Elsevier

Human Pathology

Volume 36, Issue 3, March 2005, Pages 250-261
Human Pathology

Original contribution
Assessment of the HER2 status in breast cancer by fluorescence in situ hybridization: a technical review with interpretive guidelines

https://doi.org/10.1016/j.humpath.2004.11.010Get rights and content

Summary

Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2-driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.

Introduction

The successful treatment of breast cancer is dependent upon a number of complex factors, including the discovery of the tumor early in its course of development, the biology underlying the disease, and the appropriate treatment. The decision to stratify patients newly diagnosed with breast cancer regarding the need for adjuvant therapy has traditionally been on the basis of the grade of the tumor and the anatomic extent of disease [1]. It is clear that the risk of recurrence for lymph node–positive patients is substantially higher than for node-negative patients. However, traditional pathological prognostic factors do not provide sufficient information to allow accurate individual risk assessment for patients whose tumors are discovered early in the course of their disease [2], [3]. Increasingly, management decisions regarding whether oncologists should prescribe adjuvant treatment are being based on the biology of the patient's tumor, with a consideration of the hormone receptor status [4], and more recently, the assessment of amplification and overexpression of the HER2 gene [5], [6]. The proper selection of the most appropriate therapy on the basis of biological markers is heavily dependent upon reliable and accurate laboratory determinations, and highlights the need for standardization of tissue handling, biomarker assay procedures, and proper interpretation of testing results.

The HER2 gene product is a transmembrane growth factor receptor normally expressed in secretory epithelia. It is involved in the cellular signaling that regulates growth and development [7]. In a normal cell expressing HER2, there are 2 copies of the gene and about 50 000 copies of the protein on the cell surface. However, in 20% to 30% of breast cancers, HER2 gene amplification leads to overexpressed protein and the number of receptors at the cell surface can increase to more than 1 million [8], [9], [10]. Slamon et al [9] first described the link between HER2 and its biological importance in breast cancer in 1987, and since then, more than 100 reports in the literature have examined this association. The majority has demonstrated a correlation between HER2 activity and poor prognosis [10]. In addition to an aggressive clinical course, HER2 overexpression in breast cancer correlates with lymph node positivity, high nuclear grade, negative hormone receptor status, and high proliferative activity [10], [11], [12], [13], [14]. For most cases, the molecular events that underlie HER2-driven breast cancer include gene amplification. This increase in HER2 gene dosage leads to transcriptional up-regulation, increased protein synthesis, and a proliferative drive for the subset of tumors with this molecular alteration [15], [16] (Fig. 1).

Because this protein plays a role in mediating the biological and clinical behavior of these cancers, it is an ideal therapeutic target. Herceptin (trastuzumab) is a humanized monoclonal antibody that targets the extracellular portion of the HER2 receptor. It has been shown to be effective in treating patients as a single agent [6] or in combination with more traditional chemotherapy [17], [18]. However, both clinical and in vitro studies have demonstrated that Herceptin is only active against HER2-overexpressing tumors [6], [17], [18], [19].

Therefore, the laboratory evaluation of HER2 status has become a critical element for determining the prognosis and management of breast cancer. Furthermore, the American Society of Clinical Oncology has recommended the assessment of HER2 in all breast tumors, either at the time of diagnosis or recurrence [20]. Laboratory-based methodologies such as Southern blots, which were used in the initial studies of HER2 in breast cancer, are impractical in the usual clinical setting [9], [21], [22]. The 2 biological events underlying HER2-driven breast cancer that can be assessed in routine formalin-fixed paraffin-embedded (FFPE) clinical samples include the evaluation of gene amplification by fluorescence in situ hybridization (FISH) and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). These assays have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features in tissue sections. Both the FISH and IHC methodologies have received Food and Drug Administration approval for HER2 evaluation, and Table 1 lists the current commercially available HER2 detection systems.

Studies examining HER2 overexpression in frozen sections of breast tumors have shown an excellent correlation with HER2 gene amplification assessed by Southern blotting [9], [15]. Likewise, the published HER2 gene/protein literature in FFPE clinical samples has shown a good to excellent concordance in many reports [23], [24], [25], [26], [27]. The scoring of HER2 results assessed by IHC needs to be semiquantitatively evaluated to be clinically relevant (as shown in Fig. 2). Only cases with intense circumferential thick membrane staining (scored as 3+) show a good concordance with HER2 gene amplification by FISH in most studies, and it is this group of patients who will be the most likely to benefit from Herceptin therapy [17], [18], [19], [23], [24], [25], [26], [27]. Breast tumors with absent or weak membrane staining (scored as 0 or 1+) typically demonstrate a normal HER2 gene status and are regarded as negative [25], [26], [27], whereas cases scored as 2+ show poor agreement with FISH results and are considered inconclusive [26]. These results have lead some investigators to recommend an HER2 testing algorithm, in which breast tumor samples are screened by IHC, and FISH testing is reflexively performed only on cases scored as 2+ [23], [25], [27]. This algorithm assumes that IHC assays are well standardized, rigorously quality controlled, and that there is a good concordance for testing results among different laboratories. However, recent reports have called into question these assumptions [28], [29]. Although IHC testing is appealing because it is readily accommodated in most surgical pathology laboratories, a number of preanalytical variables such as tissue handling and fixation can adversely affect immunoreactivity [30]. In addition, the subjective nature inherent in the interpretation of IHC result can lead to interobserver variability and affect the accuracy of test results [31]. Clearly, standardization of tissue handling, fixation, use of standardized assays, rigorous quantitation, quality control measures, and competency assessments are a prerequisite to this approach and will help ensure accuracy and consistency for IHC HER2 evaluation [32].

