Invasive Breast Cancer: Overexpression of HER-2 Determined by Immunohistochemistry and Multiplex Ligation-Dependent Probe Amplification

  • Paul J. van Diest
  • C. B. Moelans
  • D. Purnomosari
  • G. Pals
  • R. A. de Weger
Part of the Methods of Cancer Diagnosis, Therapy and Prognosis book series (HAYAT, volume 1)

The HER-2/neu proto-oncogene located on chromosome 17 encodes a 185-kD trans-membrane tyrosine kinase growth factor receptor belonging to the epidermal growth factor receptor family (Bargmann et al., 1986) which is involved in cell growth and development (Popescu et al., 1989). HER-2 is amplified in 20–30% of breast carcinomas. This amplification of HER-2/neu is now known to confer a poor prognosis (Slamon et al., 1987; Baak et al. 1991) and may also predict a worse response to hormonal therapy (Wright et al., 1992) and standard chemotherapy regimens (Konecny et al., 2004). HER-2/neu proto-oncogene amplification is usually accompanied by over-expression of its protein (Slamon et al., 1989) as determined by immunohistochemistry (IHC).


Breast Cancer Invasive Breast Cancer Spinal Muscular Atrophy Epidermal Growth Factor Receptor Family HER2 Testing 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arkblad, E.L., Darin, N., Berg, K., Kimber, E., Brandberg, G., Lindberg, C., Holmberg, E., Tulinius, M., and Nordling, M. 2006. Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy.Neuro-muscul. Disord. 16: 830–838CrossRefGoogle Scholar
  2. Armour, J.A., Sismani, C., Patsalis, P.C., and Cross, G. 2000. Measurement of locus copy number by hybridisation with amplifiable probes.Nucleic Acids Res. 28: 605–609PubMedCrossRefGoogle Scholar
  3. Baak, P.A., Chin, D., Van Diest, P.J., Matze-Cok, P., and Bacus, S.S. 1991. Comparative long term prognostic value of quantitative Her2/Neu protein expression, DNA ploidy, morphometric and clinical features in paraffin-embedded invasive breast cancer.Lab. Invest. 64: 215–222PubMedGoogle Scholar
  4. Bargmann, C.I., Hung, M.C., and Weinberg, R.A. 1986. The neu oncogene encodes an epidermal growth factor receptor-related protein.Nature 319: 226–230PubMedCrossRefGoogle Scholar
  5. Birner, P., Oberhuber, G., Stani, J., Reithofer, C., Samonigg, H., Hausmaninger, H., Kubista, E., Kwasny, W., Kandioler-Eckersberger, D., Gnant, M., and Jakesz, R.; Austrian Breast&Colorectal Cancer Study Group. 2001. Evaluation of the United States Food and Drug Administration-approved scoring and test system of HER-2 protein expression in breast cancer.Clin. Cancer Res. 7: 1669–1675PubMedGoogle Scholar
  6. Chorn, N. 2006. Accurate identification of HER2-positive patients is essential for superior outcomes with trastuzumab therapy.Oncol. Nurs. Forum,33: 265–272PubMedCrossRefGoogle Scholar
  7. Dikow, N., Nygren, A.O., Schouten, J.P., Hartmann, C., Kramer, N., Janssen, B., and Zschocke, J. 2007. Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.Mol. Cell Probes 21: 208–215PubMedCrossRefGoogle Scholar
  8. Di Leo, A., Dowsett, M., Horten, B., and Penault-Llorca, F. 2002. Current status of HER2 testing.Oncology 63(Suppl 1): 25–32PubMedCrossRefGoogle Scholar
  9. Durbecq, V. , Paesmans, M., Cardoso, F., Desmedt, C., Di Leo, A., Chan, S., Friedrichs, K., Pinter, T., Van Belle, S., Murray, E., Bodrogi, I., Walpole, E., Lesperance, B., Korec, S., Crown, J., Simmonds, P., Perren, T.J., Leroy, J.Y., Rouas, G., Sotiriou, C., Piccart, M., and Larsimont D. 2004. Topoisomerase-II alpha expression as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel.Mol. Cancer Ther. 3: 1207–1214PubMedGoogle Scholar
