Oxygenated metabolites of cholesterol (oxysterols) have been previously demonstrated to contribute to progression of various cancers and to modulate resistance to breast cancer endocrine therapy in vitro. We measured prognostic roles of circulating levels of seven major oxysterols in the progression of luminal subtype breast carcinoma. Liquid chromatography coupled with tandem mass spectrometry was used for determination of levels of non-esterified 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide, and cholestane-3β,5α,6β-triol in plasma samples collected from patients (n = 58) before surgical removal of tumors. Oxysterol levels were then associated with clinical data of patients. All oxysterols except cholesterol-5α,6α-epoxide were detected in patient plasma samples. Circulating levels of 7α-hydroxycholesterol and 27-hydroxycholesterol were significantly lower in patients with small tumors (pT1) and cholesterol-5β,6β-epoxide and cholestane-3β,5α,6β-triol were lower in patients with stage IA disease compared to larger tumors or more advanced stages. Patients with higher than median cholestane-3β,5α,6β-triol levels had significantly worse disease-free survival than patients with lower levels (p = 0.037 for all patients and p = 0.015 for subgroup treated only with tamoxifen). In conclusion, this study shows, for the first time, that circulating levels of oxysterols, especially cholestane-3β,5α,6β-triol, may have prognostic roles in patients with luminal subtype breast cancer.

Biomedical Centre Faculty of Medicine Pilsen Charles University alej Svobody 1655 76 Pilsen 32300 Czech Republic

Department of Biochemistry Vanderbilt University School of Medicine 2200 Pierce Avenue Nashville TN 37232 0146 United States

Department of Medicine Vanderbilt University Medical Center 2200 Pierce Avenue Nashville TN 37235 United States

Department of Oncology 2nd Faculty of Medicine Charles University and Motol University Hospital 5 Uvalu 84 1 15000 Prague 5 Czech Republic

Department of Toxicogenomics National Institute of Public Health Srobarova 48 Prague 10 10042 Czech Republic; Biomedical Centre Faculty of Medicine Pilsen Charles University alej Svobody 1655 76 Pilsen 32300 Czech Republic; 3rd Faculty of Medicine Charles University Ruska 2411 87 Prague 10 10000 Czech Republic

National Research Institute of Chinese Medicine Taipei Taiwan ROC; Department and Institute of Pharmacology School of Medicine National Yang Ming University Taipei Taiwan ROC; Institute of Medical Sciences Taipei Medical University Taipei Taiwan ROC

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