RESEARCH ARTICLE

Evaluation of HER2 Protein Expression Using 2 New Monoclonal Antibodies Pedro B. Aleixo, MD, MSc,* Rosalva T. Meurer, MSc,* Fla´via A. Vasconcellos, DSc,w Fabricio R. Conceic¸a˜o, DSc,w Jose´ A. G. Aleixo, PhD,w and Antonio A. Hartmann, MD, PhD*

Abstract: This study describes the performance of 2 new mouse anti-HER2 monoclonal antibodies (Abs), clones 33F and 410G, in evaluating HER2 overexpression in a series of 123 invasive breast carcinoma cases. In-house immunohistochemistry (IHC) was performed and the results were compared with those for the SP3 and A0485 anti-HER2 Abs. Chromogenic in situ hybridization was used to detect ERBB2 amplification and its concordance with IHC was analyzed. Comparison of IHC results for 33F with SP3 and A0485 yielded concordance rates (K) of 0.81 and 0.75, respectively; the same concordance rates were found when comparing results for 410G with SP3 and A0485. Compared with SP3 and A0485, 33F and 410G specificities were 98.6% and 98.6%, and 100% and 100%, respectively, whereas the sensitivities were 80% and 74.1%, and 78% and 72.2%, respectively. The K values between 33F and 410G HER2+ expression and chromogenic in situ hybridization-positive amplification were 1 and 0.96, respectively. These concordance rates were reproduced in another production batch (K = 0.96 and K = 0.96). Together, these results show that the tested monoclonal Abs would be well suited for detecting HER2 protein overexpression by IHC. Key Words: anti-HER2 antibody, CISH, chemistry, in situ hybridization, validation

immunohisto-

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A

ntibodies (Abs) are used in various assays to characterize the function and expression of human proteome components and to validate potential protein biomarkers in clinical studies.1 In histopathology, immunohistochemistry (IHC) tests use Abs to define cell

Received for publication December 9, 2013; accepted March 11, 2014. From the *Postgraduation Program in Pathology, Pathology Research Laboratory, Universidade Federal de Cieˆncias da Sau´de de Porto Alegre (UFCSPA), Porto Alegre; and wPostgraduation Program in Biotechnology, Technology Development Center, Universidade Federal de Pelotas (UFPEL), Pelotas, Rio Grande do Sul, Brazil. Supported by the National Council of Technological and Scientific Development (CNPq) of Brazil. The authors declare no conflict of interest. Reprints: Pedro B. Aleixo, MD, MSc, Postgraduation Program in Pathology, Pathology Research Laboratory, Universidade Federal de Cieˆncias da Sau´de de Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul CEP 90050-170, Brazil (e-mail: aleixo.pedro@ gmail.com). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.

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differentiation and protein expression in tissues. IHC assays can also contribute to determining diagnoses, setting prognoses, and predicting responses to treatments for various diseases, especially cancer.2 The fundamental concept of IHC is that antigens (Ag) present in tissue can be detected by specific Abs in situ.3 However, despite its simple concept, IHC methodology has become more complex as more stringent goals for sensitivity and specificity are established.4 Worldwide, mammary carcinoma is the leading cause of cancer-related morbidity and mortality in women.5,6 Currently, IHC plays an essential role in defining prognostic and predictive factors for breast cancer patient management.7 The status of human epidermal growth factor receptor 2 (HER2) protein overexpression is recommended for assessment in every case of invasive breast carcinoma (BC).8,9 The ERBB2 (or HER2) gene is amplified in a subset of BC, and the resulting HER2 receptor overexpression is directly linked with major changes in cell proliferation and survival in these tumors. This finding promoted the development of specific antiHER2 drugs and today the detection of BC HER2 status is a prognostic and predictive factor for response to treatments routinely used in clinical practice.10,11 Cells with HER2 amplification have cell surface HER2 receptor levels that are increased by 100-fold relative to normal cells,12 whereas the HER2 protein is overexpressed in approximately 15% to 22% of BCs.13–15 This increase in expression can be demonstrated by testing at a protein (IHC, ELISA, or Western blot) or gene level [Southern blot, QRT-PCR, or in situ hybridization (ISH)].16,17 The determination of HER2 status in patients with BC is very important because it identifies those who can benefit from anti-HER2 therapy. However, a falsenegative result can deprive patients of a treatment that offers great potential to increase survival, while falsepositive results may lead to an unnecessary risk of side effects in patients who are unlikely to respond to a given therapy. Hence, to make an accurate assessment of HER2 status, the techniques and reagents used for testing must be validated and standardized.18 IHC and ISH techniques, such as fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH), and chromogenic in situ hybridization (CISH), are the most commonly used methods for investigating HER2 status.19 These methods allow results to be

