VOLUME 32 䡠 NUMBER 35 䡠 DECEMBER 10 2014

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Human Epidermal Growth Factor Receptor 2 Testing in Primary Breast Cancer in the Era of Standardized Testing: A Canadian Prospective Study Wedad M. Hanna, Penny J. Barnes, Martin C. Chang, C. Blake Gilks, Anthony M. Magliocco, Henrike Rees, Louise Quenneville, Susan J. Robertson, Sandip K. SenGupta, and Sharon Nofech-Mozes Wedad M. Hanna and Sharon NofechMozes, Sunnybrook Health Sciences Centre, University of Toronto; Wedad M. Hanna, Martin C. Chang, and Sharon Nofech-Mozes, University of Toronto; Martin C. Chang, Mount Sinai Hospital, Toronto; Susan J. Robertson, Ottawa General Hospital and University of Ottawa, Ottawa; Sandip K. SenGupta, Kingston General Hospital and Queen’s University, Kingston, Ontario; Penny J. Barnes, Capital Health District Authority and Dalhousie University, Halifax, Nova Scotia; C. Blake Gilks, Vancouver General Hospital and University of British Columbia, Vancouver, British Columbia; Henrike Rees and Louise Quenneville, Saskatoon City Hospital and University of Saskatchewan, Saskatoon, Saskatchewan, Canada; and Anthony M. Magliocco, Esoteric Laboratory Services, Moffitt Cancer Center, Tampa, FL. Published online ahead of print at www.jco.org on November 10, 2014. Supported by F. Hoffmann-La Roche (research funding). Presented in part at the 35th Annual San Antonio Breast Cancer Symposium, San Antonio, TX, December 4-8, 2012. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article. Corresponding author: Wedad M. Hanna, MBBCh, Department of Pathology, Sunnybrook Health Sciences Centre, E432-2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5; e-mail: [email protected]. © 2014 by American Society of Clinical Oncology 0732-183X/14/3235w-3967w/$20.00 DOI: 10.1200/JCO.2014.55.6092

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Purpose Therapies that target overexpression of human epidermal growth factor receptor 2 (HER2) rely on accurate and timely assessment of all patients with new diagnoses. This study examines HER2 testing of primary breast cancer tissue when performed with immunohistochemistry (IHC) and additional in situ hybridization (ISH) for negative cases (IHC 0/1⫹). The analysis focuses on the rate of false-negative HER2 tests, defined as IHC 0/1⫹ with an ISH ratio ⱖ 2.0, in eight pathology centers across Canada. Patients and Methods Whole sections of surgical resections or tissue microarrays (TMAs) from invasive breast carcinoma tissue were tested by both IHC and ISH using standardized local methods. Samples were scored by the local breast pathologist, and consecutive HER2-negative IHC results (IHC 0/1⫹) were compared with the corresponding fluorescence or silver ISH result. Results Overall, 711 surgical excisions of primary breast cancer were analyzed by IHC and ISH; HER2 and chromosome 17 centromere (CEP17) counts were available in all cases. The overall rate of false-negative samples was 0.84% (six of 711 samples). Interpretable IHC and ISH scores were available in 1,212 cases from TMAs, and the overall rate of false-negative cases was 1.6% (16 of 978 cases). Conclusion Our observation confirms that IHC is an adequate test to predict negative HER2 status in primary breast cancer in surgical excision specimens, even when different antibodies and IHC platforms are used. The study supports the American Society of Clinical Oncology/College of American Pathologists and Canadian testing algorithms of using IHC followed by ISH for equivocal cases. J Clin Oncol 32:3967-3973. © 2014 by American Society of Clinical Oncology

INTRODUCTION

Breast cancer is a diverse disease, and its biologic behavior relates to multiple factors.1 Pathologic investigation has long been used to identify different tumor subtypes, highlighting those that may respond to specific therapeutic intervention. The most commonly assayed and well-characterized markers associated with breast cancer diagnosis are estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2).1 Testing for ER, progesterone receptor, and HER2 offers predictive information regarding therapeutic response. Therefore, it is recommended as part of the treatment planning process in every patient with breast cancer at the time of diagnosis and, more recently, at disease progression.2,3

