—  SYMPOSIUM #39  —

Molecular Targeted Therapy for Cancer
Moderators: Dr. Robert Yoshiyuki Osamura and Dr. Allen M. Gown

Section 4 - Her2/neu Testing in Breast Cancer

Wedad Hanna
Chief, Anatomic Pathology
Sunnybrook Health Sciences Centre
Professor, Department of Laboratory Medicine and Pathobiology
University of Toronto


This presentation will cover the current status regarding Her2/neu testing in breast cancer and highlight outstanding issues and future research opportunities.

The Her2/neu gene is one of four members in the Her family which encodes a 185 kilodalton glycoprotein with tyrosine kinase activity. To date the ligand for activation of the receptor has not been identified. Activation of Her2 is initiated by the formation of homodimers and heterodimers with other members of the Her family with known ligands. This leads to a cascade of events that result in tumor growth, cell motility and inhibition of apoptosis [1]. Amplification of the Her2/neu gene and over-expression of the protein occurs in about 20% of patients with breast cancer [2, 3]. Over-expression occurs as a result of amplification of the gene in 96.5% of cases.

Her2/neu as a Predictive and Prognostic Marker
Her2/neu over-expression and/or amplification have been established as a poor prognostic marker in both node positive and node negative disease [4, 5, 6]. To date testing at diagnosis is the standard of practice as numerous studies have shown the importance of Her2/neu status in managing patients with breast cancer. St. Gallen's guidelines in 2005 [7] included the Her2 status of the tumor as a factor in the risk classification of patients and subsequently in the choice of treatments. Her2/neu over-expression is a predictor of response to therapy since clinical studies have shown that these patients will benefit from anthracycline-based chemotherapy, possibly due to concomitant TopoII amplification [8, 9]. Her2 over-expression may also identify patients who may not benefit from Tamoxifen [10]. More significantly is the use of the Her2/neu oncoprotein as a target for therapy. Trastuzumab (Herceptin) is a humanized monoclonal antibody that targets the extracellular domain of the receptor.

Herceptin has shown efficacy in metastatic breast cancer in combination with chemotherapy [11] and as a single agent [12]. In 2005, the results of the HERA trial had shown improvement in disease free survival and overall survival by the addition of Herceptin following chemotherapy in the adjuvant setting [13]. Studies demonstrated that Herceptin is only active against Her2 over-expressing tumors.

Testing Methodologies
Review of testing methodologies and algorithm in clinical practice [14] showed that the most commonly used methods are immunohistochemistry (IHC) to detect over-expression of the protein and in situ hybridization (ISH) using either FISH or CISH to assess the presence of gene amplification. Both methods have the advantage of being morphologically based using formalin fixed paraffin embedded tumor samples. Because of the availability of IHC in most surgical pathology laboratories and lower cost, IHC is commonly used to identify Her2 positive and negative tumors and equivocal results are retested using ISH. This algorithm assumes excellent concordance between IHC and ISH test results. However, in clinical practice there are studies that showed interlaboratory discrepancies which might be more prevalent in low volume labs [15, 16]. Strict adherence to standardized methods, rigorous quality controls and participation in external QA initiatives can produce significant improvement and a high level of correlation between the IHC and ISH data [17, 18]. Available data does not demonstrate which method, protein expression or in-situ hybridization methods, is superior in predicting therapeutic response if the tumor's Her2/neu positive status is correctly made by either method. Thus, both pathologists and oncologists are strongly advocating the use of standardized testing methodology and participation in QA programs to properly identify the Her2/neu status of a patient's tumor. The aim is to significantly reduce both false positive and negative results to <5%, i.e. achieve 95% concordance rates between IHC and ISH test results.

The problem pathologists and oncologists are facing today in securing accurate and standardized Her2/neu testing in breast cancer, is related to the following biological and methodology-related issues:
  1. Her2/neu oncoprotein over-expression and amplification of the gene is a continuous variable and not a dichotomous status of positive and negative.

    The cutoff points clinically used today are based on the clinical trial assay (CTA) data which arbitrarily selected them [19]. In situ hybridization (ISH) methods are usually used in larger labs by expert pathologists and although more complex and costly, appear to yield more consistent results.

