HER2+ Breast Cancer Review of Biologic Relevance and Optimal Use of Diagnostic Tools

Anatomic Pathology / HER2: BIOLOGIC RELEVANCE AND DIAGNOSIS HER2+ Breast Cancer Review of Biologic Relevance and Optimal Use of Diagnostic Tools David G. Hicks, MD, 1 and Swati Kulkarni, MD 2 Key Words: HER2; Breast cancer; Trastuzumab; Immunohistochemistry; Fluorescence in situ hybridization; Concordance; Guidelines; Validation; Proficiency testing DOI: 10.1309/99AE032R9FM8WND1 Abstract Clinical laboratory testing for human epidermal growth factor receptor 2 (HER2) status in newly diagnosed breast cancer is critically important for therapeutic decision making. Unlike most pathologic testing, which serves as an adjunct to establishing a diagnosis, the results of HER2 testing stand alone in determining which patients are likely to respond to trastuzumab, a monoclonal antibody against HER2. Given the significant clinical impact of the testing results on subsequent patient management, the accuracy, precision, and reproducibility of HER2 testing are critical. At present, several preanalytic factors, including the time from tissue removal to tissue fixation, are underappreciated as important variables that have the potential to negatively impact the consistency and reliability of HER2 testing. Rigorous quality control and standardization of the testing process, from the handling of tissue samples to interpretation and reporting of results, are essential for achieving accurate and reproducible assay results. Adenocarcinoma of the breast is a leading cause of cancer morbidity and mortality among women worldwide. 1 A major challenge faced by clinicians treating patients with breast cancer is how to best assess patient outcomes and predict the clinical course of the disease so that the most appropriate treatment regimen can be identified. Clinicians carefully consider several conventional prognostic factors when making treatment decisions; these include TNM staging, surgical margin status, tumor grade, lymphovascular invasion, and hormone receptor status. Many have been validated extensively in numerous studies and in many years of clinical practice. 2,3 However, new prognostic markers that have been validated in robust studies are required to allow accurate individual risk assessment, particularly for patients with early-stage breast cancer. 4,5 It has become increasingly clear that the dysregulation of genes involved in controlling normal growth and development can drive cancer cell growth and survival. 6 In some cases, this dysregulation begins with the amplification of a gene, leading to inappropriate overexpression of the protein product of the gene and disruption of normal cellular function. Protein gene products that have direct roles in driving the biology and clinical behavior of cancer cells are potential targets for the development of novel therapeutics. Research efforts have focused on the investigation and identification of new molecular factors, which can improve the predictability of risk of metastasis and the likelihood of response to therapies. The HER2 story and the development of trastuzumab (Herceptin, Genentech, South San Francisco, CA) has been one of the more remarkable developments in medical oncology, having significant implications for treatment and outcomes in patients whose tumors harbor an HER2 molecular alteration. Am J Clin Pathol 2008;129:263-273 263 263 DOI: 10.1309/99AE032R9FM8WND1 263

Hicks and Kulkarni / HER2: BIOLOGIC RELEVANCE AND DIAGNOSIS The HER2 Receptor and Targeted Therapies An important example of the evolving usefulness of therapeutic targets in clinical oncology is the HER2 oncogene and its role in the prognosis and therapeutic management of a subset of patients with breast cancer. Normal cells have 1 copy of the ERBB2 gene (more commonly known as HER2) on each chromosome 17. Expression of this gene in normal breast epithelial cells gives rise to a 185-kd transmembrane protein growth factor receptor with cytoplasmic tyrosine kinase activity, which transmits signals regulating cell growth. 7,8 In approximately 25% of breast cancers, the HER2 gene is amplified by 2-fold to greater than 20-fold in each tumor cell nucleus relative to the number of copies of chromosome 17. 9,10 Amplification of the HER2 gene drives protein expression. 11 The resulting increase in the number of receptors at the tumor-cell surface (as many as 2 million HER2 molecules per cell instead of the normal 20,000 per cell) promotes receptor activation, leading to signaling, excessive cellular division, and the formation of tumors. 12 Therefore, over-expression of the receptor has a direct role in mediating the biologic and clinical behavior of HER2-positive tumor cells by driving their proliferation and survival. 