Abstract LB-236: Increasing global DNA hypomethylation and genomic instability in colorectal cancer development
Authors: Kalmar Alexandra , Szigeti Krisztina , Valcz Gabor , Galamb Orsolya , Tulassay Zsolt , Igaz Péter , Molnar Bela 
Published: August 2018

Background: Epigenetic changes are known to contribute to colorectal cancer (CRC) formation as gene-specific DNA hypermethylation and global hypomethylation of repetitive retrotransposon sequences of the human genome. The relationship between genomic instability and global DNA hypomethylation in colorectal tissues remains to be elucidated. Aims: Our study aimed to analyze the global DNA methylation level alterations along the colorectal normal-adenoma-carcinoma sequence progression on the basis of LINE-1 retrotransposon methylation level and in parallel to examine methylated DNA levels and DNA double-strand breaks in tissue samples.

Methods: Genomic DNA was isolated from 20 colorectal adenomas, 20 CRC and 10 healthy normal colonic biopsy samples. Bisulfite conversion of DNA samples was performed using EZ DNA Methylation-Direct Kit (Zymo). Bisulfite-specific PCR (BS-PCR) was applied for DNA methylation level quantification of LINE-1 retrotransposable element, and 146 bp long PCR products were sequenced on Pyromark Q24 system (Qiagen). FFPE tissue samples were assembled in tissue microarrays (40 normals, 40 adenomas, 40 CRCs). Epithelial DNA methylation status and DNA double-strand breaks were visualized by 5-methylcytosine (5-mC) / gamma-H2AX (γH2AX) multiplex immunofluorescent labeling using the OPAL TSA assay (Perkin Elmer Inc). Slides were digitalized with Pannoramic Confocal scanner (3DHISTECH Ltd.) at 40x 0.22 um/ pixel resolution providing high resolution for the DNA double-strand break detection. Digital slides were semiquantitatively analyzed by the Caseviewer digital microscope.

Results: According to the LINE-1 bisulfite sequencing results, significant global DNA hypomethylation was detected both in CRC (63 ± 6.7 %; p=0.0302) and adenoma samples (65 ± 3.8 %; p= 0.0093) compared to normal tissue (73 ± 1.4 %). In agreement with this strong and diffuse nuclear 5-mC staining was detected in normal epithelium (typical scoring values: +2 and +3). Epithelial components of adenomas and CRC samples showed decreased nuclear 5-mC staining (typical scoring values: +2 and +1 respectively). Significantly lower 5-mC levels and remarkable higher number of DNA double-strand breaks (&gt;5/cell) could be observed in CRCs (p&lt;0.05). The metastatic stage IV cases showed the highest degree of hypomethylation and the highest number of DNA double-strand breaks in all of the cancer cells.

Conclusion: Global DNA hypomethylation could be shown in CRC compared to healthy normal tissue samples both by LINE-1 bisulfite-sequencing and by 5-mC immunohistochemistry. In parallel with the epigenetic changes, the elevated number of DNA double-strand breaks refers to increased genomic instability along the colorectal cancer formation.

Citation Format: Alexandra Kalmar, Krisztina Szigeti, Gabor Valcz, Orsolya Galamb, Zsolt Tulassay, Péter Igaz, Bela Molnar. Increasing global DNA hypomethylation and genomic instability in colorectal cancer development [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 LB-236.