  2. Intermediate ranges of over-expression of the Her2/neu protein may be seen in about 10-15% of tumors and only one third of these are associated with amplification, mostly low level of amplification of the gene. Borderline cases or low level amplification (1.8 – 2.2 scores) can also be the source of discrepant results. However, these cases constitute about 3% of tumors.

  3. Polysomy of chromosome 17 may play a role in discrepant cases [20]

  4. Tumor heterogeneity and focal amplification of the Her2/neu gene can be seen in 1-2% of cases and may contribute to discrepancy between IHC and ISH and interlaboratory results [21].

  5. Results of IHC testing could be adversely affected by pre-analytical variables such as fixation. Also the subjective nature in interpretation of IHC, basically a qualitative rather than a quantitative test, can result in interobserver variability.

  6. Lack of stringent use of standardized methodologies and non-compulsory participation in external QA programs by testing labs.

Review of the Testing Algorithm and Pitfalls of IHC and ISH

The following points will be detailed in the presentation:
  1. Testing algorithms - definition of positive, negative and borderline cases

  2. How to achieve accurate results using immunohistochemical methods
    • fixation (fixation type and duration) [22], antigen retrieval, antibodies, controls,

    • interpretation and scoring criteria [23]

    • surrogate indicators for reflex testing with ISH
  3. Interpretation of ISH results
    • FISH [24]

    • CISH [25]

    • Validation of CISH and FISH [26, 27]
  4. Quality assurance program
    • models of QA programs in practice and results of some implemented QA programs will be discussed [18, 28]

Future Directions and Research Opportunities
  1. Clinical validation of cutoffs of positivity using response to Herceptin.

  2. Validation of the clinical benefits from Herceptin in borderline ranges of results, i.e. IHC 2+ and cases with borderline FISH scores of 1.8 – 2.2.

  3. The role of new methodologies:
    1. assay of circulating serum Her2/neu and the correlation of the level of the extracellular domain in the serum with disease status and treatment response [29]

    2. validation of image analysis in scoring IHC and ISH [30]

    3. new PCR platforms
  4. Development of sensitive and specific antibodies for IHC and probes for ISH

  5. The interaction of Her2/neu with other biological markers, for example, cMYC [31], other members of the Her family and angiogenic factors.

  6. Mechanisms of Herceptin resistance, e.g. the role of C-terminal fragments [32]

  7. Others

References:
  1. Akiyama T, Sudo C, Ogawara H, et al: The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 232:1644-6, 1986

  2. Yaziji H, Goldstein LC, Barry TS, et al: HER-2 testing in breast cancer using parallel tissue-based methods. JAMA 291:1972-7, 2004.

  3. Owens MA, Horten BC, Da Silva MM: HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer 5:63-9, 2004.

  4. Press MF, Bernstein L, Thomas PA, et al: HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas. J Clin Oncol 15:2894-904, 1997

  5. Bull S, Ozcelik H, Pinnaduwage D, Blackstein E, Sutherland D, Sidlofsky S, Hanna W, Qizilbash A, Tweeddale M, Fine S, Geddie W, McCready D, Andrulis I. The combination of p53 mutation and neu/erbB-2 amplification is associated with poor survival in node-negative breast cancer. J Clin Oncol 2004;22(1):86-96.

  6. Ross JS, Fletcher JA. The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy. Stem Cells 1998;16:413-28.

  7. Proceedings of the 9th International Conference on Primary Therapy of Early Breast Cancer, 2005. St. Gallen, Switzerland. Breast 2005 Dec;14(6):427-642.

  8. Pritchard KI, Shepherd LE, O'Malley FP, et al: HER2 and Responsiveness of Breast Cancer to Adjuvant Chemotherapy. N Engl J Med 354:2103-2111, 2006

  9. F. P. O'Malley , S. Chia, D. Tu, L. E. Shepherd, M. N. Levine, D. G. Huntsman, V. H. Bramwell, I. L. Andrulis, K. I. Pritchard. Prognostic & predictive value of topoisomerase II alpha in a randomized trial comparing CMF to CEF in premenopausal women with node positive breast cancer (NCIC CTG MA.5). Oral presentation at ASCO, June 6, 2006. Abstract #533 ASCO 2006 supplement;11S.