Overexpression or amplification of HER2 is detected in approximately 15% to 20% of breast cancers4,5 and is associated with a higher tumor grade and poorer prognosis.4,6-8 Targeting HER2 with the monoclonal antibody trastuzumab (Herceptin; F. Hoffmann-La Roche, Basel, Switzerland) is currently the standard of care for patients with HER2positive breast cancer,2,3,9 providing survival benefits in patients with HER2-positive early10-12 and metastatic disease.13,14 Thus, accurate and standardized testing algorithms, which adequately measure HER2 expression in newly diagnosed patients with breast cancer, are crucial for determining which patients may benefit from HER2-targeted therapy. The Canadian recommendations for HER2 testing,15 based on the American Society of Clinical Oncology/College of American Pathologists (ASCO/ © 2014 by American Society of Clinical Oncology

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CAP) guidelines,16 advise that breast cancers are initially screened for HER2 positivity using immunohistochemistry (IHC), followed by in situ hybridization (ISH) for samples with equivocal staining. The HER2 IHC assay is scored according to the percentage and intensity of cancer cells with membranous staining; samples are designated IHC 0/1⫹ (negative), IHC 2⫹ (equivocal), or IHC 3⫹ (positive).15,16 Samples with an equivocal IHC score are assessed by ISH where the ratio of HER2 gene copy number or the copy number of chromosome 17 centromere (CEP17) is calculated. According to 2007 ASCO/CAP guideline recommendations, an HER2 ISH ratio of more than 2.2 or HER2 gene copy number of more than 6.0 was considered positive for HER2 gene amplification, whereas an ISH ratio of less than 1.8 or HER2 gene copy number less than 4.0 was negative and a ratio between 1.8 and 2.2 was considered equivocal.15,16 Although the criteria were revised in 2013,17 patients with an ISH ratio ⱖ 2.0 were eligible for trastuzumab in both the 2007 and 2013 guidelines.16,17 Given the proven benefit of targeted therapy, there is growing concern that a subset of IHC-negative samples exhibit concomitant HER2 gene amplification. According to the treatment algorithm, these patients’ samples would not undergo confirmatory ISH testing, because they would not have equivocal IHC scores, and therefore, some patients would not receive HER2-targeted therapies. Several studies (Table 1) found substantial variation in the number of IHC-negative samples that were HER2-amplified by ISH.5,18-27 Of the 14,418 samples tested, a discrepancy between IHC and ISH results was noted in 1.1% to 11.5%. However, the majority of these studies were conducted before attempts to standardize HER2 testing, including standardization of preanalytic and postanalytic variables, and before the establishment of external quality assurance programs. The aim of this study was to determine the rate of false-negative IHC results in the context of the current practice of testing breast cancer samples first by IHC and then by reflex ISH, in samples with

equivocal HER2 protein expression. The study was carried out across eight central testing laboratories in Canada, using their local testing methods for either IHC or ISH in the setting of a routine surgical pathology laboratory. PATIENTS AND METHODS Patients and Tissue Samples Patient samples were collected either prospectively as whole sections (group 1) or retrospectively using a panel of previously described tissue microarrays (TMAs; group 2).28 Group 1 comprised formalin-fixed, paraffinembedded invasive breast carcinoma samples that were negative for HER2 overexpression by IHC (score 0 or 1⫹). Surgical resection specimens were identified by the study pathologist in seven medical laboratories across Canada (Capital Health District Authority, Halifax, Nova Scotia; Kingston General Hospital, Kingston, Ontario; Mount Sinai Hospital, Toronto, Ontario; Ottawa General Hospital, Ottawa, Ontario; Saskatoon City Hospital, Saskatoon, Saskatchewan; Sunnybrook Health Sciences Centre, Toronto, Ontario; Tom Baker Centre, Calgary, Alberta) between January 2010 and July 2011. The seven laboratories are located within academic centers that currently act as central or reference laboratories for HER2 testing both regionally and across four provinces. Laboratories serve either the whole province or a large region within the province and test an average of 800 to 3,000 samples per annum. All centers have the capability of testing breast carcinoma specimens using both IHC and ISH methods. Each center is enrolled in at least one external quality control program, and interpretation of both IHC and ISH was carried out by dedicated HER2 readers without image analysis. All centers contributed between 50 and 150 HER2-negative samples to the overall study population and provided data collection forms for documentation of IHC/ISH scores, antibody/kit used, histologic subtype, and Nottingham grade. These samples were mostly prospective and sequential in nature. Group 2 comprised 1,212 samples, which were provided by Vancouver General Hospital (Vancouver, British Columbia, Canada) and arrayed in single 0.6-mm cores as described by Turbin et al.29 The patient cohort was derived from a series of 4,150 women with newly diagnosed invasive breast

