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High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early-Onset and Familial Risk Breast Cancer Patients

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High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early-Onset and Familial Risk Breast Cancer Patients Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. High-Resolution Bisulfite-Sequencing of Peripheral Blood DNA Methylation in Early- Onset and Familial Risk Breast Cancer Patients Justin Chen1*, Maria K. Haanpää2*, Joshua J. Gruber1,2, Natalie Jäger1, James M. Ford1,2ǂ, and Michael P. Snyder1ǂ 1Department of Genetics 2Department of Medicine, Oncology Division Stanford University, Stanford, CA 94305, USA. * These authors contributed equally to this work. ǂ Co-corresponding authors Running Title: DNA Methylation and High-Risk Breast Cancer Keywords: DNA Methylation; Breast Cancer; Epigenetics; Cancer Predisposition; Allelic Methylation Additional Information: Financial Support: This work used the Genome Sequencing Service Center by Stanford Center for Genomics and Personalized Medicine Sequencing Center, supported by the grant award NIH S10OD020141. MKH is supported by grants from Sigrid Juselius Foundation, Orion Research Foundation, Päivikki ja Sakari Sohlberg Foundation and Instrumentarium Science Foundation. JJG was supported by fellowships from the Jane Coffin Childs Memorial Fund for Medical Research, Stanford Cancer Institute and Susan G. Komen Foundation, as well as funding from ASCO, the Conquer Cancer Foundation and the Breast Cancer Research Foundation. NJ was supported by an EMBO Long-Term Fellowship (ALTF 325-2014). JMF is supported by the BRCA Foundation and the Breast Cancer Research Foundation. MPS is supported by grants from the NIH including a Centers of Excellence in Genomic Science award (5P50HG00773504). Correspondence: Michael P. Snyder James M. Ford Department of Genetics 269 Campus Dr. Stanford University School of Medicine CCSR Room 1115 300 Pasteur Dr. M344 Stanford University School of Medicine Stanford, CA 94305 Stanford, CA 94305 650-736-8099 650-721-1503 [email protected] [email protected] Disclosure of Interests: MPS is cofounder and scientific advisory board member of Personalis, SensOmics, Qbio, January, Akna, Filtricine, and Tailai. MPS is on the scientific advisory board of Genapsys and Jupiter. MPS owns stock in Abcam and Epinomics. Word count (Introduction – Disclosure of Interests): 5,371 Total Pages: 37 Total Figures and Tables: 8 Main, 7 Supplemental Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Statement of Translational Relevance Personalized genomics not only provides vital information about what drives specific tumors, but can also be informative about the likelihood of developing cancer; hereditary forms constitute 5- 10% of all cancers, including breast cancer. By performing deep bisulfite sequencing of 99 early-onset, familial risk breast cancer patients, we demonstrate proof-of-principle that broader epigenetic states, in the form of allelic methylation at known cancer genes, exist in somatic DNA derived from peripheral blood in up to 9% of cancer patients. Additional studies will be required to identify the functional consequences of such epigenetic states and whether screening for such epi-alleles may help clarify unexplained risk for developing hereditary breast cancer. Chen J, et al. “DNA Methylation and High-Risk Breast Cancer” 2 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: Understanding and explaining hereditary predisposition to cancer has focused on the genetic etiology of the disease. However, mutations in known genes associated with breast cancer, such as BRCA1 and BRCA2, account for less than 25% of familial cases of breast cancer. Recently, specific epigenetic modifications at BRCA1 have been shown to promote hereditary breast cancer, but the broader potential for epigenetic contribution to hereditary breast cancer is not yet well understood. Experimental Design: We examined DNA methylation through deep bisulfite sequencing of CpG islands and known promoter or regulatory regions in peripheral blood DNA from 99 familial or early-onset breast or ovarian cancer patients, 6 unaffected BRCA-mutation carriers, and 49 unaffected controls. Results: In 9% of patients, we observed altered methylation in the promoter regions of genes known to be involved in cancer including hypermethylation at the tumor suppressor PTEN and hypomethylation at the proto-oncogene TEX14. These alterations occur in the form of allelic methylation that span up to hundreds of base-pairs in length. Conclusions: Our observations suggest a broader role for DNA methylation in early-onset, familial risk breast cancer. Further studies are warranted to clarify these mechanisms and the benefits of DNA methylation screening for early risk prediction of familial cancers. Chen J, et al. “DNA Methylation and High-Risk Breast Cancer” 3 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Breast cancer is the leading type of cancer in women world-wide, representing nearly 25% of all cases (1). Approximately 15% of invasive breast cancer can be traced to a hereditary origin, with patients having at least one first-degree female relative with the disease. The landmark discoveries of the first two major breast cancer susceptibility genes, Breast cancer 1 (BRCA1) (2) and Breast cancer 2 (BRCA2) (3), over twenty years ago revealed the power of genetic testing; over 60% of women with heterozygous germline mutations in either of these genes ultimately develop breast cancer by the age of 70. However, there have been diminishing returns with each subsequent tumor suppressor identified. Whereas mutations in BRCA1 and BRCA2 together account for around 15% of hereditary cases, deleterious genetic alterations in other genes such as Cadherin-1 (CDH1), Phosphatase and tensin homolog (PTEN), and Partner and localizer of BRCA2 (PALB2) account for less than 7% of hereditary cases combined (4). After taking into account the cumulative and putative effects of SNPs on this disease, 50% of hereditary breast cancer may still remain unexplained from a genetic perspective (4). Tumor suppressor inactivation by hypermethylation has been well described, mainly as a “second hit” in tumors (5). Since sporadic breast cancer tumors with BRCA1 hypermethylation remain histologically and molecularly similar to hereditary breast cancer tumors with BRCA1 mutations (6,7), we investigated whether somatic epigenetic alterations could exist more broadly in familial breast cancer. This hypothesis follows multiple epigenome-wide association studies that have used methylation arrays to suggest that heritable methylated marks can contribute to hereditary cancers, including breast cancer (8–11). Controversy has surrounded the potential for transgenerational epigenetic inheritance of methylated alleles that contribute to disease ever since the concept has been postulated (12,13). While the bar for irrefutable proof is beyond the experimental conditions that can be performed in human studies, observational support for this phenomenon has come from heritable hypermethylated MutL homolog 1 (MLH1) alleles that Chen J, et al. “DNA Methylation and High-Risk Breast Cancer” 4 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 7, 2019; DOI: 10.1158/1078-0432.CCR-18-2423 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. lead to Lynch syndrome (14,15), a familial predisposition to colorectal cancer (16). More recently, a study of 49 families looking for BRCA1 epimutations has identified two instances of a dominantly inherited, constitutively methylated allele at the BRCA1 promoter that leads to breast cancer (17). Despite establishing the contribution of heritable BRCA1 epimutations to breast cancer, the prominence of this occurrence across the genome remains unknown. Here we utilize an unbiased approach to investigate breast cancer predisposition by DNA methylation. Using a cohort of 99 patients with familial and early-onset breast or ovarian cancer, we assessed putative germline DNA methylation from whole blood using bisulfite sequencing at known gene promoters and CpG islands. Although we did not observe promoter epialterations at BRCA1 or BRCA2 in cancer patients, we did find other instances of allelic methylation with the potential to contribute to disease in 9% of our cohort. This includes allelic hypermethylation at known tumor suppressor loci such as PTEN as well as allelic hypomethylation at known proto-oncogenic loci such as Testis expressed 14 (TEX14). We also identify sites of recurrent allelic methylation specific to breast cancer patients, relative to a control cohort of unaffected women. These results, which are independent from chemotherapy
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