7,9,10,13,14 As a result, HER2+ breast cancers are more aggressive than HER2 tumors. Patients with HER2+ disease have a significantly shorter disease-free survival and overall survival, regardless of lymph node involvement and other prognostic factors. 9,10,13-18 HER2+ breast cancer significantly correlates with several poor prognostic factors and unfavorable pathologic tumor characteristics, including tumor size, a higher proliferative index, and a high nuclear grade. 19,20 HER2 positivity is predictive of response to trastuzumab, a humanized anti-her2 monoclonal antibody. In 1998, trastuzumab was approved by the US Food and Drug Administration (FDA) for the treatment of metastatic disease, as first-line treatment in combination with paclitaxel or as monotherapy for patients who have previously received chemotherapy. 21 The addition of trastuzumab to first-line chemotherapy significantly improves response rates, time to progression, overall survival, and quality of life compared with chemotherapy alone in patients with HER2+ metastatic breast cancer. 22-24 Trastuzumab also provides clinical benefit as monotherapy, given as first-line therapy or after 1 or more previous chemotherapy regimens for HER2+ metastatic breast cancer. 25,26 Four large clinical trials assessed the effect of adding trastuzumab to combination chemotherapy in the adjuvant setting, and significant improvements in disease-free and overall survival were observed. 27-29 In the interim joint analysis of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 and North Central Cancer Treatment Group (NCCTG) N9831 trials, the addition of trastuzumab to adjuvant chemotherapy significantly improved disease-free survival by 52% (hazard ratio, 0.48; P <.0001) compared with chemotherapy alone. 27 Based on the data from the joint analysis of NSABP B-31 and NCCTG N9831, trastuzumab was approved for use in combination with doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of HER2+, node-positive breast cancer in 2006. 21 HER2 Testing in Breast Cancer For targeted therapies to be effective, the amplification of a gene and/or overexpression of a particular protein must first be confirmed using diagnostic testing. This emphasizes the importance of optimizing assay methods to identify patients most likely to benefit from treatment with a targeted therapy. The evolution of HER2 testing, first as a prognostic marker assay and later as a diagnostic test to determine eligibility for trastuzumab-targeted therapy, has expanded the role of traditional diagnostic pathology. Unlike most testing performed by anatomic pathologists, which serves as an adjunct to establishing a diagnosis, the results of HER2 testing stand alone in determining which patients are likely to respond to trastuzumab therapy. HER2 status may also predict sensitivity to certain cytotoxic drugs and antiestrogens. 30 Eliminating false-positive HER2 results is also important for safety and cost reasons. There is a small risk of cardiac dysfunction associated with trastuzumab, 31,32 and patients should not be exposed to risks unnecessarily. The cost of cancer therapies is a consideration, and the annual cost of trastuzumab is $40,000 based on the wholesale acquisition cost; trastuzumab therapy for 52 weeks has been shown to be cost-effective. 33 Currently, 2 testing methods are approved by the FDA for HER2 testing in the laboratory: immunohistochemical analysis and fluorescence in situ hybridization (FISH). Commercially available, FDA-approved HER2 assays are available for both methods Table 1. Immunohistochemical analysis and FISH have the advantage over other assay methods (ie, those requiring homogenization) because they are morphologically driven. This allows for the direct evaluation of tumor cells, correlation with other morphologic features, and the ability to assay smaller patient samples such as needle core biopsy specimens. The FDA approval for the immunohistochemical and FISH assay methods states that the tissues should be fixed in 10% neutral buffered formalin. Formalin is an aqueous solution of formaldehyde that has been buffered to a neutral ph, most commonly with a phosphate buffer. Non formalinbased fixatives or alternative fixation methods for breast specimens are discouraged for immunohistochemical and 264 Am J Clin Pathol 2008;129:263-273 264 DOI: 10.1309/99AE032R9FM8WND1

