The clinical evaluation of HER-2 status: which test to use?

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1 Journal of Pathology J Pathol 2003; 199: Published online 21 February 2003 in Wiley InterScience ( DOI: /path.1354 Review Article The clinical evaluation of HER-2 status: which test to use? John Bartlett, 1 * Elizabeth Mallon 2 and Tim Cooke 1 1 University Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK 2 Department of Pathology, Western Infirmary, Glasgow, UK *Correspondence to: Dr John Bartlett, University Department of Surgery, Level II, Queen Elizabeth Building, Glasgow Royal Infirmary, Glasgow G31 2ER, UK. j.m.bartlett@clinmed.gla.ac.uk Received: 19 December 2002 Accepted: 20 January 2003 Abstract Accurate determination of the status of the type I receptor tyrosine kinase HER-2 in breast carcinomas provides significant insight into patient prognosis and may also inform selection of chemotherapeutic and hormonal treatments. At present, however, the single most important application of HER-2 testing is in the selection of patients for treatment with targeted therapies such as Herceptin. Although, based on current literature, fluorescence in situ hybridization (FISH) detection of HER-2 gene amplification may provide more accurate information in this context, this method is not yet widely available. Therefore, screening by immunohistochemistry (IHC) for HER-2 protein, backed by rigorous quality controls and FISH testing of equivocal cases with intermediate staining intensity, remains the current practice. In laboratories with highly standardized testing and quality assurance procedures, this protocol appears highly effective. Improvements in fixation procedures, standardization of antibodies, and use of automated image analysis may all increase the precision of IHC testing. However, on the basis of current data, there is a case to be made for the wider implementation of FISH testing to determine HER-2 status in breast cancer. Copyright 2003 John Wiley & Sons, Ltd. Keywords: evaluation HER-2; Herceptin; breast carcinoma; immunohistochemistry; FISH; diagnosis; Introduction The assessment of HER-2/c-erbB-2 /neu (hereafter HER-2) gene amplification and protein expression has become one of the central debating points in current breast cancer diagnosis and biology. The debate around whom to test, when testing should be offered, and, most importantly, which method to use is represented at most current conferences where breast cancer pathology is under discussion. Overexpression of the p185her-2 protein product of HER-2/neu is closely related to gene amplification in breast cancer [1 4]. Slamon et al first described the biological importance of HER-2 in breast cancer in 1987 [1] and many subsequent publications have confirmed the prognostic significance of HER- 2 amplification and overexpression in breast cancer [5 13]. There has been controversy regarding nodenegative carcinomas [6 8], but some of the reported differences may be methodological [14 16]. HER-2 is one of four homologous receptors which together make up the HER (or type I, or erbb) family of transmembrane receptor tyrosine kinases. These receptors form homo- or hetero-dimers following ligand binding to their external domains and activate a complete series of intracellular signalling pathways via auto-phosphorylation of tyrosines on their intracellular domains [17 19]. Recent clinical trials implicating HER-2 in modified responses to anti-oestrogens and anthracyclins [9 11,20 27] have stimulated interest in accurate and reliable identification of patients with carcinomas driven by HER-2 amplification and overexpression, although these findings remain unsubstantiated at present by prospective clinical trials. Most critically, the recent FDA approval for the first anti-her-2 therapy Herceptin [22,24,28 30], and the wide licensing of this agent throughout the world, coupled with the likelihood of further targeted therapies, has thrown the need for HER-2 testing into sharp relief and has intensified the debate [31,32]. The current debate The debate around HER-2 status centres on a series of questions. Firstly, is gene amplification or protein overexpression the primary lesion in breast cancers whose prognosis is determined by their HER-2 status? Secondly, which diagnostic system is the most accurate for the determination of the HER-2 status of a patient s tumour? Finally, which system provides the most cost-effective means of managing the HER-2- positive patient? This review briefly considers why immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently the main techniques employed and discusses the use of these techniques within Copyright 2003 John Wiley & Sons, Ltd.

