title: Comparative analysis of PCR-based biomarker assay methods for colorectal polyp detection from fecal DNA.
abstract: BACKGROUND
  Aberrantly methylated genes are promising biomarkers for the detection of colon adenomas and colorectal cancers (CRCs). The optimal assay type and specific methylated genes for these assays remain to be determined.
  
  
  METHODS
  We used genomewide microarray-based assays to identify methylated genes as candidate biomarkers for colon neoplasms. The frequency of aberrant methylation of these genes in primary tumors was assessed with methylation-specific PCR (MSP). The limits of detection and specificities for different types of PCR-based assays were then assessed with the most promising genes identified in this screen. Finally, we assessed the best-performing MSP assay as an early-detection marker using fecal DNA samples.
  
  
  RESULTS
  ITGA4 [integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)] was identified as a novel gene frequently methylated in CRC. Methylated ITGA4 is present in 75% of colon adenomas (n = 36) and 92% of colon adenocarcinomas (n = 75). Comparison of end point MSP, end point MSP with clamped primers, and quantitative fluorescent MSP (qMSP) approaches revealed that both types of end point MSP assays could routinely detect as little as 70 pg DNA, whereas the qMSP assay could routinely detect as little as 7 pg. A fecal DNA qMSP assay for methylated ITGA4 can detect 69% of individuals with colon adenomas (n = 13) with a diagnostic specificity of 79% (n = 28).
  
  
  CONCLUSIONS
  Methylated ITGA4 is a promising marker gene for the early detection of colonic neoplasms. qMSP has the lowest limit of detection of the MSP assay types tested, and a qMSP assay that detects methylated ITGA4 has potential as an early-detection assay for colon neoplasms.
authors.affiliation: Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Ausch
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Kim
authors.forename: Karen D
authors.initials: KD
authors.lastname: Tsuchiya
authors.forename: Slavomir
authors.initials: S
authors.lastname: Dzieciatkowski
authors.forename: Mary K
authors.initials: MK
authors.lastname: Washington
authors.forename: Christos
authors.initials: C
authors.lastname: Paraskeva
authors.forename: Jerry
authors.initials: J
authors.lastname: Radich
authors.forename: William M
authors.initials: WM
authors.lastname: Grady
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis* / Q000235:genetics; D000236:Adenoma / Q000175:diagnosis* / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000235:genetics; D019175:DNA Methylation; D055088:Early Detection of Cancer; D005243:Feces / Q000737:chemistry*; D006801:Humans; D039441:Integrin alpha4 / Q000235:genetics; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D039441:Integrin alpha4; D004247:DNA