Skip to main content
Log in

Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR — A comparison with immunohistochemical and FISH results

  • Article
  • Published:
Pathology & Oncology Research

Abstract

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH-group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Allred DC, Clark GM, Molina R, et al: HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 10: 599–605, 1992

    PubMed  CAS  Google Scholar 

  2. Bánkfalvi A, Boecker W, Reiner A: Comparison of automated and manual determination of HER2 status in breast cancer for diagnostic use: a comparative methodological study using Ventana BenchMark automated staining system and manual tests. Int J Oncol 25: 929–935, 2004

    PubMed  Google Scholar 

  3. Bánkfalvi A, Giuffre G, Ofner D, et al: Relationship between HER2 status and proliferation rate in breast cancer assessed by immunohistochemistry, fluorescence in situ hybridisation and standardised AgNOR analysis. Int J Oncol 23: 1285–1292, 2003

    PubMed  Google Scholar 

  4. Bánkfalvi Á: HER-2 diagnostics. Magy Onkol 46: 11–15, 2002 [In Hungarian]

    PubMed  Google Scholar 

  5. Bargmann CI, Hung MC, Weinberg RA: The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319: 226–230, 1986

    Article  PubMed  CAS  Google Scholar 

  6. Bartlett JM, Going JJ, Mallon EA, et al: Evaluating HER2 amplification and overexpression in breast cancer. J Pathol 195: 422–428, 2001

    Article  PubMed  CAS  Google Scholar 

  7. Beyser K, Reiser A, Gross C, et al: J. Real-time quantification of HER2/neu gene amplification by LightCycler Polymerase Chain Reaction (PCR) — a new research tool. Biochemica 2: 15–18, 2001

    Google Scholar 

  8. Beyser K, Reiser A, Gross C, et al: PCR is a powerful tool to assess HER2/neu positivity in breast cancer. Eur J Cancer 158: 419–429, 2001

    Google Scholar 

  9. Bilous M, Ades C, Armes J, et al: Predicting the HER2 status of breast cancer from basic histopathology data: an analysis of 1500 breast cancers as part of the HER2000 international study. Breast 12: 92–98, 2003

    Article  PubMed  CAS  Google Scholar 

  10. DiGiovanna MP: Clinical significance of HER-2/neu overexpression: Part I. Principles and Practice of Oncology 13: 1–10, 1999

    Google Scholar 

  11. Elkin EB, Weinstein MC, Winer EP, et al: HER-2 testing and trastuzumab therapy for metastatic breast cancer: a cost-effectiveness analysis. J Clin Oncol 22: 854–863, 2004

    Article  PubMed  Google Scholar 

  12. Elston CW, Ellis IO: Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long term follow up. Histopathology 19: 403–410, 1991

    Article  PubMed  CAS  Google Scholar 

  13. Esteva FJ, Sahin AA, Smith TL, et al: Prognostic significance of phosphorylated P38 mitogen-activated protein kinase and HER-2 expression in lymph node-positive breast carcinoma. Cancer 100: 499–506, 2004

    Article  PubMed  CAS  Google Scholar 

  14. Gjerdrum LM, Sorensen BS, Kjeldsen E, et al: Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma. J Mol Diagn 6: 42–51, 2004

    PubMed  CAS  Google Scholar 

  15. Gusterson BA, Machin LG, Gullick WJ, et al: Immunohisto-chemical distribution of c-erbB-2 in infiltrating and in situ breast cancer. Int J Cancer 42: 842–845, 1988

    PubMed  CAS  Google Scholar 

  16. Hammock L, Lewis M, Phillips C, et al: Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization. Hum Pathol 34: 1043–1047, 2003

    Article  PubMed  CAS  Google Scholar 

  17. Harbeck N, Ross JS, Yurdseven S, et al: HER-2/neu gene amplification by fluorescence in situ hybridization allows risk-group assessment in node negative breast cancer. Int J Oncol 14: 663–671, 1999

    PubMed  CAS  Google Scholar 

  18. Jacobs TW, Gown AM, Yaziji H, et al: HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry; a study of interlaboratory agreement. Am J Clin Pathol 113: 251–258, 2000

    Article  PubMed  CAS  Google Scholar 

  19. Jimenez RE, Wallis T, Tabasczka P, Visscher DW Determination of Her-2/Neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol 13: 37–45, 2000

    Article  PubMed  CAS  Google Scholar 

  20. Kakar S, Puangsuvan N, Stevens JM, et al: HER-2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: comparison of results and correlation with survival. Mol Diagn 5: 199–207, 2000

    PubMed  CAS  Google Scholar 

  21. Kauraniemi P, Hautaniemi S, Autio R, et al: Effects of Herceptin treatment on global gene expression patterns in HER2-amplified and nonamplified breast cancer cell lines. Oncogene 23: 1010–1013, 2004

    Article  PubMed  CAS  Google Scholar 

  22. Lebeau A, Deimling D, Kaltz C, et al: Her-2/neu analysis in archival tissue samples of human breast cancer: Comparison of immunohistochemistry and in situ hybridization. J Clin Oncol 19: 354–363, 2001

