Virchows Archiv

, Volume 470, Issue 5, pp 493–504 | Cite as

Multicolor immunofluorescence reveals that p63- and/or K5-positive progenitor cells contribute to normal breast epithelium and usual ductal hyperplasia but not to low-grade intraepithelial neoplasia of the breast

  • Werner Boecker
  • Göran Stenman
  • Tina Schroeder
  • Udo Schumacher
  • Thomas Loening
  • Lisa Stahnke
  • Catharina Löhnert
  • Robert Michael Siering
  • Arthur Kuper
  • Vera Samoilova
  • Markus Tiemann
  • Eberhard Korsching
  • Igor Buchwalow
Original Article
  • 230 Downloads

Abstract

We contend that knowledge about the cellular composition of normal breast epithelium is a prerequisite for understanding proliferative breast disease. Against this background, we used multicolor immunofluorescence to study normal breast epithelium and two types of intraepithelial proliferative breast lesion for expression of the p63, basal keratin K5, glandular keratin K8/18, SMA, ER-alpha, and Ki67. We studied eight normal breast epithelium samples, 12 cases of usual ductal hyperplasia, and 33 cases of low-grade intraepithelial neoplasia (9 flat epithelial atypia, 14 low-grade ductal carcinoma in situ and 10 cases of lobular neoplasia). Usual ductal hyperplasia showed striking similarity to normal luminal breast epithelium including p63+ and/or K5+ luminal progenitor cells and the full spectrum of luminal progeny cells. In normal breast epithelium and usual ductal hyperplasia, expression of ER-alpha was associated with lack of expression of the proliferation antigen Ki67. In contrast, we found in both types of low-grade intraepithelial neoplasia robust expression of keratin K8/18 and a positive association between ER-alpha and Ki67 expression. However, these lesions were consistently negative for p63 and/or K5. Our observational study supports the view that usual ductal hyperplasia and low-grade intraepithelial neoplasia are different entities rather than part of a spectrum of the same disease. We propose a new operational model of cell differentiation that may serve to better understand correlations between normal breast epithelium and proliferative breast diseases. From our data we conclude that p63+ and/or K5+ progenitor cells contribute to maintenance of normal epithelium and usual ductal hyperplasia, but not to low-grade intraepithelial neoplasia of the breast.

Keywords

Normal breast epithelium Usual ductal hyperplasia Low-grade intraepithelial neoplasias p63+K5+ progenitor cells Luminal cells 

