title: High mammographic density is associated with an increase in stromal collagen and immune cells within the mammary epithelium.
abstract: BACKGROUND
  Mammographic density (MD), after adjustment for a women's age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk.
  
  
  METHODS
  Tissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status.
  
  
  RESULTS
  HMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206(+) alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial-mesenchymal transition was seen; however, there was a significant increase in vimentin(+)/CD45(+) immune cells within the epithelial layer in HMD tissues.
  
  
  CONCLUSIONS
  We confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45(+)/vimentin(+) immune cells in this area.
authors.affiliation: University of Melbourne Department of Surgery, St. Vincent's Hospital, Level 2, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. wanchen.huo@gmail.com.
authors.forename: Cecilia W
authors.initials: CW
authors.lastname: Huo
authors.affiliation: University of Melbourne Department of Surgery, St. Vincent's Hospital, Level 2, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. gracelchew@gmail.com.
authors.forename: Grace
authors.initials: G
authors.lastname: Chew
authors.affiliation: Department of Pathology, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, VIC, 3065, Australia. prue.hill@svha.org.au.
authors.forename: Prue
authors.initials: P
authors.lastname: Hill
authors.affiliation: St. Vincent's Institute, 9 Princes Street, Fitzroy, VIC, 3065, Australia. dhuang@svi.edu.au.
authors.forename: Dexing
authors.initials: D
authors.lastname: Huang
authors.affiliation: Discipline of Surgery, Faculty of Health Sciences, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Adelaide, Australia. wendy.ingman@adelaide.edu.au.|Robinson Research Institute, University of Adelaide, Ground Floor, Norwich Centre, 55 King William Road, North Adelaide, SA, 5006, Australia. wendy.ingman@adelaide.edu.au.
authors.forename: Wendy
authors.initials: W
authors.lastname: Ingman
authors.affiliation: Discipline of Surgery, Faculty of Health Sciences, School of Medicine, The Queen Elizabeth Hospital, University of Adelaide, Adelaide, Australia. leigh.hodson@adelaide.edu.au.|Robinson Research Institute, University of Adelaide, Ground Floor, Norwich Centre, 55 King William Road, North Adelaide, SA, 5006, Australia. leigh.hodson@adelaide.edu.au.
authors.forename: Leigh
authors.initials: L
authors.lastname: Hodson
authors.affiliation: Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC, 3168, Australia. kristy.brown@mimr-phi.org.
authors.forename: Kristy A
authors.initials: KA
authors.lastname: Brown
authors.affiliation: Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Australia. a.magenau@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, Australia. a.magenau@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Clayton, Australia. a.magenau@garvan.org.au.
authors.forename: Astrid
authors.initials: A
authors.lastname: Magenau
authors.affiliation: Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Australia. a.allam@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, Australia. a.allam@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Clayton, Australia. a.allam@garvan.org.au.
authors.forename: Amr H
authors.initials: AH
authors.lastname: Allam
authors.affiliation: St Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, Australia. e.mcghee@beatson.gla.ac.uk.
authors.forename: Ewan
authors.initials: E
authors.lastname: McGhee
authors.affiliation: Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Australia. p.timpson@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Sydney, Australia. p.timpson@garvan.org.au.|St Vincent's Clinical School, Faculty of Medicine, University of NSW, Clayton, Australia. p.timpson@garvan.org.au.
authors.forename: Paul
authors.initials: P
authors.lastname: Timpson
authors.affiliation: University of Melbourne Department of Surgery, St. Vincent's Hospital, Level 2, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. michael.henderson@petermac.org.|Peter MacCallum Cancer Centre, 2 St. Andrews Place, East Melbourne, VIC, 3002, Australia. michael.henderson@petermac.org.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Henderson
authors.affiliation: University of Melbourne Department of Surgery, St. Vincent's Hospital, Level 2, Clinical Sciences Building, 29 Regent Street, Fitzroy, VIC, 3065, Australia. e2.thompson@qut.edu.au.|St. Vincent's Institute, 9 Princes Street, Fitzroy, VIC, 3065, Australia. e2.thompson@qut.edu.au.|Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD, 4059, Australia. e2.thompson@qut.edu.au.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
authors.affiliation: The Beatson Institute for Cancer Research, Switchback Road, Bearsden Glasgow, G61 1BD, UK. kara.britt@petermac.org.|The Sir Peter MacCallum Department of Oncology, University of Melbourne, St. Andrews Place, East Melbourne, VIC, 3002, Australia. kara.britt@petermac.org.|Department of Anatomy and Developmental Biology, Monash University, 19 Innovation Walk, Clayton, VIC, s, Australia. kara.britt@petermac.org.
authors.forename: Kara
authors.initials: K
authors.lastname: Britt
mesh_terms: D000328:Adult; D014408:Biomarkers, Tumor / Q000378:metabolism; D001940:Breast / Q000378:metabolism* / Q000473:pathology; D000071060:Breast Density; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D003094:Collagen / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition; D004848:Epithelium / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D016130:Immunophenotyping; D008264:Macrophages / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D042361:Mammary Glands, Human / Q000002:abnormalities*; D008327:Mammography; D008875:Middle Aged; D010641:Phenotype; D012307:Risk Factors; D017154:Stromal Cells / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D003094:Collagen