  10. Eldering, E., Spek, C.A., Aberson, H.L., Grummels, A., Derks, I.A., De Vos, A.F., McElgunn, C.J., and Schouten, J.P. 2003. Expression profiling via novel multiplex assay allows rapid assessment of gene regulation in defined signalling pathways.Nucleic Acids Res. 31: e153PubMedCrossRefGoogle Scholar
  11. Ginestier, C., Charafe-Jauffret, E., Penault-Llorca, F., Geneix, J., Adelaide, J., Chaffanet, M., Mozziconacci, M.J., Hassoun, J., Viens, P. , Birnbaum, D., and Jacquemier, J. 2004. Comparative multi-methodological measurement of ERBB2 status in breast cancer.J. Pathol. 202: 286–298PubMedCrossRefGoogle Scholar
  12. Gong, Y., Gilcrease, M., and Sneige, N. 2005. Reliability of chromogenic in situ hybridization for detecting HER-2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility.Mod. Pathol. 18 : 1015–1021PubMedCrossRefGoogle Scholar
  13. Hanna, W. 2001. Testing for HER2 status.Oncology 61(Suppl 2): 22–30PubMedCrossRefGoogle Scholar
  14. Hanna, W.M., and Kwok, K. 2006. Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm.Mod. Pathol.19 :481–487PubMedCrossRefGoogle Scholar
  15. Hess, C.J., Denkers, F., Ossenkoppele, G.J., Waisfisz, Q., McElgunn, C.J., Eldering, E., Schouten, J.P., and Schuurhuis, G.J. 2004. Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.Leukemia 18: 1981–1988PubMedCrossRefGoogle Scholar
  16. Hofmann, M., Stoss, O., Gaiser, T., Kneitz, H., Heinmoller, P., Gutjahr, T., Kaufmann, M., Henkel, T., and Ruschoff, J. 2008. Central HER2 IHC and FISH analysis in a trastuzumab (Herceptin® ) phase II monotherapy study: assessment of test sensitivity and impact of chromosome 17 poly-somy.J. Clin. Pathol.61: 89–94PubMedCrossRefGoogle Scholar
  17. Jacobs, T.W., Gown, A.M., Yaziji, H., Barnes, M.J., and Schnitt, S.J. 1999. Comparison of fluorescence in situ hybridization and immuno-histochemistry for the evaluation of HER-2/neu in breast cancer.J. Clin. Oncol. 17: 1974–1982PubMedGoogle Scholar
  18. Knoop, A.S., Knudsen, H., Balslev, E., Rasmussen, B.B., Overgaard, J., Nielsen, K.V., Schonau, A., Gunnarsdottir, K., Olsen, K.E., Mouridsen, H., and Ejlertsen B.; Danish Breast Cancer Cooperative Group. 2005. Retrospective analysis of topoisomer-ase IIa amplifications and deletions as predictive markers in primary breast cancer patients randomly assigned to cyclophosphamide, methotrexate, and fluorouracil or cyclophosphamide, epirubicin, and fluorouracil: Danish Breast Cancer Cooperative Group.J. Clin. Oncol. 23: 7483–7490PubMedCrossRefGoogle Scholar
  19. Konecny, G.E., Thomssen, C., Luck, H.J., Untch, M., Wang, H.J., Kuhn, W., Eidtmann, H., du Bois, A., Olbricht, S., Steinfeld, D., Mobus, V. , von Minckwitz, G., Dandekar, S., Ramos, L., Pauletti, G., Pegram, M.D., Janicke, F., and Slamon, D.J. 2004. Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer.J. Natl. Cancer Inst. 96: 1141–1151PubMedCrossRefGoogle Scholar
  20. Lebeau, A., Deimling, D., Kaltz, C., Sendelhofert, A., Iff, A., Luthardt, B., Untch, M., and Lohrs, U. 2001. Her-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization.J. Clin. Oncol. 19: 354–363PubMedGoogle Scholar
  21. Mass, R.D., Press, M.F., Anderson, S., Murphy, M., and Slamon, D. 2001. Improved survival benefit from Herceptin (trastuzumab) in patients selected by fluorescence in situ hybridization (FISH).Proc. Am. Soc. Clin. Oncol. 20: 85Google Scholar
  22. Mass, R.D., Press, M.F., Anderson, S., Cobleigh, M.A., Vogel, C.L., Dybdal, N., Leiberman, G., and Slamon, D.J. 2005. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzu-mab.Clin. Breast Cancer 6: 240–246PubMedCrossRefGoogle Scholar
