The role of azacitidine in the treatment of myelodysplastic syndromes
Haifaa Abdulhaq & James M Rossetti†
†Western Pennsylvania Cancer Institute, The Western Pennsylvania Hospital, 4800 Friendship Avenue, Suite 2303, Pittsburgh, Pennsylvania, PA 15224, USA

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders characterized by ineffective hematopoiesis and potential transformation to acute myeloid leukemia. Supportive care including transfusions and growth factors remained the mainstay of treat- ment for decades; however, further understanding of the biology behind these diseases led to the investigation of novel agents. As hypermethylation of tumor suppressor genes, such as p15, was believed to play a key role in the pathogenesis of these diseases, hypomethylating agents were investigated. Azacitidine is one of two hypomethylating agents used in the treatment of MDS, and the first approved by US FDA. In preclinical studies, azacitidine demonstrated hypomethylating/differentiating activity with low concentration, whereas high concentration was associated with cytotoxic effects. In clinical trials, azacitidine not only improved the cytopenias associated with MDS but also delayed leukemic transformation, improved quality of life and improved overall survival in many patients so treated. Azacitidine was the first agent noted to change the natural history of the disease. Further studies are underway evaluating the role of azacitidine pre- and post-transplantation, in combination with other agents, as well as in treatment of acute myeloid leukemia patients who are not good candidates for intensive chemotherapy. Azacitidine is also likely to be studied in the treatment of other malignant conditions. Although both subcutaneous and intravenous administrations have been approved, oral azacitidine is presently under investigation.

Keywords: azacitidine, methylation, myelodysplastic syndromes

Expert Opin. Investig. Drugs (2007) 16(12):1967-1975

1. Introduction
Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematologic disorders, characterized by ineffective hematopoiesis with subsequent cytopenias and potential transformation to acute myeloid leukemia (AML) [1]. In the US,  5 new cases per 100,000 of the general population are diagnosed each year. The incidence increases with age reaching 20 – 50 cases per 100,000 in individuals > 60 years. Diagnosis is established by bone marrow biopsy and aspiration to assess cellularity and detect morphologic and cytogenetic abnormalities.
Given the heterogeneity of these disorders, subcategories have been derived distinguishing between low-risk and high-risk disease. The first classification system was the French, American and British (FAB), which divided MDS into five subtypes: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-t) and chronic myelomonocytic leukemia [2].

10.1517/13543784.16.12.1967 © 2007 Informa UK Ltd ISSN 1354-3784 1967

Azacitidine

NH2

O N

HO

HO OH

Figure 1. Chemical structure of 5-azacitidine [101].

This system relies largely on the percentage of blasts within the marrow as a means for risk stratification. The World Health Organization (WHO) system recognized further categories of patients with isolated, bi-lineage and multi-lineage cytopenia, as well as patients with the 5q-abnormality who tend to have a better prognosis [3]. The International Prognostic Scoring System (IPSS), tabulated by using the percentage of bone marrow blasts, number of cytopenias and karyotype, divides the disease into low, intermediate-1, intermediate-2 and high risk categories, with marked difference in survival and transformation to AML between risk groups [4].
Allogeneic stem cell transplantation (SCT) is the only curative modality of treatment; however, the majority of patients are not candidates for SCT due to advanced age and co-morbid conditions. Therefore, supportive care measures, including the transfusion of blood products and use of hematopoietic growth factors, remained the mainstay of management for this disease for decades; these modalities, however, did not impact the natural history of the disease or survival [5]. Intensive chemotherapy on the other hand was associated with high mortality and poor survival, which resulted in the investigation of novel agents in improving outcomes such as improved cytopenias, transfusion require- ments and quality of life (QOL). Moreover, the need for agents that delay leukemic transformation and extend survival in MDS patients remained a primary focus for investigators.
Better understanding of the biology of these disorders paved the way for clinical investigation of agents with novel mechanisms of action. Epigenetic modification with DNA hypermethylation in the promoter region of some tumor suppressor genes was thought to be a possible mechanism involved in the pathogenesis of MDS. Interest in demethy- lating agents has, thus, increased over the last decade. Azacitidine (Vidaza™, Pharmion), is one of two US FDA approved demethylating agents used in treatment of these diseases. As azacitidine was the first-in-class agent approved for these disorders, much is known about its use through

both clinical trials and use in general clinical practice. Although this manuscript focuses on the role of azacitidine in the treatment of MDS, the authors also touch on other potential applications.

