Assessment of concordance between fresh-frozen and formalin-fixed paraffin embedded tumor DNA methylation using a targeted sequencing approach

// Bruce Moran 1, 10, * , Sudipto Das 1, 10, * , Dominiek Smeets 2, 3 , Gillian Peutman 2, 3 , Rut Klinger 1, 10 , Bozena Fender 4 , Kate Connor 1, 5, 10 , Matthias Ebert 6 , Timo Gaiser 6 , Jochen HM Prehn 5 , Orna Bacon 5, 7 , Elaine Kay 7 , Bryan Hennessy 7 , Verena Murphy 8 , Bauke Ylstra 9 , Diether Lambrechts 2, 3 , Annette T. Byrne 5 , William M. Gallagher 4, 10, ** and Darran P. O’Connor 1, 10, ** 1 Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland 2 Department of Oncology, Laboratory of Translational Genetics, VIB Center for Cancer Biology, Leuven, Belgium 3 Department of Oncology, Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium 4 OncoMark Ltd., NovaUCD, Belfield Innovation Park, Dublin, Ireland 5 Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland 6 Department of Internal Medicine, University of Heidelberg, Mannheim, Germany 7 Department of Pathology, Beaumont Hospital, Dublin, Ireland 8 Cancer Trials Ireland, Dublin, Ireland 9 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 10 Cancer Biology and Therapeutics Laboratory, UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College, Dublin, Ireland * These authors have contributed equally to this work ** Senior co-authors Correspondence to: Darran P. O’Connor, email: darranoconnor@rcsi.ie Keywords: targeted bisulfite sequencing, methylation, epigenetics, FFPE, tumor preservation Received: July 20, 2016      Accepted: April 03, 2017      Published: May 30, 2017 ABSTRACT DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a ‘bowing’ pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.