PhD Scientific Days 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

Methodological and Biological Factors Influence LINE-1 Pyrosequencing Assay Results in Colorectal Tissue and Peripheral Blood Cells

Text of the abstract

Long interspersed nuclear element 1 (LINE-1) methylation has a prognostic value and an important role in cancer development, hence several techniques have been introduced to measure its level. LINE-1 pyrosequencing is one of the most cost-effective and reliable techniques.

Our aim was to investigate the effects of experimental and biological factors on LINE-1 pyrosequencing to improve the comparability of the results in a potential intraindividual monitoring procedure of colorectal cancer.

LINE-1 pyrosequencing (Qiagen) was performed on colorectal tissue samples and buffy coat fractions of patients bearing colorectal tumors, and inflammatory bowel disease, along with healthy controls. Different CpG sites, sample types (fresh frozen, FF; formalin-fixed paraffin-embedded tissue, FFPE), sampling methods (endoscopic, surgical), storage times and temperatures, and kits with different LOT numbers were examined. Furthermore, biological factors such as age, gender, mutations of the methylenetetrahydrofolate reductase (MTHFR), as well as recent physical activity were also involved in this study.

Significantly decreased methylation was detected in CpG position 1 of tumorous tissue compared to normal, inflamed colorectal tissue, and buffy coat specimens. FF had altered methylation compared to FFPE samples. Slightly decreased methylation was found in surgical vs. endoscopic biopsies. Repeated measurements of the same biological samples with two years difference by using kits with different LOTs resulted in significant methylation elevation. A slight negative correlation was observed in line with aging, while significantly decreased methylation was detected in female patients over vs. under 40 years of age in colorectal tissue. Lower methylation was noticed in normal and adenoma, while elevated values were found in colorectal cancer MTHFR heterozygous vs. wildtype samples. Physical activity had no significant effect on LINE-1 methylation. Elapsed time and different storage temperatures until buffy coat separation caused no methylation alteration using K3EDTA tubes.

LINE-1 pyrosequencing is a reliable method for the investigation of LINE-1 methylation level, however methodological and biological factors could modify the results. Hence it is highly important to take the experimental details into the account, as well as to consider the application of internal controls.