Groupe dEtude des Facteurs Pronostiques par Immunohistochime dans les Cancers du Sein (GEFPICS)
Affiliations of authors: A. Vincent-Salomon, J. Couturier, Institut Curie, Paris, France; G. MacGrogan, S. Mathoulin-Pélissier, Institut Bergonié, Bordeaux, France; L. Arnould, Institut Georges-François Leclerc, Dijon, France.
Correspondence to: Anne Vincent-Salomon, M.D., Institut Curie, 26 rue dUlm, 75248 Paris Cedex 05, France (anne.salomon{at}curie.net).
We read with interest the recent studies by Paik et al. (1) and Roche et al. (2) that dealt with some of the problems with immunohistochemical (IHC) testing for the HER2 protein. We fully support the view expressed by Dr. Zujewski (3) in the editorial that accompanied these two studies regarding the need to "build quality in" when performing such assays. Because trastuzumab targets the HER2 protein, evaluation of HER2 protein status by IHC should be the most clinically relevant assay. However, the major problem with IHC for HER2 is the lack of a standardized assay among laboratories. On the basis of our recently completed study, we propose that the best way to improve the accuracy of IHC for HER2 is to calibrate it against a gold standard such as fluorescence in situ hybridization (FISH).
We performed a multicenter study to assess the value of calibrating IHC for HER2 against FISH to improve the accuracy of the IHC method. Twelve French Cancer Center laboratories participated in this study, in which 119 invasive breast carcinomas were evaluated for HER2. Each of the 12 laboratories used its own in-house IHC techniques to assess HER2 protein expression on formalin-fixed or Bouins liquid-fixed, paraffin-embedded tissue sections. All laboratories used the CB11 (Novocastra, Newcastle-upon-Tyne, U.K.) or the A0485 (Dako, Glostrup, Denmark) anti-HER2 primary antibody. To define HER2-positive cancers, we used two cutoff values: 10% and 60% of immunostained cells with moderate or strong intensity of staining. The 10% value was chosen because it was the usual cut point used to select patients for anti-HER2 therapy in clinical trials (4,5), and the 60% value was chosen because previous published data using this value reported a strong correlation between HER2IHC and HER2FISH (6). To avoid variations in fixation protocols, FISH for HER2 gene status was performed in a single laboratory on frozen sections from 116 of 119 cases. Results of IHC were collected at a central institution and compared with the FISH results. Discordances between IHC and FISH were observed in six of the 12 cancer centers. These six cancer centers then calibrated their IHC on the basis of the FISH results and performed a second IHC run. The calibration process consisted of the introduction of heat-induced epitope retrieval and an increase in the dilutions of the primary antibodies. Using the FISH results as the gold standard, the accuracy of IHC was calculated, before and after calibration, by dividing the number of true-positive and true-negative cases, which were defined by concordant results between IHC and FISH results, by the total number of samples. Forty-four of 116 cancers (37.9%) showed HER2 gene amplification. Before calibration, the accuracy of IHC was 89.6% (95% confidence interval [CI] = 84.1% to 95.1%) at the 10% cutoff value and 93.0% (95% CI = 88.3% to 97.7%) at the 60% cutoff value. After calibration, the accuracy of the IHC was 93.0% (95% CI = 88.3% to 97.7%) at the 10% cutoff value and 95.0% (95% CI = 91.1% to 98.9%) at the 60% cutoff value, which corresponded to HER2 overexpression in 47 (39.5%) and 45 (37.8%) of 119 cancers, respectively.
In conclusion, our study showed that a high accuracy rate for IHC testing of HER2 could be obtained in all 12 participating laboratories with their own in-house IHC techniques, provided that a calibration process was performed. Heat-induced epitope retrieval, high dilutions of anti-HER2 antibody, and the use of specific controls were crucial for IHC calibration. A 95% accuracy rate for IHC, using FISH as the gold standard, was obtained by considering IHC results for HER2 as a continuous variable, with a cutoff value set to 60% positively stained invasive cells. These results emphasize that, to achieve optimal accuracy, laboratories that perform an IHC assay to assess HER2 status should ideally be those able to validate the IHC assay against a standard such as FISH before IHC is introduced into routine clinical practice. As suggested by Roche et al. (2), centralized HER2 testing, in laboratories with sufficient IHC activity and access to IHC calibration procedures on FISH, should be favored.
NOTES
GEFPICS is a group affiliated with the FNCLCC (Fédération Nationale des Centres de Lutte Contre le Cancer).
REFERENCES
1 Paik S, Bryant J, Tan-Chiu E, Romond E, Hiller W, Park K, et al. Real-world performance of HER2 testingNational Surgical Adjuvant Breast and Bowel Project experience. J Natl Cancer Inst 2002;94:8524.
2 Roche PC, Suman VJ, Jenkins RB, Davidson NE, Martino S, Kaufman PA, et al. Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 2002;94:8557.
3 Zujewski JA. "Build quality in"HER2 testing in the real world. J Natl Cancer Inst 2002;94:7889.
4 Cobleigh A, Vogel C, Tripathy D, Robert N, Scholl S, Fehrenbacher L, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:263948.
5 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:78392.
6 Couturier J, Vincent-Salomon A, Nicolas A, Zafrani B, Sastre-Garau X. Strong correlation between results of fluorescent in situ hybridization and immunohistochemistry for the assessment of ERBB2 (HER-2/neu) gene status in breast carcinoma. Mod Pathol 2000;13:123843.[Medline]
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