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interpreted together with histology and eliminate contamination by dilution artifacts that can occur with PCRbased techniques.20 The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) issued guidelines to standardize and improve the accuracy and reproducibility of HER2 testing in BCs.8,9 They recommend that IHC be used as the primary test, but for equivocal results the algorithm advises a second test based on ISH. Although the gold-standard method for assessing HER2 status is still under question, FISH and CISH apparently have the best performances.21,22 Several studies showed good agreement between HER2 3+ scores by IHC and positive amplification by FISH and CISH, whereas equivocal cases showed the greatest discrepancy.14,23–33 However, this agreement may decrease in cases having more complex abnormalities of the HER2 gene and centromere 17, as well as in heterogeneous tumors.8,9,22,34,35 Anti-HER2 Abs can recognize different epitopes on the intracellular or extracellular portion of the HER2 protein. The most commonly available ready-to-use Ab clones include: 1 polyclonal rabbit Ab, clone A0485 (HercepTest; Dako, Denmark); 2 mouse monoclonal antibodies (mAbs), clones CB11 (Novocastra, England), and TAB250 (Invitrogen; San Diego, CA); and 2 rabbit mAbs, clones 4B5 (HER2/neu PATHWAY, Ventana; Tucson, AZ and Genentech; San Francisco, CA) and SP3 (NeoMarkers; Freemont, CA and Cell Marque; Rocklin, CA). Abs A0485, CB11 and 4B5 are directed to the intracellular portion of the protein, whereas Abs TAB250 and SP3 are directed to the extracellular portion.32,36 A0485 is viewed to be the most sensitive Ab, but limitations in its specificity were demonstrated in some studies.23,32,37,38 In contrast, SP3 and 4B5 demonstrated greater agreement with HIS when compared with A0485 and CB11.39 In this context, our group generated, characterized, and validated new anti-HER2 mouse mAbs to identify HER2 protein overexpression in neoplastic tumors. We previously reported the development of 5 clones of murine hybridomas that secrete mAbs against HER2. Two secreted mAbs, designated clones 33F and 410G, were characterized as IgG1 isotypes, with affinity constants of 6  108 and 1 109 L/mol and purification from ascites fluid yielding concentrations of 0.39 and 1.12 mg/mL, respectively.40,41 Both clones reacted with the native protein by indirect immunofluorescence and the denatured protein by Western blot.41 This study describes the performance characteristics of our 2 mAbs in evaluating the HER2 protein in formalinfixed, paraffin-embedded human neoplastic breast tissue. To standardize their use in HER2 status investigations, we compared performances with 2 other previously validated Abs (SP3 and A0485) in staining invasive BCs by IHC. IHC results were correlated with HER2 amplification identified by 2 CISH techniques, which were defined as our reference gold standard. A significant similarity between sensitivity, specificity, and concordance rates with all Abs and a high correlation with HIS results was obtained. Reproducibility of the tested Abs was evaluated by testing a second lot of Ab production.

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MATERIALS AND METHODS Case Selection The study cohort material consisted of 123 selected primary or recurrent invasive BC tissue samples derived from the archives of a routine pathology laboratory between January 2000 and December 2004. The specimens included incisional and excisional biopsies of formalinfixed, paraffin-embedded tissue. No information on initiation or total time of fixation was provided. Cases with tumor samples 80%) invasive tumor cells. Nonspecific cytoplasmic staining was higher with A0485 than with SP3, 33F, and 410G. SP3 and A0485 are highly Copyright