2. Azacitidine chemistry

Azacitidine, first synthesized by Sworm and co-workers in 1964 [6], is a pyrimidine nucleoside analog (ribonucleoside) that differs from cytosine by the presence of nitrogen in the C5-position (Figure 1). The chemical name is 4-amino-1--D-ribofuranosyl-S-triazin-2(1H)-one [101]. The hypomethylating effect, as in other cytidine analogs, appears to depend primarily on the presence of an altered C5-position [7].

3. Clinical pharmacology and pharmacodynamic properties

Aberrant DNA hypermethylation is thought to be relevant in leukemogenesis and has also been described in a variety of other malignancies. Aberrant methylation of CpG islands in the promoter region of several genes commonly occurs in MDS. Hypermethylation of P15, a tumor suppressor gene, is associated with loss of expression and is involved in the pathogenesis of MDS. Interestingly, this occurs more frequently in high-risk MDS [8]. Inhibition of DNA methylation has been proposed as the main mechanism responsible for azacitidine use in MDS, as this is thought to induce cellular differentiation through activation of genes silenced by hypermethylation [9].
Azacitidine is activated to triphosphate by uridine-cytidine kinase, and is degraded by cytidine deaminase. Being a ribonucleoside, azacitidine incorporates into RNA and to a lesser degree into DNA. After incorporation into DNA, azacitidine acts by non-competitively inhibiting DNA methyltransferase (termed DNMT1), the enzyme responsible for methylating DNA (Figure 2) [7]. In addition to inducing DNA methylation, DNMT1 contains two domains that can recruit histone deacetylase (HDAC1 and HDAC2) [10,11]. Drugs inhibiting histone deacetylation could further reduce DNMT1 activity.
DNMT1 inhibition by azacitidine is not seen in resting cells and occurs at azacitidine concentrations that do not cause major suppression of DNA synthesis [11]. At high concentrations azacitidine becomes highly cytotoxic and mainly exerts its actions on rapidly dividing cells.

4. Preclinical activity

Preclinical studies involving human promyelocytic leukemic HL-60 cells treated with varying concentrations of azacitidine suggested a difference in the mechanism of apoptosis depending on concentration of the drug. Low concentrations (2 – 8 µmol/l) mainly caused incorporation

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Abdulhaq & Rossetti

NH2 C
N N
C CH
O N

S-Adenosylhomocysteine DNA methyltransferase S-Adenocylmethionine

NH
2

C
N C
5-mc
C CH
O N

5-Azacytidine

NH2

C
N CH
C
C CH
O N

O
DNA
methylation C
N C
T
C CH
O N

O

C
N CH
U
C CH
O N

Figure 2. 5-Methylcytosine differs from cytosine by the presence of a methyl group at position 5 of the pyrimidine ring. DNA methyltransferases catalyze the transfer of a methyl group from the methyl-donor S-adenosyl-methionine into the 5 position in the cytosine ring. Azacitidine exerts antitumor activity through induction of DNA hypomethylation by forming a covalent complex with the major DNA methyltransferase (DNMT1).
Reproduced from LEONE G, VOSO M, TEOFILI L et al.: Inhibitors of DNA methylation in the treatment of hematological malignancies and MDS. Clin. Immunol.
(2003) 109:89-102 [7], with permission from G Leone (2007).

of the drug into RNA and induction of cytotoxicity of G1 cells. Higher concentrations (16 µmol/l) were associated with perturbation of both DNA and RNA metabolism triggering cell death in G1 and S phases [12]. Azacitidine also demonstrated differentiation-inducing activity at low concentration, and strong antileukemic effects at high concentration in cell line models. Thus, the efficacy of azacitidine as an antineoplastic agent appears to result from two distinct mechanisms: cytotoxicity (high dose) and induction of hypomethylation, leading to cellular effects that are distinct from immediate cytotoxicity (low dose) [7].
Further studies were done to assess the effect of azacitidine on different cytokines that regulate hematopoiesis, including LIF, oncostatin M, IL-6 and IL-11. Using cell culture supernatants obtained from peripheral blood mononuclear cells of healthy individuals and patients with RA, azacitidine caused downregulation of these cytokines from the mononuclear cells of RA patients but not from those of healthy subjects [13].

