The HER2 oncogene, also known as c-erbB-2, is located on chromosome 17 (17q12-21.32) and is a member of the human epidermal growth factor receptor family.1 Its tyrosine kinase activities signal cellular proliferation and indirectly affect programmed cell death.2 Between 25% and 30% of breast cancers are characterized by amplification of the HER2 gene3 that is associated with, for instance, more rapid tumor growth, increased risk of recurrence after surgery, and poor clinical outcome.4–6 Because of its location on the outside of the cell and role in the pathogenesis of breast tumors, HER2 was thought to be an ideal molecular therapeutic target. The humanized monoclonal antibody, trastuzumab (Herceptin), directed against the extracellular portion of HER2 receptor is only active against HER2 overexpressing tumors.7–10 For that, evaluation of HER2 status is a critical element for determining which patients are eligible for being treated with trastuzumab, and recently the College of American Pathologists has recommended that HER2 status should be determined for all invasive breast cancer.11
However, there is still no consensus on which method is the most predictive. Two diagnostic techniques are the tests of choice for most pathologists and are often used together on paraffin-embedded block tissue sections processed in routine practice, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). Food and Drug Administration (FDA) approved tests for assessing HER2 status, which use IHC include Hercep Test (Dako Cytomation, Carpinteria, CA) and Pathway (Ventana Medical System, Inc, Tucson, AZ), and those using FISH include PathVysion (Vysis Inc, Downers Grove, IL), Inform (Ventana Medical Systems Inc) and Dako HER2 FISH pharmDx.
IHC has the advantage of being simple to perform, cheap, and readily available in most pathology laboratories although a number of preanalytical and subjective variables, such as tissue handling and fixation, and analytical variables such as antibody sensitivity and specificity, can affect the accuracy of test results. In general, FISH is more reliable and reproducible than IHC because it is less susceptible to the adverse effects of tissue handling and fixation but FISH is more complicated, time-consuming and it requires training, experience, and expensive equipment. Comparative studies have shown high concordance rates between immunohistochemical analysis and FISH in tumors with immunohistochemical scores of 0 or 1+ (negative) and 3+ (strongly positive), and discordance rates among cases with undetermined immunohistochemical scores of 2+. In these cases, FISH is considered gold standard for confirming or excluding cases read as 2+ immunohistochemically.3 Although it would be incorrect to claim that FISH is always over IHC, nevertheless, the HER2 protein overexpression can occur without gene amplification because of, for instance, a transcriptional or posttranslational activation,6 artifactual high sensitivity of immunohistochemical assays, single copy overexpression of the HER2 gene at the messenger RNA transcription level, gene amplification below the level of detection of FISH assays,12 or the presence of chromosome 17 polysomy.13,14
The aim of this study was to systematically review the studies that compared the most frequently used test to establish HER2 status, IHC, and FISH, in an attempt to evaluate the effectiveness and provide diagnosis strategies based on evidence. We summarized the IHC and FISH HER2 testing literature. Most articles evaluated the HercepTest IHC and PathVysion FISH assay. FISH was considered the gold standard test and the FISH positivity rate was calculated in each IHC score category in each study. The review showed that there was high concordance between the FISH and IHC0, IHC1+results as well as a lack of concordance between this method and the IHC2+ and IHC3+ scores. The percentage of cases with noncorrelating IHC and FISH results were essentially the same (83% of IHC2+ cases were FISH negative; 83.5% of IHC3+cases were FISH positive). Results from IHC testing indicated it was not sufficient for appropriate selection of patients who were suitable for trastuzumab treatment. In these cases, FISH is considered the gold standard for confirming or excluding HER2 amplification.
MATERIALS AND METHODS
Search Strategy and Data Sources
Methodology was based on both a structured search using a criteria set a priori and on a critical review of the literature found, together with a summary of the results and assessment of outcomes for the health system context.
The data sources used were the following electronic databases searched from 1966 to September 2006: MedLine, Embase, Centre for Review and Dissemination (CRD), and the Cochrane Library using literature search strategies (Table 1). REFERENCES of the identified systematic reviews were checked. The search was also run on the databases of European Agency for the Evaluation of Medical Products (EMEA), the Food and Drugs Administration (FDA), and the International Network of Agencies for Healthcare Technology Assessment (INAHTA). The search terms were debated and agreed by the research team and a documentation group convened to ensure the clinical utility of the review (Table 1).
