WO2013022872A1

WO2013022872A1 - Gene methylation biomarkers and methods of use thereof

Info

Publication number: WO2013022872A1
Application number: PCT/US2012/049826
Authority: WO
Inventors: C.L. Beach; Kyle Macbeth; Tao Shi
Original assignee: Celgene Corporation
Priority date: 2011-08-10
Filing date: 2012-08-07
Publication date: 2013-02-14

Abstract

Provided herein are biomarkers, compositions, kits, and methods for diagnosis, prognosis, or monitoring of cancers and/or myelodysplasia syndromes (MDS), and for predicting or monitoring the efficacy of certain therapeutic treatment in cancer patients or in MDS patients, treated with a therapeutic agent, such as, a cytidine analog, e.g., 5-azacytidine. Also provided are methods for predicting the overall survival of certain classes of patients having MDS or cancer, and methods for selecting patients having MDS or cancer for a particular therapeutic treatment. Also provided are gene methylation biomarkers and methods of use thereof.

Description

GENE METHYLATION BIOMARKERS

AND METHODS OF USE THEREOF

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/522,167, filed August 10, 2011, which is hereby incorporated by reference in its entirety.

1. FIELD

[0002] Provided herein are biomarkers, compositions, kits, and methods, for diagnosis, prognosis, or monitoring of cancers and/or myelodysplastic syndromes (MDS), and for predicting or monitoring the efficacy or clinical benefit of certain therapeutic treatment in patients in need thereof, such as, in MDS patients treated with an agent, such as, a cytidine analog, e.g., 5-azacytidine. Also provided are methods for selecting patients for a particular therapeutic treatment. Also provided are gene methylation biomarkers and methods of use thereof.

2. BACKGROUND

[0003] Cancer is a major worldwide public health problem; in the United States alone, approximately 560,000 people died of cancer in 2006. See, e.g., U.S. Mortality Data 2006, National Center for Health Statistics, Centers for Disease Control and Prevention (2009). Many types of cancer have been described in the medical literature. Examples include cancer of blood, bone, skin, lung, colon, breast, prostate, ovary, brain, kidney, bladder, pancreas, and liver, among others. The incidence of cancer continues to climb as the general population ages and as new forms of cancer develop. A continuing need exists for effective therapies to treat subjects with cancer.

[0004] Myelodysplasia syndromes ("MDS") refers to a diverse group of hematopoietic stem cell disorders. MDS is characterized by a cellular marrow with impaired morphology and maturation (dysmyelopoiesis), peripheral blood cytopenias, and a variable risk of progression to acute leukemia, resulting from ineffective blood cell production. See, e.g., The Merck Manual 953 (17th ed. 1999); List et al, 1990, J. Clin. Oncol. 8: 1424.

[0005] The initial hematopoietic stem cell injury can be from causes such as, but not limited to, cytotoxic chemotherapy, radiation, virus, chemical exposure, and genetic predisposition. A clonal mutation predominates over bone marrow, suppressing healthy stem cells. In the early stages of MDS, the main cause of cytopenias is increased programmed cell death (apoptosis). As the disease progresses and converts into leukemia, gene mutation rarely occurs and a proliferation of leukemic cells overwhelms the healthy marrow. The disease course differs, with some cases behaving as an indolent disease and others behaving aggressively with a very short clinical course that converts into an acute form of leukemia.

[0006] An international group of hematologists, the French- American-British (FAB) Cooperative Group, classified MDS into five subgroups, differentiating them from acute myeloid leukemia. See, e.g., The Merck Manual 954 (17th ed. 1999); Bennett J. M., et al, Ann. Intern. Med. 1985 October, 103(4): 620-5; and Besa E. C, Med. Clin. North Am. 1992 May, 76(3): 599 617. An underlying trilineage dysplastic change in the bone marrow cells of the patients is found in all subtypes. Information is available regarding the pathobiology of MDS, certain MDS classification systems, and particular methods of treating and managing MDS. See, e.g., U.S. Patent No. 7,189,740 (issued March 13, 2007), which is incorporated by reference herein in its entirety.

[0007] Nucleoside analogs have been used clinically for the treatment of viral infections and proliferative disorders for decades. Most of the nucleoside analog drugs are classified as antimetabolites. After they enter cells, nucleoside analogs are successively phosphorylated to nucleoside 5 '-monophosphates, 5'-diphosphates, and 5 '-triphosphates. In most cases, nucleoside triphosphates are the chemical entities that inhibit DNA or RNA synthesis, either through a competitive inhibition of polymerases or through incorporation of modified nucleotides into DNA or RNA sequences. Nucleosides may act also as their diphosphates.

[0008] 5-Azacytidine (also known as azacitidine and 4-amino-1-P-D-ribofuranosyl-1,3 ,5- triazin-2(lH)-one; Nation Service Center designation NSC- 102816; CAS Registry Number 320-67-2) has undergone NCI-sponsored trials for the treatment of MDS. See, e.g., Komblith et al, J. Clin. Oncol. 20(10): 2441-2452 (2002); Silverman et al, J. Clin. Oncol. 20(10): 2429-2440 (2002). 5-Azacytidine may be defined as having a molecular formula of

C₈H₁₂N₄O₅, a relative molecu a structure of:

[0009] 5-Azacytidine (also referred to as azacitidine herein) is a nucleoside analog, more specifically a cytidine analog. 5-Azacytidine is an antagonist of its related natural nucleoside, cytidine. 5-Azacytidine, as well as decitabine, i.e., 5 -aza-2'-deoxy cytidine, are antagonists of decitabine's related natural nucleoside, deoxy cytidine. The only structural difference between the analogs and their related natural nucleosides is the presence of nitrogen at position 5 of the cytosine ring in place of carbon.

[0010] Other members of the class of cytidine analogs include, but are not limited to, arabinosylcytosine (Cytarabine), 2'-deoxy-2',2'-difluorocytidine (Gemcitabine), 5-aza-2'- deoxycytidine (Decitabine), 2(1H)-pyrimidine-riboside (Zebularine), 2',3'-dideoxy-5-fluoro- 3'-thiacytidine (Emtriva), N⁴-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine (Capecitabine), 2'- cyclocytidine, arabinofuanosyl-5-azacytidine, dihydro-5-azacytidine, N⁴-octadecyl- cytarabine, elaidic acid cytarabine, and cytosine Ι-β-D-arabinofuranoside (ara-C).

[0011] A need remains for more effective methods, biomarkers, compositions, and kits, which provide, e.g., increased survival to higher risk MDS patients.

[0012] Citation of any references in this Section of the application is not to be construed as an admission that such reference is prior art to the present application.

3. SUMMARY

[0013] In one embodiment, provided herein are biomarkers for diagnosis, prognosis, or monitoring of cancers and/or myelodysplastic syndromes (MDS). In one embodiment, the MDS is a higher risk MDS. In one embodiment, provided herein are biomarkers for predicting or monitoring the efficacy or clinical benefit of a therapeutic treatment in patients in need thereof, such as, in MDS patients treated with an agent, such as, a cytidine analog, e.g., 5-azacytidine.

[0014] In one embodiment, provided herein are methods for diagnosis, prognosis, or monitoring of cancers and/or myelodysplastic syndromes (MDS). In one embodiment, the MDS is a higher risk MDS. In one embodiment, provided herein are methods for predicting or monitoring the efficacy or clinical benefit of a therapeutic treatment in patients in need thereof, such as, in MDS patients treated with an agent, such as, a cytidine analog, e.g., 5- azacytidine.

[0015] In one embodiment, provided herein is a method of predicting or monitoring the efficacy or clinical benefit of a therapeutic treatment, comprising measuring the level of one or more specific biomarker(s) in cells obtained from patients having a certain disease before or during the treatment. In one embodiment, the disease is cancer. In one embodiment, the cancer is a blood-borne tumor. In one embodiment, the cancer is a solid tumor. In one embodiment, the disease is MDS, e.g., higher-risk MDS. In one embodiment, the treatment is administration of a cytidine analog provided herein. In one embodiment, the treatment is administration of 5-azacytidine. In one embodiment, provided herein is a method of predicting or monitoring the efficacy of 5-azacytidine in MDS patients (e.g., in higher-risk MDS patients), comprising measuring the level of one or more specific biomarker(s) in cells obtained from patients before or during 5-azacytidine treatment. In one embodiment, the cells are obtained from the bone marrow of patient(s). In one embodiment, the biomarker provided herein is methylation of one or more gene(s). In one embodiment, the biomarker provided herein is methylation of one or more gene(s) at one or more locus/loci (e.g., at particular CpG site(s)). In one embodiment, the biomarker provided herein is a methylation pattern or a methylation signature of a particular group of genes. In one embodiment, the biomarker provided herein is a methylation pattern or a methylation signature of a particular group of genes at particular loci. In one embodiment, the gene methylation biomarkers provided herein include, but are not limited to, methylation of one or more of the following genes: CDKN2B (pl5), SOCS1, CDHI (E-cadherin) , TP73, and/or CTNNA1 (a-catenin). In one embodiment, the gene methylation biomarkers provided herein include, but are not limited to, methylation of one or more of the following genes: ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C19orf30, Clorfl 72, Clorfi7, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDHI, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP2E1, CYP26C1, DES, DPYS, DYDCl, EGFL7, ELM03, ENTPD2, ENTPD3, ESRl, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT,

PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP 5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and/or ZNF710. In one embodiment, the gene methylation biomarkers provided herein include, but are not limited to, methylation of one or more of the following genes: WT1, CDKN2B, and CDH1. In one embodiment, the gene methylation biomarkers provided herein include, but are not limited to, methylation of one or more of the following gene: WT1. In one embodiment, the gene methylation biomarker provided herein include the methylation of one or more gene(s) provided herein at particular locus/loci. In one embodiment, the biomarkers provided herein can be used to predict whether a particular therapeutic intervention is likely to be successful in treating a cancer. In one embodiment, the biomarkers provided herein can be used to predict whether a particular therapeutic intervention is likely to be successful in treating MDS, e.g., higher-risk MDS. Further, the biomarkers provided herein can be used to monitor efficacy or progress of a drug treatment once the treatment begins.

[0016] In one embodiment, provided herein are methods for predicting the overall survival of certain classes of patients having MDS, e.g., after a therapeutic treatment. In one embodiment, the MDS is higher-risk MDS. In one embodiment, the method comprises the step of measuring the methylation level(s) of one or more gene(s) in cells obtained from patients. In one embodiment, the method comprises the step of measuring the methylation level(s) of one or more gene(s) at particular locus/loci in cells obtained from patients. In one embodiment, the method further comprises the step of grouping patients based on

methylation level(s) of one or more gene(s). In one embodiment, the method further comprises the step of grouping patients based on methylation level(s) of one or more gene(s) at particular locus/loci. In one embodiment, the method further comprises the step of selecting patients for a particular therapeutic treatment based on methylation level(s) of one or more gene(s). In one embodiment, the method further comprises the step of selecting patients for a particular therapeutic treatment based on methylation level(s) of one or more gene(s) at particular locus/loci. In one embodiment, the method further comprises the step of treating certain groups or classes of patients with a therapeutic agent. In one embodiment, the treatment is administration of a cytidine analog provided herein. In one embodiment, the treatment is administration of 5-azacytidine. In one embodiment, the patients selected based on methylation level(s) of one or more gene(s) {e.g., at particular locus/loci) exhibit better or prolonged overall survival after the therapeutic treatment. In one embodiment, the patients selected based on methylation level(s) of one or more gene(s) (e.g., at particular locus/loci) exhibit better or prolonged time to AML transformation after the therapeutic treatment.

[0017] Also provided are gene methylation biomarkers and methods of use thereof. In one embodiment, provided herein is a methylation pattern or methylation signature of a group of genes that is predictive of therapeutic efficacy or clinical benefit of a particular therapeutic agent in treating cancer. In one embodiment, provided herein is a methylation pattern or methylation signature of a group of genes that is predictive of therapeutic efficacy or clinical benefit of a particular therapeutic agent in treating MDS. In specific embodiments, provided herein is a methylation pattern or methylation signature of a group of genes that is predictive of overall survival of patients having MDS after receiving 5-azacytidine treatment. In specific embodiments, provided herein is a methylation pattern or methylation signature of a group of genes that is predictive of time to AML transformation in patients having MDS after receiving 5-azacytidine treatment. In one embodiment, the gene methylation biomarker provided herein involves the methylation of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, or at least 190 genes provided herein elsewhere. In one embodiment, the gene methylation biomarker provided herein involves the methylation level(s) of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 genes provided herein elsewhere. In one embodiment, the gene methylation biomarker provided herein involves the methylation level(s) of up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, up to 100, up to 110, up to 120, up to 130, up to 140, up to 150, up to 160, up to 170, up to 180, or up to 190 genes provided herein elsewhere. In one embodiment, the gene methylation biomarker provided herein involves the methylation level(s) of one or more gene cluster(s) provided herein. In one embodiment, the gene methylation biomarker provided herein involves the methylation level(s) of one or more genes (e.g., at particular locus/loci) selected from the group consisting of: ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CRII, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C orflO, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC8I, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP2E1, CYP26C1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESRI, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ4488I, FLVCR2, FREQ, FZD9, GAB I, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2,

HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

[0018] In particular embodiments, provide herein are methods for the treatment, prevention, and/or management of cancer using compositions comprising an effective amount of a cytidine analog, including, but not limited to, 5-azacytidine. In certain embodiments, the methods comprise treating, preventing, and/or managing certain types of cancer, including, but not limited to, blood-borne tumor or solid tumor. In certain embodiments, the methods comprise co-administering two or more active agents. In certain embodiments, the methods comprise treating, preventing, and/or managing cancer using one or more of the methods provided herein, together with one or more of the treatments including chemotherapy, immunotherapy, targeted therapy, and/or radiation therapy.

[0019] In particular embodiments, provide herein are methods for the treatment, prevention, and/or management of MDS using compositions comprising an effective amount of a cytidine analog, including, but not limited to, 5-azacytidine. In certain embodiments, the methods comprise treating, preventing, and/or managing certain types of MDS, including, but not limited to, higher-risk MDS. In certain embodiments, the methods comprise coadministering two or more active agents. In certain embodiments, the methods comprise treating, preventing, and/or managing MDS using one or more of the methods provided herein, together with one or more of the treatments including chemotherapy, immunotherapy, targeted therapy, and/or radiation therapy.

[0020] Particular embodiments provide methods for treating patients with higher risk MDS using 5-azacytidine. Particular embodiments provide methods for improving the overall survival of patients having MDS, e.g., higher risk MDS. Particular embodiments provide methods for selecting patients having better expected response to a treatment.

Particular embodiments provide alternative dosing regimens for treating MDS. Particular embodiments provide methods for treating certain subgroups of patients with higher risk MDS, e.g., patients with -7/del(7q) and/or patients with a particular gene methylation profile (or gene methylation pattern or gene methylation signature) prior to a therapeutic treatment or after initiation of a therapeutic treatment. Particular embodiments provide methods for treating elderly patients with acute myelogenous leukemia ("AML"). Particular

embodiments provide methods for ameliorating certain adverse events ("AEs") in patients with MDS, e.g., higher risk MDS. Particular embodiments provide methods for treating patients having MDS, e.g., higher risk MDS, using specific numbers of 5-azacytidine treatment cycles. Particular embodiments provide methods of treating patients who meet the WHO criteria for AML using 5-azacytidine. Particular embodiments provide methods of using IWG responses of complete remission, partial remission, hematologic improvement, and/or stable disease as predictors of overall response in patients with MDS, e.g., higher risk MDS. Particular embodiments provide using 5-azacytidine as maintenance therapy.

Particular embodiments provide using DNA and/or R A methylation as biomarkers for overall survival in patients with MDS, e.g., higher risk MDS.

[0021] In one embodiment, the cytidine analog described herein includes, but is not limited to, 5 -aza-2'-deoxy cytidine, 5-azacytidine, 5-aza-2'-deoxy-2',2'-difluorocytidine, 5- aza-2'-deoxy-2'-fluorocytidine, 2'-deoxy-2',2'-difluorocytidine, cytosine Ι-β-D- arabinofuranoside, 2(1H) pyrimidine riboside, 2'-cyclocytidine, arabinofuanosyl-5- azacytidine, dihydro-5-azacytidine, N⁴-octadecyl-cytarabine, and elaidic acid cytarabine.

[0022] In one embodiment, the cytidine analog is administered parenterally (e.g. , intravenously or subcutaneously). In one embodiment, the cytidine analog is administered orally. In specific embodiments, 5-azacytidine is administered parenterally (e.g., intravenously or subcutaneously). In specific embodiments, 5-azacytidine is administered orally. In specific embodiments, 5-azacytidine is administered in an amount of between about 75 mg/m² to about 100 mg/m² per day, e.g., for up to about 7 consecutive days followed by a resting period of about 21 days (e.g., a 28-day treatment cycle). In specific embodiments, 5-azacytidine is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20 cycles. In one embodiment, the starting dose of a 5-azacytidine treatment is about 75 mg/m² per day, e.g., for about 7 days followed by a resting period of about 21 days (a 28-day treatment cycle), e.g., for two 28-day treatment cycles. In one embodiment, after two 28-day treatment cycles, 5-azacytidine is administered in an amount of between about 75 mg/m² to about 100 mg/m² per day, e.g., for up to about 7 consecutive days followed by a resting period of up to about 21 days (e.g., a 28-day treatment cycle), e.g., for at least two, at least three, at least four, at least five, or at least six additional 28-day treatment cycles. Certain embodiments herein provide co-administration of a cytidine analog (e.g., 5-azacytidine) with one or more additional active agents to provide a synergistic therapeutic effect in subjects in need thereof. The co-administered agent(s) may be a cancer therapeutic agent, as described herein. In certain embodiments, the co-administered agent(s) may be dosed, e.g., orally or by injection (e.g., intravenous or subcutaneous injection).

[0023] In one embodiment, provided herein are compositions and kits for diagnosis, prognosis, or monitoring of cancers and/or myelodysplastic syndromes (MDS), e.g., higher risk MDS. In one embodiment, provided herein are compositions and kits for predicting or monitoring the efficacy of a therapeutic treatment in patients having a certain disease (e.g., a cancer or MDS), such as, in MDS patients treated with a cytidine analog, e.g., 5-azacytidine.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 represents a study design for the Phase III 5-azacytidine survival study.

[0025] Figure 2 represents a graph showing overall survival in the intent to treat population (higher risk MDS patients) of 5-azacytidine compared to conventional care regimens.

[0026] Figure 3 represents time to transform to AML - ITT Population comparing the 5- azacytidine group with the CCR group, showing difference of 13.7 months in time to transformation. [0027] Figure 4 represents a correlation of baseline CDH1 methylation with overall survival or with time to AML transformation, in MDS patients treated with 5-azacytidine or with conventional care regimens.

[0028] Figure 5 represents a dosing and sample collection schedule for a clinical study of MDS patients treated with 5-azacytidine.

[0029] Figure 6 represents a baseline gene methylation pattern (e.g. , pre -treatment gene methylation pattern) in a training data set of 38 MDS patients, in relation to various clinical outcomes, including overall survival, after treatment of 5-azacytidine.

[0030] Figure 7 represents a baseline gene methylation pattern (e.g., pre -treatment gene methylation pattern) in a data set of 59 MDS patients, in relation to various clinical outcomes, including overall survival, after treatment with 5-azacytidine.

5. DETAILED DESCRIPTION

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications and patents referred to herein are incorporated by reference herein in their entireties.

5.1 Definitions

[0032] As used in the specification and the accompanying claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

[0033] As used herein, and unless otherwise specified, the term "about" or

"approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%) of a given value or range.

