Azacitidine: activity and efficacy as an epigenetic treatment of myelodysplastic syndromes

Valeria Santini
UF Ematologia, AOU Careggi, via delle Oblate 1, 50141, University of Florence, Florence Italy
Tel.: +39 557 947 296
Fax: +39 557 947 343
[email protected]

5´-azacitidine is a ring analog of cytosine, differing from the natural nucleoside because it has a nitrogen in lieu of carbon in position five of the pyrimidine. Despite being synthesized approximately 40 years ago it has only recently been employed with success at low doses in the treatment of myelodysplastic syndromes (MDS). This drug has hypomethylating activity and, possibly, exerts its action by reinducing expression of genes silenced by the hypermethylation of CpG islands in their promoters. Azacitidine is administered prevalently subcutaneously (75 mg/m2/day for 7 days every 28 days) as the pharmacokinetics and pharmacoavailability are almost equivalent to the intravenous route. It was the first agent demonstrated to delay acute myeloblastic leukemia transformation and to prolong survival for patients with higher risk MDS, and it was approved in 2004 by the US FDA for treatment of all MDS risk categories. Azacitidine allows transfusion independence in more than 40% of treated MDS patients, and has opened a new era in the treatment of MDS and the use of ‘epigenetic drugs’. To correctly use this agent and obtain hematological improvements that lead to a prolonged overall survival of MDS patients, hematologists have to modify their perspective and their usual expectations from a chemotherapy-like regimen. Azacitidine may also be administered quite safely to elderly patients presenting comorbidities and it is well tolerated in an out-patient regimen. Its mode of action does not necessarily require cytotoxicity and does not induce a rapid response. Several rounds of therapy and of consequent hypomethylation of target genes are necessary to re-express silenced genes critical to differentiation and the majority of patients will respond after three to six cycles of therapy.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid disorders char- acterized by ineffective hemopoiesis, dysmorphic hematopoietic cells and low blood cell count. The worldwide incidence of MDS is approxi- mately five per 100,000 individuals in the general population. The clinical characteristics of MDS are variable and diagnosis is typically made on the basis of abnormalities in bone marrow and peripheral blood appearance. Different MDS sub- types have been identified based on shared cellular morphology, and two main classification systems have been developed based on these subtypes. The French–American–British (FAB) classifica- tion system is still commonly used, but a revised WHO classification system has been proposed. To assist in therapeutic decisions and prognostica- tion, the International Prognostic Scoring System

(IPSS) has been proposed, based on number of cytopenias, percentage of blasts and chromosomal alterations. This scoring system allows doctors to distinguish between low- and high-risk MDS, with different biological and clinical characteris- tics. Low-risk MDS (IPSS low or intermediate-1) are characterized by a high rate of marrow apop- tosis yielding severe cytopenia; high-risk MDS (IPSS high or intermediate-2) are, on the other hand, characterized by increased proliferation and a high marrow blast percentage. The clini- cal outcome of MDS patients can be evaluated using the IPSS [1]. The recently developed WHO classification-based Prognostic Scoring System is a dynamic system adding transfusion dependence as a prognostic parameter and enables predictions of survival and acute myeloid leukemia (AML) risk at any time during the course of the disease [2].

