DNA Hypomethylating Agent Decitabine Research Articles

DIPG-87. TARGETING GENE-REPRESSIVE EPIGENETIC REGULATION TO INDUCE INTERFERON SIGNALING IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brainstem tumor with no known effective treatment. DIPG is driven by the H3K27M mutant histone, which promotes epigenetic dysregulation, leading to loss of gene-repressive H3K27me3 marks, global DNA hypomethylation, and a distinct gene expression profile. DIPG cells retain histone methylation at histone H3 lysine 9 (H3K9me2/3), suggesting that the tumor may rely on these marks to repress unwanted expression. Our prior work has demonstrated that treatment with the DNA hypomethylating agent decitabine (DAC) elicits expression of cancer-specific antigens and immune activation, including activation of interferon signaling, but modulating gene-repressive H3K9 methylation in DIPG has not been extensively investigated. METHODS We treated four primary patient-derived DIPG neurosphere cell lines with BIX01294 and UNC0642, inhibitors of H3K9 methyltransferases G9a/GLP, and analyzed gene expression changes by RNA-sequencing and Western blot. We assessed the effect of combination treatments on these four cell lines with both DAC and either BIX01294 or UNC0642 and evaluated impact on gene expression, innate immune signaling, and interferon pathway activation. RESULTS We found that treatment with either of the G9a/GLP inhibitors resulted in the upregulation of genes involved in immune signaling. Given the similarities in the immune signaling response elicited from treatment with DAC and the G9a/GLP inhibitors, we evaluated the effect of combination treatments with both DAC and either BIX01294 or UNC0642. These combinations resulted in an increased induction of genes involved in immune signaling, including STING. CONCLUSIONS Pharmacologic inhibition of DNA methylation and histone H3K9 methylation elicits immune activation in DIPG cells. Upregulation of interferon and STING signaling with this combination treatment may be significant in enhancing the immunogenicity of DIPG cells to allow effective immunotherapy.

IMMU-40. DECITABINE PRIMES GLIOBLASTOMA TO NY-ESO-1 SPECIFIC T-CELL CYTOLYSIS VIA DNA DEMETHYLATION OF NY-ESO-1, CANCER-TESTIS ANTIGENS, AND PRO-INFLAMMATORY SIGNATURES

Abstract INTRODUCTION Previous work has shown that the DNA hypomethylating agent, Decitabine (DAC), can improve the immunogenicity of Glioblastoma (GBM) for immuno-therapeutic targeting through the upregulation of cancer testis antigens (CTA). Specifically, our group has published preclinical proof of principle that DAC upregulates NY-ESO-1, a well-studied and highly immunogenic CTA, in GBM for targeting by systemic adoptive T-cell therapy. However, the exact mechanisms by which this DAC-induced immunogenic enhancement occurs in GBM remains to be elucidated. We hypothesize that hypomethylating agents like DAC enable targeted immune therapy to overcome low immunogenicity in GBM cells via CpG demethylation of antigens and subsequent upregulation of inflammatory and antigen presentation gene signatures. METHODS We induced NY-ESO-1 expression in vitro in immortalized GBM lines and primary gliomaspheres using DAC and investigated the response of DAC-treatment using targeted bisulfite sequencing and quantitative real-time PCR. To functionally assess for specific immune killing of DAC treated GBM cells, we co-cultured DAC-treated cell lines with T-cells transduced to express a NY-ESO-1 specific T-cell receptor. Lastly, we performed probe-based methylation array and bulk RNA sequencing to interrogate global transcriptomic and epigenomic changes resulting from DAC treatment. RESULTS Sustained exposure to low dose (0.5 uM – 1 uM) DAC in immortalized and primary cell lines resulted in time-dependent demethylation of the CpG island of NY-ESO-1 and the subsequent robust increase in mRNA expression. Upregulation of NY-ESO-1 rendered specific T cell recognition and lysis of immortalized and primary cell lines. DAC upregulated other pro-inflammatory and immuno-modulatory signatures via CpG demethylation to enhance T-cell mediated targeted cytolysis of GBM. CONCLUSION Hypomethylating agents like DAC upregulate immunogenic signatures in primary and immortalized GBM cells, rendering them susceptible to antigen specific T-cell targeting. These pleiotropic effects demonstrate strong preclinical rationale for use of DAC to sensitize GBM for targeting by immune therapies.

