Epigenetic field cancerization in breast cancer using subject-matched tumor, ipsilateral-normal, and contralateral-normal tissues

Background Emerging work has demonstrated that histologically normal (non-tumor) tissue adjacent to breast tumor tissue shows evidence of molecular alterations related to tumorigenesis, referred to as field cancerization effects. Although changes in DNA methylation are known to occur early in breast carcinogenesis and the landscape of breast tumor DNA methylation is profoundly altered compared with normal tissue, there have been limited efforts to identify DNA methylation field cancerization effects in histologically normal breast tissue adjacent to tumor. Methods Matched tumor, histologically normal tissue of the ipsilateral breast (ipsilateral-normal), and histologically normal tissue of the contralateral breast (contralateral-normal) were obtained from nine women undergoing bilateral mastectomy. Laser capture microdissection was used to select breast epithelial cells from normal tissues, and neoplastic cells from tumor specimens for genome-scale measures of DNA methylation with the Illumina HumanMethylationEPIC array. Results We identified substantially more CpG loci that were differentially methylated between contralateral-normal breast and tumor tissue (63,271 CpG loci q < 0.01), than between ipsilateral-normal tissue and tumor (38,346 CpG loci q < 0.01). In addition, we identified differential methylation in ipsilateral-normal relative to contralateral-normal tissue (9,562 CpG loci p < 0.01). Hypomethylated loci in ipsilateral normal relative to contralateral were significantly enriched for breast cancer-relevant transcription factor binding sites including those for ESR1, FoxA1, and GATA3. Hypermethylated loci in ipsilateral-normal relative to contralateral-normal tissue were significantly enriched for CpG island shore regions. Conclusions Our results indicate that early hypermethylation events in breast carcinogenesis are more likely to occur in the regions immediately surrounding CpG islands than CpG islands per se , reflecting a field effect of the tumor on surrounding histologically normal tissue. This work offers an opportunity to focus investigations of early DNA methylation alterations in breast carcinogenesis and potentially develop epigenetic biomarkers of disease risk. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by funds of the Burroughs-Wellcome/Dartmouth Big Data in the Life Sciences Training Program to MEM. Office of the U.S. Director of the National Institutes of Health under award number T32LM012204 to AJT. COBRE Center for Molecular Epidemiology at Dartmouth P20GM104416, and R01CA216265 to BCC. NIEHS P01ES022832 and EPA RD83544201. R01CA230478-01A1 and ROH-BCR151672 to KFA. ### Author Declarations All relevant ethical guidelines have been followed and any necessary IRB and/or ethics committee approvals have been obtained. Yes All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes Any clinical trials involved have been registered with an ICMJE-approved registry such as ClinicalTrials.gov and the trial ID is included in the manuscript. Not Applicable I have followed all appropriate research reporting guidelines and uploaded the relevant Equator, ICMJE or other checklist(s) as supplementary files, if applicable. Not Applicable The datasets generated and analyzed during the current study are available in the Gene Expression Omnibus under the accession number GSE133985 (). * DCIS : ductal carcinoma in situ GO : gene ontology; OR: odds ratio CI : confidence interval TFBS : transcription factor binding site LOLA : Locus Overlap Analysis