    PubMed  CAS  Google Scholar 

  23. Lopez-Guerrero JA, Navarro S, Noguera R, et al: Histological tumor grade correlates with HER2/c-erB-2 status in invasive breast cancer: a comparative analysis between immunohistochemical (CB11 clone and Herceptest), FISH and differential PCR procedures. Arkh Patol 65: 50–55, 2003

    PubMed  CAS  Google Scholar 

  24. Lu Y, Zi X, Zhao Y, Pollak M: Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells. Biochem Biophys Res Commun 313: 709–715, 2004

    Article  PubMed  CAS  Google Scholar 

  25. Luftner D, Henschke P, Kafka A, et al: Discordant results obtained for different methods of HER-2/neu testing in breast cancer — a question of standardization, automation and timing. Int J Biol Markers 19: 1–13, 2004

    PubMed  CAS  Google Scholar 

  26. Ma Y, Lespagnard L, Durbecq V, et al: Polysomy 17 in HER-2/neu status elaboration in breast cancer: effect on daily practice. Clin Cancer Res 11: 4393–4399, 2005

    Article  PubMed  CAS  Google Scholar 

  27. Nistor A, Watson PH, Pettigrew N, et al: Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer. BMC Clin Pathol 6:2 doi:10.1186/1472-6890-6-2, 2006

  28. O’Malley FP, Parkes R, Latta E, et al: Comparison of HER2/neu status assessed by quantitative polymerase chain reaction and immunohistochemistry. Am J Clin Pathol 115: 504–511, 2001

    Article  PubMed  CAS  Google Scholar 

  29. Pavelic ZP, Pavelic L, Lower EE, et al: C-myc, c-erbB-2, and Ki-67 expression in normal breast tissue and in invasive and noninvasive breast carcinoma. Cancer Res 52: 2597–2606, 1992

    PubMed  CAS  Google Scholar 

  30. Perez EA, Roche PC, Jenkins RB, et al: 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: 148–154, 2002

    Article  PubMed  Google Scholar 

  31. Press MF, Slamon DJ, Florn KJ, et al: 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: 3095–3105, 2002

    PubMed  CAS  Google Scholar 

  32. Riben MW, Malfetano JH, Nazeer T, et al: Identification of HER-2/neu oncogene amplification by fluorescence in situ hybridization in stage I endometrial carcinoma. Mod Pathol 10: 823–831, 1997

    PubMed  CAS  Google Scholar 

  33. Ridolfl RL, Jamehdor MR, Arber JM: HER-2/neu testing in breast carcinoma: A combined immunohistochemical and fluorescence in situ hybridization approach. Mod Pathol 13: 866–873, 2000

    Article  Google Scholar 

  34. Ross JS, Fletcher JA: The HER2/neu (c-erbB-2) gene and protein in breast cancer. Am J Clin Pathol 112 (Suppl 1): 553–567, 1999

    Google Scholar 

  35. Seshadri R, Figaira FA, Horsfall DJ, et al: Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group. J Clin Oncol 11: 1936–1942, 1993

    PubMed  CAS  Google Scholar 

  36. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177–182, 1987

    Article  PubMed  CAS  Google Scholar 

  37. Slamon DJ, Godolphin W, Jones LA, et al: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707–712, 1989

    Article  PubMed  CAS  Google Scholar 

  38. Somerville JE, Clarke LA, Biggart JD: C-erbB-2 overexpression and histological type of in situ and invasive breast carcinoma. J Clin Pathol 45: 16–20, 1992

    Article  PubMed  CAS  Google Scholar 

  39. Suo Z, Daehli KU, Lindboe CF, et al: Real-time PCR quantification of c-erbB-2 gene is an alternative for FISH in the clinical management of breast carcinoma patients. Int J Surg Pathol 12: 311–318, 2004

    Article  PubMed  CAS  Google Scholar 

  40. Taylor SL, Platt-Higgins A, Rudland PS, et al: Cytoplasmic staining of c-erbB-2 is not associated with the presence of detectable c-erbB-2 mRNA in breast cancer specimens. Int J Cancer 76: 459–463, 1998

    Article  PubMed  CAS  Google Scholar 

  41. Tse C, Brault D, Gligorov J, et al: [abstract]. HER-2 gene copy number quantified by real-time quantitative PCR (qPCR) and serum HER-2 concentration by enzyme-linked immunosorbent assay (ELISA): comparison with fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). 4th European Breast Cancer Conference, Hamburg, 395, 2004

  42. Van de Vijver M, Peterse JL, Mooi WJ, et al: 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, 1988

    PubMed  Google Scholar 

  43. Woods Ignatoski KM, Grewal NK, Markwart S, et al: p38MAPK induces cell surface alpha4 integrin downregulation to facilitate erbB-2-mediated invasion. Neoplasia 5:128–134, 2003

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Janina Kulka.

Additional information

Both authors have contributed equally to this work. This work was supported by the following grants: Bio-00014/2001, NKFP-1A/0023/2002, FKFP-0129/2001, in part by EAGC-AVON 2004.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kulka, J., Tokés, AM., Kaposi-Novák, P. et al. Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR — A comparison with immunohistochemical and FISH results. Pathol. Oncol. Res. 12, 197–204 (2006). https://doi.org/10.1007/BF02893412

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02893412

Key words

Navigation