BIOM-35. IDENTIFICATION AND VALIDATION OF POTENTIAL BIOMARKERS FOR DECITABINE-MEDIATED SENSITIZATION TO TEMOZOLOMIDE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive form of brain cancer with limited treatment options and a poor prognosis. Our previous research demonstrated that the DNA hypomethylating agent decitabine (DAC) enhances the sensitivity to temozolomide (TMZ) of a subset of primary GBM cell lines derived from fresh surgical tissue specimens, and that this effect may be mediated by upregulation of DNA mismatch repair genes including MLH1. In this context, we aim to identify additional biomarkers for DAC-mediated sensitization to TMZ with potential clinical utility through in vitro and in vivo experiments. We performed single cell RNA sequencing on three DAC-sensitized and three DAC-desensitized cell lines and found 4 genes that exhibited significant differential expression: DCN, LUM, CRABP2 and NUPR1. We validated these findings using quantitative immunoblots, RT-qPCR and immunohistochemistry, finding that NUPR1 protein expression levels varied most significantly between DAC-sensitized and desensitized GBM tissues. Long-range direct methylation sequencing of non-amplified genomic DNA enriched using CRISPR/Cas9 demonstrated several CpG sites within the 4 genes that may be predictive of TMZ sensitization response after treatment with DAC. Finally, we performed mouse survival experiments using orthotopic patient-derived xenografts (PDXs) of the six GBM cell lines, with preliminary results showing that in selected PDX survival was significantly increased with combined DAC-TMZ versus TMZ monotherapy as predicted by in vitro assays. In conclusion, our study demonstrates that combining DAC with TMZ may be a potential treatment strategy for GBM in a subset of patients. There likely exist several biomarkers that can be used at the time of diagnosis, alone or in combination, to predict the response to epigenetic preconditioning using DAC for TMZ sensitization, which will be necessary for optimal patient selection in an early phase clinical trial. Further data and validation studies are necessary to strengthen these findings and to confirm their clinical relevance.

Fast proliferating and slowly migrating non-small cell lung cancer cells are vulnerable to decitabine and retinoic acid combinatorial treatment.

Non-small cell lung cancer (NSCLC) patients are often elderly or unfit and thus cannot tolerate standard aggressive therapy regimes. In our study, we test the efficacy of the DNA-hypomethylating agent decitabine (DAC) in combination with all-trans retinoic acid (ATRA), which has been shown to possess little systemic adverse effects. Screening a broad panel of 56 NSCLC cell lines uncovered a decrease in cell viability after the combination treatment in 77% of the cell lines. Transcriptomics, proteomics, proliferation and migration profiling revealed that fast proliferating and slowly migrating cell lines were more sensitive to the drug combination. The comparison of mutational profiles found oncogenic KRAS mutations only in sensitive cells. Additionally, different cell lines showed a heterogeneous gene expression response to the treatment pointing to diverse mechanisms of action. Silencing KRAS, RIG-I or RARB partially reversed the sensitivity of KRAS-mutant NCI-H460 cells. To study resistance, we generated two NCI-H460 cell populations resistant to ATRA and DAC, which migrated faster and proliferated slower than the parental sensitive cells and showed signs of senescence. In summary, this comprehensive dataset uncovers a broad sensitivity of NSCLC cells to the combinatorial treatment with DAC and ATRA and indicates that migration and proliferation capacities correlate with and could thus serve as determinants for drug sensitivity in NSCLC.

