ALBERT

Description: The suppressor of cytokine signaling (SOCS) family of proteins has been implicated in the negative regulation of several cytokine pathways, particularly the receptor-associated tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathways of transcriptional activation. SOCS-1 (also known as JAB and SSI-1) inhibits signaling by many cytokines. Because of the previously observed hypermethylation-associated inactivation of SOCS-1in hepatocellular carcinoma and the critical role of interleukin-6 (IL-6) as a survival factor in multiple myeloma (MM), we examined CpG island methylation of the SOCS-1 gene in MM cell lines and primary MM samples. Aberrant SOCS-1methylation was found in the IL-6–dependent MM cell lines U266 and XG1, which correlated with transcriptional silencing. Treatment of these cell lines with the demethylating agent 5-aza-2′-deoxycytidine (DAC) up-regulated SOCS-1 expression. Methylation-associated inactivation of SOCS-1 in hematopoietic cell lines correlated with greater sensitivity to the chemical JAK inhibitor AG490. Using methylation-specific polymerase chain reaction (MSP), we found that SOCS-1 is hypermethylated in 62.9% (23/35) of MM patient samples. In contrast, methylation analysis of malignant lymphomas of various histologies revealed SOCS-1 hypermethylation in only 3.2% (2/62), and there was no methylation of SOCS-1 in normal peripheral blood leukocytes or bone marrow cells. We conclude thatSOCS-1 is frequently inactivated by hypermethylation in MM patients. Silencing of the SOCS-1 gene may impair negative regulation of the Jak/STAT pathway and therefore result in greater responsiveness to cytokines, thus supporting survival and expansion of MM cells.

Description: Background: Loss of the long arm of chromosome (Ch) 5 or complete loss of Ch 5 is frequent in de novo MDS and AML. Epigenetic modifications of tumor suppressor genes, including aberrant DNA methylation, may play an important role in the progression of MDS and AML. Cell signaling is regulated by a-catenin, which forms a trimolecular complex with E-cadherin (ECAD) and b-catenin that links with actin-containing filaments of the cytoskeleton. Studies have reported reduced expression of a-catenin in MDS and AML patients with 5q deletion as compared to those without 5q deletion. Whether promoter methylation of the a-catenin gene is responsible for this decreased expression is controversial. To explore the potential role of a-catenin in the pathogenesis of AML transformation, we (a) determined the tumor specificity of methylation in AML and non- myeloid malignancies and (b) frequency of methylation in patients with −5/del(5q) MDS/AML and those with normal cytogenetics; (c) performed bioinformatics and experimental analysis of the a-catenin adhesion complex and local 5q31.1 genes to investigate mechanisms of epigenetic silencing; and (d) performed a detailed analysis of primary AML samples correlating promoter methylation, a-catenin expression, and chromatin conformation. Methods and Results: Using methylation sensitive PCR, we found that methylation of the a-catenin promoter gene was specific for myeloid malignancy. No methylation was observed in 19 acute lymphocytic leukemia cases, 20 chronic myelogenous leukemia cases, or in 99 primary gastric and esophageal samples where a-catenin has been implicated as a tumor suppressor gene. In those patients with AML and an associated 5q deletion the frequency was 31% (8/26) as compared to those without a 5q deletion with 13% (16/120). Bioinformatics analyses of the Valk et al., 2004 leukemia database provide supportive data for under expression of a-catenin in non-5/del (5q) AML cases. We quantitated a-catenin mRNA expression by Q-PCR in our cohort. Expression was lowest in AML patients with a-catenin methylation (n=9), but also in a subset of patients without promoter methylation (n=17), suggesting alternative mechanisms of inactivation. In contrast to AML, methylation of a-catenin was rare in myelodysplastic syndrome (MDS). Although p15 was methylated in over 50% of these cases as a positive control, only 2/18 MDS cases with 5q deletion (11%) and 1/13 MDS cases with 5q intact (8%) were methylated at the a-catenin promoter. The three positive cases were RAEB-2 (1) or RAEB-t (2), suggesting that a-catenin methylation may be most important in promoting transformation from MDS to AML. To explain a potential lack of correlation of methylation with decreased a-catenin in MDS and AML, we investigated a-catenin chromatin in a myeloid stem cell progression model and in primary AML samples. We performed chromatin immunoprecipitation on the CTNNA1 promoter using two active chromatin histone marks, H3K9Ac and H3K4me2 and two inactive marks, H3K9me2 and H3K27me3. In cell lines and primary leukemia samples where a-catenin was highly expressed (N=4), activation marks were present and repression marks absent. In cell lines and primary samples with low a-catenin expression and methylation of the promoter (N=4), the opposite pattern was observed. So called “bivalent” chromatin with mixed marks, no a-catenin methylation, and intermediated mRNA expression levels were observed with 7 additional cases (2 cell lines, 5 primary AML). Conclusions: Our data indicate that methylation of a-catenin is common in AML patients with 5q deletion but also observed in cases with normal chromosome 5 copy number. We propose a model of progressive inactivation of the a-catenin locus with AML transformation, with methylation representing a late stage event. The tissue-specificity of our results and in vivo chromatin observations in primary AML samples have implications for the timing and combinatorial therapy of MDS/AML with HDAC inhibitors and methylation inhibitors. Additionally, it appears that inactivation of adhesion molecules (ECAD, a-catenin) are frequent events overall in AML, suggesting a new pathway of transformation from MDS to AML.

