticipated that the incidence and prevalence of MDS will increase, making this condition a much more serious healthcare concern. Because of their age, most patients are not considered eligible for ablative allogeneic stem cell transplant (alloSCT).^{7, 8} Many patients are treated with best supportive care (BSC) measures, which include erythropoietin, granulocyte-colony-stimulating factor, and blood transfusions, and represent the traditional therapeutic approach of choice.⁷

Over the past decade, a growing body of clinical evidence is beginning to define the role that DNA methyltransferase (DNMT) inhibitors play in the treatment of MDS.⁹ The rationale behind the research involving therapeutics that inhibit methyltransferase emanates from the knowledge that EPIGENETIC SILENCING—through promoter methylation of a number of genes, in particular CDKN2B (encoding p15^INK4b)—is present in poor-risk subtypes of MDS and often predicts transformation to acute myeloid leukemia (AML). DNMT inhibitors that represent promising treatment modalities include azacitidine (5-azacytidine; Vidaza®, Pharmion Corp., Boulder, CO, USA) and decitabine (Dacogen™, SuperGen Inc., Dublin, CA, USA, and MGI Pharma Inc., Bloomington, MN, USA).¹⁰Azacitidine is approved by the FDA for the treatment of all FAB MDS subtypes, including low- and high-risk diseases.^{11, 12, 13} Decitabine is currently undergoing regulatory review and additional information is needed regarding transfusion requirements.¹⁰

Given the clinical availability of the DNMT inhibitors, it becomes both prudent and timely to examine the present clinical experience and trials involving their use for MDS. Furthermore, this review examines several key issues to assist clinicians in appropriately evaluating how to use these treatments in the management of MDS patients.

Clinical trial experience with methyltransferase inhibitors

Azacitidine and decitabine have undergone significant clinical trial testing. Azacitidine was approved by the FDA for MDS based upon the phase III Cancer and Leukemia Group B (CALGB) 9221 study¹⁴ published in the Journal of Clinical Oncology in May 2002. Decitabine has been presently undergoing FDA review for treatment of MDS.¹⁰ The experience with decitabine has included a number of phase I/II studies.^{15, 16, 17, 18, 19} Phase III results have been recently presented at the American Society of Hematology and American Society of Clinical Oncology but have not yet been published in the peer-reviewed literature.

Azacitidine experience in myelodysplastic syndrome

Background

During the 1960s, researchers in Czechoslovakia were the first to synthesize azacitidine, then a novel chemotherapeutic agent, which was soon evaluated in clinical trials.²⁰ Originally thought to represent a promising cytotoxic chemotherapy with antileukemic activity,²¹ azacitidine was never approved by the FDA for the treatment of active cancers. However, interest in recent years brought it back to the forefront of research activity, this time for the treatment of MDS.

The CALGB performed two phase II evaluations^{22, 23} of the efficacy of azacitidine in patients with refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, or chronic myelomonocytic leukemia, a classification that was included in the second phase II trial only.²³ In the first study, patients received azacitidine 75 mg/m² intravenously for 7 consecutive days, repeated in 28-day cycles. For the second study, the same dose and schedule were used but administration was subcutaneous in an ambulatory regimen—in contrast to the administration of decitabine, which has to be intravenous. A subsequent evaluation involved a randomized, open-label, crossover multicenter study by Marcucci and colleagues,²⁴ which assessed the relative bioavailability of azacitidine given subcutaneously. Six patients with MDS received 75 mg/m² subcutaneously and intravenously at least 1 week apart. Bioavailability of 89% was observed for the subcutaneous route, on the basis of the ratio of the subcutaneous to the intravenous area-under-the-curve geometric least-squares mean. The original intravenous azacitidine protocol of the CALGB phase II trial produced a response rate of 49% without serious adverse effects.²² The modified protocol for subcutaneous administration increased the total response rate to 53%. Bone marrow hypoplasia occurred in only 10% of patients, despite pre-existing cytopenias.²³