Table 1. False-Negative IHC Rates (IHC negative and FISH positive) in 11 Previously Published Investigations

Study

IHC-Negative/ FISH-Positive Samples (%)ⴱ

No. of Samples

Antibody for IHC

21

1.1

561

HercepTest (DAKO, Carpinteria, CA)

Kobayashi et al20

2.0

173

Polyclonal antibody (Nitirei, Tokyo, Japan)

Lottner et al25

2.3

215

HercepTest (DAKO)

Yaziji et al23 Birner et al19

2.8 2.9

2,963 303

Hammock et al22 Prati et al26 Mass et al18 Lal et al24

4.0 9.9 10.0 10.0

102 199 529 2,279

Kovacs and Stenman27

10.7

538

Owens et al5

11.5

6,556

Lal et al

A0845 polyclonal antibody to HER2 (DAKO) Polyclonal rabbit anti-HER2/neu antibody (DAKO), HercepTest (DAKO), and monoclonal antibody CB-11 from either BioGenex (San Ramon, CA) or Ventana (Frankfurt, Germany) HercepTest (DAKO) HercepTest kit (DAKO) HercepTest kit (DAKO) HercepTest kit (DAKO) and A0485 polyclonal antibody kit (DAKO) HercepTest kit (DAKO)

ISH Method PathVysion HER2/neu probe kit (Vysis, Downers Grove, IL) Dual color (FISH) using the c-erbB-2–specific probe and the chromosome 17 centromere–specific probe from Vysis Combined fluorescence IHC and FISH method was developed by using the PathVysion HER2 DNA probe kit (Vysis) Vysis reagents PathVysion gene detection system (Vysis)

PathVysion kit (Vysis) PathVysion kit (Vysis) PathVysion kit (Vysis) PathVysion HER2 probe kit (dual and single FISH; Vysis) PathVysion HER2 DNA probe kit (Vysis) PathVysion kit (Vysis)

Abbreviations: FISH, fluorescent in situ hybridization; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization. ⴱ IHC score of 0 or 1⫹ but with gene amplification showed by FISH.

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Table 2. IHC 0 and 1⫹ Samples Grouped According to the Antibody Used for the HER2 Status Test

0 No. of Samples

No. of Samples

HercepTest SP3 4B5 Total

251 175 285 711

70 90 147 307

No. of Samples Tumor Grade

IHC Score

Antibody Used

Table 3. Breakdown of IHC 0/1⫹ Samples by Tumor Grade

1⫹ %

No. of Samples

%

9.80 12.70 20.70 43.20

181 85 138 404

25.50 12.00 19.40 56.80

1 2 3 Total no. Total No.

IHC 0

IHC 1⫹

71 124 108 303

91 195 117 403 706ⴱ

Abbreviation: IHC, immunohistochemistry. ⴱ Five samples had missing grades.

Abbreviations: HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry.

