HER-2/neu Protein Expression in Breast Cancer Evaluated by Immunohistochemistry A Study of Interlaboratory Agreement

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1 Anatomic Pathology / INTERLABORATORY AGREEMENT OF HER-2/NEU EXPRESSION BY IMMUNOHISTOCHEMISTRY HER-2/neu Protein Expression in Breast Cancer Evaluated by Immunohistochemistry A Study of Interlaboratory Agreement Timothy W. Jacobs, MD, 1 Allen M. Gown, MD, 2 Hadi Yaziji, MD, 2 Melissa J. Barnes, 1 and Stuart J. Schnitt, MD 1 Key Words: Immunohistochemistry; HER-2/neu; c-erbb-2; Breast cancer Abstract Immunohistochemistry (IHC) is used commonly for evaluating HER-2/neu protein expression in breast cancer. Given the potential clinical importance of HER- 2/neu status in patient management, interlaboratory variability in HER-2/neu IHC results is a matter of legitimate concern. We compared the results from 2 laboratories for HER-2/neu determined by IHC on paraffin sections of the same 100 consecutive invasive breast cancers. Both laboratories used the same primary antibody; however, different methods for heatinduced epitope retrieval (microwave or steam) and immunostaining (automated equipment from different manufacturers) and different scoring systems (positivenegative and 0-4+) were used. Slides were read in a blinded fashion and the results from the 2 laboratories were compared. Of the 93 cases evaluable in both laboratories, 24% were scored as HER-2/neu positive at 1 laboratory, and 23% were scored as positive at the other. Complete concordance in categorization of HER- 2/neu status between the 2 laboratories was achieved in 90 of 93 cases. Excellent interlaboratory agreement for HER-2/neu IHC was attained using the same primary antibody to HER-2/neu, even without standardization of assay method or scoring criteria. However, standardization of these parameters remains an important objective to optimize interlaboratory agreement. A significant relationship between amplification of the HER-2/neu (c-erbb-2) oncogene and adverse clinical outcome in patients with breast cancer was first noted by Slamon et al 1 in Several subsequent studies have confirmed this association in patients with node-positive disease However, the role of HER-2/neu gene status as an independent prognostic factor in patients with node-negative breast cancer remains controversial, with positive 10,11,13,14,19,22-32 and negative 6,7,9,15,17,33-37 studies reported to date. More recently, interest has focused on the role of HER-2/neu status in breast cancers as a predictor of response or resistance to various forms of systemic therapy. In particular, recent clinical trials have indicated that treatment with a monoclonal antibody to the HER- 2/neu protein (trastuzumab; Herceptin) may prolong the survival of patients with metastatic disease In addition, some studies have shown that tumors that overexpress HER- 2/neu may be resistant to certain forms of cytotoxic chemotherapy (such as cyclophosphamide/methotrexate) 14,21,35,41-43 and sensitive to others (such as doxorubicin) Some studies also have suggested that HER-2/neu overexpression may be associated with resistance to tamoxifen. 43,48-51 Therefore, there is a growing clinical demand for analysis of the HER-2/neu status of current and archived breast cancer specimens. Immunohistochemistry (IHC) is the most commonly used method for evaluating HER2/neu protein expression on formalin-fixed paraffin-embedded samples of breast cancers However, because various tissue fixation protocols, assay methods, HER-2/neu antibodies, and scoring systems are in use, variability in HER-2/neu IHC results has become a matter of legitimate concern A standardized IHC kit for the evaluation of HER-2/neu protein expression (HercepTest; Dako, Carpinteria, CA) has recently been approved by the US Food and Drug Administration. 64 However, recent data suggest that American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

2 Jacobs et al / INTERLABORATORY AGREEMENT OF HER-2/NEU EXPRESSION BY IMMUNOHISTOCHEMISTRY the HercepTest has low specificity for HER-2/neu protein expression when the manufacturer s Food and Drug Administration approved scoring system is used. 65,66 Therefore, development of standardized methods and scoring for HER-2/neu IHC remains an important goal. The purpose of the present study was to compare the results of IHC for HER-2/neu performed on a single cohort of invasive breast cancers in 2 laboratories that routinely use HER-2/neu IHC in clinical practice. Materials and Methods Study Design The study population consisted of 100 consecutive cases of invasive breast cancer accessioned at Beth Israel Deaconess Medical Center, Boston, MA (BIDMC) between July 24, 1997, and February 18, These specimens had been fixed initially in alcoholic formalin (Anatech, LTD, Battle Creek, MI) followed by fixation in 10% neutral buffered formalin. Only cases with sufficient invasive carcinoma for multiple assays were included in the