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Home , Cytogenetics, Epigenetic Priming, Epigenetic therapy, Hypomethylating agent

Leukemia (2013) 27, 1803–1812 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www..com/leu

SPOTLIGHT REVIEW in clinical practice: the examples of and in myelodysplasia and

EH Estey

Randomized trials have clearly demonstrated that the hypomethylating agents azacitidine and decitabine are more effective than ‘best supportive care’(BSC) in reducing transfusion frequency in ‘low-risk’ myelodysplasia (MDS) and in prolonging survival compared with BSC or low-dose ara-C in ‘high-risk’ MDS or acute myeloid leukemia (AML) with 21–30% blasts. They also appear equivalent to conventional induction in AML with 420% blasts and as conditioning regimens before allogeneic transplant (hematopoietic transplant, HCT) in MDS. Although azacitidine or decitabine are thus the standard to which newer therapies should be compared, here we discuss whether the improvement they afford in overall survival is sufficient to warrant a designation as a standard in treating individual patients. We also discuss pre- and post-treatment covariates, including assays of methylation to predict response, different schedules of administration, combinations with other active agents and use in settings other than active , in particular post HCT. We note that rational development of this class of awaits delineation of how much of their undoubted effect in fact results from hypomethylation and reactivation of expression.

Leukemia (2013) 27, 1803–1812; doi:10.1038/leu.2013.173 Keywords: epigenetics; MDS; azacitidine; AML; decitabine; hypomethylating agents

‘Epigenetics’ refers to changes in occurring promoting . More direct evidence comes from findings without alterations in DNA sequence.1 For this paper, the relevant that AML blasts, particularly those obtained from patients with epigenetic events are DNA methylation and, to a less extent, secondary AML, have DNA methylation patterns distinct from modification.2 DNA methylation typically occurs at those seen in normal CD34 þ cells3 and that specific types of AML, the 50-position of cytosine in areas of the rich in for example, those associated with AML1-ETO, CBFb-MYH11, PML- CpG dinucleotides (CpG islands), results in silencing of gene RARa and mutated CEBPA or NPM1 have distinctive methylation expression and is fundamental to normal development. DNA patterns.4 Furthermore, an increase in aberrant DNA methylation methyltransferase 1 (DNMT1) maintains existing methylation was uniformly observed in 184 patients in whom MDS patterns following DNA replication, whereas DNMT3A and 3B ‘progressed’ to AML.5 The latter study did not include a group in methylate unmethylated CpGs. In contrast, members of the TET whom such progression did not occur and the above data are (‘10–11 translocation’) family of proteins remove methyl groups consistent with epigenetic changes as secondary, rather than from CpGs, a process requiring a-ketoglutarate, which is derived primary, events. However, experiments transferring leukemia from from isocitrate in a reaction catalyzed by isocitrate dehydrogenase mice with reduced DNMT1 activity suggest that constitutive 1 and 2. undergo many post-translational modifications, methylation is needed for proper renewal of leukemia stem including acetylation (which increases gene expression), cells,6,7 thus suggesting that epigenetic alterations can be primary deacetylation, methylation and demethylation, with different events. SPOTLIGHT combinations of this ‘histone code’ leading, via Unlike genetic aberrations, epigenetic changes, are reversible, modification, to activation or repression of gene expression.1,2 prompting interest in drugs that affect the ‘’. The Relative to their normal counterparts, cells generally ‘epigenetic drugs’ that have found widest use and which we will exhibit genome-wide hypomethylation and CpG island hyper- emphasize are the DNMT inhibitors azacitidine and decitabine. methylation.1 The former leads to the genetic instability Here we will first discuss their use in (a) lower-risk MDS and (b) characteristic of cancer, whereas the latter silences tumor higher-risk MDS and AML, recognizing the distinctive therapeutic suppressor genes. There is considerable evidence that abnormal goals in a and b. We will emphasize comparisons between these methylation has an important role in pathogenesis of acute drugs and other therapeutic options and discuss the possibility myeloid leukemia (AML) and myelodysplasia (MDS), the that, although used essentially interchangeably, azacitidine and of interest to us here. Circumstantially, mutations in DNMT3a and decitabine may differ qualitatively. Concluding that neither is isocitrate dehydrogenase 1 and 2, and aberrations in the TET 2 on average satisfactory treatment, particularly for high-risk MDS or gene predicted to disturb its normal function, occur not AML, we will discuss means to predict response in individual infrequently in AML and MDS while many commonly observed patients. Of particular relevance, we will examine the strength of abnormal fusions of the mixed lineage leukemia gene affect the evidence that the undoubted clinical activity of DNMT histone methyl transferases presumably activating leukemia inhibitors and histone decateylase inhibitors results from their