2 412 JBartlettet al clinical diagnostic laboratories, paying particular attention to issues of reproducibility and accuracy. Her-2 assay methods Within the scientific literature, a number of different methods have been used to assess HER-2 expression or gene amplification status. As mentioned above, Slamon et al s original report made use of Southern, northern, and western blotting techniques. In the intervening years, HER-2 expression has been most frequently assessed using IHC in formalin-fixed and, more rarely, frozen material, with a bewildering array of antibodies. The lack of standardization of these immunohistochemical methods accounts, in large part, for the fact that overexpression of HER-2 in breast cancers is reported in anywhere from 5% to 85% of all cases and errors relating to antibody selection, fixation, and interpretation have been widely documented [16,33 36]. Only a very small number of reports have made any attempt to quantify HER-2 expression accurately and objectively, since, in the absence of an endogenous HER-2 ligand, this method requires the use of antibodies as surrogate ligands [3,4]. Furthermore, analyses of tumour samples are often based on the assumption, often false, that all the material present represents tumour cells. In only one series of reports was any attempt at a quantitative measurement of expression in situ performed and these data have proved invaluable as a gold standard for current molecular diagnostic tests [3,4]. Similarly, mrna/dna has been assessed by northern or Southern blots, and by quantitative and semi-quantitative PCR and RT-PCR [37 41]. All of the above methods have value in investigations of the biology of HER-2 in clinical material, but many of them are limited by the need to utilize frozen tissues. Current clinical diagnostic tests are predominantly restricted to those that can be applied to fixed tissues, usually those fixed in buffered formalin and embedded in paraffin wax. This limits the tests that can currently be applied in clinical diagnostic practice to those that evaluate protein expression, such as IHC, or gene copy/gene amplification, such as FISH techniques, and can be applied to fixed material. Alternative methods of testing for HER-2 expression, such as the use of the shed external domain of the HER-2 antigen, have been shown to be of value in monitoring HER-2-positive patients but are unproven as diagnostic screening methods for HER-2 expression in tumours. PCR-based methods are also being increasingly evaluated, but are at present not widely used for the diagnosis of HER-2 status. Although these methods show promise, careful evaluation of PCR-based techniques, with particular attention to the need for microdissection of tumour tissues and the discrimination between carcinoma in situ and invasive tumour components, will be required before their true value is established [37 42]. At present, therefore, diagnostic approaches to the evaluation of HER-2 status rely, in most cases, on immunohistochemical and in situ hybridization techniques. These have the distinct advantages, firstly, of largely conserving the tissue architecture, allowing a clear distinction between invasive and noninvasive (eg DCIS) components, and, secondly, of being readily applicable in many routine histology laboratories. Within this context, however, there remains considerable debate as to which of these approaches is the most appropriate for implementation in routine clinical practice. A cursory scan of the literature will reveal a significant number of publications addressing this issue, and yet the debate continues [32,43 46]. It is perhaps important to review some of the key criteria for selection of a diagnostic test and ask what evidence is required to aid in the selection of a diagnostic approach to HER-2 testing. Clinical diagnostic tests must satisfy a number of criteria to be made available within a clinical diagnostic setting. Firstly, the test must provide an accurate determination of the marker being evaluated. Secondly, the assay must be reproducible and precise, both within laboratories and, where appropriate (such as for FISH and IHC), between observers. These considerations are key for the biochemical evaluation of a test; however, where the technique is used for the selection of patients for particular therapies, information on the relative response rates for different test approaches may also be of value in assessing diagnostic approaches. Once these key criteria are met, secondary considerations may be applied, such as cost, availability, and ease of application, but these considerations should never override objective measurements of test performance. Evaluation of clinical diagnostic procedures: accuracy, reproducibility, and inter-observer variation How, then, can we assess the accuracy of FISHbased or IHC-based assays for HER-2? Despite the large number of publications in this area, there is relatively little information available on this specific topic. Concordance does not imply accuracy Most series have concentrated on demonstrating concordance rates between IHC and FISH [7,47 51]. These serve to highlight a significant problem in selecting between tests by using concordance rates, since any pair of tests that are reasonably accurate will display a high degree of concordance. For example, if test A was 95% accurate and test B was also 95% accurate, the predicted concordance would be 90%. However, if test A was 99.5% accurate while test B was only 90% accurate, the predicted concordance would also be 90%. Concordance rates between

