J Clin Oncol 24: by American Society of Clinical Oncology INTRODUCTION

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1 VOLUME 24 NUMBER 19 JULY JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T HER2 Testing by Local, Central, and Reference Laboratories in Specimens From the North Central Cancer Treatment Group N9831 Intergroup Adjuvant Trial Edith A. Perez, Vera J. Suman, Nancy E. Davidson, Silvana Martino, Peter A. Kaufman, Wilma L. Lingle, Patrick J. Flynn, James N. Ingle, Daniel Visscher, and Robert B. Jenkins From the Mayo Clinic, Jacksonville, FL; Mayo Clinic, Rochester, MN; Sidney Kimmel Cancer Center at Johns Hopkins, Baltimore, MD; John Wayne Cancer Institute, Santa Monica, CA; Norris Cotton Cancer Center at Dartmouth-Hitchcock Medical Center, Lebanon, NH; and the Metro Minnesota Clinical Community Oncology Program, St Louis Park, MN. Submitted September 7, 2005; accepted April 12, Supported by grants from the Breast Cancer Research Foundation, New York, NY; Genentech Inc, South San Francisco, CA; and the National Cancer Institute (CA25224), National Institutes of Health, Bethesda, MD. Presented in part at the 40th Annual Meeting of the American Society of Clinical Oncology, New Orleans, LA, June 5-8, Authors disclosures of potential conflicts of interest and author contributions are found at the end of this article. Address reprint requests to Edith A. Perez, MD, Mayo Clinic, 4500 San Pablo Rd, Jacksonville, FL 32224; perez.edith@mayo.edu by American Society of Clinical Oncology X/06/ /$20.00 DOI: /JCO A B S T R A C T Purpose To evaluate concordance between local and central laboratory HER2 testing results in patients from the North Central Cancer Treatment Group (NCCTG) N9831 adjuvant trial of trastuzumab. Patients and Methods NCCTG N9831 is a randomized, phase III clinical trial comparing three drug regimens: doxorubicin/ cyclophosphamide followed by paclitaxel with trastuzumab added concurrently, sequentially, or not at all as adjuvant therapy for women with HER2-positive resected breast cancer. Originally, patients were eligible if their tumors were HER2 positive by either local laboratory immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). A protocol modification in 2002 made central laboratory testing mandatory, with additional testing of discordant cases conducted by a reference laboratory. Concordance between local and central laboratory, and level of agreement between central and reference laboratory HER2 findings in discordant cases were examined. Results HER2 positivity was confirmed in 85.8% of 2,535 patients registered since March When local and central evaluation used the same methodology, concordance was 88.1% for FISH and 81.6% for a diagnostic test for presence of the HER2 protein. Among discordant cases examined at the reference laboratory, there was 94.3% agreement for IHC (0, 1, 2 ) and 95.2% agreement for FISH (not gene amplified). Conclusion There was a high degree of discordance between local and central testing for IHC and FISH, but a high degree of agreement between central and reference laboratories. These findings support the importance of using high-volume, experienced laboratories for HER2 testing to improve the process of selecting patients likely to benefit from trastuzumab therapy. J Clin Oncol 24: by American Society of Clinical Oncology INTRODUCTION Amplification of the HER2 gene is the underlying molecular abnormality that causes overexpression of the HER2 protein on the cell surface of 25% to 30% of all breast tumors. 1 HER2-positive breast cancer has a significantly worse prognosis than breast cancer with normal HER2 levels, with an increased risk of recurrence and more aggressive disease course. 1,2 Trastuzumab (Herceptin; Genentech Inc, South San Francisco, CA), a recombinant monoclonal antibody that specifically targets the HER2 receptor, is a proven therapy for HER2- positive metastatic breast cancer. 3-5 Concomitant with the development of trastuzumab, assays were developed to determine which patients had HER2-positive breast cancer and might benefit from this drug. Initial clinical trials were performed using immunohistochemistry (IHC), which measures HER2 protein levels on the cell surface, to determine HER2-positivity in breast cancer specimens. IHC scoring is performed on a subjective basis by a pathologist, who grades HER2 immunostaining as 0, 1, 2, or3 on the basis of the percentage of malignant cells stained and the degree of membrane staining present in these malignant cells. Two types of IHC test to determine HER2 status are approved by the US Food and Drug Administration (FDA): HercepTest(Dako, Carpinteria, CA) andpathway(ventana Medical Systems Inc, Tucson, AZ). More recently, fluorescence in situ hybridization (FISH), which directly measures HER2 gene amplification, has been added to the armamentarium of tests that can be used to identify patients with 3032