5. Pharmacokinetic properties

The bioavailability of subcutaneously (s.c.) administered azacitidine was investigated and compared to that of intravenous (i.v.) administration using a single dose of 75 mg/m2 given either s.c. or i.v. in a randomized study of 6 MDS patients. A minimum of 7 days and a maximum of

28 days were permitted between treatments. The study demonstrated good bioavailability of s.c. azacitidine dose with area under the curve values within 89% of those measured following i.v. administration. Maximum plasma concentration was 750 ng/ml and occurred in 0.5 h with a mean half-life of 41 min. Mean volume of distribution following i.v. dosing was 76 l [14].
Urinary excretion is the primary route of elimination of azacitidine and its metabolites [101]. A study in 5 cancer patients revealed mean excretion of radioactivity in the urine following s.c. administration of 14C-azacitidine (50%) to be substantially lower than excretion after i.v. administration (85%).
A pharmacokinetic study in dogs given oral azacitidine, which was recently presented at the American Society of Clinical Oncology annual meeting, demonstrated rapid absorption with absolute bioavailability of 67%. When com- paring a single parenteral dose of 75 mg/m2 ( 2 mg/kg) given s.c. to humans versus a single oral dose of 16 mg/m2 (0.8 mg/kg) given to dogs, plasma concentrations of azaciti- dine were similar despite the 4- to 5-fold difference in
dose as calculated by body surface area (BSA). A 14-day toxicology study in dogs evaluated the oral doses of 0.2, 0.4 and 0.8 mg/kg/day. The high dose is the previously identified maximum tolerated dose (MTD) of 0.55 mg/kg/day based on an oral bioavailability of 67% (approximately equal to 16 mg/m2/day). Hematologic toxicity, a known and expected effect of azacitidine administered in a repeat-dose

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Azacitidine

regimen was observed at the two highest doses. The oral MTD was determined to be 0.2 mg/kg/day for 14 consecu- tive days followed by a 21-day recovery period. This provides a cumulative MTD of 2.8 mg/kg for the 14-day dosing regimen, which is similar to that seen with i.v. dosing (2.75 mg/kg over 5 days) [15]. Based on preclinical studies, a multicenter, single-treatment study of oral azacitidine is underway in subjects with MDS, AML or solid tumors. The trial assesses the safety, tolerability and pharmacokinetics of escalating single doses of orally administered azacitidine.
Drug interaction studies with azacitidine have not been conducted [101], and the potential of azacitidine to inhibit CYP450 is not known.

6. Clinical efficacy

The Cancer and Leukemia Group B (CALGB) conducted a Phase I/II study (CALGB 8421) of patients with largely RAEB and RAEB-t, using azacitidine at 75 mg/m2/day continuous i.v. infusion for 7 days every 28 days. Responses were seen in 21 out of 43 evaluable patients (49%);
5 patients (12%) achieved a complete response (CR), 11 patients (25%) had partial response (PR) and 5 (12%) had hematologic improvement (HI). Patients who did not respond after 4 months were discontinued from the study. Median survival for all patients was 13.3 months, whereas median remission duration was 14.7 months. Interestingly, the best response was observed after a mean of 3.8 cycles (range: 2 – 11), indicating that azacitidine may need repeated applications to achieve maximum efficacy [16]. The most frequent side effects were nausea and/or vomiting (63%), followed by diarrhea (30%). Myelosuppression occurred in 33% of patients.
A second Phase II study (CALGB 8921), using s.c. bolus injection of azacitidine at the same dose, also produced a response in  50% of patients, with 27% achieving CR/PR and 13% demonstrating HI [17]. Other studies using a lower dose of azacitidine (15 mg/m2/day for 14 days) resulted in modest activity, without significant myelosuppression [18].
Encouraged by results of prior Phase II studies, Silverman et al. conducted a Phase III study (CALGB 9221), which randomized 191 patients to s.c. azacitidine at 75 mg/m2/day for 7 days every 28 days, versus supportive care. Patients in the supportive care arm whose disease worsened were permitted to crossover to receive azacitidine. Nearly half of the supportive care group met crossover criteria and thus received azacitidine treatment. The FAB classification was used at study entry, as the IPSS had not yet been described. Patients with RA and RARS were required to meet additional criteria including symptomatic anemia requiring red blood cell transfusions for at least 3 months before study entry, thrombocytopenia with two or more platelet counts
 50  109/l, a significant clinical hemorrhage requiring