Anatomic Pathology / REVIEW ARTICLE Table 1 US Food and Drug Administration Approved, Commercially Available HER2 Assays Immunohistochemical Assays HercepTest, DAKO, Carpinteria, CA; polyclonal antibody PATHWAY, Ventana Medical Systems, Tucson, AZ; murine monoclonal CB11 Fluorescence In Situ Hybridization Assays PathVysion, Vysis, Downers Grove, IL; HER2/CEP17 INFORM, Ventana Medical Systems; HER2 FISH assays. This is because performance data are limited and extrapolation from formalin-fixed data is unreliable. 34 Immunohistochemical Analysis The eligibility criteria for the initial pivotal clinical trials of trastuzumab required the development of an assay to document HER2 protein expression. 22,26 As a result, a research-useonly clinical trials assay (CTA) using immunohistochemical analysis was developed to determine HER2 status and to identify patients eligible for entry into the pivotal trials. 35 The CTA incorporated 2 mouse monoclonal antibodies, 4D5 (the murine precursor of trastuzumab) and CB11, that are specific for different portions of the HER2 protein. To be clinically relevant, the HER2 status of tumors was subjectively, semiquantitatively evaluated on a 0 to 3+ scoring scale that reflected increasing levels of HER2 protein expression Table 2 and Image 1. Patients were eligible for inclusion in the initial trials if their tumors had an HER2 immunohistochemical score of 2+ or 3+ with either antibody. This initial scoring criterion for this trial required that the immunohistochemical 2+ or 3+ staining pattern be seen in more than 10% of tumor cells. 35 The pivotal clinical trial in metastatic breast cancer demonstrated that for patients selected with an immunohistochemical score of 2+ or 3+, there was a significant improvement in median overall survival if they received trastuzumab plus chemotherapy compared with chemotherapy alone. 22 Subgroup analysis of data from the phase 2 monotherapy trial showed that patients with the highest HER2 protein overexpression levels (scored immunohistochemically as 3+) had a better response rate than patients with an immunohistochemical score of 2+ (18% vs 6%). 26 An immunohistochemical score of 2+ is now considered an equivocal result requiring confirmation as discussed subsequently. The CTA was the prototype for the development and eventual FDA approval of 2 HER2 immunohistochemical assays, the DAKO HercepTest assay (DAKO, Carpinteria, CA), which uses a rabbit polyclonal antibody, and the Ventana PATHWAY assay (Ventana Medical Systems, Tucson, AZ), which uses a mouse monoclonal antibody (CB11). The HercepTest results were compared with those obtained using the CTA and demonstrated a concordance rate of greater than 75%. Similarly, the PATHWAY assay using CB11 (part of the original CTA) was compared with HercepTest and demonstrated equivalent results to gain FDA approval. Several assessments of the accuracy, reproducibility, and concordance rates between different immunohistochemical reagents across different populations of patients have been reported. 16,36-44 Fluorescence In Situ Hybridization FISH is a clinically validated and approved alternative to immunohistochemical analysis for the assessment of HER2 status. It uses fluorescent-labeled nucleic acid probes to enumerate the average number of HER2 gene copies within the nuclei of tumor cells. Like immunohistochemical analysis, FISH testing is performed on formalin-fixed, paraffin-embedded (FFPE) clinical samples and is also morphologically driven. In the FDA-approved PathVysion assay (Vysis, Downers Grove, IL), 2 differently colored fluorescent probes are used to enumerate the number of HER2 gene signals and the number of copies of chromosome 17 (CEP17) in tumor cell nuclei. The HER2 probe binds to the DNA of the HER2 gene and the CEP17 probe binds to the centromeric region of chromosome 17. A ratio of the average number of copies of HER2/CEP17 is calculated that helps to distinguish true gene amplification (HER2/CEP17, 2) from Table 2 Scoring Criteria for the Evaluation of HER2 Immunohistochemical Results Score/HER2 Level of Expression Tumor Cell Membrane Staining Pattern (see Image 1) 0/Negative No staining 1+/Negative Faint incomplete membrane staining, >10% of cells * 2+/Weak positive Weak to moderate complete membrane staining, >10% of cells * 3+/Strong positive Strong complete membrane staining, >10% of cells * * Initial scoring criterion required for enrollment in the original pivotal clinical trials required that the staining pattern seen be more than 10% of tumor cells. The revised scoring criterion from the updated HER2 testing guidelines jointly issued by the American Society of Clinical Oncology and College of American Pathologists are given in Figures 1 and 2. Am J Clin Pathol 2008;129:263-273 265 265 DOI: 10.1309/99AE032R9FM8WND1 265

