title: Methylation profiling of ductal carcinoma in situ and its relationship to histopathological features.
abstract: BACKGROUND
  DNA methylation is a well-studied biomarker in invasive breast cancer, but its role in ductal carcinoma in situ (DCIS) is less well characterized. The aims of this study are to assess the methylation profile in DCIS for a panel of well-characterized genes that are frequently methylated in breast cancer, to investigate the relationship of methylation with pathological features, and to perform a proof-of-principle study to evaluate the practicality of methylation as a biomarker in diagnostic DCIS material.
  
  
  METHODS
  Promoter CpG island methylation for a panel of 11 breast cancer-related genes was performed by methylation-sensitive high resolution melting (MS-HRM). Formalin-fixed, paraffin-embedded (FFPE) biopsies from 72 samples of pure DCIS (DCIS occurring in the absence of synchronous invasive carcinoma), 10 samples of mixed DCIS (DCIS adjacent to invasive carcinoma), and 18 samples of normal breast epithelium adjacent to a DCIS lesion were micro-dissected prior to DNA extraction.
  
  
  RESULTS
  Methylation was seen for all the tested genes except BRCA1. RASSF1A was the most frequently methylated gene (90% of DCIS samples) and its methylation was associated with comedo necrosis (p = 0.018). Cluster analysis based on the methylation profile revealed four groups, the highly methylated cluster being significantly associated with high nuclear grade, HER2 amplification, negative estrogen receptor (ER) α status, and negative progesterone receptor (PgR) status, (p = 0.038, p = 0.018, p <0.001, p = 0.001, respectively). Methylation of APC (p = 0.017), CDH13 (p = 0.017), and RARβ (p <0.001) was associated with negative ERα status. Methylation of CDH13 (p <0.001), and RARβ (p = 0.001) was associated with negative PgR status. Methylation of APC (p = 0.013) and CDH13 (p = 0.026) was associated with high nuclear grade. Methylation of CDH13 (p = 0.009), and RARβ (p = 0.042) was associated with HER2-amplification.
  
  
  CONCLUSIONS
  DNA methylation can be assessed in FFPE-derived samples using suitable methodologies. Methylation of a panel of genes that are known to be methylated in invasive breast cancer was able to classify DCIS into distinct groups and was differentially associated with phenotypic features in DCIS.
authors.forename: Jia-Min B
authors.initials: JM
authors.lastname: Pang
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Deb
authors.forename: Elena A
authors.initials: EA
authors.lastname: Takano
authors.forename: David J
authors.initials: DJ
authors.lastname: Byrne
authors.forename: Nicholas
authors.initials: N
authors.lastname: Jene
authors.forename: Alice
authors.initials: A
authors.lastname: Boulghourjian
authors.forename: Anne
authors.initials: A
authors.lastname: Holliday
authors.forename: Ewan
authors.initials: E
authors.lastname: Millar
authors.forename: C Soon
authors.initials: CS
authors.lastname: Lee
authors.forename: Sandra A
authors.initials: SA
authors.lastname: O'Toole
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Fox
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008875:Middle Aged; D010641:Phenotype; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA