Abstract 1428: Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples
Authors: Xiao Jianfeng , Tang Jiajie , Liu Xiangzhi , Tian Xiaoyan 
Published: August 2018

Introduction:

The ONCO/REVEAL Lung&Colon Caner Panel (LC103, Pillar Biosciences Inc.) interrogates regions in 22 genes that are frequently mutated in NSCLC and CRC. To evaluate the performance of ONCO/REVEAL Lung&Colon Cancer Panel, we assessed a cohort of 207 colorectal cancer FFPE samples collected by a top-tier hospital in Shanghai between 2015 and 2016. Among them, 27 FFPE samples showed abnormally high numbers of low frequency variants (&lt;2%) and were further investigated to assess the effect of DNA damage in somatic variant detection.

Materials and Methods:

DNA library preparation and sequencing: 10-20 ng of FFPE DNA (Quantitated by Qubit, Life Tech.) was used to prepare libraries using the LC103 panel. All libraries were subsequently sequenced on Illumina MiSeq sequencer.

FFPE DNA repair: DNA extracted from 27 FFPE samples with a high number of low VAF (&lt;2%) variants was treated with NEBNext FFPE DNA Repair Mix prior to library preparation. FFPE and DNA repaired FFPE were compared to analyze the effect of DNA damage in variant detection. Matched fresh frozen tumor tissue samples were also tested.

Data analysis: PiVATTM (Pillar Biosciences Inc.) was used for data analysis. PCR errors and sequencing errors are reduced to be well below 1% VAF through the PiVAT error correction algorithm.

Results:

100% successful rate of library preparation and sequencing: All of 207 FFPE samples yielded high quality sequencing data that detected mutant alleles at frequencies as low as 1%.

Variant detection: A total of 414 somatic variants, including SNV and small indels, were identified above 2% VAF in 193 out of 207 samples. TP53 (38%), KRAS (24%), PIK3CA (12%), FBXW7 (9%) and PTEN (4%) were the most frequently mutated genes. CNVs were identified in EGFR, MET, ERBB2, KRAS and FGFR1 genes.

The effect of DNA damage: The majority of detected variants between 1% and 2% VAF, are not known hotspot mutations. C:G&gt;T:A mutations account for 73.8% of variants between 1% and 2% VAF. DNA repair by NEBNext FFPE DNA Repair Mix or other UDG enzymes reduced 1-2% VAF calls significantly, indicating that these variants are false positive calls. However, DNA repair enzymes used in our study could not eliminate false positive calls completely. Other mechanisms are suspected to contribute to the remaining low frequency calls.

Variant between 2% and 5% VAF: In total, 33 somatic variants were detected at 2-5% VAF. There are many clinically actionable mutations or common driver mutations, including KRAS G12C (2.10%), KRAS G12D (2.21%), KRAS Q61L (2.74%), BRAF G469E (2.51%), PIK3CA E545K (2.08%, 2.39%, 4.78%), PIK3CA E545G (4.61%), PIK3CA Q546K (4.51%), PIK3CA H1047R (2.80%).

Conclusions:

The ONCO/REVEAL Lung&Colon Caner Panel is a robust and sensitive NGS Assay for the detection of somatic variants. DNA damage confounds variant identification in FFPE samples between 1 and 5% frequency.

Citation Format: Jianfeng Xiao, Jiajie Tang, Xiangzhi Liu, Xiaoyan Tian. Application of ONCO/REVEAL Lung & Colon Cancer Panel in CRC samples and effect of DNA damage in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1428.