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Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making


The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard. In this study of ten melanoma patients, we compared WES data produced from analysis of gDNA isolated from FFPE tumour tissue with that isolated from fresh-frozen tumour tissue from the same specimen. FFPE samples were sequenced using both Illumina's Nextera and NimbleGen SeqCap exome capture kits. To examine mutations between the two tissue sources and platforms, somatic mutations in the FFPE exomes were called using the matched fresh tissue sequence as a reference. Of the 10 FFPE DNA samples, seven Nextera and four SeqCap samples passed library preparation. On average, there were 5341 and 2246 variants lost in FFPE compared to matched fresh tissue utilising Nextera and SeqCap kits, respectively. In order to explore the feasibility of future clinical implementation of WES, FFPE variants in 27 genes of important clinical relevance in melanoma were assessed. The average concordance rate was 43.2% over a total of 1299 calls for the chosen genes in the FFPE DNA. For the current clinically most important melanoma mutations, 0/3 BRAF and 6/8 (75%) NRAS FFPE calls were concordant with the fresh tissue result, which was confirmed using a Sequenom OncoCarta Panel. The poor performance of FFPE WES indicates that specialised library construction to account for low quality DNA and further refinements will be necessary before this approach could be used for routine clinical decision making over currently preferred techniques.

Type Journal
ISBN 1465-3931 (Electronic) 0031-3025 (Linking)
Authors De Paoli-Iseppi, R. ; Johansson, P. A. ; Menzies, A. M. ; Dias, K. R. ; Pupo, G. M. ; Kakavand, H. ; Wilmott, J. S. ; Mann, G. J. ; Hayward, N. K. ; Dinger, M. E. ; Long, G. V. ; Scolyer, R. A.;
Publisher Name PATHOLOGY
Published Date 2016-01-01
Published Volume 48
Published Issue 3
Published Pages 261-6
Status Published in-print
URL link to publisher's version
OpenAccess link to author's accepted manuscript version