platelet transfusions, or an absolute neutrophil count
< 1  109/l and an infection requiring intravenous antibiotics.
CALGB response criteria were used in this study because patient enrollment predated IWG criteria. Responses occurred in 60% of patients in the azacitidine arm (7% CR, 16% PR, 37% HI) compared with 5% (improved) receiving supportive care (p < 0.001). As seen previously in other trials, most responses were seen in the third or fourth month. Median time to leukemic transformation or death was 21 months for azacitidine versus 13 months for supportive
care (p  0.007). Transformation to AML occurred as the
first event in 15% of patients treated with azacitidine and 38% of patients receiving supportive care (p < 0.001), suggesting a significant delay in leukemic transformation in azacitidine treated patients. Eliminating the confounding effect of early crossover to azacitidine, a landmark analysis after 6 months showed median survival of an additional
18 months for azacitidine and 11 months for supportive care (p  0.03) [19]. Quality of life assessment found signifi- cant advantages for patients initially randomized to azacitidine. Patients in the azacitidine arm experienced significantly greater improvement in fatigue (p  0.001), dyspnea (p  0.0014), physical functioning (p  0.0002), positive affect (p  0.0077), and psychologic distress (p  0.015) than those in the supportive care arm [18,19]. In conclusion, this study found that azacitidine treatment results in significantly higher response rates, improved quality of life, reduced risk of leukemic transformation, and improved survival compared with supportive care [20].
As a result of this study, s.c. azacitidine was the first drug to be approved for the treatment of MDS of all FAB subtypes in May 2004 [102]. The FDA recently approved
i.v. administration of azacitidine (January 2007), with a similar dosing schedule to the subcutaneous route [102].
In 2006, Silverman et al. reported further analysis of CALGB studies using the WHO classification system and IWG response criteria [21]. Response rates in azacitidine- treated patients were consistent across studies, with 40 – 47% of patients demonstrating a response. Ten to 17% of patients achieved a CR, PR was rare, and 23 – 36% of patients had HI. Median duration of response was
13.1 months (range: 2 – 166 months). In all three studies, the median number of cycles from first treatment with azacitidine to any response (CR, PR or HI) was three cycles (range: 1 – 17 cycles). The majority of responders (90%) achieved a response by cycle 6 (Table 1).
Encouraging results were noted in patients with WHO-defined AML ( 20% marrow blasts). Between 35 and 48% of the azacitidine-treated patients in protocols 8421, 8921 and 9221 experienced CR, PR or HI. Among the 33 AML patients who responded in the 3
studies, the median duration of response was 7.3 months (range: 2.2 – 25.9 months). In protocol 9221, 7% of patients with WHO AML in the azacitidine group achieved CR or PR, compared with zero in the supportive care group.

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Table 1. Response to azacitidine in Phase II and III trials per IWG response criteria [20].

IWG response Protocol 8421
(N  48)

Protocol 8921
(N  70)

Azacitidine (N  99)

Protocol 9221

Observation alone (N  41)

Observation then azacitidine (N  51)

No. of % Pts No. of % Pts No. of % Pts No. of % Pts No. of % Pts

CR 7 15 12 17 10 10 0 0 3 6
PR 1 2 0 0 1 1 0 0 2 4
HI 13 27 16 23 36 36 7 17 13 25
OR 21 44 28 40 47 47 7 17 18 35
CR: Complete response; HI: Hematologic improvement; IWG: International Working Group; N: Number of patients; OR: Overall response (CR+ PR + HI); PR: Partial response; Pts: Patients.

Table 2. Most frequently observed adverse events (NCI CTC grades 1 – 4) by patient-years of exposure in CALGB 9221 [20].
Adverse events Azacitidine patients (N  150) Observation patients (N  92)

Total exposure, patient-years

138.2 43.2

Patients with events per patient-year of exposure Number of patients Patients with events per Number of patient-year of exposure patients
Nausea 0.72 100 0.37 16
Vomiting 0.52 72 0.12 5
Fatigue 0.42 58 0.53 23
Injection site erythema 0.35 49 0 0
Diarrhea 0.39 54 0.30 13
Injection site pain 0.26 36 0 0
Pyrexia 0.56 77 0.65 28
Ecchymosis 0.32 44 0.32 14
Constipation 0.42 58 0.14 6
Thrombocytopenia 0.74 102 0.97 42
Anemia 0.77 107 1.37 59
Leukopenia 0.55 76 0.63 27
Anorexia 0.23 32 0.14 6
Pharyngitis 0.23 32 0.16 7
Weakness 0.32 44 0.44 19
Arthralgia 0.26 36 0.07 3
CALGB: Cancer and Leukemia Group B; CTC: Common Terminology Criteria; N: Number of patients; NCI: National Cancer Institute.