2 HER2 Testing in N9831 Adjuvant Trial HER2-positive tumors for trastuzumab therapy. FISH testing results are semiquantitative, based on the average ratio of HER2 signals to CEP17 signals in non-overlapping interphase nuclei of the lesion. Three types of FISH test that determine HER2 status are FDA approved: PathVysion (Vysis Inc, Downers Grove, IL; 2.0 ratio HER2:CEP17 considered amplified), INFORM (Ventana Medical Systems Inc; 5.0 gene copies of HER2 considered amplified), and HER2 FISH pharmdx (Dako; 2.0 ratio HER2/CEN-17 considered amplified). A strongly HER2-positive tumor obtains the most clinical benefit from trastuzumab. 3-5 Subgroup analysis of a multinational, phase III trial in 469 patients showed that patients with IHC 3 tumors (using the noncommercially available Clinical Trials Assay) experienced significant improvements in all clinical outcomes considered (response rate, duration of response, time to disease progression, and overall survival) with trastuzumab therapy. In contrast, benefit in patients with IHC 2 tumors was minimal, 5 an observation that prompted a retrospective analysis of tumor samples from the pivotal trial that showed clinical benefit from trastuzumab was restricted to patients with HER2-amplified tumors. 6 HER2 status not only predicts potential benefit from trastuzumab, 7 but also may predict sensitivity to certain cytotoxic drugs and antiestrogens. 8 Studies suggest that patients with HER2-positive, lymph node positive breast cancer may benefit more from anthracyclinecontaining chemotherapy than non anthracycline-based regimens compared with patients who are not HER2 positive Although more data are needed, evidence also suggests that patients whose tumors overexpress HER2 may have worse outcomes with antiendocrine therapy and may not benefit from tamoxifen to the same extent as patients without HER2 gene amplification Trastuzumab is being investigated in the adjuvant setting for primary breast cancer in four large, randomized phase III clinical trials involving more than 13,000 patients. The trials offer the opportunity to examine HER2 testing by IHC and FISH in a large population. Concordance between testing methods and between testing laboratories using the same methods is important for interpreting results from clinical trials and selecting treatment. Eliminating false-positive and false-negative results is an important objective for clinical trial enrollment. Because patients with false-positive HER2 results may receive little benefit from an HER2-targeted agent, excluding them from clinical trials will help better evaluate the true effect of trastuzumab therapy. 15 The North Central Cancer Treatment Group (NCCTG) Intergroup trial N9831 is a randomized phase III clinical trial conducted by the Breast Cancer Intergroup of North America evaluating trastuzumab as adjuvant therapy for patients with HER2-positive resected early breast cancer. The trial compares three treatment regimens: doxorubicin/cyclophosphamide followed by (1) paclitaxel; (2) paclitaxel followed by trastuzumab; or (3) paclitaxel plus trastuzumab followed by trastuzumab alone (Fig 1). Details of the trial protocol have been published previously. 16,17 Patients were originally eligible for NCCTG N9831 if they had strong HER2 positivity according to a local laboratory s IHC or FISH test. A strongly positive IHC result was defined as IHC 3 with HercepTest or any other IHC assay. A positive FISH result was defined as at least 2.0 HER2 probe/control probe ratio with PathVysion. A preliminary, protocol-specified review of the first 119 patients revealed poor concordance between local and central laboratory assessments of Fig 1. Treatment schedule. Doxorubicin 60 mg/m 2 and cyclophosphamide 600 mg/m 2 (AC); paclitaxel, 80 mg/m 2 every week (qw); trastuzumab (H), 4 mg/kg loading dose followed by 2 mg/kg qw for 52 weeks. Radiation and/or tamoxifen for premenopausal women and radiation and/or hormonal therapy (tamoxifen or aromatase inhibitor) for postmenopausal women were administrated, if indicated, after 6 months of chemotherapy. q3w, every 3 weeks. HER2 status. 