An unexpected but consistent finding across studies was that the median time to initial response was in excess of 90 days, or after at least three cycles of treatment. Investigators in trials evaluating the efficacy of oncologic and chemotherapeutic pharmacotherapy often withdraw treatment from patients who do not respond after two cycles. Had this trial similarly concluded that a lack of response after two cycles equated to therapeutic failure, a significant number of eventual responders would have been removed from the study before exhibiting a response, suggesting that the drug was inactive. This observation, that the optimal duration of DNMT inhibitor therapy is longer than two cycles, has since been corroborated in other clinical trials.^{15, 19}

Current experience: phase III and beyond

As a result of the promising efficacy and safety results of early azacitidine trials, the Cancer and Leukemia Group B conducted a randomized phase III study (CALGB 9221) comparing azacitidine with BSC.¹⁴ In this study, 191 patients with all FAB subtypes of MDS were recruited and received azacitidine subcutaneously 75 mg/m² for 1 week every 28 days and were assessed for response after four treatment cycles. Patients who achieved a complete response (CR) received azacitidine for three additional treatment cycles; those who achieved a partial response (PR) or hematologic improvement (HI) continued to receive azacitidine until they reached CR or relapse.

Patients randomized to the BSC study arm received treatment for a minimum of 4 months unless any of the following occurred: they demonstrated evidence of progression to AML, succumbed to disease, or had a platelet count of 20 10⁹/l after week 8. After the 4-month initial phase of the study, patients were allowed to cross over to the azacitidine arm if their MDS worsened or transformed to AML.¹⁴

Responses to azacitidine were seen in patients with all MDS FAB subtypes (Table 3). The overall response rates in patients treated with this drug versus those given BSC were 60% versus 5% (P < 0.0001), with a median response duration of 14 months. These data represented the first evidence of azacitidine efficacy in MDS patients with low-risk disease (i.e. those with RA/RARS; Table 3). The median time to initial response was 64 days and that to best response was 93 days. The median time to AML or death was 21 months in patients receiving azacitidine, compared with 12 months in patients receiving BSC (P = 0.007; Figure 1). Transformation to AML as the first event occurred in 38% of those patients in the BSC group, as compared with 15% for those in the azacitidine group. Overall, patients treated with azacitidine survived longer than patients receiving supportive care (a median of 20 months versus 14 months, P = 0.1). By definition, those patients who exhibited CR or PR became transfusion-independent. Of the patients who experienced HI, 73% had a red blood cell (RBC) response, 35% (n = 13) had 50% amelioration of RBC deficit, 22% (n = 8) had an elimination of all RBC transfusion requirements, and 16% (n = 6) had 50% decrease in RBC transfusions. Of the 65 patients who were receiving RBC transfusions at the beginning of the study, 29 (45%) experienced sustained transfusion-independence and another six (9%) had a reduction in transfusions by 50%.¹⁴

Figure 1 CALGB (Cancer and Leukemia Group B) 9221: Median time to transformation to acute myeloid leukemia or death in patients with myelodysplastic syndrome treated with azacitidine or given best supportive care

Measured from entry on study to the time of first event, either transformation to acute myeloid leukemia or death, and estimated according to the Kaplan–Meier method. Adapted from Silverman LR et al. (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the Cancer and Leukemia Group B. J Clin Oncol 20: 2429–2440. Reprinted with permission from the American Society of Clinical Oncology.

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Table 3 CALGB 9221: Responses to azacitidine in patients with myelodysplastic syndromes, according to FAB subtype 14

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To overcome the confounding effect of the crossover design, a LANDMARK ANALYSIS was performed in patients alive at 6 months. Patients randomized initially to azacitidine had a higher additional median survival, 18 months, than the 11 months observed in patients who did not cross over or who crossed over after 6 months (P = 0.03; Figure 2).