Division of Hematology, University of Washington School of and Fred Hutchinson Center, Seattle Cancer Care Alliance, Seattle, WA, USA. Correspondence: Dr EH Estey, Division of Hematology, University of Washington School of Medicine and Fred Hutchinson Cancer Research Center, Seattle Cancer Care Alliance, 825 Eastlake Ave East, G3-204, Seattle, WA 98109, USA. E-mail: [email protected] Received 24 April 2013; revised 24 May 2013; accepted 3 June 2013; accepted article preview online 12 July 2013; advance online publication, 23 July 2013 Epigenetics in clinical practice EH Estey 1804 undoubted epigenetic effects, as examining this hypothesis seems patients who received a median of six cycles (up to 32) after a fundamental to rational development of this class of drugs. Lastly, single 75 mg m À 2 dose of s.c. drug. Although, at its maximum we will discuss newer approaches to epigenetic therapy, involving tolerated dose (480 mg daily  7 days every 28 days) exposure to use (a) as post remission therapy, particularly after allogeneic oral azacitidine was less than with s.c. preparation (as was hematopoietic cell transplantation (HCT) (b) of different schedules hypomethylation), clinical activity was observed, which likely of administration, (c) of pharmacologically improved congeners of owed in part to the oral preparation, as responses are unusual decitabine and (d) of combinations with other active agents. after a single dose of s.c. azacitidine.16 More frequent dosing and/or more extended dosing are planned. Although initial studies with decitabine used a three times intravenous daily AZACITIDINE AND DECITABINE IN ‘LOWER-RISK’ MDS schedule, it appears that results are similar with a more By ‘risk’ I refer to overall survival (OS), the outcome of most convenient once daily 5-day schedule.17 interest to patients. Perhaps the most useful means to assess To date, there have been no randomized trials comparing these expected OS in MDS are the revised International Prognostic various means of administration. More striking is the absence of Scoring System (IPSS-R) based on an exceptionally large database randomized trials comparing azacitidine with decitabine, or either (7012 patients with untreated de novo MDS),8 the WPSS (World drug with lenalidomide, which has activity in MDS patients who Health Organization -based Prognostic Scoring System),9 which lack deletion of the long arm of 5q.18 Suggestion includes patients with secondary MDS and which addresses that the lack of a formal decitabine–azacitidine comparison may changes in prognosis over time, and the MD Anderson prognostic be more than an academic issue comes from an analysis, which, system for lower-risk MDS,10 which removes some such patients using propensity scoring to account for known prognostic factors from the lower-risk category. By lower-risk MDS, I mean an expected (such as age, blast percent, IPSS score and ), found median OS of X3 years corresponding to the IPSS-R or WPSS superior survival (P ¼ 0.02) in patients aged X65 given azacitidine groups ‘very low’, ‘low’ and ‘intermediate’, and the MD Anderson rather than decitabine;19 it was not noted whether as seems category 1. It is generally accepted that the principal therapeutic likely this finding reflected results in the 40% of patients who goal in such patients is to decrease, particularly red blood cell (RBC), were higher risk. Such a finding may reflect observed differences 20,21

SPOTLIGHT transfusions, in the process improving ‘quality of life’. in the epigenetic effects of azacitidine and decitabine. Randomized trials have shown that azacitidine and decitabine Thus, regarding these drugs as interchangeable epigenetic or are superior to a transfusion-only approach (‘best supportive care’, hypomethylating agents may be an oversimplification. BSC) in this regard. RBC transfusion independence for X8 weeks was observed in 45% of 65 RBC transfusion-dependent patients randomized to subcutaneous (s.c.) azacitidine,11 and B35% of a AZACITIDINE AND DECITABINE IN HIGHER RISK MDS OR AML similar number of transfusion-dependent patients given The natural history of higher-risk MDS as defined, for example, by decitabine12 versus only a trivial number of BSC patients. Both IPSS-R or WPSS bears more resemblance to AML than to lower-risk drugs improve quality of life more than BSC.12,13 These results MDS. Indeed European cooperative groups, such as the Medical establish azacitidine or decitabine as the preferred options in RBC Research Council and Hemato-Oncologie voor Volwassenen transfusion-dependent nondeletion 5q low-risk patients who Nederland, and American centers, such as MD Anderson and either have failed or are predicted to fail treatment with Fred Hutchinson, now extend eligibility for AML trials to patients erythropoietic-stimulating agents, and in patients who are with 10–20% blasts, even though these patients are considered to receiving platelet as well as RBC transfusions. It appears that, at have high-risk MDS rather than AML (the World Health Organiza- least with azacitidine, results similar to those reported from tion criterion for which is 420% blasts). Perhaps most importantly academic centers can be obtained in community settings even in in both higher-risk MDS and AML, the principal goal of therapy is very elderly patients (median age 75).14 to improve OS, whereas this aim is less explicit in lower-risk MDS. Considerable attention has been devoted to making adminis- Hence, I will discuss higher-risk MDS and AML together and focus tration of azacitidine and decitabine as convenient as possible. on OS. As such ‘lumping’ is not necessarily standard, I will Thus, there have been suggestions that 5 days of azacitidine are as comment on similarities or differences in outcome between effective as the initially described 7-day schedule15 and that s.c. higher-risk MDS and AML in various studies (Table 1). and intravenous schedules are equivalent. Of most interest is an Randomized trials have reported that either azacitidine or oral azacitidine preparation,16 which was administered to 41 decitabine produce better OS than BSC or low-dose ara-C (LDAC)

Table 1. Randomized trials involving decitabine or azacitidine

Drug Median Control Patients Marrow Deaths median Months median OS at 2 years OS ‘AML’ vs MDS number group median blasts follow-up OS (c) cycles age

