Abstract 3741: Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients
Authors: Nagy Zsófia Brigitta , Barták Barbara Kinga , Fodor Tamás , Karvaly Gellért Balázs , Zsigrai Sára , Szigeti Krisztina Andrea , Kalmár Alexandra , Dank Magdolna , Takács István , Molnár Béla 
Published: June 2022

The main function of the single-carbon metabolic cycle is maintaining nucleotide pool and DNA methylation which is highly influenced by the chemotherapy protocols. This study aimed to evaluate the ultrashort effect of reduced folate combined anticancer treatment on peripheral blood parameters of colorectal cancer (CRC) patients. Post-operative CRC patients were treated with chemotherapy using the combination of oxaliplatin, 5-FU, leucovorin, and capecitabine. Blood samples were taken until the beginning of the treatment and immediately after the therapy. Plasma fractions were separated and the levels of S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH) and the simultaneous quantification of nucleotides (adenine, cytosine, thymine, guanine and uracil) were detected by HPLC-MS/MS. Homocysteine (HCY) was also determined from plasma specimens. Cell-free DNA (cfDNA) was isolated from plasma and DNA methylation was analyzed by LINE-1 bisulfite pyrosequencing. Moreover, LINE-1 methylation statuses were also examined from PBMC cells that were separated from whole blood by a density gradient centrifugation. In each patient, the HCY level of plasma was decreased by an average of 17% after oxaliplatin treatment with the combination of capecitabine (XELOX) and also with 5-FU + leucovorin (FOLFOX) agents. Our study also confirmed that cancer patients have an elevated cfDNA level (avg. 20ng/plasma mL); however, its concentration decreased significantly (p=0.02) on average by 50% after treatment compared to the baseline. The same tendency was observed in the case of SAM after XELOX and FOLFOX adjuvant therapy in plasma samples (p=0.03). Moreover, mostly elevated SAM/SAH ratios were observed in parallel with reduced SAH level after the therapy. Significant (p=0.0001) mean LINE-1 hypomethylation was detected in PBMC (81.84%) compared to cfDNA of plasma (73.76%), furthermore, slightly DNA hypermethylation was noticed in CpG1 position of LINE-1 after chemotherapy in both mononuclear cells and circulating DNA. Lower level of plasma nucleotides was identified in case of adenine, cytosine and uracil after treatment, while thymine and guanine were below the detection limit. The present study demonstrates that the DNA-damaging chemotherapy results in a detectable decrease in cfDNA immediately after the treatment, that was supported by the decrease in the level of the nucleotides measured in the plasma fraction. The methionine cycle is responsible for the DNA methylation maintenance, and though the amount of members of this pathway was reduced, only a moderately increased DNA methylation could be detected even in such a short period time. Co-administration of high dose leucovorin as a folate derivative and a methyl donor with further chemotherapeutic agents may actively contribute to genome alterations.

Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Tamás Fodor, Gellért Balázs Karvaly, Sára Zsigrai, Krisztina Andrea Szigeti, Alexandra Kalmár, Magdolna Dank, István Takács, Béla Molnár. Folic acid combined chemotherapy has an immediate effect on C1 methionine cycle and consequential DNA methylation in liquid biopsy samples of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3741.