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sensitive Abs and in our series accounted for the large number of 2+ scored cases identified (15.44% and 27.64%, respectively). 33F and 410G detected fewer 2+ scored cases (5.69% and 7.31%, respectively), with most of the discrepant cases scored as 0 or 1+. No staining in normal epithelium was seen with any Ab. Several studies demonstrated that most HER2 2+ scored cases identified by IHC did not show HER2 positive amplification,14,24,26,31,46,47 and due to this finding we classified these cases as negative in our statistical analysis. The selection of breast cancer patients for targeted treatment with anti-HER2 drugs requires a method of HER2 testing that prevents unnecessary treatment and accurately identifies potential responsive patients. Assays using Abs are performed on a daily basis in research and diagnostic pathology laboratories. When testing formalin-fixed paraffin-embedded tissue samples for protein expression, IHC is frequently the first method of choice. However, differences in protein expression results by IHC can occur depending on the assay protocols, reagents used and interpretation criteria.2,9,19,48–52 Because these analytical features can influence HER2 expression testing by IHC, methods that evaluate HER2 gene amplification in tissue samples, primarily ISH techniques, are considered to be more reliable for determining HER2 status.21,22 Nonetheless, discrepant results between ISH techniques (FISH, CISH, or SISH) and IHQ have also been reported.14,23–33 These discordant findings are mostly due to tumor heterogeneity34 and chromosome 17 abnormalities (aneusomy, centromere 17 coamplification) in breast cancer.22 The use of CISH in molecular pathology is relatively recent, and like FISH its reliability in HER2 gene evaluation has been well established.19,53–63 CISH allows for simultaneous signal interpretation with morphology visualization, provides staining of an internal positive control in normal cells and can be evaluated with a light microscope. The critical steps identified in some studies to ensure quality CISH reactions include proper tissue fixation, tissue pretreatment, and digestion time optimization.64–67 In our series we performed CISH using 2 techniques, 1 involving a single probe against the HER2 gene and the other using 1 probe against HER2 and 1 reference probe against CEN17. The rate of DC-CISH nonevaluable cases was high (19.5%) when compared with SC-CISH (8.9%). However, when we combined DCCISH and SC-CISH results, only 3 cases (2.4%) were lost in the analysis. The causes of test failure included lack of viable invasive tumor cells, lack of signals, or presence of nonspecific signals. Two equivocal cases by DC-CISH (1.6%) and 3 cases by SC-CISH (2.4%) were found and intratumoral heterogeneity or polysomy 17 could explain these. To date, the clinical significance of the relationship between intratumoral genetic heterogeneity or aneusomy of chromosome 17 and possible benefits from anti-HER2 therapy has not been established,68 so in our analysis these cases were considered as negative. As an alternative method for identifying the same target as IHC, we used 2 CISH techniques to determine www.appliedimmunohist.com |

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FIGURE 1. HER2 overexpression and gene amplification detected with immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). A and B, IHC images in a case with HER2 overexpression (3+) with first lot production of 33F (A,  200) and 410G (B,  400). C, Dual-color CISH image showing HER2 probe amplification (green signals) in relation to the CEN17 reflex probe (red signals) on the same case as in images A/B ( 1000). D and E, IHC images for another case with HER2 overexpression (3+) with first lot production of 33F (D, 200) and 410G (E, 200). F, Single-color CISH image showing HER2 probe amplification (brown signals) in the same case as images (D/E) ( 1000). G and H, IHC images for a case with HER2 overexpression (3+) with second lot production of 33F (G,  200) and 410G (H, 400). I, IHC image showing HER2 overexpression (3+) detected by SP3 on the same case as for images (G/H). J and K, IHC images for a case with HER2 overexpression (3+) detected by 410G (J, 200) and A0485 (L,  200). L, IHC image of a control case demonstrating HER2 overexpression (3+) detected with SP3 where ImmunoMembrane classification was 3+ (red = complete and strong, green = incomplete or weak) ( 400).

the HER2 gene amplification status, and they served as a reference standard test to correlate with HER2 expression. Similarly to SP3 and A0485, 33F and 410G demonstrated excellent sensitivity, specificity, and concordance rates with ISH techniques (Tables 6 and 7). As observed in other reports,14,23–33 when comparing 33F and 410G expression levels with combined CISH results we found a high concordance rate (100% and 96%, respectively). The sensitivity and specificity was high with all Abs. After excluding equivocal cases, the sensitivity of