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References

  1. 1.
    Asselin-Labat ML, Shackleton M, Stingl J et al (2006) Steroid hormone receptor status of mouse mammary stem cells. J Natl Cancer Inst 98:1011–1014CrossRefPubMedGoogle Scholar
  2. 2.
    Aulmann S, Braun L, Mietzsch F et al (2012) Transitions between flat epithelial atypia and low-grade ductal carcinoma in situ of the breast. Am J Surg Pathol 36:1247–1252CrossRefPubMedGoogle Scholar
  3. 3.
    Azzopardi JG (1979) Epitheliosis and in situ carcinoma. Problems in breast pathology. W.B. Saunders, London, pp 113–149Google Scholar
  4. 4.
    Azzopardi JG (1979) Problems in breast pathology. W.B. Saunders, LondonGoogle Scholar
  5. 5.
    Boecker W, Bier B, Ludwig A et al (1993) Benign proliferative lesions and in situ carcinoma of the breast: new immunohistological findings and their biological implications. Eur J Cancer Prev 2:41–49CrossRefGoogle Scholar
  6. 6.
    Boecker W, Buerger H (2003) Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept. Cell Prolif 36(Suppl 1):73–84CrossRefPubMedGoogle Scholar
  7. 7.
    Boecker W, Buerger H, Schmitz K et al (2001) Ductal epithelial proliferations of the breast: a biological continuum? Comparative genomic hybridisation and high-molecular-weight cytokeratin expression patterns. J Path 195:415–421CrossRefPubMedGoogle Scholar
  8. 8.
    Boecker W, Moll R, Dervan P et al (2002) Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ. J Pathol 198:458–467CrossRefPubMedGoogle Scholar
  9. 9.
    Boecker W, Stenman G, Loening T et al. (2014) Squamous/epidermoid differentiation in normal breast and salivary gland tissues and their corresponding tumors originate from p63/K5/14-positive progenitor cells. Virchows ArchGoogle Scholar
  10. 10.
    Boecker WJ, Bier B, Freytag G et al (1992) An immunohistochemical study of the breast using antibodies to basal and luminal keratins, alpha-smooth muscle actin, vimentin, collagen IV and laminin. Part II: epitheliosis and ductal carcinoma in situ. Virchows Archiv A 421:323–330CrossRefGoogle Scholar
  11. 11.
    Brown JK, Pemberton AD, Wright SH et al (2004) Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem 52:1219–1230CrossRefPubMedGoogle Scholar
  12. 12.
    Buchwalow I, Samoilova V, Boecker W et al (2011) Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Sci Rep 1(28):2011. doi:10.1038/srep00028 Google Scholar
  13. 13.
    Buchwalow IB, Boecker W (2010) Immunohistochemistry: basics and methods. Springer, Heidelberg, Dordrecht, London, New YorkCrossRefGoogle Scholar
  14. 14.
    Buchwalow IB, Minin EA, Boecker W (2005) A multicolor fluorescence immunostaining technique for simultaneous antigen targeting. Acta Histochem 107:143–148CrossRefPubMedGoogle Scholar
  15. 15.
    Buerger H, Otterbach F, Simon R et al (1999) Comparative genomic hybridization of ductal carcinoma in situ of the breast-evidence of multiple genetic pathways. J Pathol 187:396–402CrossRefPubMedGoogle Scholar
  16. 16.
    Buerger H, Simon R, Schafer KL et al (2000) Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast. MolPathol 53:118–121Google Scholar
  17. 17.
    Buono KD, Robinson GW, Martin C et al (2006) The canonical Notch/RBP-J signaling pathway controls the balance of cell lineages in mammary epithelium during pregnancy. Dev Biol 293:565–580CrossRefPubMedGoogle Scholar
  18. 18.
    Chivukula M, Bhargava R, Tseng G et al (2009) Clinicopathologic implications of “flat epithelial atypia” in core needle biopsy specimens of the breast. Am J Clin Pathol 131:802–808CrossRefPubMedGoogle Scholar
  19. 19.
    Clarke RB, Howell A, Potten CS et al (1997) Dissociation between steroid receptor expression and cell proliferation in the human breast. Cancer Res 57:4987–4991PubMedGoogle Scholar
  20. 20.
    Clarke RB, Spence K, Anderson E et al (2005) A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol 277:443–456CrossRefPubMedGoogle Scholar
  21. 21.
    Dabbs DJ, Carter G, Fudge M et al (2006) Molecular alterations in columnar cell lesions of the breast. Mod Pathol 19:344–349CrossRefPubMedGoogle Scholar
  22. 22.
    Daniely Y, Liao G, Dixon D et al (2004) Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. Am J Physiol Cell Physiol 287:C171–C181CrossRefPubMedGoogle Scholar
  23. 23.
    Ellis IO (2010) Intraductal proliferative lesions of the breast: morphology, associated risk and molecular biology. Mod Pathol 23(Suppl 2):S1–S7CrossRefPubMedGoogle Scholar
  24. 24.
    Goldstein NS, Lacerna M, Vicini F (1998) Cancerization of lobules and atypical ductal hyperplasia adjacent to ductal carcinoma in situ of the breast. AmJ Clin Pathol 110:357–367CrossRefGoogle Scholar
  25. 25.