Figure 2 CALGB (Cancer and Leukemia Group B) 9221: Survival from landmark data according to crossover status (Kaplan–Meier method)

Patients with myelodysplastic syndrome were analyzed in three subgroups: those given supportive care who either never crossed over or crossed over after 6 months, those given supportive care who crossed over before 6 months, and those who were initially randomized to treatment with azacitidine. From Silverman LR et al. (2002) Randomized controlled trial of azacitidine in patients with the myelodysplastic syndrome: a study of the Cancer and Leukemia Group B. J Clin Oncol 20: 2429–2440. Reprinted with permission from the American Society of Clinical Oncology.

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With regard to safety and tolerability, the most frequent adverse effect of azacitidine therapy was exacerbation of pre-existing cytopenias. After adjustment for pre-existing, disease-related myelosuppression, azacitidine-attributed side effects included grade 3 or 4 leukopenia (43%), granulocytopenia (58%), and thrombocytopenia (52%). Azacitidine-related death occurred in 1% of patients.¹⁴

To measure quality of life (QOL) in the CALGB 9221 patient sample, Kornblith et al.²⁵ used two measures: the European Organization for Research and Treatment of Cancer (EORTC) Questionnaire C30, a 30-item survey that generates a total score on a scale of 0 to 100; and the Mental Health Inventory, a 38-item test measuring psychological state. There was a clear divergence in the severity of symptoms experienced by patients in the two treatment groups: fatigue (P = 0.001), dyspnea (P = 0.0014), and physical functioning (P = 0.0002) improved significantly for patients in the azacitidine group compared with patients in the BSC group. The Mental Health Inventory similarly demonstrated azacitidine's efficacy: patients in the azacitidine arm were significantly more likely to improve with regard to positive affect (P = 0.0077) and psychological distress (P = 0.015). Patients who crossed over from supportive care to azacitidine therapy also benefited: dyspnea (P = 0.018), fatigue (P = 0.0031), physical functioning (P = 0.0003; Figure 3), and psychological wellbeing (P = 0.04; Figure 4) significantly improved after initiation of azacitidine. It is noteworthy that these QOL parameters were stable or worsening in the BSC patients before crossover even as the patients were continuing to receive RBC transfusion support.

Figure 3 CALGB (Cancer and Leukemia Group B) 9221: Fatigue, dyspnea, and physical functioning, as recorded on the EORTC (European Organization for Research and Treatment of Cancer) questionnaire, of patients with myelodysplastic syndrome who crossed over from supportive care to treatment with azacitidine (5-Aza-C) (n = 30)

^*Higher scores indicate better functioning. ^**Lower scores indicate symptom improvement. From Kornblith AB et al. (2002) Impact of azacitidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol 20: 2441–2452. Reprinted with permission from the American Society of Clinical Oncology.

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Figure 4 CALGB (Cancer and Leukemia Group B) 9221: Physiological distress and well-being, as measured by the Mental Health Inventory (MHI), of patients who crossed over from supportive care to treatment with azacytidine (5-Aza-C) (n = 30)

^*Higher scores indicate better well-being. ^**Lower scores indicate less distress. From Kornblith AB et al. (2002) Impact of azacitidine on the quality of life of patients with myelodysplastic syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin Oncol 20: 2441–2452. Reprinted with permission from the American Society of Clinical Oncology.

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Recent observations by Najfeld et al.²⁶ have suggested that specific cytogenetic abnormalities may be associated with a better rate of response to azacitidine. Of 159 MDS patients who were treated with azacitidine, those with normal cytogenetic characteristics at baseline survived a median of 29.1 months as compared with those with overall karotype abnormalities, who survived a median of 15.7 months (P = 0.004). However, those with TRISOMY 8 survived a median of 28.7 months, nearly comparable with the patients who had had no cytogenetic abnormalities at baseline. These observations have been corroborated by an ongoing trial by Mufti and colleagues at King's College Hospital, London (GJ Mufti, personal communication). After a median of four azacitidine cycles in 21 patients, Mufti and colleagues observed an overall 24% rate of CR. However, three of the six patients with monosomy 7 and two of the three patients with trisomy 8 experienced a CR, suggesting a potential benefit for patients with these specific cytogenetic defects. Historically, subgroups such as patients with monosomy 7 and trisomy 8 have had a very poor prognosis. These recent findings are the subject of ongoing investigations to further expand on these initial observations.