Hypomethylating agent decitabine sensitizes diffuse large B-cell lymphoma to venetoclax.

Despite recent advances in the therapy of diffuse large B-cell lymphoma (DLBCL), many patients are still not cured. Therefore, new therapeutic strategies are needed. The anti-apoptotic B-cell lymphoma 2 (BCL2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32;q21) and copy number alterations; however, targeting BCL-2 with the selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a rational combination partner of venetoclax to improve its activity against DLBCL cells. Utilizing a functional assay, dynamic BH3 profiling, we found that the DNA hypomethylating agent decitabine increased mitochondrial apoptotic priming and BCL-2 dependence in DLBCL cells. RNA-sequencing analysis revealed that decitabine suppressed the pro-survival PI3K-AKT pathway and altered the mitochondria membrane composition in DLBCL cell lines. Additionally, it induced a DNA damage response and increased BAX and BAK activities. The combination of decitabine and venetoclax synergistically suppressed proliferation of DLBCL cells both in vitro and in vivo in a DLBCL cell line-derived xenograft mouse model. Our study suggests that decitabine plus venetoclax is a promising combination to explore clinically in DLBCL.

DIPG-42. MAPPING THE DNA METHYLATION LANDSCAPE OF DIPG REVEALS NOTABLE METHYLOME VARIABILITY RESPONSIVE TO PHARMACOLOGIC MODULATION

Abstract Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brainstem tumor, driven by the H3K27M mutation leading to epigenetic dysregulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation and altered gene expression. However, most investigations of the epigenetic landscape of DIPG only provide a static view of the epigenome. Such studies are not capable of adequately capturing the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed Whole-Genome Bisulfite Sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls. To analyze this data set, we applied a novel framework for analysis of DNA methylation variability, called informME analysis, which permits the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increased stochasticity localizing to key regulatory elements and genes. We demonstrate that epigenetic instability maps onto key genes regulating pluripotency and developmental identity and hypothesize that these changes might enable tumor cells to sample a diverse range of transcriptional programs and phenotypes. Given this, we evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. We found that treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment also elicited changes in gene expression, including upregulation of genes related to immune signaling. Finally, treatment with DAC sensitized DIPG cells to the effects of histone deacetylase inhibition. This study suggests the application of epigenetic therapies to constrain cellular heterogeneity and plasticity in DIPG.

Pre-Clinical Evaluation of the Hypomethylating Agent Decitabine for the Treatment of T-Cell Lymphoblastic Lymphoma.

T-cell lymphoblastic lymphoma (T-LBL) is a rare and aggressive lymphatic cancer, often diagnosed at a young age. Patients are treated with intensive chemotherapy, potentially followed by a hematopoietic stem cell transplantation. Although prognosis of T-LBL has improved with intensified treatment protocols, they are associated with side effects and 10-20% of patients still die from relapsed or refractory disease. Given this, the search toward less toxic anti-lymphoma therapies is ongoing. Here, we targeted the recently described DNA hypermethylated profile in T-LBL with the DNA hypomethylating agent decitabine. We evaluated the anti-lymphoma properties and downstream effects of decitabine, using patient derived xenograft (PDX) models. Decitabine treatment resulted in prolonged lymphoma-free survival in all T-LBL PDX models, which was associated with downregulation of the oncogenic MYC pathway. However, some PDX models showed more benefit of decitabine treatment compared to others. In more sensitive models, differentially methylated CpG regions resulted in more differentially expressed genes in open chromatin regions. This resulted in stronger downregulation of cell cycle genes and upregulation of immune response activating transcripts. Finally, we suggest a gene signature for high decitabine sensitivity in T-LBL. Altogether, we here delivered pre-clinical proof of the potential use of decitabine as a new therapeutic agent in T-LBL.