Decitabine (‘dec’)22 4 BSC(a) 233/70 Up to 30% 195/2.5 years Dec 10.1 BSC 8.5 10% in each Effect dec same in IPSS high risk as in IPSS int-2 Decitabine (‘dec’)24 Dec 4 88% LDAC, 485/73 420% 385/not noted Dec 7.7LDAC or 10% in each Not applicable LDAC(b)2 12% BSC BSC 5.0 Azacitidine (‘aza’)23 Aza 9 59% BSC 358/69 Up to 30% 195//21 mos Aza 21.1 BSC 11.5 Aza 50–60% Effect aza same for LDAC 5 28% LDAC Aza 24.5 LDAC 15.3 BSC, LDAC, 11–20% and for 14% 3 þ 7 3 þ 7 25–30% 21–30% blasts Azacitidine (‘aza’)25 Aza 8 47% BSC, 113/70 20–30% Not noted/20 mos Aza 19.1 BSC 13.4 Aza 46% BSC 0 Not applicable LDAC 5 34% LDAC, Aza 24.5 LDAC 17 Aza 56% LDAC 19% 3 þ 7 32% Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplasia; LDAC, low-dose ara-C. (a) Best supportive care, (b) low-dose ara-C, (c) overall survival and (d) International Prognostic Scoring System.27 Doses: decitabine 15 mg m À 2 TID  3 days every 6 weeks in Lubert et al.,22 20 mg m À 2 daily  5 days every 4 weeks in Kantarjian et al.,24 azacitidine 75 mg m À 2 daily  7 days 854 in Fenaux et al.23–25

Leukemia (2013) 1803 – 1812 & 2013 Macmillan Publishers Limited Epigenetics in clinical practice EH Estey 1805 in patients with either higher-risk MDS or AML22,23 or in decitabine at the 15 mg m À 2 tid dose (Table 1) produces longer patients with AML per se24,25 (Table 1). Lubbert et al.22 noted OS than BSC, and that azacitidine does the same compared with that OS was shorter in patients with (original) IPSS scores26 of BSC or LDAC in higher-risk MDS (o21% blasts). Azacitidine seems ‘high’ than in patients with scores of ‘intermediate 2’, but it is not better than BSC or LDAC in patients with AML and 21–30% blasts clear whether patients with high scores had a blast count with extension of these results to patients with 430% blasts 420% (AML), rather than blast counts o20% (MDS) but with awaiting analysis of NCT01074047. Decitabine was not superior to ‘unfavorable’ cytogenetics or lower blood counts, the other LDAC or BSC in patients with 21–30% blasts but was superior in components of the original IPSS in any event, the effect of patients with 430% blasts,24 although in the absence of a decitabine versus control was the same regardless of the multivariate analysis, it is hard to know whether this reflected blast distinction between IPSS high or intermediate 2. Azacitidine per cent per se. Comparison of azacitidine and decitabine is reduced the risk of death to the same extent regardless of complicated by the administration of fewer cycles of decitabine whether the blast count was 11–20% or 21–30%.23 An ongoing than azacitidine (Table 1), the limited (azacitidine) or nonexistent trial (NCT01074047 at clinicaltrials.gov) is addressing whether the (decitabine) use of 3 þ 7 in the respective control groups despite same is true in patients with 430% blasts. the likelihood that many older patients can likely tolerate this In neither of the decitabine studies22,24 was the difference in OS regimen,29 use of a lower dose of LDAC28 given for fewer courses ‘statistically significant’, whereas such significance was observed in in the decitabine trials (Table 1), and perhaps most importantly by the azacitidine trials.23,25 Rather than merely a function of sample the seeming different nature of the patients in trials of the two size, the difference between azacitidine and decitabine reflected agents. Whether either drug is, as often stated, the ‘standard of larger differences between azacitidine and BSC or LDAC than care’ for older patients with higher-risk MDS or AML depends on between decitabine and these treatments (Table 1). Interpretation, what is meant by standard of care. It seems reasonable to however, is complicated because the control group had better OS compare new therapies for these diseases to azacitidine or in the azacitidine than in the decitabine studies. For example, decitabine rather than, as often currently, LDAC. However, it is less median OS with LDAC was 15–17 months in the former versus 5 clear that for at least some patients the statistically significant months in the latter with this difference greater than that between improvement in OS with, especially, azacitidine translates into LDAC and azacitidine in the azacitidine studies. Hence, the equivalent medical significance. For example, the US Social patients given azacitidine and those given decitabine may not Security data (http://www.socialsecurity.gov/OACT/population/ have been strictly comparable despite similarities in diagnosis. The longevity.html) indicate that a 65-year-old male will on average proportion of patients considered for enrollment but not enrolled live another 18 years. The azacitidine data in Table 1 indicate that was 0%22 and 23%24 in the decitabine trials, whereas comparable a diagnosis of high-risk MDS would result in a loss of 93% of this figures were not provided for the azacitidine trials.23,25 The life expectancy with LDAC versus 89% with azacitidine. Given patients in the azacitidine trials also tended to receive more cycles these data, I would expect some patients would prefer a clinical of therapy (Table 1) and, as discussed later, the number of cycles trial, possibly including azacitidine (or decitabine) to azacitidine administered appears to be important in determining response to alone. epigenetic drugs, although the fewer cycles generally, given the Although there have been no randomized trials, it seems quite decitabine patients, may have been a result, rather than a cause, plausible that at least in older patients with AML (20–100% blasts) of relatively poor outcomes in some patients. results with azacitidine or decitabine rival, not only those with The control group in the azacitidine trials consisted of BSC, LDAC but those with ‘more intense’ therapies. For example, after LDAC or standard AML induction therapy (‘3 þ 7’). While accounting for prognostic covariates, although not the year of statistically significant differences were seen in favor of azacitidine treatment, Quintas-Cardama et al.30 reported similar OS among compared to BSC or LDAC, the number of patients receiving 3 þ 7 557 patients aged X65 treated with ara-C at doses of was almost certainly too small to detect statistical significance 1–2 g m À 2± or fludarabine±other drugs and 114 absent very great superiority in favor of azacitidine, although the patients given decitabine (n ¼ 67) or azacitidine (n ¼ 47) despite hazard ratios in favor of azacitidine were less impressive with 3 þ 7 higher CR rates with the more intense therapies (Figure 1).30 (0.76 in the MDS þ AML trial,23 0.97 in the AML trial25 than with Although the former drugs are undoubtedly more convenient BSC (0.58 and 0.48) or LDAC (0.36 and 0.37). The control group in than the latter, being able to be administered to outpatients, it is the decitabine trials received BSC in the high-risk MDS study22 and not clear that they can be viewed as ‘satisfactory’. either BSC or LDAC in the AML study, with 88% of the latter It is not clear how many of the patients the Quintas-Cardama SPOTLIGHT patients receiving LDAC. However, as noted by Ferrara and et al. study30 received HCT after the initial therapy. HCT is thought Musto,27 the LDAC dose employed (20 mg m À 2 once daily  10 to be the therapy most likely to cure high-risk MDS or AML. days) was lower than that used (20 mg m À 2 daily  14 days) in the Accordingly, a French study examining the effect of azacitidine azacitidine studies, and this may have served to exaggerate the OS versus more intense induction chemotherapy on outcome after differences between decitabine and LDAC. Nonetheless, the subsequent HCT in 163 patients is of considerable interest.31 complete response (CR) rates (8%) were essentially identical with Ninety-eight patients received induction chemotherapy alone, 48 LDAC in the decitabine and azacitidine studies,23,24 although, as patients received azacitidine alone and 17 patients received both noted below, the relation between CR and OS is not necessarily as modalities, in either order. After accounting for age (median 57), obvious as once believed. This CR rate was less than that observed the IPSS score (58% intermediate 2 or high,26 cytogenetics, CR, (18%) with LDAC (20 mg twice daily  10 days) in the MRC’s AML partial response (PR) or marrow CR (mCR) versus none of these at 14 trial, eligibility for which was limited to ‘unfit’ older patients;28 time of HCT (such responses pre-HCT were seen in 78% with furthermore, although the Medical Research Council found that induction chemotherapy, 69% with azacitidine and 53% with half the patients receiving LDAC needed three to four courses to both), time from diagnosis to HCT and type of HCT (80% reduced achieve CR the median number of courses received in the intensity and 54% matched unrelated) OS was similar in the decitabine trial was only two. Nonetheless, median OS (5–6 azacitidine and induction chemotherapy groups and worse in the months) seemed approximately the same in the LDAC arm of the group that received both modalities pre-HCT (Figure 2),31 perhaps decitabine trial and in the LDAC arm of AML 14, recalling the latter because of unknown prognostic factors not detectable via was limited to unfit patients, whereas the 23% exclusion rate multivariate analysis. It seems likely, however, that azacitidine suggests this was unlikely the case in the decitabine trial.24 alone will be unable to substitute for HCT if OS is the aim. Thus, What then are the roles of decitabine and azacitidine in higher- although recognizing the risks of selection bias and limiting their risk MDS and AML? Randomized trials certainly suggest that multivariate analysis to patients aged 60–70 years with high-risk