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SP3, A0485, and 410G all increased to 100%, whereas specificity declined for SP3 (85.5%) and A0485 (74.5%). IHC results from the second lot of 33F and 410G maintained similar correlations with CISH results, which shows that both mAbs have good reproducibility. In generating and validating Abs, they should be shown to be specific, selective, and reproducible in the context in which they will be used.69 In IHC, the final result can be influenced by preanalytical, analytical, and postanalytical factors. For example, the cold ischemia Copyright

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TABLE 7. Results of Dual-Color Chromogenic In Situ Hybridization and Single-Color Chromogenic In Situ Hybridization for HER2 Amplification in 123 Cases

TABLE 9. Sensitivity and Specificity of Antibodies Compared With HER2 Amplification by Chromogenic In Situ Hybridization in 120 Cases* Antibody

Sensitivity (%)

95% CI

Specificity (%)

95% CI

j

SP3 A0485 33F 410G 33F-2L 410G-2L

97.5 97.5 100 95 95 95

88.3-99.9 88.3-99.9 92.8-100 84.4-99.2 84.4-99.2 84.4-99.2

88.8 82.5 100 100 100 100

80.4-94.4 73-89.7 96.3-100 96.3-100 96.3-100 96.3-100

0.82 0.74 1 0.96 0.96 0.96

N (%) Result

DC-CISH*

Negative Equivocal Amplified Not evaluable Total

68 2 29 24 123

(55.3) (1.6) (23.6) (19.5) (100)

SC-CISHw 69 3 40 11 123

DC+SC-CISH

(56.1) (2.4) (32.5) (8.9) (100)

78 2 40 3 123

(63.4) (1.6) (32.5) (2.4) (100)

*DC-CISH criteria: HER2/CEP17 ratior1.8 (negative); Z1.8- < 2.2 (equivocal); Z2.2 (amplified). wSC-CISH: average HER2 signals/cellr4.0 (negative); Z4- < 6 (equivocal); Z6 (amplified). DC-CISH indicates dual-color chromogenic in situ hybridization; SC-CISH, single-color chromogenic in situ hybridization.

time before fixation, the duration of fixation, and the use of different types of fixative and tissue processing are factors that may affect epitope preservation.70,71 In addition, the Ab clone and dilution used, the type of Ag retrieval, the method of detection, and the interpretation of results also influence IHC staining measurements.72,73 TABLE 8. Relationship Between Immunohistochemical Results and HER2 Gene Amplification by Chromogenic In Situ Hybridization in 123 Cases HER2 Gene Amplification Status by CISH* IHQ

Not Amplified Equivocal Amplified Not Evaluable Total

SP3 0/1+ 2+ 3+ Total A0485 0/1+ 2+ 3+ Total 33F 0/1+ 2+ 3+ Total 410G 0/1+ 2+ 3+ Total 33F-2L 0/1+ 2+ 3+ Total 410G-2L 0/1+ 2+ 3+ Total

53 16 9 78

0 2 0 2

0 1 39 40

1 0 2 3

54 19 50 123

35 31 12 78

0 0 2 2

0 1 39 40

0 2 1 3

35 34 54 123

72 6 0 78

2 0 0 2

0 0 40 40

1 1 1 3

75 7 41 123

73 5 0 78

1 1 0 2

0 2 38 40

1 1 1 3

75 9 39 123

74 4 0 78

2 0 0 2

0 2 38 40

1 1 1 3

77 7 38 123

74 4 0 78

2 0 0 2

0 2 38 40

1 1 1 3

77 7 38 123

*Dual-color CISH prevailed over single-color CISH results in the analysis. 2L indicates second lot; CISH, chromogenic in situ hybridization; IHC, immunohistochemistry.

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*Nonevaluable cases by both dual-color and single-color CISH were excluded. Dual-color CISH prevailed over single-color CISH results in the analysis. 2L indicates second lot; CI, confidence interval.

