Chemotherapy induces microssatelite instability (MIS) in plasma free DNA (pfDNA), peripheral blood mononuclear fraction (PBMNF) cells, and urine free DNA (ufDNA) of breast cancer (BC) patients as well as in normal PBMNF cells in vitro in the absence of amifostine (A)

Abstract

e11505 Background: We have previously shown that alkylating agent based chemotherapy regimens (AQT) could induce MIS in the PBMNF of BC patients in parallel to a decrease in the expression of the protein hMSH2 in these cells (Fonseca et al., 2005, Breast Cancer Res, 7, R28–32). Since plasma DNA derives mainly from tumor cells, we wanted to know if chemotherapy would also produce MIS in tumor DNA and if this phenomenon could be reproduced in vitro. Methods: 33 previously untreated female BC patients with a mean age of 51 years received AQT(16 ACT;3FAC;2 TAC;1FEC;10AC). Samples from 3 additional patients who received Fulvestrant only as neoadjuvant therapy were also included. Blood (pfDNA and PBMNNF) and urine (ufDNA) were evaluated at time 0,3 and 6 months with 6 MIS markers (BAT40,BAT26, MR2,TP53 PCR15.1, APC and ALU). Levels of fpDNA and fuDNA were measured by spectrophotometry. We incubated in vitro cultures of MCF- 7 cells and PBMNF cells with M at a dose of 0.7μg/ml for 30 minutes with and without A at 20% of the M dose and evaluated serially for 48 hours for MIS and hMH2 expression by immunohistochemistry. Results: We observed at least one MIS event in the PBMNF, fpDNA or fuDNA in 87%, 80% and 80% of the patients, respectively, mainly in BAT40 and BAT 26 markers. There was only 14.74% of concordance of MIS alterations between PBMNF and fpDNA and 8. 42% between fpDNA and fuDNA. Patients receiving Hormones also exhibited MIS. Interestingly, fpDNA levels increased significantly in patients with measurable disease who responded to therapy (47.4 ± 13.34 vs 14.37± 5.32; p = 0.021). In vitro, incubating MCF-7 cells and normal PBMNF cells with M ±A, we observed that we could induce MIS in both MCF-7 cells and normal PBMNF cells but A prevented MIS only in normal PBMNF cells. In normal PBMNF cells without A that sustained MIS there was a significantly decreased percentage of cells expressing hMSH2 ( 96% vs 57% p < 0.001). Conclusions: We conclude that Chemotherapy as well as Fulvestran can induce MIS in normal and malignant cells and that in vitro these effects could be reproduced by treatment with M and prevented in normal cells by A. No significant financial relationships to disclose.