18 Only 67% of samples classified as HER2-positive by local FISH testing could be confirmed by central FISH testing, and overall concordance between local and central IHC testing was only 74%. For these reasons, eligibility criteria were modified in March 2002 to require central laboratory confirmation of HER2 status. 18 This article presents results of HER2 testing concordance between local and central laboratory testing after protocol amendment and examines the level of agreement between the central and reference laboratory testing results for cases with discordant findings. PATIENTS AND METHODS NCCTG N9831 HER2 Testing Algorithm According to the protocol amendment, results of local HER2 testing could qualify a patient for inclusion in the NCCTG N9831 trial, however HER2 status had to be confirmed centrally (at Mayo Medical Laboratories; Rochester, MN) by either IHC (HercepTest) or FISH (PathVysion) before completing the first chemotherapy regimen. If strong HER2 positivity was not confirmed by central testing, an independent reference laboratory (high-throughput commercial facility) examined the case (Laboratory Corporation of America, Research Triangle Park, NC [LabCorp]) to determine HER2 status using IHC (HercepTest) and FISH (PathVysion). If the tumor was not HER2 IHC 3 and not HER2 gene amplified by the reference laboratory, the patient and physician were informed, and adjuvant therapy was continued at the physician s discretion (Fig 2). HER2 Testing Methodologies at Central and Reference Laboratories A paraffin-embedded breast tumor block or 15 unstained 4- sections were submitted for central laboratory testing for each patient. Identical methods were used at Mayo Medical Laboratories and the reference laboratory. IHC analysis for HER2 protein overexpression was performed on paraffinembedded tissue sections using the HercepTest according to manufacturer s recommendations. To validate the staining procedure, both positive and negative control cell lines were tested in parallel; these are components of the HercepTest. Benign epithelial cells are also routinely examined as another control. In addition, the central laboratory subscribes to the College of American Pathologists (CAP) proficiency HER2 IHC testing program. 19 FISH analysis for HER2 amplification was performed on deparaffinized tissue sections using the PathVysion HER2 DNA probe kit. Detailed methodology is published elsewhere. 18,20,21 The central laboratory performs several measures to assess the quality control of the HER2 FISH test, following standard CAP guidelines as well as the guidelines of the American College of Medical Genetics. 19,22 Each lot of HER2 probe is tested against normal metaphases as well as amplified and nonamplified paraffin-embedded primary breast cancer specimens to assure that the probe is hybridizing correctly. In addition, each FISH assay includes a known positive control paraffin-embedded primary breast cancer specimen with HER2 amplification. Every month, the central laboratory assesses the prevalence of

3 Perez et al Fig 2. HER2 testing algorithm for North Central Cancer Treatment Group N9831. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; FISH, gene amplified; FISH, not gene amplified. HER2 amplification as well as other HER2 anomalies. As assessed by standard Westgard rules, 23 the performance of the assay has been stable. The central laboratoryalsoparticipatesinthecap-proficiencyher2fishtestingprogramaswell as biannual CAP and New York State laboratory inspections. 19 Local laboratories were not required to participate in external quality control programs. Statistical Analyses The study cohort included all patients enrolled in NCCTG N9831 from March 11, 2002, through to the completion of enrollment on April 30, 2005, who met all registration requirements and did not withdraw before treatment. Local laboratory testing was performed by Mayo Medical Laboratories in 99 cases. After registration, the 99 cases underwent central laboratory testing and, if appropriate, reference laboratory testing, in the same manner as all other patients. Statistical analyses were performed with and without the patients who used Mayo Medical Laboratories as their local laboratory. Because the results were similar in both instances, the results presented are those associated with the entire postaddendum study population. A patient s breast cancer was considered strongly HER2-positive if the tumor met local laboratory criteria for strong HER2 expression by IHC or HER2 gene amplification by FISH. Concordance between local and central laboratory HER2 findings was defined as the number of study patients whose tumors were found, on central laboratory testing, to be HER2 gene