Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied.
Highlights
Due to the rapid progress in next-generation sequencing (NGS), cancer genomics is revealing the somatic variants and driver mutations of genes in various cancers [1, 2]
We determined DNA quality using a relative Quantitative PCR (qPCR) ratio of FFPE tissue DNA to fresh-frozen tissue DNA and compared between FFPE samples fixed with 10% formalin and 20% formalin, considering various fixation time periods (Fig 1A)
When using 10% formalin, the DNA quality was decreased by a fixation period of longer than 3 days (p
Summary
Due to the rapid progress in next-generation sequencing (NGS), cancer genomics is revealing the somatic variants and driver mutations of genes in various cancers [1, 2]. 4) Immunohistochemistry (IHC) of FFPE sections helps in extracting specific target cells by LCM to elucidate genetic alterations in specific marker-positive cells. To precisely compare DNA quality via pair analysis, matched samples of fresh-frozen tissues and FFPE tissues were prepared from a single specimen. It has been reported that such artifactual mutations are caused by FFPE preparation [5,6,7], whereas other studies report that this does not occur [8,9,10] To resolve this disagreement, we compared a pair of fresh-frozen and FFPE specimens from the same tissue by comprehensive genetic sequencing. We extracted DNA from 4 pairs of fresh-frozen and FFPE tissues of a single normal liver with no mutation and compared NGS results in each case by pair analysis
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