Abstract 5316: DNA methylation index of lifetime estrogen exposure in breast cancer
Authors: Johansson Annelie , Palli Domenico , Masala Giovanna , Grioni Sara , Agnoli Claudia , Tumino Rosario , Giurdanella Maria Concetta , Fasanelli Francesca , Sacerdote Carlotta , Panico Salvatore , Mattiello Amalia , Swerdlow Anthony , Schoemaker Minouk , Jones Michael , Orr Nick , Fletcher Olivia , Johnson Nichola , Tomczyk Katarzyna , Vineis Paolo , Flanagan James M. 
Published: August 2018

Abstract
Breast cancer is the most common female cancer worldwide and makes up 25% of all cancers diagnosed in women. There is a need for improved risk assessment methods to identify women at high risk before the disease develops. It is well established that estrogens are involved in estrogen receptor positive (ER+) breast cancer carcinogenesis, however the mechanisms are not fully understood. We hypothesize that lifetime estrogen exposure accumulates changes in DNA methylation detectable in blood as a surrogate for other tissues. Here, we have identified a methylation signature of estrogen exposure measured in blood DNA that could improve risk assessment for breast cancer.
Our Estimated Lifetime Estrogen Exposure (ELEE) model, taking into account reproductive time (age at menopause minus age at menarche), number of pregnancies and breastfeeding duration, shows a 5% increased risk of developing breast cancer per unit increase in ELEE (range 18 – 44) in the European Prospective into Cancer and Nutrition (EPIC)-Italy study cohort (30671 healthy controls, 1193 cases, age at diagnosis ≥ 50). An Epigenome-Wide Association Study (EWAS) of ELEE was conducted on 450K methylation data in EPIC-Italy (199 controls, 97 cases) and the Generations Study (GS) cohort (n=65 controls). For validation targeted bisulfite sequencing using the Fluidigm 48.48 Access Array was performed on independent DNA samples from the GS cohort (440 matched ER+ case-control pairs). The Methylation Index (MI) of ELEE was developed on 450K data using ridge regression and includes DNA methylation levels at selected CpG sites.
DNA methylation levels at 694 CpG sites show significant (false discovery rate q &lt; 0.05) association with ELEE in the EWAS and of these 42 CpG sites from the top-probes were selected for validation. The MI was developed on 28 CpG sites passing quality control and shows high correlation with ELEE in 450K training data (r=0.69) and significant association with breast cancer risk in EPIC-Italy with an OR of 1.38 per unit MI (range 24 – 43, 95% CI: 1.23 – 1.57, P=2.0E-07). Validation step is yet to be finalized; initial analysis on the first 318 case-control pairs suggests a modest but significant association with ER+ breast cancer risk (unadjusted OR=1.04 per unit MI, 95% CI: 1.00 – 1.07, P=0.030, MI range 20 – 53).
In summary, we show that changes in DNA methylation following estrogen exposure are detectable in blood and we have developed a Methylation Index of ELEE that is significantly increased in breast cancer cases compared to controls in EPIC-Italy, and modestly increased in the GS validation cohort. This molecular measure of estrogen exposure could potentially improve risk assessment methods and be used to identify women at high risk of developing breast cancer.
Citation Format: Annelie Johansson, Domenico Palli, Giovanna Masala, Sara Grioni, Claudia Agnoli, Rosario Tumino, Maria Concetta Giurdanella, Francesca Fasanelli, Carlotta Sacerdote, Salvatore Panico, Amalia Mattiello, Anthony Swerdlow, Minouk Schoemaker, Michael Jones, Nick Orr, Olivia Fletcher, Nichola Johnson, Katarzyna Tomczyk, Paolo Vineis, James M. Flanagan. DNA methylation index of lifetime estrogen exposure in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5316.