cancer within the catchment area of the British Columbia Cancer Agency between 1986 and 1992; clinical details of these patients have been extensively described previously.30-38 Briefly, this series consisted of all samples tested at a central laboratory for biochemical ER determination. During the specified period, approximately 75% of breast cancer specimens in the province were referred to this laboratory,35,36 and formalin-fixed, paraffin-embedded tissue blocks from these specimens served as donor blocks. Samples were not preselected, and those with available tissue were included in the microarray; only those with staining for both HER2 protein expression (IHC) and HER2 copy number (fluorescent ISH [FISH]) were included in this study. IHC HER2 protein expression was assessed in paraffin-embedded sections using HercepTest (DAKO, Carpinteria, CA), SP3 (NeoMarkers, Freemont, CA), or 4B5 (Ventana Medical Systems, Tucson, AZ) antibodies (Table 2). All sections were fixed in phosphate-buffered formalin, and sections were collected on charged slides for IHC. Each center performed IHC testing according to local clinical practices, and staining was scored as per the national Canadian recommendations15; this was in line with both the 2007 and 2013 update of the ASCO/CAP recommendations for HER2 testing in breast cancer.16,17 Samples that scored 0/1⫹ and met the inclusion criteria were further tested by ISH. ISH HER2 copy number was determined using either FISH or silverenhanced ISH (SISH) according to the standard local protocol of each participating center. FISH was performed using the PathVysion HER2 DNA Probe Kit (Vysis, Downers Grove, IL), and SISH was carried out with an INFORM SISH kit (Ventana). Testing was performed according to both the manufacturer’s specifications and the US Food and Drug Administration testing guidelines.39 Specimens were scored visually by the local breast pathologist and were considered positive if the HER2/CEP17 ratio was ⱖ 2.0. Whole sections were scanned, and in sections where the signal for HER2 and CEP17 was homogeneous, 20 to 40 cells were scored. In sections with low scores, 40 to 60 cells were counted. Designation of False-Negative Samples Sections of samples with HER2-negative IHC scores were compared with the corresponding ISH (either SISH or FISH) result. False-negative samples were defined as IHC-negative with an ISH ratio of ⱖ 2.0, because these patients are eligible for trastuzumab therapy.

RESULTS

Sample Characteristics Group 1 comprised 711 samples, and histologic analysis confirmed that 162 patients (22.8%) had grade 1 disease, 320 (45.0%) had www.jco.org

grade 2 disease, and 225 (31.7%) had grade 3 tumors. Tumor grade was unavailable for five patients (0.7%). Within group 1, 651 samples (91.6%) were ductal type, 44 (6.2%) were lobular, and 11 (1.5%) had mixed ductal and lobular features. Of the 706 samples with an evaluable tumor grade, 162 (22.9%) were grade 1 (IHC 0, n ⫽ 71; IHC 1⫹, n ⫽ 91), 319 (45.2%) were grade 2 (IHC 0, n ⫽ 124; IHC 1⫹, n ⫽ 195), and 225 (31.9%) were grade 3 (IHC 0, n ⫽ 108; IHC 1⫹, n ⫽ 117; Table 3). Within group 2, 1,412 samples had available FISH data, with the majority of missing cases being a result of the detachment of cores from slides after prehybridization digestion or, in some cases, a result of there being too few tumor cells for FISH analysis. A further 200 samples were excluded as a result of either core drop-off or insufficient tumor material in the cores after immunostaining. Overall, 1,212 samples had data for both HER2 FISH and HER2 IHC and were included in the analysis. Incidence of False-Negative Samples Of the 711 samples in group 1, 307 (43.2%) were scored as IHC 0, and 404 (56.8%) were scored as IHC 1⫹ (Table 2). ISH results were available for each sample. Overall, six (0.84%) of 711 samples had discrepant IHC and ISH results (Table 4), none of which were heterogeneous for HER2 expression. Five (83.3%) of the six false-negative samples were invasive ductal carcinomas of no special type, and one discordant sample (16.7%) was an invasive breast carcinoma with mixed ductal and lobular features. One of the false-negative samples showed a high-grade invasive ductal carcinoma with a retraction artifact, indicative of poor fixation (Fig 1A). This sample had an IHC score of 0 (Fig 1B). SISH testing demonstrated an average of five copies of the HER2 gene per cell and a centromere count of 2 (ratio, 2.5; Figs 1C

Table 4. Discrepant IHC-Negative/ISH-Positive Samples Histologic Type

Tumor Grade

Antibody

IHC Score

ISH Ratioⴱ

IDC IDC IDC IDC IDC Mixed IDC/ILC

3 2 3 3 2 2

HercepTest SP3 4B5 4B5 4B5 4B5

1 1 0 0 0 1

2.00 2.07 2.45 3.30 2.07 2.08

Abbreviations: IDC, invasive ductal carcinoma; IHC, immunohistochemistry; ILC, invasive lobular carcinoma; ISH, in situ hybridization. ⴱ Four cases were tested with fluorescent ISH and two by silver-enhanced ISH.

