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Tumor Biology

, Volume 36, Issue 11, pp 9083–9091 | Cite as

Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

  • G. P. Burrai
  • A. Tanca
  • M. R. De Miglio
  • M. Abbondio
  • S. Pisanu
  • M. Polinas
  • S. Pirino
  • S. I. Mohammed
  • S. Uzzau
  • M. F. Addis
  • E. Antuofermo
Research Article

Abstract

Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22 % of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer.

Keywords

Antibody specificity Canine mammary tumors HER2 IHC Proteomics RT-PCR 

Notes

Acknowledgments

The authors gratefully acknowledge Dr. Tiziana Cubeddu for help in the interpretation of results.

This work was supported by Fondazione Banco di Sardegna 2010 and by PhD fellowship to G.P.B (P.O.R. Sardegna F.S.E. 2007–2013) - Obiettivo competitività regionale e occupazione, Asse IV Capitale umano, Linea di Attività l.3.1.

References

  1. 1.
    Sorenmo KU, Rasotto R, Zappulli V, Goldschmidt MH. Development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms. Vet Pathol. 2011;48(1):85–97. doi: 10.1177/0300985810389480.
  2. 2.
    Rivera P, von Euler H. Molecular biological aspects on canine and human mammary tumors. Vet Pathol. 2011;48:132–46.CrossRefPubMedGoogle Scholar
  3. 3.
    Klopfleisch R, Gruber AD. Transcriptome and proteome research in veterinary science: what is possible and what questions can be asked? Sci World J. 2012. doi: 10.1100/2012/254962.
  4. 4.
    Pinho SS, Carvalho S, Cabral J, Reis CA, Gartner F. Canine tumors: a spontaneous animal model of human carcinogenesis. Transl Res. 2012;159(3):165–72.CrossRefPubMedGoogle Scholar
  5. 5.
    Weigel MT, Dowsett M. Current and emerging biomarkers in breast cancer: prognosis and prediction. Endocrinol Relat Cancer. 2010;17(4):R245–62.CrossRefGoogle Scholar
  6. 6.
    Yamauchi H, Stearns V, Hayes DF. When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer. J Clin Oncol. 2001;19(8):2334–56.CrossRefPubMedGoogle Scholar
  7. 7.
    Popescu NC, King CR, Kraus MH. Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32. Genomics. 1989;4(3):362–6.CrossRefPubMedGoogle Scholar
  8. 8.
    Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T. The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science. 1986;232(4758):1644–6.CrossRefPubMedGoogle Scholar
  9. 9.
    Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177–82.CrossRefPubMedGoogle Scholar
  10. 10.
    Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist. 2009;14(4):320–68.CrossRefPubMedGoogle Scholar
  11. 11.
    Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM, Allison KH, et al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. J Clin Oncol. 2013;31(31):3997–4013.CrossRefPubMedGoogle Scholar
  12. 12.
    Rungsipipat A, Tateyama S, Yamaguchi R, Uchida K, Miyoshi N, Hayashi T. Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors. J Vet Med Sci. 1999;61(1):27–32.CrossRefPubMedGoogle Scholar
  13. 13.
    Martin de las Mulas J, Ordas J, Millan Y, Fernandez-Soria V, Ramon y Cajal S. Oncogene HER-2 in canine mammary gland carcinomas: an immunohistochemical and chromogenic in situ hybridization study. Breast Cancer Res Treat. 2003;80(3):363–7.CrossRefPubMedGoogle Scholar
  14. 14.
    Dutra AP, Granja NV, Schmitt FC, Cassali GD. c-erbB-2 expression and nuclear pleomorphism in canine mammary tumors. Braz J Med Biol Res. 2004;37(11):1673–81.CrossRefPubMedGoogle Scholar
  15. 15.
    Hsu WL, Huang HM, Liao JW, Wong ML, Chang SC. Increased survival in dogs with malignant mammary tumours overexpressing HER-2 protein and detection of a silent single nucleotide polymorphism in the canine HER-2 gene. Vet J. 2009;180(1):116–23.CrossRefPubMedGoogle Scholar
  16. 16.
    Muhammadnejad A, Keyhani E, Mortazavi P, Behjati F, Haghdoost IS. Overexpression of her-2/neu in malignant mammary tumors; translation of clinicopathological features from dog to human. Asian Pac J Cancer Prev. 2012;13(12):6415–21.CrossRefPubMedGoogle Scholar
  17. 17.
    Singer J, Weichselbaumer M, Stockner T, Mechtcheriakova D, Sobanov Y, Bajna E, et al. Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting. Mol Immunol. 2012;50(4):200–9.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ressel L, Puleio R, Loria GR, Vannozzi I, Millanta F, Caracappa S, et al. HER-2 expression in canine morphologically normal, hyperplastic and neoplastic mammary tissues and its correlation with the clinical outcome. Res Vet Sci. 2013;94(2):299–305.CrossRefPubMedGoogle Scholar
  19. 19.
    Murua Escobar H, Becker K, Bullerdiek J, Nolte I. The canine ERBB2 gene maps to a chromosome region frequently affected by aberrations in tumors of the dog (Canis familiaris). Cytogenet Cell Genet. 2001;94(3–4):194–5.PubMedGoogle Scholar
  20. 20.
