Identification of 5 novel genes methylated in breast and other epithelial cancers

Using this approach we identified numerous CpG islands that demonstrated aberrant DNA methylation in breast cancer cell lines. Using a combination of COBRA and sequencing of bisulphite modified DNA, we confirmed 5 novel genes frequently methylated in breast tumours; EMILIN2, SALL1, DBC1, FBLN2 and CIDE-A. Methylation frequencies ranged from between 25% and 63% in primary breast tumours, whilst matched normal breast tissue DNA was either unmethylated or demonstrated a much lower frequency of methylation compared to malignant breast tissue DNA. Furthermore expression of the above 5 genes was shown to be restored following treatment with a demethylating agent in methylated breast cancer cell lines. We have expanded this analysis across three other common epithelial cancers (lung, colorectal, prostate). We demonstrate that the above genes show varying levels of methylation in these cancers. Lastly and most importantly methylation of EMILIN2 was associated with poorer clinical outcome in breast cancer and was strongly associated with estrogen receptor as well as progesterone receptor positive breast cancers.The combination of the MIRA assay with CpG island arrays is a very useful technique for identifying epigenetically inactivated genes in cancer genomes and can provide molecular markers for early cancer diagnosis, prognosis and epigenetic therapy.In most Western countries breast cancer is a leading cause of cancer related deaths in women. Breast cancer accounts for over one million of the estimated 10 million cancers that are diagnosed globally each year in both males and females and claimed about 375,000 deaths in the year 2000 [1]. The use of screening mammography and MRI has led to a decline in breast cancer-related mortality. But these screening procedures can also lead to overdiagnosis and false positive cases leading to unnecessary treatment. Therefore there is a critical need to develop molecular biomarkers that can detect early stage disease so that treatment c