& 2013 Macmillan Publishers Limited Leukemia (2013) 1803 – 1812 Epigenetics in clinical practice EH Estey 1806 de novo MDS or secondary AML, who either received HCT (n ¼ 105; with at least intermediate intensity conditioning in about half the cases after induction chemotherapy) or who received azacitidine but not HCT(n ¼ 75) because of lack of a donor or institutional guidelines, Platzbecker et al.32 for the German and French MDS Groups and the Cooperative Transplant Study Group found that after accounting for performance status, cytogenetics,time from diagnosis and blast %, HCT was associated with superior OS than azacitidine without HCT, with the difference in OS becoming apparent only from 1 year after initiation of treatment (2 year OS 39 vs 23%).

CLINICAL PREDICTORS OF OUTCOME WITH AZACITIDINE OR DECITABINE Are there patients who might have results better than the averages described above? Efforts to identify such patients have examined both clinical and basic parameters. Analyzing 282 consecutive patients with higher-risk MDS (of whom 22% had ‘AML’) given azacitidine in a compassionate use program and verifying their conclusions using an independent data set, Itzykson et al.33 found that intermediate and poor-risk cytogenetics,26 performance status Z2, circulating blasts and requirement for at

SPOTLIGHT least four units of RBCs/8 weeks were independent predictors of shorter OS and could be used to define ‘low-’, ‘intermediate-’ and ‘high-risk’ groups with median OS of not reached, 15 and 6 months, respectively. However, only 11% of patients were in the best group (15% in the validation population). Combining 163 patients, 50% original IPSS intermediate 2 or high,26 treated in two decitabine studies.12,17 Jabbour et al.34 noted that and/or 7 abnormalities and low hemoglobin and platelet levels were independently associated with shorter OS and concluded Figure 1. Shown are the OS (a) and relapse-free survival (b)of that the factors for poor outcome with decitabine and with MDS patients treated with standard intensive AML chemotherapy 30 in general were the same. A similar conclusion was reached (Chemo) or with epigenetic therapy. when testing the value of the IPSS-R in patients treated with

Figure 2. Kaplan–Meier estimates of (a) 3-year OS, (b) 3-year event-free survival, (c) cumulative incidence of 3-year relapse and (d) nonrelapse mortality (NRM) in 163 patients, according to the before-transplantation treatment received. Abbreviations: AZA, azacitidine; HR, hazard ratio; ICT, induction chemotherapy; ns, not significant.31