Hicks and Kulkarni / HER2: BIOLOGIC RELEVANCE AND DIAGNOSIS B C D Image 1 Immunohistochemical (IHC) assessment of the level of HER2 protein expression at the tumor cell membrane using the US Food and Drug Administration approved DAKO HercepTest kit according to the manufacturer s instructions. A, Cases with no membrane staining are scored as IHC 0. B, Cases scored as IHC 2+ demonstrate circumferential membrane staining in more than 10% of tumor cells, but the staining ring is thin. C, Cases scored as IHC 1+ demonstrate partial weak membrane staining in more than 10% of tumor cells with no complete circumferential staining. D, Cases scored as IHC 3+ demonstrate circumferential membrane staining in more than 10% of tumor cells, but the staining ring is thick and has a retractile quality (A-D, 400). pseudoamplification owing to multiple copies of chromosome 17 (polysomy), which would have an HER2/CEP17 ratio of less than <2. A pathologist is required to examine samples using a fluorescence microscope and correlate the number of HER2 gene signals from tumor cells within areas of invasive tumor Image 2. FISH assays received FDA approval as a method for the assessment of HER2 status after a retrospective analysis of samples from the pivotal clinical trials showed high concordance between the CTA and FISH.35 In addition, this analysis also showed that for patients with tumors that 266 266 Am J Clin Pathol 2008;129:263-273 DOI: 10.1309/99AE032R9FM8WND1 demonstrated HER2 gene amplification, median overall survival was significantly improved in patients who received trastuzumab.45 Equivocal Immunohistochemical Results Only cases with strong circumferential membrane staining using an immunohistochemical score of 3+ show a good concordance with HER2 gene amplification by FISH, and it is patients with these findings who are most likely to benefit from trastuzumab therapy. Cases that demonstrate an HER2 immunohistochemical score of 0 or 1+ have shown a good A

Anatomic Pathology / REVIEW ARTICLE A B Image 2 Fluorescence in situ hybridization (FISH) assay for HER2 quantitatively measures the level of HER2 gene amplification This image shows the appearance of the dual-color FISH assay (PathVysion assay). Infiltrating tumor cell nuclei are highlighted by the blue fluorescence of the 4',6-diamidino-2-phenylindole (DAPI) DNA counterstain. A, A nonamplified breast carcinoma with an HER2 (red signals) to CEP17 (green signals) ratio of <2 (DAPI counterstain, 1,000). B, An HER2-amplified breast carcinoma with increased numbers of HER2 gene signals (red signals) and an HER2/CEP17 ratio of 2 (DAPI counterstain, 1,000). correlation with no amplification of the HER2 gene, and these cases are considered HER2. An immunohistochemical score of 2+ is an equivocal result; these cases demonstrate a poor correlation with HER2 gene status in most published reports and should be considered inconclusive. All breast cancer cases with an HER2 immunohistochemical score of 2+ should be reflex tested for HER2 gene amplification using FISH, as outlined in the updated American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) HER2 testing guidelines detailed subsequently. 46 Equivocal Fluorescence In Situ Hybridization Results The original evaluation of clinical outcomes according to HER2 status detected by FISH considered specimens with an HER2/CEP17 ratio of 2.0 or more to be positive for HER2 gene amplification, dividing patients into 2 categories, FISH+ or FISH. Although this approach works well for most patients with breast cancer, approximately 3% of cases 46 will have ratios very close to this cutoff point. The most appropriate HER2 status for such patients falls in this gray zone (now defined as a FISH ratio of 1.8-2.2) and remains unclear. To address this, the new ASCO/CAP HER2 guidelines have added equivocal categories for FISH results, similar to those for immunohistochemical analysis, in the hope that this will help prompt further study to better define the potential benefit from HER2-directed therapy for patients with FISH results that fall within this equivocal or borderline category. 46 HER2 Testing Issues Concordance Between Methods Immunohistochemical and FISH assays provide complementary results, each evaluating a different facet of the biologic events underlying HER2-driven breast cancer. In addition, each assay has important methodological and interpretive advantages and disadvantages that need to be considered when interpreting HER2 test results. Retrospective analyses of patient outcomes and survival from the trastuzumab pivotal clinical trials and other series have suggested that FISH analysis is superior to immunohistochemical analysis in predicting response to trastuzumab. 39,45 This is despite the fact that other studies examining HER2 overexpression in frozen sections of breast tumors have found a high degree of concordance with HER2 gene amplification. 