[0034] As used herein, and unless otherwise specified, the terms "treat," "treating" and "treatment" refer to the eradication or amelioration of a disease or disorder, or of one or more symptoms associated with the disease or disorder. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or disorder resulting from the

administration of one or more prophylactic or therapeutic agents to a subject with such a disease or disorder. In some embodiments, the terms refer to the administration of a compound or dosage form provided herein, with or without one or more additional active agent(s), after the diagnosis or the onset of symptoms of the particular disease.

[0035] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0036] As used herein, and unless otherwise specified, the terms "manage," "managing" and "management" refer to preventing or slowing the progression, spread or worsening of a disease or disorder, or of one or more symptoms thereof. Often, the beneficial effects that a subject derives from a prophylactic and/or therapeutic agent do not result in a cure of the disease or disorder. In this regard, the term "managing" encompasses treating a subject who had suffered from the particular disease in an attempt to prevent or minimize the recurrence of the disease.

[0037] As used herein, and unless otherwise specified, "amelioration" of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with the administration of the composition.

[0038] As used herein, and unless otherwise specified, the term "therapeutically effective amount" or "effective amount" of a compound means an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A "therapeutically effective amount" or "effective amount" of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a therapeutic benefit in the treatment or management of the disease or disorder. The terms "therapeutically effective amount" and "effective amount" can encompass an amount that improves overall therapy, reduces, delays, or avoids symptoms or causes of disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. [0039] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0040] As used herein, and unless otherwise specified, the term "subject" is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In specific embodiments, the subject is a human. The terms "subject" and "patient" are used

interchangeably herein in reference, for example, to a mammalian subject, such as a human. In particular embodiments, a subject having MDS is a subject who has been previously diagnosed as having MDS.

[0041] As used herein, and unless otherwise specified, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. As used herein, and unless otherwise specified, "neoplastic" refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells having dysregulated or unregulated cell growth.

[0042] As used herein, and unless otherwise specified, the terms "cancer" and

"cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, lymphoma, leukemia, and solid tumors, such as, for example, lung cancer. In one

embodiment, the term "cancer" as used herein includes, but is not limited to, solid tumors and blood-borne tumors. The term "cancer" refers to disease of skin tissues, organs, blood, and vessels, including, but not limited to, cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, and uterus. Specific cancers include, but are not limited to, advanced malignancy, amyloidosis, neuroblastoma, meningioma, atypical meningioma, hemangiopericytoma, multiple brain metastase, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karyotype acute myeloblasts leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma, metastatic melanoma (localized melanoma, including, but not limited to, ocular melanoma), malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressiva, hormone refractory prostate cancer, resected high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen independent prostate cancer, androgen dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy- insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma. In a specific embodiment, the cancer is metastatic. In another embodiment, the cancer is refractory or resistant to chemotherapy or radiation.

[0043] As used herein, and unless otherwise specified, the term "proliferative" disorder or disease refers to unwanted cell proliferation of one or more subset of cells in a

multicellular organism resulting in harm (i.e., discomfort or decreased life expectancy) to the multicellular organism. For example, as used herein, proliferative disorder or disease includes neoplastic disorders and other proliferative disorders.

[0044] As used herein, and unless otherwise specified, the term "relapsed" refers to a situation where a subject, that has had a remission of cancer after a therapy, has a return of cancer cells.

[0045] As used herein, and unless otherwise specified, the term "refractory" or "resistant" refers to a circumstance where a subject, even after intensive treatment, has residual cancer cells in the body.

[0046] As used herein, and unless otherwise specified, the term "drug resistance" refers to the condition when a disease does not respond to the treatment of a drug or drugs. Drug resistance can be either intrinsic, which means the disease has never been responsive to the drug or drugs, or it can be acquired, which means the disease ceases responding to a drug or drugs that the disease had previously responded to. In certain embodiments, drug resistance is intrinsic. In certain embodiments, the drug resistance is acquired. [0047] As used herein, and unless otherwise specified, the term "anticancer agent" or "cancer therapeutic agent" is meant to include anti-proliferative agents and chemotherapeutic agents, including, but not limited to, antimetabolites (e.g., 5-fluoro uracil, methotrexate, fludarabine, cytarabine (also known as cytosine arabinoside or Ara-C), and high dose cytarabine), antimicrotubule agents (e.g., vinca alkaloids, such as vincristine and vinblastine; and taxanes, such as paclitaxel and docetaxel), alkylating agents (e.g., mechlorethamine, chlorambucil, cyclophosphamide, melphalan, melphalan, ifosfamide, carmustine, azacitidine, decitabine, busulfan, cyclophosphamide, dacarbazine, ifosfamide, and nitrosoureas, such as carmustine, lomustine, bischloroethylnitrosurea, and hydroxyurea), platinum agents (e.g., cisplatin, carbop latin, oxaliplatin, satraplatin (JM-216), and CI-973), anthracyclines (e.g., doxorubicin and daunorubicin), antitumor antibiotics (e.g. , mitomycin, bleomycin, idarubicin, adriamycin, daunomycin (also known as daunorubicin, rubidomycin, or cerubidine), and mitoxantrone), topoisomerase inhibitors (e.g. , etoposide and camptothecins), purine antagonists or pyrimidine antagonists (e.g., 6-mercaptopurine, 5-fluorouracil, cytarabine, clofarabine, and gemcitabine), cell maturing agents (e.g., arsenic trioxide and tretinoin), DNA repair enzyme inhibitors (e.g., podophyllotoxines, etoposide, irinotecan, topotecan, and teniposide), enzymes that prevent cell survival (e.g. , asparaginase and pegaspargase), histone deacetylase inhibitors (e.g., vorinostat), any other cytotoxic agents (e.g., estramustine phosphate, dexamethasone, prednimustine, and procarbazine), hormones (e.g.,

dexamethasone, prednisone, methylprednisolone, tamoxifen, leuprolide, flutamide, and megestrol), monoclonal antibodies (e.g., gemtuzumab ozogamicin, alemtuzumab, rituximab, and yttrium-90-ibritumomab tiuxetan), immuno-modulators (e.g., thalidomide and

lenalidomide), Bcr-Abl kinase inhibitors (e.g., AP23464, AZD0530, CGP76030, PD 180970, SKI-606, imatinib, BMS354825 (dasatinib), AMN107 (nilotinib), and VX-680), hormone agonists or antagonists, partial agonists or partial antagonists, kinase inhibitors, surgery, radiotherapy (e.g., gamma-radiation, neutron bean radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biological response modifiers (e.g., interferons, interleukins, and tumor necrosis factor), hyperthermia and cryotherapy, and agents to attenuate any adverse effects (e.g., antiemetics).

[0048] As used herein, and unless otherwise specified, the terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents simultaneously, concurrently or sequentially within no specific time limits unless otherwise indicated. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), essentially concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.

[0049] As used herein, and unless otherwise specified, the terms "composition,"

"formulation," and "dosage form" are intended to encompass products comprising the specified ingredient(s) (in the specified amounts, if indicated), as well as any product(s) which result, directly or indirectly, from combination of the specified ingredient(s) in the specified amount(s).

[0050] As used herein, and unless otherwise specified, the term "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. In one embodiment, by "pharmaceutical" or "pharmaceutically acceptable" it is meant that any diluent(s), excipient(s) or carrier(s) in the composition, formulation, or dosage form are compatible with the other ingredient(s) and not deleterious to the recipient thereof. See, e.g., Remington, The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins:

Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al, ed., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash ed., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson ed., CRC Press LLC: Boca Raton, FL, 2004. [0051] As used herein, and unless otherwise specified, the term "hydrate" means a compound provided herein or a salt thereof, which further includes a stoichiometric or non- stoichiometric amount of water bound by non-covalent intermolecular forces.

[0052] As used herein, and unless otherwise specified, the term "solvate" means a solvate formed from the association of one or more solvent molecules to a compound provided herein. The term "solvate" includes hydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and the like).

[0053] As used herein, and unless otherwise specified, a compound described herein is intended to encompass all possible stereoisomers, unless a particular stereochemistry is specified. Where structural isomers of a compound are interconvertible via a low energy barrier, the compound may exist as a single tautomer or a mixture of tautomers. This can take the form of proton tautomerism; or so-called valence tautomerism in the compound, e.g., that contain an aromatic moiety.

[0054] As used herein, and unless otherwise specified, in one embodiment, a compound described herein is intended to encompass isotopically enriched analogs. For example, one or more hydrogen position(s) in a compound may be enriched with deuterium and/or tritium. Other suitable isotopes that may be enriched at particular positions of a compound include, but are not limited, C-13, C-14, N-15, 0-17, and/or 0-18. In one embodiment, a compound described herein may be enriched at more than one position with isotopes, that are the same or different.

[0055] As used herein, and unless otherwise specified, a cytidine analog referred to herein is intended to encompass the free base of the cytidine analog, or a salt, solvate, hydrate, cocrystal, complex, prodrug, precursor, metabolite, and/or derivative thereof. In certain embodiments, a cytidine analog referred to herein encompasses the free base of the cytidine analog, or a salt, solvate, hydrate, cocrystal or complex thereof. In certain embodiments, a cytidine analog referred to herein encompasses the free base of the cytidine analog, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

5.2 Cytidine Analogs

[0056] In one embodiment, the methods provided herein comprise administration or coadministration of one or more cytidine analogs. In certain embodiments, the cytidine analog is 5-azacytidine (azacitidine). In certain embodiments, the cytidine analog is 5-aza-2'- deoxy cytidine (decitabine). In certain embodiments, the cytidine analog is 5-azacytidine (azacitidine) or 5 -aza-2'-deoxy cytidine (decitabine). In certain embodiments, the cytidine analog is, for example: Ι-β-D-arabinofuranosylcytosine (Cytarabine or ara-C); pseudoiso- cytidine (psi ICR); 5-fluoro-2'-deoxycytidine (FCdR); 2'-deoxy-2',2'-difluorocytidine

(Gemcitabine); 5-aza-2'-deoxy-2',2'-difluorocytidine; 5-aza-2'-deoxy-2'-fluorocytidine; Ι-β-D- ribofuranosyl-2(1H)-pyrimidinone (Zebularine); 2',3'-dideoxy-5-fluoro-3'-thiacytidine (Emtriva); 2'-cyclocytidine (Ancitabine); l-P-D-arabinofuranosyl-5-azacytosine (Fazarabine or ara-AC); 6-azacytidine (6-aza-CR); 5,6-dihydro-5-azacytidine (dH-aza-CR);

N⁴-pentyloxy-carbonyl-5'-deoxy-5-fluorocytidine (Capecitabine); N⁴-octadecyl-cytarabine; or elaidic acid cytarabine. In certain embodiments, the cytidine analogs provided herein include any compound which is structurally related to cytidine or deoxycytidine and functionally mimics and/or antagonizes the action of cytidine or deoxycytidine.

[0057] In certain embodiments, exemplary cytidine analogs have the structures provided below:

Azacitidine ocytidine (psi ICR)

Gemcitabine Emtriva

[0058] Certain embodiments herein provide salts, cocrystals, solvates (e.g., hydrates), complexes, prodrugs, precursors, metabolites, and/or other derivatives of the cytidine analogs provided herein. For example, particular embodiments provide salts, cocrystals, solvates (e.g., hydrates), complexes, precursors, metabolites, and/or other derivatives of 5-azacytidine. Certain embodiments herein provide salts, cocrystals, and/or solvates (e.g., hydrates) of the cytidine analogs provided herein. Certain embodiments herein provide salts and/or solvates (e.g. , hydrates) of the cytidine analogs provided herein. Certain embodiments provide cytidine analogs that are not salts, cocrystals, solvates (e.g., hydrates), or complexes of the cytidine analogs provided herein. For example, particular embodiments provide 5- azacytidine in a non-ionized, non-solvated (e.g., anhydrous), non-complexed form. Certain embodiments herein provide a mixture of two or more cytidine analogs provided herein.

[0059] Cytidine analogs provided herein may be prepared using synthetic methods and procedures referenced herein or otherwise available in the literature. For example, particular methods for synthesizing 5-azacytidine are disclosed, e.g., in U.S. Patent No. 7,038,038 and references discussed therein, each of which is incorporated herein by reference. Other cytidine analogs provided herein may be prepared, e.g. , using procedures known in the art, or may be purchased from a commercial source. In one embodiment, the cytidine analogs provided herein may be prepared in a particular solid form (e.g., amorphous or crystalline form). See, e.g., U.S. Patent Application No. 10/390,578, filed March 17, 2003 and U.S. Patent Application No. 10/390,530, filed March 17, 2003, both of which are incorporated herein by reference in their entireties.

[0060] In one embodiment, the compound used in the methods provided herein is a free base, or a pharmaceutically acceptable salt or solvate thereof. In one embodiment, the free base or the pharmaceutically acceptable salt or solvate is a solid. In another embodiment, the free base or the pharmaceutically acceptable salt or solvate is a solid in an amorphous form. In yet another embodiment, the free base or the pharmaceutically acceptable salt or solvate is a solid in a crystalline form. For example, particular embodiments provide 5-azacytidine in solid forms, which can be prepared, for example, according to the methods described in U.S. Patent Nos. 6,943,249, 6,887,855 and 7,078,518, and U.S. Patent Application Publication Nos. 2005/027675 and 2006/247189, each of which is incorporated by reference herein in their entireties. In other embodiments, 5-azacytidine in solid forms can be prepared using other methods known in the art.

[0061] In one embodiment, the compound used in the methods provided herein is a pharmaceutically acceptable salt of the cytidine analog, which includes, but is not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, 1 ,2-ethanedisulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate,

glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate (mesylate), 2-naphthalenesulfonate (napsylate), nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, or undecanoate salts.

5.3 Pharmaceutical Compositions

[0062] In one embodiment, provided herein are pharmaceutical compositions, which comprise one or more cytidine analogs, or a pharmaceutically acceptable salt or solvate thereof, as an active ingredient, in combination with one or more pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition comprises at least one nonrelease controlling excipient or carrier. In one embodiment, the pharmaceutical composition comprises at least one release controlling and at least one nonrelease controlling excipients or carriers.

[0063] In certain embodiments, the cytidine analog used in the pharmaceutical compositions provided herein is in a solid form. Suitable solid forms include, but are not limited to, solid forms comprising the free base of the cytidine analog, and solid forms comprising salts of the cytidine analog. In certain embodiments, solid forms provided herein include polymorphs, solvates (including hydrates), and cocrystals comprising the cytidine analog and/or salts thereof. In certain embodiments, the solid form is a crystal form of the cytidine analog, or a pharmaceutically acceptable salt or solvate thereof.

[0064] In one embodiment, the pharmaceutical compositions provided herein may be formulated in various dosage forms for oral, parenteral, and topical administration. The pharmaceutical compositions may also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsed-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, e.g., Remington, The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Modified-Release Drug Delivery Technology, Rathbone et al., eds., Drugs and the Pharmaceutical Science, Marcel Dekker, Inc.: New York, NY, 2003; Vol. 126).

[0065] In one embodiment, the pharmaceutical compositions are provided in a dosage form for oral administration. In another embodiment, the pharmaceutical compositions are provided in a dosage form for parenteral administration. In yet another embodiment, the pharmaceutical compositions are provided in a dosage form for topical administration.

[0066] In one embodiment, the pharmaceutical compositions provided herein may be provided in a unit-dosage form or multiple-dosage form. A unit-dosage form, as used herein, refers to a physically discrete unit suitable for administration to human and animal subjects, and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the active ingredient(s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carriers or excipients. Examples of a unit- dosage form include an ampoule, syringe, and individually packaged tablet and capsule. A unit-dosage form may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dosage form. Examples of a multiple-dosage form include a vial, bottle of tablets or capsules, or bottle of pints or gallons.

[0067] In one embodiment, the pharmaceutical compositions provided herein may be administered at once or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.

5.3.1 Oral Administration

[0068] In one embodiment, the pharmaceutical compositions provided herein may be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, capsules, pills, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, granules, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, solutions, wafers, sprinkles, elixirs, and syrups. In addition to the active ingredient(s), the pharmaceutical compositions may contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, and flavoring agents.

[0069] In one embodiment, binders or granulators impart cohesiveness to a tablet to ensure the tablet remaining intact after compression. Suitable binders or granulators include, but are not limited to, starches, such as corn starch, potato starch, and pre-gelatinized starch (e.g., STARCH 1500); gelatin; sugars, such as sucrose, glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as acacia, alginic acid, alginates, extract of Irish moss, panwar gum, ghatti gum, mucilage of isabgol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone (PVP), Veegum, larch arabogalactan, powdered tragacanth, and guar gum; celluloses, such as ethyl cellulose, cellulose acetate,

carboxymethyl cellulose calcium, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC); microcrystallme celluloses, such as AVICEL-PH-101, AVICEL-PH-103, AVICEL RC-581, AVICEL-PH-105 (FMC Corp., Marcus Hook, PA); and mixtures thereof. Suitable fillers include, but are not limited to, talc, calcium carbonate, microcrystallme cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre- gelatinized starch, and mixtures thereof. The binder or filler may be present from about 50 to about 99% by weight in the pharmaceutical compositions provided herein.

[0070] In one embodiment, suitable diluents include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, sorbitol, sucrose, inositol, cellulose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar. Certain diluents, such as mannitol, lactose, sorbitol, sucrose, and inositol, when present in sufficient quantity, can impart properties to some compressed tablets that permit disintegration in the mouth by chewing. Such compressed tablets can be used as chewable tablets.

[0071] In one embodiment, suitable disintegrants include, but are not limited to, agar; bentonite; celluloses, such as methylcellulose and carboxymethylcellulose; wood products; natural sponge; cation-exchange resins; alginic acid; gums, such as guar gum and Veegum HV; citrus pulp; cross-linked celluloses, such as croscarmellose; cross-linked polymers, such as crospovidone; cross-linked starches; calcium carbonate; microcrystallme cellulose, such as sodium starch glycolate; polacrilin potassium; starches, such as corn starch, potato starch, tapioca starch, and pre-gelatinized starch; clays; aligns; and mixtures thereof. The amount of a disintegrant in the pharmaceutical compositions provided herein varies upon the type of formulation, and is readily discernible to those of ordinary skill in the art. The pharmaceutical compositions provided herein may contain from about 0.5 to about 15% or from about 1 to about 5% by weight of a disintegrant.

[0072] In one embodiment, suitable lubricants include, but are not limited to, calcium stearate; magnesium stearate; mineral oil; light mineral oil; glycerin; sorbitol; mannitol; glycols, such as glycerol behenate and polyethylene glycol (PEG) (e.g. , PEG400 and PEG6000); stearic acid; sodium lauryl sulfate; talc; hydrogenated vegetable oil, including peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; zinc stearate; ethyl oleate; ethyl laureate; agar; starch; lycopodium; silica (silicone dioxide) or silica gels, such as AEROSIL® 200 (W.R. Grace Co., Baltimore, MD) and CAB-O-SIL® (Cabot Co. of Boston, MA); and mixtures thereof. The pharmaceutical compositions provided herein may contain about 0.1 to about 5% by weight of a lubricant.

[0073] In one embodiment, suitable glidants include colloidal silicon dioxide, CAB-O- SIL® (Cabot Co. of Boston, MA), and asbestos-free talc. Coloring agents include any of the approved, certified, water soluble FD&C dyes, and water insoluble FD&C dyes suspended on alumina hydrate, and color lakes and mixtures thereof. A color lake is the combination by adsorption of a water-soluble dye to a hydrous oxide of a heavy metal, resulting in an insoluble form of the dye. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic blends of compounds which produce a pleasant taste sensation, such as peppermint and methyl salicylate. Sweetening agents include sucrose, lactose, mannitol, syrups, glycerin, and artificial sweeteners, such as saccharin and aspartame.