Overall, clinical trial evidence¹⁴ provides the clinician with a clear picture of azacitidine's effectiveness in treating MDS. The drug produces a significant effect on hematopoietic cell function, with a high response rate (60%, versus 5% in patients given only BSC, P < 0.0001), and also significantly delays or prevents the transformation to AML (time to AML or death with azacitidine is 21 months, versus 12 months in patients receiving supportive care, P = 0.007). Overall survival favored patients in the azacitidine group (median 20 months with azacitidine, versus 14 with supportive care, P = 0.1), but in the intent-to-treat analysis, this finding is confounded by the crossover design. However, the landmark analysis suggests an overall survival advantage for patients in the azacitidine group, or at least a significant benefit when treatment is given early. Another benefit is that those patients who had experienced CR or PR in the azacitidine group and who had been transfusion dependent became transfusion independent.^{13, 14} Transfusion dependence has become an increasingly important standard, as evidenced by the FDA's lengthened review of decitabine.¹⁰ Najfeld et al.²⁶ and GJ Mufti and colleagues (personal communication) further define the role of azacitidine in the treatment of MDS by identifying cytogenetic abnormalities that may be associated with or predictive of a response to azacitidine. Further analysis (GJ Mufti, personal communication) of toxicity and adverse event data also strongly suggest that azacitidine may be be safely administered to patients with cytopenia and concomitant infection, in the knowledge that it does not worsen outcomes.

Decitabine MDS experience

Background

Decitabine's efficacy has been observed over the last decade.^{27, 28, 29} Several clinical trials in recent years have continued to define its place in therapy in patients with intermediate-risk to high-risk MDS (as defined by the International Prognostic Scoring System).⁶ Much of the published experience has been in phase I/II studies.^{15, 16, 17, 18}

In a study comparing three different decitabine dose schedules in 52 patients (90 were enrolled, of which 38 dropped out) with intermediate- to high-risk MDS, O'Brien et al.¹⁶ observed a CR in 18 (34.8%), a PR in 4 (7.7%), marrow CR plus clinical benefit (CB) in 13 (25%), and CB alone in 8 (13%), constituting an overall response rate of 83%. The definition of CB was similar to the definition used for HI in other studies,^{14, 15, 17, 30} but with the addition of a few clauses, particularly a decrease by 50% in splenomegaly or monocyte count (Table 4).^{14, 15, 16, 17, 30}

Table 4 Variable definitions^a of response between trials of DNA methyltransferase inhibitors

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Wijermans et al.¹⁷ reported a phase II trial of 66 patients with intermediate-risk to high-risk MDS who received decitabine 45 mg/m² per day for 3 consecutive days every 6 weeks. The total response rate, inclusive of CR (20%), PR (5%), and HI (24%), was 49%. The median duration of response to decitabine was 31 weeks. Three patients died from infection during therapy-associated neutropenic episodes.

Some of the participants in the Wijermans et al.¹⁷ study who relapsed were enrolled in a trial conducted by Rüter et al.,¹⁵ who re-treated 22 patients with decitabine, 45 mg/m² per day for 3 consecutive days every 6 weeks for a maximum of six cycles. One patient achieved CR with re-treatment, two patients achieved PR, and seven experienced HI.

Garcia-Manero et al.¹⁸ conducted a phase II study of decitabine in combination with the histone deacetylase inhibitor valproic acid. Forty patients with AML or MDS received a fixed dose of decitabine, 15 mg/m² intravenously, for 10 days, with valproic acid at 20, 35, or 50 mg/kg by mouth. One of the three patients receiving valproic acid (20 mg/kg) exhibited a CR. At a dose of 35 mg/kg, one of nine patients showed CR.

