Promoter hypermethylation of p16 INK4A , p14 ARF , CyclinD2 and Slit2 in serum and tumor DNA from breast cancer patients Gayatri Sharma a , Sameer Mirza a , Chandra P. Prasad a , Anurag Srivastava b , Siddhartha Dutta Gupta c , Ranju Ralhan a, ⁎ a Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India b Department of Surgery, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India c Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Received 13 August 2006; accepted 16 February 2007 Abstract Epigenetic mechanisms such as DNA methylation play important role in cancer. Epigenetic alterations involved in the onset and progression of breast cancer may serve as biomarkers for early detection and prediction of disease prognosis. Furthermore, using body fluids such as serum offers a non-invasive method to procure multiple samples for biomarker analyses. The aim of this study is to determine the correlation between methylation status of multiple cancer genes, p16 INK4A , p14 ARF , Cyclin D2 and Slit2 in invasive ductal carcinoma of the breast and paired serum DNA and clinicopathological parameters. Of the 36 breast cancer patients investigated, 31 (86%) tumors and 30 (83%) paired sera showed methylation of at least one of these 4 genes. Methylation frequencies varied from 27% for CyclinD2, 44% for p16 INK4A , 47% for p14 ARF to 58% for Slit2. There was concordance between DNA methylation in tumor and paired serum DNA of each gene. This study underscores the potential utility of DNA methylation based screening of serum as a surrogate marker for tumor DNA methylation status of these genes in breast cancer. Further, expression profile of p16 INK4A could be linked to epigenetic events, thus suggesting this pathway as a potential target for therapeutic strategies based on reversal of epigenetic silencing. © 2007 Elsevier Inc. All rights reserved. Keywords: Breast cancer; Methylation; Serum Introduction Breast cancer is the most common malignancy among women in most western countries, where women have an overall lifetime risk of N 10% for developing invasive breast cancer (Feuer et al., 1993). In India, this is the second most common cancer among females; while in the metropolitan cities of Delhi and Mumbai it ranks as the most common cancer (Chopra, 2001; ICMR, 2001). The traditional triple test for breast cancer diagnosis includes physical examination, mammography and aspiration cytology. Unfortunately, all these methods are either not adequate or require an expert to identify breast cancer in early stages (Mitika, 2003). Accu- mulation of genetic and epigenetic alterations results in de- velopment of breast carcinoma (Welch et al., 2000). It is becoming increasingly recognized that methylation of CpG dinucleotide-rich areas in promoter regions of tumor suppres- sor genes is a major epigenetic mechanism leading to inacti- vation of these genes (Asch and Barcellos-Hoff, 2001; Lehmann et al., 2002; Widschwendter and Jones, 2002; Szyf et al., 2004a,b). Methylation mediated silencing of genes involved in DNA repair, cell-cycle regulation, cell adhesion and cell signaling has been observed in breast cancer (Yang et al., 2001; Szyf et al., 2004a,b). Detection of these epigenetic changes in serum or other body fluids has been reported in different cancers (Hibi et al., 1998; Esteller et al., 1999; Ichikawa et al., 2000; Sanchez- Cespedes et al., 2000; Muller et al., 2003; Dulaimi et al., 2004; Hoque et al., 2004; Ibanez de Caceres et al., 2004; Fujiwara Life Sciences 80 (2007) 1873 – 1881 www.elsevier.com/locate/lifescie Abbreviations: MSP, Methylation Specific PCR; ERα, Estrogen receptor α. ⁎ Corresponding author. Tel.: +91 11 2659 3478; fax: +91 11 26588663. E-mail addresses: ralhanr@rediffmail.com, ralhanr@hotmail.com (R. Ralhan). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.02.026