Leukemia (2013) 1803 – 1812 & 2013 Macmillan Publishers Limited Epigenetics in clinical practice EH Estey 1807 azacitidine,35 and heterogeneity in benefit in different subgroups needed to achieve a given response. A landmark analysis was has not been reported when comparing azacitidine with LDAC or performed in a French azacitidine compassionate use program for BSC,23,25 or compare azacitidine/decitabine with more ‘intensive’ patients with AML by the World Health Organization criteria AML regimens.30 (420% blasts). The 138 patients, median age 73 years and 50% Post-treatment predictors include number of courses adminis- with ‘unfavorable’ cytogenetics had a CR rate of 14% but an tered and response to initial courses. It is often felt that patients overall response rate (including CR, CRp, PR and HI) of 54%.38 should receive at least six cycles of treatment before being CR, PR and mCR on the one hand and HI on the other were each declared resistant to azacitidine or decitabine. However, Steensma associated with 1 year OS of 55% versus 19% for no response.38 et al.17 noted that with the commonly employed 20 mg m À 2 Using time-dependent covariates, Gore et al.39 found that even daily  5 decitabine schedule 82% of 50 patients who achieved stable, rather than progressive, disease improved OS. Thus, the CR, mCR or ‘hematologic improvement’ (HI)36 had shown an initial need for CR to obtain longer OS, seen with traditional anti-AML response after two cycles and 90% after three cycles17 (Figure 3). therapy,40 is unlikely to apply with azacitidine and decitabine; the Likewise, in the Fenaux et al. trial,23 median time to first response same may apply with regard to survival beyond 2 years.41 Whether (CR, PR and HI) was two cycles, with 80% of first responses seen by these relations would apply in patients who subsequently receive four cycles (Figure 4).33 Firty-eight percent of patients who achieve allogeneic HCT, which would often employ reduced intensity such a first response with azacitidine have been found to proceed conditioning remains to be seen; particularly, given the general to have a better response after a median of another 3.5 cycles.37 reluctance to perform reduced intensity conditioning –HCT in The relation between courses needed to achieve the first or best patients with 45% marrow blasts. response and subsequent OS or duration of response seems worthy of further investigation, and given their shorter OS the question of cycles needed to properly evaluate azacitidine or BIOLOGIC PREDICTORS OF RESPONSE TO EPIGENETIC decitabine is considerably more consequential in higher-risk MDS THERAPY or AML than in lower-risk MDS. DNMT3a and TET 2 A related issue is whether the type of response (CR, CRp PR, DNMT and TET2 have important roles in epigenetic pathways.1,2 mCR, HI, stable disease with mCR and HI defined originally for Metzler et al.42 found that 6/8 patients with DNMT3a mutations MDS)36 affects outcomes presumably of more interest to patients, (5 single and 3 together with NPM1 mutations) achieved CR with a such as OS or the duration of freedom from transfusions. Proper 10-day schedule of decitabine (described below) versus 13/38 analysis of the former of course needs a ‘landmark’ analysis or one without these mutations.42 Higher levels of microRNA (miR)-29b, incorporating time-dependent covariates to account for the time which decrease levels of messenger RNA for the DNMT3a gene, also correlated with better response to decitabine.42 TET2 mutations did not affect response to this decitabine schedule, although such mutations were associated with a higher CR þ PR þ mCR rate (9/13 vs 23/70) in patients given a standard azacitidine regimen.43

Decitabine triphosphate Decitabine must be converted to its triphosphate (DAC-TP) to become active. Assays have been developed to measure DAC-TP concentrations in marrow or blood mononuclear cells obtained from patients before and after treatment with decitabine. Two reports, one in seven, and the other in three patients, have found higher DAC-TP levels in responders.44,45

DNA methylation Despite their description as ‘hypomethylating agents’, it has

proven difficult to consistently demonstrate correlations between SPOTLIGHT response to azacitidine or decitabine and pre- or post-treatment Figure 3. Time to first response and best response by cycle (n ¼ 50). DNA methylation status. The proclivity of different authors to Abbreviations: CR, complete response; mCR, marrow CR; PR, partial analyze different genes complicates such attempts. A few response; HI, hematologic improvement.17 examples will suffice. Selecting 10 genes to analyze Shen et al.46 found no correlation between their pre-treatment methylation status and response to decitabine on the 15 m À 2 TID Â 3 days Q 4-week schedule.22 However, methylation averaged over multiple time points after therapy decreased more in patients given decitabine than BSC, and, in decitabine-treated patients, more with CR or PR than with HI, and more with HI than with stable or progressive disease. Examining phosphoinositide–phospholipase b1C (PI-PLb1C), a gene thought important in MDS evolution to AML, but this not examined by Shen et al., 46 Follo et al.47 found a decrease in methylation and/or increase in PI-PL C b 1 expression in 10/10 high-risk MDS patients achieving CR or HI with azacitidine but in 0/8 nonresponders. Ettou et al.48 found that low expression of FAS (a pro-apoptotic protein) at diagnosis, reflecting hypermethylation of the Fas gene promoter, correlated with response rate (CR þ PR þ HI) to azacitidine independently of age, Figure 4. Time to first response CR, PR and HI in patients who IPSS, or prior therapy, although the derivation of the cutpoint used achieved a response during treatment with azacitidine is shown.37 to define low Fas expression is not obvious, nor did Fas expression

