Fibroadenomas are the most common breast tumors in women under 30 (refs. 1,2). Exome sequencing of eight fibroadenomas with matching whole-blood samples revealed recurrent somatic mutations solely in MED12, which encodes a Mediator complex subunit. Targeted sequencing of an additional 90 fibroadenomas confirmed highly frequent MED12 exon 2 mutations (58/98, 59%) that are probably somatic, with 71% of mutations occurring in codon 44. Using laser capture microdissection, we show that MED12 fibroadenoma mutations are present in stromal but not epithelial mammary cells. Expression profiling of MED12-mutated and wild-type fibroadenomas revealed that MED12 mutations are associated with dysregulated estrogen signaling and extracellular matrix organization. The fibroadenoma MED12 mutation spectrum is nearly identical to that of previously reported MED12 lesions in uterine leiomyoma but not those of other tumors. Benign tumors of the breast and uterus, both of which are key target tissues of estrogen, may thus share a common genetic basis underpinned by highly frequent and specific MED12 mutations.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Krishnamurthy, S., Ashfaq, R., Shin, H.J.C. & Sneige, N. Distinction of phyllodes tumor from fibroadenoma. Cancer 90, 342–349 (2000).
Fine, R.E. et al. Low-risk palpable breast masses removed using a vacuum-assisted hand-held device. Am. J. Surg. 186, 362–367 (2003).
Bernardes, J.R.M. Jr., Seixas, M.T., Lima, G.R., Marinho, L.C. & Gebrim, L.H. The effect of tamoxifen on PCNA expression in fibroadenomas. Breast J. 9, 302–306 (2003).
Coriaty Nelson, Z., Ray, R.M., Gao, D.L. & Thomas, D.B. Risk factors for fibroadenoma in a cohort of female textile workers in Shanghai, China. Am. J. Epidemiol. 156, 599–605 (2002).
Noguchi, S., Motomura, K., Inaji, H., Imaoka, S. & Koyama, H. Clonal analysis of fibroadenoma and phyllodes tumor of the breast. Cancer Res. 53, 4071–4074 (1993).
Dupont, W.D. et al. Long-term risk of breast cancer in women with fibroadenoma. N. Engl. J. Med. 331, 10–15 (1994).
Liu, X.F. et al. A clinical study on the resection of breast fibroadenoma using two types of incision. Scand. J. Surg. 100, 147–152 (2011).
The Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 490, 61–70 (2012).
Stephens, P.J. et al. The landscape of cancer genes and mutational processes in breast cancer. Nature 486, 400–404 (2012).
Millikan, R. et al. p53 mutations in benign breast tissue. J. Clin. Oncol. 13, 2293–2300 (1995).
Franco, N., Picard, S.-F., Mege, F., Arnould, L. & Lizard-Nacol, S. Absence of genetic abnormalities in fibroadenomas of the breast determined at p53 gene mutations and microsatellite alterations. Cancer Res. 61, 7955–7958 (2001).
Vorkas, P.A. et al. PIK3CA hotspot mutation scanning by a novel and highly sensitive high-resolution small amplicon melting analysis method. J. Mol. Diagn. 12, 697–704 (2010).
Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).
Mäkinen, N. et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science 334, 252–255 (2011).
Forbes, S.A. et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 39, D945–D950 (2011).
Sherry, S.T. et al. dbSNP: the NCBI database of genetic variation. Nucleic Acids Res. 29, 308–311 (2001).
1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).
Harper, P.S. Mary Lyon and the hypothesis of random X chromosome inactivation. Hum. Genet. 130, 169–174 (2011).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Barbieri, C.E. et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat. Genet. 44, 685–689 (2012).
Assié, G. et al. Integrated genomic characterization of adrenocortical carcinoma. Nat. Genet. 10.1038/ng.2953 (2014).
Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615 (2011).
Je, E.M., Kim, M.R., Min, K.O., Yoo, N.J. & Lee, S.H. Mutational analysis of MED12 exon 2 in uterine leiomyoma and other common tumors. Int. J. Cancer 131, E1044–E1047 (2012).
Kämpjärvi, K. et al. Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer. Br. J. Cancer 107, 1761–1765 (2012).
Zhu, B.T. & Conney, A.H. Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19, 1–27 (1998).
Kang, Y.K., Guermah, M., Yuan, C.-X. & Roeder, R.G. The TRAP/Mediator coactivator complex interacts directly with estrogen receptors α and β through the TRAP220 subunit and directly enhances estrogen receptor function in vitro. Proc. Natl. Acad. Sci. USA 99, 2642–2647 (2002).