Suitable emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants, such as polyoxyethylene sorbitan monooleate (e.g., TWEEN® 20), poloxamers (e.g., PLURONIC® F68), polyoxyethylene sorbitan monooleate 80 (e.g., TWEEN® 80), and triethanolamine oleate. Suspending and dispersing agents include sodium

carboxymethylcellulose, pectin, tragacanth, Veegum, acacia, sodium carbomethylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, and lauroyl polyoxylglycerides (e.g. , GELUCIRE® 44/14). Preservatives include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. [0074] In one embodiment, suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, β-cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether- -cyclodextrin, and sulfobutylether 7- -cyclodextrin (CAPTISOL^®, CyDex, Lenexa, KS).

[0075] It should be understood that many carriers and excipients may serve several functions, even within the same formulation.

[0076] In one embodiment, the pharmaceutical compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets. In one embodiment, enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach. Enteric- coatings include, but are not limited to, fatty acids, fats, phenyl salicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates. Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation. Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material. Film coatings include, but are not limited to, hydroxyethylcellulose, sodium

carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. In one embodiment, film coating imparts the same general characteristics as sugar coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.

[0077] In one embodiment, the tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled- release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

[0078] In one embodiment, the pharmaceutical compositions provided herein may be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate. The hard gelatin capsule, also known as the dry-filled capsule (DFC), consists of two sections, one slipping over the other, thus completely enclosing the active ingredient. The soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol. The soft gelatin shells may contain a preservative to prevent the growth of microorganisms. Suitable preservatives are those as described herein, including methyl- and propyl-parabens, and sorbic acid. The liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule. Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Patent Nos. 4,328,245; 4,409,239; and 4,410,545. The capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.

[0079] In one embodiment, the pharmaceutical compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups. An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in- water or water-in- oil. Emulsions may include a pharmaceutically acceptable non-aqueous liquid or solvent, emulsifying agent, and preservative. Suspensions may include a pharmaceutically acceptable suspending agent and preservative. Aqueous alcoholic solutions may include a

pharmaceutically acceptable acetal, such as a di(lower alkyl) acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxyl groups, such as propylene glycol and ethanol. Elixirs are clear, sweetened, and

hydroalcoholic solutions. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative. For a liquid dosage form, for example, a solution in a polyethylene glycol may be diluted with a sufficient quantity of a

pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.

[0080] In one embodiment, other useful liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient(s) provided herein, and a dialkylated mono- or poly-alkylene glycol, including, 1 ,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol. These formulations may further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates. [0081] In one embodiment, the pharmaceutical compositions provided herein may be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form. Pharmaceutically acceptable carriers and excipients used in the non- effervescent granules or powders may include diluents, sweeteners, and wetting agents.

Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.

[0082] Coloring and flavoring agents can be used in all of the above dosage forms.

[0083] In one embodiment, the pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

[0084] In one embodiment, the pharmaceutical compositions provided herein may be co- formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.

[0085] In one embodiment, active ingredients provided herein can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active agents provided herein. In one embodiment, provided are single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release.

[0086] In one embodiment, controlled-release pharmaceutical products improve drug therapy over that achieved by their non-controlled counterparts. In another embodiment, the use of a controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.

[0087] In another embodiment, the controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic or prophylactic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In one embodiment, in order to maintain a constant level of drug in the body, the drug can be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.

[0088] In one embodiment, provided herein is an oral formulation of a cytidine analog. See, e.g., U.S. Patent Application No. 12/466,213, filed May 14, 2009 and U.S. Patent Application No. 11/849,958, filed September 4, 2007, both of which are incorporated herein by reference in their entireties.

5.3.2 Parenteral Administration

[0089] In one embodiment, the pharmaceutical compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

[0090] In one embodiment, the pharmaceutical compositions provided herein may be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, e.g., Remington, The Science and Practice of Pharmacy, supra).

[0091] In one embodiment, the pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

[0092] In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl- 2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0093] In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, β-cyclodextrin,

hydroxypropyl- -cyclodextrin, sulfobutylether- -cyclodextrin, and sulfobutylether 7-β- cyclodextrin (CAPTISOL^®, CyDex, Lenexa, KS).

[0094] In one embodiment, the pharmaceutical compositions provided herein may be formulated for single or multiple dosage administration. The single dosage formulations are packaged in an ampoule, a vial, or a syringe. The multiple dosage parenteral formulations may contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.

[0095] In one embodiment, the pharmaceutical compositions are provided as ready-to-use sterile solutions. In another embodiment, the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use. In yet another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile suspensions. In yet another embodiment, the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use. In still another embodiment, the pharmaceutical compositions are provided as ready-to-use sterile emulsions.

[0096] In one embodiment, the pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.

[0097] In one embodiment, the pharmaceutical compositions may be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the pharmaceutical compositions provided herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.

[0098] In one embodiment, suitable inner matrixes include polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers,

polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.

[0099] In one embodiment, suitable outer polymeric membranes include polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyloxyethanol copolymer, and ethylene/vinyl acetate/vinyl alcohol terpolymer.

[00100] In specific embodiments, the pharmaceutical composition provided herein comprise 5-azacytidine and mannitol. In specific embodiments, the pharmaceutical composition provided herein is a lyophilized powder. In specific embodiments, the pharmaceutical composition provided herein is a lyophilized powder comprising 5- azacytidine and mannitol. In specific embodiments, the pharmaceutical composition provided herein comprises 5-azacytidine and mannitol with a relative weight ratio of about 1 : 1 w/w. In specific embodiments, the pharmaceutical composition provided herein comprises about 100 mg of 5-azacytidine. In specific embodiments, the pharmaceutical composition provided herein comprises about 100 mg of 5-azacytidine and about 100 mg of mannitol.

5.3.3 Topical Administration

[00101] In one embodiment, the pharmaceutical compositions provided herein may be administered rectally, urethrally, vaginally, or perivaginally in the forms of suppositories, pessaries, bougies, poultices or cataplasm, pastes, powders, dressings, creams, plasters, contraceptives, ointments, solutions, emulsions, suspensions, tampons, gels, foams, sprays, or enemas. These dosage forms can be manufactured using conventional processes as described in, e.g., Remington, The Science and Practice of Pharmacy, supra.

[00102] In one embodiment, rectal, urethral, and vaginal suppositories are solid bodies for insertion into body orifices, which are solid at ordinary temperatures but melt or soften at body temperature to release the active ingredient(s) inside the orifices. Pharmaceutically acceptable carriers utilized in rectal and vaginal suppositories include bases or vehicles, such as stiffening agents, which produce a melting point in the proximity of body temperature, when formulated with the pharmaceutical compositions provided herein; and antioxidants as described herein, including bisulfite and sodium metabisulfite. Suitable vehicles include, but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white and yellow wax, and appropriate mixtures of mono-, di- and triglycerides of fatty acids, hydrogels, such as polyvinyl alcohol, hydroxyethyl methacrylate, polyacrylic acid; glycerinated gelatin. Combinations of the various vehicles may be used. Rectal and vaginal suppositories may be prepared by the compressed method or molding. The typical weight of a rectal and vaginal suppository is about 2 to about 3 g.

[00103] In one embodiment, the pharmaceutical compositions provided herein may be administered intranasally or by inhalation to the respiratory tract. The pharmaceutical compositions may be provided in the form of an aerosol or solution for delivery using a pressurized container, pump, spray, atomizer, such as an atomizer using

electrohydrodynamics to produce a fine mist, or nebulizer, alone or in combination with a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. The pharmaceutical compositions may also be provided as a dry powder for insufflation, alone or in combination with an inert carrier such as lactose or phospholipids; and nasal drops. For intranasal use, the powder may comprise a bioadhesive agent, including chitosan or cyclodextrin.

[00104] In one embodiment, solutions or suspensions for use in a pressurized container, pump, spray, atomizer, or nebulizer may be formulated to contain ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active ingredient provided herein, a propellant as solvent; and/or a surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

[00105] In one embodiment, the pharmaceutical compositions provided herein may be micronized to a size suitable for delivery by inhalation, such as about 50 micrometers or less, or about 10 micrometers or less. Particles of such sizes may be prepared using a

comminuting method known to those skilled in the art, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[00106] In one embodiment, capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the pharmaceutical compositions provided herein; a suitable powder base, such as lactose or starch; and a performance modifier, such as Z-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate. Other suitable excipients or carriers include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. The pharmaceutical compositions provided herein for inhaled/intranasal administration may further comprise a suitable flavor, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium.

[00107] In one embodiment, the pharmaceutical compositions provided herein for topical administration may be formulated to be immediate release or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted, and programmed release.

5.3.4 Kits

[00108] In one embodiment, active ingredients provided herein are not administered to a patient at the same time or by the same route of administration. In another embodiment, provided are kits which can simplify the administration of appropriate amounts of active ingredients. [00109] In one embodiment, a kit comprises a dosage form of a compound provided herein. Kits can further comprise one or more second active ingredients as described herein, or a pharmacologically active mutant or derivative thereof, or a combination thereof.

[00110] In other embodiments, kits can further comprise devices that are used to administer the active ingredients. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers.

[00111] In one embodiment, kits can further comprise cells or blood for transplantation as well as pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

5.4 Method of Use

[00112] Without being limited to a particular theory, in one embodiment, 5-azacytidine is approved for treatment of patients with higher-risk MDS. In certain clinical studies, treatment with 5-azacytidine increases median overall survival by about 9.5 months in patients with higher-risk MDS in comparison to conventional care regimens (CCR). In these studies, the median overall survival for 5-azacytidine-treated patients having higher-risk MDS is about two years.

[00113] Without being limited to a particular theory, in one embodiment, development of biomarkers predictive of maximal clinical benefit with 5-azacytidine would allow

identification of those patients particularly suited for 5-azacytidine therapy. Thus, in one embodiment, provided herein are biomarkers that could be used, for example, in the management of therapeutic choices for patients with MDS. In other embodiments, provided herein is a method of using a biomarker provided herein in selecting cancer patients for a particular therapy, e.g., 5-azacytidine therapy for a particular cancer, to derive maximal clinical benefits from that therapy. [00114] Without being limited to a particular theory, DNA methylation plays an essential role in the regulation of gene expression. See, e.g., Esteller M., New England Journal of Medicine, 2008, 358: 1148-59. For example, CpG islands are generally unmethylated in normal cells, and demethylated state allows gene transcription. Methylation of CpG islands may be associated with gene silencing, including imprinted genes and X-chromosome inactivation. On the other hand, CpG sites outside CpG islands are generally methylated in normal cells, and methylation helps prevent mutations and genomic instability, and helps prevent recombination and activation of transposable elements. Large alternations in DNA methylation patterns are observed in most human cancers. CpG islands may become hypermethylated, while CpG sites outside of CpG islands may become hypomethylated. Thus, DNA methylation may be used as biomarkers for cancers or for MDS.

[00115] In one embodiment, provided herein are predictive biomarkers for assessing potential clinical benefit of a cancer therapy. In one embodiment, provided herein are predictive biomarkers for assessing potential clinical benefit of an MDS therapy. In one embodiment, provided herein are predictive biomarkers for assessing potential clinical benefit of 5-azacytidine therapy. In one embodiment, provided herein are methods of using a predictive biomarker provided herein {e.g., baseline patterns or levels of DNA methylation in pre-treatment bio-samples from patients). In one embodiment, the clinical benefit includes, but is not limited to, prolonged survival, delayed progression to AML, and/or other beneficial clinical responses. In one embodiment, the biomarker provided herein is nucleic acid methylation of one or more particular gene(s) or one or more particular locus/loci. In one embodiment, the biomarker provided herein is DNA methylation of one or more particular gene(s) or one or more particular locus/loci. In one embodiment, the predictive or response biomarkers provided herein include, but are not limited to, one or more of the following: DNA methylation, RNA methylation, previous LD AraC treatment, bone marrow blasts, abnormal karyotype, performance status, intermediate and poor risk cytogenetics, presence of circulating blasts, and/or RBC transfusion dependency. In one embodiment, the predictive or response biomarkers provided herein can be used to assess or predict response rate, overall survival, or other clinical responses.

[00116] In some embodiments, high baseline methylation of specific genes, such as, e.g., CDKN2B (pl5), SOCS1, CDH1, TP73, and/or CTNNA1, may be indicative of poor overall survival as compared to subjects with lower methylation levels.

[00117] In one embodiment, methylation pattern of specific group of genes may be indicative of better or worse overall survival after a particular therapeutic treatment. In one embodiment, provided herein is a methylation pattern of particular genes, selected from the group consisting of: ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orfi2, Cl9orfl0,

Clorfl 72, 01θΓβ7, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRCl 7, LTF, MBD3L1, MEGFIO, MICALl, MRPL28, MTMR9, MTNRIB, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLN1, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAPl, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINTl, SRD5A2, SRRT, SSTR4, STMNl, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

[00118] In one embodiment, provided herein are response biomarkers for assessing clinical benefit or predicting long-term clinical response, after the initiation of a 5-azacytidine treatment {e.g., assessing clinical benefit or potential long-term clinical response in a patient after or during treatment with 5-azacytidine). In one embodiment, provided herein are methods of using a response biomarker provided herein {e.g., changes in DNA methylation of specific genes). In one embodiment, the clinical benefit includes, but is not limited to, prolonged survival, delayed progression to AML, and/or other beneficial clinical responses. For example, DNA methylation in post-treatment samples may be compared to baseline samples {e.g., after a treatment cycle of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or greater than about 12 months; or after a treatment cycle of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38, about 40, about 42, about 44, about 46, about 48, about 50, about 52, about 54, about 56, or greater than about 56 weeks). In one embodiment, the methylation levels of particular genes, shown to be methylated in a high proportion at baseline, are monitored periodically after the initiation of a 5-azacytidine treatment.

[00119] In one embodiment, provided herein are biomarkers that could be used to predict which cancer patients will have the most, or least, clinical benefit from a particular cancer therapy. In one embodiment, the methods or biomarkers provided herein may be applied to cancers, such as, e.g., hematological cancers, blood-borne cancers, and/or solid cancers, or a type of cancer described herein elsewhere. See, e.g., International Patent Application No. PCT/US2010/000361, filed February 9, 2010, published as WO2010/093435, incorporated herein by reference in its entirety. In one embodiment, provided herein are biomarkers that could be used to predict which MDS patients will have the most, or least, clinical benefit from a particular MDS therapy, including a therapy for higher-risk MDS. In one

embodiment, the methods or biomarkers provided herein may be applied to MDS, e.g., higher-risk MDS. In one embodiment, the biomarkers provided herein are gene methylation biomarkers (e.g., DNA methylation of a particular gene or a particular locus). In other embodiments, methylation level or methylation pattern of other type(s) of nucleic acid(s), e.g., RNA methylation, may be used as a biomarker in a method described herein.

[00120] In one embodiment, provided herein is a method of using DNA methylation (e.g. , multiple loci or various single locus) as a pre-treatment biomarker to distinguish patients having potentially greater or lesser response(s) to a particular therapy. In one embodiment, provided herein is a method of using DNA methylation (e.g., multiple loci or various single locus) as a biomarker to predict or monitor the efficacy of a particular cancer treatment. In one embodiment, provided herein is a method of using DNA methylation (e.g., multiple loci or various single locus) as a biomarker to predict or monitor the efficacy of a particular MDS treatment. In one embodiment, provided herein is a method of using DNA methylation (e.g. , multiple loci or various single locus) as a biomarker to predict or monitor patient response to a particular cancer treatment. In one embodiment, provided herein is a method of using DNA methylation (e.g., multiple loci or various single locus) as a biomarker to predict or monitor patient response to a particular MDS treatment.

[00121] For example, bio-samples can be obtained from patients having a certain disease (e.g., bone marrow samples obtained from patients having higher-risk MDS; however, it is understood that other bio-samples, e.g., blood or tissue samples, may be used in a method provided herein). In one embodiment, DNA methylation levels of one or more gene(s) or locus/loci are measured for a particular patient and compared with reference values. In one embodiment, DNA methylation levels of a group of genes are measured for a particular patient and compared with one or more reference methylation pattern(s). In one embodiment, patients are grouped or selected based on DNA methylation level(s) of one or more gene(s) (e.g., a gene or a group of genes described herein elsewhere). In one embodiment, selected patients are further treated with a particular therapy to derive maximal response or clinical benefit, e.g., prolonged overall survival and/or time to AML transformation in MDS.

[00122] In specific embodiments, provided herein is a method of using DNA methylation of one or more gene(s) (e.g., multiple loci or various single locus) as a pre-treatment biomarker to distinguish MDS patients having potentially greater or lesser response to or overall survival benefit from 5-azacytidine therapy.

[00123] In one embodiment, bio-samples (e.g., unpurified bone marrow aspirates) are obtained from patients pre-treatment (e.g., from higher-risk MDS patients before receiving certain treatment). In one embodiment, DNA methylation level of one or more gene(s) provided herein is measured. In one embodiment, DNA methylation of particular gene(s) of a patient is compared with reference value(s). In one embodiment, DNA methylation of a particular group of genes is measured. In one embodiment, DNA methylation pattern of a particular group of genes of a patient is compared with reference pattern(s). In one embodiment, a particular methylation level or a particular methylation pattern is used to distinguish patients having potentially greater or lesser response to or overall survival benefit from a particular therapy (e.g., 5-azacytidine therapy). In one embodiment, a particular group of MDS patients selected based on a method provided herein is treated with 5-azacytidine.

[00124] In one embodiment, provided herein is a method of using gene methylation (e.g., DNA methylation of specific gene(s)) as a predictive biomarker of clinical response or overall survival in MDS patients (e.g., higher-risk MDS patients) treated with a cytidine analog provided herein. In one embodiment, provided herein is a method of using gene methylation as a predictive biomarker of clinical response or overall survival or time to AML transformation in higher-risk MDS patients treated with 5-azacytidine.

[00125] In one embodiment, provided herein is a method of using increased DNA methylation of one or more gene(s), including, but not limited to, CDKN2B (pi 5), SOCS1, CDH1 (E-cadherin) , TP73, and CTNNA1 (a-catenin) , as a predictive biomarker for worse overall survival in patients. In one embodiment, provided herein is a method of using lower levels of methylation of one or more gene(s), including, but not limited to, CDKN2B (pi 5), SOCS1, CDH1 (E-cadherin), TP73, and CTNNA1 (a-catenin), as a predictive biomarker for better overall survival benefit from 5-azacytidine therapy. In one embodiment, the biomarkers of the methods provided herein relate to DNA methylation of one or more gene(s), including, but not limited to, CDKN2B (pi 5), SOCS1, CDH1 (E-cadherin) , TP73, and CTNNA1 (a-catenin), in pre-treatment baseline bone marrows of MDS. In one embodiment, provided herein is a method of selecting patients having better predicted overall survival benefit using a biomarker provided herein. In one embodiment, the method further comprises administering a therapeutically effective amount of 5-azacytidine to the selected patients.

