title: Characterization of in vitro and in vivo hypomethylating effects of decitabine in acute myeloid leukemia by a rapid, specific and sensitive LC-MS/MS method.
abstract: DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2'-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5 microM decitabine, GDM decreased to approximately 50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo.
authors.affiliation: College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Zhongfa
authors.initials: Z
authors.lastname: Liu
authors.forename: Shujun
authors.initials: S
authors.lastname: Liu
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Xie
authors.forename: William
authors.initials: W
authors.lastname: Blum
authors.forename: Danilo
authors.initials: D
authors.lastname: Perrotti
authors.forename: Peter
authors.initials: P
authors.lastname: Paschka
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klisovic
authors.forename: John
authors.initials: J
authors.lastname: Byrd
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
mesh_terms: D000208:Acute Disease; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology* / Q000627:therapeutic use; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D017321:Clinical Trials, Phase I as Topic; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000032:analysis; D003849:Deoxyguanosine / Q000032:analysis; D006801:Humans; D007951:Leukemia, Myeloid / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000964:Antimetabolites, Antineoplastic; D003841:Deoxycytidine; D000077209:Decitabine; C016569:5-methyldeoxycytidine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D003849:Deoxyguanosine; D001374:Azacitidine