amplified and/or IHC 3 by HercepTest, divided by the total number of study patients. A 95% CI for this proportion was constructed using the properties of the binomial distribution. For patients with discordant local and central laboratory findings, agreement between central and reference laboratory findings was defined as the proportion of patients who the central and reference laboratories concluded did not have strongly HER2-positive breast cancer. Fisher s exact test was used to compare testing method concordance between local and central laboratories. RESULTS Patient Population From March 11, 2002, through April 30, 2005, 2,601 patients were registered onto NCCTG N9831. Thirty-eight patients were found to be ineligible, and 16 patients cancelled participation before the start of study treatment. Tumor specimens from the remaining 2,547 patients were submitted to the central HER2 testing laboratory at Mayo Medical Laboratories to confirm HER2 status and eligibility. Of these 2,547 patients, 12 (0.5%) had specimens lacking sufficient invasive tumor for central laboratory testing. Of the remaining 2,535 patients, 2,446 (96.5%) had sufficient invasive tumor for the central laboratory to perform IHC and FISH; 78 patients (3.1%) either lacked sufficient invasive tumor or had hybridization failure so that only central IHC results were available, and 11 patients (0.4%) either lacked sufficient invasive tumor or had poor IHC staining so that only central FISH results were available. Central laboratory testing of the 2,535 patients confirmed strong HER2 positivity (IHC 3 and/or gene amplified by FISH) in 2,176 cases (85.8%; 95% CI, 84.4% to 87.2%). Concordance Between Local Laboratory and Central Laboratory HER2 Findings To identify strongly HER2-positive breast cancer in these 2,535 patients, local laboratories used HercepTest (42.1%), other non- HercepTest IHC methods (25.2%), and FISH (32.7%; Table 1). In 23 patients (1%), central laboratory HER2 testing results were unavailable from the same method employed by the local laboratory because of insufficient tumor tissue, hybridization failure, or poor staining (HercepTest, five patients; non-herceptest, two patients; FISH, 16 patients). Concordance between local and central laboratory testing was examined in 2,512 remaining patients when the central laboratory findings were limited to the same technique used at the local laboratory to establish HER2 positivity (Table 2). Of the 1,063 patients whose local laboratory used the HercepTest to establish IHC 3 overexpression, 81.6% had IHC 3 overexpression confirmed by central laboratory testing with HercepTest. Of the 636 patients whose local laboratory used a non-herceptest technique to establish IHC 3 overexpression, 75.0% had IHC 3 overexpression confirmed by central laboratory testing with HercepTest. Of the 813 patients whose local laboratory used FISH to establish HER2 gene amplification, 88.1% had HER2 gene amplification confirmed by central laboratory testing with FISH (Fig 3). Concordance between local and central laboratory HER2 findings when FISH was used by both laboratories was greater than the concordance between local and central laboratory HER2 findings when HercepTest was used by both laboratories (Fisher s exact test P.001). It is not known how many local laboratories were of high volume ( 99 HER2 assays per month) or low volume ( 99 HER2 assays per month), and whether samples were sent to more-experienced/high-volume laboratories for IHC and/or FISH testing. Reference Laboratory Testing of Discordant Cases A total of 305 cases (12%) were not found to be strongly HER2- positive (Fig 4). Results from the central laboratory showed that HER2 Table 1. Testing Methods Used by Local Laboratories to Determine HER2 Status Specimens Test No. % HercepTest 1, Non-HercepTest FISH Total 2, NOTE. HercepTest, DAKO, Carpinteria, CA. Abbreviation: FISH, fluorescence in situ hybridization JOURNAL OF CLINICAL ONCOLOGY