& 2013 Macmillan Publishers Limited Leukemia (2013) 1803 – 1812 Epigenetics in clinical practice EH Estey 1808 correlate with OS after azacitidine. An increase in Fas expression inhibitor included phenylbutyrate,56 valproic acid (±ATRA,57,58 after azacitidine correlated with response.48 However, in patients MGCD0103 (Garcia-Manero et al.59) and etinostat.60 With the given azacitidine þ the histone deaetylase inhibitor etinostat and exception of the last study, these have been single-arm trials and sequentially analyzing, during cycle 1, four presumed tumor the number of patients treated has been too small, and their suppressor genes, not including Fas but including two genes that prognoses too heterogeneous to allow a meaningful comparison were studied by Shen et al.,46 Fandy et al.49 could find no relation with single-agent azacitidine or decitabine. Prebet et al.60 between clinical response, baseline methylation, or changes in randomized 150 patients (65% MDS and 35% AML) to 10 days methylation or gene expression in any of the four genes in either of azacitidine ±etinostat. Although the 10-day schedule seemed the bulk or the CD34 þ population despite induction of histone superior to a 7-day schedule, addition of etinostat had no effect acetylation. Klco et al.50 used a novel means to expand primary on response rate or OS (18 months without and 13 months with AML cells in culture and studied a vast number of genes etinostat). Although one group found a relation between changes 3 days after treatment with decitabine in clinically relevant in p15 methylation and response,56 others also examining the p15 concentrations, noting that longer exposure, as often done, results gene did not.57,58 in differentiation and resultant changes in methylation and expression independent of decitabine. They found only limited Azacitidine þ lenalidomide correlation between decitabine-induced changes in methylation 61 and gene expression. Rather than affecting a specific group of Sekeres et al. gave 36 patients, median age 68 years, with genes, in particular those most commonly monitored after higher-risk MDS (87% with original IPSS intermediate 2 or high, the remainder with 5–9% blasts, none with AML as defined by the decitabine or azacitidine treatment, decitabine affected a range À 2 of genes, typically those most methylated pre-treatment. The World Health Organization) azacitidine 75 mg m daily days authors conclude that ‘the mechanism of action of decitabine is 1–5 þ lenalidomide 10 mg daily days 1–10 repeated every 28 days.61 The median number of cycles was five contrasted with more complex than previously recognized’. The implications for 23 rational development of epigenetic drugs are obvious. nine in the Fenaux et al. study, which produced similar results in patients with high-risk MDS and those with 20–30% blast AML.23,25 Although Sekeres et al.61 observed a CR rate of 44% versus 17% in SPOTLIGHT 23 NEWER DIRECTIONS: EXTENDED DURATION DECITABINE AND the Fenaux et al. study, median OS was less (13 vs 22 months), AZACITIDINE again pointing out the possible disconnect between CR and OS, and stressing the need for the planned North American Although decitabine has generally been given at 20 mg m À 2 daily 51 randomized trial comparing azcitidine±lenalidomide in MDS/ for 5 days, Blum et al. extended the duration to 10 days in 53 Pollyea et al.62 gave 42 patients aged X60 years (median 74 years) patients with untreated AML, median age 74 years. The CR rate with untreated AML azacitidine 75 mg m À 2 daily  7 followed on was 47% and adding in mCRs gave a response rate of 64%. Also, 52 day 8 by escalating doses of lenalidomide (5, 10, 25, 50 mg)  21 using a 10-day schedule, Ritchie et al. reported a CR rate of 40%. days with cycles repeated every 42 days.62 The 50-mg dose was Although median OS was only about 1 year in the Blum et al. 51 52 considered the maximum tolerated dose and patients received a study and 10 months in the Ritchie et al. study, these results median of five cycles. The CR rate was 19% and the CR þ CRi þ PR seem superior to those obtained in the Kantarjian et al.24 À 2 rate 41%. Although OS did not differ according to CR þ CRi versuss randomized trial using 20 mg m daily  5. All these patients PR, and was not worse in patients with ‘unfavorable’ prognosis are typically described as having ‘poor prognostic features’. according to the European Leukemia Net Classification,63 the Nonetheless, it is not implausible that various biases might have median OS was only 20 weeks versus 40 weeks in the French been operating in each study, emphasizing the need for a compassionate azacitidine program for 138 patients (median age randomized trial comparing the 5- and 10-day schedules. The 38 53 73 years) with untreated AML. It is similarly unclear whether the same applies to reports in Prebet et al. that a 10-day schedule of 1 CR, 1 PR and 3 CRi seen in 20 patients with high-risk MDS or AML azacitidine is superior to the conventional 7-day schedule. together with del5q and generally a complex karyotype, who received azacitidine 75 mg m À 2 daily  5 days and lenalidomide in escalating doses (10–25 mg daily) days 6–19 (Platzbecker NEWER DIRECTIONS—‘SECOND-GENERATION’ et al.64) is superior to what might have occurred with HYPOMETHYLATING AGENTS lenalidomide 10 mg daily alone, given a report of a response SGI-110 is a dinucleotide of decitabine and deoxyguanosine that is rate of 27% in 47 patients with high-risk MDS in such patients given s.c. It was administered on either a weekly or daily  5 Ades et al.65 schedule to 78 patients (64 AML and 14 MDS).54 Assuming a relation between hypomethylation and clinical outcome, the Decitabine biologically effective dose was declared to be 60 mg m À 2 þ The association between higher levels of miR-29b and response to daily  5, at which hypomethylation reached a plateau of 25%, 51 which compared ‘favorably’ with that seen with 20 mg m À 2 a 10-day course of decitabine, and the ability of bortezomib to induce miR-29b expression motivated combination of these drugs decitabine given intravenous. All responses (2CR, 1 CRp, 1 CRi in 66 AML, 1 mCR and 1 HI in MDS patients previously given in AML. Although the number of patients treated was small azacitidine), occurred in patients with 410% hypomethylation. (10 untreated and 9 relapsed/refractory), there was a trend to The s.c. exposure resulted in a prolonged half-life compared with increased miR-29b expression post treatment. Neurotoxicity due that seen with intravenous decitabine (2.4 vs 0.6 h). to bortezomib occurred with repetitive cycles. A phase 2 trial randomizing patients to decitabine±bortezomib is underway.

