RESEARCH ARTICLE Mitochondrial DNA Mutations and Mitochondrial DNA Depletion in Breast Cancer Ling-Ming Tseng, 1 Pen-Hui Yin, 2,3 Chin-Wen Chi, 2,4 Chih-Yi Hsu, 1 Chew-Wun Wu, 1 Liang-Ming Lee, 5 Yau-Huei Wei, 3 and Hsin-Chen Lee 4,6 * 1 Department of Surgery,Taipei Veterans General Hospital, and National Yang-Ming University,Taiwan,Republic of China 2 Department of Medical Research and Education,Taipei Veterans General Hospital,Taiwan,Republic of China 3 Department of Biochemistry and Molecular Biology, School of Medicine, National Yang-Ming University,Taiwan,Republic of China 4 Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University,Taiwan,Republic of China 5 Department of Urology,Taipei Medical University-Aff|liated Taipei Municipal Wan-Fang Hospital,Taiwan,Republic of China 6 Department of Education and Research,Taipei City Hospital,Taipei,Taiwan,Republic of China Somatic mutations in mitochondrial DNA (mtDNA) have been demonstrated in various tumors, including breast cancer. How- ever, it still remains unclear whether the alterations in mtDNA are related to the clinicopathological features and/or the progno- sis in the breast cancer. We analyzed somatic mutations in the D-loop region, the common 4,977-bp deletion, and the copy num- ber of mtDNA in breast cancer and paired nontumorous breast tissues from 60 Taiwanese patients. We found that 18 of the 60 (30%) breast cancers displayed somatic mutations in mtDNA D-loop region. The incidence of the 4,977-bp deletion in nontumo- rous breast tissues (47%) was much higher than that in breast cancers (5%). The copy number of mtDNA was significantly decreased in 38 of the 60 (63%) breast cancers as compared to their corresponding nontumorous breast tissues (P ¼ 0.0008). The occurrence of D-loop mutations was associated with an older onset age (50 years old, P ¼ 0.042), and tumors that lacked expressions of estrogen receptor and progesterone receptor (P ¼ 0.024). Patients with mtDNA D-loop mutation and breast can- cer had significantly poorer disease-free survival than those without mutation, when assessed by Kaplan–Meier curves and log- rank test (P ¼ 0.005). Multivariate Cox regression analysis indicated that a D-loop mutation is a significant marker that is inde- pendent of other clinical variables and that it can be used to assess the prognosis of patients. Our findings suggest that somatic mutations in mtDNA D-loop can be used as a new molecular prognostic indicator in breast cancer. V V C 2006 Wiley-Liss, Inc. INTRODUCTION Breast cancer is the fourth leading cause of can- cer death among Taiwanese women. Its incidence has rapidly increased over the past decade both in Taiwan and in other areas of Asia (Seow et al., 1996; Chen et al., 2002). According to epidemiolog- ical studies, prolonged exposure to estrogen, including a reduced fertility rate, an earlier men- arche, and prolonged reproductive stimulation dur- ing lifetime, is significantly associated with an increased risk of female breast cancer (Pike et al., 1993; Shen et al., 2005). The oxidized metabolites of estrogen, E2,3,4-semi-quinones and E2,3,4-qui- nones, have been shown to bind to DNA to form adducts, which may lead to genetic damage (Yager, 2000). In addition, the generation of reactive oxy- gen species (ROS) during the conversion of E2,3,4-semi-quinones to E2,3,4-quinones can also lead to oxidative DNA damage. Estrogen metabo- lites-induced oxidative stress has thus been thought to play an important role in the initiation of breast carcinogenesis (Yager, 2000). Mitochondria are cytoplasmic organelles and have a variety of important roles to play, including the generation of ATP through oxidative phospho- rylation (OXPHOS), the production of ROS, and the initiation of apoptosis (Wallace, 1999). Human mitochondrial DNA (mtDNA) is a 16.6-kb double- stranded circular DNA molecule, and multiple copies of mtDNA are present in each mitochond- rion. The human mitochondrial genome encodes 13 polypeptides that form part of the respiratory chain, together with 22 transfer RNAs and two ri- bosomal RNAs that are required for protein syn- thesis (Anderson et al., 1981). mtDNA is more sus- ceptible to oxidative damage and has a higher mutation rate than nuclear DNA due to a lack of protective histone proteins, limited DNA repair *Correspondence to: Dr. Hsin-Chen Lee, Department and Insti- tute of Pharmacology, School of Medicine, National Yang-Ming Uni- versity, Taipei, Taiwan 112,Republic of China. E-mail: hclee2@ym.edu.tw Supported by: Taipei Veterans General Hospital, Grant numbers: V93-234 and V94-248; National Science Council, Taiwan, Republic of China, Grant numbers: NSC 93-2320-B-010-058, NSC 94-2314-B- 075-035, and NSC 94-2320-B-010-063; Chen Shuyi Cancer Foundation. Received 21 December 2005; Accepted 17 February 2006 DOI 10.1002/gcc.20326 Published online 27 March 2006 in Wiley InterScience (www.interscience.wiley.com). V V C 2006 Wiley-Liss, Inc. GENES, CHROMOSOMES & CANCER 45:629–638 (2006)