Description: Abstract 5089 Background: Multiple myeloma (MM) is a B-cell neoplasm characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene expression. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP2) gene as a tumor suppressor. Overexpression of TIMP2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP2 in primary MM samples and investigated correlations between TIMP2 methylation and clinical parameters. Methods: Methylation of the promoter-associated CpG island of TIMP2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 76 MM patients (median age 65 years [range 40–94]; 44 males, 32 females; 71 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher's exact two-sided test and independent t-test, respectively. Results: MSP analysis revealed that there was aberrant methylation of the TIMP2 promoter region in 4/76 MM patient samples. We found an association of TIMP2 hypermethylation with plasma cell leukemia (p

Description: Introduction: In older patients with AML in whom conventional chemotherapy is not indicated (comorbidities, performance status, poor cytogenetics etc.), treatment with low-dose azanucleoside DNA demethylating agents may be a less intensive alternative. In MDS, these drugs need to be administered over a prolonged time period in order to gain full benefit, since delayed responses are common, and thus occur not infrequently after more than 6 months of treatment. In a phase II multicenter trial (00331) of low-dose decitabine (DAC, 135 mg/m2 administered over 3 days in 9 intravenous 3-hour infusions, with 15 mg/m2/infusion, repeated every 6 weeks) patients benefiting from this treatment were offered an outpatient maintenance with the drug given at an even lower dose and as one-hour infusions on 3 consecutive days. Patients and Methods: Patients having completed 4 courses of DAC according to the study protocol and being in complete or partial remission (CR, PR), having achieved an antileukemic affect or stable disease were offered continued DAC treatment with 20 mg/m2 given intravenously over one hour on 3 consecutive days, repeated every 6 to 8 weeks. Maintenance treatment was continued until relapse or progression. Results: Of the 235 patients included in the 00331 study, 57 (25%) received the 3-day DAC maintenance. Median age of these 57 patients before study inclusion was 71 years (range 60 – 81), the median white blood count 4200/μl (range 0.2–285,000/μl), 59% had preceding MDS, with a median of 16 months duration. Performance status before initial treatment with DAC: ECOG 0, 1 and 2 in 29%, 58% and 13% of the patients, respectively. 60% had intermediate-risk cytogenetics, 28 % poor-risk cytogenetics, no metaphases were obtained or cytogenetics not done in 12 %. Remission status at maintenance start, i.e. response to 4 courses of DAC, was CR+PR in 71 % of the patients. A median number of 4 maintenance courses was administered (range, 1–16), with 24 patients (42 %) receiving 5 or more courses. Treatment was overall very well tolerated, with dose reductions because of cytopenia necessary in

