Poster Presentation 25th Lorne Cancer Conference 2013

Genome-wide DNA methylation analysis of archival formalin-fixed paraffin-embedded tissue (FFPET) using MBDCap-Seq identifies novel epigenetic diagnostic biomarker loci in breast cancer  (#414)

Elena Zotenko* , Clare Stirzaker* 1 , Jenny Z Song 2 , Wenjia Qu 2 , S. Nair 2 , Kelly Keijida 3 , Rodney Scott 3 , Melissa Brown 4 , Matt Trau 5 , John Forbes 6 , Sandra Stein 7 , Susan J Clark 8
  1. St Vincent's Clinical School, University of NSW, Sydney, NSW, Australia
  2. Epigenetics Group, Cancer Program, Garvan Institute of Medical Research, Sydney, NSW, Australia
  3. School of Biomedical Sciences, Faculty of Health, University of Newcastle, Newcastle, NSW, Australia
  4. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia
  5. Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia
  6. Surgical Oncology, Universitry of New South Wales, Sydney, NSW, Australia
  7. Queensland Health Pathology Service, Princess Alexandra Hospital, Brisbane, QLD, Australia
  8. St Vincent's Clinical School, University of New South Wales, Sydney, NSW, Australia

DNA methylation is an important epigenetic modification of DNA in mammalian genomes and plays a critical role in cellular differentiation and normal development and its deregulation underpins many disease states, including cancer.  Advances in genome-wide DNA methylation sequencing technology has enabled new strategies for the identification of novel DNA methylation loci for potential cancer diagnosis and prognosis biomarkers.  Archival cancer samples are commonly stored as formalin-fixed paraffin embedded tissue (FFPET) and this provides an invaluable resource for identification of epigenetic events that were associated with the cancer subtypes and progression.  However, DNA extracted from FFPET is often limiting and degraded previously presenting challenges for genome-wide DNA methlyation studies. 

 

Here, we describe a detailed protocol using MBDCap-Seq to perform genome-wide DNA methylation profiling on a cohort of matched tumour and lymph node positive triple-negative breast cancer samples from archival formalin-fixed paraffin embedded tissue (FFPET) clinical DNA samples. We show this to be equivalent if not more sensitive than profiling from fresh frozen samples. We detail the computational pipeline for analysis of the Illumina sequencing data for detecting differentially methylated regions and show the identification of novel loci displaying differentially methylated cancer-specific marks from matched tumour and lymph node positive triple-negative breast cancer samples and an independent cohort validation of these loci.  In addition, we show that MBDCap-Seq interrogates genomic sequences not covered by 450K Illumina array technology, in particular, the coverage of enhancers and insulators, enabling the discovery of novel genomic loci.  MBDCap-Seq therefore enables genome-wide DNA methylation profiling of clinical samples, and has merit in being a simple comprehensive approach that does not involve bisulphite treatment of DNA.