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Myelodysplastic syndromes are characterized by chromosomal numerical alterations or, more rarely, by chromosomal trans- locations, involving genes critical in controlling hematopoiesis. Epigenetic alterations may also be influential in disease progres- sion. Among these, the best characterized is CpG island hyper- methylation [3]. Epigenetic alterations significantly contribute to neoplastic transformation and have prognostic significance in MDS. Alterations may affect the pattern of histone acetylation, histone methylation and DNA methylation and are extremely common in high-risk MDS.
Epigenetic modifications are potentially reversible DNA and chromatin modifications transmitted from a cell to its progeny, able to induce altered gene expression without changing the DNA sequence and without any new genetic information. DNA methy- lation is a covalent chemical modification, adding a CH3 group at the carbon five position of cytosine situated in the sequence contest 5´-CG-3´. CpG islands are generally unmethylated in normal cells and their methylation determines downstream gene silencing. DNA methylation is regulated by DNA methyltransfer- ases (DNMTs). Alterations of DNA methylation in cancer yields global hypomethylation, DNMT1 hyperexpression and regional hypermethylation in normally unmethylated CpG islands. Many genes belonging to different functional classes have been demon- strated to be hypermethylated in MDS. DNA methylation induces gene silencing by interference with transcription factors binding at respective promoters and by regional recruitment of specific transcription repressors. Reversal of abnormalities in DNA methy- lation may restore expression of genes with tumor-suppressive function and provide a novel approach to cancer therapy [3].
Patients affected by MDS are generally elderly and the presence of comorbidities does not allow the use of aggressive chemotherapy or bone marrow transplantation. The lack of therapeutic options has, in the past, discouraged even a correct and complete diagno- sis. At present, new agents effective in the treatment of MDS have been identified and, as treatment options for MDS depend on the diagnostic classification of the disease, improved individual risk assessment is essential. Earliest among these agents was azacitidine (Vidaza®; Celgene), the first drug approved specifically for the treatment of MDS. The results obtained with this agent have been such to determine what can be described as
a ‘Copernican’ revolution in the approach to MDS, which was beforehand a neglected disease even from a diagnostic point of view because of the lack of effective treatment and hopeless management.

Chemistry
5´-azacitidine (4-amino-1--d-ribofura- nosyl-1,3,5-triazine-2-one or 1--d-ribo- furanosyl-5-azacytosine) (Figure 1), and the analog 5´-aza-deoxycytidine-decitabine, were synthesized in what was then Czechoslovakia more than 40 years ago as cytotoxic drugs that are analogs of cytosine

cytosine, differing from the natural nucleoside because it has a nitrogen in lieu of carbon in position five of the pyrimidine. This peculiarity seems fundamental for a part of its biological activity. The chemical stability of azacitidine is considered to be limited; in fact, in alkaline solutions this nucleoside undergoes a rapid and reversible opening of the 5-azacitosine ring, followed by irreversible decomposition. In acidic solutions the glycosidic bond is cleaved. Recently, evidence of stability of azacitidine in water solution has been produced, indicating high stability at 4°C and a half-life of 37 h at 20°C. At 37°C the half-life is shortened to 7 h. These data secure the general handling and clinical use of the drug [4]. In vivo, azacitidine has a wide distribution in body fluids and rapid clear- ance from systemic circulation. As systemic clearance exceeds the glomerular filtration rate and total renal blood flow, it is suggested that nonrenal elimination of azacitidine has an important role [4]. Azacitidine is rapidly absorbed following both intravenous and subcutaneous injection, with peak plasma concentrations achieved subcutaneously within 30 min of administration. Its subcutane- ous bioavailability, based on AUC, is 89% relative to intravenous administration [5]. It has been shown that subcutaneous adminis- tration of azacitidine results in twofold higher  (substance elimi- nation) half-life compared with intravenous administration. In the USA, in contrast to Europe, it is quite common to administer azacitidine as a continuous intravenous infusion, seemingly to avoid local reaction at the site of injection. As there are no direct comparisons on the efficacy of the two routes of administra- tion, it is impossible to give an opinion based on evidence on the preferable one.

Regulatory affairs
On 19 May, 2004, azacitidine for injectable suspension received regular approval by the US FDA for the treatment of all FAB subtypes of MDS [6,7].
Azacitidine received expanded FDA approval last August to reflect the prolongation in overall survival achieved in the AZA-001 survival study of patients with higher risk MDS. This observa- tion led to new data being inserted in the prescription label and further supported the 2004 FDA authorization of azacitidine as the first therapy approved for the treatment of patients with

arabynoside. Azacitidine is a ring analog of

Figure 1. Cytidine and 5-azacitidine.

