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Next-generation sequencing of formalin-fixed, paraffin-embedded tumor biopsies: navigating the perils of old and new technology to advance cancer diagnosis

Formalin-fixed, paraffin-embedded (FFPE) tumor biopsies are a primary pathological specimen for oncology diagnostics. Hundreds of millions of such biopsies exist, and the method for creating them persists due to the ease and stability of sample fixation and the clarity of morphological features of disease that is revealed. These advantages, however, come at a cost that could not have been anticipated when formalin fixation was first described by Blum 120 years ago [1]. Formaldehyde readily crosslinks both proteins and nucleic acids, and it ignites a series of chemical modifications that compromise enzymatic treatments in downstream assays. The consequences can be dire for nucleic acid testing. Every step along the path from sample to signal – extraction, amplification, detection and interpretation – is influenced by the FFPE process. Most concerning is the lack of standardization for preparing FFPE blocks, leading to highly variable results [2]. Although a number of DNAand RNA-friendly preservation methods have been proposed [3,4], the entrenched use of FFPE procedures, coupled with the vast repository of archived specimens, has ensured its place in molecular pathology. By comparison, next-generation sequencing (NGS) technology emerged less than a decade ago. Yet, its trajectory has been meteoric: the price of sequencing per raw-base pair decreased by approximately 100,000-fold from 2000 to 2010 [5], and much of this gain was realized through massively parallel methods that have rapidly displaced Sanger sequencing. Like PCR, it only took about 5 years for NGS to find its way into the clinic. Now that it has, there can be little doubt of its revolutionary nature. With capabilities to accurately detect point mutations, structural variation, copy number changes, methylation status and gene expression, NGS is a multifaceted and versatile tool for molecular investigations.