title: Tools for translational epigenetic studies involving formalin-fixed paraffin-embedded human tissue: applying the Infinium HumanMethyation450 Beadchip assay to large population-based studies.
abstract: BACKGROUND
  Large population-based translational epigenetic studies are emerging due to recent technological advances that have made molecular analyses possible. For example, the Infinium HumanMethylation450 Beadchip (HM450K) has enabled studies of genome-wide methylation on a scale not previously possible. However, application of the HM450K to DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour material has been more challenging than application to high quality DNA extracted from blood. To facilitate the application of this assay consistently across a large number of FFPE tumour-enriched DNA samples we have devised a modification to the HM450K protocol for FFPE that includes an additional quality control (QC) checkpoint.
  
  
  RESULTS
  QC checkpoint 3 was designed to assess the presence of DNA after bisulfite conversion and restoration, just prior to application of the HM450K assay. DNA was extracted from 474 archival FFPE breast tumour material. Five samples did not have a detectable amount of DNA with an additional 42 failing to progress past QC checkpoint 3. Genome-wide methylation was measured for the remaining 428 tumour-enriched DNA. Of these, only 4 samples failed our stringent HM450K data criteria thus representing a 99% success rate. Using prior knowledge about methylation marks associated with breast cancer we further explored the quality of the data. Twenty probes in the BRCA1 promoter region showed increased methylation in triple-negative breast cancers compared to Luminal A, Luminal B and HER2-positive breast cancer subtypes. Validation of this observation in published data from The Cancer Genome Atlas (TCGA) Network (obtained from DNA extracted from fresh frozen tumour samples) confirms the quality of the data obtained from the improved protocol.
  
  
  CONCLUSIONS
  The modified protocol is suitable for the analysis of FFPE tumour-enriched DNA and can be systematically applied to hundreds of samples. This protocol will have utility in population-based translational epigenetic studies and is applicable to a wide variety of translated studies interested in analysis of methylation and its role in the predisposition to disease and disease progression.
authors.affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. emwong@unimelb.edu.au.
authors.forename: Ee Ming
authors.initials: EM
authors.lastname: Wong
authors.affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. ji.joo@unimelb.edu.au.
authors.forename: JiHoon E
authors.initials: JE
authors.lastname: Joo
authors.affiliation: Anatomical Pathology, Alfred Health, The Alfred Hospital, Melbourne, VIC, 3181, Australia. Catriona.McLean@monash.edu.
authors.forename: Catriona A
authors.initials: CA
authors.lastname: McLean
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. laura.baglietto@inserm.fr.
authors.forename: Laura
authors.initials: L
authors.lastname: Baglietto
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. dallas.english@cancervic.org.au.
authors.forename: Dallas R
authors.initials: DR
authors.lastname: English
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. gianluca.severi@hugef-torino.org.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Severi
authors.affiliation: Centre for Epidemiology and Biostatistics, The University of Melbourne, Melbourne, VIC, 3010, Australia. johnlh@unimelb.edu.au.
authors.forename: John L
authors.initials: JL
authors.lastname: Hopper
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. roger.milne@cancervic.org.au.
authors.forename: Roger L
authors.initials: RL
authors.lastname: Milne
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. liesel.fitzgerald@cancervic.org.au.
authors.forename: Liesel M
authors.initials: LM
authors.lastname: FitzGerald
authors.affiliation: Cancer Epidemiology Centre, Cancer Council Victoria, 615 St Kilda Road, Melbourne, VIC, 3004, Australia. graham.giles@cancervic.org.au.
authors.forename: Graham G
authors.initials: GG
authors.lastname: Giles
authors.affiliation: Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia. msouthey@unimelb.edu.au.
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Southey
mesh_terms: D000328:Adult; D000368:Aged; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D015331:Cohort Studies; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D044127:Epigenesis, Genetic; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D015870:Gene Expression; D015894:Genome, Human; D006801:Humans; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016612:Paraffin Embedding; D016707:Tissue Fixation; D064726:Triple Negative Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde