Abstract P4-05-11: Methylation profiling of mucinous breast cancer
Authors: Pareja Fresia , Ferrando Lorenzo , da Silva Edaise M , Paula Arnaud Da Cruz , Stylianou Anthe , Brown David N , Selenica Pier , Serrano Jonathan , Wen Hannah Y , Zhang Hong , Brogi Edi , Norton Larry , Snuderl Matjia , Reis-Filho Jorge S , Weigelt Britta 
Published: March 2020

Abstract
Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer characterized by tumor cells floating in lakes of mucin. As compared to invasive ductal carcinoma of no special type (IDC-NST), we have recently shown that MCBs harbor a lower frequency of PIK3CA mutations and lack concurrent 1q gains and 16q losses, hallmark genetic alterations of ER-positive breast cancer. Despite their distinctive phenotype and previous efforts to characterize their genomic landscape by whole-genome, whole-exome and RNA-sequencing, no pathognomonic somatic mutation or fusion gene underpinning MCBs or the mucinous phenotype have been identified. In this study we sought to determine whether MCBs would be defined by specific epigenetic changes.
Materials and methods: Thirty-six MCBs were subjected to DNA methylation profiling using Infinium MethylationEPIC arrays. Following quantile normalization, low quality probe filtering, and data background and dye bias correction using the ‘noob’ algorithm, methylation profiling data of MCBs were compared to those of ER-positive/HER2-negative IDC-NSTs from The Cancer Genome Atlas (TCGA) lacking concurrent 1q gains/16q losses or PIK3CA hotspot mutations and matched according to age and menopausal status at a 1:2 ratio (n=72). An enrichment analysis of the differentially methylated targets in gene promoters and enhancers was conducted using Minfi and MethylGSEA R packages. A subset of 12 MCBs of this cohort was subjected to RNA-sequencing and compared to age, menopausal status and molecular features-matched ER-positive/HER2-negative IDC-NSTs from TCGA (1:2 ratio; n=24) using gene set enrichment analysis (GSEA).
Results: Enrichment analysis of differentially methylated probes revealed a significant enrichment of targets in promoters and enhancers of mucin-encoding genes and in genes of the mTOR signaling pathway between MCBs and matched IDC-NSTs from TCGA (P&lt;0.01). Compared to matched IDC-NSTs, MCBs displayed promoter/enhancer hypomethylation of mucin-encoding genes, such as MUC1, MUC2 and MUCL1. We also observed promoter/enhancer hypomethylation of mTOR signaling pathway genes including MTOR, which encodes for the catalytic subunit of the mTORC complex, RPTOR, which codes for an mTOR binding protein that positively regulates the downstream effector S6 Kinase 1, EIF4E and EIF4B, key downstream effectors of the mTOR signaling pathway, and the oncogenes PIK3R2 and PIK3R3, which encode for regulatory isoforms of PI3K, in MCBs. Our gene expression analysis revealed that the mucin-encoding genes MUC1, MUC2 and MUCL1, and the mTOR signaling genes MTOR, RPTOR, EIF4E, EPIF4B, PIK3R1 and PIK3R2, found to be hypomethylated in MCBs, displayed a significantly higher gene expression in MCBs (P&lt;0.001, each) compared to clinically and molecularly matched IDC-NSTs.
Conclusions: Taken together, our data suggest that MCBs display a high expression of mucin-encoding genes, due to hypomethylation of promoters and enhancers and could provide the basis for their distinctive phenotype. Moreover, our findings suggest that despite the lack of genetic alterations affecting genes of the PI3K/mTOR signaling pathway in MCBs, mTOR signaling might be constitutively active in these tumors via epigenetic mechanisms, supporting the notion that, in the absence of pathognomonic genetic alterations, a disruption of the epigenetic landscape is a critical driver in the development of this rare breast cancer type.
Citation Format: Fresia Pareja, Lorenzo Ferrando, Edaise M da Silva, Arnaud Da Cruz Paula, Anthe Stylianou, David N Brown, Pier Selenica, Jonathan Serrano, Hannah Y Wen, Hong Zhang, Edi Brogi, Larry Norton, Matjia Snuderl, Jorge S Reis-Filho, Britta Weigelt. Methylation profiling of mucinous breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-11.