16 Furthermore, the target of trastuzumab is the protein product of the gene and not the gene itself. 47 Concordance rates similar to those seen in frozen tumors have not been reported when using immunohistochemical analysis for HER2 evaluation in FFPE tissue samples. Although the published HER2 protein/gene results in FFPE clinical samples have been shown to have a good correlation by many authors, 37,38 others have reported significant discordant results between FISH and immunohistochemical analysis and have questioned the accuracy of immunohistochemical HER2 testing in formalin-fixed tissues. 15,35,39,40,44,48 Am J Clin Pathol 2008;129:263-273 267 267 DOI: 10.1309/99AE032R9FM8WND1 267

Hicks and Kulkarni / HER2: BIOLOGIC RELEVANCE AND DIAGNOSIS It remains unclear whether discordances between these 2 methods reflect real biologic differences between tumors or whether they represent an artifact due to analytic and preanalytic variables that can affect assay performance. In this regard, several hypotheses have been proposed to account for discordant results. Proposed explanations include adverse effects of fixation, 41,49 polysomy for chromosome 17, 11,50-52 subjectivity and reproducibility of scoring criteria, 49,53,54 and widely varying sensitivities and specificities among antibodies used for immunohistochemical detection of HER2. 16,49,55 Many authors believe that in most cases, the lack of concordance associated with immunohistochemical analysis can be attributed to variability in testing methods rather than problems inherent to a particular method or true, biologically meaningful discordances 16,49,55 ; however, this remains to be fully defined. Discordant results cannot be entirely attributable to an artifact of immunohistochemical HER2 testing, as some authors have suggested, given that in subset analysis of data from the pivotal clinical trials, the patients with tumors characterized immunohistochemically as 3+ and FISH seem to have also received some benefit from trastuzumab therapy; however, the number of patients in this subgroup was small. 56 Despite the lack of perfect concordance between these 2 methods, patients with tumors characterized immunohistochemically as 3+ independent of FISH status or FISH+ independent of immunohistochemical status generally had similar times to progression and overall survival after receiving treatment with a trastuzumab-containing treatment regimen. 56 Furthermore, it has become increasingly clear that rigorous quality control, together with standardization of reagents, assay performance, and test interpretation, can lead to improved agreement between immunohistochemical and FISH results for HER2. Concordance Between Laboratories The results of HER2 assay testing must be reliable and consistent so that valid comparisons can be made between institutions and laboratories. However, recent studies evaluating the concordance of HER2 testing between laboratories have identified potential concerns in this regard. 57-59 One of the aims of the NSABP B-31 clinical trial was to assess the concordance between local and central laboratories engaged in HER2 testing. 57 The original eligibility for the trial was based on the HER2 assay results submitted by the accruing institution. A HercepTest immunohistochemical score of 3+ and gene amplification by FISH were both acceptable inclusion criteria for participation in the trial. A central review of the first 104 cases entered in the trial based on their immunohistochemical results showed that, of the 104 cases, 82 were found to have immunohistochemical scores of 3+ with the HercepTest (21.2% discordant); 82 out of 104 cases were also found to be FISH+ using the PathVysion assay (21.2% discordant). 57 Interestingly, there was less discrepancy between results when a high-volume laboratory (defined as performing at least 100 cases per month on average) performed the assay compared with laboratories with lower volumes. Subsequently, NSABP amended the eligibility criteria for the NSABP B-31 trial to include only patients whose tumors had an immunohistochemical score of 3+ according to an NSABP-approved reference laboratory or tumors demonstrating gene amplification by FISH from any laboratory. Based on these revised eligibility criteria, the rate of immunohistochemical discordance on central review dropped to about 3% in 240 patient samples assessed. A similar analysis was performed on the first 119 specimens from patients enrolled in the NCCTG N9831 clinical trial. This demonstrated remarkably similar results, with a 26% discordance rate between the local laboratory and central testing. 