    Andelfinger G, Hitte C, Etter L, Guyon R, Bourque G, Tesler G, et al. Detailed four-way comparative mapping and gene order analysis of the canine ctvm locus reveals evolutionary chromosome rearrangements. Genomics. 2004;83(6):1053–62.CrossRefPubMedGoogle Scholar
  21. 21.
    Kim JH, Im KS, Kim NH, Yhee JY, Nho WG, Sur JH. Expression of HER-2 and nuclear localization of HER-3 protein in canine mammary tumors: histopathological and immunohistochemical study. Vet J. 2011;189(3):318–22.CrossRefPubMedGoogle Scholar
  22. 22.
    Pena L, Gama A, Goldschmidt MH, Abadie J, Benazzi C, Castagnaro M, et al. Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry. Vet Pathol. 2014;51(1):127–45.CrossRefPubMedGoogle Scholar
  23. 23.
    Misdorp W. Tumors of the mammary gland. In: Meuten DJ, editor. Tumors in domestic animals. Iowa: Blackwell; 2002. p. 575–606.Google Scholar
  24. 24.
    Antuofermo E, Miller MA, Pirino S, Xie J, Badve S, Mohammed SI. Spontaneous mammary intraepithelial lesions in dogs—a model of breast cancer. Cancer Epidemiol Biomarkers Prev. 2007;16(11):2247–56.CrossRefPubMedGoogle Scholar
  25. 25.
    Mouser P, Miller MA, Antuofermo E, Badve SS, Mohammed SI. Prevalence and classification of spontaneous mammary intraepithelial lesions in dogs without clinical mammary disease. Vet Pathol. 2010;47(2):275–84.CrossRefPubMedGoogle Scholar
  26. 26.
    Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method. Methods. 2001;25(4):402–8.CrossRefPubMedGoogle Scholar
  27. 27.
    Brinkhof B, Spee B, Rothuizen J, Penning LC. Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem. 2006;356(1):36–43.CrossRefPubMedGoogle Scholar
  28. 28.
    Etschmann B, Wilcken B, Stoevesand K, von der Schulenburg A, Sterner-Kock A. Selection of reference genes for quantitative real-time PCR analysis in canine mammary tumors using the GeNorm algorithm. Vet Pathol. 2006;43(6):934–42.CrossRefPubMedGoogle Scholar
  29. 29.
    Burrai GP, Mohammed SI, Miller MA, Marras V, Pirino S, Addis MF, et al. Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor- and progesterone receptor-negative breast lesions. BMC Cancer. 2010;10:156.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Tanca A, Addis MF, Pagnozzi D, Cossu-Rocca P, Tonelli R, Falchi G, et al. Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives. J Proteome. 2011;74(3):359–70.CrossRefGoogle Scholar
  31. 31.
    Tanca A, Biosa G, Pagnozzi D, Addis MF, Uzzau S. Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome. Proteomics. 2013;13(17):2597–607.CrossRefPubMedGoogle Scholar
  32. 32.
    Zhang EY, Cristofanilli M, Robertson F, Reuben JM, Mu ZM, Beavis RC, et al. Genome wide proteomics of ERBB2 and EGFR and other oncogenic pathways in inflammatory breast cancer. J Proteome Res. 2013;12(6):2805–17.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Matsuyama S, Nakamura M, Yonezawa K, Shimada T, Ohashi F, Takamori Y, et al. Expression patterns of the erbB subfamily mRNA in canine benign and malignant mammary tumors. J Vet Med Sci. 2001;63(9):949–54.CrossRefPubMedGoogle Scholar
  34. 34.
    Nistor A, Watson PH, Pettigrew N, Tabiti K, Dawson A, Myal Y. Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer. BMC Clin Pathol. 2006;6:2.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Klopfleisch R, Klose P, Weise C, Bondzio A, Multhaup G, Einspanier R, et al. Proteome of metastatic canine mammary carcinomas: similarities to and differences from human breast cancer. J Proteome Res. 2010;9(12):6380–91.CrossRefPubMedGoogle Scholar
  36. 36.
    Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet. 2012;13(4):227–32.PubMedPubMedCentralGoogle Scholar
  37. 37.
    HercepTest™ Test Interpretation Manual. Available from: URL: http://www.dako.com.
  38. 38.
    Rasotto R, Caliari D, Castagnaro M, Zanetti R, Zappulli V. An immunohistochemical study of HER-2 expression in feline mammary tumours. J Comp Pathol. 2011;144(2–3):170–9.Google Scholar
  39. 39.
    Schrohl AS, Pedersen HC, Jensen SS, Nielsen SL, Brunner N. Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies. Histopathology. 2011;59(5):975–83.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • G. P. Burrai
    • 1
  • A. Tanca
    • 2
  • M. R. De Miglio
    • 3
  • M. Abbondio
    • 2
  • S. Pisanu
    • 2
  • M. Polinas
    • 1
  • S. Pirino
    • 1
  • S. I. Mohammed
    • 4
  • S. Uzzau
    • 2
  • M. F. Addis
    • 2
  • E. Antuofermo
    • 1
  1. 1.Dipartimento di Medicina VeterinariaUniversità degli Studi di SassariSassariItaly
  2. 2.Porto Conte Ricerche SrlAlgheroItaly
  3. 3.Dipartimento di Medicina Clinica e SperimentaleUniversità degli Studi di SassariSassariItaly
  4. 4.Department of Comparative Pathobiology and Purdue University Centre for Cancer ResearchPurdue UniversityWest LafayetteUSA

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