DNA methylation atlas and machinery in the developing and regenerating annelid Platynereis dumerilii

BackgroundMethylation of cytosines in DNA (5mC methylation) is a major epigenetic modification that modulates gene expression and constitutes the basis for mechanisms regulating multiple aspects of embryonic development and cell reprogramming in vertebrates. In mammals, 5mC methylation of promoter regions is linked to transcriptional repression. Transcription regulation by 5mC methylation notably involves the nucleosome remodeling and deacetylase complex (NuRD complex) which bridges DNA methylation and histone modifications. However, less is known about regulatory mechanisms involving 5mC methylation and their function in non-vertebrate animals. In this paper, we study 5mC methylation in the marine annelid worm Platynereis dumerilii, an emerging evolutionary and developmental biology model capable of regenerating the posterior part of its body post-amputation.ResultsUsing in silico and experimental approaches, we show that P. dumerilii displays a high level of DNA methylation comparable to that of mammalian somatic cells. 5mC methylation in P. dumerilii is dynamic along the life cycle of the animal and markedly decreases at the transition between larval to post-larval stages. We identify a full repertoire of mainly single-copy genes encoding the machinery associated with 5mC methylation or members of the NuRD complex in P. dumerilii and show that this repertoire is close to the one inferred for the last common ancestor of bilaterians. These genes are dynamically expressed during P. dumerilii development and regeneration. Treatment with the DNA hypomethylating agent Decitabine impairs P. dumerilii larval development and regeneration and has long-term effects on post-regenerative growth.ConclusionsOur data reveal high levels of 5mC methylation in the annelid P. dumerilii, highlighting that this feature is not specific to vertebrates in the bilaterian clade. Analysis of DNA methylation levels and machinery gene expression during development and regeneration, as well as the use of a chemical inhibitor of DNA methylation, suggest an involvement of 5mC methylation in P. dumerilii development and regeneration. We also present data indicating that P. dumerilii constitutes a promising model to study biological roles and mechanisms of DNA methylation in non-vertebrate bilaterians and to provide new knowledge about evolution of the functions of this key epigenetic modification in bilaterian animals.

Tracking Decitabine Incorporation into Malignant Myeloid Cell DNA in vitro and in vivo by LC-MS/MS with Enzymatic Digestion

The DNA hypomethylating agents decitabine and 5-azacytidine are the only two drugs approved for treatment of all subtypes of the myeloid malignancy myelodysplastic syndromes (MDS). The key to drug activity is incorporation into target cell DNA, however, a practical method to measure this incorporation is un-available. Here, we report a sensitive and specific LC-MS/MS method to simultaneously measure decitabine incorporation and DNA hypomethylation. A stable heavy isotope of 2′-deoxycytidine was used as an internal standard and one-step multi-enzyme digestion was used to release the DNA bound drug. Enzyme-released decitabine along with other mononucleosides were separated by a reverse-phase C18 column and quantified by mass spectrometry using multiple-reaction-monitoring (MRM) mode, with a lower limit of quantitation at 1.00 nM. In vitro studies demonstrated dosage and time-dependent incorporation of decitabine into myeloid leukemia cell DNA that correlated with extent of DNA hypomethylation. When applied to clinical samples serially collected from MDS patients treated with decitabine, the method again demonstrated correlation between decitabine DNA-incorporation and DNA hypomethylation. This novel assay to measure the intended molecular pharmacodynamic effect of decitabine therapy can therefore potentially provide insights into mechanisms underlying sensitivity versus resistance to therapy.