The specificity of our anti-HER2 mAbs was previously demonstrated on MCF-7 breast cancer cells by testing their reactions with the native protein by indirect immunofluorescence and with the denatured protein by Western blot.41 In this report, the first step in evaluating the selectivity of clones 33F and 410G was to test them in controlled HER2+ and HER2 formalin-fixed paraffinembedded cases. When titrated in tissue sections, our mAbs showed membrane staining only in the HER2+ case, and a decrease in staining intensity with increasing Ab dilution was seen. In addition, the level of target expression was quantified on our controls by digital analysis using ImageJ software with the plug-in ImmunoMembrane.74 The HER2+ and HER2 controls were, respectively, scored as 3+ (Fig. 1L) and 0 in the analysis. Another indication that IHC staining with 33F and 410G is reproducible was seen when different batches tested in multiple reactions, and tests performed on different days produced similar results, which, according to some authors, are characteristics of a good Ab.69,75–77 As another control step, we tested our mAbs in 5 samples of nonneoplastic breast tissue derived from reduction mammoplasty and all cases showed no labeling (data not shown). In summary, we report here the performance of 2 new mAbs relative to other validated Abs and CISH in determining HER2 status in invasive BC tumors. Our study limitations included lack of information on the time when fixation was initiated or the total fixation time. In addition, our study was interpreted by consensus microscopy in TABLE 10. Sensitivity and Specificity of Antibodies Compared With HER2 Amplification by Chromogenic In Situ Hybridization With Equivocal Cases Excluded* Antibody

Sensitivity (%)

95% CI

Specificity (%)

95% CI

j

SP3 A0485 33F 410G 33F-2L 410G-2L

100 100 100 100 100 100

92.6-100 92.6-100 92.8-100 92.4-100 92.4-100 92.4-100

85.5 74.5 100 100 100 100

75-92.7 60.6-85.4 95.9-100 96-100 96-100 96-100

0.82 0.73 1 1 1 1

*Nonevaluable cases by both dual-color and single-color CISH were excluded. Dual-color CISH prevailed over single-color CISH results in the analyses. 2L indicates second lot; CI, confidence interval.

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difficult cases and lacked an interobserver agreement evaluation. Despite these limitations, our 2 mAbs proved to be reliable and showed excellent performance characteristics for the detection of HER2 overexpression by IHC in human tissue fixed in formalin and embedded in paraffin. Importantly, we observed a >95% concordance rate between HER2 expression with 33F/410G and CISH in determining HER2 amplification status. The identification of HER2 status provides information for researchers, clinicians, and cancer patients. We demonstrated through specific laboratory techniques that mAbs 33F and 410G are suitable candidates for this intended use. As such, clinical validation of HER2 expression assessment using our mAbs and response to targeted therapy with anti-HER2 drugs can be examined. ACKNOWLEDGMENTS The authors are grateful to the staff and technicians at the UFSCP Postgraduation Program and Pathology Research Laboratory for their support and cooperation. REFERENCES 1. Strachan T, Read A. Analyzing the structure and expression of genes and genomes. In: Strachan T, Read A, eds. Human Molecular Genetics. 4th ed.. New York: Garland Science; 2011:213–223. 2. Taylor CT, Shi SR, Barr NJ. Techniques of immunohistochemistry: principles, pitfalls, and standardization. In: Dabbs DJ, ed. Diagnostic Immunohistochemistry. Theranostic and Genomic Applications. 3rd ed. Philadelphia: Elsevier; 2011:1–41. 3. Kampf C, Andersson AC, Wester K, et al. Antibody-based tissue profiling as a tool for clinical proteomics. Clin Proteomics. 2004;1: 285–299. 4. Mighell AJ, Hume WJ, Robinson PA. An overview of the complexities and subtleties of immunohistochemistry. Oral Dis. 1998;4:217–223. 5. Bray F, Ren JS, Masuyer E, et al. Estimates of global cancer prevalence for 27 sites in the adult population in 2008. Int J Cancer. 2013;132:1133–1145. 6. Soerjomataram I, Lortet-Tieulent J, Parkin DM, et al. Global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in 12 world regions. Lancet. 2012;380:1840–1850. 7. Ly A, Lester SC, Dillon D. Prognostic factors for patients with breast cancer: traditional and new. Surg Pathol Clin. 2012;5: 775–785. 8. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol. 2007;25:118–145. 9. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131:18–43. 10. Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61(suppl 2):1–13. 11. Citri A, Yarden Y. EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol. 2006;7:505–516. 12. Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–137. 13. Yaziji H, Goldstein LC, Barry TS, et al. HER-2 testing in breast cancer using parallel tissue-based methods. JAMA. 2004;291: 1972–1977. 14. 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. 2004;5:63–69. 15. Choritz H, Bu¨sche G, Kreipe H. Quality assessment of HER2 testing by monitoring of positivity rates. Virchows Arch. 2011;459: 283–289.

