Abstract 155: Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression
Authors: Szigeti Krisztina A. , Galamb Orsolya , Kalmár Alexandra , Valcz Gábor , Zsigrai Sára , Barták Barbara K. , Nagy Zsófia B. , Tulassay Zsolt , Igaz Péter , Molnár Béla 
Published: September 2020

Backgrounds: Global DNA hypomethylation is characteristic in various cancer types including colorectal cancer. Alterations of DNA methylation related enzymes expression and decreased level of methyl-donor molecules (folic acid (FA), S-adenosylmethionine (SAM)) can lead to aberrant DNA methylation pattern and elevated homocysteine level. Aims: Our aim was to examine global DNA methylation changes during aging and colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease in tissue and liquid biopsy samples for diagnostic purposes. Moreover, we aimed to explore the reasons of global hypomethylation on gene expression level and methyl-donor molecule content.

Methods: Bisulfite treatment was performed on DNA isolated from 30 normal (N), 10 adenoma (Ad), 10 colorectal carcinoma (CRC), 10 colitis ulcerosa (UC) tissue samples and on 11 N, 10 Ad, 15 CRC, 12 UC plasma specimens. 30 N samples contained different age groups derived from under 20 to 70 years old healthy controls for examination of aging process. LINE-1 PCR product was generated and pyrosequenced. Whole genome expression level of 60 biopsy samples was evaluated by HTA 2.0 RNA microarraychip (Affymetrix). In situ tissue appearance of 5-methylcytosine, FA, SAM, homocysteine, and expression of DNA methyltransferases (DNMTs) were analyzed by immunohistochemistry staining (IHC).

Results: According to LINE-1 bisulfite sequencing results, DNA methylation was 72.6±1% in samples of healthy controls under 50 years old and 71.6±1.8% in specimens of patients over 50 years old. Significant DNA hypomethylation was found in CRC (62.9±8.7%; p&lt;0.001, early stages: 67.4±7.8%, late stages: 58.3±0.1%), Ad (66.7±5.1%; p&lt;0.001) tissue samples in comparison with N samples (72±1.4%). Significant decrease of DNA methylation was observed in CRC (78.8±1.7%; p&lt;0.02), and Ad (80.1±1.7%; p&lt;0.02) plasma samples compared to N specimens (82.2±1.8%). Global DNA hypomethylation was not detected in UC samples. Significantly elevated RNA expression of enzymes connected to nucleotide synthesis was observed in Ad and CRC samples compared to N (p&lt;0.05), while no changes were detected in the RNA levels of DNA methylation-related proteins. The intensity of 5-mC labeling of CRC and Ad samples was lower than in N tissue samples. Decreased FA, SAM, and increased homocysteine levels were noticed in CRC compared to N specimens; however no expression changes of DNMT enzymes were observed.

Conclusion: Significant decrease in DNA methylation level was found in tissue and liquid biopsy samples of colorectal normal-adenoma-carcinoma sequence, but not in UC specimens. Our results suggest that determination of global DNA hypomethylation could have prognostic and diagnostic value as well, and reduction of DNA methylation level could be linked to decreased FA and SAM availability and not to methylation related ezymes activity.

Citation Format: Krisztina A. Szigeti, Orsolya Galamb, Alexandra Kalmár, Gábor Valcz, Sára Zsigrai, Barbara K. Barták, Zsófia B. Nagy, Zsolt Tulassay, Péter Igaz, Béla Molnár. Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression [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 155.