Epigenetic Silencing of SPINT2 Promotes Cancer Cell Motility via HGF-MET Pathway Activation in Melanoma

Aberrant HGF-MET signaling activation via interactions with surrounding stromal cells in tumor microenvironment plays significant roles in malignant tumor progression. However, extracellular proteolytic regulation of HGF activation which is influenced by the tumor microenvironment and its consequential effects on melanoma malignancy remain uncharacterized. In this study we identified SPINT2: a proteolytic inhibitor of hepatocyte growth factor activator (HGFA), which plays a significant role in the suppression of the HGF-MET pathway and malignant melanoma progression. SPINT2 expression is significantly lower in metastatic melanoma tissues compared to those in early stage primary melanomas which also corresponded with DNA methylation levels isolated from tissue samples. Treatment with the DNA hypomethylating agent decitabine in cultured melanoma cells induced transcriptional reactivation of SPINT2, suggesting that this gene is epigenetically silenced in malignant melanomas. Furthermore, we show that ectopically expressed SPINT2 in melanoma cells inhibits HGF induced MET-AKT signaling pathway and decreases malignant phenotype potential such as cell motility, and invasive growth of melanoma cells. These results suggest that SPINT2 is associated with tumor suppressive functions in melanoma by inhibiting an extracellular signal regulator of HGF which is typically activated by tumor-stromal interactions. These findings indicate that epigenetic impairment of the tightly regulated cytokine-receptor communications in tumor microenvironment may contribute to malignant tumor progression.

Abstract LB-158: P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC)

Abstract Intrinsic and/or acquired resistance to cisplatin is a major obstacle in the treatment of advanced bladder cancer (BC). It is important to find markers that predict tumor responsiveness to cisplatin-based chemotherapy. P73, a p53 homologue, plays an important role in cisplatin sensitivity. TAp73, a major isoform of p73, utilizes an extrinsic promoter (P1) and is pro-apoptotic. We utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples and found 9 distinct CpGs in the P1 promoter region that were hypermethylated in tumors compared to adjacent normal tissues (p<.05). To determine the functional link between promoter methylation and transcriptional regulation of TAp73, we treated BC cell lines (253J, HT1376, and T24) with DNA hypomethylating agent decitabine (DAC) at various doses (0,1,5 μM). We found that a 10-20% decrease in methylation across 5 interrogated CpG sites resulted in a two fold increase in TAp73 expression. We hypothesize that demethylation of the P1 promoter increases TAp73 expression, sensitizing BC cells to cisplatin. To test this, T24 cells were pretreated with DAC (2μM), followed by cisplatin treatment (0.5μg/mL). Clonogenicity of T24 was measured, as well as changes in p73 methylation and expression. Results show a decrease in clonogenic potential following combination therapy. T24 treated with DAC alone or in combination with cisplatin showed a 15% decrease in P1 promoter methylation, while the same was not seen in cells treated with cisplatin alone. Treatment with DAC and cisplatin did not have a clear effect on TAp73 mRNA expression. We are investigating whether TAp73 protein expression reflects the effects of DAC. To determine the clinical value of TAp73 protein expression we utilized tissue microarrays (TMAs) of 354 BC patients. Immunohistochemical staining was assigned H-score indicating intensity of TAp73 expression and percentage of positive nuclei. Results showed lower TAp73 expression to be associated with patient samples of higher tumor grade and staging (p<.001), suggesting that TAp73 expression is lost as the disease progresses from superficial to advanced BC. Consequently, low TAp73 protein expression was associated with shorter overall survival (p<.05). To further investigate clinical utility of TAp73 promoter methylation and expression, we analyzed tumor samples from 24 patients treated with cisplatin in a neoadjuvant setting. Results showed hypermethylation in tumor compared with non-tumor samples (p<.05), but no significant difference in mRNA expression was observed. Studies continue to focus on understanding the mechanism of cisplatin resistance in the context of epigenetic dysregulation and utilization of DAC as a potentiating agent of cispatin-based chemotherapy in muscle invasive BC. Supported by NCI grants CA067267 and CA016056. Citation Format: Rebecca D. Greenspan, Nithya Krishnan, Carl Morrison, Angela Omilian, Wiam Bshara, Amber Worral, Kristopher Attwood, Donald L. Trump, Anna Woloszynska-Read, Candace S. Johnson. P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2015-LB-158

