Genome-Wide DNA Methylation Profiles of Breast Tumors Reveal Loci Associated with Relapse Risk.
Authors: Varadan V. , Kamalakaran S. , Giercksky Russnes H. , Giercksky Russnes H. , Levy D. , Kendall J. , Janevski A. , Riggs M. , Banerjee N. , Synnestvedt M. , Schlichting E. , Kåresen R. , Lucito R. , Wigler M. , Dimitrova N. , Naume B. , Hicks J. , Borresen-Dale A. , Borresen-Dale A. 
Published: December 2010

Abstract
Background:Breast cancer prognosis is used in determining the course of adjuvant therapy for patients. Clinical prognostic indices like the Nottingham Prognostic Index have poor specificity, overestimate the risk of disease recurrence and necessitate more specific and robust prognostic markers. Prognostic gene expression markers are already in clinical use and show improved decision support. Methylation of CpG islands, an important regulator of gene expression, is reported to be disregulated in tumors, thus making methylation markers an important alternative to gene expression markers. We present the results of a genome-wide study that explored loci whose methylation status was significantly associated with recurrence risk.Methods:We used 108 frozen primary breast cancer specimens with ten year follow-up and extensive clinical data including histopathological measurements to identify potential epigenetic markers associated with recurrence risk. Using a previously validated array based method (Kamalakaran et. al., Nucleic Acids Research, 2009) we performed genome-wide measurements of differential CpG island methylation covering over 150,000 loci. We evaluated each locus for its ability to stratify patients into good or poor prognosis groups depending on its methylation status. Statistical significance was established using permutation analysis with appropriate multiple testing corrections. Prognostic markers independent of histopathological factors (ER, PR, HER2, tumor size, grade, node status, age) were identified using multivariate Cox regression analysis.Results:The methylation status of several loci proximal to genes significantly stratified samples independent of other clinical variables. Demethylation of several loci were associated with poor prognosis including ADAMTS4 (Hazard Ratio = 17.5, p-value&lt;&lt;0.001), a metalloproteinase previously implicated in the progression of glioma; DNA Topoisomerase I (HR = 3.81; 95% CI = 1.953-7.462; p&lt;&lt;0.001), implicated in chemotherapy resistance; and JMJD2C (HR = 3.7; 95% CI = 1.828-7.519; p&lt;&lt;0.001), which was found to be frequently amplified in esophageal cancers. The methylation of several loci also had significant association with poor prognosis, such as several members in the forkhead box family (FOXF1, FOXG1B, FOXJ1, FOXL2) and FHL1 (HR = 4.78; 95% CI = 2.38-9.62; p&lt;&lt;0.001). We selected several loci to form an ensemble classifier with statistically significant performance on our dataset. We show that this classifier achieved much higher specificity when compared to the Nottingham Prognostic Index, while maintaining high sensitivity.Discussion:Our retrospective study of genome-wide DNA methylation in breast cancer has identified several novel markers for prognosis. We found methylation deregulation of CpG islands proximal to genes implicated in metastasis and chemotherapy resistance is associated with poor prognosis. Furthermore, the potential for clinical benefit of these markers is their ability to jointly identify significantly larger number of low-risk patients compared to the Nottingham Prognostic Index.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4046.