3 The clinical evaluation of HER-2 status: which test to use? 413 tests therefore provide a poor indicator for selection of tests for clinical use, since they may mask significant differences in test performance. The optimum approach to the evaluation of clinical diagnostic tests is to use an accepted external standard against which the performance of such tests can be evaluated. Accuracy Is such an approach possible for HER-2 testing? A recent report from our own group [52] correlated HER-2 status determined by IHC and FISH to quantitative expression data from assays of frozen tissue [1 4]. To achieve this, we selected tumours previously analysed using a precise quantitative in situ radio-immunohistochemical assay [3,4], adapted from methods used for the analysis of EGF receptor expression [4,53]. Using this data set, we evaluated current protocols for HER-2 testing. The conclusion of this study was that FISH-based tests (PathVysion) were more accurate in determining expression levels of HER-2, when compared with quantitative expression data, than the IHC approaches used (CB11 and Herceptest) [52]. More recently, a report by Press et al [54] has, using a similar approach, produced broadly similar findings showing higher FISH positivity rates in tumours unequivocally positive for gene amplification and overexpression than those observed with the Herceptest. Further studies using this approach should be undertaken to validate this finding. Reproducibility and observer variation Reproducibility is also critically important to patient selection, since an accurate test that cannot be reproduced in other laboratories cannot be widely applied. Fixation is widely recognized as a major source of variability in the immunohistochemical staining of breast samples [55]. Furthermore, all FDA-approved tests of HER-2 status are open to scorer bias, due to the subjective nature of the interpretation of results. The effect of observer bias and variability in IHC is widely documented and is a recognized problem in the interpretation of quantitative expression levels using colour-grading approaches [56 58]. Various different scoring systems have been introduced to seek to improve reproducibility between observers, for example the Allred categorization for ER status [59] and, more relevantly, the scoring system for HER-2 status published by DAKO Ltd. IHC requires the scorer to determine the proportion of cells with positive membrane staining and to categorize the colour intensity. Evaluation of colour intensity is subjective and scorer bias is well documented [56 58]. It is difficult to see how such bias can be avoided across multiple testing centres. While automated image analysis systems may quantify immunohistochemical staining intensity objectively, such systems are unable fully to compensate for variance arising from differences in fixation, tissue processing, antigen retrieval, and expression of an immunohistochemical signal. By contrast, analysis of gene and chromosome copy number by FISH requires simple counting of two coloured signals and for such analysis, inter-scorer errors are below 10% [52,60 62]. The greater simplicity of scoring and robustness of signal are likely to explain the better inter-scorer consistency of FISH over IHC described in our previous study [52]. However, it must be stressed that these advantages only apply to studies which quantitate FISH signals numerically; the practice of estimating FISH ratios or scores, while possibly useful in some contexts, should be avoided in studies seeking to evaluate the accuracy of this technique versus IHC and other approaches. Response and survival: surrogate measures of test performance? Overall, there appears to be significant evidence to support the tenet that FISH, when applied correctly, provides a more accurate and precise indicator of the HER-2 expression status than IHC performed on fixed tissues. However, this evidence is often submerged beneath the large number of studies arguing that concordance rates of 80 90% support the use of one or other approach for testing. Are there therefore other strands of data that can be used to support this argument prior to the publication of further evaluation studies performed as suggested above? The central problem surrounding the evaluation of FISH and IHC tests is the infrequent use of a standard measurement of HER-2 