Description: Interstitial loss of the long arm of chromosome(Ch) 5 or complete loss of Ch 5 is seen in de novo MDS and AML, and more frequently in MDS and AML arising after previous cancer treatments with alkylating agents or radiotherapy. The recurrent nature of these chromosomal deletions suggests that 5q contains tumor suppressor gene(s) important to hematological transformation. Although the common deleted 5q31 region has been delineated for some time, no genes have been identified that explain the poor prognosis of these patients. While tumor suppressor genes altered in solid tumors are infrequently mutated in MDS or AML, several studies indicate that epigenetic changes, including aberrant DNA methylation, can also play important roles in the progression of MDS or AML. Two demethylating agents have been approved by the FDA for the treatment of MDS, further providing support for the involvement of DNA methylation changes in this disease. In this work we have analyzed previously implicated Ch 5 tumor suppressor genes for epigenetic silencing in the myeloid malignancies. Genes analyzed for hypermethylation were a-catenin, Egr1, Smad5, and IRF1 within the minimally deleted 5q31 region, and NPM1 at 5q35.1. Methylation of a-catenin was found in myeloid cell lines associated with lack of gene expression and its expression restored with 5-aza-2′-deoxycytidine. No methylation of the other four 5q genes was detected in MDS and AML. No a-catenin methylation was found in mononuclear cells from 15 normal healthy individuals. In 27 MDS (15 with 5q deletion) and 140 AML cases tested (26 with 5q deletion), we found that methylation of a-catenin is common and more frequent in AML patients with 5q deletion (50%) than those without 5q deletion (16%), a-catenin methylation also occurs more frequently in MDS/AML patients (37%) than in AML patients without preexisting MDS (14%). Although methylation of a-catenin was found more frequently in AML patients with an unfavorable karyotype, secondary AML or in patients 〉60 years of age, in multivariate analysis only 5q deletion was associated with methylation of a-catenin. We also examined other hematological malignancies including CLL, CML, ALL. Methylation was observed only in two CLL patients of these 40 cases. Surprisingly, since a-catenin has been identified as under expressed in MDS (Liu et al., 2007), and 5q loss is a common occurrence in MDS, methylation of a-catenin was not found in 5q deletion or 5q intact MDS patients, with the exception of two case of RAEB-t (currently reclassified by WHO as AML). The methylation frequency of a-catenin was frequent in leukemia with p15 methylation, but and was also observed in those with CDH1 methylation, although these trends did not reach statistical significance. These results suggest that a-catenin methylation may be most important in promoting transformation from MDS to AML. Our data indicate that methylation of a-catenin is very common in AML patients with 5q deletion and in AML patients progressing from MDS. a-catenin methylation is correlated with unfavorable karyotype and poor prognosis. The accumulation of epigenetic events affecting genes which are involved in regulating cell cycle inhibition and cell adhesion may contribute to the progression of AML.

Description: Chronic idiopathic myelofibrosis (CIMF) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, angiogenesis and extramedullary hematopoiesis. No specific genetic defect underlying the disease has been identified so far. The spectrum of cytogenetic abnormalities in CIMF includes del (13q), del (20q) and partial trisomy 1q as well as abnormalities of chromosomes 1, 7 and 9. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function in cancerogenesis. In order to investigate the role of DNA methylation changes in the pathogenesis of CIMF, we have analyzed the methylation status of the promoter-associated CpG islands of 13 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in peripheral blood cells from 20 adult patients with CIMF. The frequency of aberrant methylation among the patient samples was 25.0 % (5/20) for SOCS-1 and 5.0 % (1/20) for E-cadherin, MGMT, p73 as well as TIMP-2. There was no hypermethylation of p15, p16, RARbeta, DAP kinase 1, SOCS-3, hMLH1, TIMP-3 and RASSF1A. We detected at least one hypermethylated gene promoter region in 35.0 % (7/20) of the primary patient samples. Our data indicate that hypermethylation of tumor suppressor genes is a common event in CIMF. Epigenetic modification of genes regulating growth factor signaling, cell adhesion and DNA repair may, in addition to genetic aberrations, contribute to the malignant phenotype in CIMF. The exploration of our growing knowledge about epigenetic aberrations in tumorigenesis may help develop novel strategies in diagnosis and treatment of CIMF for the future.

Description: Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.

