Supplementary Material for: Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer

<b><i>Objective:</i></b> Resistance to trastuzumab is a problem that remains to be solved in HER2-positive breast cancer. We aimed to characterize profiles of genetic and epigenetic alterations in cancer-related pathways in HER2-positive breast cancers, using biopsy tissue samples obtained from patients enrolled in a prospective neoadjuvant clinical trial. <b><i>Methods:</i></b> HER2-positive breast cancer tissue samples were collected and processed with the PAXgene Tissue System. A total of 24 breast cancers were analyzed. Genetic alterations of 409 cancer-related genes were analyzed by a bench-top next-generation sequencer. DNA methylation statuses were analyzed by a bead array with 485,512 probes. <b><i>Results:</i></b> The WNT pathway was potentially activated by aberrant methylation of its negative regulators, such as <i>DKK3</i> and <i>SFRP1</i>, in 9 breast cancers. The AKT/mTOR pathway was activated by mutations of <i>PIK3CA</i> in 5 breast cancers. The Notch pathway was potentially activated by mutations of <i>NOTCH1</i> and <i>NOTCH2</i> in 4 breast cancers. The p53 pathway was inactivated by mutations of <i>TP53</i> in 13 breast cancers and potentially by aberrant methylation of its downstream genes in 10 breast cancers. Cell adhesion was affected by mutations of <i>CDH1</i> in 1 breast cancer. <b><i>Conclusion:</i></b> Genes involved in cancer-related pathways were frequently affected not only by genetic but also by epigenetic alterations in HER2-positive breast cancer.