Epigenetic inactivation of the candidate tumor suppressor USP44 is a frequent and early event in colorectal neoplasia

In mouse models, loss of the candidate tumor suppressor gene Ubiquitin Specific Protease 44 (USP44) is associated with aneuploidy and cancer. USP44 is also transcriptionally silenced in human cancers. Here we investigated the molecular mechanism of USP44 silencing and whether this correlated with aneuploidy in colorectal adenomas. DNA methylation at the USP44 CpG island (CGI) promoter was measured using combined bisulfite restriction analysis (COBRA) in colorectal cancer (CRC) cell lines (n = 18), and with COBRA and bisulfite sequencing in colorectal adenomas (n = 89) and matched normal colonic mucosa (n = 51). The USP44 CGI was hypermethylated in all CRC cell lines, in most colorectal adenomas (79 of 89, 89%) but rarely in normal mucosa samples (3 of 51, 6%). USP44 expression was also compared between normal mucosa and paired hypermethylated adenomas in six patients using qRT-PCR. Hypermethylation of the USP44 CGI in adenomas was associated with a 1.8 to 5.5-fold reduction in expression compared with paired normal mucosa. Treatment of CRC cell lines with the DNA hypomethylating agent decitabine resulted in a 14 to 270-fold increase in USP44 expression. Whole genome SNP array data showed that gain or loss of individual chromosomes occurred in adenomas, but hypermethylation did not correlate with more aneuploidy. In summary, our data shows that USP44 is epigenetically inactivated in colorectal adenomas, but this alone is not sufficient to cause aneuploidy in colorectal neoplasia.

Epigenetic Priming of AML Blasts for All-trans Retinoic Acid-Induced Differentiation by the HDAC Class-I Selective Inhibitor Entinostat

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This “priming” effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a “priming” agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

Outcome of Older AML Patients with Adverse Cytogenetics, Including Single or Multiple Monosomies, Treated with the DNA Hypomethylating Agent Decitabine

