Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next-generation sequencing.

ABSTRACT

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR 4.30, 95% CI 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR 0.23, 95% CI 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR 4.43, 95% CI 1.87-10.49; and P=0.0003, OR 5.51, 95% CI 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.