    Goldstein NS, O’Malley BA (1997) Cancerization of small ectatic ducts of the breast by ductal carcinoma in situ cells with apocrine snouts: a lesion associated with tubular carcinoma. Am J Clin Pathol 107:561–566CrossRefPubMedGoogle Scholar
  26. 26.
    Gong G, DeVries S, Chew KL et al (2001) Genetic changes in paired atypical and usual ductal hyperplasia of the breast by comparative genomic hybridization. Clin Cancer Res 7:2410–2414PubMedGoogle Scholar
  27. 27.
    Hackett TL, Shaheen F, Johnson A et al (2008) Characterization of side population cells from human airway epithelium. Stem Cells 26:2576–2585CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Iqbal M, Davies MP, Shoker BS et al (2001) Subgroups of non-atypical hyperplasia of breast defined by proliferation of oestrogen receptor-positive cells. JPathol 193:333–338CrossRefGoogle Scholar
  29. 29.
    Jahn SW, Kashofer K, Thuringer A et al (2016) Mutation profiling of usual ductal hyperplasia of the breast reveals activating mutations predominantly at different levels of the PI3K/AKT/mTOR pathway. Am J Pathol 186:15–23CrossRefPubMedGoogle Scholar
  30. 30.
    Jarasch ED, Nagle RB, Kaufmann M et al (1988) Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins. Hum Pathol 19:276–289CrossRefPubMedGoogle Scholar
  31. 31.
    Jones C, Merrett S, Thomas VA et al (2003) Comparative genomic hybridization analysis of bilateral hyperplasia of usual type of the breast. J Pathol 199:152–156CrossRefPubMedGoogle Scholar
  32. 32.
    Lakhani SR (1998) The transition from hyperplasia to invasive carcinoma of the breast. J Pathol 187:272–278CrossRefGoogle Scholar
  33. 33.
    Lakhani SR, Collins N, Stratton MR (1995) Loss of heterozygosity in lobular carcinoma in situ of the breast. J Clin Pathol: Mol Pathol 48:M74–M78Google Scholar
  34. 34.
    Lakhani SR, Ellis IO, Schnitt SJ et al. (2012) WHO classification of tumors of the breast. IARC LyonGoogle Scholar
  35. 35.
    Lakhani SR, Slack DN, Hamoudi RA et al (1996) Detection of allelic imbalance indicates that a proportion of mammary hyperplasia of usual type are clonal neoplastic proliferations. Lab Investig 74:129–135PubMedGoogle Scholar
  36. 36.
    Lee S, Mohsin SK, Mao S et al (2006) Hormones, receptors, and growth in hyperplastic enlarged lobular units: early potential precursors of breast cancer. Breast Cancer Res 8:R6CrossRefPubMedGoogle Scholar
  37. 37.
    Lim E, Vaillant F, Wu D et al (2009) Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med 15:907–913CrossRefPubMedGoogle Scholar
  38. 38.
    Mills AA, Zheng B, Wang XJ et al (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398:708–713CrossRefPubMedGoogle Scholar
  39. 39.
    Moinfar F (2009) Flat ductal intraepithelial neoplasia of the breast: a review of diagnostic criteria, differential diagnoses, molecular-genetic findings, and clinical relevance–it is time to appreciate the Azzopardi concept! Arch Pathol Lab Med 133:879–892PubMedGoogle Scholar
  40. 40.
    Mukherjee A, Ellis IA, Rakha A (2015) Molecular pathology of precancerous lesions of the breast. In: Khan A et al (eds) Precision molecular pathology of breast cancer. Springer, New York, Heidelberg, Dordrecht, London, pp 51–62CrossRefGoogle Scholar
  41. 41.
    Mukherjee AK, Saviola AJ, Burns PD et al (2015) Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production. Apoptosis 20:1358–1372CrossRefPubMedGoogle Scholar
  42. 42.
    Mommers EC, van Diest PJ, Leonhart AM et al (1998) Expression of proliferation and apoptosis-related proteins in usual ductal hyperplasia of the breast. Hum Pathol 29:1539–1545CrossRefPubMedGoogle Scholar
  43. 43.
    Nagle RB, Bocker W, Davis JR et al (1986) Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells. J Histochem Cytochem 34:869–881CrossRefPubMedGoogle Scholar
  44. 44.
    Nylander K, Vojtesek B, Nenutil R et al (2002) Differential expression of p63 isoforms in normal tissues and neoplastic cells. J Pathol 198:417–427CrossRefPubMedGoogle Scholar
  45. 45.
    O’Connell P, Pekkel V, Fuqua SA et al (1998) Analysis of loss of heterozygosity in 399 premalignant breast lesions at 15 genetic loci. J NatlCancer Inst 90:697–703Google Scholar
  46. 46.
    Perou CM, Sorlie T, Eisen MB et al (2000) Molecular portraits of human breast tumours. Nature 406:747–752CrossRefPubMedGoogle Scholar
  47. 47.
    Reis-Filho JS, Simpson PT, Martins A et al (2003) Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray. Virchows Arch 443:122–132CrossRefPubMedGoogle Scholar
  48. 48.
    Rock JR, Onaitis MW, Rawlins EL et al (2009) Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 106:12771–12775CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Rock JR, Randell SH, Hogan BL (2010) Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis Model Mech 3:545–556CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Rosen PP (1999) Columnar cell hyperplasia is associated with lobular carcinoma in situ and tubular carcinoma. Am J Surg Pathol 23:1561CrossRefPubMedGoogle Scholar