4 HER2 Testing in N9831 Adjuvant Trial Table 2. Concordance Between Central and Local Laboratories Specimens Confirmed Agreement With Central Laboratory Test at Local Laboratory by Central Testing (No.) % 95% CI Method HercepTest 1, % to 83.9% HercepTest Non-HercepTest % to 78.3% HercepTest FISH % to 90.2% FISH NOTE. HercepTest, DAKO, Carpinteria, CA. Abbreviation: FISH, fluorescence in situ hybridization. Testing using the same method at both laboratories was not possible for 23 specimens. expression was not IHC 3 by HercepTest and the HER2 gene was not amplified by FISH (288 cases), or the central laboratory reported that the HER2 expression level was not IHC 3 by HercepTest and FISH results were not available because of hybridization failure or insufficient invasive tumor tissue (17 cases). In two (0.7%) of the 305 patients, central testing completely depleted the invasive component of the tumor. As a result, 303 (99.3%) local/central laboratory discordant cases were tested for HER2 at the reference laboratory. Agreement in terms of a lack of strong HER2 positivity was first examined considering only the IHC findings from the central and reference laboratories. Of the 303 specimens, IHC results were not obtained in three cases because of a lack of sufficient invasive tumor in the specimen. Among the remaining 300 cases, there were 17 cases with an HER2 IHC 0, 1,or2 expression level by central laboratory testing for which the reference laboratory reported an IHC 3 expression level. Thus, agreement between the central and reference laboratories in a lack of IHC 3 overexpression among the cases was 94.3% (283 of 300 cases). Agreement was then examined considering only FISH findings from the central and reference laboratories. Of the 303 specimens, 32 cases lacked central laboratory FISH results and/or reference laboratory FISH results. The central laboratory reported no HER2 gene amplification, and the reference laboratory reported HER2 gene amplification in 13 of the remaining 271 local/central laboratory discordant cases. Thus, agreement between central and reference laboratories in a lack of FISH gene amplification among cases was 95.2% (258 of 271 cases). Lastly, agreement in terms of a lack of strong HER2 positivity was examined considering IHC and FISH findings from the central and reference laboratories. Central and reference laboratories were said to agree if the reference laboratory did not find IHC 3 overexpression or HER2 gene amplification by FISH. There were two cases that lacked both reference laboratory IHC and FISH results. Among the 301 cases with reference laboratory IHC and/or FISH results, 28 cases (9.3%) were IHC 3 and/or FISH amplified. Thus, agreement between central and reference laboratories in a lack of both HER2 gene amplification and IHC 3 expression was 90.7% (273 of 301 cases). DISCUSSION Results from this study show that among patients found to be strongly HER2 positive by local laboratories, a considerable proportion of cases were unconfirmed when tested at a central laboratory: 18.4% for IHC HercepTest and 11.9% for FISH. When discordant cases were retested at a reference laboratory, the central (Mayo Medical Laboratories) and reference laboratories showed a high level of agreement: 94.3% for IHC (0, 1, and 2 ) and 95.2% for FISH (not gene amplified). The Fig 3. Central laboratory testing results. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; FISH, gene amplified; FISH, not gene amplified; NA, not available. HercepTest: DAKO, Carpinteria, CA

5 Perez et al Fig 4. HER2 status of local/central laboratory discordant cases. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; FISH, gene amplified; FISH, not gene amplified; NA, not available. reference laboratory did not retest local and central laboratory HER2- positive (FISH gene amplified or IHC 3 ) tumor samples. After the establishment of central HER2 testing in all patients in NCCTG N9831, 305 (12.0%) of 2,535 cases were discordant. There were 28 cases among the discordant cases found to be HER2 positive by the reference laboratory. Thus, 2,258 (89.1%) of the 2,535 patients registered after establishment of central testing were declared to have HER2-positive disease. This is a marked improvement compared with the 26% discordance rate among the 119 patients enrolled in NCCTG N9831 before mandatory central laboratory testing. 17 If central testing had not been implemented, 277 patients (8% of the total study cohort) unlikely to benefit from trastuzumab therapy would have been included in the efficacy analysis of this trial. For this reason, statistical power to detect differences in disease-free survival between patients randomly assigned to receive a trastuzumab-containing regimen and those randomly assigned not to receive a trastuzumab-containing regimen may have been diminished. Data are not available from our study concerning whether any of the patients found not to be HER2 positive by local laboratory testing would have been found to be HER2-positive by central laboratory testing, because these patients could not register onto NCCTG N9831. Other studies, however, highlight potential consequences of falsenegative HER2 testing. Previously, we