Median survival for the 27 WHO AML patients in the azacitidine group was 19.3 months compared with
12.9 months for the 25 WHO AML patients randomly assigned to supportive care [21].
Improvement in overall survival (OS) was recently demonstrated from a large, multi-institutional, international Phase III study using azacitidine versus conventional care

regimens (CCR) in high-risk MDS patients. CCR included best supportive care (BSC), low-dose cytarabine plus BSC, or standard chemotherapy plus BSC. The median number of treatment cycles for azacitidine was nine. Azacitidine was associated with a median survival of 24.4 months versus 15 months for those receiving CCR (p  0.0001). Two-year survival rates were 50.8 versus 26.2%, respectively (p < 0.0001).

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Compared with CCR, azacitidine extended survival by 74%. Complete results of this study have not yet been presented [102].
Investigators at the Western Pennsylvania Cancer institute (WPCI) have had considerable experience with azacitidine in MDS. A total of 57 patients were registered in the initial azacitidine program. Of 48 evaluable patients, 46 were transfusion dependent before treatment. Of them, 18 (39%) became transfusion independent. The majority of patients
required  3 cycles to respond. The median duration of
response was 7 months, with 3 responses continuing beyond 2 years. FAB classification and IPSS score did not predict response to azacitidine. Interestingly, a decrease in the white blood cell count during the initial cycle of azacitidine correlated with a higher response rate [22].
Another retrospective analysis of 80 MDS patients treated with azacitidine at the authors’ institution showed no statistical difference in response rate between patients with simple ( 2 cytogenetic abnormalities) and complex karyotype ( 3 cytogenetic abnormalities) (38 versus 66%; p  0.15) [23].
Encouraging results were also noted retrospectively in
20 patients who had bone marrow blast counts  20% (WHO AML). These patients had an overall response rate of 60% (20% CR, 25% PR and 15% HI). The median survival of responders was 15+ months compared with
2.5 months for non-responders (p  0.009). During therapy,
responders had an Eastern Cooperative Oncology Group performance status of 1 or 0. Eight patients were hospitalized for complications during treatment. Only four patients were hospitalized during the first cycle of treatment [24].

7. Combination with histone deacetylase inhibitors

The synergistic effects of demethylating agents and HDAC inhibitors in reactivating silent genes encouraged clinical studies of this combination in MDS. Two studies of azaciti- dine combined with phenylbuterate (PB) were presented at the American Society of Hematology 2001 meeting. Investigators at Memorial Sloan Kettering Cancer Center initiated a study using subcutaneous injections of azacitidine for 7 consecutive days (75 mg/m2/day) followed by 5 days of i.v. PB (200 mg/kg/day), repeated on a 21 – 28-day schedule, contingent on tolerability and response. At that time, six patients with MDS/secondary AML had received at least one cycle of therapy (range: 1 – 3 cycles). Reduction in bone marrow blast counts as well as increased myeloid maturation was observed in four patients. One patient with relapsed leukemia following bone marrow transplantation (BMT), had complete elimination of bone marrow blasts after one cycle of therapy, and subsequently underwent a second allogeneic BMT. An increase in histone acetylation was consistently detected in peripheral blood and bone marrow samples collected after PB administration. Selected genes commonly silenced (e.g., p15 in myelogenous leukemia)