  23. Moelans, C., De Weger, R.A., Van Blokland, M., Ezendam, C., Griesdoorn, V., Elshof, S.M., Rozemuller, E., Tilanus, M., and Van Diest, P.J. 2007. HER-2/neu amplification testing in breast cancer by multiplex ligation-dependent probe amplification in comparison with immunohisto-chemistry and in situ hybridization.Cell. Oncol. in pressGoogle Scholar
  24. Moerland, E., Van Hezik, R.L.H.P.M., Van der Aa, T.C.J.M., Van Beek, M.W.P.M., and Van den Brule, A.J.C. 2006. Detection of HER2 amplification in breast carcinomas: comparison of Multiplex Ligation-dependent Probe Amplification (MLPA) and Fluorescence In Situ Hybridization (FISH) combined with automated spot counting.Cell. Oncol. 28: 151–159PubMedGoogle Scholar
  25. Nygren, A.O., Ameziane, N., Duarte, H.M., Vijzelaar, R.N., Waisfisz, Q., Hess, C.J., Schouten, J.P., and Errami, A. 2005. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences.Nucleic Acids Res. 33: e128PubMedCrossRefGoogle Scholar
  26. Pauletti, G., Dandekar, S., Rong, H., Ramos, L., Peng, H., Seshadri, R., and Slamon, D.J. 2000. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry.J. Clin. Oncol. 18: 3651–3664PubMedGoogle Scholar
  27. Penault-Llorca, F., Adelaide, J., Houvenaeghel, G., Hassoun, J., Birnbaum, D., and Jacquemier, J. 1994. Optimization of immunohistochemical detection of ERBB2 in human breast cancer: impact of fixation.J. Pathol. 173: 65–75PubMedCrossRefGoogle Scholar
  28. Perez, E.A., Roche, P.C., Jenkins, R.B., Reynolds, C.A., Halling, K.C., Ingle, J.N., and Wold, L.E. 2002. HER2 testing in patients with breast cancer: poor correlation between weak positivity by immunohistochemistry and gene amplification by fluorescence in situ hybridization.Mayo Clin. Proc. 77: 148–154PubMedCrossRefGoogle Scholar
  29. Popescu, N.C., King, C.R., and Kraus, M.H. 1989. Localization of the human erbB-2 gene on normal and rearranged chromosomes 17 to bands q12-21.32.Genomics 4: 362–366PubMedCrossRefGoogle Scholar
  30. Press, M.F., Slamon, D.J., Flom, K.J., Park, J., Zhou, J.Y., and Bernstein, L. 2002. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens.J. Clin. Oncol. 20: 3095–3105PubMedGoogle Scholar
  31. Procter, M., Chou, L.S., Tang, W., Jama, M., and Mao, R. 2006. Molecular diagnosis of Prader-Willi and Angelman syndromes by methylation-specific melting analysis and methylation-specific multiplex ligation-dependent probe amplification.Clin. Chem. 52: 1276–1283PubMedCrossRefGoogle Scholar
  32. Purnomosari, D., Aryandono, T., Setiaji, K., Nugraha, S.B., Pals, G., and Va n Diest, P.J. 2006. Comparison of multiplex ligation probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer.Biotechnic&Histochemistry.81(2–3) : 79–85Google Scholar
  33. Raju, U., Lu, M., Sethi, S., Qureshi, H., Wolman, S.R., and Worsham, M.J. 2006. Molecular classification of breast carcinoma in situ.Curr. Genomics 7: 523–532PubMedCrossRefGoogle Scholar
  34. Rhodes, A., Jasani, B., Anderson, E., Dodson, A.R., and Balaton, A.J. 2002a. Evaluation of HER-2/ neu immunohistochemical assay sensitivity and scoring on formalin-fixed and paraffin-processed cell lines and breast tumors: a comparative study involving results from laboratories in 21 countries.Am. J. Clin. Pathol. 118: 408–417CrossRefGoogle Scholar
  35. Rhodes, A., Jasani, B., Couturier, J., McKinley, M.J., Morgan, J.M., Dodson, A.R., Navabi, H., Miller, K.D., and Balaton, A.J. 2002b. A formalin-fixed, paraffin-processed cell line standard for quality control of immunohistochemical assay of HER-2/neu expression in breast cancer.Am. J. Clin. Pathol. 117: 81–89CrossRefGoogle Scholar