MDS. Azacitidine is also the first and only drug to show a sta- tistically significant and clinically meaningful extension of sur- vival in higher risk MDS patients. The European Committee for Medicinal Products for Human Use (CHMP) has given azacitidine a positive opinion for the treatment of high-risk MDS patients who are not candidates for stem cell transplants. The CHMP’s posi- tive opinion is based on the results of the AZA-001 trial, which demonstrated an overall survival benefit for higher risk MDS patients. In December 2008, Vidaza has received final approval by the European Medicines Agency (EMEA) for intermediate-2 and high-risk MDS patients [101].

Preclinical studies
The efficacy of 5-azacitidine as an antineoplastic agent is attributed to two distinct mechanisms: cytotoxicity and induction of DNA hypomethylation, as its direct activity on RNA still needs evalu- ation in terms of antineoplastic significance. Azacitidine is trans- formed into the active nucleotide for DNA methylation inhibition, 5-aza-2´-deoxycytidine-5´-triphosphate, through ATP-dependent phosphorylation by uridine–cytidine kinase. Early incorpora- tion studies in L1210 leukemic cells have shown that 80–90% of azacitidine is incorporated directly into RNA. Decitabine, quite differently, incorporates primarily into DNA [3]. There are no direct comparisons of the two agents in vivo or in vitro, but their difference in metabolism and DNA incorporation suggests pos- sible diverse mechanisms of action. A rate-limiting step for the conversion of azacitidine ribonucleotide to deoxyribonucleotide is the activity of the ribonucleotide reductase enzyme. This enzyme is inhibited by hydroxyurea. Although this is irrelevant for patients with MDS, it may become crucial if AML patients are treated with azacitidine and need previous cytoreduction. Azacitidine incorpo- ration into RNA produces disassembly of polyribosomes, defective methylation and acceptor function of transfer RNA, alterations of messenger RNA and inhibition of protein production. The subse- quent, more limited incorporation of azacitidine into DNA leads to covalent linking with DNMT and blockage of DNA synthe- sis, ultimately resulting in direct cytotoxicity. The formation of a DNA–DNMT adduct by itself could cause DNA damage and apoptosis. In fact, 5-azacitidine is highly cytotoxic to neoplas- tic cells in S phase and exerts its action mainly on rapidly divid- ing cells; however, it also impairs cell progression to S phase [4]. Azacitidine’s anti-tumor effect can be exerted through induction of DNA hypomethylation and re-expression of silenced genes situated downstream of hypermethylated CpG islands, which are observed frequently in a vast array of human neoplasms, especially in hema- tological malignancies, such as MDS. Regional hypomethylation is thought to be the principal action responsible for the clinical efficacy of azacitidine in the treatment of MDS. By covalently trapping DNMT, azacitidine depletes the treated dysplastic cells of functionally active DNMT, resulting in reversible but profound hypomethylation after several rounds of DNA replication. In vitro, it was demonstrated in several solid tumor and leukemic cell mod- els that azacitidine can induce cellular differentiation, expression of fetal hemoglobin, hepatic enzymes, re-expression of DR antigens and erythroid and granulocytic maturation markers [3].

Clinical studies
Notwithstanding the amount of evidence for a biological activity of azacitidine in epithelial neoplasms, relatively few clinical trials have concentrated on assessing its clinical efficacy in solid tumors [3]. This was probably due to the fact that early trials for colon, breast and ovarian cancer performed in the 1970s and early 1980s did not lead to encouraging results. Azacitidine is now being investigated in solid tumors in association with other chemotherapeutics.
5´-azacitidine was first used in the therapy of hematological malig- nancies as salvage treatment for AML in the 1980s, as a single drug and in polychemotherapy regimens [3]. Azacitidine was employed in a substantial number of AML patients, at doses ranging from 100 to 400 mg/m2, for 5 days, intravenously. At these high doses, azaciti- dine had antileukemic efficacy with complete and partial responses obtained in 9–36% of AML patients. Indeed, side effects, especially myelosuppression, were quite relevant. As first-line therapy, 5-azaciti- dine was used in consolidation therapy with amsacrine and etopo- side or as maintenance in AML patients. Used in these schedules, azacitidine did not modify AML patient clinical outcome.

High-risk myelodysplastic syndromes
In an early Phase I study by Silverman et al., 43 MDS patients were treated with azacitidine intravenous continuous infusion 75–150 mg/m2 for 7 days every month, with 49% hematological improvement [8]. The drug was used in subsequent Phase II trials subcutaneously, with a rate of trilineage response and hematological improvement of 40% (Table 1) [8]. When patients were treated with azacitidine 75 mg/m2, plasma levels of the drug were in the range of 3–11 µM and peak plasma concentrations were comparable to the concentrations used to achieve DNA demethylation in vitro [5]. A milestone study was the Phase III trial conducted by the Cancer and Leukemia Group B (CALGB) in 191 MDS patients of all FAB subtypes randomized to receive subcutaneous aza- citidine 75 mg/m2/day for 7 days every 28 days or best supportive care [9]. While therapy-related mortality was negligible, overall response reached 60%, compared with 5% in the control arm. Recent re-evaluation of the results of all azacitidine trials, with the

Table 1. Hematological response and improvement based on the International Working Group criteria for myelodysplastic syndromes.
Trial Response (%)
Overall CR PR HI
AZA-001 sc. (n = 179) 51 17 12 49
CALGB 9221 (Aza sc. arm 47 10 1 36
[n = 99])
CALGB 9221 (Aza after 35 6 4 18
crossover [n = 51])
CALGB 8921 sc. (n = 72) 40 17 0 23
CALGB 8421 (Aza iv.; n = 48) 44 15 2 27
Aza: Azacitidine; CALGB: Cancer and Leukemia Group B; CR: Complete response; HI: Hematological improvements; iv.: Intravenous; PR: Partial response; sc.: Subcutaneous.
Data from [33].

application of the revised International Working Group criteria of response to treatment, has confirmed the efficacy of the drug [10], indicating a 10–17% complete remission (CR) rate and validating an overall response of 44–47%. In all the studies, achievement of response was obtained after at least four cycles of therapy in 75% of MDS-responding patients, but in some cases there were very late responders. Mean response duration was 15 months (Table 1) [9,11]. The CALGB trial did not succeed in assessing the possible effi- cacy of azacitidine on prolongation of survival because of the cross- over allowed to nonresponding patients. For EMEA approval of the drug, which requires survival data, a prospective, randomized, Phase III, clinical trial was established to assess azacitidine’s effect on overall survival in patients with higher risk MDS compared with a range of routinely used MDS treatment regimens [12]. In the AZA-001 trial, 358 patients were randomized to receive azacitidine (75 mg/m2/day for 7 days every 28 days) or conventional care regi- mens (Table 1), consisting of best supportive care, low-dose ara-C or intensive chemotherapy. Patients were assigned to one specific treatment arm before randomization and upon physician judgment, in order to evaluate patients with homogeneous clinical charac- teristics and avoid preselection bias. The primary end point was overall survival and secondary end points included time to trans- formation to AML, red blood cell transfusion independence and hematologic responses. Kaplan–Meier median overall survival was
24.4 months versus 15.0 months for the azacitidine and combined
conventional-care regimen groups, respectively, determining an overall survival at 24 months of 50.8% for the azacitidine-treated patients versus 26.2%. Median time to transformation to AML was
26.1 and 12.4 months, respectively, with azacitidine versus com- bined conventional-care regimens [12]. Data suggest achievement of International Working Group-defined hematologic response is suf- ficient but not necessary to prolong overall survival in the AZA-001 trial, as hematological improvement gives 71% survival at 2 years and even patients with stable disease experienced a survival ben- efit [13]. Moreover, a subgroup analysis conducted on MDS patients included in the AZA-001 study indicated that prolongation of over- all survival shown with azacitidine in the AZA-001 study extended to MDS patients with unfavorable cytogenetics, in particular with 53.6% hematological improvement in azacitidine-treated -7/del(7q) MDS patients versus 18.5% hematological improvements achieved by -7/del7 patients treated with the conventional-care regimens [14]. Prolongation of treatment with azacitidine has been demon- strated to be essential to achieve optimal response, as 48% of MDS patients may benefit and improve the quality of response
upon additional cycles of therapy [15].

