Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark？) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens
We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH). 1. Introduction The human epidermal growth factor receptor 2 (HER2) is a transmembrane protein tyrosine kinase receptor that is important in initiating signal transduction pathways in normal and abnormal cells [1–5]. HER2 is overexpressed/amplified in approximately 15%–30% of human breast tumors and is a biomarker of poor prognosis in patients demonstrating either high protein levels and/or gene amplification on chromosome 17 [4, 6, 7]. For this reason, HER2 testing is recommended for all newly diagnosed breast cancer patients for the selection of individuals that may benefit from treatment with the humanized monoclonal antibody Trastuzumab [8–11]. Despite confirmed overexpression of HER2, the current response rates to Trastuzumab are less than 50% in the metastatic setting and many of the patients that respond initially will eventually develop resistance and subsequent recurrence of their disease [12–14]. Standardization of both IHC and ISH methodologies across laboratories remains a major problem [15]. because of this approximately 20% of HER2 testing performed in the field may be inaccurate [16]. The ability to accurately and reproducibly quantify the level of HER2 protein expression in tumors is critical to the appropriate selection of patients for Trastuzumab and other HER2 targeted therapies. Most laboratories in North America and Europe use IHC to determine HER2 protein status, with equivocal category results confirmed by indirectly measuring HER2 gene amplification by Fluorescence In Situ
References
[1]
A. Citri, K. B. Skaria, and Y. Yarden, “The deaf and the dumb: the biology of ErbB-2 and ErbB-3,” Experimental Cell Research, vol. 284, no. 1, pp. 54–65, 2003.
[2]
P. P. Di Fiore, J. H. Pierce, M. H. Kraus, O. Segatto, R. King, and S. A. Aaronson, “erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells,” Science, vol. 237, no. 4811, pp. 178–182, 1987.
[3]
Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nature Reviews Molecular Cell Biology, vol. 2, no. 2, pp. 127–137, 2001.
[4]
C. A. Hudis, “Trastuzumab—mechanism of action and use in clinical practice,” The New England Journal of Medicine, vol. 357, no. 1, pp. 39–51, 2007.
[5]
J. S. Ross, E. A. Slodkowska, W. F. Symmans, L. Pusztai, P. M. Ravdin, and G. N. Hortobagyi, “The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine,” Oncologist, vol. 14, no. 4, pp. 320–368, 2009.
[6]
R. W. Carlson, S. J. Moench, M. E. H. Hammond, et al., “HER2 testing in breast cancer: NCCN task force report and recommendations,” Journal of the National Comprehensive Cancer Network, vol. 4, no. 3, pp. S1–S22, 2006.
[7]
D. J. Slamon, G. M. Clark, S. G. Wong, et al., “Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene,” Science, vol. 235, no. 4785, pp. 177–182, 1987.
[8]
S. Amar, A. Moreno-Aspitia, and E. A. Perez, “Issues and controversies in the treatment of HER2 positive metastatic breast cancer,” Breast Cancer Research and Treatment, vol. 109, no. 1, pp. 1–7, 2008.
[9]
A. Di Leo, M. Dowsett, B. Horten, and F. Penault-Llorca, “Current status of HER2 testing,” Oncology, vol. 63, pp. 25–32, 2002.
[10]
D. G. Hicks and S. Kulkarni, “Trastuzumab as adjuvant therapy for early breast cancer: the importance of accurate human epidermal growth factor receptor 2 testing,” Archives of Pathology and Laboratory Medicine, vol. 132, no. 6, pp. 1008–1015, 2008.
[11]
A. Rhodes, B. Jasani, J. Couturier, et al., “A formalin-fixed, paraffin-processed cell line standard for quality control of immunohistochemical assay of HER-2/neu expression in breast cancer,” American Journal of Clinical Pathology, vol. 117, no. 1, pp. 81–89, 2002.
[12]
C. L. Arteaga, A. O'Neill, S. L. Moulder, et al., “A phase I-II study of combined blockade of the ErbB receptor network with trastuzumab and gefitinib in patients with HER2 (ErbB2)-overexpressing metastatic breast cancer,” Clinical Cancer Research, vol. 14, no. 19, pp. 6277–6283, 2008.
[13]
E. de Alava, A. Ocana, M. Abad, et al., “Neuregulin expression modulates clinical response to trastuzumab in patients with metastatic breast cancer,” Journal of Clinical Oncology, vol. 25, no. 19, pp. 2656–2663, 2007.
[14]
M. Piccart, C. Lohrisch, A. Di Leo, and D. Larsimont, “The predictive value of HER2 in breast cancer,” Oncology, vol. 61, supplement 2, pp. 73–82, 2001.
[15]
A. C. Wolff, M. E. Hammond, J. N. Schwartz, et al., “American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer,” Archives of Pathology and Laboratory Medicine, vol. 131, p. 18, 2007.
[16]
S. Paik, J. Bryant, E. Tan-Chiu, et al., “Real-world performance of HER2 testing—National Surgical Adjuvant Breast and Bowel Project experience,” Journal of the National Cancer Institute, vol. 94, no. 11, pp. 852–854, 2002.
[17]
E. D. Hsi and R. R. Tubbs, “Guidelines for HER2 testing in the UK,” Journal of Clinical Pathology, vol. 57, no. 3, pp. 241–242, 2004.
[18]
O. Hameed, A. L. Adams, A. C. Baker, et al., “Using a higher cutoff for the percentage of HER2+ cells decreases interobserver variability in the interpretation of HER2 immunohistochemical analysis,” American Journal of Clinical Pathology, vol. 130, no. 3, pp. 425–427, 2008.