study. For each case, 4-µm-thick tissue sections were cut from a representative paraffin block and applied to positively charged slides. IHC for HER-2/neu was performed in 2 separate laboratories: BIDMC and PhenoPath Laboratories, Seattle, WA (PPL). Although both laboratories used the same primary antibody to HER-2/neu (Dako rabbit anti-human polyclonal antibody to c-erbb-2 oncoprotein, code number A0485; Dako), each laboratory used the assay method and scoring system used in its routine clinical practice (see following sections). The primary antibody used in the present study is the same anti-her-2/neu antibody currently available in the Dako HercepTest kit. However, in the HercepTest kit, the antibody is provided in a prediluted form. In addition, the scoring system required for the HercepTest kit differs from both of the scoring systems used in the present study. Interpretation of each IHC assay was performed by investigators blinded to the results of the other assay. IHC at Beth Israel Deaconess Medical Center Tissue sections were deparaffinized in two 5-minute changes of xylene and were rehydrated through alcohols to distilled water. Subsequently, sections were subjected to heatinduced epitope retrieval by heating in a microwave oven in a 0.01-mol/L concentration of citrate buffer (ph 6) for a total of 10 minutes (ie, two 5-minute periods with replacement of evaporated buffer between). Immunostaining for HER-2/neu was performed using the Ventana 320 automated immunostainer (Ventana Medical Systems, Tucson, AZ). The primary rabbit polyclonal antibody to the HER-2/neu oncoprotein (Dako) was used at a 1:500 dilution. Diaminobenzidine (DAB; Sigma Chemicals, St Louis, MO) was used as the chromogen, and the sections were lightly counterstained with hematoxylin. Positive controls were included in each staining run and consisted of freshly cut breast cancer specimens known to express HER-2/neu. Negative controls consisted of substituting normal rabbit serum for HER-2/neu antibody. HER- 2/neu staining was considered positive when the tumor cells showed intense circumferential cell membrane staining, easily identified with a 10 objective. In all of these cases, staining was observed in the majority (>50%) of the tumor cells. Tumors in which there was cytoplasmic staining without distinct cell membrane staining were scored as negative Image 1. All slides at BIDMC were reviewed simultaneously by 2 pathologists (T.W.J. and S.J.S.). IHC at Phenopath Laboratories Four-micron-thick sections from each case were deparaffinized and rehydrated in graded alcohols. The slides were subjected to heat-induced epitope retrieval by immersing them in a 0.01-mol/L concentration of citrate buffer (ph 6.0) preheated to more than 90 C and then heated in a Black and Decker (Towson, MD) vegetable steamer for 20 minutes, followed by a 20-minute cooldown period at room temperature. Slides were then incubated with an anti-her-2/neu polyclonal antibody (1:1,000 dilution, Dako) on a Dako Autostainer (Dako) for 30 minutes at room temperature. For each case, 1 slide was incubated with phosphate-buffered saline instead of the primary antibody as a negative control. For positive controls, a composite slide composed of formalin-fixed cell pellets of the following 4 cell lines (obtained from Nora Disis, MD, Fred Hutchinson Cancer Research Center, Seattle, WA): MCF-7 (a cell line negative for HER-2/neu overexpression) and 3 human carcinoma cell lines showing increasing levels of overexpression of HER-2/neu: BT-20 (low overexpressor), SKBR-3 (intermediate overexpressor), and SKOV-3 (high overexpressor). Antibody was localized using the LSAB+ Detection System (labeled streptavidin biotin immunoperoxidase; Dako) according to the manufacturer s instructions using the Dako Autostainer (Dako) and counterstained with hematoxylin. Membrane staining intensity and pattern were evaluated using a 0 to 4+ scale (0, completely negative; 1+, faint membranous positivity; 2+, moderate membranous positivity; 3+, strong, circumferential membranous positivity; 4+, extremely strong, circumferential membranous positivity) Image 2. For a score of 2+ to 4+, membrane staining in the majority of the tumor cells had to be present. Cytoplasmic immunostaining was noted but not incorporated into the final scoring. For each case, infiltrating carcinoma and adjacent normal epithelium (if available) were scored separately. A final subtracted score of 252 Am J Clin Pathol 2000;113: American Society of Clinical Pathologists