were analyzed for methylation and expression. Changes in methylation of the p15 promoter were assessed using real-time PCR. Treatment was relatively well tolerated, with mild adverse reactions including fatigue, nausea, vomiting and local tenderness at injection sites (associated with azacitidine) and transient somnolence and drowsiness (associated with PB) [25].
The Johns Hopkins Oncology Group reported a Phase I study using sequential administration of azacitidine and PB in patients with MDS and AML. The initial azacitidine dose was 75 mg/m2/day s.c. given for 5 days, followed by PB at
375 mg/kg/day continuous i.v. infusion on days 5 – 12 repeated every 28 days. Dose de-escalation to determine the minimal azacitidine dose associated with significant demethylation was performed. A total of 11 patients were treated with 39 courses. The combination was well tolerated with 1 out of 6 patients at the highest dose level developing dose-limiting toxicity (fatigue). No unexpected clinical toxicities were observed. Two patients had significant hematopoietic improvement. The primary laboratory end point was inhibition of methylation. Baseline methylation activity was highly variable. Histone acetylation was increased over baseline in 4 out of 11 patients investigated. In addition, two patients had significant detectable acetylation that persisted during the PB infusion. In 9 patients, sequential measurements of p15 promoter methylation by a newly developed PCR-based assay were performed. All had measurable methylation of the p15 promoter exceeding 10% of available CpG sites (normal < 2%). P15 methylation density was higher in patients with AML or RAEB-t compared with patients with lower-grade MDS. During azacitidine/PB treatment, 3 patients had p15 methylation levels decreased to 19, 45 and 56%, respectively. P15 methylation increased in one patient (in association with disease progression) and was stable in five patients. Baseline methylation density did not predict for the extent of demethylation in response to azacitidine/PB [26]. Both of these studies demonstrate that the sequential administration of a demethylating agent and HDAC inhibitor is feasible with preliminary evidence of impact on the targeted gene promoter.
Another Phase I/II study was presented at American
Society of Hematology 2006, using the combination of azacitidine at 75 mg/m2 s.c. daily  7 days every 28 days, with the oral HDAC inhibitor MGCD0103. MGCD0103 was started on day 5 of azacitidine and was given 3 times a week without adjusting for body weight. The Phase I design
followed a 3 + 3 model and only MGCD0103 was dose escalated. Three dose levels have been studied thus far: 35,
60 and 90 mg. Twelve patients have been registered in the study. All but one patient with MDS had AML. All patients had relapsed or refractory disease. A total of 24 cycles have been administered (mean  2). One patient had dose-limiting toxicity of grade 3, vomiting at a dose of
90 mg. Otherwise the combination was well tolerated.

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The majority of patients exhibited substantial reduction in HDAC activity during treatment. Analysis of DNA methylation is ongoing. Two patients achieved a CR but one died of pneumonia. This study is still ongoing, and will continue as a Phase II study once the MTD of the combination is documented [27].

8. Safety and tolerability

In CALGB 9221, azacitidine was associated with worsening of pre-existing cytopenias in  78% of patients. In general, the percentage of patients with shifts from grade 0 – 2 to grade 4 cytopenias was greatest during cycle 1 and then decreased with subsequent cycles. Azacitidine, however, was well tolerated and treatment did not increase the rate of infection or bleeding.
The most commonly occurring adverse reactions by s.c. route were nausea (70.5%), anemia (69.5%), thrombocytopenia (65.5%), vomiting (54.1%), pyrexia
(51.8%), leukopenia (48.2%), diarrhea (36.4%), fatigue (35.9%), injection site erythema (35.0%), constipa- tion (33.6%), neutropenia (32.3%) and ecchymosis (30.5%). Febrile neutropenia occurred at a rate of 16.4%. The most common adverse reactions by i.v. route also included petechiae (45.8%), weakness (35.4%), rigors (35.4%) and
hypokalemia (31.3%) [102].
Because azacitidine is potentially hepatotoxic in patients with severe pre-existing hepatic impairment, caution is needed in patients with liver disease.

9. Expert opinion

Recent years have witnessed remarkable advances in our understanding of the biology of MDS. Many novel agents have been introduced into clinical trials with encouraging results. Azacitidine, the first demethylating agent to be approved by the FDA for treatment of MDS, was the first drug to prove that a change in the natural history of these diseases could be accomplished. Another demethylating agent, decitabine (Dacogen, MGI Pharma), has since been approved by the FDA and has similar indications to azacitidine. There is at present no clear data to suggest a significant difference between azacitidine and decitabine in terms of efficacy and safety profile. Importantly, however, the recent survival data favoring azacitidine over CCR, may play a role in determining first-line therapy for MDS patients who are candidates for demethylating agents.
Although the dosing schedule approved for azacitidine is 75 mg/m2/day for 7 consecutive days every 28 days, many centers are delaying the last 2 doses of each cycle until the following week. Alternative dosing schedules are under investigation, with some centers beginning to report experience with a 5-day schedule [28]. Recent data suggests that various dosing schedules may be equally well tolerated and efficacious. As a practical matter, azacitidine is given as

