Abstract 1276: Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up
Authors: Bartak Barbara K. , Szigeti Krisztina Andrea , Kalmar Alexandra , Galamb Orsolya , Nagy Zsófia Brigitta , Zsigrai Sara , Tulassay Zsolt , Igaz Peter , Dank Magdolna , Molnar Bela 
Published: September 2020

Cell-free DNA (cfDNA) level increases in several physiological conditions, such as pregnancy, aging or high physical activity. Furthermore, alterations of cfDNA concentration and genetic or epigenetic (e.g. DNA methylation) patterns can also be observed in pathological processes, as inflammatory diseases or different cancer types including colorectal cancer (CRC). However, studies about quality changes of cfDNA aimed to analyze its fragment length profile and DNA methylation pattern are lacking. We aimed to define the quantity and quality changes of cfDNA, including concentration, fragment length distribution and global DNA methylation pattern during high physical exercise, and in colorectal adenoma and cancer patients. Moreover, we aimed to investigate the treatment response in plasma of metastatic CRC (mCRC) patients analyzing the above-mentioned parameters and the DNA methylation level of SFRP2 gene during therapy. Plasma fraction was separated from blood samples of 6 healthy athletes before, during and after physical training, and from healthy (n=16), adenoma (n=13), IBD (n=19), and CRC (n=16) patients. Moreover, plasma longitudinal assessment was performed in the case of 20 mCRC patients treated with chemotherapy. CfDNA level was quantified with Qubit 1.0 (Thermo Fisher Scientific), fragment length distribution was examined by 2100 Bioanalyzer instrument (Agilent), and global DNA methylation level was measured with bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) (Qiagen) in all samples. Besides, DNA methylation status of SFRP2 gene was determined with droplet digital PCR (Bio-Rad) in mCRC plasma samples. Increased cfDNA amount was observed during physical exercise (198.5±90ng), in comparison with control phase (86.3±40.6ng), and then in restitution period, lower cfDNA level (155.6±119.3ng) was detected. Elevated cfDNA amounts have been found in plasma of adenoma (72.1±37.5ng), IBD (78.0±50.9ng) and CRC (84.5±70.1ng) patients compared to controls (36.2±11.3ng). CfDNA fragment length distribution showed different patterns in each sample group, longer fragments (200-600bp) enriched in AD and CRC samples in addition to short (&lt;200bp) fragments. Slightly lower level of global DNA methylation was noticed in AD (79,5%) and CRC (79,2%) compared to normal (83,8%) samples. Interestingly, the amount and global methylation level alteration of cfDNA revealed negative correlation in CRC patients during therapy and progression. Furthermore, the amount of methylated SFRP2 copies (5-500copies) was increased in the case of progressive disease, while were undetectable in patients achieved complete remission. Characteristic cfDNA level, fragment length, and global DNA methylation level were identified in the different sample groups. The above-mentioned parameters can be applied for the follow-up of mCRC patients during chemotherapy and could be integrated into current follow-up methods.

Citation Format: Barbara K. Bartak, Krisztina Andrea Szigeti, Alexandra Kalmar, Orsolya Galamb, Zsófia Brigitta Nagy, Sara Zsigrai, Zsolt Tulassay, Peter Igaz, Magdolna Dank, Bela Molnar. Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up [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 1276.