Pathology Patterns Reviews

Measuring HER-2 in Breast Cancer Immunohistochemistry, FISH, or ELISA?

I-Tien Yeh, MD
Key Words: Breast cancer; HER-2; Immunohistochemistry; Fluorescent in situ hybridization; Enzyme-linked immunosorbent assay

Abstract
Measuring HER-2 is important for selecting optimal therapy and predicting prognosis in breast cancer patients. Current methods for evaluating HER-2 include measuring protein overexpression by immunohistochemistry (IHC), measuring gene copy number by fluorescent in situ hybridization (FISH), and measuring shed antigen in the serum by enzyme-linked immunosorbent assay (ELISA). This review compares these 3 methods and analyzes the current literature pertaining to this subject. In comparing IHC with FISH, the negative predictive value is excellent for commonly used commercial antibodies but the positive predictive value is highly variable. However, by considering only strongly staining cases as positive by IHC, the positive predictive value is markedly improved. ELISA is useful in the follow-up care of patients with breast cancer. An algorithm for using all 3 methods is presented.

The HER-2 (neu, or c-erbB-2) gene encodes a transmembrane receptor of the epidermal growth factor (tyrosine kinase) receptor family, notably the only one without a known ligand. About 20% to 30% of breast cancers show increased activity of this membrane receptor, either by gene amplification or by protein overexpression. HER-2 was first described to be important as a prognostic marker of breast cancers in 1987,1 and subsequently measurement of HER-2 has gained prominence in the ancillary laboratory testing of breast cancer, second only to estrogen and progesterone receptor testing. Patients with HER-2 gene amplification, protein overexpression, or both have decreased overall survival and a higher probability of disease recurrence.2 Perhaps even more important than prognosis, measurement of HER-2 became crucial in predicting response to therapy when trastuzumab (Herceptin, Genentech, San Francisco, CA), a humanized monoclonal antibody that targets the HER-2 receptor, became available in 1998. Other therapies commonly used in breast cancer also may be related to HER2 levels: HER-2 may predict response to anthracyclinecontaining chemotherapy regimens, and HER-2 may predict response to hormonal therapy.3,4 In addition to breast cancer, HER-2 gene amplification and protein overexpression may have a role in a variety of other carcinomas, including colon, salivary gland, and gynecologic carcinomas. The best method for measuring HER-2 is controversial. The existing methods include the following: (1) measuring the gene copy number by Southern blot analysis, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), or chromogenic in situ hybridization (CISH); (2) measuring messenger RNA by Northern blot analysis or PCR; and (3) measuring protein expression by Western blot or immunohistochemical analysis. Shed antigen may be
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Table 1 Comparison of Immunohistochemical Analysis, FISH, and ELISA

Immunohistochemical Analysis Specimen type Technical complexity Target Interpretation Clinical applications Tissue Moderate Protein P185 Subjective Trastuzumab therapy, prognosis FISH Tissue High DNA Quantitative Trastuzumab therapy, prognosis ELISA Serum Low Protein extracellular domain, P105 Quantitative Follow-up monitoring

ELISA, enzyme-linked immunosorbent assay; FISH, fluorescent in situ hybridization.

measured in the serum by enzyme-linked immunosorbent assay (ELISA). Evaluation of HER-2 can be performed on paraffin-embedded tissue sections when using immunohistochemical and FISH methods. A tissue approach permits assessment of tumor morphologic features and correlation,

permits the use of archival material, and avoids the dilutional artifacts of most of the other approaches. Of the aforementioned methods, 3 have gained US Food and Drug Administration (FDA) approval for patient care purposes: immunohistochemical analysis, FISH, and ELISA. As of this writing, there are 2 approved immunohistochemical methods: the HercepTest (DAKO, Carpinteria, CA), a kit using a polyclonal antibody to the internal domain of the HER-2 receptor, and CB11 (Ventana Medical Systems, Tucson, AZ), a monoclonal antibody to the internal domain of the HER-2 receptor. There also are 2 FDA-approved FISH kits, INFORM (Ventana Medical Systems, Tucson, AZ), which uses a single probe for the HER-2 gene, and Pathvysion (Vysis, Downers Grove, IL), which uses 2 probes, 1 for the HER-2 gene and 1 for the centromeric region of chromosome 17. Only 1 FDA-approved ELISA assay is available: HER-2/neu serum assay ECD (Oncogene Science, Bayer, Cambridge, MA). Table 1 enumerates some basic comparisons of these methods. Sites of action of the different methods on the cell are pictured in Figure 1.

Immunohistochemical Analysis
Immunohistochemical analysis detects the actual HER-2 receptor on the cell membrane through the use of antibodies against the HER-2 receptor Image 1. This receptor also is the target of trastuzumab therapy, and it would seem to follow that that actual detection of the HER-2 receptor would predict response to trastuzumab therapy. However, there are a number of variables that affect the results of HER-2 testing by immunohistochemical analysis, and the consequent inconsistencies in results probably contribute to lack of correlation between trastuzumab treatment and immunohistochemical findings. These variables include fixation, storage, antigen retrieval, reagent optimization, the specific antibody and its domain, controls, the scoring system, and interobserver variability in interpretation. Prolonged fixation will cross-link antigenic sites and make them unavailable for immunohistochemical analysis.5,6 Requests for testing of archival cases are sometimes made
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Figure 1 HER-2 receptor. The HER-2 receptor is a transmembrane receptor, denoted by P185, with an external domain and an internal domain. Immunohistochemical analysis (IHC) uses an antibody against either the internal or external domain. The cleaved portion of the HER-2 receptor is detected by enzyme-linked immunosorbent assay (ELISA), whereas fluorescent in situ hybridization (FISH) analyzes the gene copy number on chromosome 17 CISH, chromogenic in . situ hybridization; ECD, extracellular domain. For proprietary information, see the text.