Abstract 2609 Background:The monosomal karyotype (MK+) has recently been identified as the single most adverse cytogenetic prognosticator of AML outcome (1, 2). We made the recurrent - and seemingly paradoxical - observation that MDS/AML patients (pts) with complex karyotypes (CK+) including monosomy 7 showed encouraging responses to decitabine (DAC, ref.s 3, 4). We now evaluated a large cohort of AML pts >60 years ineligible for induction chemotherapy treated on a single phase II multicenter DAC study (trial 00331) for the effect of the MK+ genotype upon outcome. Methods:Comparisons of response rate (RR), i.e. attainment of complete and partial remissions (CR/PR) and overall survival (OS) were performed in 179 treatment-naive AML pts with available cytogenetics (median age 72 years) treated with DAC as described (ref. 3, 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 white blood cell counts prior to treatment was 7.500/μl (range 0.5–241,000), 79% had a performance status ECOG >1. Cytogenetic risk groups are shown in Table 1. All karyotypes were centrally reviewed and scored for MK (A.H.). Analyses were adjusted for the parameters which had a strong effect on outcome in multivariate analyses of RR and OS, i.e., performance status (on RR and OS) and platelet counts (on OS). Results:By the established definition of MK+, i.e either 1 autosomal monosomy and at least one structural change (MK1) or 2 or more autosomal monosomies (MK2), 39/179 patients were scored as MK+, with chromosomes 7 (n=17), 17 (n=17), and 5 (n=8) being most frequently lost. 17 pts had a single monosomy, as part of a median of 5 abnormalities (range, 2–21), 22 pts had multiple monosomies (median 3, range 2–11), as part of a median of 9.5 abnormalities (range, 4–17). As shown in Table 1, abnormal cytogenetics (AA) were associated with a lower RR and OS compared to pts with normal karyotype. When analyzing pts with AA (n=123) according to the presence (n=39) or absence of MK+ (n=84), MK+ patients had a higher RR than MK-, irrespective of a CK+ genotype. Since it has been shown by several groups that multiple monosomies may herald an even worse prognosis of AML than single monosomy, it was notable that pts with multiple monosomies (MK2, n=22) had a 45% RR and 25% 1-year OS (Fig. 1) compared with 24% and 12% in those with a single monosomy (MK1, n=17), both comparisons p=0.11. Conclusions:Patients with normal-karyotype AML had a better outcome with DAC treatment than those with chromosomal abnormalities. Patients with single or even multiple monosomies still appeared to respond to this treatment, supporting our previous, surprising observation that pts with highly complex karyotype including monosomies may benefit from DAC. Because of a lack of control group in our trial, it is difficult to hypothesize if this encouraging result may be related to modes of action of DAC which differ from low-dose chemotherapy, e.g., with cytarabine. The role of MK will be further studied prospectively in a randomized clinical trial in the same patient population (NCT00867672), and retrospectively in a randomized trial of higher-risk MDS patients treated with either DAC or Best Supportive Care (EORTC 06011).Table 1CharacteristicResponse (CR/PR)Overall survival (OS)NCR/PR rate (%)Odds Ratio95%- Confidence Intervalp-value1-year OS rate (%)Hazard Ratio95%- Confidence Intervalp-valueAll patients with known cytogenetics17928260.0006Cytogenetics0.13normal56381.00−431.00−abnormal123241.72[0.85,3.48]191.86[1.30,2.65]MK/CK0.0530.38MK+39361.00−211.00−MK-/CK+17124.75[0.88,25.6]181.43[0.79,2.59]MK-/CK-67192.83[1.08,7.41]180.96[0.63,1.45]MK+0.110.11MK1 (1 monosomy)17241.00−121.00−MK2 (>2 monosomies)22450.27[0.05,1.35]270.57[0.29,1.14] [Display omitted] Disclosures:Off Label Use: Decitabine is approved for treatment of different types of MDS, in the present study its activity in AML of older patients was studied. Germing:Celgene: Consultancy, Research Funding.

Retrospective analysis of effects of transfusion status on response to decitabine and survival in patients with myelodysplastic syndromes.

6617 Background: The DNA-hypomethylating agent decitabine is used to treat myelodysplastic syndromes (MDS). Methods: This retrospective analysis pooled a subset of patients (pts) with MDS from 2 decitabine trials: 3-day (phase III) and 5-day (phase II). Excluded pts had received an erythropoiesis-stimulating agent. Pts were stratified as RBC or PLT transfusion dependent (TD) or independent (TI) at baseline (BL) and on study. Response rate (RR, CR+PR+mCR) to decitabine (Fisher exact test) and overall survival (OS) were compared (log-rank test). Results: Pt characteristics, transfusion status, RR, OS, and time to TI are in the table. 23/62 (37%) RBC TD pts at BL became TI on study, and 10/18 (56%) PLT TD pts at BL became TI on study. RBC TI pts at BL and on study (24/34, 70%) had the highest RR (54%, p<.0001) and longest OS (median 19.8 mo; p=.0032).RBC TI pts, regardless of BL status, had significantly longer survival probability (HR 0.33; p=.0122). RBC or PLT TD pts at BL who did not achieve TI on study had the lowest RR and OS. Conclusions: In this retrospective analysis, MDS patients treated with decitabine who were TI at BL or achieved TI on study appeared to have better clinical response and better survival compared with patients who were TD at BL and did not achieve TI on study. Transfusion status N Age, mean, y IPSS high risk, n (%) ORR (CR+PR+mCR)n (%) CRn (%) PRn (%) mCRn (%) Overall survival, median (95% CI), mo Time from TD to TI, median (95% CI), mo Red blood cells BL TD/ on-study TD 39 71 11 (28) 1 (3) 0 1 (3) 0 9.4 (5.8, 13.0) NE BL TD/ on-study TI 23 69 3 (13) 9 (39) 8 (35) 1 (4) 0 19.2 (14.7, 21.6) 2.3 (0.99, 2.6) BL TI/ on-study TD 10 68 2 (20) 2 (20) 0 0 2 (20) 6.8 (0.99, NE) NE BL TI/ on-study TI 24 69 5 (21) 13 (54) 9 (38) 3 (13) 1 (4) 19.8 (12.9, NE) NE P <.0001* <.0001* .2994* .0156* .0032† <.0001† Platelets BL TD/ on-study TD 8 70 3 (38) 0 0 0 0 2.1 (0.9, 6.7) NE BL TD/ on-study TI 10 72 2 (20) 3 (30) 2 (20) 1 (1) 0 14.2 (5.0, 22.3) 2.9 (0.6, 15.5) BL TI/ on-study TD 19 69 4 (21) 1 (5) 0 0 1 (5) 3.1 (1.6, 12.6) NE BL TI/ on-study TI 59 69 12 (20) 21 (36) 15 (25) 4 (7) 2 (3) 19.2 (15.7, 25.7) NE P .0120* .0234* .5038* 1.000* <.0001† <.0001† Abbreviations: CI, confidence interval; NE, not evaluable. * Fisher exact test. † Log-rank test.