Current phase III experience

Results from a phase III study with decitabine were presented during 2004 at the American Society of Hematology and earlier this year at the American Society of Clinical Oncology, but have not been published in peer-reviewed literature. Saba and colleagues¹⁹ conducted an open-label phase III trial of decitabine versus BSC alone in 170 patients with intermediate- (INT-1 and INT-2) to high-risk MDS. Patients in the decitabine group received 45 mg/m² per day for 3 consecutive days every 6 weeks and were evaluated for response after every two cycles of therapy. The study was designed to meet co-primary end points of significant effect on response and on time to AML or to death. The investigators found a significant difference between total response in the decitabine group versus those given supportive care (30% versus 7%, P < 0.001), with a median time to response to decitabine therapy of 3.2 months. There was no difference overall in time to AML or death between the two groups (P = 0.19). Grade 4 neutropenia occurred in 77% of patients in the decitabine arm and in 25% of those in the group given supportive care; grade 4 thrombocytopenia occurred in 63% versus 16% of patients given decitabine or supportive care, respectively.

Important therapy considerations with DNMT inhibitors

Dose and dosing regimens

DNMT inhibitors were first studied between the 1970s^{20, 21} and the early 1990s for the treatment of leukemia at doses 20 times those for azacitidine tested in the MDS studies.^{22, 31} The strategy for how to best use these agents has evolved significantly during this past decade. Lower-dose regimens,^{14, 24, 30} in which the drugs are given over several days and multiple cycles,¹⁴ have been used to reduce the cytotoxic effect while still inhibiting DNMT. These regimens thus reverse the epigenetic silencing that leads to a premalignant or a potentially neoplastic phenotype.

At higher doses, the mechanism of action with DNMT inhibitor therapy has been observed to be cytotoxic rather than an effect of hypomethylation.^{32, 33} With azacitadine, this cytotoxicity occurs as a result of primary incorporation into RNA and secondary incorporation into DNA, inhibiting genetic synthesis.¹¹ Decitabine appears to be incorporated only into DNA. While some suggest differences in safety, response rates, or potency due to differential incorporation of DNMT inhibitors into the DNA and RNA, there are no clinical data to support such assertions.^{34, 35} Clinicians should also keep in mind that, at lower doses, these agents inhibit DNA hypermethylation and do not suppress DNA synthesis. Thus, the suggestion that such potential differences related to site of activity cannot be substantiated based upon present clinical experience.

Data from several trials appear to support the use of lower doses in MDS. Based on CALGB trial evidence,¹⁴ azacitidine is approved¹² at a dose schedule of 75 mg/m² per day for 7 days every 4 weeks. For decitabine, of seven dose schedules assessed in a phase I study of 63 patients with MDS, Issa and colleagues³⁰ observed that the schedule eliciting the best total response (65%) was 15 mg/m² for 10 days.³¹ Interestingly, in a dose-ranging, decitabine trial²⁸ with a larger sample size, O'Brien et al.¹⁶ observed a more favorable CR rate in the treatment arm using 20 mg/m² intravenously once daily for 5 days (48% CR) than in the treatment arms using 10 mg/m² intravenously once daily for 10 days (24% of CR) or 10 mg/m² subcutaneously twice daily for 5 days (28% CR). These observations have stimulated questions regarding optimal dose and schedule, which still need further exploration in future trials.

With respect to number of treatment cycles, this area is still evolving. Evidence currently points to the necessity of multiple treatment cycles, probably more than four, before an appropriate clinical^{14, 23} (GJ Mufti, personal communication) or QOL²⁵ response is seen or can be evaluated.