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Fig 1. Immunohistochemical analysis of a discordant high-grade invasive ductal carcinoma. The section was poorly fixed, resulting in poor morphology. Paraffin-embedded sections were stained for (A) hematoxylin and eosin, (B) human epidermal growth factor receptor 2 (HER2) protein expression with the antibody 4B5, (C) chromosome 17 amplification (silver-enhanced in situ hybridization [SISH]), or (D) HER2 amplification (SISH). This sample was classified as immunohistochemistry 0, with discordant SISH localization and chromosome 17 staining visualized using silver reagent.

and 1D). Nine hundred seventy-eight samples in group 2 were scored as IHC 0 or 1⫹, with 16 samples (1.6%) amplified by FISH with a mean score of 2.9. DISCUSSION

The current HER2 testing protocol in patients with breast cancer is to assess samples initially with IHC and follow up with ISH for samples with equivocal results.15-17 Using this system, samples with an IHC score of 0 or 1⫹ would not be evaluated with ISH, even though they may have HER2 gene amplification. These patients with false-negative IHC results would thus not receive anti-HER2 therapy. We evaluated the presence of false-negative IHC results in eight central laboratories that use three commercially available HER2 antibodies and two commercially available ISH kits, all of which are routinely used in clinical practice. The aim was to identify the number of IHC-negative/ISHpositive samples present in a cohort of consecutive Canadian patients undergoing resection for breast cancer at eight separate medical facilities. Overall, false-negative results were infrequent, occurring in less than 1% of the surgical excision samples examined (group 1; n ⫽ 711). The TMA samples (group 2), in comparison, showed a slightly higher false-negative rate of 1.6%. For the latter analysis, one biopsy of 0.6 mm was examined per patient, and the difference in false-negative rates between full sections and cores was most likely a result of issues with sampling or tumor heterogeneity. In addition, these samples were collected between 1986 and 1992, and thus, poor fixation may 3970

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also have contributed to variability. A limitation of our study was that all discordant samples had low levels of amplification, making them technically challenging to score. However, although TMA analysis is not used in routine clinical testing, the false-negative rate was much lower than the 5% rate of discordance recommended when comparing new and previously validated assays.17 Previous evidence suggests that discordance between IHC negativity and ISH positivity occurs in 1.1% to 11.5% of samples; a 5-year review of discordant ISH and IHC 0/1⫹ for HER2 assessment gave similar results (0% to 10.7% discordance).27,40-45 Because IHC and ISH measure different features, namely protein expression and gene copy number, respectively, some degree of discordance might be expected.46,47 However, the large variability between different laboratories may be caused by a number of other factors at the preanalytic (differences in fixation or processing practices between different centers, resulting in protein degradation or reduced sensitivity), analytic (standardization of laboratory practices and staff competency), and postanalytic (interpretation of results and quality assurance procedures) stages.16 Thus, the strict use of standardized methodology and quality assurance programs by laboratories participating in this study and the experience of breast pathologists may account for the high concordance rate. Few false-negative results were shown to occur in high-grade tumors. Therefore, to further reduce false-negative IHC results, highgrade tumors with apparently poor fixation and negative hormone receptor status may be considered for further analysis by ISH at the pathologist’s discretion. The current ASCO/CAP guidelines detail a JOURNAL OF CLINICAL ONCOLOGY