Decitabine Improves Clinical Outcomes in Severely Ill Sickle Cell Disease Patients Who Have Exhausted Standard of Care Options.

Interventions such as immunization, penicillin prophylaxis, hydroxyurea and transfusion have extended life in patients with sickle cell disease (SCD). Nonetheless, these interventions are limited by toxicity or effectiveness; continued substantial morbidity and mortality in SCD indicates the need for better disease modification. In previous phase I/II clinical trials, 13 of 13 patients treated with the DNA hypomethylating agent decitabine responded with clinically significant fetal hemoglobin and total hemoglobin elevation and improvement in surrogate clinical end-points. However, in these early studies, no clinical end-points were measured and further studies have been delayed by funding issues. We describe an off-label experience in four patients with severe SCD that suggestsi.remarkable clinical effectiveness in patients who have exhausted standard of care and are severely ill;ii.tolerability even in the severely ill;iii.a mechanism of action based on increased reticulocytosis in addition to increased fetal hemoglobin.All four patients had multiple alloantibodies and red-cell auto-antibodies that limited availability and increased risks of transfusion, and had previously been treated with hydroxyurea with continued clinical deterioration. Three of the four patients had relative reticulocytopenia (absolute reticulocyte count <250x109/L and hemoglobin <9g/dl) and were receiving erythropoietin or darbopoietin for more than 8 weeks with continued progressive anemia and progressive congestive heart failure. All four patients were ECOG performance status 3 and ineligible for available clinical trials. Based on the clinical trial experience conducted at our institution, decitabine therapy at 0.1–0.2 mg/kg 1–2X/week was initiated in these patients not for research purposes but with the intent to produce direct clinical benefit. The limited clinical data and potential for unanticipated toxicity was discussed in full with each patient and family members. IRB approval was obtained for a retrospective chart review. No decitabine related adverse events occurred. All patients demonstrated >2g/dl increases in hemoglobin levels with an associated improvement in clinical status - decrease in pain, improvement in performance status, improvement in congestive heart failure symptoms/signs. Upward trends in the platelet and reticulocyte counts concurrent with downward trends in the neutrophil counts were consistent with previously observed effects of low dose decitabine or the related compound 5-azacytidine. Clinically significant neutropenia was avoided by dose reductions that did not reverse the improved hemoglobin levels. The differentiation altering effects of low dose decitabine relieve SCD anemia by decreasing hemolysis (through elevated HbF) and increasing reticulocytosis. Previous clinical trials, and this off-label experience, suggest that decitabine holds remarkable promise as a disease modifying agent for SCD and β-thalassemia. Further clinical trials to confirm this impression should be supported.