58 A more recent report cited potential contributing factors to explain these discrepancies, including lack of standardization of testing protocols and inexperienced test interpretation. 59 Standardization and Quality Control for HER2 Testing The results of these studies indicate a need for specific guideline standardization for the assessment of HER2 status. Adherence to strict laboratory quality control, improvements in tissue fixation and embedding procedures, standardization of immunohistochemical methods, and the use of automated image analysis to remove some of the subjective nature of assay interpretation may all increase the precision of immunohistochemical testing for HER2 status. 44,60 Computerized automated cellular imaging systems (ACIS; DAKO) are now commercially available and can improve the accuracy and reliability of HER2 immunohistochemical analysis. The high degree of correlation between immunohistochemical analysis using ACIS and results obtained using FISH indicates that errors in manual immunohistochemical interpretation are a contributing factor to discrepant results. 61 However, image analysis should not be considered a substitute for the standardization of tissue handling, fixation, processing, reagents, and assay procedures or for rigorous training to improve proficiency in HER2 testing. HER2 Testing Guidelines A joint ASCO/CAP Task Force recently released updated guidelines and recommendations for HER2 testing, which were simultaneously published in the Journal of Clinical Oncology 46 and the Archives of Pathology and Laboratory Medicine. 62 The purposes of these new guidelines were to 268 Am J Clin Pathol 2008;129:263-273 268 DOI: 10.1309/99AE032R9FM8WND1

Anatomic Pathology / REVIEW ARTICLE Breast cancer specimen (invasive component) HER2 testing by validated IHC assay for HER2 protein expression Positive for HER2 protein expression IHC 3+ (defined as uniform intense membrane staining of >30% of invasive tumor cells) Positive for HER2 gene amplification address issues relevant to improving HER2 testing accuracy and to reduce the variation in results between local laboratories. The intent was to provide concrete and practical solutions to the real-world problems encountered in HER2 testing in breast cancer. Current guidelines recommend that HER2 overexpression or amplification be evaluated in all cases of invasive breast cancer. The elements of these guidelines include testing algorithms for immunohistochemical analysis and FISH, specifying how these tests should be used in various circumstances and how results should be interpreted and reported Figure 1 and Figure 2. Based on the new guidelines, an immunohistochemical result of 3+ requires uniform, intense membrane staining in more than 30% of cells, compared with the original scoring criterion of more than 10% of cells. 35 The guidelines did not recommend one testing method over another. Interestingly, the ASCO/CAP Task Force recognized that there is an equivocal gray zone or borderline category that is occasionally encountered when interpreting the results from immunohistochemical and FISH assays for HER2. These equivocal categories recognize that the HER2 test results represent a continuous rather than a categorical variable and can no longer be reported simply as binary. The equivocal category for immunohistochemical assays must be confirmed by reflex FISH analysis of the sample, whereas equivocal FISH results can be confirmed by counting additional cells, repeating the FISH assay, or confirmatory immunohistochemical testing. Equivocal for HER2 protein expression IHC 2+ Test with validated assay for HER2 gene amplification Equivocal HER2 gene amplification result (patients with HER2/CEP17 ratio 2.0 were eligible for the adjuvant trastuzumab trials) Negative for HER2 protein expression IHC 0 or 1+ Negative for HER2 gene ampification Figure 1 Recommended algorithm for immunohistochemical analysis (IHC). From Wolff et al. 46 Reprinted with permission from the American Society of Clinical Oncology. An important caveat for applying the HER2 algorithm is that it is dependent on assurance that the laboratory has established high concordance rates between FISH and immunohistochemical analysis of at least 95% for positive and negative Positive for HER2 gene amplification (FISH ratio >2.2 or HER2 gene copy >6.0) Breast cancer specimen (invasive component) HER2 testing by validated FISH assay for HER2 gene amplification Equivocal for HER2 gene amplification (FISH ratio 1.8-2.2 or HER2 gene copy 4.0-6.0) Count additional cells for FISH or retest, or test with HER2 IHC Equivocal HER2 gene amplification result (patients with HER2/CEP17 ratio 2.0 were eligible for the adjuvant trastuzumab trials) Negative for HER2 gene amplification (FISH ratio <1.8 or HER2 gene copy <4.0) Figure 2 Recommended algorithm for fluorescence in situ hybridization (FISH). From Wolff et al. 46 Reprinted with permission from the American Society of Clinical Oncology. Am J Clin Pathol 2008;129:263-273 269 269 DOI: 10.1309/99AE032R9FM8WND1 269