Combination with histone deacetylase inhibitors
DNA methylation and histone acetylation are interdependent and modulate gene expression. In fact, chromatin status is determined by chemical modifications occurring directly on DNA, on cyti- dine within CpG islands and on lysine of histones. Methylation of CpG islands recruits methyl-binding proteins locally and the latter bind protein complexes containing corepressors, DNMTs and histone deacetylases (HDACs). HDACs deacetylate histone lysines locally and this modification is accompanied by changes

in lysine methylation in the same histone, promoted by histone methyltransferases. In cancer, repression of gene expression is maintained by corepressor complexes of proteins recruited by regionally hypermethylated DNA [3,16].
On this biological background, combination therapy with azaciti- dine and HDAC inhibitors (HDACis) has been attempted: asso- ciation with phenylbutyrate [17,18] or , in several studies, associa- tion with valproic acid at very different doses (from 500 mg/day to 50–75 mg/kg/day) and all-trans retinoic acid 45 mg/m2/day [19–23]. The scheme with high-dose valproic acid was applied in a cohort of pretreated, elderly patients, with 83% of abnormal cytogenetics, but responses occurred early (after one to three cycles) [19]. Responses accounted for 39% of patients, with an overall rate of 56% observed in patients over 60 years of age and previously untreated. Dose- limiting toxicity in this trial was neurological and due to valproic acid dosage [19]. Owing to the unusually rapid onset of response and the high percentage of cytogenetic complete responses, this kind of combination seemed the most powerful therapy in MDS and prompted a Gruppo Italiano Malattie Ematologiche Maligne Dell’ Adulto (GIMEMA) multicenter Phase II study on the com- bination of 5-azacitidine, valproic acid and all-trans retinoic acid. A total of 62 patients with intermediate-2/high-risk MDS were treated and 58.9% of patients are alive at 12 months. Disease pro- gression occurred in 15 patients. Red blood cell transfusion needs significantly decreased [20]. Similar studies were performed by other authors with a smaller number of patients [21–23]. All the studies published to date demonstrate feasibility of the association of the two types of agents but the efficacy of HDACi addition has to be evalu- ated further. Indeed, intriguing results have been obtained treating higher risk MDS and AML patients with azacitidine combined with vorinostat, with more than 80% early responses [24].
Of note, no consensus has been reached (due to the scarce and
sparse data available) on the in vivo significance of DNA hyper- methylation in MDS patients prior to and after azacitidine (but also decitabine) treatments in terms of prediction of achievement and duration of response [25].

Acute myeloid leukemias
In the AZA-001 trial, patients with 20–30% marrow blasts were enrolled. This group of patients is, at present, classified accord- ing to the WHO as AML. None of the patients studied presented hyperleukocytosis or highly proliferative disease. In this cohort of patients, who currently have limited therapeutic options, azaciti- dine significantly prolongs overall survival with improvements in important patient outcomes [26]. Other authors demonstrated a high efficacy of azacitidine in elderly AML patients, with induction of significant responses in approximately a third of AML patients; the standard dose of 75 mg/m2/day seems to be more effective than 100 mg/day (one single vial) fixed dose and the drug appears more effective in de novo compared with pretreated (refractory and/or relapsed) disease [27]. Azacitidine has also been used in maintenance treatment in AML patients who have achieved complete remission after conventional chemotherapy regimens [28]. New studies are needed to confirm the effect of azacitidine in elderly AML patients with more proliferative disease.