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16. Pfeifer JD. Molecular Genetic Testing in Surgical Pathology. Philadelphia: Lippincott Williams & Wilkins; 2006. 17. Ross JS, Slodkowska EA, Symmans WF, et al. The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist. 2009;14:320–368. 18. Bilous M, Dowsett M, Hanna W, et al. Current perspectives on HER2 testing: a review of national testing guidelines. Mod Pathol. 2003;16:173–182. 19. Bhargava R, Esposito NN, Dabbs DJ. Immunohistology of the breast. In: Dabbs DJ, ed. Diagnostic Immunohistochemistry. Theranostic and Genomic Applications. 3rd ed. Philadelphia: Elsevier; 2011:1–41. 20. Shah S, Chen B. Testing for HER2 in breast cancer: a continuing evolution. Pathol Res Int. 2010;2011:903202. 21. Cuadros M, Villegas R. Systematic review of HER2 breast cancer testing. Appl Immunohistochem Mol Morphol. 2009;17:1–7. 22. Mansfield AS, Sukov WR, Eckel-Passow JE, et al. Comparison of fluorescence in situ hybridization (FISH) and dual-ISH (DISH) in the determination of HER2 status in breast cancer. Am J Clin Pathol. 2013;139:144–150. 23. Jacobs TW, Gown AM, Yaziji H, et al. Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol. 1999;17:1983–1987. 24. Jimenez RE, Wallis T, Tabasczka P, et al. Determination of Her-2/ Neu status in breast carcinoma: comparative analysis of immunohistochemistry and fluorescent in situ hybridization. Mod Pathol. 2000;13:37–45. 25. 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. 2000;5:199–207. 26. Ridolfi RL, Jamehdor MR, Arber JM. HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach. Mod Pathol. 2000;13:866–873. 27. Wang S, Saboorian MH, Frenkel E, et al. Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays. J Clin Pathol. 2000;53: 374–381. 28. Lebeau A, Deimling D, Kaltz C, et al. Her-2/neu analysis in archival tissue samples of human breast cancer: comparison of immunohistochemistry and fluorescence in situ hybridization. J Clin Oncol. 2001;19:354–363. 29. McCormick SR, Lillemoe TJ, Beneke J, et al. HER2 assessment by immunohistochemical analysis and fluorescence in situ hybridization: comparison of HercepTest and PathVysion commercial assays. Am J Clin Pathol. 2002;117:935–943. 30. Ogura H, Akiyama F, Kasumi F, et al. Evaluation of HER-2 status in breast carcinoma by fluorescence in situ hybridization and immunohistochemistry. Breast Cancer. 2003;10:234–240. 31. Dybdal N, Leiberman G, Anderson S, et al. Determination of HER2 gene amplification by fluorescence in situ hybridization and concordance with the clinical trials immunohistochemical assay in women with metastatic breast cancer evaluated for treatment with trastuzumab. Breast Cancer Res Treat. 2005;93:3–11. 32. Nunes CB, Rocha RM, Reis-Filho JS, et al. Comparative analysis of six different antibodies against Her2 including the novel rabbit monoclonal antibody (SP3) and chromogenic in situ hybridisation in breast carcinomas. J Clin Pathol. 2008;61:934–938. 33. Mayr D, Heim S, Weyrauch K, et al. Chromogenic in situ hybridization for Her-2/neu-oncogene in breast cancer: comparison of a new dual-colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization. Histopathology. 2009;55:716–723. 34. Wu JM, Halushka MK, Argani P. Intratumoral heterogeneity of HER-2 gene amplification and protein overexpression in breast cancer. Hum Pathol. 2010;41:914–917. 35. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–892.