Hicks and Kulkarni / HER2: BIOLOGIC RELEVANCE AND DIAGNOSIS test results. These high concordance rates for HER2 testing need to be established according to new test validation requirements. By 2008, all CAP-accredited pathology laboratories must have validated their HER2 assays against another previously validated assay. The laboratory has a choice between using a validated in-house assay or a validated similar assay performed by another laboratory. The samples used for the validation need to be handled and fixed in a manner similar to the clinical material analyzed by the laboratory. A range of 25 to 100 invasive breast cancers is required for validation, and the concordance between methods or laboratories must be at least 95% for positive and negative test results. An important goal of these new guidelines was to identify specific elements in the process of HER2 testing that contribute to variation and provide specific recommendations to standardize these elements of the procedure. Specific areas in which recommendations on standardization were made included specimen handling and fixation requirements, method validation, reporting elements, internal quality assurance efforts, and participation in mandatory proficiency testing. These recommendations state that the gross examination of the samples be performed as soon as possible and that these tissues need to be fixed in 10% neutral buffered formalin for no less than 6 hours and not more than 48 hours. If alternative fixatives are used, the test must be validated against buffered formalin fixation using the same HER2 assay, and concordance must be 95%. An important question raised by these new guidelines is How will laboratories and pathologists be evaluated and judged on their performance in meeting these new higher standards for HER2 testing? Mandatory proficiency testing will occur twice a year. Laboratories will receive unknown test cases using tissue arrays (40 cases per challenge for immunohistochemical analysis and 20 cases per challenge for FISH). A satisfactory performance requires a 90% correct response on the graded challenges for either test. In addition, inspectors will review the laboratory validation process, procedures, and documentation of quality assurance results (including proficiency testing) as a part of the standard laboratory inspection process. The ASCO/CAP guidelines are incomplete with respect to how pathologists and oncologists must deal with a result that falls within the equivocal ranges for immunohistochemical analysis and FISH. What is clear is that equivocal results of a single test require some additional action in an attempt to resolve the HER2 status for the patient. The authors of the guidelines note that patients with equivocal HER2 test results constitute a poorly studied subgroup with uncertainty as to the potential benefit from HER2-directed therapy. 46 The hope is that as sufficient detailed data are accumulated from clinical trials with trastuzumab, a subset analysis of the equivocal group could be performed to determine the clinical usefulness and potential benefit of this treatment for these patients. Another important area not covered by the ASCO/CAP guidelines, but in need of clarification, is the significance of finding genotypic abnormalities other than gene amplification with HER2 FISH assays. Polysomy for chromosome 17 is not infrequently encountered when performing FISH assays for HER2 with the Vysis PathVysion kit; however, the exact incidence is dependent on the criterion used to define the presence of a polysomic state. Reports from the literature suggest that this incidence ranges from 7.5% to more than 50%. 11 These differences in incidence are most likely due to the different criteria that have been used to define chromosome 17 polysomy in these reports, 11 and they make it difficult to determine the clinical significance of this result. Even less commonly encountered are findings related to genotypic heterogeneity for HER2 amplification, monosomy for chromosome 17, and monoallelic deletions of the HER2 gene. In addition to clinical studies, better and more uniform criteria are needed to help define these abnormalities and thereby establish their relevance in terms of potential benefit from trastuzumab therapy. Despite these omissions, it is hoped that when the new ASCO/CAP HER2 testing guidelines are fully implemented, they will