Retrieved articles were independently assessed for inclusion by 2 reviewers and were selected for inclusion in the systematic review of the evidence if they met the following inclusion criteria: (1) Study design: health technology assessment reports, evaluation studies, systematic reviews, randomised clinical trials, cohorts studies, case series, and prospective studies; (2) Population: breast cancer patients; (3) Intervention: HER2 status testing (FISH and IHC), the studies must describe the comparison between at least these 2 techniques; (4) Comparison: any alternative; (5) Outcomes: specificity, sensitivity, concordance, detection limit, validity, reliability, and reproducibility; and (6) published in any language.
The studies were excluded if they were narrative and descriptive reviews, studies on other diseases, letters to the editor, editorials, commentaries, conference communications and panel of experts though we review them for any new information.
A critical appraisal was conducted using an ad hoc scale, which had been based on the CASPe scale (Critical Appraisal Skills Programme Spanish), available on http://www.redcaspe.org/. The quality of the studies included was examined by using this checklist, which contained the following questions about internal and external validity: did the trial address a clearly focused issue?; was the assignment of patients to HER2 testing randomized?; were all of the patients enrolling in the trial properly accounted for at its conclusion?; was the study personnel blinded to the intervention?; were the groups similar at the beginning of the trial?; were the groups treated the same?; what outcomes were measured?; how precise/accurate were the results?; how can the results from the study be applied to other populations?; and were all the clinically important outcomes considered? If the answer to all these questions is “yes,” we can conclude that the document is of quality. Taking into account these aspects, the researchers reviewed unblinded studies. Finally, the findings from individual studies were formally compared with establishing generalizability of the findings and the consistency of the results.
Assuming FISH is the gold standard, IHC was evaluated by comparison with FISH. The concordance between IHC and FISH was calculated as FISH positivity rate in IHC score category x (number of FISH positives in IHC score category x/number of patients in IHC category x), where IHC score category refers to the score categories 0, 1+, 2+, and 3+.15 The concordance ranged from 0% (no agreement) to 100% (perfect agreement).
A cost-effectiveness analysis realized by the Centre Universitarie de Santé McGill, Technology Assessment Unit (TAU) of the McGill University Health Centre (MUHC) de Montreal (Canada) entitle “Testing for HER2 positive breast cancer: a cost-effectiveness analysis” May, 2006 (available in http://www.mcgill.ca/tau/publications/2006/) was found.15 According to CASPe scale, we estimated that the methodology of work was clear, explicit, and answered the formulated question. Thus, our systematic review updated the above-mentioned report with the latter date being September, 2005 (date of the most recent included article).
One hundred ninety-three documents were located in the databases of Embase and Medline; 31 of them were published since September, 2005. The first selection was realized on discarding the abstract initially of 11 articles for not fulfilling the inclusion criteria related to the population, intervention, comparison, and/or outcomes, or for fulfilling some of the exclusion criteria. Twenty articles were selected to read full text. After reading, 3 documents were eliminated for not comparing IHC and FISH directly despite using both technologies. In conclusion, 176 publications were excluded from the systematic review (Fig. 1) and 17 publications were analyzed within this review (Table 2).
The major methodologic problems were the following:
* The tumor samples were classified by at least 2 independent pathologists and/or reference laboratory in 6 studies18,19,22,25,29,31 and the results from both HER2 assays were blinded in 2 reports.16,19
* There are several HER2 IHC antibodies available. In 5 articles, HER2 IHC testing was carried out using more than 1 antibody.18,25,28,29,31. In addition, 2 studies used several antibodies but their results were not separated clearly.23,24
* To obtain the maximum possible benefit is essential to standardize and validate analytic procedures, and also to use diagnostic tests approved by the FDA and/or the European Community. The Hercep Test (IHC) and Pathway (FISH) kits were used in 12 studies. The strict controls of quality, which should be used in the development of the nonstandard procedures, were not detailed in the other documents that were analyzed.
* FISH test was carried out only in the category IHC2+in 4 studies17,20–22 and in another, the FISH results did not specify the IHC category in which the tests were performed.29
* FISH was performed in all IHC categories but FISH scores appeared grouped.23 Therefore, it was not possible to classify the results according to each IHC category.
* The objectives of the 3 reports were HER2 status diagnostic techniques16,19,20 different from IHC and FISH. Nevertheless, they were included in the systematic review because of showing IHC and FISH results. Similarly, 3 clinical randomized essays were included. The first one established as inclusion criteria the HER2 status confirmation by a central laboratory,23 in the second one only HER2-positive breast cancer patients were selected,31 and in the last one the impact of the HER1 and HER2 amplification in the endocrine therapy was evaluated.16
The number of samples needed for a trustworthy validation has not been defined clearly, although it depends on the number of possible results and on the variation in the results found.33 The number of samples ranged between 42 (Willmore et al32) and 2535 (Perez et al23).