[19]
D. G. Hicks and S. Kulkarni, “HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools,” American Journal of Clinical Pathology, vol. 129, no. 2, pp. 263–273, 2008.
[20]
A. M. Gown, “Current issues in ER and HER2 testing by IHC in breast cancer,” Modern Pathology, vol. 21, no. 2, pp. S8–S15, 2008.
[21]
E. A. Perez, V. J. Suman, N. E. Davidson, et al., “HER2 testing by local, central, and reference laboratories in specimens from the north central cancer treatment group N9831 intergroup adjuvant trial,” Journal of Clinical Oncology, vol. 24, no. 19, pp. 3032–3038, 2006.
[22]
W. M. Hanna and K. Kwok, “Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm,” Modern Pathology, vol. 19, no. 4, pp. 481–487, 2006.
[23]
Y. Shi, W. Huang, Y. Tan, et al., “A novel proximity assay for the detection of proteins and protein complexes: quantitation of HER1 and HER2 total protein expression and homodimerization in formalin-fixed, paraffin-embedded cell lines and breast cancer tissue,” Diagnostic Molecular Pathology, vol. 18, no. 1, pp. 11–21, 2009.
[24]
W. Huang, M. Reinholz, J. Weidler, et al., “Comparison of Central HER-2 tests with quantitative HER-2 expression and HER-2 homodimer measurements using a novel proximity assay ,” American Journal of Pathology. In press.
[25]
G. R. Baehner, T. Maddala, B. Childs, et al., “HER2 concordance between Central Laboratory Immunohistochemistry, FISH and Quantitative RT-PCR in Intergroup Trial E2197,” in Proceedings of the United States and Canadian Academy of Pathology Annual Meeting (USCAP '09), Boston, Mass, USA, 2009, Abstract no. 1421.
[26]
J. M. Rae, C. J. Creighton, J. M. Meck, B. R. Haddad, and M. D. Johnson, “MDA-MB-435 cells are derived from M14 Melanoma cells—a loss for breast cancer, but a boon for melanoma research,” Breast Cancer Research and Treatment, vol. 104, no. 1, pp. 13–19, 2007.
[27]
M. M. Moasser, A. Basso, S. D. Averbuch, and N. Rosen, “The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells,” Cancer Research, vol. 61, no. 19, pp. 7184–7188, 2001.
[28]
G. E. Konecny, M. D. Pegram, N. Venkatesan, et al., “Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells,” Cancer Research, vol. 66, no. 3, pp. 1630–1639, 2006.
[29]
M. Dowsett, W. M. Hanna, M. Kockx, et al., “Standardization of HER2 testing: results of an international proficiency-testing ring study,” Modern Pathology, vol. 20, no. 5, pp. 584–591, 2007.
[30]
C. Desmedt, J. Sperinde, F. Piette, et al., “Quantitation of HER2 expression or HER2:HER2 dimers and differential survival in a cohort of metastatic breast cancer patients carefully selected for trastuzumab treatment primarily by FISH,” Diagnostic Molecular Pathology, vol. 18, no. 1, pp. 22–29, 2009.
[31]
M. Toi, J. Sperinde, W. Huang, et al., “Differential survival following trastuzumab treatment based on quantitative HER2 expression and HER2 homodimers in a clinic-based cohort of patients with metastatic breast cancer,” BMC Cancer, vol. 10, article 56, 2010.
[32]
A. Lipton and S. Ali, “HER2 protein expression and homodimer levels predict response to Trastuzumab in centrally tested FISH-positive metastatic breast cancer patients,” in Proceedings of the 31st CTRC-AACR San Antonio Breast Cancer Symposium, 2008, Abstract no. 550749.
[33]
M. H. Joensuu, J. Weidler, Y. Lie, et al., “Quantitative measurements of HER2 expression and HER2 homodimer using a novel proximity based assay: comparison with HER2 status by immunohistochemistry and chromogenic in situ hybridization in the FinHer study,” in Proceedings of the 31st Annual San Antonio Breast Cancer Symposium, 2008, Poster no. 2071.
[34]
J. Sperinde, S. Ali, K. Leitzel, et al., “Identification of a subpopulation of metastatic breast cancer patients with very high HER2 expression levels and possible resistance to trastuzumab,” Journal of Clinical Oncology, vol. 27, supplement, no. 15, 2009, abstract no. 1059.
[35]
S. Rauser, R. Weis, H. Braselmann, et al., “Significance of HER2 low-level copy gain in Barrett's cancer: implications for fluorescence in situ hybridization testing in tissues,” Clinical Cancer Research, vol. 13, no. 17, pp. 5115–5123, 2007.
[36]
A. McCabe, M. Dolled-Filhart, R. L. Camp, and D. L. Rimm, “Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis,” Journal of the National Cancer Institute, vol. 97, no. 24, pp. 1808–1815, 2005.
[37]
H. Joensuu, J. Weidler, Y. Lie, et al., “Quantitative measurements of HER2 expression and HER2 homodimer using a novel proximity based assay: comparison with HER2 status by immunohistochemistry and chromogenic in situ hybridization in the FinHer study,” in Proceedings of te 31st Annual San Antonio Breast Cancer Symposium, 2008, Poster no. 2071.
[38]
H. Joensuu, P.-L. K. Lehtinen, and P. Bono, “Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer,” The New England Journal of Medicine, vol. 354, pp. 809–820, 2006.
