E. J. Zmuda, K. R. Covington, C. Kandoth, C. K. Y. Ng, D. J. Weisenberger, R. Bowlby, A. Chu, R. Corbett, D. Brooks, A. D. Cherniack, B. Murray, S. Ling, W. Zhao, E. Shinbrot, R. S. Lim, M. S. Bootwalla, T. Hinoue, J. Hu, N. Kakkar, M. Lawrence, M. McLellan, C. Miller, D. Morton, A. J. Mungall, D. Muzny, C. Sougnez, P. Stojanov, L. Xi, N. Schultz, R. Akbani, C. Curtis, B. Fulton, R. A. Gibbs, G. Getz, P. Spellman, M. Marra, M. Ladanyi, R. Tarnuzzer, H. Sofia, The Cancer Genome Atlas Research Network, C. Hutter, D. A. Wheeler, J. Gastier-Foster, Lair
Genomic, transcriptomic and epigenomic characterization has accelerated the discovery of clinically-relevant alterations in cancer, predominantly using fresh frozen (FF) specimens. However, clinical molecular pathology laboratories prefer formalin-fixed paraffin-embedded (FFPE) methods, known to introduce artifacts at the nucleic acid level, over fresh frozen methods. Extending the multi-platform analysis to FFPE specimens for comprehensive clinical molecular diagnosis requires a thorough understanding of the consequence of formalin-fixation. We present a detailed multi-platform characterization of FFPE preservation using paired FF specimens as the 'gold standard'. DNA and RNA were obtained from 38 patients across 6 cancer types using a FFPE optimized co-isolation. The impact of FFPE on exome sequencing was dependent on filtering, where a minimum coverage or supporting read filter can mitigate FFPE-specific false positives. Copy number alterations, MSI assessment, mutational signatures, and DNA methylation were comparable between FFPE and FF. FFPE biases in RNA expression can be overcome when using biology-relevant genes and we describe a novel consequence of FFPE on miRNA species diversity. Collectively, this data provides a broad view of FFPE artifact and offers best practices for overcome these biases.