overexpression, such as that described above, against which these tests can be readily evaluated. There are, however, two surrogates available that can provide further information to inform the selection process. Response to HER-2-directed therapies is assumed, rightly, to be related to expression of the HER-2 protein. Patients with normal expression levels of HER-2 would not be expected to respond to anti-her-2- directed agents. Therefore, the predictive power of individual tests in determining response to agents such as Herceptin may provide an indicator of the accuracy of these tests in determining HER-2 status. Similarly, data gathered in the absence of HER-2- directed therapies can be of use where patient survival, known to be adversely affected in HER-2- overexpressing cancers, can be a surrogate for HER-2 overexpression. Data on comparative response rates to Herceptin are limited to a single series of reports summarized at ASCO in 2000/1 and recently updated [63 65]. These studies compared the response rates to Herceptin in the pivotal clinical trials. All patients treated were positive by IHC and overall response rates were of the order of 20%. However, gene amplification was identified by FISH (PathVysion) in only approximately two-thirds of patients and this included all bar one of

4 414 JBartlettet al the responders [63 65]. The conclusion of the study was that patients with FISH-positive tumours were more likely to respond than patients whose tumours were IHC-positive, but with no demonstrable HER-2 gene amplification. The study suffers from being a retrospective analysis and, as pointed out by the authors themselves, prospective trials are required to address this issue [11]. Also, the clinical trials assay used for IHC is not widely used in the diagnosis of HER-2 status and comparisons between this and other IHC approaches are lacking. Finally, no patients with IHCnegative/FISH-positive tumours were included within the trial and therefore the importance of this group could not be assessed [63 65]. A more recent report by Seidman and co-workers [66] again demonstrated that FISH was amongst the most accurate predictors of response to Herceptin. Notwithstanding these minor flaws, these studies provide a strong argument that selection of patients using FISH-based approaches may provide a more effective means of identifying those patients likely to benefit from treatment with Herceptin [63 65]. This view is substantiated by a recent analysis by Fornier et al [67] suggesting that the improved patient selection offered by FISH testing may provide a more cost-effective means of managing the HER-2-positive patient. The effect of testing by either IHC or molecularbased approaches (including FISH) has been reviewed in an extensive meta-analysis by Ross and Fletcher [5] and more recently by Mitchell and Press [68]. Both studies reached similar conclusions, namely that FISH-related studies more consistently identify a negative association between HER-2 amplification and adverse prognosis than the widely variable IHC approaches reviewed. While the meta-analysis by Ross and Fletcher, due to its inclusive approach, may have been biased by the inclusion of studies where the IHC was clearly sub-optimal, the focus of Mitchell and Press s overview was on more recent studies [5,68]. More recently, direct comparisons between diagnostic IHC and FISH approaches have introduced comparisons focusing on survival of FISH-amplified/IHCnegative and FISH-negative/IHC-positive tumours, ie those tumours where the tests applied give discordant results. While these cases are relatively uncommon, they may provide significant insight into testing methods, since they provide the opportunity to identify the discriminatory power of individual tests. In Pauletti et al s recent report, patients with FISHpositive/IHC-negative tumours showed a 10-year survival of around 50%, equivalent to that for patients whose tumours were positive by both methods [49]. Conversely, patients with FISH-negative/IHC-positive tumours exhibited a 10-year survival comparable to that of patients whose cancers were negative by both IHC and FISH [49]. These data indicate that samples with discordant IHC and FISH results may reflect false-negative/positive results between assay systems. They do support the findings by Ross and Fletcher and by Mitchell and Press that FISH provides a more powerful prognostic indicator [5,68]. They may, however, also be interpreted to suggest that patients with overexpression of HER-2 in the absence of gene amplification represent a subgroup of HER-2 overexpressors with a prognosis similar to that of HER-2-negative patients. The precise biology of those cases with no detectable expression of HER-2 but with FISH amplification would, however, in this case remain unexplained. Current practice A consensus is emerging within the literature that the optimal means of detecting patients whose tumours overexpress HER-2 is to use molecular testing; this