Abstract 3616: Fix the fixation: effect of formalin fixation on targeted sequencing, variant calling and gene expression
Authors: Alla Ravi , Luo Shujun , Helman Elena , Boyle Sean M. , Clark Michael J. , Scott Kirk , Sripakdeevong Parin , Karbelashvili Mirian , Church Deanna M. , Snyder Michael , West John , Chen Richard 
Published: July 2016

Abstract
Tumor biopsies are often Formalin-Fixed and Paraffin-Embedded (FFPE) for histological staining, genetic testing and archival purposes. Formalin treatment preserves tissue by crosslinking proteins, but can lead to mutation of the nucleic acid bases that can be detected by next-generation sequencing (NGS) methods despite being unrelated to the cancer. Studies have shown that certain fixation protocols are compatible with high quality nucleic acid isolation and variant calling by NGS. These studies used tissue fixed with 10% neutral buffered formalin for 24 hours, a standard protocol in the pathology field. In practice, however, FFPE sample handling varies from site-to-site. As we processed FFPE samples from various sites, we found that the quality of the isolated nucleic acids and subsequent sequencing results varied substantially. We hypothesized that this is due to deviations from the standard protocol such as improperly buffered reagents, variation in the fixation time and temperature, microwaving, and varied storage conditions of the samples.
To understand the role of formalin fixation on the quality of the isolated nucleic acids and subsequent NGS analyses, we subjected the widely studied cell line NA12878 to various formalin fixation conditions. We then performed an augmented target enrichment and sequencing assay on both the DNA and RNA isolated from fresh frozen (FF) and formalin fixed (FFPE) NA12878 samples. We assessed raw nucleic acid quality, library quality, alignment rate, duplication rate, on-target efficiency and variant concordance between FF and FFPE.
We noted substantial negative effects on sequencing library quality associated with sample incubation in unbuffered formalin, at high temperatures, and long periods of time (e.g. 3 days rather than 1 day). These harshly treated samples also showed poor alignment qualities, higher duplication rates, and lower mapping qualities. At the small variant level, they showed an increase in global C&gt;T deamination (CG-&gt;TA, 50% FFPE vs 35% in FF) and oxidation (CG-&gt;AT, 15% FFPE vs 10% FF) rates. Variants caused by formalin fixation were most commonly detected at low (&lt;5%) allele frequencies (AFs). When compared to gold set mutations in NA12878, we observed 80% false negative and 60% false positive variants in harshly treated samples. We are performing a similar analysis with RNA isolated from FF and FFPE NA12878 samples to understand the role of formalin on RNA variant and gene expression assays.
Using the results of this study, we intend to provide guidance on optimal fixation conditions for NGS applications. It is crucial to fix tissues in buffered formalin for less than 24hr at room temperature to preserve nucleic acid integrity and amenability for downstream NGS assays. We also demonstrate how a deeper understanding of the effects of formalin can improve variant calling from FFPE tissues, especially at lower AFs where formalin-related errors have the greatest impact.
Citation Format: Ravi Alla, Shujun Luo, Elena Helman, Sean M. Boyle, Michael J. Clark, Kirk Scott, Parin Sripakdeevong, Mirian Karbelashvili, Deanna M. Church, Michael Snyder, John West, Richard Chen. Fix the fixation: effect of formalin fixation on targeted sequencing, variant calling and gene expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3616.