[00126] In one embodiment, provided herein is a method of using DNA methylation of one or more gene(s), including, but not limited to, ABHD14A, ABO, ADAMTS18, ADRA2B, AD KB 3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C orflO, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orfi7, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB,

CKMTIB, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPTIB, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALDl, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT,

PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP 5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710, as a predictive biomarker of better or worse overall survival in patients {e.g., after treatment with 5-azacytidine). In one embodiment, provided herein is a method of using DNA methylation of one or more genes(s), including, but not limited to, WT1, CDKN2B, and/or CDH1, as a predictive biomarker of better or worse overall survival in patients {e.g., after treatment with 5-azacytidine). In one embodiment, provided herein is a method of using DNA methylation of one or more genes(s), including, but not limited to, WT1, as a predictive biomarker of better or worse overall survival in patients {e.g., after treatment with 5- azacytidine).

[00127] In one embodiment, provided herein is a method of using DNA methylation of one or more gene(s), including, but not limited to, ABHD14A, ABO, ADAMTS18, ADRA2B, AD KB 3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C19orf30, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orfi7, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB,

PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMHl, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP 5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710, as a predictive biomarker of better or worse overall survival benefit from a drug therapy {e.g., administering a cytidine analog to a subject in need thereof, in one

embodiment, the cytidine analog is 5-azacytidine). In one embodiment, provided herein is a method of using DNA methylation of one or more gene(s), including, but not limited to, WT1, CDKN2B, and/or CDH1, as a predictive biomarker of better or worse overall survival benefit from a drug therapy {e.g., administering a cytidine analog to a subject in need thereof, in one embodiment, the cytidine analog is 5-azacytidine). In one embodiment, provided herein is a method of using DNA methylation of one or more gene(s), including, but not limited to, WT1, as a predictive biomarker of better or worse overall survival benefit from a drug therapy (e.g., administering a cytidine analog to a subject in need thereof, in one embodiment, the cytidine analog is 5-azacytidine).

[00128] In one embodiment, provided herein is a method of treating MDS, comprising determining DNA methylation of one or more gene(s), including, but not limited to,

ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C19orf30, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB I, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2,

HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINTl, SRD5A2, SRRT, SSTR4, STMNl, TBCIDI, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNTl, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710, in pre-treatment MDS patients (e.g., gene methylation in baseline bone marrows); and selecting patients having better predicted overall survival benefit. In one embodiment, provided herein is a method of treating MDS, comprising determining DNA methylation of one or more gene(s), including, but not limited to, WT1, CDKN2B, and/or CDH1, in pre-treatment MDS patients (e.g., gene methylation in baseline bone marrows); and selecting patients having better predicted overall survival benefit. In one embodiment, provided herein is a method of treating MDS, comprising determining DNA methylation of one or more gene(s), including, but not limited to, WT1, in pre-treatment MDS patients (e.g., gene methylation in baseline bone marrows); and selecting patients having better predicted overall survival benefit. In one embodiment, the method further comprises administering a therapeutically effective amount of a cytidine analog (e.g., 5-azacytidine) to the selected patients. In one embodiment, DNA methylation of one or more gene(s) is measured in patients after receiving drug treatment (e.g., 5-azacytidine treatment). In one embodiment, provided herein is a method of using DNA methylation of one or more genes(s) in treated patient as responsive biomarker to assess clinical response and/or predict long term clinical benefit (e.g., overall survival). In one embodiment, DNA methylation is used as biomarker in a method provided herein. In other embodiments, RNA methylation is used as biomarker in a method provided herein.

[00129] In one embodiment, the biomarkers provided herein comprise methylation of one or more gene(s), such as, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 genes, selected from the group consisting of ABHD14A, ABO,

ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C19orf30, Clorfl 72, C1orf87, C3orfl5, C1QTNF6,

C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427,

KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAPIGAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

[00130] In one embodiment, the biomarkers provided herein comprise methylation of one or more gene(s), such as, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, or more than 100 genes, selected from the group consisting of ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, AVP, BHMT, C18orf22, C19orf30, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FU44881, FLVCR2, FREQ, FZD9, GABl, GAS2L2, GATA4, GBGTl, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINTl, SRD5A2, SRRT, SSTR4, STMNl, TBCIDI, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNTl, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

5.4.1 Treatment, Prevention, and/or Management

[00131] In one embodiment, provided herein is a method of treating, preventing, or managing cancer, comprising measuring gene methylation of a patient having cancer, and administering a therapeutic agent {e.g., a cytidine analog, e.g. 5-azacytidine) to a patient in need thereof. In one embodiment, the cancer is a blood-borne tumor. In one embodiment, the cancer is a solid tumor.

[00132] In one embodiment, provided herein is a method of treating, preventing, or managing MDS, comprising measuring gene methylation of a patient having MDS, and administering a therapeutic agent {e.g., a cytidine analog, e.g. 5-azacytidine) to a patient in need thereof. In one embodiment, the MDS is higher-risk MDS.

[00133] In one embodiment, patients are screened prior to enrollment in a clinical study or prior to treatment by a physician, for DNA or RNA methylation levels. In one embodiment, patients are monitored during a clinical study or during a treatment course, for DNA or RNA methylation levels. In one embodiment, DNA or R A methylation levels of particular gene(s) (e.g. , at particular loci/locus) are indicative of a potential response to a treatment (e.g., treatment comprising a cytidine analog, e.g., 5-azacytidine).

[00134] Embodiments further provided herein are methods of treatments with a

pharmaceutical composition comprising a cytidine analog, particularly, 5-azacytidine, providing particular benefit to the population of patients stratified into the higher risk groups of myelodysplastic syndromes (MDS) by conventional scoring systems, as measured by improved survival of this population upon treatment with a cytidine analog, e.g., 5- azacytidine. See, e.g., U.S. Patent Application No. 12/740,636, filed November 3, 2008, incorporated herein by reference in its entirety.

[00135] Accordingly, in one embodiment, provided herein is a method of treating a patient diagnosed with a higher risk MDS, the method comprising treating the patient diagnosed with a higher risk MDS with an effective amount of a composition comprising a cytidine analog. In one embodiment, the method further comprises the step of selecting patients using a predictive biomarker provided herein elsewhere, and treating the selected patients with an effective amount of a composition comprising a cytidine analog (e.g., 5-azacytidine).

[00136] In one embodiment, the cytidine analog includes any moiety which is structurally related to cytidine or deoxycytidine and functionally mimics and/or antagonizes the action of cytidine or deoxycytidine. These analogs may also be called cytidine derivatives herein. In one embodiment, cytidine analog includes 5 -aza-2'-deoxy cytidine (decitabine), 5-azacytidine, 5-aza-2'-deoxy-2',2'-difluorocytidine, 5-aza-2'-deoxy-2'-fluorocytidine, 2'-deoxy-2',2'- difluorocytidine (also called gemcitabine), or cytosine Ι-β-D-arabinofuranoside (also called ara-C), 2(lH)-pyrimidine-riboside (also called zebularine), 2'-cyclocytidine, arabinofuanosyl- 5-azacytidine, dihydro-5-azacytidine, N⁴-octadecyl-cytarabine, and elaidic acid cytarabine. In one embodiment, cytidine analog includes 5-azacytidine and 5 -aza-2'-deoxy cytidine. The definition of cytidine analog used herein also includes mixtures of cytidine analogs.

[00137] Cytidine analogs may be synthesized by methods known in the art. In one embodiment, methods of synthesis include methods as disclosed in U.S. Serial No.

10/390,526 (U.S. Patent No. 7,038,038); U.S. Serial No. 10/390,578 (U.S. Patent No.

6,887,855); U.S. Serial No. 11/052615 (U.S. Patent No. 7,078,518); U.S. Serial No.

10390530 (U.S. Patent No. 6,943,249); and U.S. Serial No. 10/823,394, all incorporated by reference herein in their entireties. [00138] In one embodiment, an effective amount of a cytidine analog to be used is a therapeutically effective amount. In one embodiment, the amounts of a cytidine analog to be used in the methods provided herein and in the oral formulations include a therapeutically effective amount, typically, an amount sufficient to cause improvement in at least a subset of patients with respect to symptoms, overall course of disease, or other parameters known in the art. Therapeutic indications are discussed more fully herein below. Precise amounts for therapeutically effective amounts of the cytidine analog in the pharmaceutical compositions will vary depending on the age, weight, disease, and condition of the patient. For example, pharmaceutical compositions may contain sufficient quantities of a cytidine analog to provide a daily dosage of about 10 to 150 mg/m² (based on patient body surface area) or about 0.1 to 4 mg/kg (based on patient body weight) as single or divided (2-3) daily doses. In one embodiment, dosage is provided via a seven-day administration of 75 mg/m² subcutaneously, once every twenty-eight days, for as long as clinically necessary. In one embodiment, dosage is provided via a seven-day administration of 100 mg/m² subcutaneously, once every twenty- eight days, for as long as clinically necessary. In one embodiment, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or more 28-day cycles are administered. Other methods for providing an effective amount of a cytidine analog are disclosed in, for example, "Colon-Targeted Oral Formulations of Cytidine Analogs", U.S. Serial No. 11/849,958, and "Oral Formulations of Cytidine Analogs and Methods of Use Thereof, U.S. Serial No. 12/466,213, both of which are incorporated by reference herein in their entireties.

[00139] Hematologic disorders include abnormal growth of blood cells which can lead to dysplastic changes in blood cells and hematologic malignancies such as various leukemias. Examples of hematologic disorders include but are not limited to acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, the myelodysplastic syndromes, and sickle cell anemia.

[00140] Acute myeloid leukemia (AML) is the most common type of acute leukemia that occurs in adults. Several inherited genetic disorders and immunodeficiency states are associated with an increased risk of AML. These include disorders with defects in DNA stability, leading to random chormosomal breakage, such as Bloom's syndrome, Fanconi's anemia, Li-Fraumeni kindreds, ataxia-telangiectasia, and X-linked agammaglobulinemia.

[00141] Acute promyelocytic leukemia (APML) represents a distinct subgroup of AML. This subtype is characterized by promyelocytic blasts containing the 15; 17 chromosomal translocation. This translocation leads to the generation of the fusion transcript comprised of the retinoic acid receptor and a sequence PML. [00142] Acute lymphoblastic leukemia (ALL) is a heterogenerous disease with distinct clinical features displayed by various subtypes. Reoccurring cytogenetic abnormalities have been demonstrated in ALL. The most common cytogenetic abnormality is the 9;22 translocation. The resultant Philadelphia chromosome represents poor prognosis of the patient.

[00143] Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of a pluripotent stem cell. CML is characterized by a specific chromosomal abnormality involving the translocation of chromosomes 9 and 22, creating the Philadelphia chromosome. Ionizing radiation is associated with the development of CML.

[00144] The myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell disorders grouped together, because of the presence of dysplastic changes in one or more of the hematopoietic lineages including dysplastic changes in the myeloid, erythroid, and megakaryocytic series. These changes result in cytopenias in one or more of the three lineages. Patients afflicted with MDS typically develop complications related to anemia, neutropenia (infections), or thrombocytopenia (bleeding). Generally, from about 10% to about 70% of patients with MDS develop acute leukemia. MDS affects approximately 40,000-50,000 people in the U.S. and 75,000-85,000 patients in Europe. The majority of people with higher risk MDS eventually experience bone marrow failure. Up to 50% of MDS patients succumb to complications, such as infection or bleeding, before progressing to acute myeloid leukemia (AML). MDS patients have a median survival of four months to five years depending on risk stratification. Higher risk patients have a median survival of five to 14 months. Altering the natural history of the disease and providing increased survival is one of the most important treatment goals in higher risk MDS.

[00145] In one embodiment, MDS is a condition to be treated with methods provided herein, and includes the following MDS subtypes: refractory anemia, refractory anemia with ringed sideroblasts (if accompanied by neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia. In another embodiment, the condition to be treated is higher risk MDS.

[00146] In classifying a patient's disease as "higher risk MDS" (also referred to herein as, e.g., "higher-risk MDS," "high risk MDS" and "high-risk MDS"), methods known in the art can be used by the skilled person in order to classify a patient's disease as "higher risk" MDS. Such methods include, e.g., the FAB system, the WHO system, and IPSS, as discussed herein below {See, e.g., Bennett J.M., A comparative review of classification systems in myelodysplastic syndromes (MDS), Semin. Oncol. 2005 Aug; 32(4 Suppl 5):S3- 10; Bennett et al, Br. J. Haematol. 1982, 51 : 189-99; Harris et al, J. Clin. Oncol. 1999, 17(12):3835-49; Greenberg et al, Blood 1997, 89(6), 2079-98). Other methods for such assessment may lie within the knowledge or expertise of the skilled person, and methods provided herein include such a skilled person's assessment.

[00147] The skilled person knows that experience has shown that certain disease factors affect a person's prognosis— his or her chances of long-term survival and risk of developing AML. Researchers use these factors to classify MDS into types. In one embodiment, the system to classify MDS is the FAB system, so-called because it was developed by a team of French, American and British researchers. In the FAB system, there are five types of MDS. The FAB system uses several disease factors to classify MDS. One important factor is the percent of blasts in the bone marrow (Table 1). A higher percent of blasts is linked to a higher likelihood of developing AML and a poorer prognosis. The two more common types of MDS are refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS). These are also the less severe forms of MDS. They have a lower risk of turning into AML. Some patients with these forms of MDS may live with few symptoms and need little treatment for many years.

[00148] The other types of MDS tend to be more severe and more difficult to treat successfully. The refractory anemia with excess blasts (RAEB) and refractory anemia with excess blasts in transformation (RAEB-t) forms of MDS also have a high risk of turning into AML.

[00149] In another embodiment, a system for defining types of MDS is the newer World Health Organization (WHO) system which divides MDS into eight types. (See, e.g., Muller- Berndorff, et ah, Ann. Hematol. 2006 Aug; 85(8):502-13.) In certain embodiments, a skilled person may use either the FAB or WHO system to determine the type of MDS .

[00150] In another embodiment, individual prognosis is determined using the international prognostic scoring system (IPSS). The IPSS risk score describes the risk that a person's disease will develop into AML or become life-threatening. A doctor may use the IPSS risk score along with the MDS type to plan treatment. The IPSS risk score is based on three factors that have been shown to affect a patient's prognosis:

(1) The percent of cells in the bone marrow that are blasts.

(2) Whether one, two or all three types of blood cells are low (also called cytopenias). The three types are red blood cells, white blood cells, and platelets.

(3) Changes in the chromosomes of bone marrow blood cells. This may be called cytogenetics (the study of chromosome abnormalities). It may also be called the karyotype (a picture of the chromosomes that shows whether they are abnormal).

[00151] A person may have an IPSS risk score of low, intermediate- 1, intermediate-2 or high risk. Doctors can use the risk score to plan treatment. Someone with low-risk disease may be likely to survive for years with few symptoms. That person may need less intense treatment. Someone with intermediate- 1, intermediate-2 or high-risk disease may be likely to survive only if he or she receives aggressive treatment, such as a transplant.

[00152] In one embodiment, a higher risk patient is treated by the methods provided herein. In one embodiment, a patient defined as a higher risk MDS patient includes those whose disease is assessed as any one or more of the following: RAEB, RAEB-T, or CMML (10-29% marrow blasts) under FAB or with an IPSS of Intermediate-2 or High.

[00153] In one embodiment, dosing schedules for the compositions and methods provided herein, for example, can be adjusted to account for the patient's characteristics and disease status. Appropriate dose will depend on the disease state being treated. In some cases, dosing schedules include daily doses, and in others, selected days of a week, month or other time interval. In one embodiment, the drug will not be given more than once per day. In one embodiment, dosing schedules for administration of pharmaceutical compositions include the daily administration to a patient in need thereof. Dosing schedules may mimic those that are used for non-oral formulations of a cytidine analog, adjusted to maintain, for example, substantially equivalent therapeutic concentration in the patient's body. [00154] In certain embodiments, appropriate biomarkers may be used to evaluate the drug's effects on the disease state and provide guidance to the dosing schedule. For example, particular embodiments herein provide a method of determining whether a patient diagnosed with MDS has an increased probability of obtaining a greater benefit from treatment with a cytidine analog by assessing the patient's nucleic acid methylation status. In particular embodiments, the cytidine analog is 5-azacytidine. In particular embodiments, the nucleic acid is DNA or R A. In particular embodiments, the greater benefit is an overall survival benefit. In particular embodiments, the methylation status is examined in one or more genes, e.g., genes associated with MDS or AML. In particular embodiments, the methylation status is examined in one or more genes, e.g., genes described herein elsewhere. Specific embodiments involve methods for determining whether baseline DNA methylation levels influence overall survival in patients with MDS (e.g., higher risk MDS) treated with 5- azacytidine. Specific embodiments provide methods for determining whether gene methylation levels influence overall survival in patients with MDS (e.g., higher risk MDS). Specific embodiments provide methods for determining whether gene promoter methylation levels influence overall survival in patients with MDS (e.g., higher risk MDS).

[00155] For example, specific embodiments herein provide methods for evaluating the influence of gene methylation on prolonged survival in patients with MDS (e.g., higher risk MDS). In particular embodiments, such evaluation is used to predict overall survival in patients with MDS (e.g., higher risk MDS), e.g., upon treatment with 5-azacytidine. In particular embodiments, such evaluation is used for therapeutic decision-making. In specific embodiments, such therapeutic decision-making includes planning or adjusting a patient's treatment, e.g., the dosing regimen, amount, and/or duration of 5-azacytidine administration.

[00156] Certain embodiments provide methods of identifying individual patients diagnosed with MDS having an increased probability of obtaining an overall survival benefit from 5-azacytidine treatment, using analysis of methylation levels, e.g., in particular genes.

[00157] In some embodiments, lower levels of nucleic acid methylation (e.g., of certain genes) are associated with an increased probability of obtaining improved overall survival following 5-azacytidine treatment.

[00158] In some embodiments, particular patterns of nucleic acid methylation (e.g., particular gene methylation signature) are associated with an increased probability of obtaining improved overall survival following 5-azacytidine treatment.

[00159] In particular embodiments, the increased probability of obtaining improved overall survival following 5-azacytidine treatment is at least a 5% greater probability, at least a 10% greater probability, at least a 20% greater probability, at least a 30% greater probability, at least a 40% greater probability, at least a 50% greater probability, at least a 60% greater probability, at least a 70% greater probability, at least an 80% greater

probability, at least a 90% greater probability, at least at least a 100% greater probability, at least a 125% greater probability, at least a 150% greater probability, at least a 175% greater probability, at least a 200% greater probability, at least a 250% greater probability, at least a 300% greater probability, at least a 400% greater probability, or at least a 500% greater probability of obtaining improved overall survival following 5-azacytidine treatment. In particular embodiments, the greater probability of obtaining improved overall survival following 5-azacytidine treatment is a greater probability as compared to the average probability of a particular comparison population of patients diagnosed with MDS. In specific embodiments, the comparison population is a group of patients classified with a particular myelodysplastic subtype, as described herein. In one embodiment, the comparison population consists of patients having higher risk MDS. In particular embodiments, the comparison population consists of a particular IPSS cytogenetic subgroup.

[00160] In particular embodiments, nucleic acid (e.g., DNA or RNA) hypermethylation status may be determined by any method known in the art. In certain embodiments, DNA hypermethylation status may be determined using the bone marrow aspirates of patients diagnosed with MDS, e.g., by using quantitative real-time methylation specific PCR

("qMSP"). In certain embodiments, the methylation analysis may involve bisulfite conversion of genomic DNA. For example, in certain embodiments, bisulfite treatment of DNA is used to convert non-methylated CpG sites to UpG, leaving methylated CpG sites intact. See, e.g., Frommer, M., et al., Proc. Nat Ί Acad. Sci. USA 1992, 89: 1827-31.