  51. 51.
    Ross AJ, Dailey LA, Brighton LE et al (2007) Transcriptional profiling of mucociliary differentiation in human airway epithelial cells. Am J Respir Cell Mol Biol 37:169–185CrossRefPubMedGoogle Scholar
  52. 52.
    Sahoo S, Recant WM (2005) Triad of columnar cell alteration, lobular carcinoma in situ, and tubular carcinoma of the breast. Breast J 11:140–142CrossRefPubMedGoogle Scholar
  53. 53.
    Samanta S, Khan A, Mercurio AM (2015) Breast cancer stem cells: role of tumor initiation, progression, and targeted therapy. In: Khan A et al (eds) Precision molecular pathology of breast cancer. Springer, New York Heidelberg Dodrecht London, pp 63–78CrossRefGoogle Scholar
  54. 54.
    Schnitt SJ (2003) The diagnosis and management of pre-invasive breast disease: flat epithelial atypia–classification, pathologic features and clinical significance. Breast Cancer Res 5:263–268CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Schnitt SJ (2004) Columnar cell lesions of the breast: pathological features and clinical significance. Curr Diagn Pathol 10:193–203CrossRefGoogle Scholar
  56. 56.
    Senoo M, Pinto F, Crum CP et al (2007) p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129:523–536CrossRefPubMedGoogle Scholar
  57. 57.
    Shoker BS, Jarvis C, Clarke RB et al (1999) Estrogen receptor-positive proliferating cells in the normal and precancerous breast. Am J Pathol 155:1811–1815CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Shoker BS, Jarvis C, Clarke RB et al (2000) Abnormal regulation of the oestrogen receptor in benign breast lesions. J Clin Pathol 53:778–783CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Shoker BS, Jarvis C, Sibson DR et al (1999) Oestrogen receptor expression in the normal and pre-cancerous breast. J Pathol 188:237–244CrossRefPubMedGoogle Scholar
  60. 60.
    Signoretti S, Waltregny D, Dilks J et al (2000) p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 157:1769–1775CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Simpson PT, Gale T, Reis-Filho JS et al (2005) Columnar cell lesions of the breast: the missing link in breast cancer progression? A morphological and molecular analysis. Am J Surg Pathol 29:734–746CrossRefPubMedGoogle Scholar
  62. 62.
    Sleeman KE, Kendrick H, Ashworth A et al (2006) CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Cancer Res 8:R7CrossRefPubMedGoogle Scholar
  63. 63.
    Sorlie T, Perou CM, Tibshirani R et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci U S A 98:10869–10874CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Vos CB, ter Haar NT, Rosenberg C et al (1999) Genetic alterations on chromosome 16 and 17 are important features of ductal carcinoma in situ of the breast and are associated with histologic type. Br J Cancer 81:1410–1418CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Warner SM, Hackett TL, Shaheen F et al. (2013) Transcription factor p63 regulates key genes and wound repair in human airway epithelial basal cells. Am J Respir Cell Mol BiolGoogle Scholar
  66. 66.
    Wellings SR, Jensen HM, Marcum RG (1975) An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J Natl Cancer Inst 55:231–273PubMedGoogle Scholar
  67. 67.
    Xu S, Wei B, Zhang H et al (2008) Evidence of chromosomal alterations in pure usual ductal hyperplasia as a breast carcinoma precursor. Oncol Rep 19:1469–1475PubMedGoogle Scholar
  68. 68.
    Yeh IT, Mies C (2008) Application of immunohistochemistry to breast lesions. Arch Pathol Lab Med 132:349–358PubMedGoogle Scholar
  69. 69.
    Yu Q, Niu Y, Yu Y et al (2009) Analysis of the progression of intraductal proliferative lesions in the breast by PCR-based clonal assay. Breast Cancer Res Treat 114:433–440CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Werner Boecker
    • 1
    • 4
  • Göran Stenman
    • 2
  • Tina Schroeder
    • 3
  • Udo Schumacher
    • 3
  • Thomas Loening
    • 4
  • Lisa Stahnke
    • 1
  • Catharina Löhnert
    • 1
  • Robert Michael Siering
    • 1
  • Arthur Kuper
    • 1
  • Vera Samoilova
    • 5
  • Markus Tiemann
    • 5
  • Eberhard Korsching
    • 6
  • Igor Buchwalow
    • 5
  1. 1.Gerhard-Domagk-Institute of PathologyUniversity of MuensterMuensterGermany
  2. 2.Sahlgrenska Cancer CenterUniversity of GothenburgGothenburgSweden
  3. 3.Institute for Anatomy and Experimental MorphologyUniversity of HamburgHamburgGermany
  4. 4.Gerhard-Seifert ReferenzzentrumHamburgGermany
  5. 5.Institute for HematopathologyHamburgGermany
  6. 6.Institute of BioinformaticsUniversity of MuensterMuensterGermany

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