Safety & tolerability
Azacitidine is generally very well tolerated, as demonstrated by the numerous studies conducted so far. The most common adverse reac- tions by the subcutaneous route were hematological and due to the myelosuppressive effect of azacitidine, which is more pronounced during the first two to three cycles of therapy. Nonhematological adverse events were not frequent throughout all studies, but the most common are injection-site erythema (35.0%) and gastrointestinal side effects. By the intravenous route, the most common adverse reactions included myelosuppression and hypokalemia (31.3%) [102]. In the AZA-001 survival trial, the most commonly occurring adverse reactions were thrombocytopenia (69.7%), neutropenia (65.7%), anemia (51.4%), constipation (50.3%), nausea (48.0%), injection-site erythema (42.9%) and pyrexia (30.3%). The most commonly occurring grade 3/4 adverse reactions were due to
myelosuppression [12].
It is important to know that myelosuppression is particularly relevant after the first cycles of therapy and then tends to decrease as a result of improved hematopoiesis. During the first months of therapy, it is important to check blood counts regularly and possibly perform antibacterial and antimycotic prophylaxis. Transfusion needs may increase as a consequence of myelo- suppression during the first weeks of treatment with azacitidine. Time to response is 3–6 months, that is three to six cycles of ther- apy. For this reason, long-term treatment is required and therapy with azacitidine should not be interrupted until at least six cycles have been completed, unless serious adverse events or disease pro- gression occur. The majority of the most common adverse events (20%) in the AZA-001 study were transient (median duration: 13 days), nonserious and managed by either dose delays for hema- tological eventsor supportive care measures. Clinicians should be alert to the onset, duration and management of these events to allow patients to achieve maximum therapeutic benefit. Several MDS patients have been treated for years with azacitidine, main- taining a stable disease and not presenting major side effects [29]. Moreover, stopping azacitidine treatment has almost invariably led to relapse of the disease; therefore, it is advisable to prolong therapy for as long as the patients show response.
Since azacitidine is potentially hepatotoxic, MDS patients with
severe pre-existing hepatic impairment should be carefully evalu- ated for liver function before treatment. In addition, azacitidine and its metabolites are excreted substantially by the kidneys and the risk of toxic reactions to this drug may be greater in patients with impaired renal function and consequent higher plasma levels of azacitidine. There are anecdotal reports of dialyzed MDS patients who have received azacitidine. There are no reports of fetus mal- formation or of teratogenicity of the drug but, of course, pregnancy is a counterindication to the use of azacitidine and male patients should be discouraged from procreating. Nursing mothers should discontinue nursing while they are treated with azacitidine [102].

Expert commentary
Azacitidine has opened a new era in the treatment of MDS and in the use of epigenetic drugs. To use azacitidine optimally and obtain hematological improvements that lead to prolonged overall

survival of MDS patients, hematologists should modify their per- spective and their usual ‘expectance’ from a chemotherapy-like regimen. Azacitidine may be administered quite safely to elderly patients presenting comorbidities and it is well tolerated in an out-patient regimen. The activity of azacitidine is supposedly dependent upon its hypomethylating effect, quite clearly present both in vitro and in vivo in cells obtained from MDS patients after treatment. Regarding this point, it has already been men- tioned that at present no predictive role has been attributed to DNA hypermethylation status. Since azacitidine action does not necessarily require cytotoxicity, we do not have a rapid response in terms of improved hematopoiesis, as we are used to seeing in AML treatment. Several rounds of therapy and of consequent hypo- methylation of target genes are necessary to re-express silenced genes critical to differentiation. The reversibility of hypomethyl- ation justifies the use of long-term therapy to consolidate and maintain the hematological improvement. Myelosuppression caused by the first cycles of azacitidine requires strict monitor- ing, but is quite easily manageable and demands hospitalization only in sporadic cases.
Myelodysplastic syndrome patients with poor prognosis, such as those presenting with -7/del7 mutations, appear to gain the same advantage in terms of response and of significant prolongation of survival from azacitidine treatment. This fact may indeed pre- lude to a modification of prognostication in these cases, similarly to what has happened in a different setting for chronic myeloid leukemia, but also in analogy to 5q- syndrome. Moreover, pro- longation of survival and improvement of general health status allow time for hematopoietic stem cell transplant planning and realization, especially for younger patients.