Selection of patients

Consistent with the National Comprehensive Cancer Network³⁶ and International Working Group guidelines,³⁷ the first goal of therapy in patients with MDS is to ameliorate cytopenias, improve QOL, and reduce the risk of leukemic transformation. In patients classified with low- or intermediate-1 (INT-1)-risk prognoses, the first step in the treatment algorithm after determination of clinical stability is assessment of the patient's symptomatology related to MDS. Low- or INT-1-risk patients without symptoms and with well conserved bone marrow function not requiring transfusion can be observed. Patients with significantly impaired bone marrow function with anemia requiring transfusion support can receive therapy with erythroid cytokines. Patients with significant cytopenias involving myeloid and megakaryocytic lineage or failing erythropoietic cytokine support in those who are RBC transfusion-dependent may be considered for DNMT inhibitor therapy. Those low-risk patients for whom BSC fails and patients with intermediate- to high-risk MDS are all candidates for DNMT inhibitor therapy, which has been shown to reduce transformation to AML, prolong survival,¹⁴ and improve QOL.²⁵ DNMT inhibitors may be dosed subcutaneously,²⁴ permitting treatment in an outpatient, ambulatory setting. Despite exacerbation of pre-existing cytopenias in the first 1–2 months of therapy before the onset of response in most patients, treatment with azacitidine does not increase mortality of the underlying disease, nor increase the risk of infection or hemorrhage in comparison with those receiving BSC¹⁴ (GJ Mufti, LR Silverman, personal communications).

Other than DNMT inhibitor therapy, the only other treatment that prolongs survival in the MDS population is alloSCT. This strategy typically is not considered in low-risk patients until disease progression occurs,³⁶ because it carries a significant risk of morbidity and mortality.³⁸ The vast majority of MDS patients are 65 years of age or older^{1, 39} and are ineligible for alloSCT.^{7, 8, 36} However, it is worth noting that research by Ho et al.⁴⁰ suggests that reduced-intensity alloSCT may extend the age at which patients can survive alloSCT procedures. For a small percentage of intermediate-risk to high-risk patients, alloSCT is an option. Nonetheless, even in these cases, reduction of marrow blasts to <5% offers significant improvement in post-alloSCT outcomes. Although intensive AML chemotherapies have been used in the MDS population to induce remissions before alloSCT, studies have recently been initiated to evaluate the role of DNMT inhibitors for this purpose. If DNMT inhibitor therapy proves successful, it should obviate the toxicity associated with chemotherapy and the need for inpatient admission.

DNMT inhibitors do represent a promising therapy for a large number of MDS patients, particularly adults aged 65 and older,^{1, 7, 8} a subpopulation that often does not receive due attention. It is also an ever-growing segment of the population; in the US alone, the number of people aged 65 and older is expected to grow to nearly 2.5 times its present size over the next 45 years and is anticipated to represent 20.7% of the population by the year 2050.⁴¹

Further research continues to refine present understanding of which patients are the best candidates for DNMT inhibitor therapy. Cytogenetics in particular is an important area of research, as illustrated by the trials of Najfeld et al.²⁶ and Mufti and colleagues (GJ Mufti, personal communicaton), which suggest specifically that patients without cytogenetic abnormalities and those with monosomy 7 and trisomy 8 may benefit from DNMT inhibitors.

Comparing azacitidine and decitabine responses

The wealth of published clinical trials,^{14, 15, 16, 17, 18, 19, 23, 24, 25, 28, 30} particularly phase I and II, and recently presented data makes it tempting to draw comparisons between azacitidine and decitabine. However, it would be premature to surmise that either agent is clinically better, for a number of reasons. Comparing results between studies fails to reconcile differences in methodology that, however minute, may have a profound additive impact on the results of a study. Comparisons between trials also fail to take into account differences in populations studied by the different trials. For example, in trials conducted by Garcia-Manero et al.¹⁸ and Issa et al.,³⁰ the majority of patients had already progressed to active leukemia, limiting the extent to which the results can be generalized and applied to patients with MDS. Some patients also received adjunctive experimental therapy, as was the case in the Garcia-Manero et al. trial,¹⁸ in which patients received valproic acid in addition to decitabine.

