Advertisement

Breast Cancer Research and Treatment

, Volume 158, Issue 1, pp 21–28 | Cite as

Stromal cells in phyllodes tumors of the breast are enriched for EZH2 and stem cell marker expression

  • Yanhong Zhang
  • Adam L. Liss
  • Eugene Chung
  • Lori J. Pierce
  • Celina G. KleerEmail author
Preclinical study

Abstract

Phyllodes tumors (PTs) of the breast are fibroepithelial neoplasms with stromal hypercellularity, which is the basis for their classification as benign, borderline, and malignant. The histologic diagnosis of PTs is often difficult, and the pathological features may not always predict clinical behavior. The pathobiology of PT remains poorly understood. Enhancer of Zeste 2 (EZH2) epigenetically regulates cell-type identity, cellular differentiation, and breast cancer stem cells. EZH2 exerts oncogenic functions in breast cancer and is associated with metastasis. We hypothesized that in PTs, EZH2 and the stem cell marker ALDH1 may be expressed in stromal cells and may be associated with their degree of differentiation. Forty PTs were histologically characterized at our institution following the World Health Organization criteria. We investigated the expression of EZH2 and ALDH1 by immunohistochemistry and recorded as percentage of positive epithelial and stromal cells. EZH2 was positive when over 10 % of cells exhibited nuclear staining; ALDH1 was positive when over 5 % of cells had cytoplasmic staining. Of the 40 PTs, 24 (60 %) were histologically benign, 8 (20 %) borderline, and 8 (20 %) malignant. Stromal EZH2 was significantly associated with the diagnosis of malignant PT, as it was detected in 1 of 24 (4 %) benign, 3 of 8 (37.5 %) borderline, and 5 of 8 (62.5 %) malignant tumors. Stromal EZH2 was significantly associated with stromal overgrowth (p = 0.01), atypia (p = 0.01), hypercellularity (p = 0.01), and mitoses (p = 0.02), all features of malignant PT. Stromal EZH2 and ALDH1 were significantly associated with grade of PT (p = 0.01 and p < 0.05 respectively). In conclusion, EZH2 and ALDH1 expression in the stroma of PT may mark malignant progression and may be helpful to distinguish histologically benign from borderline and malignant tumors in challenging cases. Our study also suggests that PTs contain mesenchymal stem cells, shedding light into the pathogenesis of these tumors.

Keywords

EZH2 ALDH1 Phyllodes tumors Breast cancer Stromal cells Stem cell 

Notes

Funding

This work was supported by NIH Grants R01 CA107469 and CA125577, and the National Institutes of Health through the University of Michigan’s Cancer Center Support Grant (5 P30 CA46592).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