Commercially available kits may be used for such bisulfite treatment. In certain

embodiments, to facilitate methylation PCR, primers are designed as known in the art, e.g. , outer primers which amplify DNA regardless of methylation status, and nested primers which bind to methylated or non-methylated sequences within the region amplified by the first PCR. See, e.g., Li et al, Bioinformatics 2002, 18: 1427-31. In certain embodiments, probes are designed, e.g., probes which bind to the bisulfite-treated DNA regardless of methylation status. In certain embodiments, CpG methylation is detected, e.g., following PCR

amplification of bisulfite-treated DNA using outer primers. In certain embodiments, amplified product from the initial PCR reaction serves as a template for the nested PCR reaction using methylation- specific primers or non-methylation-specific primers. In certain embodiments, a standard curve is established to determine the percentage of methylated molecules in a particular sample. Methods for detecting nucleic acid methylation (e.g., RNA or DNA methylation) are known in art. See, e.g., Laird, P.W., Nature Rev. Cancer 2003, 3:253-66; Belinsky, S.A., Nature Rev. Cancer 2004, 4: 1-11.

[00161] In certain embodiments, statistical analyses are performed to assess the influence of particular methylation levels with the potential benefit of treatment with a particular cytidine analog. In certain embodiments, the influence of methylation on overall survival is assessed, e.g. , using Cox proportional hazards models and Kaplan-Meier (KM) methodology.

[00162] In one embodiment, a gene associated with MDS and/or AML may be examined for its methylation status in a patient. In one embodiment, particular genes include, but are not limited to, CDKN2B (pi 5), SOCS1, CDH1 (E-cadherin) , TP73, and CTNNA1 (alpha- catenin). In some embodiments, some of the genes associated with MDS and/or AML, which may be suitable for use in the methods disclosed here, are known in the art.

[00163] In one embodiment, particular genes that may be examined for its methylation status in a patient, include, but are not limited to, ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C orflO, Clorfl 72, Clorfi7, C3orfl5, C1QTNF6, C22orfi7, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB,

PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMD11, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP 5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710. In one embodiment, particular genes that may be examined for its methylation status in a patient, include, but are not limited to, WT1, CDKN2B, and CDH1. In one embodiment, particular gene that may be examined for its methylation status in a patient, includes, but is not limited to, WT1. In certain embodiments, particular gene methylation patterns or methylation signatures as disclosed herein elsewhere are suitable for use in the methods disclosed herein.

[00164] In another embodiment, provided herein is a method of selecting a patient diagnosed with MDS for treatment with 5-azacytidine, comprising assessing a patient diagnosed with MDS for having a particular gene methylation profile or pattern, and selecting a patient for treatment with 5-azacytidine where the patient's gene methylation profile is predicted or assessed as having greater clinical response to 5-azacytidine {e.g., prolonged overall survival or prolonged time to AML transformation). In another embodiment, provided herein is a method to improve survival in a patient population with higher risk MDS, the method comprising treating at least one patient diagnosed with a higher risk MDS with an effective amount of a composition comprising a cytidine analog. In one embodiment, the method comprises selecting patients based on their gene methylation profiles, prior to the initiation of treatment.

[00165] Certain embodiments herein provide methods for the treatment of MDS. In certain embodiments, the methods comprise providing for the survival of an MDS patient beyond a specific period of time by administering a specific dose of 5-azacytidine for at least a specific number of cycles of 5-azacytidine treatment. In particular embodiments, the contemplated specific period of time for survival is, e.g., beyond 10 months, beyond 11 months, beyond 12 months, beyond 13 months, beyond 14 months, beyond 15 months, beyond 16 months, beyond 17 months, beyond 18 months, beyond 19 months, or beyond 20 months. In particular embodiments, the contemplated specific number of cycles administered is, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, at least 40, at least 42, at least 44, at least 46, at least 48, or at least 50 cycles of 5-azacytidine treatment. In particular embodiments, the contemplated treatment is administered, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days out of a 28-day period. In particular embodiments, the contemplated specific 5-azacytidine dose is, e.g., at least at least 10 mg/day, at least 20 mg/day, at least 30 mg/day, at least 40 mg/day, at least 50 mg/day, at least 55 mg/day, at least 60 mg/day, at least 65 mg/day, at least 70 mg/day, at least 75 mg/day, at least 80 mg/day, at least 85 mg/day, at least 90 mg/day, at least 95 mg/day, or at least 100 mg/day. In particular embodiments, the dosing is performed, e.g., subcutaneously or intravenously. In particular embodiments, the contemplated specific 5-azacytidine dose is, e.g., at least 50 mg/m²/day, at least 60 mg/m²/day, at least 70 mg/m²/day, at least 75 mg/m²/day, at least 80 mg/m²/day, at least 90 mg/m²/day, or at least 100 mg/m²/day. One particular embodiment herein provides a method for obtaining the survival of an MDS patient beyond 15 months by administering at least 9 cycles of 5-azacytidine treatment. One particular embodiment herein provides administering the treatment for 7 days out of each 28- day period. One particular embodiment herein provides a dosing regimen of 75 mg/m² subcutaneously or intravenously, daily for 7 days. One particular embodiment herein provides a dosing regimen of 100 mg/m² subcutaneously or intravenously, daily for 7 days.

[00166] Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with surgery. Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with chemotherapy. Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with immunotherapy. Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with targeted therapy. Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with radiation therapy. Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with two or more of the treatments selected from surgery, chemotherapy, immunotherapy, targeted therapy, and radiation therapy.

Particular embodiments provide treating a subject having MDS using one or more of the methods provided herein, together with two or more of the treatments selected from chemotherapy, immunotherapy, radiation, and targeted therapy.

[00167] In certain embodiments, the subject to be treated with one of the methods provided herein has not been treated with an anticancer or anti-MDS therapy prior to the administration of the cytidine analog. In certain embodiments, the subject to be treated with one of the methods provided herein has been treated with one or more anticancer or anti- MDS therapies prior to the administration of the cytidine analog. In certain embodiments, the subject to be treated with one of the methods provided herein has been treated with a cancer therapeutic agent or a MDS therapeutic agent. In certain embodiments, the subject to be treated with one of the methods provided herein has developed drug resistance to anticancer or anti-MDS therapy. In certain embodiments, the subject to be treated with the methods provided herein has a relapsed cancer. In certain embodiments, the subject to be treated with the methods provided herein has a refractory cancer. In certain embodiments, the subject to be treated with the methods provided herein has a metastatic cancer. In certain embodiments, the subject to be treated with the methods provided herein has a high-risk MDS. In certain embodiments, the subject to be treated with the methods provided herein has a higher-risk MDS.

[00168] In one embodiment, the methods provided herein encompass treating a subject regardless of patient's age, although some diseases or disorders are more common in certain age groups. Further provided herein is a method for treating a subject who has undergone surgery in an attempt to treat the disease or condition at issue. Further provided herein is a method for treating a subject who has not undergone surgery as an attempt to treat the disease or condition at issue. In some embodiments, because the subjects with cancer have heterogeneous clinical manifestations and varying clinical outcomes, the treatment given to a particular subject may vary, depending on his/her prognosis. In some embodiments, the skilled clinician may be able to readily determine without undue experimentation, specific secondary agents, types of surgery, and types of non-drug based standard therapy that can be effectively used to treat an individual subject with cancer or MDS.

[00169] In each embodiment provided herein, the method may further comprise one or more diagnostic steps, to determine, e.g., the type of MDS or cancer, the presence of particular gene methylation profile, and/or the staging of the disease in a subject.

[00170] In each embodiment provided herein, the method may further comprise a disease evaluation step after the cytidine analog has been administered to the subject, to determine, e.g., changes in one or more molecular markers as described herein elsewhere, and/or other benchmarks used by those skilled in the art to determine the prognosis of MDS in a subject. In one embodiment, the evaluation step is carried out after a treatment period or treatment cycle {e.g. , time from administration of first dose) of about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7, weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 16 weeks, about 18 weeks, about 20 weeks, about 22 weeks, about 24 weeks, about 26 weeks, about 28 weeks, about 30 weeks, about 32 weeks, about 34 weeks, about 36 weeks, about 38 weeks, about 40 weeks, about 42 weeks, about 44 weeks, about 46 weeks, about 48 weeks, about 50 weeks, or greater than 50 weeks. In one embodiment, the evaluation step may be repeated periodically (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 24, or greater than 24 weeks).

[00171] In certain embodiments, appropriate biomarkers may be used to determine or predict the effect of the methods provided herein on the disease state and to provide guidance as to the dosing schedule. For example, particular embodiments herein provide a method for determining whether a patient diagnosed with MDS has an increased probability of obtaining a greater benefit from treatment with a pharmaceutical composition comprising a cytidine analog by assessing the patient's nucleic acid methylation status. In particular embodiments, the cytidine analog is 5-azacytidine. In particular embodiments, the nucleic acid is DNA or RNA. In particular embodiments, the greater benefit is an overall survival benefit. In particular embodiments, the methylation status is examined in one or more genes, e.g., genes associated with MDS, or genes described herein elsewhere. Specific embodiments involve methods for determining whether baseline DNA methylation levels influence overall survival in patients with MDS treated with 5-azacytidine. Specific embodiments provide methods for determining whether gene promoter methylation levels influence overall survival in patients with MDS.

[00172] In one embodiment, provided herein is a method for determining whether a patient diagnosed with MDS has an increased probability of obtaining a greater benefit from treatment with a pharmaceutical composition comprising a cytidine analog by assessing the gene expression profile in the patient. In one embodiment, provided herein is a method for determining whether a patient diagnosed with MDS has an increased probability of obtaining a greater benefit from treatment with a pharmaceutical composition comprising a cytidine analog by assessing molecular markers, including one or more cell cycle markers, apoptosis markers, and DNA damage markers. In one embodiment, provided herein is a method for determining whether a patient diagnosed with MDS has increased probability of obtaining a greater benefit from treatment with a pharmaceutical composition comprising a cytidine analog by assessing methylation of one or more gene(s), including, but not limited to, ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orf22, C orflO, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB I, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2,

HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRC17, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNR1B, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINTl, SRD5A2, SRRT, SSTR4, STMNl, TBCIDI, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNTl, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and/or ZNF710. In one embodiment, provided herein is a method for determining whether a patient diagnosed with MDS has increased probability of obtaining a greater benefit from treatment with a pharmaceutical composition comprising a cytidine analog by assessing methylation of one or more gene(s), including, but not limited to, WT1. In particular embodiments, the cytidine analog is 5-azacytidine. In particular embodiments, the greater benefit is an overall survival benefit.

5.4.2 Administration of Cytidine Analogs

[00173] Certain methods herein provide administration of the cytidine analog by, e.g., intravenous (IV), subcutaneous (SC) or oral routes administration. Certain embodiments herein provide co-administration of a cytidine analog (e.g., 5-azacytidine) with one or more additional active agents to provide a synergistic therapeutic effect in subjects in need thereof. The co-administered agent(s) may be a cancer therapeutic agent, as described herein. In certain embodiments, the co-administered agent(s) may be dosed, e.g., orally or by injection (e.g., IV or SC).

[00174] Certain embodiments herein provide methods for treating disorders of abnormal cell proliferation comprising administering a cytidine analog using, e.g., IV, SC and/or oral administration methods. In certain embodiments, treatment cycles comprise multiple doses administered to a subject in need thereof over multiple days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or greater than 14 days), optionally followed by treatment dosing holidays (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or greater than 28 days). Suitable dosage amounts for the methods provided herein include, e.g., therapeutically effective amounts and prophylactically effective amounts. For example, in certain embodiments, the amount of the cytidine analog (e.g., 5-azacytidine) administered in the methods provided herein may range, e.g., between about 50 mg/m²/day and about 2,000 mg/m²/day, between about 100 mg/m²/day and about 1,000 mg/m²/day, between about 100 mg/m²/day and about 500 mg/m²/day, between about 50 mg/m²/day and about 500 mg/m²/day, between about 50 mg/m²/day and about 200 mg/m²/day, between about 50 mg/m²/day and about 100 mg/m²/day, between about 50 mg/m²/day and about 75 mg/m²/day, or between about 120 mg/m²/day and about 250 mg/m²/day. In certain embodiments, particular dosages are, e.g., about 50 mg/m²/day, about 60 mg/m²/day, about 75 mg/m²/day, about 80 mg/m²/day, about 100 mg/m²/day, about 120 mg/m²/day, about 140 mg/m²/day, about 150 mg/m²/day, about 180 mg/m²/day, about 200 mg/m²/day, about 220 mg/m²/day, about 240 mg/m²/day, about 250 mg/m²/day, about 260 mg/m²/day, about 280 mg/m²/day, about 300 mg/ m²/day, about 320 mg/m²/day, about 350 mg/m²/day, about 380 mg/m²/day, about 400 mg/m²/day, about 450 mg/m²/day, or about 500 mg/m²/day. In certain embodiments, particular dosages are, e.g., up to about 100 mg/m²/day, up to about 120 mg/m²/day, up to about 140 mg/m²/day, up to about 150 mg/m²/day, up to about 180 mg/m²/day, up to about 200 mg/m²/day, up to about 220 mg/m²/day, up to about 240 mg/m²/day, up to about 250 mg/m²/day, up to about 260 mg/m²/day, up to about 280 mg/m²/day, up to about 300 mg/ m²/day, up to about 320 mg/m²/day, up to about 350 mg/m²/day, up to about 380 mg/m²/day, up to about 400 mg/m²/day, up to about 450 mg/m²/day, up to about 500 mg/m²/day, up to about 750 mg/m²/day, or up to about 1000 mg/m²/day.

[00175] In one embodiment, the amount of the cytidine analog (e.g., 5-azacytidine) administered in the methods provided herein may range, e.g., between about 5 mg/day and about 2,000 mg/day, between about 10 mg/day and about 2,000 mg/day, between about 20 mg/day and about 2,000 mg/day, between about 50 mg/day and about 1,000 mg/day, between about 100 mg/day and about 1,000 mg/day, between about 100 mg/day and about 500 mg/day, between about 150 mg/day and about 500 mg/day, or between about 150 mg/day and about 250 mg/day. In certain embodiments, particular dosages are, e.g., about 10 mg/day, about 20 mg/day, about 50 mg/day, about 75 mg/day, about 100 mg/day, about 120 mg/day, about 150 mg/day, about 200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day, about 400 mg/day, about 450 mg/day, about 500 mg/day, about 600 mg/day, about 700 mg/day, about 800 mg/day, about 900 mg/day, about 1,000 mg/day, about 1,200 mg/day, or about 1,500 mg/day. In certain embodiments, particular dosages are, e.g., up to about 10 mg/day, up to about 20 mg/day, up to about 50 mg/day, up to about 75 mg/day, up to about 100 mg/day, up to about 120 mg/day, up to about 150 mg/day, up to about 200 mg/day, up to about 250 mg/day, up to about 300 mg/day, up to about 350 mg/day, up to about 400 mg/day, up to about 450 mg/day, up to about 500 mg/day, up to about 600 mg/day, up to about 700 mg/day, up to about 800 mg/day, up to about 900 mg/day, up to about 1,000 mg/day, up to about 1,200 mg/day, or up to about 1,500 mg/day.

[00176] In one embodiment, the amount of the cytidine analog (e.g., 5-azacytidine) in the pharmaceutical composition or dosage form provided herein may range, e.g. , between about 5 mg and about 2,000 mg, between about 10 mg and about 2,000 mg, between about 20 mg and about 2,000 mg, between about 50 mg and about 1,000 mg, between about 50 mg and about 500 mg, between about 50 mg and about 250 mg, between about 100 mg and about 500 mg, between about 150 mg and about 500 mg, or between about 150 mg and about 250 mg. In certain embodiments, particular amounts are, e.g., about 10 mg, about 20 mg, about 50 mg, about 75 mg, about 100 mg, about 120 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,200 mg, or about 1,500 mg. In certain embodiments, particular amounts are, e.g., up to about 10 mg, up to about 20 mg, up to about 50 mg, up to about 75 mg, up to about 100 mg, up to about 120 mg, up to about 150 mg, up to about 200 mg, up to about 250 mg, up to about 300 mg, up to about 350 mg, up to about 400 mg, up to about 450 mg, up to about 500 mg, up to about 600 mg, up to about 700 mg, up to about 800 mg, up to about 900 mg, up to about 1,000 mg, up to about 1,200 mg, or up to about 1,500 mg.

[00177] In one embodiment, depending on the disease to be treated and the subject's condition, the cytidine analog (e.g., 5-azacytidine) may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, CIV, intracistemal injection or infusion, subcutaneous injection, or implant), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g. , transdermal or local) routes of administration. The cytidine analog may be formulated, alone or together with one or more active agent(s), in suitable dosage unit with

pharmaceutically acceptable excipients, carriers, adjuvants and vehicles, appropriate for each route of administration. In one embodiment, the cytidine analog (e.g., 5-azacytidine) is administered orally. In another embodiment, the cytidine analog (e.g., 5-azacytidine) is administered parenterally. In yet another embodiment, the cytidine analog (e.g., 5- azacytidine) is administered intravenously.

[00178] In one embodiment, the cytidine analog (e.g., 5-azacytidine) can be delivered as a single dose such as, e.g., a single bolus injection, or oral tablets or pills; or over time such as, e.g., continuous infusion over time or divided bolus doses over time. In one embodiment, the cytidine analog (e.g., 5-azacytidine) can be administered repetitively if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. For example, stable disease for solid tumors generally means that the perpendicular diameter of measurable lesions has not increased by 25% or more from the last measurement. See, e.g., Response Evaluation Criteria in Solid Tumors (RECIST) Guidelines, Journal of the National Cancer Institute 92(3): 205-216 (2000). Stable disease or lack thereof is determined by methods known in the art such as evaluation of patient's symptoms, physical examination, visualization of the tumor that has been imaged using X-ray, CAT, PET, or MRI scan and other commonly accepted evaluation modalities.

[00179] In one embodiment, the cytidine analog {e.g., 5-azacytidine) can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), three times daily (TID), and four times daily (QID). In one embodiment, the administration can be continuous {i.e., daily for consecutive days or every day), intermittent, e.g., in cycles {i.e., including days, weeks, or months of rest when no drug is administered). In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered daily, for example, once or more than once each day for a period of time. In one embodiment, the cytidine analog {e.g., 5- azacytidine) is administered daily for an uninterrupted period of at least 7 days, in some embodiments, up to 52 weeks. In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered intermittently, i.e., stopping and starting at either regular or irregular intervals. In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered for one to six days per week. In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered in cycles {e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week; or e.g., daily administration for one week, then a rest period with no administration for up to three weeks). In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered on alternate days. In one

embodiment, the cytidine analog {e.g., 5-azacytidine) is administered in cycles {e.g., administered daily or continuously for a certain period interrupted with a rest period).

[00180] In one embodiment, the frequency of administration ranges from about daily to about monthly. In certain embodiments, the cytidine analog {e.g., 5-azacytidine) is administered once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks. In one embodiment, the cytidine analog {e.g., 5-azacytidine) is

administered once a day. In another embodiment, the cytidine analog {e.g., 5-azacytidine) is administered twice a day. In yet another embodiment, the cytidine analog {e.g., 5- azacytidine) is administered three times a day. In still another embodiment, the cytidine analog (e.g., 5-azacytidine) is administered four times a day.

[00181] In one embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks. In certain

embodiments, the cytidine analog (e.g., 5-azacytidine) is administered once per day for one week, two weeks, three weeks, or four weeks. In one embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day for one week. In another embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day for two weeks. In yet another embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day for three weeks. In still another embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day for four weeks.