Description: Abstract 2185 Background: Secondary (s)AML from MDS is more frequent in older AML patients, and associated with an overall worse outcome with standard chemotherapy than de novo AML, particularly after MDS of longer duration (1). The azanucleoside hypomethylating agents 5-azacytidine (Vidaza) and 5-aza-2′-deoxycytidine (Decitabine, DAC) are active in MDS and, as recently shown, also AML. Compared to other predictors of response to these drugs, MDS duration prior to treatment thus far has received only limited attention, with two recent publications reporting conflicting results (2, 3). To independently validate our finding that shorter duration of MDS prior to DAC treatment may be a novel predictor of poor outcome (2, 4), we now applied this parameter to a large trial of low-dose DAC in AML pts (aged 〉60 years and judged ineligible for standard induction chemotherapy), about half of them with sAML from MDS with variable disease duration. Patients and Methods: Comparisons of response rate (RR, i.e. CR or PR) and overall survival (OS) from start of treatment according to MDS duration (pre-specified categorization according to quartiles) were performed post-hoc in 109 patients (pts) with previously untreated sAML (median age 72 years) treated with DAC (given over 72 hours, every 6 weeks, for up to 4 courses, followed by “maintenance” with 3 daily 1-hour infusions of DAC 20 mg/m2 every 4–6-weeks). Median WBC prior to treatment was 5.200/μl, median serum LDH 279U/l, 31.2% of pts had adverse cytogenetics, 82.6% had a performance status 〉 1, and 80.7% had a comorbidity index (HCT-CI) 〉=1. Comparisons by logistic regression and Cox regression (univariate and multivariate, adjusted for other prognostic factors showing an effect in this population of sAML pts) were performed. Results: Of the 227 AML patients treated within the 00331 trial, 109 (48%) had prior MDS with known MDS duration, with a median duration of 8 (25% quartile 3, 75% quartile 25, range 1–101) mths. The overall RR in these pts was 26/109 (24%), the overall 1 yr OS rate was 31% (94 deaths). A comparison of RR according to MDS duration revealed a trend to an increase in RR with longer duration of MDS [=25 mths: 10/28 (36%), test for heterogeneity p=0.29, test for trend p=0.06]. Similarly, when OS from start of DAC was analyzed according to this parameter, for pts with previous MDS of longer duration there was a trend to better outcome [=25 mths: 46%, test for heterogeneity p=0.17, test for trend p=0.16]. When these analyses were adjusted for other prognostic factors showing an effect in this population of sAML pts (comorbidity index, sLDH with respect to RR, and performance status, comorbidity index, and white blood count with respect to OS), the results were similar (effect of MDS duration with respect to RR: test for heterogeneity p=0.35, test for trend p=0.06, and effect of MDS duration with respect to OS: test for heterogeneity p=0.04, test for trend p=0.11). Conclusion: In this large cohort of uniformly treated pts with sAML, MDS of longer duration appeared to be associated with a better outcome, even after adjusting for important other prognostic factors. These results are supported by a similar analysis of MDS pts randomized in the 06011 EORTC intergroup trial (which compares DAC to Best Supportive Care), where MDS patients with longer (〉=3 mths) disease duration prior to treatment also had better outcome (4). They warrant application of this discriminator in the evaluation also of other non-intensive AML treatment modalities. References 1. Estey et al., Blood 90:2969-77, 1997 2. Wijermans et al., Ann. Hematol. 84 Suppl 1:9-14, 2005 3. Kantarjian et al., Cancer 109:265-73, 2007 4. Lübbert, Suciu et al., Abstract submitted, ASH 2010 Disclosures: Off Label Use: decitabine is FDA-approved for treatment of MDS and AML with up to 30% blasts. In the present study, patients with AML and higher blast percentage were treated. Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Döhner: Pfizer: Research Funding.

Description: Abstract 4141 Medically non-fit AML patients (pts), often with adverse cytogenetics, have few therapeutic options. We performed a large phase II trial in untreated AML pts 〉60 years ineligible for induction. Primary endpoint was best response: complete (CR) or partial remission (PR) or an antileukemic effect (ALE, 〉25% bone marrow [BM] blast reduction). Secondary endpoints: overall survival (OS) and safety. DAC was given at 15 mg/m2 q8 hours on day 1-3, total 135 mg/m2, q6 weeks for up to 4 cycles, with ATRA (45 mg/m2/day p.o. for 28 days in cycle 2) to be administered to pts not attaining a CR or PR after course 1, i.e. those with ALE or stable disease (SD). Maintenance with 20 mg/m2 DAC i.v. over 1 hour on 3 days (total 60mg/m2, outpatient administration) q 6-8 weeks was offered to pts completing all 4 cycles. 227 pts were recruited (median age 72 yrs, range 56-86). 38% of pts were ≥75 yrs of age, 23% had a performance status of ECOG 〉 2, and by comorbidity scoring (HCT-CI) 80% of pts had at least one, and 36% had three or mor comorbidities. Adverse cytogenetics or preceding MDS were present in 34% and 54%, respectively. Median WBC before treatment was 4400/μl (range, 500-241 000, 〉50 000/μl in 10%). Median BM blasts were 55% (10-100). A median of 2 DAC cycles was given (range 1-4). 100 pts received ATRA during course 2, and 51 pts (out of 79 who completed 4 cycles) received a total of 306 DAC maintenance cycles (median 5, range 1-19). Best response was CR in 30 pts (13%) and PR in 29 pts (13%). An ALE occurred in 60 pts (26%), resulting in a 52% overall response rate. SD was seen in 57 pts (25%), 19 pts (8%) had progressive disease, 29 (13%) had early death and 3 pts (1%) were inevaluable for response. Median OS from start of treatment was 5.5 months (range, 0-57.5+), the 1-year survival 28% (95%CI: 22%,34%). Pts with adverse cytogenetics did not differ from those with non-adverse cytogenetics in CR+PR rate nor in median OS. The addition of ATRA to DAC during cycle 2 resulted in similar survival (from start of cycle 2) despite the initial modest response to DAC of these 100 pts. Toxicities of DAC (alone or in combination with ATRA) were predominantly hematologic, no differentiation syndrome was observed. Conclusions: DAC is very well tolerated by older, medically non-fit AML pts, with myelosuppression being the major toxicity. CR+PR was attained by 26% of pts. A frequent ALE and the good feasibility of outpatient maintenance support continued DAC treatment, and the response to DAC combined with ATRA warrants a randomized study. Disclosures: Off Label Use: Decitabine (FDA approved for MDS and AML with up to 30% bone marrow blasts) is used here (at the approved dose and schedule)in AML patients with 〉30% blasts. ATRA (approved for APL) is used here in non M3 AML patients.