conducted a retrospective analysis of 216 centrally tested specimens scored as IHC 2 by HercepTest and found that 12% had gene amplification by FISH. 20 Comparing local laboratory IHC and central laboratory FISH test results from 865 patient samples screened for entry in a community-based study, Anderson et al 24 found that 17 (5%) of 363 samples identified locally asihc0or1 were FISH amplified by central testing. Data are needed to determine the clinical benefit of trastuzumab in patients whose tumors are negative for protein overexpression (0, 1,or2 ) using HercepTest but have gene amplification by FISH analysis; an important issue in view of the lack of prospective data related to the benefit of trastuzumab in this subset and the fact that some tumors may be tested by only FISH and not IHC. The Cancer and Leukemia Group B (CALGB) 9840 trial randomly assigned patients with HER2- normal tumors (classified as IHC 0, 1,or2 determined by a local laboratory) to receive paclitaxel with or without trastuzumab and found that the addition of trastuzumab did not improve the efficacy of paclitaxel in this patient population. 25 Additional analyses, including correlation based on FISH status and central HER2 testing, in this population are ongoing. 25 False-positive and false-negative HER2 test results can partly be attributed to the lack of concordance between IHC and FISH, 26 particularly when IHC testing is performed locally and FISH testing is performed at a central laboratory. Anderson et al 24 found concordance between FISH results performed at a reference laboratory and IHC tests performed locally were 66% (IHC 2 /not amplified by FISH) and 73% (IHC 3 / FISH amplified). IHC 2 is the category most likely to be discordant with FISH HER2 status. In a study comparing FISH with HercepTest in central laboratories (IMPATH Inc, New York, NY), Owens et al 27 showed that only 23.3% of IHC 2 samples could be confirmed as HER2- positive by FISH. When IHC and FISH tests were performed centrally by Dybdal et al, 15 overall concordance was more than 80%, but accuracy varied according to IHC score. Agreement rates between FISH and IHC were only 24% for IHC 2 compared with 97% for IHC 0, 93% for IHC 1, and 89% for IHC 3. AnIHC2 test result is therefore a potentially unreliable indicator of HER2 status, regardless of whether the test is performed locally or centrally. Findings from this prospective study showing poor concordance between local and central HER2 testing confirm previous pilot results and are supported by other studies. 18,28 In the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 adjuvant trial of trastuzumab plus chemotherapy, Paik et al 28 found that 18% of the first 104 enrolled patients had community-based IHC assays that could not be confirmed by central HercepTest. In this study, greater concordance was observed between local and central laboratory IHC results for HercepTest assays than non-herceptest assays (81.6% v 75.0%, respectively). However, the higher concordance of HercepTest compared with non-herceptest assays in this study could be because HercepTest has been subjected to comprehensive standardization of IHC scoring criteria, and internal controls are a component of the test. Despite consensus with some investigators that FISH may be more reliable than IHC in testing HER2 gene amplification, concordance between local and central FISH results was only 88.1% 3036 JOURNAL OF CLINICAL ONCOLOGY

6 HER2 Testing in N9831 Adjuvant Trial compared with 95.2% between the central and reference laboratories. Dybdal et al 15 reported a similar FISH HER2 concordance (92%) between two reference laboratories. Therefore, discordance between local and central FISH testing in this study suggest that specific characteristics of local laboratories may contribute to discordance between local and central HER2 FISH and IHC testing. Concordance was significantly higher between local and central laboratories when both laboratories used FISH than when both laboratories used HercepTest (88.1% v 81.6%; P.001). However, possible reasons for the difference may be that (a) small local laboratories (performing 99 HER2 assays per month) send specimens to larger laboratories (performing 99 HER2 assays per month) for FISH analysis, which would be more cost-effective than setting up the assay in-house if only a few patients required the test each month; or (b) local laboratories that perform FISH may be more experienced than those that perform IHC, although this cannot be quantified in this study, because local laboratories were not required to adhere to specific quality control procedures. Although the central and reference laboratories have active quality-assurance and -control programs for their HER2 FISH and IHC studies, we did not canvas the local laboratories concerning their participation in such programs, as mentioned herein. The relative discordance between the central or reference laboratories with the local laboratory suggest that such programs should be strongly encouraged. The recent development of control cell lines with known levels of HER2 amplification and expression should facilitate the standardization of HER2 testing between institutions. 