Next-generation sequencing technologies and formalin-fixed paraffin-embedded tissue: Application to clinical cancer research

Billions of tissue samples are now archived by formalin fixation paraffin embedding (FFPE) in tissue banks and hospitals around the world. For thosebiomarkers measured by immunohistochemistryand used today as a standard of care in cancer treatment, this method of preservation works well. However, the heterogeneous nature of the disease means that many patients do not respond or relapse under standard treatment.Next-generation sequencing (NGS) technologies now provide extensive genome analyses at the level of gene expression, identification of somatic copy number aberration, somatic single nucleotide variants, fusion transcripts, and epigenetic modification. Successful application of this technologyto the large volumes of archival FFPE material with long-term follow-up data will be ahugely powerful tool in identifying new biomarkers of disease outcome, disease recurrence, and treatment response.The major hurdle for NGS application to archival material is the effect of formalin fixation on nucleic acids. The process resultsin chemical modification, cross-linking, and fragmentation. Chemical modification can result in false-positive mutation calls, and fragmentation can result inoverrepresentation of the 3’ end of genes creating bias in gene expression. There are now a number of NGS kits and protocols which are marketed specifically for use with FFPE material. Laboratories are beginning to validate and apply these methods. Inthis chapter, we review the progress in theadaption of NGS technologies to FFPE tissuefor clinical cancer research.