Five-year view
Although the efficacy of azacitidine in treating high-risk MDS has been confirmed in many patients, both in controlled trials and in spontaneous studies, we still need to learn more about this drug.

Optimal dose & schedule finding
The use of azacitidine, as indicated in the prescriptive indication, is 75 mg/m2/day subcutaneously every 28 days. However, at least one study has shown that doses and schedules different than the 7-day schedule may be efficacious in terms of inducing transfusion independence, at least in lower risk MDS, although there has not been an evaluation of overall survival [30]. Therefore, in the next few years, it will be necessary to assess which is the best dose of the drug, refreshing pharmacokinetic studies but also performing pharmacoeconomic evaluations.

Response predictive criteria
The most important point remains the possibility of predicting the response to azacitidine. In other words, we still do not know which are the biological, clinical and cytogenetic parameters determining the optimal response to the drug. How do we select the ideal patient candidate for hypomethylating treatment? At present, the only major lead is a high IPSS score, as indicated by recently published guidelines [103]. This field deserves further study. The answer to

this question could come from a careful evaluation of the methy- lation pattern of the DNA of MDS patients who have undergone azacitidine treatment. Methods to study hypermethylation and the number of genes to be scanned are still uncertain and major expectations are held for ongoing investigations exploring these subjects [25,31].

Combination therapy
Azacitidine has shown an outstanding clinical efficacy as a single drug, but its potential in association with other chemotherapeutics or to other agents has not been ascertained completely.
As mentioned earlier, the most logical combination attempted in the clinic has been the one determined by the activity of azacitidine to influence epigenetic alterations. HDACis have been reasoned to be the ideal partner to revert the block of gene expression determined by CpG island hypermethylation and regional histone deacetylation. Azacitidine has thus been used together with phenylbutyrate, valproic acid, MGCD0103 and SNDX-275 (previously MS-275). The results have been interesting, although they are in need of wider confirmation. The influence of this combination of agents on the epigenome of MDS cells has not been directly correlated to their efficacy in vivo. Other combinations with thalidomide, lenalidomide, cytosine arabynoside and gemtuzamab ozagamicin have been planned and trials are ongoing.

Acute myeloid leukemia
New studies with better characterized de novo and second- ary AML patients should clarify the activity of azacitidine at ‘hypomethylating doses’.

Lower risk MDS
Finally, the efficacy of azacitidine in low–intermediate-1 risk MDS has been observed within the CALGB trial in a limited number of patients and interesting data have been presented recently in a retrospective study for erythropoietin-resistant MDS patients [32]. A perspective trial of azacitidine in lower risk MDS is needed to confirm these observations.
Analysis of the results of past and future studies and a better biological understanding of the modulation of the epigenome of MDS cells would undoubtedly help in targeting therapy and optimizing the efficacy of azacitidine.

Financial & competing interests disclosure
This work was supported by Ente Cassa di Risparmio di Firenze (ECR), and Ministero per la Istruzione, l’Università e la Ricerca (MIUR). Valeria Santini received honoraria for lectureship from Celgene. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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Websites
101EPARs for authorised medicinal products for human use www.emea.europa.eu/humandocs/ Humans/EPAR/vidaza/vidaza.htm
102Vidaza prescribing information www.celgene.com/pdfs/VID08043_ Vidaza%20Aug%202008%20PI_FINAL. pdf
103Greenberg PL, Attar E, Battiwalla M et al. NCCN clinical practice guidelines in oncology: myelodysplastic syndromes (2009) www.nccn.org/professionals/physician_gls/ PDF/mds.pdf
•• Most recent guidelines concerning therapy for myelodysplastic syndromes. Very useful and straightforward.

Affiliation
•Valeria Santini
Associate Professor of Hematology,
UF Ematologia, AOU Careggi, via delle Oblate 1, 50141, University of
Florence, Florence, Italy Tel.: +39 557 947 296
Fax: +39 557 947 343
[email protected]