3 Anatomic Pathology / ORIGINAL ARTICLE A B C D Image 1 Scoring system for HER-2/neu protein immunohistochemistry used at Beth Israel Deaconess Medical Center, Boston, MA. A, No tumor cell membrane staining seen using a 10 objective (negative) (original magnification 100). B, Intense, circumferential tumor cell membrane staining seen using a 10 objective (positive) (original magnification 100). C, Faint, barely perceptible staining seen using a 10 objective (original magnification 100). D, Weak membrane staining in some of the tumor cells (original magnification 600). Cases with this type of staining were scored as negative. the tumor minus normal epithelium was used to correct for variability in background staining of normal epithelium (which should not overexpress the HER-2/neu protein). A final subtracted score of 2 or more or tumor cell staining of 3+ or greater was required to categorize a case as HER- 2/neu positive. All slides at PPL were reviewed by 1 pathologist (A.M.G.). Statistical Methods Statistical significance was set at P <.05. Data were analyzed by the Mann-Whitney rank sum test and Fisher exact test, where appropriate. Computations were performed with the SigmaStat for Windows software (V.2.03, SPSS, Chicago, IL). Results Patient Data and Histologic Features of Carcinomas The median age of the patients was 53.5 years (range, years). Seventy-two carcinomas were of infiltrating ductal type, 11 were infiltrating lobular, 9 were invasive cancers with ductal and lobular features, 3 were mucinous (colloid) carci- American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

4 Jacobs et al / INTERLABORATORY AGREEMENT OF HER-2/NEU EXPRESSION BY IMMUNOHISTOCHEMISTRY A B C D E Image 2 Scoring system for HER-2/neu protein immunohistochemistry used at PhenoPath Laboratories. A, Completely negative, 0 (original magnification 400). B, Faint membranous positivity, 1+ (original magnification 400). C, Moderate membranous positivity, 2+ (original magnification 400). D, Strong, circumferential membranous positivity, 3+ (original magnification 400). E, Extremely strong, circumferential membranous positivity, 4+ (original magnification 400). 254 Am J Clin Pathol 2000;113: American Society of Clinical Pathologists

5 Anatomic Pathology / ORIGINAL ARTICLE nomas, 3 were tubular carcinomas, 1 was an invasive micropapillary carcinoma, and 1 was a metaplastic carcinoma. The median size of the tumors was 15 mm (range, mm). Histologic grading was performed using the Elston and Ellis modification of the Bloom-Richardson grading system. 67 Twenty-seven carcinomas were grade 1, 37 were grade 2, and 35 were grade 3. The metaplastic carcinoma was not graded, since there are no universally accepted criteria for the grading of such lesions. Forty-two of the cases were axillary lymph node negative and 33 were node-positive. Axillary lymph node status was not available for 25 patients. Seventy-eight cases were estrogen receptor positive and 20 were negative. The estrogen receptor status was not determined in 2 cases. HER-2/neu Status At BIDMC, 97 of the 100 cases were evaluable for HER-2/neu, with 23 (24%) of these 97 cases interpreted as positive. All 3 unevaluable cases had no invasive carcinoma present on the immunostained slides. At PPL, 96 of the 100 cases were evaluable for HER-2/neu, with 22 (23%) of these 96 cases interpreted as positive. All 4 unevaluable cases had tumor cell membrane staining of 1+ or 2+ but no normal breast ducts or lobules on the slide. A total of 93 cases were evaluable in both laboratories. There was complete concordance with regard to categorization of HER-2/neu status between the 2 laboratories in 90 of 93 cases (97%; P <.001). Of 23 cases positive at BIDMC, 2 were negative at PPL, and of 70 cases negative at BIDMC, 1 was positive at PPL Table 1. Discussion In the present study, excellent interlaboratory agreement for HER-2/neu IHC was achieved by using the same primary antibody to the HER-2/neu protein, even without standardization of the assay method or scoring criteria. The assays used were those used by the respective laboratories in routine clinical practice. The levels of HER-2/neu protein overexpression found in the present study (24% at BIDMC and 23% at PPL) are in keeping with the published Table 1 Comparison of HER-2/neu Status of 93 Invasive Carcinomas as Determined by Immunohistochemistry in Two Laboratories BIDMC Positive BIDMC Negative PPL positive 21 1 PPL negative 2 69 BIDMC, Beth Israel Deaconess Medical Center, Boston, MA; PPL, PhenoPath Laboratories, Seattle, WA. range of 20% to 30%. 1,68 In addition, we previously have shown a high level of concordance (91%) between HER- 2/neu gene amplification as determined by fluorescence in situ hybridization and one of these IHC assays. 69 Although we demonstrated an interlaboratory concordance rate of 97% between the 2 assays used, several previous studies have highlighted a number of potential problems in the use of IHC for HER-2/neu. These include variability in tissue fixation and processing, variable sensitivity and specificity of commercially available antibodies, and differences in scoring criteria with considerable interobserver variability in interpretation of results ,63 The largest and perhaps the most widely cited study with regard to sensitivity of HER-2/neu IHC is that of Press and coworkers. 