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36. Dekker TJ, Borg ST, Hooijer GK, et al. Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue microarray approach. Breast Cancer Res. 2012;14:R93. 37. Roche PC, Ingle JN. Increased HER2 with US Food and Drug Administration-approved antibody. J Clin Oncol. 1999;17:434. 38. Ricardo SA, Milanezi F, Carvalho ST, et al. HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas. J Clin Pathol. 2007; 60:1001–1005. 39. Rhodes A, Jasani B, Anderson E, et al. Evaluation of HER-2/neu immunohistochemical assay sensitivity and scoring on formalinfixed and paraffin-processed cell lines and breast tumors: a comparative study involving results from laboratories in 21 countries. Am J Clin Pathol. 2002;118:408–417. 40. Vasconcellos FA. Monoclonal antibodies 410G and 33F against human and canine HER2 protein. Hybridoma. 2011;30:498–498. 41. Vasconcellos FA, Aleixo PB, Stone SC, et al. Generation and characterization of new HER2 monoclonal antibodies. Acta Histochem. 2013;115:240–244. 42. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007;25:5287–5312. 43. Bartlett JM, Starczynski J, Atkey N, et al. HER2 testing in the UK: recommendations for breast and gastric in-situ hybridisation methods. J Clin Pathol. 2011;64:649–653. 44. Powell WC, Roche PC, Tubbs RR. A new rabbit monoclonal antibody (4B5) for the immuno-histochemical (IHC) determination of the HER2 status in breast cancer: comparison with CB11, fluorescence in situ hybridization (FISH), and interlaboratory reproducibility. Appl Immunohistochem Mol Morphol. 2008;16:569. 45. Thomson TA, Hayes MM, Spinelli JJ, et al. HER-2/neu in breast cancer: interobserver variability and performance of immunohistochemistry with 4 antibodies compared with fluorescent in situ hybridization. Mod Pathol. 2001;14:1079–1086. 46. Tubbs RR, Pettay JD, Roche PC, et al. Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message. J Clin Oncol. 2001;19:2714–2721. 47. Rossi E, Ubiali A, Cadei M, et al. HER-2/neu in breast cancer: a comparative study between histology, immunohistochemistry, and molecular technique (FISH). Appl Immunohistochem Mol Morphol. 2006;14:127–131. 48. Penault-Llorca F, Adelaı¨ de J, Houvenaeghel G, et al. Optimization of immunohistochemical detection of ERBB2 in human breast cancer: impact of fixation. J Pathol. 1994;173:65–75. 49. Press MF, Hung G, Godolphin W, et al. Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression. Cancer Res. 1994;54:2771–2777. 50. O’Leary TJ. Standardization in immunohistochemistry. Appl Immunohistochem Mol Morphol. 2001;9:3–8. 51. Khoury T, Sait S, Hwang H, et al. Delay to formalin fixation effect on breast biomarkers. Mod Pathol. 2009;22:1457–1467. 52. Lourenc¸o HM, Pereira TP, Fonseca RR, et al. HER2/neu detection by immunohistochemistry: optimization of in-house protocols. Appl Immunohistochem Mol Morphol. 2009;17:151–157. 53. Tanner M, Gancberg D, Di Leo A, et al. Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples. Am J Pathol. 2000;157:1467–1472. 54. Dandachi N, Dietze O, Hauser-Kronberger C. Chromogenic in situ hybridization: a novel approach to a practical and sensitive method for the detection of HER2 oncogene in archival human breast carcinoma. Lab Invest. 2002;82:1007–1014. 55. Arnould L, Denoux Y, MacGrogan G, et al. Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer. Br J Cancer. 2003; 88:1587–1591. 56. Isola J, Tanner M, Forsyth A, et al. Interlaboratory comparison of HER-2 oncogene amplification as detected by chromogenic and fluorescence in situ hybridization. Clin Cancer Res. 2004;10:4793–4798.

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Evaluation of New Anti-HER2 Antibodies

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Evaluation of HER2 Protein Expression Using 2 New Monoclonal Antibodies.

This study describes the performance of 2 new mouse anti-HER2 monoclonal antibodies (Abs), clones 33F and 410G, in evaluating HER2 overexpression in a...
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