29 Methodologic factors may also contribute to discordance between local and central test results. 18 For example, IHC testing can yield false-negative results when the HER2 epitope is destroyed by formalin fixation. This process may occur in up to 20% of HER2- positive samples 30,31 and can be problematic when testing archive samples that have been stored long term (eg, when testing primary tumor samples at relapse). Conversely, tissue processing, reagent variability, antigen retrieval, and scoring can contribute to false-positive IHC results. 28,32 Potential concerns with FISH include difficulties with tumor heterogeneity between and within paraffin blocks; problems with slide pretreatment (eg, too much or too little enzymatic digestion); the semiquantitative nature of the test; differences in cutoff criteria; and diluted amplification when cancer cells are widely distributed among stroma. 33 In laboratories performing a small number of HER2 assays ( 99 per month), higher levels of discordance with central test results have been reported compared with high-volume laboratories. 28 In NSABP B-31, the first 104 cases submitted for eligibility had a 24% falsepositive rate in small-volume laboratories compared with 3% in larger-volume laboratories. Paik et al 28 postulated that bias introduced by pathologist overestimation of HER2-positive cases in small-volume laboratories may cause the high degree of discordance. This supports the importance of using a high-volume experienced laboratory for HER2 testing. In this study, local laboratories were not divided into small- and high-volume centers, and the impact of assay volume on accuracy was not determined. Sources of error in this study are most likely to be primarily methodologic, particularly in local laboratories that are not subject to quality-control procedures. However, interpretation of results may also be a key factor, especially in low-volume testing laboratories. Results from this study and other detailed analyses of HER2 testing emphasize the need for rigorous application of standardized procedures. All primary breast cancers should be tested for HER2 status using IHC and/or FISH. 34,35 For this reason, an algorithmic approach based on a quality-assurance program conducted in nearly 3,000 breast tumor specimens tested by FISH and IHC at a reference laboratory (PhenoPath Laboratories, Seattle, WA), has been proposed. 36 Similar to the proposal published by Perez et al 20 in 2002, the algorithm suggests using FISH when HER2 status is indeterminate (defined as 2 with IHC). The most recent National Comprehensive Cancer Network guidelines recommend that all IHC 2 results be confirmed by FISH testing. 35 Accurate determination of HER2 status will accrue savings by eliminating inappropriate treatment of patients with false-positive results and will increase aggregate effectiveness by minimizing falsenegative results. 37 Overall effectiveness of HER2-targeted therapy will be maximized with appropriate testing to identify the pathologic target in tumor specimens. 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Hum Pathol 35: , Elkin EB, Weinstein MC, Winer EP, et al: HER-2 testing and trastuzumab therapy for metastatic breast cancer: A cost-effectiveness analysis. J Clin Oncol 22: , 2004 Acknowledgment Writing assistance for this manuscript was provided by Genentech Inc, and additional editorial assistance was provided by Evelyn Bass and Daythel Williams. Authors Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. Author Contributions Data analysis and interpretation: Edith A. Perez, Vera J. Suman, Nancy E. Davidson, Silvana Martino, Peter A. Kaufman, Wilma L. Lingle, Patrick J. Flynn, James N. Ingle, Daniel Visscher, Robert B. Jenkins Manuscript writing: Edith A. Perez, Vera J. Suman, Nancy E. Davidson, Silvana Martino, Peter A. Kaufman, Wilma L. Lingle, Patrick J. Flynn, James N. Ingle, Daniel Visscher, Robert B. Jenkins 3038 JOURNAL OF CLINICAL ONCOLOGY