[00182] In one embodiment, the cytidine analog (e.g., 5-azacytidine) is administered once per day for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 9 weeks, about 12 weeks, about 15 weeks, about 18 weeks, about 21 weeks, or about 26 weeks. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered intermittently. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered intermittently in the amount of between about 50 mg/m²/day and about 2,000 mg/m²/day. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered continuously. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is

administered continuously in the amount of between about 50 mg/m²/day and about 1,000 mg/m²/day.

[00183] In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered to a patient in cycles (e.g., daily administration for one week, then a rest period with no administration for up to three weeks). Cycling therapy involves the administration of an active agent for a period of time, followed by a rest for a period of time, and repeating this sequential administration. Cycling therapy can reduce the development of resistance, avoid or reduce the side effects, and/or improves the efficacy of the treatment.

[00184] In one embodiment, 5-azacytidine is administered to a patient in cycles. In one embodiment, a method provided herein comprises administering 5-azacytidine in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or greater than 40 cycles. In one embodiment, the median number of cycles administered in a group of patients is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or greater than about 30 cycles.

[00185] In one embodiment, 5-azacytidine is administered to a patient at a dose provided herein over a cycle of 28 days which consists of a 7-day treatment period and a 21 -day resting period. In one embodiment, 5-azacytidine is administered to a patient at a dose provided herein each day from day 1 to day 7, followed with a resting period from day 8 to day 28 with no administration of 5-azacytidine. In one embodiment, 5-azacytidine is administered to a patient in cycles, each cycle consisting of a 7-day treatment period followed with a 21 -day resting period. In particular embodiments, 5-azacytidine is administered to a patient at a dose of about 50, about 60, about 70, about 75, about 80, about 90, or about 100 mg/m²/d, for 7 days, followed with a resting period of 21 days. In one embodiment, 5- azacytidine is administered intravenously. In one embodiment, 5-azacytidine is administered subcutaneously.

[00186] In other embodiments, 5-azacytidine is administered orally in cycles.

[00187] Accordingly, in one embodiment, the cytidine analog (e.g., 5-azacytidine) is administered daily in single or divided doses for about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about eight weeks, about ten weeks, about fifteen weeks, or about twenty weeks, followed by a rest period of about 1 day to about ten weeks. In one embodiment, the methods provided herein contemplate cycling treatments of about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about eight weeks, about ten weeks, about fifteen weeks, or about twenty weeks. In some embodiments, the cytidine analog (e.g., 5-azacytidine) is administered daily in single or divided doses for about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks with a rest period of about 1, 3, 5, 7, 9, 12, 14, 16, 18, 20, 22, 24, 26, 28, 29, or 30 days. In some embodiments, the rest period is 1 day. In some embodiments, the rest period is 3 days. In some

embodiments, the rest period is 7 days. In some embodiments, the rest period is 14 days. In some embodiments, the rest period is 28 days. The frequency, number and length of dosing cycles can be increased or decreased.

[00188] In one embodiment, the methods provided herein comprise: i) administering to the subject a first daily dose of the cytidine analog (e.g., 5-azacytidine); ii) optionally resting for a period of at least one day where the cytidine analog (e.g., 5-azacytidine) is not administered to the subject; iii) administering a second dose of the cytidine analog (e.g., 5-azacytidine) to the subject; and iv) repeating steps ii) to iii) a plurality of times. In certain embodiments, the first daily dose is between about 50 mg/m²/day and about 2,000 mg/m²/day. In certain embodiments, the second daily dose is between about 50 mg/m²/day and about 2,000 mg/m²/day. In certain embodiments, the first daily dose is higher than the second daily dose. In certain embodiments, the second daily dose is higher than the first daily dose. In one embodiment, the rest period is 2 days, 3 days, 5 days, 7 days, 10 days, 12 days, 13 days, 14 days, 15 days, 17 days, 21 days, or 28 days. In one embodiment, the rest period is at least 2 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 2 days and steps ii) through iii) are repeated at least five times. In one embodiment, the rest period is at least 3 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 3 days and steps ii) through iii) are repeated at least five times. In one embodiment, the rest period is at least 7 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 7 days and steps ii) through iii) are repeated at least five times. In one embodiment, the rest period is at least 14 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 14 days and steps ii) through iii) are repeated at least five times. In one embodiment, the rest period is at least 21 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 21 days and steps ii) through iii) are repeated at least five times. In one embodiment, the rest period is at least 28 days and steps ii) through iii) are repeated at least three times. In one embodiment, the rest period is at least 28 days and steps ii) through iii) are repeated at least five times. In one embodiment, the methods provided herein comprise: i) administering to the subject a first daily dose of the cytidine analog (e.g., 5-azacytidine) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; ii) resting for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days; iii) administering to the subject a second daily dose of the cytidine analog (e.g., 5-azacytidine) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; and iv) repeating steps ii) to iii) a plurality of times. In one embodiment, the methods provided herein comprise: i) administering to the subject a daily dose of the cytidine analog (e.g., 5-azacytidine) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days; ii) resting for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days; and iii) repeating steps i) to ii) a plurality of times. In one embodiment, the methods provided herein comprise: i) administering to the subject a daily dose of the cytidine analog (e.g., 5-azacytidine) for 7 days; ii) resting for a period of 21 days; and iii) repeating steps i) to ii) a plurality of times. In one embodiment, the daily dose is between about 50 mg/m²/day and about 2,000 mg/m²/day. In one embodiment, the daily dose is between about 50 mg/m²/day and about 1,000 mg/m²/day. In one embodiment, the daily dose is between about 50 mg/m²/day and about 500 mg/m²/day. In one embodiment, the daily dose is between about 50 mg/m²/day and about 200 mg/m²/day. In one embodiment, the daily dose is between about 50 mg/m²/day and about 100 mg/m²/day.

[00189] In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered continuously for between about 1 and about 52 weeks. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered continuously for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In certain embodiments, the cytidine analog (e.g., 5-azacytidine) is administered continuously for about 14, about 28, about 42, about 84, or about 112 days. It is understood that the duration of the treatment may vary with the age, weight, and condition of the subject being treated, and may be determined empirically using known testing protocols or according to the professional judgment of the person providing or supervising the treatment. The skilled clinician will be able to readily determine, without undue

experimentation, an effective drug dose and treatment duration, for treating an individual subject having a particular type of cancer.

5.4.3 Co- Administered Therapeutic Agents

[00190] In certain embodiments, methods provided herein for treating a cancer or an MDS, comprise co-administering a cytidine analog, such as, for example, 5-azacytidine, with one or more therapeutic agents, such as, for example, cancer therapeutic agents, to yield a synergistic therapeutic effect. The co-administered therapeutic agents include, but are not limited to, e.g., cytotoxic agents, anti-metabolites, antifolates, HDAC inhibitors such as MGCD0103 (a.k.a. N-(2-aminophenyl)-4-((4-(pyridin-3-yl)pyrimidin-2- ylamino)methyl)benzamide), DNA intercalating agents, DNA cross-linking agents, DNA alkylating agents, DNA cleaving agents, topoisomerase inhibitors, CDK inhibitors, JAK inhibitors, anti-angiogenic agents, Bcr-Abl inhibitors, HER2 inhibitors, EGFR inhibitors, VEGFR inhibitors, PDGFR inhibitors, HGFR inhibitors, IGFR inhibitors, c-Kit inhibitors, Ras pathway inhibitors, PI3K inhibitors, multi-targeted kinase inhibitors, mTOR inhibitors, anti-estrogens, anti-androgens, aromatase inhibitors, somatostatin analogs, ER modulators, anti-tubulin agents, vinca alkaloids, taxanes, HSP inhibitors, Smoothened antagonists, telomerase inhibitors, COX-2 inhibitors, anti-metastatic agents, immunosuppressants, biologies such as antibodies, and hormonal therapies. In particular embodiment, the co- administered therapeutic agent is thalidomide, lenalidomide, or pomalidomide. The coadministered agent may be dosed, e.g., orally or by injection.

[00191] In one embodiment, the route of the administration of the cytidine analog {e.g., 5- azacytidine) is independent of the route of the administration of a second therapy. In one embodiment, the cytidine analog {e.g., 5-azacytidine) is administered orally. In another embodiment, the cytidine analog {e.g., 5-azacytidine) is administered intravenously. In accordance with these embodiments, the cytidine analog {e.g., 5-azacytidine) is administered orally or intravenously, and the second therapy can be administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form. In one embodiment, the cytidine analog {e.g. , 5-azacytidine) and a second therapy are administered by the same mode of administration, e.g., orally or intravenously. In another embodiment, the cytidine analog {e.g., 5-azacytidine) is administered by one mode of administration, e.g. , intravenously, whereas the second agent {e.g. , an anticancer agent) is administered by another mode of administration, e.g. , orally. In another embodiment, the cytidine analog {e.g., 5-azacytidine) is administered by one mode of administration, e.g. , orally, whereas the second agent {e.g. , an anticancer agent) is

administered by another mode of administration, e.g. , intravenously.

[00192] In one embodiment, each method provided herein may independently, further comprise the step of administering a second therapeutic agent. In one embodiment, the second therapeutic agent is an anticancer agent. In one embodiment, the anticancer agent is an antimetabolite, including, but not limited to, 5-fluoro uracil, methotrexate, cytarabine, high dose cytarabine, and fludarabine. In one embodiment, the anticancer agent is an

antimicrotubule agent, including, but not limited to, vinca alkaloids {e.g., vincristine and vinblastine) and taxanes {e.g., paclitaxel and docetaxel). In one embodiment, the anticancer agent is an alkylating agent, including, but not limited to, cyclophosphamide, melphalan, carmustine, and nitrosoureas {e.g., hydroxyurea and bischloroethylnitrosurea). In one embodiment, the anticancer agent is a platinum agent, including, but not limited to, cisplatin, carboplatin, oxaliplatin, satraplatin (JM-216), and CI-973. In one embodiment, the anticancer agent is an anthracycline, including, but not limited to, doxrubicin and daunorubicin. In one embodiment, the anticancer agent is an antitumor antibiotic, including, but not limited to, mitomycin, idarubicin, adriamycin, and daunomycin (also known as daunorubicin). In one embodiment, the anticancer agent is a topoisomerase inhibitor, e.g. , etoposide and camptothecins. In one embodiment, the anticancer agent is selected from the group consisting of adriamycin, busulfan, cytarabine, cyclophosphamide, dexamethasone, fludarabine, fluorouracil, hydroxyurea, interferons, oblimersen, platinum derivatives, taxol, topotecan, and vincristine.

[00193] In one embodiment, other therapies or anticancer agents that may be used in combination with the cytidine analog (e.g., 5-azacytidine) include surgery, radiotherapy (e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes), endocrine therapy, biologic response modifiers (e.g., interferons, interleukins, and tumor necrosis factor (TNF)), hyperthermia and cryotherapy, agents to attenuate any adverse effects (e.g., antiemetics), and other approved chemotherapeutic drugs, including, but not limited to, alkylating drugs (mechlorethamine, chlorambucil, cyclophosphamide, melphalan, and ifosfamide), antimetabolites (cytarabine, high dose cytarabine, and methotrexate), purine antagonists and pyrimidine antagonists (6- mercaptopurine, 5 -fluorouracil, cytarabine, and gemcitabine), spindle poisons (vinblastine, vincristine, vinorelbine, and paclitaxel), podophyllotoxins (etoposide, irinotecan, and topotecan), antibiotics (daunorubicin, doxorubicin, bleomycin, and mitomycin), nitrosoureas (carmustine and lomustine), inorganic ions (cisplatin and carboplatin), enzymes

(asparaginase), and hormones (tamoxifen, leuprolide, flutamide, and megestrol), imatinib, adriamycin, dexamethasone, and cyclophosphamide. For additional available cancer therapies, see, e.g, http://www.nci.nih.gov/; for a list of FDA approved oncology drugs, see, e.g., http://www.fda.gov/, The Merck Manual, 18th Ed. 2006, and PDR: Physician Desk Reference 2010, 64th Ed. 2009; the contents of each of which are hereby incorporated by reference in their entireties.

[00194] In one embodiment, provided herein is a kit incorporating a biomarker provided herein. In some embodiments, the kit is a diagnostic kit. For example, single gene epigenetics biomarkers may be used in the diagnosis of certain cancers. See, e.g., www.epigenomics.com. Such gene signature may be developed into a diagnostic kit, for use in a high-throughput methylation assay. In other embodiments, employing gene array technology, customized gene chip for a particular gene signature or pattern may be used in a kit provided herein. See, e.g., www.pathworkdx.com.

6. EXAMPLES

[00195] The following examples are provided by way of illustration, not limitation. 6.1 Example 1

[00196] This phase III randomized trial assessed the effect of 5-azacytidine on prolonging overall survival in patients with higher risk MDS compared with 3 other frequently used conventional care regimens.

[00197] A phase III, international, multi-center, prospective, randomized, controlled, parallel group trial was conducted and demonstrated prolonged overall survival in higher risk MDS patients as compared to conventional care regimens and best supportive care. (This study is referred to herein as the "AZA-001" study). See, e.g., Fenaux et ah, Lancet

Oncology, 2009, 10:223-32. The primary study objective and endpoint were overall survival (OS), comparing 5-azacytidine and conventional care regimens. Secondary objectives and endpoints included time to transformation to acute myeloid leukemia (AML), red blood cell transfusion independence, hematologic responses and improvement, infections requiring IV therapy, and safety.

[00198] Eligible patients were 18 years or older with higher risk MDS, defined as an IPSS of Intermediate-2 or High and FAB-defmed RAEB, RAEB-T, or non-myeloproliferative chronic myelomonocytic leukemia (CMML), using modified FAB criteria (blood monocytes greater than 1 x 10⁹/L, dysplasia in 1 or more myeloid cell lines, 10%-29% marrow blasts, and a white blood count below 13 x 10⁹/L). Patients were to have an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 and life expectancy of 3 months or more. Patients with secondary therapy-related MDS, prior 5-azacytidine treatment, or eligibility for allogenetic stem cell transplantation were excluded.

[00199] The Phase III, international, multi-center, randomized, controlled, parallel-group trial was conducted in accordance with the Declaration of Helsinki. All patients provided written informed consent, and the study was approved by the institutional review boards at all participating study sites. Enrollment to the trial and monitoring was conducted by site investigators and central pathology reviewers with standardized central review of cytogenetic data. An independent Data Safety Monitoring Board reviewed safety data and conducted blinded review of a scheduled interim analysis.

[00200] Patients were randomized to 1 of 2 treatment groups: 5-azacytidine plus best supportive care (BSC) or conventional care regimens (CCR) plus BSC. Patients were randomized 1 : 1 to receive 5-azacytidine or CCR. Prior to randomization, investigators preselected (based on age, health and disease status, co-morbidities, etc.) the most appropriate one of three conventional CCR groups for higher risk MDS patients, which the patients then received if randomized to CCR. Patients randomized to 5-azacytidine received 5-azacytidine regardless of CCR selection. This pre -randomization step was performed to enable meaningful comparisons of CCR subgroups with relevant 5-azacytidine-treated subgroups. No crossover was allowed in this trial and administration of erythropoietin or darbepoetin was prohibited. Balanced enrollment across treatments was ensured using blocked randomization with patients stratified by FAB subtype and IPSS risk group.

[00201] During the treatment phase of the trial, all regimens were continued until study end or patient discontinuation due to relapse, disease progression, unacceptable toxicity, or transformation to AML (defined as 30% or greater bone marrow blasts). 5-Azacytidine was administered subcutaneously at 75 mg/m²/day for 7 days every 28 days (delayed as needed until cell line recovery), which constituted one cycle of therapy, for at least 6 cycles until study end unless treatment was discontinued due to unacceptable toxicity, relapse after response, or disease progression. The CCR group consisted of 3 treatment regimens administered until study end or treatment discontinuation: BSC only (including blood product transfusions, antibiotics, with G-CSF for neutropenic infection); low-dose ara-C (LDara-C): 20 mg/m²/day subcutaneously for 14 days, every 28-42 days (delayed as needed until cell line recovery) for at least 4 cycles; or intensive chemotherapy, i.e. induction with ara-C 100-200 mg/m²/day by continuous intravenous infusion for 7 days plus 3 days of intravenous daunorubicin (45-60 mg/m²/day), idarubicin (9-12 mg/m²/day), or mitoxantrone (8-12 mg/m²/day). Patients with complete or partial remission after induction (defined by IWG criteria for AML, see e.g., J. Clin. Oncol. 2003, 21(24):4642-9) received 1-2 consolidation courses with reduced doses of the cytotoxic agents used for induction, followed by BSC only. All patients could receive BSC as needed. After treatment discontinuation, all patients were followed until death or end of study (12 months following randomization of the last patient). Figure 1 shows the study design.

[00202] All efficacy analyses used the intent-to-treat (ITT) population. Safety analyses were performed on the safety population (all patients who received at least 1 dose of study drug and 1 or more post-dose safety assessments). The primary trial endpoint was overall survival (time from randomization until death from any cause), analyzed for the ITT group comparing the 5-azacytidine group and the CCR group, and for predefined subgroups based on age, gender, FAB, IPSS (Int-2, high), IPSS cytogenetics (good, intermediate, and poor) and -7/del(7q) cytogenetic abnormality, IPSS cytopenias (0/1 and 2/3), WHO classification, karyotype, and lactic dehydrogenase (LDH). The primary assessment of overall survival used the ITT population and compared 5-azacytidine with the combined CCR group. A secondary analysis compared overall survival of 5-azacytidine subgroups (the 3 CCR subgroups of patients who were randomized to 5-azacytidine) with the corresponding CCR subgroups (patients in the corresponding CCR subgroups, who were randomized to CCR).

[00203] Secondary efficacy endpoints were transformation to AML (from randomization until AML transformation [30% bone marrow blast count or greater]), hematologic response and improvement assessed using IWG 2000 criteria for MDS (See e.g., Cheson et al., Blood 2000, 96(12):3671-4), red blood cell (RBC) transfusion independence (absence of transfusions during 56 consecutive days), infections requiring intravenous antimicrobials (analyzed from randomization to 28 days post last study visit), and adverse events. Bone marrow samples were collected every 16 weeks during active treatment and as clinically indicated during follow-up. Infections requiring intravenous antimicrobials were counted from randomization to last study visit. Adverse events were assessed using the National Cancer Institute's Common Toxicity Criteria, Version 2.0.

[00204] Time to event was studied using the Kaplan-Meier method; treatment comparisons were made using stratified log-rank tests and Cox proportional-hazards models. All statistical tests were two-sided without correction for multiple testing.

[00205] Efficacy analyses included all patients randomized according to the ITT principle. Overall survival was defined as the time from randomization until death from any cause. Patients for whom death was not observed were censored at the time of last follow-up. Time to transformation to AML was measured from randomization to development of 30% or greater bone marrow blasts. Patients for whom AML transformation was not observed were censored at the time of last adequate bone marrow sample. Randomization and analyses were stratified on FAB subtype and IPSS risk group. Time-to-event curves were estimated according to the Kaplan-Meier method (See e.g., Kaplan et al., J. Am. Stat. Assoc. 1958, 53;457-81) and compared using stratified log-rank tests (primary analysis). Stratified Cox proportional hazards regression models (See e.g., Cox, J. Royal Stat. Soc. B, 1972, 34;184- 92) were used to estimate hazard ratios and associated 95% confidence intervals (CI). The primary analysis of overall survival between the 5-azacytidine and combined CCR groups used the stratified Cox proportional hazards model without any covariate adjustments to estimate the hazard ratio. Cox proportional hazards regression with stepwise selection was used to assess the baseline variables of sex, age, time since original MDS diagnosis, ECOG performance status, number of RBC transfusions, number of platelet transfusions, hemoglobin, platelets, absolute neutrophil count, LDH, bone marrow blast percentage, and presence or absence of cytogenetic -7/del(7q) abnormality. The final model included ECOG performance status, LDH, hemoglobin, number of RBC transfusions and presence or absence of cytogenetic -7/del(7q) abnormality. Secondary analyses used the final Cox proportional hazards model. The consistency of treatment effect across subgroups was assessed by the difference in likelihood ratio between the full model with treatment, subgroup and treatment- by-subgroup interaction, and the reduced model without the interaction.