lead to improvement in the quality of HER2 testing, better patient selection for trastuzumab and other HER2-targeted therapies, and, ultimately, to improved patient outcomes. Conclusions The assessment of HER2 status in breast cancer aids in the identification of patients who are the most likely to benefit from receiving trastuzumab. This approach to clinical management is critically dependent on accurate and reproducible assay results. Among the most important lessons learned from using HER2 testing is the need for standardization of all aspects of the handling of clinical samples. This includes all aspects of preanalytic tissue sample handling, the type and duration of fixation, tissue processing, assay performance, interpretation, and reporting. Regardless of the laboratory method used, specific guidelines and training to gain proficiency in HER2 testing are needed to ensure accuracy, specificity, and reproducibility of the test results. Current guidelines recommend that HER2 overexpression or amplification be evaluated for every patient with newly diagnosed breast cancer. Immunohistochemical analysis and FISH are considered equally appropriate testing methods for clinical laboratories, provided that high concordance rates are established and 270 Am J Clin Pathol 2008;129:263-273 270 DOI: 10.1309/99AE032R9FM8WND1

Anatomic Pathology / REVIEW ARTICLE appropriate quality control procedures are in place. A rigorous quality control program, experience, and proper training for accurate interpretation, together with proficiency testing, are not only required, but also will soon be mandatory for all local laboratories engaged in HER2 testing. The goal should be to achieve a greater than 95% concordance between immunohistochemically negative/fish and between immunohistochemically 3+/FISH+ results. This concordance should be established for each laboratory by rigorous test validation. For cases in which the immunohistochemical result is indeterminate (scored as 2+), the tissue sample should be reflex tested by FISH for HER2 gene amplification. Immunohistochemical assays for HER2 testing are appealing for several practical reasons, including availability, lower cost, turnaround-time, and adaptability to most pathology practices. However, legitimate concerns about the effects of fixation on antigenicity, false-positive and false-negative results, and the subjectivity of scoring remain. Studies have shown that laboratories with greater experience and higher testing volumes produce more reliable results than those that test samples less frequently. This, again, emphasizes the importance of standardization, proper assay performance, and experience with and training in HER2 test interpretation. It follows that a commitment to reliable and reproducible test results is essential for any laboratory performing HER2 determinations. The use of FDA-approved, clinically validated test kits that contain high-quality reagents of known sensitivity and specificity, together with meticulous attention to quality control, interpretation, and reporting, will, in all likelihood, improve concordance rates between immunohistochemical and FISH results, which will ultimately result in selecting the most appropriate treatment regimen for patients diagnosed with breast cancer. From the Departments of 1 Pathology and Laboratory Medicine and 2 Surgical Oncology, Roswell Park Cancer Institute, Buffalo, NY. Address reprint requests to Dr Hicks: Dept of Pathology and Laboratory Medicine, Roswell Park Cancer Institute, Elm and Carlton Sts, Buffalo, NY 14263. Writing assistance for this manuscript was provided by Genentech. References 1. Greenlee RT, Murray T, Bolden S, et al. Cancer statistics, 2000. CA Cancer J Clin. 2000;50:7-34. 2. Esteva FJ, Sahin AA, Cristofanilli M, et al. Molecular prognostic factors for breast cancer metastasis and survival. Semin Radiat Oncol. 2002;12:319-328. 3. Singletary SE, Allred C, Ashley P, et al. Staging system for breast cancer: revision for the 6th edition of the AJCC Cancer Staging Manual. Surg Clin North Am. 2003;83:803-819. 4. Hicks DG, Dawson A, Mattingly S, et al. Unmet need for new prognostic and predictive markers in breast cancer. Arch Pathol Lab Med. 2005;129:1372-1374. 5. Hayes DF. Prognostic and predictive factors revisited. Breast. 2005;14:493-499. 6. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med. 2004;10:789-799. 7. Yarden Y. Biology of HER2 and its importance in breast cancer. Oncology. 2001;61(suppl 2):1-13. 8. Yarden Y, Sliwkowski MX: Untangling the ErbB signaling network. Nat Rev Mol Cell Biol. 2001;2:127-137. 9. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. 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