Abstract 2945: Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients
Authors: Kalmar Alexandra , Szabo Gitta , Galamb Orsolya , Bartak Barbara Kinga , Nagy Zsofia Brigitta , Zsigrai Sara , Szigeti Krisztina Andrea , Kothalawala William , Igaz Peter , Takacs Istvan , Molnar Bela 
Published: June 2022

Abstract
Background: Analysis of circulating cell-free DNA (cfDNA) of colorectal cancer (CRC) patients provides an ideal approach to explore genetic alterations in a minimally invasive way.
Aims: We aimed to perform a comprehensive analysis of tissue-originated genomic DNA and plasma-derived cfDNA from CRC patients by whole-exome and targeted panel sequencing.
Materials & methods: DNA was isolated from tissue and plasma samples of 55 [7 healthy (N), 16 adenomas (AD), and 32 CRC] patients using the High Pure PCR Template Kit (Roche) and the Quick cfDNA Serum & Plasma Kit (Zymo). cfDNA quality was assessed with the High Sensitivity DNA kit on BioAnalyzer 2100 and quantified by the Qubit dsDNA HS assay. Exome libraries from tissue samples were constructed by the Nextera DNA Exome Kit (Illumina). For the cfDNA samples, we used the QIAseq cfDNA All-in-One kit combined with the QIASeq Human Exome Kit, and in the case of 12 patients, CRC-specific oncogenes were further analyzed with a solution developed by QIAGEN for reliable calling of low frequency variants in cfDNA samples. Exome libraries were quantified with the KAPA Library Quantification Kit and the QIAseq™ Library Quant Assay Kit (Qiagen) and were sequenced using the NextSeq 500/550 High Output v2 kit on a NextSeq 500 Instrument (Illumina). Raw data analysis and demultiplexing were completed on the BaseSpace Sequence Hub. Variants of the tissue and cfDNA samples were determined by the Mutect2 and Haplotype caller algorithms of GATK 4.1.4.1, respectively. Clinical significance was evaluated according to the OncoKB database.
Results: The most frequently mutated genes were APC, KRAS, DYNC1H1, KCNO5, and MARCH6 in the colorectal adenoma tissue samples, while those in CRC samples were APC, TP53, TTN, KRAS, and DYNCC2H1. CfDNA quantity was significantly higher in the CRC group compared to the AD (p&lt;0.02) and N (p&lt;0.005) patients. Based on the plasma exome results, 4.4-59.4% of the tumor somatic variants could be found in 12.5% of the patients. These patients were above 60 years of age and had Dukes D stage CRC. Panel sequencing of the circulating DNA samples detected tumor somatic variants in 8 out of the 12 enrolled patients. This method could identify 60% of all tumor somatic variants falling on its targeted regions, while whole-exome sequencing recovered only 20% of tumor somatic variants in the respective regions in cfDNA of the same patients.
Conclusion: We have performed a comprehensive genetic analysis on CRC tissue and cfDNA samples in the Hungarian population. Exome sequencing offers a broad overview of the coding regions, however, targeted panel sequencing with a higher coverage depth can detect tumor somatic variants more reliably in cfDNA, therefore, it can hold a relevant clinical potential.
Citation Format: Alexandra Kalmar, Gitta Szabo, Orsolya Galamb, Barbara Kinga Bartak, Zsofia Brigitta Nagy, Sara Zsigrai, Krisztina Andrea Szigeti, William Kothalawala, Peter Igaz, Istvan Takacs, Bela Molnar. Comprehensive analysis of tissue and plasma-related genetic alterations in Hungarian colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2945.