a deep subcutaneous injection with a needle change after mixing with diluent to minimize erythema at the injection site. A cool or warm compress to the injection site roughly four hours after injection may also reduce local reactions.
Early myelosuppression with azacitidine is common and tends to regress as a response is seen. Once a response ensues, improvement in all three cell lines may be seen. The typical nadir period is roughly 2 weeks after the start of therapy. As most trials demonstrate that initial response occurred after an average of 3 cycles, it would be reasonable to expect cytopenias for the first 3 months, requiring close monitoring of counts. In this regard, we recommend not stopping azacitidine prematurely as long as the patient tolerates the drug well, as late responses can be seen after as many as seven cycles of therapy. The administration of granulocyte-colony stimulating factor (G-CSF) during early cycles of azacitidine is recommended as it may limit the degree and duration of neutropenia, thus allowing one to stay on schedule. Moreover, we recently reported that G-CSF increases the hematologic response to
azacitidine (84 versus 51%, p  0.003) [29]. There also
appears to be a survival advantage when these two agents are used together [30].
In general, azacitidine is indicated in MDS patients with low and Int-1 IPSS scores who have failed growth factors, or in such patients who are significantly thrombocytopenic. Patients with low-risk disease and the 5q-abnormality, however, may be best served with first-line lenalidomide, as this agent is associated with a high rate of transfusion independence and cytogenetic response in this group of individuals. For patients with Int-2 and high IPSS scores who are not candi- dates for SCT, azacitidine is a preferred agent as it may delay leukemic transformation and improve survival. In addition, it is known to impact all three cell lines. Patients with hypo- cellular MDS are best treated by immunomodulatory therapy consisting of anti-thymocyte globulin, ciclosporin and steroids. Azacitidine is not recommended for this group of patients as cytopenias may worsen for prolonged periods.
For patients who are SCT candidates, current thinking is that high-risk patients should be transplanted as early as possible, whereas low-risk patients are best served if transplanted at disease progression [31]. Recent studies evaluating administration of azacitidine prior to SCT have shown a lower post-transplant relapse rate in high-risk MDS patients [32].
Investigators at MD Anderson also reported the use of low-dose azacitidine post transplant at 8 mg/m2/day for 5 days starting on day 42 post transplant, with a good safety profile and detectable levels of hypomethylation without inversely affecting the graft. At five months of follow-up, none of the high-risk patients in this study had relapsed [33]. Further studies are required to evaluate whether this effect will translate into long-term reductions in relapse rate and improved survival.

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Azacitidine has been shown to impact all three cell lines, delay leukemic transformation and improve quality of life and survival in many patients so treated. The availability of this agent has significantly impacted the way clinicians approach the treatment of MDS. Azacitidine has given much hope to patients afflicted with these diseases. We continue to learn important information on alternative dosing and combination therapy with other agents. The promise of azacitidine treatment in MDS (and AML) will probably

result in future studies using this agent for the treatment other malignant conditions.

Declaration of interest

JM Rossetti’s institution has received research funds for azacitidine clinical trials from Pharmion. JM Rossetti is a member of the Pharmion speaker’s bureau and has received speaker’s honoraria.

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Websites
101. http://www.fda.gov FDA website.
102. http://www.pharmion.org Pharmion Corp. website.

Affiliation
Haifaa Abdulhaq1 MD & James M Rossetti†2 DO
†Author for correspondence
1Fellow, Hematology & Oncology Western Pennsylvania Cancer Institute, The Western Pennsylvania Hospital, 4800 Friendship Avenue, Suite 2303, Pittsburgh, Pennsylvania, PA 15224, USA 2Associate Director,
Cell Transplantation Program,
Western Pennsylvania Cancer Institute, The Western Pennsylvania Hospital,
4800 Friendship Avenue, Suite 2303, Pittsburgh, Pennsylvania, PA 15224, USA
Tel: +1 412 578 4355;
Fax: +1 412 578 4391;
E-mail: [email protected]

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