has been supported by the recent recognition by the FDA of FISH-based testing to select patients for Herceptin therapy. Although FISH is being increasingly established within routine diagnostic laboratories, its application remains limited by the small number of centres where such expertise is available. The development of such services through training and the implementation of quality assurance schemes is a prerequisite for the wider application of this technology. In the interim, the use of IHC, with standardized antibodies and antigen retrieval protocols, has been recommended as a pre-screen for determining HER-2 status, particularly for cases with intermediate staining by IHC [5,13,32,45,46,68]. To what extent improvements in the normalization of fixation methods and the standardization of scoring methods, perhaps using automated image analysis systems, may improve the accuracy of this approach remains to be seen. These developments may well be paralleled by further improvements in molecular testing through the use of colorimetric as opposed to fluorescent detection systems and the automation of FISH analysis and processing, both of which are currently being evaluated. Quality control and reference laboratories There is a current debate, fuelled by two recent reports on the divergence of IHC test results between central reference laboratories and those referring samples for inclusion in clinical trials, on the sample work load and quality assurance procedures to be recommended for laboratories undertaking HER-2 testing. Two reports by Paik et al [69] and Roche et al [70] highlighted discrepancies between local and central laboratory IHC results in the context of two major clinical trials. Interestingly, a recent update by Paik et al [71] presented at the 2002 San Antonio Breast Cancer Symposium suggested that local laboratory FISH results were accurate in over 97% of cases. This may reflect the greater centralization of FISH methodologies as opposed to those involving IHC. Within this issue of The Journal of Pathology, an article from the UK reference laboratories by Dowsett

5 The clinical evaluation of HER-2 status: which test to use? 415 et al [72] illustrates the value of centralized testing in reference laboratories. Results from three reference laboratories show that where frequent quality control and communication between laboratories are combined with a significant sample throughput, the current testing protocols appear both robust and effective. Very few FISH-positive cases were included in the weakly staining group. Conversely, few cases demonstrated strong IHC positivity without gene amplification. However, even within the controlled environment of these reference laboratories, marked differences in the HER-2 FISH positivity rates for equivocal (IHC 2+) cases were observed, ranging from 30% to 80%. This may highlight both the difficulty in identifying this group and the potential effect of even slight variations in performance and scoring of IHC results. Dowsett et al [72] point out that these results are achieved within the context of three experienced laboratories with extensive quality assurance experience. In the light of these results and those of Paik et al [69,71] and Roche et al [70], Dowsett et al recommend participation in training and quality assurance schemes, which will undoubtedly be of value to both experienced and novice diagnosticians alike. The final recommendation in Dowsett et al s paper that testing with such modern methodologies is likely to be most accurate when performed in laboratories with significant caseloads ( some hundreds of cases per year ) is in agreement with both Paik et al s and Roche et al s [69 71] observations that larger laboratories tended to perform better in their experience. Extrapolating this recommendation to routine clinical practice would result in the establishment of a network of reference laboratories within the current clinical framework. Within the UK, individual, or indeed groups of, cancer networks may identify those laboratories either with existing expertise or where training may most appropriately be focused, as candidates to fill this role. The wider development of the National EQA Scheme (NEQAS) IHC quality assurance scheme and the development of such a scheme for FISH-based methods will be essential for the wider development of future HER-2 testing within the UK. Conclusion Accurate determination of the type I receptor tyrosine kinase HER-2 status provides significant insight into patient prognosis and may also inform selection of chemotherapeutic and hormonal treatments. At present, however, the single most important application of HER-2 testing is in the selection of patients for treatment with targeted therapies such as Herceptin. Experience to date suggests that testing may be optimally performed in larger centres with a clear need for a rigorous EQA scheme for both FISH and IHC. 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