[00206] Response rates (overall response, transfusion independence, and hematological improvement) were compared between the 5-azacytidine and CCR groups using Fisher's exact test. The rate of infection requiring intravenous antimicrobials was computed as the number of observed infections requiring intravenous antimicrobials divided by the total number of patient-years of follow-up. The relative risk was computed by dividing the 5- azacytidine rate by the CCR rate. The relative risks across the 4 strata were tested for homogeneity using the Breslow-Day test {See e.g., Breslow et al., Chapter 3: Comparisons Among Exposure Groups. In: Heseltine E. ed. Statistical Methods in Cancer Research Volume II - The Design and Analysis of Cohort Studies. Lyon: IARC Scientific Publications; 1987:82-119). The Mantel-Haenszel estimate of the common relative risk, the associated 95% CI, and the test that it equals unity were computed {See e.g., Mantel, Cancer

Chemotherapy Reports, 1966, 50(3): 163-70). This study was designed with 90% power - based on a log rank analysis - to detect a hazard ratio of 0.60 for overall survival in the 5- azacytidine group compared with the CCR group with a two-sided alpha of 0.05. The protocol specified that approximately 354 patients were to be randomized over 18 months and then monitored for at least 12 months of treatment and follow-up, resulting in at least 167 deaths over the 30 month trial period. Recruitment, however, necessitated a longer study period that lasted 42 months with 195 deaths that resulted in a 95% power under the design assumptions of the study. The interim analysis was conducted using an O'Brien-Fleming monitoring boundary and Lan-DeMets alpha spending function to control the overall alpha at 0.05 {See e.g., Lan et al. , Biometrika 1983, 70(3):659-63).

[00207] 358 Patients (ITT population, 98% Caucasian, 70% male) at 79 sites were randomized: 179 to 5-azacytidine and 179 to CCR (105 to BSC 59%, 49 to LDara-C 27%, and 25 to intensive chemotherapy 14%). Median age was 69 years (range: 38-88) with 258 (72%>) patients aged 65 years or older. Baseline demographic and disease characteristics were well balanced between the 5-azacytidine and CCR combined and between 5-azacytidine and the 3 CCR regimens (Table 2 A and 2B). As expected, patients in the intensive chemotherapy group were younger. At baseline, 95% of patients were higher risk: RAEB (58%), RAEB-T (34%), CMML (3%), and other (5%). By IPSS, 87% were higher risk: Int- 2 (41%), High (47%)), and 13% indeterminate/other. Additionally, 32% of patients were classified as WHO AML (marrow blast count, 20%-30%). Upon IRC review, 10 and 5 patients, respectively, in the 5-azacytidine and CCR groups had received prior radiation, chemotherapy, or cytotoxic therapies for non-MDS conditions, which constituted protocol deviations. 5-Azacytidine was administered for a median of 9 cycles (range 1 to 39) with 86%) of patients remaining on the 75 mg/m²/day dose throughout the study with no adjustments. The median 5-azacytidine cycle length was 34 days (range 15 to 92). LDara-C was administrated for a median of 4.5 cycles (range 1 to 15), BSC only patients for a median of 7 cycles (range 1 to 26, 6.2 months), and intensive chemotherapy for 1 cycle (range 1 to 3, i.e. induction plus 1 or 2 consolidation cycles, with cytarabine and anthracycline). Median follow-up for the overall survival analysis was 21.1 months. Overall analysis (ITT): AZA (N=179 vs. CCR (N=179). Analysis by CCR treatment selection: AZA (N=l 17) vs. BSC (n=105); AZA (N=45) vs. LD Ara-C (N=49); AZA (N=17) vs. Intensive Chemo (N=25). Four patients in the 5-azacytidine group and 14 in the CCR group never received but were followed for overall survival and were included in the ITT analysis. Eight patients went on to transplant after treatment (4 in the 5-azacytidine group and 4 in the CCR group: BSC [n=2], LDara-C [n=l], intensive chemotherapy [n=l]) and were also included in the ITT analysis.

Overall Survival

[00208] 5-Azacytidine demonstrated statistically superior overall survival vs. conventional care regimens. After a median follow-up of 21.1 months (range 0 to 38.4), median Kaplan- Meier overall survival was 24.4 months in the 5-azacytidine group compared with 15 months in the CCR group, for a difference of 9.4 months (stratified log-rank p=0.0001) (Figure 2). The hazard ratio (Cox Model) was 0.58 (95% CI: 0.43-0.77) indicating a 42% reduction in risk of death in the 5-azacytidine group and a 74% overall survival advantage. At two year, 50.8%) (95%o CI: 42.1-58.8) of patients in the 5-azacytidine group were alive compared with 26% (95% CI: 18.7-34.3) in the CCR group (p<0.0001). After approximately 100 days (about 3 months), with 78%> (140/179) of 5-azacytidine patients completing 3 cycles of therapy, the Kaplan-Meier curves for the 5-azacytidine and CCR groups separated for the remainder of the trial.

[00209] Results in the predefined patient subgroups (based on age, gender, FAB classification, IPSS, WHO classification, karyotype, and LDH) also showed a consistent overall survival benefit for the 5-azacytidine group (Figure 2). In particular, IPSS

cytogenetic subgroups showed significant overall survival differences favoring the 5- azacytidine group versus the CCR group (hazard ratio; log-rank p): Poor, 11.2 months (0.52, p=0.011); Intermediate, 9.3 months (0.43, p=0.017); and Good, median not reached (0.62, log-rank p=0.038). In patients with -7/del(7q), median Kaplan-Meier overall survival was 13.1 months (95% CI, 9.9 to 24.5) in the 5-azacytidine group (n=30) compared with 4.6 months (95%> CI, 3.5 to 6.7) in the CCR group (n=27) (stratified log-rank p=0.002, hazard ratio, 0.33 (95%> CI, 0.16 to 0.68). Additionally, sensitivity analyses exploring the influence of the 8 transplanted patients included in the ITT analyses above did not influence the significance of the overall survival results for 5-azacytidine.

[00210] The survival benefits of 5-azacytidine were consistent regardless of the CCR treatment options. Differences in median overall survival (hazard ratio; log-rank p) between the 5-azacytidine subgroups and the CCR subgroups of BSC, LDara-C, and intensive chemotherapy were 9.6 months (0.58; p=0.005), 9.2 months (0.36; p=0.0006), and 9.4 months (0.76, 95% CI: 0.33 to 1.74), respectively. Similar to the primary overall survival comparison (5-azacytidine vs. CCR), results from the investigator pre-selection subgroup analysis of overall survival showed significant differences between 5-azacytidine (n=l 17) and BSC (n=105) (p=0.005) and 5-azacytidine (n=45) and LDara-C (n=49) (p=0.0006). The difference in the comparison between 5-azacytidine (n=17) and intensive chemotherapy (n=25), however, was not significant (0.51).

[00211] The significant prolongation of overall survival observed with 5-azacytidine compared with CCR was not dependent on the achievement of complete remission (HR=0.39 [95% CI: 0.14-1.15], log rank p = 0.078). The achievement of hematologic improvement, partial remission, or complete remission contributed to but was not required for improvement in overall survival with 5-azacytidine treatment.

[00212] To date, 5-azacytidine is the only agent to demonstrate survival benefit in MDS compared to conventional care regimens, and the only epigenetic modifier to show survival benefits in cancer. The study described herein represented the largest study ever conducted in higher risk MDS. These results, showing a significant improvement in survival in the most advanced MDS patients, demonstrated the benefit 5-azacytidine can provide to treat the disease. Building on the established data from earlier clinical studies, which showed that 5- azacytidine offers transfusion independence benefits to patients with MDS to improve the overall quality of life, the present study showed that 5-azacytidine not only improves patient's life, but extends it as well. Secondary Efficacy Endpoints

[00213] Red blood cell transfusion independence, hematologic remission, and hematologic improvement were also significantly increased with 5-azacytidine as compared with combined conventional care regimens. 5-Azacytidine was well tolerated.

Time to AML Transformation

[00214] Assessed over the entire trial, median time to transformation to AML or death was 13.0 months (95% CI: 9.9-15.0) in the 5-azacytidine group compared with 7.6 months (95% CI: 5.4-9.8) in the CCR group (hazard ratio: 0.68, log-rank p<0.003).

[00215] Time to AML transformation was assessed during treatment with a median of 26.1 months (95% CI: 15.0-28.7) in the 5-azacytidine group compared with 12.4 months (95% CI: 10.4-15.4) in the CCR group (log-rank p=0.004, Figure 3).

[00216] Median time to AML transformation was 17.8 months (95% CI, 13.6 to 23.6) in the 5-azacytidine group compared with 11.5 months (95%> CI, 8.3 to 14.5) in the CCR group (hazard ratio, 0.50 (95% CI, 0.35 to 0.70), log rank pO.0001).

Hematologic Response and Improvement Rates

[00217] Complete and partial remission rates were significantly higher in the 5-azacytidine group than in the CCR group. Using the investigator pre-selection analysis, remission rates were generally significantly higher with 5-azacytidine compared with either BSC or LDara- C, but no significant differences in remission rates were observed when comparing 5- azacytidine with intensive chemotherapy. Time to disease progression, relapse after complete or partial remission, or death was significantly longer in the 5-azacytidine group (median, 14.1 months) than in the CCR group (median, 8.8 months, log-rank P=0.047).

Erythroid and platelet improvement rates were significantly higher in the 5-azacytidine group compared with the CCR group. Major erythroid improvement was observed in 39.5% (62 of 157) vs. 10.6%) (17 of 160) of patients in the 5-azacytidine vs. CCR groups, respectively, (p<0.0001). Major platelet improvement was observed in 32.6% (46 of 141) vs. 14% (18 of 129) of patients in the 5-azacytidine vs. CCR groups, respectively (p=0.0003). No significant differences for major neutrophil improvement were observed between groups. Duration of hematologic improvement was significantly longer in the 5-azacytidine group (median, 13.6 months, 95% CI, 10.1 to 16.3) than in the CCR group (median, 5.2 months, 95% CI, 4.1 to 9.7, P=0.0002). 50 of 111 (45%, 95% CI, 35.6 to 54.8) baseline RBC transfusion-dependent patients in the 5-azacytidine group became transfusion independent compared with 13 of 114 (11.4%, 95% CI, 6.2 to 18.7) in the CCR group (PO.0001). [00218] Overall, 51 of 179 (28.5%) patients in the 5-azacytidine group achieved complete + partial remission compared with 21 of 179 (11.7%, p=0.0001) in the CCR group, including 5 of 105 (5%), 6 of 49 (12.2%), and 10 of 25 (40%) in the BSC, LDara-C, and intensive chemotherapy subgroups, respectively. 17% (30 of 179) and 8% (14 of 179) of patients in the 5-azacytidine and CCR groups, respectively, had a complete remission (p=0.02). The proportion of patients showing any hematologic improvement was significantly higher in the 5-azacytidine group (87 of 177, 49.2%) compared with the CCR group (51 of 178, 28.7%, p<0.0001).

Transfusion Independence

[00219] 45% (95% CI: 35.6-54.8) of patients in the 5-azacytidine group became RBC transfusion independent after being baseline dependent compared with 11.4% (95% CI: 6.2- 18.7) in the CCR group (p=0.0001). The effect on platelet transfusions showed no significant differences between the 5-azacytidine and CCR groups, which was likely due to the small numbers of patients with baseline platelet transfusion dependence in the 5-azacytidine (n=38) and CCR (n=27) groups.

Infections Requiring Intravenous Antimicrobials

[00220] The rate of infections requiring intravenous antimicrobials per patient year in the 5-azacytidine group was 0.60 (95% CI, 0.49 to 0.73) compared with 0.92 (95% CI, 0.74 to 1.13) in the CCR group, indicating a 34% reduction (hazard ratio, 0.66, 95% CI, 0.49 to 0.87, P=0.003). Using the investigator pre-selection analysis, per patient year rates were similar when comparing 5-azacytidine (0.66) and BSC (0.61) (hazard ratio: 1.1, 95% CI, 0.74 to 1.65, P=0.68), but significantly lower with 5-azacytidine (0.44) compared with LDara-C (1.00) (hazard ratio: 0.44, 95% CI, 0.25 to 0.86, P=0.017) or with 5-azacytidine (0.64) versus intensive chemotherapy (2.30) (hazard ratio: 0.28, 95% CI, 0.13 to 0.60, P=0.0006).

Safety

[00221] Discontinuations prior to study closure due to adverse events were observed in 12.6% of patients in the 5-azacytidine group compared with (7.3%) in the CCR group. The 2 active therapies in the CCR group showed similar rates with 5-azacytidine but BSC had a much lower rate of discontinuations due to adverse events (3.9%). The most frequently observed treatment-related adverse events (including Grade 3-4 events) were peripheral blood cytopenias, frequently observed across all treatments, which led to discontinuation prior to study closure in 4.6% in the 5-azacytidine group and 2.4% in the CCR group. The most common treatment-related non-hemato logic adverse events included injection site reactions with 5-azacytidine, and nausea, vomiting, fatigue, and diarrhea with 5-azacytidine, LDara-C, and intensive chemotherapy. During the first 3 cycles of treatment, deaths occurred in 14 (8%) of patients in the 5-azacytidine group and 25 (14%) in the CCR group. The most common causes of death in either group were related to underlying disease,

thrombocytopenia, sepsis/infection, hemorrhage, and respiratory complications.

Transformation to AML was also a cause of death during the first 3 cycles of treatment but observed only in the CCR group. Deaths considered to be related to treatment during the first 3 cycles were observed in 4 patients in the 5-azacytidine group (septic shock, cerebral hemorrhage, hematemesis, respiratory tract infection) and 1 patient in the CCR group

(receiving LDara-C) (cerebral ischemia).

[00222] In the higher risk MDS population, the most frequently observed treatment-related adverse events (including Grade 3 and 4 events) were blood cytopenias, frequently observed across all treatments, which led to early withdrawal in 4.6%, 4.5%, and 2% of patients in the 5-azacytidine, LDara-C, and BSC treatment groups, respectively.

[00223] The most common treatment-related non-hematologic adverse events included injection site reactions with 5-azacytidine, and nausea, vomiting, fatigue, and diarrhea across the 5-azacytidine, low-dose ara-C, and intensive chemotherapy treatment groups. During treatment and follow-up, deaths were reported in 45% of patients in the 5-azacytidine group, and 62%), 59%, and 79% of patients, respectively, in the BSC, LDara-C, and intensive chemotherapy subgroups. The major causes of death were infection and AML (>30%> blasts).

Discussion:

[00224] Results of the phase III, randomized, controlled comparative trial showed that 5- azacytidine was the first drug treatment to prolong overall survival in higher risk MDS patients. While allogeneic stem cell transplantation is potentially curative in MDS, its use is limited by older age, a lack of donors, and increased transplant-related mortality. In a previous randomized phase III CALGB trial comparing 5-azacytidine with BSC (See, e.g., J. Clin. Oncol. 2002, 20(10):2429-40), the 5-azacytidine group showed a trend for improved overall survival over BSC. The finding was possibly limited by a heterogeneous patient population and a cross-over trial design, with 51% of BSC patients subsequently receiving 5- azacytidine. Findings of the CALGB trial were also lessened by the use of BSC, a treatment not considered as intensive care in higher risk MDS by many clinicians.

[00225] No crossover was allowed in the present study. The present study included only patients with higher risk MDS. Additionally, the study compared azacytidine to three frequently used treatments (LDara-C, intensive chemotherapy, or BSC) for higher risk MDS including two active therapties (LDara-C, or intensive chemotherapy). As there is no current consensus on the use of those three regimens, their allocation for patients was made by the investigators based on patient age, general condition, presence of co-morbidities, and personal choice.

[00226] Overall survival in the present study showed an advantage of 9.4 months for the 5- azacytidine group over the CCR group, corresponding to a 42% reduction in risk of death. The robustness of this overall survival benefit was further shown in the nearly 2-fold higher proportion of patients in the 5-azacytidine group surviving at two years compared with those in the CCR group. This overall survival advantage with 5-azacytidine in the primary, ITT analysis was highly similar to that seen using the secondary, investigator-selection analysis with median survival differences ranging from 9.2 months to 9.6 months between 5- azacytidine and the three CCR subgroups.

[00227] The onset of the significant survival benefit occurred early in the present study with the Kaplan Meier curves for the 5-azacytidine and CCR groups separating permanently at approximately 3 months with nearly 80% of patients in the 5-azacytidine group having completed more than three cycles of treatment. Results obtained in the subgroup analyses for age, gender, FAB and WHO classification, karyotype; and LDH confirmed the robustness of the overall survival results achieved in the ITT population. The survival advantage in the 5- azacytidine group was maintained irrespective of IPSS cytogenetic risk group (favorable, intermediate, and poor), an important finding as abnormal karyotype is a frequent finding in MDS and a strong prognostic factor for a poorer outcome.

[00228] Findings in the secondary efficacy endpoints support the overall survival advantage demonstrated in the 5-azacytidine group. 5-Azacytidine treatment significantly prolonged the time to AML transformation or death and the time to transformation to AML compared with CCR. Significantly higher IWG-defmed response rates were observed in the 5-azacytidine group compared with the CCR group, including complete or partial remission and major erythroid hematologic improvement. The superior response rates observed in the 5-azacytidine group were driven by notably lower rates in the LDara-C and BSC subgroups. Response rates in the small intensive chemotherapy subgroup were higher than those seen in the 5-azacytidine group. Remission and hematologic improvement rates also endured longer in the 5-azacytidine group than the CCR group.

[00229] RBC transfusion independence after baseline dependence was significantly higher in the 5-azacytidine group than in the CCR group, an important finding as transfusion dependency had been shown to be an significant marker of poorer outcome in MDS. No differences were observed between the 5-azacytidine and CCR group for platelet transfusion independence, which was likely due to the small number of patients with baseline dependency. Additionally, although 5-azacytidine treatment was not associated with an increase in the proportion of patients with neutrophil improvement compared with the CCR group, a 33% reduction in the risk of infection requiring intravenous antimicrobials was observed in the 5-azacytidine group.

[00230] Grade 3 and 4 neutropenia was observed more frequently in the 5-azacytidine group than in the BSC subgroup, and at a similar rate compared with the LDara-C or intensive chemotherapy subgroups. Thrombocytopenia was also observed more commonly with 5-azacytidine than with BSC but less frequently than with LDara-C and intensive chemotherapy. However, despite the higher frequency of thrombocytopenia and neutropenia observed with 5-azacytidine compared with BSC, the overall occurrence of bleeding and infection was similar in both treatments.

[00231] Nonhemato logic adverse events more commonly reported in the 5-azacytidine group than with the BSC subgroup, such as injection site reactions, nausea, and vomiting, were largely Grade 1-2 in severity, were well recognized events observed with 5-azacytidine treatment, and caused no patients to discontinue therapy. Generally, injection site reactions were easily managed by varying injection sites and by applying a post-injection cool or warm compress for 15 minutes.

