**11. Analysis of DNA methylation in breast cancer**

Moreover, much of the research effort to date has concentrated on the identification of silenced genes implicated in breast tumorigenesis. Evron et al. successfully used a three-gene panel (Cyclin D2, RARβ and TWIST) to detect malignant breast cancer cells in ductal fluid from routine operative breast endoscopy (ROBE) and ductal lavage (Evron et al., 2001). Fackler et al. improved this method and tested a four-gene panel (RASSF1A, TWIST, HIN1 and Cyclin D2) using the QM-MSP assay to examine clinical tissue samples (Fackler et al., 2004). The cumulative methylation of these four genes is commonly observed to be higher in primary invasive breast cancers compared with reduction mammoplasty specimens from healthy women (Fackler et al., 2004). Fackler et al. further used the same technique but adopted a ninegene panel (RASSF1A, TWIST, HIN1, Cyclin D2, RARβ, APC, BRCA1, BRCA2 and p16) to examine ductal lavage samples from women with or without breast cancer. This trial demonstrated that methylation-marker detection was twice as sensitive as cytological diagnosis of ductal lavage cells (Fackler et al., 2006). In addition to biopsied tissue sections and ductal fluid, methylated DNA is also detected in blood since the blood of patients with manifest breast cancer contains detectable amounts of circulating methylated DNA (Widschwendte & Menon, 2006). The blood detection of tumor-specific methylated DNA has been pursued for its potential for prognostic prediction and monitoring relapse of breast cancer after therapy (Widschwendte & Menon, 2006; Muller et al., 2003; Silva et al., 2002).

The analysis of methylation profiles in human cancer indicates that hypermethylation of some of the CpG islands is shared by multiple tumour types, whereas others are methylated in a tumour type-specific manner (Bae et al., 2004; Costello et al., 2000; Esteller et al., 2001; Nass et al., 2000; Parrella et al., 2004; Parrella, 2010). Promoter-aberrant methylation seems to be an early event in tumorigenesis, and an increase in the number of methylated genes during progression has been observed in several tumour types including breast cancer (Lehmann et al., 2002; Subramaniam et al., 2009). Hoque et al (2009) have shown there were differences in the patterns of methylation in pre-invasive breast lesions (atypical ductal hyperplesia and

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