  36. Ridolfi, R.L., Jamehdor, M.R., and Arber, J.M. 2000. HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.Mod. Pathol. 13: 866–873PubMedCrossRefGoogle Scholar
  37. Roche, P.C., and Ingle, J.N. 1999. Increased HER2 with U.S. Food and Drug Administration-approved antibody.J. Clin. Oncol. 17: 434PubMedGoogle Scholar
  38. Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwijnenburg, D., Diepvens, F., and Pals, G. 2002. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 30: e57PubMedCrossRefGoogle Scholar
  39. Sellner, L.N., and Taylor, G.R. 2004. MLPA and MAPH: new techniques for detection of gene deletions.Hum. Mutat. 23: 413–419PubMedCrossRefGoogle Scholar
  40. Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A., and McGuire, W.L. 1987. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science 235: 177–182PubMedCrossRefGoogle Scholar
  41. Slamon, D.J., Godolphin, W., Jones, L.A., Holt, J.A., Wong, S.G., Keith, D.E., Levin, W.J., Stuart, S.G., Udove, J., and Ullrich, A. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.Science 244: 707–712PubMedCrossRefGoogle Scholar
  42. Sparano, J.A. 2001. Cardiac toxicity of trastuzumab (Herceptin): implications for the design of adjuvant trials.Semin Oncol 28(1 Suppl 3): 20–27PubMedCrossRefGoogle Scholar
  43. Tanner, M., Gancberg, D., Di, L.A., Larsimont, D., Rouas, G., Piccart, M.J., and Isola, J. 2000. Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples.Am. J. Pathol. 157: 1467–1472PubMedGoogle Scholar
  44. Tanner, M., Isola, J., Wiklund, T., Erikstein, B., Kellokumpu-Lehtinen, P., Malmstrom, P., Wilking, N., Nilsson, J., and Bergh, J. 2006. Topoisomerase IIalpha gene amplification predicts favorable treatment response to tailored and dose-escalated anthracycline-based adjuvant chemotherapy in HER-2/neu-amplified breast cancer: Scandinavian Breast Group Trial 9401.J. Clin. Oncol. 24: 2428–2436PubMedCrossRefGoogle Scholar
  45. Tubbs, R.R., Pettay, J.D., Roche, P.C., Stoler, M.H., Jenkins, R.B., and Grogan, T.M. 2001. Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.J. Clin. Oncol. 19: 2714–2721PubMedGoogle Scholar
  46. Van de Vijver, M.J., Peterse, J.L., Mooi, W.J., Wisman, P., Lomans, J., Dalesio, O., and Nusse, R. 1988. Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer.N. Engl. J. Med. 319: 1239–1245. Vorstman, J.A., Jalali, G.R., Rappaport, E.F., Hacker, A.M., Scott, C., and Emanuel, B.S. 2006. MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q.Hum. Mutat. 27: 814–821PubMedGoogle Scholar
  47. Vorstman, J.A., Jalali, G.R., Rappaport, E.F., Hacker, A.M., Scott, C., and Emanuel, B.S. 2006. MLPA: a rapid, reliable, and sensitive method for detection and analysis of abnormalities of 22q.Hum. Mutat. 27: 814–821PubMedCrossRefGoogle Scholar
  48. Wright, C., Nicholson, S., Angus, B., Sainsbury, J.R., Farndon, J., Cairns, J., Harris, A.L., and Horne, C.H. 1992. Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer.Br. J. Cancer 65: 118–121PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media B.V. 2008

Authors and Affiliations

  • Paul J. van Diest
    • 1
  • C. B. Moelans
    • 1
  • D. Purnomosari
    • 2
  • G. Pals
    • 3
  • R. A. de Weger
    • 1
  1. 1.Department of PathologyUniversity Medical Center UtrechtGA UtrechtThe Netherlands
  2. 2.Department of Histology and Cell Biology Faculty of MedicineGadjah Mada UniversityJogjakartaIndonesia
  3. 3.Department of Clinical GeneticsVU University Medical CenterAmsterdamThe Netherlands

Personalised recommendations