Description: Few therapeutic options exist for older, unfit AML patients (pts) who often have poor cytogenetics. We initiated a phase II trial in untreated AML pts 〉60 years ineligible for induction. 1° endpoint was best response: complete (CR) or partial remission (PR) or an antileukemic effect (ALE, 〉25% bone marrow [BM] blast reduction). 2° endpoints: overall survival (OS), toxicity. Low-dose DAC was given as for MDS (i.e. 135 mg/m2 i.v. over 72 hrs), repeated q 6 weeks for up to 4 courses, with all-trans retinoic acid (ATRA, 45mg/m2/day for 28 days) given during course 2 in pts with ALE or stable disease (SD). Maintenance with 20 mg/m2 DAC i.v. over 1 hour on 3 days (total dose 60mg/m2, outpatient administration) q 6–8 weeks was offered to pts completing all 4 courses. Pts with a WBC of 〉20 000/μl received a short course of hydroxyurea (HU) prior to DAC. Pts requiring HU beyond day 28 of course 1 and/or showing blast increase had progressive disease (PD). In pts with high absolute peripheral blood blasts, RNA from these cells was isolated sequentially (day 0, 2, 5), with CD34 selection in 2 pts, for global expression studies. At time of this analysis, 155 fully evaluable pts have been recruited (median age 72.5 yrs, range 56–85). 39% of pts were 〉74 yrs. Poor-risk cytogenetics and/or preceding MDS were present in 32 and 49%, respectively. Median WBC before treatment was 4800/μl (range, 400-241,000, 〉20,000/μl in 25%, 〉50,000/μl in 10%). Median BM blasts were 56% (25–100). A median of 2 courses was given. 69 pts received ATRA. 41 pts received a total of 162 maintenance courses (median 3, range 1–11). Best response was CR in 23 pts (15%) and PR in 15 pts (10%). An ALE occurred in 45 pts (29%), resulting in a 54% overall response rate. SD was seen in 37 pts (24%), 15 had PD (10%), 20 pts early death (13%). CR+PR rate by cytogenetic subgroups: with normal karyotype 18/51 (35%), with poor-risk 9/46 (20%) and with other abnormalities 6/36 (17%). Toxicities of inpt DAC were very similar to those described for MDS (neutropenia, fever/infection, pancytopenia). No unexpected toxicities or ATRA syndrome were observed with the combination of DAC+ATRA. Median OS from start of treatment was 5.5 months (range, 0.3–38+), the 1-year survival 26%. Cytogenetic subgroups did not differ in median OS. Affymetrix HG-U133 Plus 2.0 microarrays of 9 pts revealed transcription changes (both induction and repression of large sets of genes): between 53 and 256 previously silent genes were reexpressed. Conclusions: low-dose DAC is very well tolerated by older AML pts ineligible for more aggressive treatment, with myelosuppression being the major toxicity. In vivo reexpression of genes was noted in leukemic blasts. Complete and partial remissions occured in 25% of pts. A frequent antileukemic effect and the good feasibility of outpatient maintenance support continued DAC treatment.