Changes in active DNA demethylation pathway components during neoadjuvant chemotherapy and their prognostic significance in breast cancer

Abstract Background Neoadjuvant chemotherapy (NACT) for early breast cancer is a systemic treatment administered before surgery to achieve regression of the primary lesion, perform breast-conserving surgery, if possible, and ensure long-term recurrence-free survival. The main aim of this study was to investigate the impact of NACT on the active DNA demethylation process and the relationship between the compounds involved in this path and disease-free survival (DFS) and overall survival (OS) during a six-year follow-up. Methods This study included 71 patients with breast cancer who were eligible for NACT consisting of 4 cycles of doxorubicin and cyclophosphamide regimens at doses of 60 mg/m 2 and 600 mg/m 2 , respectively, on the first day every 21 days, as well as paclitaxel administered at a dose of 80 mg/m 2 weekly for 12 weeks. Peripheral blood and urine samples were collected from breast cancer patients at four time points. Additionally, tissue samples from a breast cancer tumor and regional metastatic lymph nodes were collected during surgery. The levels of 5-methylcytosine (5-mCyt) and its derivatives in DNA and urine were determined via two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The qRT‒PCR technique was used to determine the transcript levels of the genes involved in active DNA demethylation. Results During chemotherapy, increases in the levels of 5-methyl-2ʹ-deoxycytidine (5-mdC), 5-formyl-2ʹ-deoxycytidine (5-fdC), and 5-carboxyl-2ʹ-deoxycytidine (5-cadC) and decreases in the expression of genes encoding TET (ten-eleven translocation) proteins, TDG (thymine DNA glycosylase), and AID (activation-induced cytidine deaminase) were observed in peripheral blood leukocytes. The Kaplan‒Meier curves revealed that patients with a lower level of 5-mdC in leukocyte DNA and urine before NACT, higher levels of 5-cadC, and lower TDG expression in tumor cells after treatment had a more favorable prognosis. Furthermore, significant correlations were found between the levels of 5-fdC in the DNA of leukocytes, 5-cadC in DNA tumor cells, the expression of TET2 , and decreased Ki-67 after NACT. Conclusions Our results indicate that NACT can alter active DNA demethylation markers. Breast cancer patients with prolonged DFS had lower leukocyte 5-mdC and higher urinary 5-mdC levels before chemotherapy. Thus, assessing 5-mdC in these minimally invasive biospecimens may provide prognostic information for breast cancer patients undergoing NACT.