  10. Konecny G, Pauletti G, Pegram M, et al: Quantitative Association Between HER-2/neu and Steroid Hormone Receptors in Hormone Receptor-Positive Primary Breast Cancer. J Natl Cancer Inst 95:142-153, 2003

  11. Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783-92., 2001

  12. Vogel CL, Cobleigh MA, Tripathy D, et al: Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719-26, 2002

  13. The Herceptin Adjuvant (HERA) Trial Study Team: Trastuzumab following adjuvant chemotherapy in HER2-positive early stage breast cancer (HERA trial): Disease-free and overall survival after 2 year follow-up. Proc Am Soc Clin Oncol 24, 2006

  14. Bilous M, Dowsett M, Hanna W, et al: Current perspectives on HER2 testing: a review of national testing guidelines. Mod Pathol 16:173-82, 2003

  15. 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 77:148-54, 2002

  16. Paik S, Bryant J, Tan-Chiu E, et al: Real-world performance of HER2 testing-National Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst 94:852-4, 2002

  17. Ellis IO, Bartlett J, Dowsett M, et al: Best Practice No 176: Updated recommendations for HER2 testing in the UK. J Clin Pathol 57:233-7, 2004

  18. Hanna W, O'Malley FM. Updated recommendations from the HER2/neu consensus meeting – Toronto , Ontario , 7 September, 2001. Current Oncology 2002;9(1):S18-S19.

  19. Press MF, Slamon DJ, Flom KJ, et al. Evaluation of HER2 gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 2002;20:3095-104.

  20. Downs-Kelly E, Yoder BJ, Stoler M, et al: The influence of polysomy 17 on HER2 gene and protein expression in adenocarcinoma of the breast: a fluorescent in situ hybridization, immunohistochemical, and isotopic mRNA in situ hybridization study. Am J Surg Pathol 29:1221-7, 2005

  21. Hanna W , Nofech-Mozes S, Kahn H. Intratumoral heterogeneity of Her2/neu in breast cancer – a rare event or an overlooked phenomenon? United States and Canadian Academy of Pathology, February 2006, Mod Pathol 2006, abstract #123.

  22. Penault-Llorca F, Adelaide J, Houvenaeghel G, et al. Optimization of immunohistochemical detection of ERBB2 in human breast cancer: impact of fixation. J Pathol 1994 May;173(1):65-75.

  23. Ross JS, Fletcher JA, Linette GP, et al. The HER-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. The Oncologist 2003;8:307-325.

  24. Hicks DG, Tubbs RR: Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines. Hum Pathol 36:250-61, 2005

  25. Hanna WM, Kwok K: Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm. Mod Pathol. 2006 Apr;19(4):481-7.

  26. Joensuu H, Kellokumpu-Lehtinen PL, Bono P et al. Adjuvant Docetaxel or Vinorelbine with or without Trastuzumab for Breast Cancer. NEJM 354:809-820, February 23, 2006.

  27. Mass RD, Press MF, Anderson S, et al: Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab. Clin Breast Cancer 6:240-6, 2005

  28. Persons DL, Cooley LD, Dewald GW, Dowling PK, Du E, Mascarello JT, Rao KW, Tubbs RR, Wilson KS, Wolff DJ, Vance GH. HER-2 Fluorescence In Situ Hybridization (FISH): Results from the Survey Program of the College of American Pathologists (CAP). Arch Pathol Lab Med 2006; 130:325-331.

  29. Kostler WJ, Steger GG, Soleiman A, et al. Monitoring of Serum Her-2/neu Predicts Histopathological Response to Neoadjuvant Trastuzumab-based Therapy for Breast Cancer. Anticancer Res 2004;24:1127-1130.

  30. Bloom K, Harrington D. Enhanced Accuracy and Reliability of HER-2/neu Immunohistochemical Scoring Using Digital Microscopy. Am J Clin Pathol 121:620-630, 2004.

  31. Kim C, Bryant J, Horne Z, et al. Trastuzumab sensitivity of breast cancer with co-amplification of HER2 and cMYC suggest pro-apoptotic function of deregulated cMYC in vivo. Proceedings of 28th San Antonio Breast Cancer Symposium 2005, abstract #46.

  32. Anido J, Scaltriti M, Bech Serra JJ, et al. Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation. EMBO J 2006 Jul 12;25(13):3224-3244.