PO-384 Manual and automated detection of DNA methylation alterations in colorectal neoplasia in circulating cell-free DNA fraction

Introduction During colorectal cancer (CRC) development several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumour cells that can be utilised for the diagnosis of CRC. Cell-free DNA (cfDNA) in the circulatory system can originate from tumour tissue; therefore the evaluation of tumor-related methylated DNA in plasma can be a simple and promising minimally invasive method for early cancer screening, especially using automated isolation techniques. Our aim was to analyse the methylation level alterations of 4 selected genes along the healthy-adenoma-CRC sequence in plasma samples. Moreover, we aimed to test different preservative blood collection tubes and automated cfDNA isolation method to determine whether these factors influence the DNA methylation patterns. Material and methods 121 blood samples were collected in K3EDTA tubes, and manual cfDNA isolation was performed. The methylation pattern of SFRP1 , SFRP2 , SDC2 and PRIMA1 was determined using MethyLight PCR containing multiplex preamplification step. Further 30 blood samples were collected in Roche Cell-Free DNA Collection Tubes, which was developed for stabilisation of cfDNA with preservative reagents. In case of these samples, in addition to manual isolation, MagNA Pure 96 Instrument was used for automated cfDNA extraction. Results and discussions Elevated methylation of the genes was observed in 70% to 90% of adenoma and CRC patients. Average DNA methylation level was SFRP1, SFRP2, SDC2 and PRIMA1 was observed with methylation percentage values of 21.77%±33.32%; 6.82%±17.1%; 12.06%±24.37% and 13.66%±25.14%. Using combined analysis, the 4 markers were able to distinguish CRC patients from normal with 91.5% sensitivity and 97.3% specificity and could differentiate AD samples from controls with 89.2% sensitivity and 86.5% specificity. Interestingly, Roche blood collection tube and automated cfDNA isolation method influence the detected methylation levels; however, methylation differences still have been found between the clinical groups, especially in case of PRIMA1 and SFRP1 genes. Conclusion Our findings suggest that the 4 markers can be promising epigenetic biomarkers for colorectal adenoma and CRC detection with high sensitivity. Automated isolation procedures may contribute to wide-spread, screening applications of methylation markers; however, further developments are needed.