title: Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma.
abstract: BACKGROUND
  Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy.
  
  
  METHODS
  We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing.
  
  
  RESULTS
  Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively.
  
  
  CONCLUSIONS
  Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Gengming
authors.initials: G
authors.lastname: Huang
authors.affiliation: Molecular Genetic Technology Program, School of Health Sciences, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Krocker
authors.affiliation: Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Kirk
authors.affiliation: Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Shehzad N
authors.initials: SN
authors.lastname: Merwat
authors.affiliation: Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Hyunsu
authors.initials: H
authors.lastname: Ju
authors.affiliation: Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Roger D
authors.initials: RD
authors.lastname: Soloway
authors.affiliation: Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Lucas R
authors.initials: LR
authors.lastname: Wieck
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Li
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Okorodudu
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Petersen
authors.affiliation: Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Nihal E
authors.initials: NE
authors.lastname: Abdulla
authors.affiliation: Internal Medicine, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Duchini
authors.affiliation: Surgery, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Cicalese
authors.affiliation: Surgery, University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Rastellini
authors.affiliation: Molecular Genetic Technology Program, School of Health Sciences, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Hu
authors.affiliation: Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0743, USA.
authors.forename: Jianli
authors.initials: J
authors.lastname: Dong
mesh_terms: D000483:Alleles; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000235:genetics*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004247:DNA / Q000097:blood* / Q000235:genetics; D019175:DNA Methylation; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D004247:DNA