Another factor making it difficult to draw effective comparisons between studies is the variability of criteria used to define response rates between the DNMT inhibitor trials (Table 4). These differences are important to keep in mind when comparing, for example, the results of Silverman et al.¹⁴ and O'Brien et al.¹⁶ At the superficial level, decitabine might be considered a more effective DNMT inhibitor, based upon a general comparison of total response rate—83% versus azacitidine's 60%. However, several of the studies, such as that of O'Brien and colleagues, might have used less stringent response criteria, particularly with respect to the responses of CR and CB (Table 4) and defining total response as the sum of patients experiencing CR, PR, marrow CR plus CB, and CB (other studies^{14, 17, 19} define total response as CR, PR, and HI). One other factor is duration of therapy. Patients treated with azacitidine have received therapy to the point of CR and then for an additional three cycles or on an open-ended basis until relapse or progression for those with PR or HI. Patients have received treatment for several years, up to 14 years or more in the longest-running study (LR Silverman, personal communication). Treatment with decitabine has generally been administered for fewer numbers of cycles, usually lasting from 2 to 6 months. There, duration of response has been consistently been longer in studies of azacitidine compared with decitabine. This observation raises a question for a potential role of maintenance therapy in MDS with these agents.

Monitoring and evaluating responses

A further question that has yet to be answered is how to best define an overall therapeutic success with DNMT inhibitors. While it is easy to characterize stable disease or progression as treatment failure, it is difficult to determine the relative importance of the many outcomes that comprise success, which include response rates, duration of response, QOL, and safety outcomes. Clinicians should bear in mind that the majority of patients eligible for DNMT inhibitor therapy are 65 years of age, and that this group need not be relegated to receiving BSC only. It is also important to consider the mechanism of action of DNMT inhibitors⁴² when monitoring for response. These inhibitors must be incorporated into DNA, either directly, or indirectly through a reduced metabolite, before methyltransferase is inhibited; the resulting DNA strand after one cycle of replication is hemimethylated. It takes two cycles of replication before the DNA may be entirely demethylated, and even then, some concomitant remethylation occurs (Figure 5). Thus, it takes several doses and cycles before a clinical response may be seen.

Figure 5 Mechanism of action of DNA methyltransferase inhibitors and their incorporation into DNA

After methylation, one of two outcomes can occur during DNA replication: (1) hemimethylated sites are remethylated by maintenance methyltransferase, or (2) methylation is lost because methyltransferase is unable to remethylate hemimethylated sites, as when they are covalently bound to DNA methyltransferase inhibitors. C, cytosine; G, guanine; M, methylated. Reproduced with permission from reference 44 © (2002) Nature Publishing Group.

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Despite the questions that remain about how to define total response or overall therapeutic success that make it difficult to compare the two agents meaningfully, there are also some areas of consensus in the literature with respect to monitoring response. A CR generally requires the presence of <5% myeloblasts with platelets (PLT) 100 10⁹/l^{14, 15, 16, 17, 19, 37} and hemoglobin (Hb) >11 g/dl.^{15, 17, 19, 37} The International Working Group guidelines³⁷ also recommend that the definition of complete response should include a neutrophil count of 1.5 10⁹/l. A PR response usually involves a 50% decrease in myeloblasts^{14, 15, 17, 19, 37} and a trilineage response, as defined by blood counts specified in a given CR definition.^{14, 15, 17, 19} HI is broadly classified as a 50% reduction in a patient's blood transfusion requirements^{14, 15, 17, 19, 37} and a monolineage or bilineage response, as defined by a minimum increment in a specific lineage.^{14, 15, 17}

Because these agents must be incorporated into DNA to exert their actions, they work slowly, and several cycles of treatment may be required before responses are observed. As observed in the CALGB trial, the best response with azacitidine occurs after at least 93 days, or three cycles of treatment.¹⁴ Similarly, Saba et al.¹⁹ observed a median time to first response to decitabine therapy after at least 3.2 months, or three cycles of treatment. Thus, clinicians and their patients may need to wait several months before they can evaluate response rate and overall success.