References

  1. 1.
    Rosen PP, Oberman HA (1993) Tumors of the mammary gland, vol 7., Atlas of tumor pathology Third seriesArmed Forces Institute of Pathology, WashingtonGoogle Scholar
  2. 2.
    Lakhani SR, Ellis. IO, Schnitt, SJ, Tan, PH, van de Vijver, M.J. eds (2012) World Health Organization Classification of Tumours of the Breast. IARC LyonGoogle Scholar
  3. 3.
    Sawyer EJ, Hanby AM, Ellis P, Lakhani SR, Ellis IO, Boyle S, Tomlinson IP (2000) Molecular analysis of phyllodes tumors reveals distinct changes in the epithelial and stromal components. Am J Pathol 156(3):1093–1098CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Sawyer EJ, Hanby AM, Rowan AJ, Gillett CE, Thomas RE, Poulsom R, Lakhani SR, Ellis IO, Ellis P, Tomlinson IP (2002) The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours. J Pathol 196(4):437–444. doi: 10.1002/path.1067 CrossRefPubMedGoogle Scholar
  5. 5.
    Kwon JE, Jung WH, Koo JS (2012) Molecules involved in epithelial-mesenchymal transition and epithelial-stromal interaction in phyllodes tumors: implications for histologic grade and prognosis. Tumour Biol 33(3):787–798. doi: 10.1007/s13277-011-0296-9 CrossRefPubMedGoogle Scholar
  6. 6.
    Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, Popkin D, Pillus L, Jenuwein T (1997) Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J 16(11):3219–3232CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ringrose L, Paro R (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38:413–443CrossRefPubMedGoogle Scholar
  8. 8.
    Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK, Bell GW, Otte AP, Vidal M, Gifford DK, Young RA, Jaenisch R (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441(7091):349–353CrossRefPubMedGoogle Scholar
  9. 9.
    Alford SH, Toy K, Merajver SD, Kleer CG (2012) Increased risk for distant metastasis in patients with familial early-stage breast cancer and high EZH2 expression. Breast Cancer Res Treat 132(2):429–437. doi: 10.1007/s10549-011-1591-2 CrossRefPubMedGoogle Scholar
  10. 10.
    Gonzalez ME, Moore HM, Li X, Toy KA, Huang W, Sabel MS, Kidwell KM, Kleer CG (2014) EZH2 expands breast stem cells through activation of NOTCH1 signaling. Proc Natl Acad Sci USA 111(8):3098–3103. doi: 10.1073/pnas.1308953111 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF, Sabel MS, Livant D, Weiss SJ, Rubin MA, Chinnaiyan AM (2003) EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci USA 100(20):11606–11611. doi: 10.1073/pnas.1933744100 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kunju LP, Cookingham C, Toy KA, Chen W, Sabel MS, Kleer CG (2011) EZH2 and ALDH-1 mark breast epithelium at risk for breast cancer development. Mod Pathol 24(6):786–793. doi: 10.1038/modpathol.2011.8 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, Hsu JM, Hortobagyi GN, Hung MC (2011) EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-beta-catenin signaling. Cancer Cell 19(1):86–100. doi: 10.1016/j.ccr.2010.10.035 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17(10):1253–1270CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 Is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lin JJ, Huang CS, Yu J, Liao GS, Lien HC, Hung JT, Lin RJ, Chou FP, Yeh KT, Yu AL (2014) Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells. Breast Cancer Res 16(2):R29. doi: 10.1186/bcr3631 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K (2003) EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J 22(20):5323–5335CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kamminga LM, Bystrykh LV, de Boer A, Houwer S, Douma J, Weersing E, Dontje B, de Haan G (2006) The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood 107(5):2170–2179CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM (2002) The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419(6907):624–629CrossRefPubMedGoogle Scholar
  20. 20.
    Ciarapica R, Miele L, Giordano A, Locatelli F, Rota R (2011) Enhancer of zeste homolog 2 (EZH2) in pediatric soft tissue sarcomas: first implications. BMC Med 9:63. doi: 10.1186/1741-7015-9-63 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ciarapica R, Russo G, Verginelli F, Raimondi L, Donfrancesco A, Rota R, Giordano A (2009) Deregulated expression of miR-26a and Ezh2 in rhabdomyosarcoma. Cell Cycle 8(1):172–175CrossRefPubMedGoogle Scholar
  22. 22.
    Richter GH, Plehm S, Fasan A, Rossler S, Unland R, Bennani-Baiti IM, Hotfilder M, Lowel D, von Luettichau I, Mossbrugger I, Quintanilla-Martinez L, Kovar H, Staege MS, Muller-Tidow C, Burdach S (2009) EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation. Proc Natl Acad Sci USA 106(13):5324–5329. doi: 10.1073/pnas.0810759106 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Charytonowicz E, Cordon-Cardo C, Matushansky I, Ziman M (2009) Alveolar rhabdomyosarcoma: is the cell of origin a mesenchymal stem cell? Cancer Lett 279(2):126–136. doi: 10.1016/j.canlet.2008.09.039 CrossRefPubMedGoogle Scholar
  24. 24.