NEWER DIRECTIONS: COMBINATION THERAPY Combinations with AML-type therapy Combinations of epigenetic drugs This is likely to be an area of increasing interest. To date, The possibility of additive or synergistic effects resulting from most of the trials have been small, intended to establish combined epigenetic therapy (see, for example, Follo et al.55) has ‘feasibility’ with limited comparison with other therapy. I will motivated numerous studies examining this possibility. These discuss two studies. Garcia-Manero et al.67 administered generally have involved addition of inhibitors 500 mg tid for 3 days and then gave idarubicin þ ara-C at standard to azacitidine or decitabine. The investigated histone deacetylase MD Anderson doses (idarubicin + ara-C: 12 mg m À 1 daily days

Leukemia (2013) 1803 – 1812 & 2013 Macmillan Publishers Limited Epigenetics in clinical practice EH Estey 1809 4–6, 1.5 g m À 2 daily  3–4 days by continuous infusion). Seventy- AML effect by increasing the immunogenicity of AML blasts71 or five newly diagnosed patients aged o65 years were treated and demethylating KIR regions on donor natural killer cells,72 and CR rate was 76% (85% CR þ CRp) and median OS was 82 weeks.67 (b) suppress acute graft versus host disease by increasing As part of an effort to identify the ‘experimental’ arm for the next US numbers of regulatory T cells73,74 and reducing production of intergroup phase 3 trial in patients aged o65 years, who do not proinflammatory cytokines.75 have core-binding factor or FLT3ITD þ AML, a multivariate analysis, Schroeder et al.76 gave azacitidine (100 mg m À 2 daily  5 days accounting for age, cytogenetics and de novo versus secondary every 28 days) with donor lymphocyte infusions on day 6 of cycles AML, was undertaken and identified IA þ vorinostat as associated 2, 4 and 6 as first salvage therapy to 30 patients with relapsed with a higher CR rate than IA without vorinostat, as well as several (median 34% blasts) AML (n ¼ 28) or MDS after HCT (reduced other single arm regimens (Othus, personal communication). intensity in 26 patients). CR rate was 23% and CR þ PR rate was Accordingly, IA þ vorinostat will be the experimental induction 30% with median time to best response three cycles and median arm to be compared with IA and 3 þ 7 in this phase 3 trial. OS 117 days.77 The authors noted that this outcome was not Scandura et al.68 gave decitabine for varying times immediately obviously different than what might be expected in similar before standard 3 þ 7 therapy and reported a CR rate of 25/30 patients. However, they also commented that the rates of acute (with 8 of the CRs occurring with the patients’ next therapy) in a graft versus host disease and of grade 2–4 acute graft versus host population in which 83% had at least one ‘high-risk’ feature; disease (37% and 17%) were lower than typically reported, median OS had yet to be reached (lower bound of 95% perhaps reflecting an increase in regulatory T cells, particularly in confidence interval, 15 months). patients with early relapse. Perhaps of more interest are studies in patients with less apparent disease. de Lima et al.77 administered four cycles of s.c. NEWER DIRECTIONS—USE IN PATIENTS IN REMISSION azacitidine at varying doses  5 days starting on day 43 after OR AFTER HCT reduced intensity HCT, provided the patient was in CR on day 30. The discussion so far has focused on patients with ‘active disease’. Thirty patients had active AML or high-risk MDS at the time of HCT However, it is quite plausible that agents that are relatively and 15 were in CR1, but at high risk of relapse. The ‘optimal’ dose ineffective in this setting will be more active in patients with less was 32 mg m À 2 given for four cycles with thrombopenia dose obvious disease. With this in mind, the US Alliance (formerly limiting. With median 20.5 months follow-up, 1-year event-free CALGB) gave decitabine (20 mg m À 2 daily  4–5 days every 6 survival (58%) and OS (77%) were felt to warrant a currently weeks for a planned eight cycles) to AML patients who had ongoing randomized trial. A better indicator of the potential completed post remission therapy (three cycles of ‘high-dose’ ara- efficacy of this approach comes from a study of Platzbecker et al.78 C or an autologous transplant) and were within 90 days of their They had previously established that a loss of CD34 þ donor last therapy. One hundred and thirty four out of 412 CR patients chimerism below 80% after HCT was a very strong predictor of received decitabine, with 75% of the 134 receiving at least 4 relapse after a median of 2 months, prompting the administration cycles. Although it is not clear whether the 134 were representa- of four cycles of azacitidine 75 mg m À 2 daily  7 every 4 weeks to tive of the 412, OS and disease-free survival from registration were 20 patients with such loss of donor chimerism. Ten patients had similar to that observed in a previous Alliance study in which an increase in donor chimerism to 480%; four of these remain in patients, given identical induction and post-remission therapy, remission at median of 11 months and the remaining six relapsed received no further therapy or interleukin-2 (Blum et al.69). at a median of 12 months. Three out of ten patients who did not Boumber et al. randomized 45 patients (35 in first, 10 in second achieve an increase in donor chimerism to 480% remain in and CR or CRp) between decitabine at 20 mg m À 2 daily  5 every remission at a median of 10 months, with the remaining seven 4–8 weeks (median number of cycles ¼ 4.5) and either observa- relapsing but at a median of only 6 months. Thus, it seems fair to tion, low-dose ara-C, or ‘intensive’ chemotherapy.70 Multivariate infer that azacitidine delayed relapse compared with historical analysis found that presence of minimal residual disease at time of data with perhaps more delay in patients in whom donor randomization reduced OS and disease-free survival, whereas chimerism increased 480%. treatment with decitabine had no effect. Although the number of patients randomized may have been too small to detect any but very powerful decitabine effects, it appears fair to say that, as SUMMARY often the case, disease characteristics (here minimal residual There is no doubt that azacitidine and decitabine, the two most SPOTLIGHT disease) dominate treatment as predictors of outcome. prominent epigenetic drugs, have altered the therapeutic land- There are considerable pre-clinical data, suggesting that scape in MDS and AML. However, as often the case, the very epigenetic drugs, particularly azacitidine, might be effective in success of a therapy raises further questions (Table 2). Beginning the post-HCT setting due to its ability to (a) augment graft versus with perhaps the most mundane, are decitabine and azacitidine

