title: Comprehensive genome methylation analysis in bladder cancer: identification and validation of novel methylated genes and application of these as urinary tumor markers.
abstract: OBJECTIVE
  Epigenetic alterations are common and can now be addressed in a parallel fashion. We investigated the methylation in bladder cancer with respect to location in genome, consistency, variation in metachronous tumors, impact on transcripts, chromosomal location, and usefulness as urinary markers.
  
  
  METHODS
  A microarray assay was utilized to analyze methylation in 56 samples. Independent validation was conducted in 63 samples by a PCR-based method and bisulfite sequencing. The methylation levels in 174 urine specimens were quantified. Transcript levels were analyzed using expression microarrays and pathways were analyzed using dedicated software.
  
  
  RESULTS
  Global methylation patterns were established within and outside CpG islands. We validated methylation of the eight tumor markers genes ZNF154 (P < 0.0001), HOXA9 (P < 0.0001), POU4F2 (P < 0.0001), EOMES (P = 0.0005), ACOT11 (P = 0.0001), PCDHGA12 (P = 0.0001), CA3 (P = 0.0002), and PTGDR (P = 0.0110), the candidate marker of disease progression TBX4 (P < 0.04), and other genes with stage-specific methylation. The methylation of metachronous tumors was stable and targeted to certain pathways. The correlation to expression was not stringent. Chromosome 21 showed most differential methylation (P < 0.0001) and specifically hypomethylation of keratins, which together with keratin-like proteins were epigenetically regulated. In DNA from voided urine, we detected differential methylation of ZNF154 (P < 0.0001), POU4F2 (P < 0.0001), HOXA9 (P < 0.0001), and EOMES (P < 0.0001), achieving 84% sensitivity and 96% specificity.
  
  
  CONCLUSIONS
  We initiated a detailed mapping of the methylome in metachronous bladder cancer. Novel genes with tumor, chromosome, as well as pathway-specific differential methylation in bladder cancer were identified. The methylated genes were promising cancer markers for early detection of bladder cancer.
authors.affiliation: Department of Molecular Medicine and Urology, Aarhus University Hospital, Aarhus N, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinert
authors.forename: Charlotte
authors.initials: C
authors.lastname: Modin
authors.forename: Francisco M
authors.initials: FM
authors.lastname: Castano
authors.forename: Philippe
authors.initials: P
authors.lastname: Lamy
authors.forename: Tomasz K
authors.initials: TK
authors.lastname: Wojdacz
authors.forename: Lise Lotte
authors.initials: LL
authors.lastname: Hansen
authors.forename: Carsten
authors.initials: C
authors.lastname: Wiuf
authors.forename: Michael
authors.initials: M
authors.lastname: Borre
authors.forename: Lars
authors.initials: L
authors.lastname: Dyrskjøt
authors.forename: Torben F
authors.initials: TF
authors.lastname: Orntoft
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics / Q000652:urine*; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D052138:Genes, Neoplasm; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008297:Male; D008745:Methylation; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000652:urine
chemical_list: D014408:Biomarkers, Tumor; D007633:Keratins