    Juan AH, Kumar RM, Marx JG, Young RA, Sartorelli V (2009) Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. Mol Cell 36(1):61–74. doi: 10.1016/j.molcel.2009.08.008 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lenhard MS, Kahlert S, Himsl I, Ditsch N, Untch M, Bauerfeind I (2008) Phyllodes tumour of the breast: clinical follow-up of 33 cases of this rare disease. Eur J Obstet Gynecol Reprod Biol 138(2):217–221CrossRefPubMedGoogle Scholar
  26. 26.
    Karim RZ, Gerega SK, Yang YH, Spillane A, Carmalt H, Scolyer RA, Lee CS (2009) Phyllodes tumours of the breast: a clinicopathological analysis of 65 cases from a single institution. Breast 18(3):165–170. doi: 10.1016/j.breast.2009.03.001 CrossRefPubMedGoogle Scholar
  27. 27.
    Tan PH, Thike AA, Tan WJ, Thu MM, Busmanis I, Li H, Chay WY, Tan MH (2012) Predicting clinical behaviour of breast phyllodes tumours: a nomogram based on histological criteria and surgical margins. J Clin Pathol 65(1):69–76. doi: 10.1136/jclinpath-2011-200368 CrossRefPubMedGoogle Scholar
  28. 28.
    Belkacemi Y, Bousquet G, Marsiglia H, Ray-Coquard I, Magne N, Malard Y, Lacroix M, Gutierrez C, Senkus E, Christie D, Drumea K, Lagneau E, Kadish SP, Scandolaro L, Azria D, Ozsahin M (2008) Phyllodes tumor of the breast. Int J Radiat Oncol Biol Phys 70(2):492–500CrossRefPubMedGoogle Scholar
  29. 29.
    Barth RJ Jr (1999) Histologic features predict local recurrence after breast conserving therapy of phyllodes tumors. Breast Cancer Res Treat 57(3):291–295CrossRefPubMedGoogle Scholar
  30. 30.
    Pezner RD, Schultheiss TE, Paz IB (2008) Malignant phyllodes tumor of the breast: local control rates with surgery alone. Int J Radiat Oncol Biol Phys 71(3):710–713. doi: 10.1016/j.ijrobp.2007.10.051 CrossRefPubMedGoogle Scholar
  31. 31.
    Grimes MM (1992) Cystosarcoma phyllodes of the breast: histologic features, flow cytometric analysis, and clinical correlations. Mod Pathol 5(3):232–239PubMedGoogle Scholar
  32. 32.
    Noronha Y, Raza A, Hutchins B, Chase D, Garberoglio C, Chu P, Weiss L, Wang J (2011) CD34, CD117, and Ki-67 expression in phyllodes tumor of the breast: an immunohistochemical study of 33 cases. Int J Surg Pathol 19(2):152–158. doi: 10.1177/1066896910382009 CrossRefPubMedGoogle Scholar
  33. 33.
    Tan PH, Jayabaskar T, Yip G, Tan Y, Hilmy M, Selvarajan S, Bay BH (2005) p53 and c-kit (CD117) protein expression as prognostic indicators in breast phyllodes tumors: a tissue microarray study. Mod Pathol 18(12):1527–1534PubMedGoogle Scholar
  34. 34.
    Tse GM, Lui PC, Vong JS, Lau KM, Putti TC, Karim R, Scolyer RA, Lee CS, Yu AM, Ng DC, Tse AK, Tan PH (2009) Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors. Breast Cancer Res Treat 114(3):441–448CrossRefPubMedGoogle Scholar
  35. 35.
    Karim RZ, Gerega SK, Yang YH, Spillane A, Carmalt H, Scolyer RA, Lee CS (2010) p16 and pRb immunohistochemical expression increases with increasing tumour grade in mammary phyllodes tumours. Histopathology 56(7):868–875. doi: 10.1111/j.1365-2559.2010.03562.x CrossRefPubMedGoogle Scholar
  36. 36.
    Kleer CG, Giordano TJ, Braun T, Oberman HA (2001) Pathologic, immunohistochemical, and molecular features of benign and malignant phyllodes tumors of the breast. Mod Pathol 14(3):185–190CrossRefPubMedGoogle Scholar
  37. 37.
    Tan WJ, Thike AA, Bay BH, Tan PH (2014) Immunohistochemical expression of homeoproteins Six1 and Pax3 in breast phyllodes tumours correlates with histological grade and clinical outcome. Histopathology 64(6):807–817. doi: 10.1111/his.12329 CrossRefPubMedGoogle Scholar
  38. 38.
    Khosravi-Shahi P (2011) Management of non metastatic phyllodes tumors of the breast: review of the literature. Surg Oncol 20(4):e143–e148. doi: 10.1016/j.suronc.2011.04.007S0960-7404(11)00038-7 CrossRefPubMedGoogle Scholar
  39. 39.
    Chen WH, Cheng SP, Tzen CY, Yang TL, Jeng KS, Liu CL, Liu TP (2005) Surgical treatment of phyllodes tumors of the breast: retrospective review of 172 cases. J Surg Oncol 91(3):185–194. doi: 10.1002/jso.20334 CrossRefPubMedGoogle Scholar
  40. 40.
    Mitus J, Reinfuss M, Mitus JW, Jakubowicz J, Blecharz P, Wysocki WM, Skotnicki P (2014) Malignant phyllodes tumor of the breast: treatment and prognosis. Breast J 20(6):639–644. doi: 10.1111/tbj.12333 CrossRefPubMedGoogle Scholar
  41. 41.
    August DA, Kearney T (2000) Cystosarcoma phyllodes: mastectomy, lumpectomy, or lumpectomy plus irradiation. Surg Oncol 9(2):49–52CrossRefPubMedGoogle Scholar
  42. 42.
    Kondo Y (2014) Targeting histone methyltransferase EZH2 as cancer treatment. J Biochem 156(5):249–257. doi: 10.1093/jb/mvu054 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yanhong Zhang
    • 1
  • Adam L. Liss
    • 2
  • Eugene Chung
    • 2
  • Lori J. Pierce
    • 2
    • 4
  • Celina G. Kleer
    • 3
    • 4
    Email author
  1. 1.Department of PathologyUniversity of California DavisSacramentoUSA
  2. 2.Departments of Radiation OncologyUniversity of MichiganAnn ArborUSA
  3. 3.Department of PathologyUniversity of MichiganAnn ArborUSA
  4. 4.Comprehensive Cancer CenterUniversity of MichiganAnn ArborUSA

Personalised recommendations