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range of histopathologic features suggestive of possible discordance, in addition to high grade, which should be considered before retesting.9 In addition, ISH testing should be considered, particularly in cases where there is uncertainty about the preanalytic conditions. A prospective observational study, which compared IHC and FISH assays in samples scored as IHC 1⫹ (n ⫽ 492), reported that in 13% of the patients with adverse prognostic features (n ⫽ 84), FISH-positive staining was detected.48 This was approximately twice as many falsenegative cases as reported in a separate concordance study where FISH positivity in IHC 0 and IHC 1⫹ samples was 3.3% and 6.7%, respectively (n ⫽ 244).49 Thus, IHC 1⫹ samples associated with adverse prognostic characteristics, such as high grade and elevated Ki-67 staining, should also be considered for ISH testing. It is possible that some of the IHC/ISH-discordant samples are falsely positive for ISH staining, rather than falsely negative for IHC, particularly in tumors with disorganized genomes as a result of chromosomal instability in high-grade or basal-like tumors. Analysis of samples with unresolved HER2 status, using a clinically validated genomic array, identified artificial pericentromeric copy number changes, which altered the HER2/CEP17 ratio in breast cancer with chromosome 17 complexity.50 The array used for this analysis contained 127 probes covering the HER2 amplicon, the pericentromeric regions, and both arms of chromosome 17; the results suggested that FISH analysis of samples with chromosome 17 abnormalities should be interpreted cautiously.50 Development of and strict adherence to testing guidelines and mandatory participation in external quality assurance programs ensure that all IHC laboratories are concordant with FISH testing 95% of the time.51 The current ASCO/CAP recommendations for HER2 testing advise additional ISH analysis in patients with breast cancer in whom IHC staining is equivocal (IHC 2⫹).17 Expansion of reflex testing, where patients would receive ISH testing irrespective of their IHC status, may be a potentially expensive approach, because although the number of false-negative patients would decrease, there is an increased risk of treating false-positive patients.52 A recent study estimated the incremental cost-effectiveness ratio of expanded reflex testing and projected that this strategy is cost effective.52 The assessment, based on a decision-analytic model, examined the potential expense of retesting IHC-negative samples (IHC 0 or IHC 1⫹) by ISH and vice versa,52 and estimated that the probability of a FISH-positive result after an initial IHC-negative score was 1.6% or 4.9% when samples were IHC 0 or IHC 1⫹, respectively.53 We report an overall ISH positivity in IHC-negative cases from whole tissue sections of less than 1%, which REFERENCES 1. Bertos NR, Park M: Breast cancer: One term, many entities? J Clin Invest 121:3789-3796, 2011 2. Cardoso F, Harbeck N, Fallowfield L, et al: Locally recurrent or metastatic breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 23:vii11-vii19, 2012 3. National Comprehensive Cancer Network: NCCN Clinical Practice Guidelines in Oncology: Breast Cancer, V3. Fort Washington, PA, National Comprehensive Cancer Network, 2013 4. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer: Correlation of relapse and survival www.jco.org

is considerably lower than the estimated false-negative probability of 2.27% projected by Garrison et al.52 In conclusion, the Canadian or ASCO/CAP guidelines, which suggest that all invasive carcinomas be tested with IHC followed by ISH for equivocal samples, are a safe and appropriate approach for determining HER2 status. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Although all authors completed the disclosure declaration, the following author(s) and/or an author’s immediate family member(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Anthony M. Magliocco, Ventana Medical Systems (C) Stock Ownership: None Honoraria: Anthony M. Magliocco, Ventana Medical Systems, Genoptix Research Funding: Wedad M. Hanna, Roche; Penny J. Barnes, F. Hoffman-La Roche; Anthony M. Magliocco, Roche, Ventana Medical Systems; Sharon Nofech-Mozes, F. Hoffmann-La Roche Expert Testimony: None Patents, Royalties, and Licenses: None Other Remuneration: None AUTHOR CONTRIBUTIONS Conception and design: Wedad M. Hanna, C. Blake Gilks, Anthony M. Magliocco, Sharon Nofech-Mozes Financial support: Anthony M. Magliocco Administrative support: Anthony M. Magliocco Provision of study materials or patients: Penny J. Barnes, Anthony M. Magliocco, Henrike Rees, Louise Quenneville, Sandip K. SenGupta Collection and assembly of data: Wedad M. Hanna, Penny J. Barnes, Martin C. Chang, C. Blake Gilks, Anthony M. Magliocco, Henrike Rees, Louise Quenneville, Susan J. Robertson, Sharon Nofech-Mozes Data analysis and interpretation: Wedad M. Hanna, C. Blake Gilks, Anthony M. Magliocco, Sandip K. SenGupta, Sharon Nofech-Mozes Manuscript writing: All authors Final approval of manuscript: All authors

with amplification of the HER-2/neu oncogene. Science 235:177-182, 1987 5. 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 5:63-69, 2004 6. Kallioniemi OP, Holli K, Visakorpi T, et al: Association of c-erbB-2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer. Int J Cancer 49:650-655, 1991 7. 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: 2894-2904, 1997