Like IHC, FISH testing for HER2 is morphologically driven and uses a fluorescent-labeled probe to enumerate the HER2 gene copy number within the nuclei of tumor cells (Fig. 3). Although FISH assays are viewed with a fluorescence microscope, the use of fluorescent DNA counterstains (either 4′,6-Diamidino-2-phenylindole [DAPI] or propidium iodide [PI]) allows for identification of nuclei, and when viewed in conjunction with a standard hematoxylin and eosin (H&E) section from the same block, enables morphologic correlations and identification of invasive tumor (Fig. 4). DNA is a more stable target compared to the HER2 protein and is not as susceptible to the adverse effects of tissue handling or fixation [10]. Another advantage of FISH testing is that the quantitative interpretation of results with experience is relatively straightforward, and concordance rates among observers are higher than with IHC in some studies [16], [33], [34], [35]. However, despite the excellent concordance between IHC and FISH reported in some series [27], a number of retrospective clinical studies have suggested that FISH results were superior in predicting a benefit from Herceptin therapy, leading some authors and clinicians to call for wider implementation of primary FISH testing in breast cancer [6], [11], [17], [19], [24], [34], [36]. The results of prospective clinical trials should shed additional light on which methodology is most suitable for predicting benefit from Herceptin treatment in the clinical setting.

In spite of the above advantages and the need for FISH testing at the very least in tumors scored as 2+ by IHC, pathologists have been slow to adopt FISH for the clinical laboratory testing of HER2. The reasons for this are related to a number of factors including the technical challenges associated with FISH assays [37], the expense and time necessary for interpretation [27], and the relative lack of community-based experience with this technology. The development of automated platforms for probe/target gene hybridization, image analysis for signal enumeration, and increased familiarity with FISH assays and their interpretation should broaden the availability of this technology to the greater laboratory community.

Two commercial FISH assays are currently available (Table 1). The Inform test (Ventana Medical Systems, Tucson, Ariz) enumerates the HER2 gene copy number (nuclear criterion for gene amplification), and the PathVysion test (Abbott-Vysis, Downers Grove, IL) in addition includes a second probe for the centromeric region of chromosome 17 (CEP17). The CEP17 probe allows for a correction of the HER2 gene copy number to the number of copies of chromosome 17 (HER2/CEP17 ratio) (chromosomal criterion for gene amplification). Published reports have indicated that the results of these 2 FISH detection systems are highly correlative [10], [38], [39]. Lal et al demonstrated a 96% concordance rate between the 2 assays. Discrepant results were mainly attributable to increased chromosome 17 copy number (polysomy for chromosome 17), which would have been scored as negative by the Vysis assay (ratio of HER2/CEP < 2), and positive by the Ventana assay (mean signal number of >4). A recent report has recommended changing the cutoff mean HER2 signal number to 6 or greater for the Inform assay to account for the discrepant cases due to chromosome 17 polysomy [40].

We have recently reviewed 743 consecutive cases of breast cancer assessed at our institution by the Abbott-Vysis HER2 FISH assay, paying particular attention to the incidence of polysomy for chromosome 17 and its effect on HER2 transcriptional up-regulation and protein overexpression in these cases [41]. If polysomy was defined as a mean CEP17 of 3 or more, we found 56 polysomic cases (7.5% of the total), 5% of the total were polysomic without HER2 amplification, whereas 2.5% were polysomic and low-level HER2 amplified. If the following criterion for the mean HER2 signal counts was applied to this series of tumors (1 to <4 [nonamplified], 4 to <6 [polysomic for chromosome 17], and 6 or greater [amplified]), no cases with HER2 amplification would have been misclassified, and less than 1% of nonamplified cases would have been incorrectly classified as low-level amplified.

Immunohistochemistry for HER2 protein (CB11) in the polysomy 17 cases was scored as 0/1+ in 68% (38/56), 2+ in 27% (15/56), and 3+ in 4% (2/56) of the polysomic cases. A subset of 26 of these cases with IHC results ranging from 0 to 3+ was also evaluated by a sensitive isotopic assay for HER2 messenger RNA (mRNA). The results of these studies failed to show any increase in mRNA expression (normalized to actin mRNA) for any of the polysomic cases, in agreement with other reports that suggest polysomy for chromosome 17 is probably not significant in HER2 gene and protein expression [42].

Other series have suggested a higher incidence of polysomy for chromosome 17 in up to or greater than one third of cases [27], [34], which may reflect different criterion for defining polysomy or an institutional bias for our patient population. Currently, the clinical significance of polysomy for chromosome 17 in term of response to Herceptin therapy in breast cancer is unknown and awaits clinical trial results and outcome studies.

Section snippets

Technical considerations for FISH testing

Fig. 3 schematically illustrates the principles of the FISH assay including denaturation of DNA, hybridization of the probe to target, and detection. Interested readers are referred to 2 excellent recent reviews on the technical aspects of FISH assay procedures and interpretation by Bartlett [37] and Bartlett and Forsyth [43].

The standardization of all preanalytic variables including tissue handling, fixation, and processing are important aspects of HER2 FISH testing, and will help ensure that

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