[00232] The results demonstrated the first finding of an overall survival benefit in the treatment of MDS. Significantly longer overall survival was clearly shown with 5- azacytidine treatment compared with the CCR group, which comprised three other commonly used treatments in patients with higher risk MDS. The overall survival advantage was demonstrated irrespective of the CCR regimen (BSC, LDara-C, or intensive chemotherapy) and regardless of a good, intermediate, or poor IPSS cytogenetic risk. The results showing the overall survival benefit demonstrated with 5-azacytidine, given for a median of 9 cycles, was supported by a significant prolongation in time to AML transformation as well as increases in transfusion independence, complete and partial remissions, and major hematologic improvements. The significant increases in transfusion independence and hematologic improvement particularly suggested that decreasing cytopenias reduces the risk of their lethal complications, thus altering the natural disease course of MDS. These findings strongly established 5-azacytidine as the reference treatment in higher risk MDS, against which newer treatments will have to be compared or combined with in future trials in these patients.

6.2 Example 2

[00235] This study evaluates gene methylation biomarkers and prolonged survival in patients with certain MDS (e.g., higher risk MDS) treated with 5-azacytidine.

Hypomethylation is believed to be a molecular mechanism of action of 5-azacytidine;

accordingly, research on the effect of methylation status particular genes, and of gene combinations, is conducted. Both DNA methylation and RNA methylation are contemplated as potential biomarkers.

[00236] In one embodiment, a study is performed to examine whether baseline DNA and/or RNA methylation levels influence overall survival (OS) as well as the interaction between gene promotor methylation levels and treatment (e.g., 5-azacytidine or CCR). For example, methylation is determined for 5 genes: CDKN2B (pi 5), SOCS1, CDH1 (E- cadherin), TP73, and CTNNA1 (alpha-catenin), in pre-treatment bone marrow aspirates of patients enrolled in a clinical study using quantitative real-time methylation specific PCR (qMSP). The influence of methylation on OS is assessed using Cox proportional hazards models and Kaplan-Meier (KM) methodology.

[00237] The number of patients (for 5-azacytidine and CCR) having nucleic acid sufficient for analysis of these genes is determined. For example, methylation is detected in a specific percentage of patients for CDKN2B, SOCS1, CDH1, TP73, and CTNNA1. Differences in methylation levels between the treatment arms are determined. The OS benefit for 5- azacytidine treatment is determined for patients who are positive and negative for

methylation at these genes. It is determined whether the presence of methylation is associated with improvement in OS in the CCR group (prognostic indicator of good outcome). The existence and magnitude of any effect is compared to the 5-azacytidine group, which may suggest an interaction between DNA and/or RNA methylation and treatment.

[00238] OS improvement is assessed with 5-azacytidine treatment in patients with methylation at any of these genes, and HR of death for methylation is determined. The frequency of methylation of particular genes allows for examination of the influence of methylation level on OS and treatment effect. For example, for particular genes, lower levels of methylation may be associated with the longest OS and the greatest OS benefit from 5- azacytidine treatment, compared with the absence of methylation. Influence of methylation level on OS may be assessed in each IPSS cytogenetic subgroup (good, intermediate, and poor). For example, the influence of methylation on OS may be strongest in the "poor" risk group, where risk of death is greatest. [00239] Such data and analysis may indicate, e.g. , that patients with lower levels of methylation may derive greater benefit from 5-azacytidine. Molecular biomarkers may be important in MDS, e.g., as indicators of disease prognosis and predictors of response to epigenetic therapy.

6.3 Example 3

[00240] Studies were done to determine if baseline DNA methylation influenced overall survival in MDS subjects from the AZA 001 study (Example 1). Potential correlations between gene promoter methylation levels and treatment efficacy (AZA or conventional care regimens (CCR)) were evaluated. Specifically, the DNA methylation of 5 genes, CDKN2B (pi 5), SOCSI, CDHI (E-cadherin) , TP73, and CTNNAI (a-catenin), in pre-treatment baseline bone marrow aspirates of 303 patients were evaluated, using quantitative real-time methylation specific PCR. The influence of methylation levels on overall survival was assessed using Cox proportional hazards models and Kaplan-Meier methodology.

[00241] In one embodiment, the DNA methylation of baseline bone marrows from patients were measured using the llumina Infinium Methylation27 DNA methylation arrays (e.g., Beadarray). In one embodiment, other known technologies for measuring DNA methylation are used in a method provided herein.

[00242] Two hundred and seventy-one patients (AZA [n=136] and CCR [n=135]) had sufficient DNA for analysis of all 5 genes. DNA methylation was detected in 82% of patients' samples for CDKN2B, 54% for SOCSI, 87% for CDHI, 2% for TP73, and 10% for CTNNAI. Overall, methylation levels did not differ between the two treatment arms. There was an OS benefit for AZA vs. CCR treatment in patients regardless of methylation status at these five genes. The presence of abnormal methylation at any locus was associated with a slightly improved OS in both the AZA and CCR groups (prognostic indicator of good outcome); however, this effect was more pronounced in the AZA group. This suggests an interaction between DNA methylation and treatment. OS improved to a greater extent with AZA treatment in patients with methylation of any one of 4 of these 5 genes (SOCSI, CDHI, TP73, and CTNNAI), with HR of death for methylation 0.54-0.93 (HR for CDKN2B is 1.01). Two genes were infrequently methylated (TP73, CTNNAI), but the frequency of methylation of CDKN2B, SOCSI, and CDHI allowed examination of the influence of methylation level on OS and treatment effect. For these three genes, the presence of methylation was associated with significant improvement in OS with AZA treatment compared with no methylation. Lower levels of methylation (Table 3) were associated with the greatest OS benefit from AZA; this benefit diminished as methylation levels increased (data for CDH1 are shown in Table 3; this pattern was similar for the other genes). This influence of methylation level on OS was seen in all IPSS cytogenetic subgroups, but was strongest in the poor risk group where risk of death was greatest.

[00243] The overall survival benefit observed with AZA versus CCR appeared to be independent of pre-treatment baseline methylation status of the five genes analyzed.

However, increased methylation in pre-treatment baseline bone marrow was associated with worse overall survival, and patients with lower levels of methylation treated with AZA had the best overall survival, suggesting that these patients may derive greater benefit from AZA.

[00244] In one embodiment, the relationship between CDH1 gene methylation and prolonged overall survival was analyzed and summarized in Table 3. The hazard ratios and 95% CI of the analysis of CDH1 gene methylation and prolonged overall survival and AML transformation are summarized in Figure 4.

[00245] Additional studies were done, for example, with CDKN2B promoter DNA methylation density in pre-treatment baseline bone marrows from patients in the AZA 001 study, looking at association of overall survival and response with AZA or CCR treatments. Specifically, the percentage of DNA methylation was determined for 18 CpG units in the CDKN2B promoter of genomic DNA isolated from 295 pre-treatment bone marrow aspirates of consenting patients using the Sequenom EpiTYPER® platform. A weighted average of methylation levels in these 18 CpG units was used to evaluate association with clinical outcome, with the number of individual CpG dinucleotides comprising each CpG unit used as the weight for that unit. Kaplan-Meier methods were used for survival estimates. Cox proportional hazards models, stratified by the randomization factors FAB subtype and international prognostic scoring system (IPSS) group, were used to estimate hazard ratios (HRs). It appeared that the overall survival and hematologic response benefits observed with 5-azacytidine vs. CCR were independent of baseline methylation status of CDKN2B, and that a similar proportion of patients experienced increased hematologic response rates with 5- azacytidine in each methylation group.

[00246] In sum, it appeared that the survival benefit of 5-azacytidine treatment of MDS was observed in all methylation levels, but was greatest for patients with lower levels of DNA methylation pre-treatment. This correlation with methylation level was also seen for time to AML progression, which often showed a stronger correlation than the correlation for overall survival. It appeared that the OS benefit observed with AZA vs. CCR was independent of methylation status of the five genes analyzed. Increasing methylation was associated with worse OS. Patients with lower levels of methylation treated with AZA had the best OS and may derive greater benefit from AZA. These results underscore the complexity of using molecular biomarkers to predict response to epigenetic therapy.

6.4 Example 4

[00247] In this study, analysis of DNA methylation patterns in the baseline bone marrow (BM) of MDS patients from the AZA-001 trial was expanded to a greater group of genes to identify a DNA methylation-based predictive signature or pattern, predictive of overall survival (OS) or other clinical benefits in AZA-treated patients.

[00248] Air-dried drops of bone marrow aspirate were collected on unstained slides at baseline (pre-treatment), and at every 16 weeks post-therapy (Figure 5). DNA methylation levels of 27,578 genomic loci were measured in pre-treatment unpurified bone marrow aspirates of 129 patients (AZA [n=59] and CCR [n=70]) using Illumina Infinium Methylation27 Beadarray . The data were randomly divided into training data set (n=95 : AZA [n=38] and CCR [n=57]) and validation data set (n=34: AZA [n=21] and CCR [n=13]). A DNA methylation signature predictive of OS in AZA-treated patients was identified using uniCox algorithm (Tibshirani, Stat Appl Genet Mol Biol. 2009) first in the training data and then re-evaluated in the validation data (Figure 5). The influence of the signature on OS was assessed using Cox proportional hazards models. All statistical analyses were carried out in R (R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org).

[00249] To identify baseline DNA methylation signature that correlates with overall survival in the 38 patients treated with 5-azacytidine, 'uniCox' R package was used. See, e.g., Tibshirani, "Univariate Shrinkage in the Cox Model for High Dimensional Data," Statistical Applications in Genetics and Molecular Biology, 8(1), 2009, for a description of an algorithm. The identified signature contained a total of 214 loci mapped to 187 genes. A heatmap of the signature in the baseline samples is shown in Figure 6. The coefficients for each locus within the signature and their mean methylation ratios in the training data are listed in Table 4 (the first column of the table lists ID numbers from Illumina BeadArray and each ID represents one CpG site which is subject to DNA methyaltion). A predicted score for a new sample was calculated as follows:

where β^* was the methylation ratio for the 1^th locus in the new sample and β . was the mean methylation ratio for the 1^th locus in the training data. In the validation data set, this predicted score was used to assess its correlation with overall survival, e.g., in a Cox regression model.

[00250] A baseline DNA methylation signature predictive of overall survival in AZA- treated patients was identified based on a training set of 38 AZA-treated patients and reevaluated in a test set of 21 AZA-treated patients. The signature contains 214 genomic loci representing 187 genes, including, ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orfi2,

C19orf30, Clorfl 72, C1orf87, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, DES, DPYS, DYDC1, EGFL7, ELM03, ENTPD2, ENTPD3, ESR1, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FU44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HISTIHIA, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDRl, IRF6, KAZALDl, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRCl 7, LTF, MBD3L1, MEGFIO, MICALl, MRPL28, MTMR9, MTNRIB, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROG1, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOPl, OXT, PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRFl, RASIPl, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAPl, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WTI, ZFP41, ZNF205, and ZNF710. A list of enriched bio-groups for predicting overall survival is summarized in Table 5.

[00251] A DNA methylation score was calculated for each patient as the weighted average of the DNA methylation levels of these 214 genomic loci, with weights optimized in the training data set. In the training set, the DNA methylation score was significantly (hazard ratio [HR]=2.34 [95% confidence interval: 1.54, 3.54]; p = 5.8e-5) associated with overall survival (OS) and remained significant (HR=3.25 [1.65, 6.38]; p = 0.00062) in a multivariate analysis, adjusting for the clinical co-variates: sex, cytogenetic score, ECOG grade, and baseline RBC transfusion status. The DNA methylation score was not predictive of OS in patients treated with conventional care (p = 0.81), demonstrating the specificity of this marker to 5-azacytidine therapy. When applied to the validation set, the association of the DNA methylation score with OS remained marginally significant (HR=1.66 [0.99, 2.79]; p = 0.053) in a univariate analysis, but became less significant (p = 0.52) in a multivariate analysis. For the majority of the loci in the signature, lower DNA methylation correlated with better survival outcome. A predictive signature was also generated using the entire dataset (training and test sets combined), resulting in 53 loci, representing 39 genes, including WTI (Figure 7).

[00252] DNA methylation of WTI loci alone was predictive of OS in both univariate (HR=1.47 [1.00, 2.16]; p = 0.048) and multivariate (HR=2.04 [1.14, 3.64]; p = 0.016) analyses of AZA-treated patients in the training data set. DNA methylation of WTI loci remained as a significant (HR=2.87 [1.24, 6.64]; p = 0.014) predictor of OS in the test data set in univariate analysis, but became less significant (p = 0.35) in a multivariate analysis. As with the signature containing 214 loci, DNA methylation of WTI loci was not predictive of OS in patients treated with conventional care (p = 0.64). All performance results are summarized in Table 6.

[00253] In the combined dataset, the DNA methylation signature was significantly (p = 0.000278) associated with OS in a multivariate analysis, including addition of clinical co- variates. Methylation of WTI loci had predictive value in univariate and multivariate analyses.

[00254] In sum, specific DNA methylation patterns were observed in certain higher-risk MDS patients, which were predictive of overall survival or other clinical benefits in patients upon 5-azacytidine treatment. For example, a DNA methylation signature was identified in the unpurified pre -treatment bone marrow aspirate samples, which was shown to be predictive of OS of 5-azacytidine -treated higher-risk MDS patients.

[00255] While the examples have been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All patents, publications, and other references cited herein are incorporated by reference herein in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having a disease, comprising:

(i) obtaining cells from the subject;

(ii) measuring the level of methylation of one or more gene(s) in the cells;

(iii) comparing the methylation level of one or more gene(s) with a reference methylation value or a methylation pattern and selecting a therapeutic agent to be administered to the subject; and

(iv) administering the therapeutic agent to the subject in need thereof.

2. The method of claim 1, wherein the disease is a myelodysplastic syndrome.

3. The method of claim 1, wherein the disease is higher-risk

myelodysplastic syndrome.

4. The method of claim 1, wherein the disease is cancer.

5. The method of claim 1, wherein the disease is a blood-born tumor.

6. The method of claim 1, wherein the disease is a solid tumor.

7. The method of claim 1 , wherein the cells are obtained from bone marrow.

8. The method of claim 1, wherein the gene(s) is/are selected from the group consisting of ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CR11, AMT, ANKRD33, APC2, A VP, BHMT, C18orfi2, C19orfl0, Clorfl 72, ΟΙθΓβ7, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPTIB, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDCl, EGFL7, ELM03, ENTPD2, ENTPD3, ESRl, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FLJ44881, FLVCR2, FREQ, FZD9, GAB1, GAS2L2, GATA4, GBGT1, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRCl 7, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNRIB, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROGl, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOPl, OXT, PACSINl, PAOX, PARP3, PAXl, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMH1, SCUBE3, SEMA3B, SGPP2, SHROOM1, SKAP1, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINT1, SRD5A2, SRRT, SSTR4, STMN1, TBC1D1, TCEA2, TCF15, TFAP2E, TGFBI, TIAM1, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNTl, TP53INP1, TRPC4, TRPM3, UNC80, VAMP5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

9. The method of claim 1, wherein the gene(s) is/are selected from the group consisting of WT1, CDH1, and CDKN2B.

10. The method of claim 1, wherein the gene is WT1.

11. The method of claim 1 , wherein the methylation pattern corresponds to a methylation signature provided in Figure 6.

12. The method of claim 1, wherein the therapeutic agent is a cytidine analog.

13. The method of claim 1, wherein the therapeutic agent is 5-azacytidine.

14. The method of claim 1, wherein the therapeutic agent is administered parenterally.

15. The method of claim 1, wherein the therapeutic agent is administered orally.

16. The method of claim 1, wherein the therapeutic agent is administered in a therapeutically effective amount.

17. The method of claim 16, wherein the therapeutically effective amount is between about 50 mg/m² and about 200 mg/m² per day.

18. The method of claim 17, wherein the therapeutic agent is administered parenterally.

19. A method for identifying a patient diagnosed with a myelodysplastic syndrome having an increased probability of obtaining improved overall survival following 5-azacytidine treatment.

20. The method of claim 19, which comprises analyzing methylation levels of the patient's nucleic acid.

21. The method of claim 20, wherein the nucleic acid is DNA.

22. The method of claim 20, wherein the nucleic acid is RNA.

23. The method of claim 20, which comprises analyzing the methylation level of a gene selected from the group consisting of ABHD14A, ABO, ADAMTS18, ADRA2B, ADRB3, AIRE, AKAP12, ALOX15B, ALS2CRII, AMT, ANKRD33, APC2, A VP, BHMT, C18orfi2, C19orf30, Clorfl 72, C1orf87, C3orfl5, C1QTNF6, C22orf27, C7orfl6, C7orf41, CBX7, CCDC19, CCDC81, CD164L2, CDH1, CDKN2B, CHAD, CHRNG, CIDEB, CKMT1B, CKMT2, CLCN6, CLDN6, CLDN9, CNTN4, CPT1B, CRHBP, CXCL5, CYP26C1, CYP2E1, DES, DPYS, DYDC1, EGFL7, ELM03,

ENTPD2, ENTPD3, ESRI, EYA4, F2RL2, FAM57B, FBLN1, FBX02, FKBP1B, FU44881, FLVCR2, FREQ, FZD9, GABl, GAS2L2, GATA4, GBGTl, GDF5, GHSR, GNAS, GNMT, GNPNAT1, GP1BA, GPR25, GRM6, GSTM5, HCN4, HIST1H1A, HOXD4, HSPA2, HTATIP2, HTR7, HYDIN, IGDCC3, ILDR1, IRF6, KAZALD1, KCNA6, KCNK3, KCNQ1, KIAA0427, KIR3DX1, KRT25, KRT7, KRT72, LAD1, LAMA4, LAMC2, LGTN, LRRCl 7, LTF, MBD3L1, MEGF10, MICALl, MRPL28, MTMR9, MTNRIB, NALCN, NCAN, NCOR2, NDRG2, NDUFAF3, NEUROGl, NGB, NPFFR2, NPM2, NPPB, NPR2, NXN, OBFC2B, OGFR, ONECUT2, OTOP1, OXT, PACSIN1, PAOX, PARP3, PAX1, PCDH8, PCDHAC2, PDE4C, PF4V1, PKDREJ, PM20D1, POMC, POU3F1, PPAPDC3, PRIC285, PRLH, PSMDll, PTGIS, RAB36, RAP 1 GAP, RASGRF1, RASIP1, RBPJL, RLNl, RPL36, RPL36AL, RPUSD3, SCG5, SCMHl, SCUBE3, SEMA3B, SGPP2, SHROOMl, SKAPl, SLC12A8, SLC5A8, SNN, SORBS3, SPG7, SPINTl, SRD5A2, SRRT, SSTR4, STMNl, TBCIDI, TCEA2, TCF15, TFAP2E, TGFBI, TIAMl, TMEM125, TMEM151A, TMEM184A, TMEM189, TMOD3, TNNT1, TP53INP1, TRPC4, TRPM3, UNC80, VAMP 5, VHL, VSTM1, WBSCR27, WDR52, WT1, ZFP41, ZNF205, and ZNF710.

24. The method of claim 20, which comprises analyzing the methylation level of a gene selected from the group consisting of WT1, CDH1, and CDKN2B.

25. The method of claim 20, which comprises analyzing the methylation level of a gene, which is WT1.

26. The method of claim 20, in which the patient's increased probability of obtaining improved overall survival following 5-azacytidine treatment is used to plan or adjust the patient's 5-azacytidine treatment.

27. The method of claim 19, in which the increased probability is a 10% greater probability, a 50% greater probability, a 100% greater probability, or a 200% greater probability.