Evaluation of Available Data
The studies using Hercep Test were finally analyzed. They are summarized in Table 3 in which the number of neoplasia, the percentage of cases, and the percentage of FISH positive (PathVysion) in each IHC category are included.
The lowest FISH positivity rate was found in IHC0 category, increasing to the highest FISH positivity rate in the IHC3+ category. The percentage of FISH positives in the cases scored as negative (IHC0 and IHC1+) ranged from 0% to 20% for IHC0 and from 3.13% to 50% for IHC1+, for the positives (3+) from 50% to 96.8%, and for the undetermined (2+) from 8.3% to 92.3%. Nevertheless, the quality of the evidence was moderated in some of the works.
FISH positivity rate in each IHC category was not estimated in 4 studies.17–19,23 A study did not report sufficient FISH data in each IHC category (losses higher than 30%).26
Interobserver variability may occur in manually grading HER2 protein expression by IHC. A study compared the HER2 expression by IHC using a computer-based image analysis system with that of the gene amplification by FISH.27 The results showed that HER2 amplification correlated better with overexpression of the HER2 protein by IHC when the score was either <1.5 or >2.6 by automatic analysis system.
The study that included more number of cases was the one published by Reddy et al,24 with more than a thousand cases of breast cancer. This study was a clinical essay with trastuzumab, where HER2-positive breast cancer patients were selected. HER2 status was established by the same reference laboratory using IHC (Hercep Test) and FISH (PathVysion). The degrees of conformity between IHC and FISH improved sensitively owing to the variables related to the technology (fixation, used antibodies, automation, etc) and evaluation (training and homogeneous criteria of diagnosis) was minimized.
The distributions of the IHC scores and FISH positivity rate were determined using the data median of 6 articles (Table 4). FISH positive rate was 3.5% for IHC0, 5.8% for IHC1+, 17% for IHC2+, and 83.5% for IHC3+. These results revealed a very high concordance rate between IHC and FISH assessment of HER2 status in those tumors scored as 0 and +1 by IHC.
Accurate detection of the HER2 status in human breast tumors has become the most important criterion for appropriate selection of patients for HER2 target therapy with trastuzumab. There is a uniform acceptance of the need for HER2 testing in all patients with invasive breast carcinoma. Indeed, increasing numbers of investigators are suggesting that HER2 status is more important than histologic type.
IHC, which measures HER2 protein overexpression, and FISH, which measures HER2 gene amplification, are the test of choice for many practicing pathologies. Although FISH assays have higher sensitivity and higher accuracy and more often correctly identify altered HER-2/neu status (amplification/overexpression),34 because there is a good correlation between HER2 gene amplification and protein overexpression, IHC and FISH assays are often used together. However, the optimal method for assessing immunohistochemically HER2 status in the clinical settings has remained somewhat controversial. Differences in sensitivities and specificities among the various commercially available antibodies, variability in IHC interpretation, and technical artifact have proven problematic.35
Many laboratories are not subject to quality control procedures and the interpretation of results may also be a key factor, especially in low volume testing laboratories.23 For that, we considered as high quality reports that performed IHC and FISH assays using kits approved by FDA and European Union. Most of them used Hercep Test and PathVysion kits. Based on the systematic review of the published literature, we observed that IHC and FISH results were closely associated. These findings also agree with previous reports.36–38 However, among other considerations, lack of standardization across studies, different origin, and number of included patients and publication bias should be considered.
IHC tests were evaluated in terms of validity by comparison to a reference standard, FISH. The percentage of positive FISH results (median) was 3.5 for IHC0, 5.8 for IHC1+, 17 for IHC2+, and 83.5 for IHC3+, confirming high levels of consensus between IHC and FISH in the negative IHC scores. However, the percentage of cases with noncorrelating IHC and FISH results was essentially the same between IHC2+ and IHC3+cases (83% of IHC 2+ tumors were FISH negative; 83.5% of IHC 3+ tumors were FISH positive). Results from IHC testing indicated IHC was not sufficient for appropriate selection of patients who are suitable for trastuzumab treatment. Patients could not be treated with life-saving therapy or could be over-treated with a very expensive therapy, which has the possibility of fatal adverse events.
Among different HER2 testing strategies based on IHC and FISH, IHC is the suitable method for obviously negative cases (0 and 1+), whereas FISH is the method that is more accurate, reliable, and a precise test for confirming or excluding the test in indeterminate 2+ and 3+ cases.3,39
The authors thank the Andalusian Agency for Health Technology Assessment. The authors also thank Victor Sarmiento, MD, Sandra Flores, Leda Ojeda, and José Luis Cortés for their expert assistance and Ángel Concha, PhD, MD, for his dedication and contribution to this report.
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