Abstract 3623: Differences in the frequency of CRC-associated pathogen variants in colorectal adenomas and cancers from the Hungarian population
Authors: Kalmar Alexandra , Galamb Orsolya , Wichmann Barnabas , Bartak Barbara B , Nagy Zsofia B , Zsigrai Sara , Szigeti Krisztina , Pipek Orsolya , Medgyes-Horváth Anna , Csabai Istvan , Tulassay Zsolt , Igaz Peter , Molnar Bela 
Published: September 2020

Abstract
Background: Colorectal cancer (CRC) has high incidence in Eastern-Europe, especially in Hungary. In addition to environmental factors, the genetic and epigenetic background of this phenomenon still remains unknown. Aims: We aimed to perform whole exome sequencing along the colorectal adenoma-carcinoma sequence on tissue samples in order to identify characteristic germline and somatic variants.
Materials & methods: Genomic DNA was isolated from 111 colonic tissue samples including 20 adenomas (AD) with matched normal adjacent tissue (NAT) pairs, 18 CRC with matched NAT pairs, 3 normal (N) young donors, 1 CRC and 1 AD and from 2 colorectal cancer cell lines (HT29, SW480), furthermore, 20 buffy coat (from 10 healthy, 5 adenoma and CRC patients). Exome enrichment was done with the Nextera DNA Exome Kit and sequencing was performed with the NextSeq 500/550 High Output v2 kit using the NextSeq 500 Instrument. Raw data analysis and demultiplexing was completed on BaseSpace Sequence Hub. Germline and somatic variants were determined by Genome Analysis Toolkit (GATK) and MuTect2 algorithms by using the default filter settings and were confirmed by ddPCR.
Results: The total number of detected variants increased along the adenoma-carcinoma sequence and NAT samples showed higher number of variants compared to healthy tissues. On the basis of the somatic variant analysis, certain genes with the most frequent CRC-associated pathogenic mutations could be detected in relatively higher [e.g. APC (89% of AD samples, 74% of CRC samples)], with relatively lower [e.g. KRAS (47% of ADs, 33% CRC), TP53 (26% of ADs, 14% of CRCs), or with average frequencies [PIK3CA (4.8% of AD samples, 11% of CRC samples), NRAS (5% of ADs, 4.7% of CRCs)]. One adenoma sample (4.7%) exerted BRAF mutation. On the basis of the COSMIC database, the expected variant profile of the CRC cell lines could be confirmed (HT-29: BRAF, TP53; SW480: KRAS, TP53). Among the germline variants, MTHFR C677T (rs1801133) were detected in 4 N (40% CT) 8 AD (37% CT, 5% TT) 9 CRC samples (37% CT, 11% TT), and MTHFR A1298C (rs1801131) were detected in 7 N (50% AC, 20% CC) 12 AD (47% AC, 16% CC) and 10 CRC samples (42% AC, 11% CC) representing a relatively higher frequency in the Hungarian population compared to the European and worldwide statistics.
Conclusion: Identification of CRC-associated pathogen variant patterns in the Hungarian population revealed a higher tumor mutation burden already in pre-malignant colorectal adenoma samples, as well as in CRC. Alterations in the frequencies of CRC-associated somatic variants compared to the European averages might contribute to the high CRC incidence observed in the Hungarian population. The germline variants of MTHFR gene were observed with a relatively higher frequency in our cohort having an effect on the DNA methylation regulation system that might be a link between the genetic and epigenetic alterations of the CRC development.
Citation Format: Alexandra Kalmar, Orsolya Galamb, Barnabas Wichmann, Barbara B Bartak, Zsofia B Nagy, Sara Zsigrai, Krisztina Szigeti, Orsolya Pipek, Anna Medgyes-Horváth, Istvan Csabai, Zsolt Tulassay, Peter Igaz, Bela Molnar. Differences in the frequency of CRC-associated pathogen variants in colorectal adenomas and cancers from the Hungarian population [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3623.