Mäkinen, N., Vahteristo, P., Bützow, R., Sjöberg, J. & Aaltonen, L.A. Exomic landscape of MED12 mutation–negative and –positive uterine leiomyomas. Int. J. Cancer 134, 1008–1012 (2014).
Chan-On, W. et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non–infection-related bile duct cancers. Nat. Genet. 45, 1474–1478 (2013).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Wilson, C.L. & Miller, C.J. Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis. Bioinformatics 21, 3683–3685 (2005).
Irizarry, R.A. et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003).
Dai, M. et al. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data. Nucleic Acids Res. 33, e175 (2005).
Smyth, G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
Edgar, R., Domrachev, M. & Lash, A.E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002).
Hänzelmann, S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-Seq data. BMC Bioinformatics 14, 7 (2013).
This work was supported in part by funding from the Singapore National Medical Research Council (NMRC/STAR/0006/2009), the Singapore Millennium Foundation, the Lee Foundation, the Tanoto Foundation, the Singapore National Cancer Centre Research Fund, the Duke-NUS Graduate Medical School, the Cancer Science Institute, Singapore and the Verdant Foundation, Hong Kong. We thank the Duke-NUS Genome Biology Facility for sequencing services rendered, as well as the Advanced Molecular Pathology Laboratory, Institute of Molecular and Cell Biology, A*STAR, Singapore for providing LCM resources. We also thank the SingHealth Tissue Repository for frozen tissue samples.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Confirmation of somatic MED12 mutations in fresh frozen fibroadenoma samples by Sanger sequencing.
Genomic DNA Sanger sequencing of MED12 variants in eight fresh frozen fibroadenomas and their matched whole-blood. Variant allele frequency as determined by ultra-deep targeted amplicon sequencing is provided on the left of each sample.
Supplementary Figure 2 Sanger sequencing of MED12 using cDNA from fresh frozen fibroadenomas confirms transcription of mutant MED12.
Complementary DNA Sanger sequencing of MED12 variants in eight fresh frozen fibroadenomas and their matched whole-blood. Variant peaks were unambiguous except for Sample002, possibly due to RNA degradation (the sample had a RNA integrity number of 7.3; the lowest among eight samples included in our microarray study). Other explanations include the mutant allele simply not being expressed, or PCR bias towards the wild-type allele.
Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample004. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 aberrant splice site mutation is exclusive to the stromal compartment.
Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample006. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.
Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample007. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.
Laser capture microdissection (LCM) followed by Sanger sequencing. The image is a hematoxylin and eosin (H&E) stain of a section of Sample008. Epithelial compartments are marked in green. Sanger sequencing of MED12 using two different PCR primer sets show that the MED12 p.Gly44Asp mutation is exclusive to the stromal compartment.
GSEA analysis similar to Fig. 2b, but against genes upregulated 2x in UL instead of 4x.
Supplementary Figures 1-7 and Supplementary Tables 2, 5 and 7 (PDF 18985 kb)
Clinical characteristics of fibroadenoma patients. (XLS 38 kb)
List of confirmed somatic mutations identified from whole-exome sequencing of eight fibroadenomas. (XLS 35 kb)
Mutations detected in ultra-deep targeted amplicon sequencing of MED12 exon 2 in 98 fibroadenoma samples. (XLS 36 kb)
Top 50 enriched MSigDB curated (c2) gene sets for upregulated and downregulated genes in MED12 mutant fibroadenomas. Gene sets of interest are highlighted. ES: Enrichment Score, NES: Normalized Enrichment Score, FDR: False Discovery Rate (XLS 42 kb)
Differentially expressed genes between mutant and wild-type MED12 fibroadenoma samples. (XLS 38 kb)
About this article
Cite this article
Lim, W., Ong, C., Tan, J. et al. Exome sequencing identifies highly recurrent MED12 somatic mutations in breast fibroadenoma. Nat Genet 46, 877–880 (2014). https://doi.org/10.1038/ng.3037
High‐grade uterine sarcoma with osteosarcomatous differentiation arising from a MED12‐ mutated leiomyoma, a case report
Pathology International (2021)
Modern Pathology (2021)
Droplet-digital PCR reveals frequent mutations in TERT promoter region in breast fibroadenomas and phyllodes tumours, irrespective of the presence of MED12 mutations
British Journal of Cancer (2021)
Human Pathology (2021)
Insights into the regulatory role and clinical relevance of mediator subunit, MED12, in human diseases
Journal of Cellular Physiology (2021)