8. Andrulis IL, Bull SB, Blackstein ME, et al: Neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer: Toronto Breast Cancer Study Group. J Clin Oncol 16:1340-1349, 1998 9. Goldhirsch A, Winer EP, Coates AS, et al: Personalizing the treatment of women with early breast cancer: Highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Ann Oncol 24:2206-2223, 2013 10. Goldhirsch A, Gelber RD, Piccart-Gebhart MJ, et al: 2 years versus 1 year of adjuvant trastuzumab for HER2-positive breast cancer (HERA): An openlabel, randomised controlled trial. Lancet 382:10211028, 2013 11. Perez EA, Romond EH, Suman VJ, et al: Four-year follow-up of trastuzumab plus adjuvant

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© 2014 by American Society of Clinical Oncology

JOURNAL OF CLINICAL ONCOLOGY

HER2 Testing in Primary Breast Cancer

GLOSSARY TERMS

fluorescent in situ hybridization (FISH): in situ hybridization is a sensitive method generally used to detect specific gene sequences in tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. FISH uses a fluorescent probe to increase the sensitivity of in situ hybridization. HER2/CEP17: the number of copies of the HER2 gene divided by the number of copies of chromosome 17 (strictly the number of copies of the pericentric region of chromosome 17 to which the CEP17 fluorescent in situ hybridization probe hybridizes).

HER2/neu (human epidermal growth factor receptor 2): also called ErbB2. HER2/neu belongs to the epidermal

immunohistochemistry: the application of antigen-antibody interactions to histochemical techniques. Typically, a tissue section is mounted on a slide and incubated with antibodies (polyclonal or monoclonal) specific to the antigen (primary reaction). The antigen-antibody signal is then amplified using a second antibody conjugated to a complex of peroxidase-antiperoxidase, avidin-biotin-peroxidase, or avidinbiotin alkaline phosphatase. In the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibodyantigen binding. Immunofluorescence is an alternate approach to visualize antigens. In this technique, the primary antigen-antibody signal is amplified using a second antibody conjugated to a fluorochrome. On ultraviolet light absorption, the fluorochrome emits its own light at a longer wavelength (fluorescence), thus allowing localization of antibody-antigen complexes.

growth factor receptor (EGFR) family and is overexpressed in several solid tumors. Like EGFR, it is a tyrosine kinase receptor whose activation leads to proliferative signals within the cells. On activation, the human epidermal growth factor family of receptors are known to form homodimers and heterodimers, each with a distinct signaling activity. Because HER2 is the preferred dimerization partner when heterodimers are formed, it is important for signaling through ligands specific for any members of the family. It is typically overexpressed in several epithelial tumors.

tissue microarray: microarray used to analyze the expression of

HercepTest: an immunohistochemical assay for human epidermal growth factor receptor overexpression in carcinomas to determine whether patients could benefit from treatment with trastuzumab.

trastuzumab: a humanized anti-ErbB2 monoclonal antibody ap-

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genes of interest simultaneously in multiple tissue samples. Tissue microarrays consist of hundreds of individual tissue samples placed on slides ranging from 2 to 3 mm in diameter. Using conventional histochemical and molecular detection techniques, tissue microarrays are powerful tools for evaluating the expression of genes of interest in tissue samples. In cancer research, tissue microarrays are used to analyze the frequency of a molecular alteration in different tumor types, to evaluate prognostic markers, and to test potential diagnostic markers.

proved for treating patients whose breast cancers overexpress the ErbB2 protein or demonstrate ErbB2 gene amplification. It is currently being tested in combination with other therapies.

© 2014 by American Society of Clinical Oncology

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Human epidermal growth factor receptor 2 testing in primary breast cancer in the era of standardized testing: a Canadian prospective study.

Therapies that target overexpression of human epidermal growth factor receptor 2 (HER2) rely on accurate and timely assessment of all patients with ne...
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