J Cancer Res Clin Oncol (2010) 136:203–210 DOI 10.1007/s00432-009-0649-6 123 ORIGINAL PAPER Aberrant methylation of RASSF1A is associated with poor survival in Tunisian breast cancer patients Sondes Karray-Chouayekh · Fatma Trifa · Abdelmajid Khabir · Nouredine Boujelbane · Tahia Sellami-Boudawara · Jamel Daoud · Mounir Frikha · Rachid Jlidi · Ali Gargouri · Raja Mokdad-Gargouri Received: 14 January 2009 / Accepted: 17 July 2009 / Published online: 6 August 2009  Springer-Verlag 2009 Abstract Introduction Epigenetic gene silencing is one of the major causes of inactivation of tumor-suppressor genes in many human cancers. Materials and methods The aim of the present study was to determine the methylation status of the promoter region CpG islands of four cancer-related genes RASSF1A, RAR2, CDH1, and p16 INK4a in 78 breast cancer specimens and to evaluate whether the methylation status is associated with estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu) together with the major clinico-pathological parameters. Results We showed that the methylation frequencies ranged from 19.6% (p16 INK4a ) to 87% (RASSF1A) in pri- mary breast tumors of Tunisian patients. Aberrant methyla- tion of RAR2 was observed in 66.6% of cases and associated with age at diagnosis (P = 0.043), while CDH1 was methylated in 47.4% of tumors and was correlated with tumor size (P = 0.013). RASSF1A presented the highest percentage of methylation (87%) and was strongly associ- ated with poor survival (P = 0.014), with age (P = 0.048), and tumor stage (P = 0.033). Loss of ER and PR was strongly associated with GIII tumors (P = 0.000 and 0.037 respectively) while HER2/neu was associated with lymph node involvement (P = 0.026) and 5-year survival rate (P = 0.028). Conclusions Our preliminary Wndings suggested that aberrant methylation of RASSF1A and RAR2 occurs fre- quently in Tunisian breast cancer patients compared with others. Furthermore, RASSF1A hypermethylation could be used as a potential marker of poor prognosis. Keywords Promoter methylation · Methylation-speciWc PCR · Tumor suppressor genes · Breast cancer · Disease-free survival Introduction Breast cancer is the most common cancer worldwide in women and the overall lifetime risk for developing invasive breast cancer is estimated to 10% (Feuer et al. 1993). In Tunisia, breast cancer is the second most cancer among females, with an incidence of 30/100,000 in the south (Mourali et al. 1980). Tunisian breast cancer is character- ized by a particular aggressive proWle compared with west- ern countries, the incidence being more prevalent in young (<35 years) than older patients (Parkin et al. 2003). Breast cancer onset and progression is a multistep process resulting from a series of epigenetic and genetic changes. The loss of tumor suppressor gene function, which nor- mally acts as a negative regulator of cell proliferation is one of the mechanisms involved in the carcinogenesis process. It is becoming increasingly recognized that methylation of CpG dinucleotides is a major epigenetic mechanism S. Karray-Chouayekh · F. Trifa · A. Gargouri · R. Mokdad-Gargouri (&) Unité de Recherche Génétique du Cancer et Production de Protéines Thérapeutiques, Centre de Biotechnologie de Sfax, BP 1177, Route Sidi Mansour, Sfax, Tunisia e-mail: raja.gargouri@cbs.rnrt.tn A. Khabir · N. Boujelbane · T. Sellami-Boudawara · J. Daoud · M. Frikha Centre Hospitalo-Universitaire Habib Bourguiba, Sfax, Tunisia R. Jlidi Laboratoire privé d’Anatomo-pathologie, Sfax, Tunisia