Table 2. Questions possibly worthy of further exploration19,21

In lower-risk MDS In higher-risk MDS or AML General questions

Are 5 and 7 day Are 10 days of decitabine superior to 5, or 10 days of Are decitabine and azacitidine interchangeable? schedules of azacitidine azacitidine superior to 7? equivalent? What is role of oral What is role for combination with other ‘epigenetic Can clinical marker(s) be found to permit earlier azacitidine? drugs’, lenalidomide, bortezomib or more traditional AML discontinuation in patients unlikely to respond to therapy? continued therapy? Might epigenetic drugs be more relatively effective in CR How much of the activity of hypomethylating drugs post HCT than in active disease? results from hypomethylation? Abbreviations: AML, acute myeloid leukemia; MDS, myelodysplasia; CR, complete response; HCT, hematopoietic cell transplant.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1803 – 1812 Epigenetics in clinical practice EH Estey 1810 19 interchangeable? Some patients receive the former, others elderly patients with myelodyspastic syndromes (MDS) in the Avida registry in a the latter, but the reasons for this choice do not appear totally community setting. Haematologica 2010; 95: s2. based on comparative data. In lower-risk MDS patients, the role of 15 Lyons RM, Cosgriff TM, Modi SS, Gersh RH, Hainsworth JD, Cohn AL et al. oral azacitidine awaits definition and there is uncertainty as to the Hematologic response to three alternative dosing schedules of azacitidine merits of a 5 day, permitting weekends off, versus a 7-day in patients with myelodysplastic syndromes. JClinOncol2009; 27: schedule of the parenteral drug, again reflecting a dearth of 1850–1856. comparative data. Given the short OS of patients with higher-risk 16Garcia-ManeroG,GoreSD,CogleC,WardR,ShiT,MacbethKJet al. MDS or AML questions here, such as the role of 10 days Phase I study of oral azacitidine in myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia. JClinOncol2011; 29: (or perhaps longer) of administration and of combinations with 2521–2527. other active drugs, are in need of quicker resolution. Although 17 Steensma DP, Baer MR, Slack JL, Buckstein R, Godley LA, Garcia-Manero G et al. understandably enough, emphasis to date has been on patients Multicenter study of decitabine administered daily for 5 days every 4 weeks to with frank MDS or AML, are there situations that would inherently adults with myelodysplastic syndromes: the alternative dosing for outpatient lend themselves to more successful use of epigenetic therapies, treatment (ADOPT) trial. JCO 2009 3842–3848. for example, in remission after HCT? Identification of clinical 18 Estey E. Fairest of them all for myelodysplastic syndromes. Blood 2008; 111:6. indicators of early response would spare patients from continuing 19 Lee YG, Kim I, Yoon SS, Park S, Cheong JW, Min YH et al. Comparative analysis therapy with only a small chance of success. Best would be between azacitidine and decitabine for the treatment of myelodysplastic biologic markers to guide therapy. Currently, there appears to be syndromes. Br J Haematol 2013; 161: 339–347. 20 Flotho C, Claus R, Batz C, Schneider M, Sandrock I, Ihde S et al. The DNA uncertainty as to how much of the activity of hypomethylating 49–50 methyltransferase inhibitors azacitidine, decitabine and zebularine exert agents owes to hypomethylation. The view that they differential effects on cancer gene expression in acute myeloid leukemia cells. merely allow re-expression of tumor suppressor genes may be Leukemia 2009; 23: 1019–1028. an oversimplification. Although a complex task, understanding 21 Hollenbach PW, Nguyen AN, Brady H, Williams M, Ning Y, Richard N et al. how these drugs work appears fundamental to their rational A comparison of azacitidine and decitabine activities in acute myeloid leukemia development. cell lines. PLoS One 2010; 5: e9001. 22 Lubbert M, Suciu S, Baila L, Ruter BH, Platzbecker U, Giagounidis A et al. Low-dose decitabine versus best supportive care in elderly patients with intermediate- or SPOTLIGHT high-risk (MDS) ineligible for intensive chemotherapy: CONFLICT OF INTEREST final results of the randomized phase III study of the European Organisation for The author declares no conflict of interest. Research and Leukemia Group and the German MDS Study Group. J Clin Oncol 2011; 29: 1987–1996. 23 Fenaux P, Mufti GJ, Hellstrom-Lindberg E, Santini V, Finelli C, Giagounidis A et al. 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