58 These investigators performed HER-2/neu IHC on formalin-fixed paraffin-embedded tissue, comparing the sensitivity and specificity of 28 different antibodies. The results were compared with known Southern blot, Northern blot, Western blot, and frozen section IHC results for the cases. The sensitivity of the various antibodies studied ranged from 6% to 82%, and with 1 antibody, the rate of tumor positivity was as low as 2%. However, there are several important limitations to the IHC assays used in that study. First, epitope retrieval methods were not used for 27 of the 28 antibodies evaluated. Formalin fixation is associated with loss of immunoreactivity for HER-2/neu, 56 and it is now clear that epitope retrieval is important for obtaining optimal staining of formalin-fixed paraffin-embedded tissue with at least some of the commercially available HER-2/neu antibodies. 70 Second, the peroxidase-antiperoxidase method was used as the detection system. This method has substantially lower sensitivity than the avidin-biotin complex systems currently in widespread use. Finally, only a very small tissue sample was evaluated for each case, since the authors used multitumor tissue blocks. This could have resulted in erroneously categorizing as negative the cases that exhibited regional variation in HER-2/neu staining. Therefore, it is difficult to extrapolate the results of the study of Press et al 58 to studies of HER-2/neu IHC that use current reagents and methods, such as the present study. There are few available data about reproducibility of HER-2/neu immunostains. Kay and coworkers 59 analyzed interobserver variability of HER-2/neu IHC interpretation of various malignant tumors among 3 pathologists. Overall, the level of interobserver agreement with regard to the presence or absence of membrane staining was high (89%). However, there was marked disagreement as to the intensity of tumor membrane staining, with only 60% and 47% agreement using a 3-point or 4-point scoring scale, respectively. There was only 40% agreement on the extent of staining, and only 27% agreement was observed when American Society of Clinical Pathologists Am J Clin Pathol 2000;113:

6 Jacobs et al / INTERLABORATORY AGREEMENT OF HER-2/NEU EXPRESSION BY IMMUNOHISTOCHEMISTRY intensity and extent were interpreted together. In contrast, intraobserver agreement was excellent, suggesting that if these pathologists studied a standardized training slide set before interpretation of the test cases, interobserver agreement could have been improved. Another study found significant differences between HER-2/neu positivity when comparing formalin-fixed tissue and cytologic specimens, with a positive rate ranging from 47% to 73%, respectively. 63 However, these investigators used different primary antibodies for the histologic and cytologic specimens. Moreover the scoring system used in that study has been reported to have low specificity. 65,66 It must be emphasized that our results are based on the evaluation of breast cancer cases accessioned at a single institution. These cases were subjected to relatively uniform tissue fixation and processing protocols. Whether a similar level of interlaboratory agreement can be obtained in a study population consisting of breast cancer specimens obtained from a variety of laboratories that use different methods of tissue fixation and processing is an important but unresolved issue. In addition, our study used one of several commercially available HER-2/neu antibodies used in clinical practice. Whether a similar high level of interlaboratory agreement can be achieved with other primary antibodies to HER-2/neu also is unknown and is an issue worthy of investigation. Finally, in the present study, a high level of interlaboratory concordance was achieved despite differences in assay methods and scoring between the 2 laboratories. In contrast with the scoring system used at BIDMC, the system used at PPL considered the level of staining of nonneoplastic breast epithelium. This was done to avoid interpreting as HER-2/neu positive the cases in which the level of tumor cell staining was not appreciably greater than the level of staining of nonneoplastic epithelium. However, one potential disadvantage of the PPL method is the lack of nonneoplastic epithelium in association with some primary tumors and in metastatic lesions. Therefore, the BIDMC method may be more generally applicable. A high level of interlaboratory agreement in the assessment of HER-2/neu immunostaining was achieved in the present study despite differences in methods and scoring systems. However, the development of standards for assay methods and, particularly, for scoring of HER-2/neu immunohistochemistry results, is clearly an important goal. From the 1 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, and 2 PhenoPath Laboratories and IRIS, Seattle, WA. Dako, Carpinteria, CA, supplied the reagents for the immunohistochemistry assays. 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