As there are many considerations when evaluating response to DNMT inhibitor therapy based upon clinical parameters, ongoing translational research may be able to provide newer and more effective ways to assess response to and the extent of demethylation with these agents. This may be accomplished by measurement of CDKN2B promoter methylation density measured before and after treatment by methods such as the COMBINED BISULFITE RESTRICTION ANALYSIS.^{30, 43, 44} Continued research is being conducted to better understand this aspect and how it can be best incorporated within the clinical setting.

Finally, monitoring for safety is also very important with these agents. Clinicians need to remember that while these agents treat the cytopenias inherent in MDS, they may also cause myelosuppression. Side effects attributed to azacitidine include grade 3 or 4 leukopenia (43%), granulocytopenia (58%), and thrombocytopenia (52%), although toxicity is usually brief and resolves before the next treatment cycle.¹⁴ Decitabine therapy is also commonly associated with myelosuppressive side effects, such as grade 3 or 4 neutropenia (87%), febrile neutropenia (23%), thrombocytopenia (85%), and anemia (12%).¹⁹ Thus, the need to monitor, both for efficacy after several cycles of treatment and for toxicity after each cycle of therapy, is paramount. This consideration is important particularly in the geriatric patient population, where MDS is most common. Clinicians and support staff need to be vigilant and supportive to patients to help them through therapy.

Conclusion

Therapy with DNMT inhibitors provides hematologists and oncologists with a novel therapeutic option in the treatment of patients with MDS. Evidence from phase III clinical trials^{14, 19} demonstrates that azacitidine elicits a 60% total response and decitabine, a 30% total response. Both responses are relatively long-lasting, with the median duration being 14 months for azacitidine and 9 months for decitabine. Azacitidine improves QOL,²⁵ particularly by ameliorating fatigue, dyspnea, physical functioning, affect, and psychological distress, while decitabine has also demonstrated a statistically significant improvement of global health status (P < 0.05)¹⁹ on the EORTC scale after cycles 2 and 4.

Azacitidine significantly prevents or delays transformation to AML overall and significantly prolongs AML-free survival globally.¹⁴ Decitabine does not have an effect on frequency of transformation to AML and prolongs AML-free survival in only the high-risk patient population. The effect of decitabine on overall survival has not been reported, while early treatment with azacitidine appears to confer significant survival advantage.

Decitabine shows promise on the basis of a number of phase I and II studies.^{15, 16, 17, 18} Recent phase III experience by Saba and colleagues¹⁹ offers further encouraging observations, including a significant difference between total response as compared with supportive care (30% versus 7%, P < 0.001), and a median time to response to decitabine therapy of 3.2 months. However, further investigation is needed to better evaluate the impact of this agent upon time to AML or death.

Clinical trial evidence is continuing to elucidate the ways in which physicians and patients may maximize therapeutic response. A number of important considerations must be taken into account in order to optimize use of DNMT inhibitors. The picture that begins to emerge is that these agents are most effective in MDS when used at a low dose^{14, 30} and given for more than four cycles^{23, 30} (GJ Mufti, personal communication), and that DNMT inhibitor therapy may be likely to succeed in patients with specific cytogenetic abnormalities (monosomy 7 and trisomy 8)²⁶ (GJ Mufti, personal communication). Although many of these considerations, particularly duration of therapy and cytogenetics, represent areas of ongoing research, clinical trial evidence has firmly established the place of DNMT inhibitors in the pharmacotherapeutic management paradigm of MDS.⁸

Future studies are needed to better define optimal dosing regimens, MDS treatment algorithms, strategies for selecting patients, and whether adjunctive therapies with agents such as histone deacetylase inhibitors^{18, 45} improve response. In addition, prospective, comparator-controlled trials are needed to fairly compare the efficacy and safety profiles between individual DNMT inhibitors and to best understand each agent's appropriate place in therapy.

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