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for patients with recurrent or metastatic disease, but with prolonged storage, antigens may degrade, leading to falsenegative assay results. Antigen-retrieval methods have largely solved these problems, but there still is variability in methods and, thus, variability in the retrieval of antigens. In fact, false-positive assay results are possible, since HER-2 is a normal gene, and if antigen retrieval is successful enough, normal expression will be seen. The difference between normal expression (tens of thousands of receptors per cell) and overexpression (millions of receptors per cell) is substantial. Jacobs et al7 tried to take this factor into account in their scoring system, which subtracts out the normal staining, but normal tissue is not always available in a small needle biopsy specimen for comparison. Individual laboratories optimize immunoassays for their own conditions (eg, temperature, humidity, manual vs automated), so antibody and other reagent concentrations may vary. Numerous antibodies against the HER-2 receptor are available commercially, including both polyclonal and monoclonal antibodies. In general, monoclonal antibodies are considered more specific. Of the 2 FDA-approved antibodies, the HercepTest uses a polyclonal antibody, and CB11 is a monoclonal antibody. The antibody used in the HercepTest, A0485, is also available as a stand-alone product and is used by some laboratories in lieu of the kit. The antigenic domain of both FDA-approved antibodies is for the internal domain of the HER-2 receptor. Some of the commercially available antibodies are against the external domain of HER-2. Probably the most widely used external domain antibody is TAB250 (Zymed, South San Francisco, CA). There is some concern that the external domain of HER-2 may be cleaved (this is the portion measured by the serum ELISA assay), and, thus, antibodies against the external domain may not be as sensitive as antibodies against the internal domain.8 However, this concern is not borne out in comparison of antibodies (see FISH vs Immunohistochemical Analysis). DAKO, in the HercepTest, recommends using cell line controls that are known to have negative, low, and high levels of HER-2 expression. While in theory this is an excellent idea, in reality, a control really should have undergone the same processing steps as the tissue in question, and, therefore, Jacobs et al7 proposed using a method of analysis in which the level of staining of the background benign breast tissue is recorded and subtracted from the staining level of the tumor. This proposal is based on normal tissues always having a normal HER-2 gene copy number and normal expression. Stark et al9 demonstrated rare cases of increased HER-2 gene copy number by PCR in archival benign breast biopsy specimens of patients in whom breast carcinoma subsequently developed, although in that study, no overexpression was seen in benign cells by immunohistochemical analysis.
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Image 1 Invasive breast cancer showing strong, complete membranous staining by immunohistochemical analysis (400).

The scoring system in widest use is based on the scoring system recommended by the HercepTest: 0, no staining; 1+, weak partial membranous staining in more than 10% of cells; 2+, weak to moderate complete membranous staining in more than 10% of cells; and 3+, strong complete membranous staining in more than 10% of cells. Note that granular cytoplasmic staining is nonspecific (some antibodies result in cytoplasmic staining that may be misinterpreted by inexperienced observers). A negative assay is reported with 0 and 1+ staining, whereas 2+ and 3+ are reported as positive. This scoring system has been strongly critiqued in the last few years because in 2+ cases, gene amplification could be demonstrated in only about a quarter of the cases in the initial trastuzumab trials using the clinical trial assay. 10 For trastuzumab response, cases with immunohistochemical results interpreted as 2+ responded to treatment only 4% of the time to treatment, whereas 3+ cases responded 17% of the time (trastuzumab product insert). In addition, only a minority of 2+ cases showed gene amplification. Negative and positive cases had good correlation. These data have been interpreted to indicate that a 2+ level of staining should not be interpreted as positive, but rather regarded as a borderline value, and that confirmatory testing by FISH should be performed. Other scoring systems, using 0 to 211 and 0 to 4+7 also have been used, as well as one using an additive score of 0 to 8, with staining intensity and proportion of cells stained separately recorded.12 Interobserver variability has been a well-documented problem with pathologic interpretation and, indeed, has been documented in reading immunohistochemical results. In the
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Table 2 HercepTest vs FISH*

Sensitivity Authors Bnkfalvi et al15 Birner et al16 Hoang et al14 Jacobs et al17 Lebeau et al18 Pauletti et al19 Thomson et al13 Tubbs et al27 No. of Cases 40 207 100 48 79 80 127 144 FISH Method Ventana Vysis Vysis Ventana Vysis Vysis Vysis Vysis 100 97 100 100 78 59-77 83 3+ Only 100 97 100 90 81 77 76 91 42 79 80 91-93 76 Specificity 3+ Only 100 97 97 74 100 93 50 45 71 65 73 46 PPV 3+ Only 100 89 89 100 89 75 100 99 100 100 84 93-97 (0); 50-70 (1+) 95 NPV 3+ Only 100 99 100 96 95 81 80 93 42 85 79 77 Efficiency 3+ Only 100 97 98 74 97 90

FISH, fluorescent in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells. * For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. Skewed population, cases negative by FISH only, using data presented in paper, without subtraction of benign staining. Limited analysis on subgroup of 900 cases; 5 cases with low levels of gene amplification excluded from analysis. Three observers, using a modified statistical method outlined in reference.

Table 3 A0485 vs FISH*

Sensitivity Authors Birner et al16 Jacobs et al7 Jimenez et al21 Lebeau et al18 Onody et al22 Ridolfi et al23 Thomson et al13 Wang et al24 Wang et al20|| No. of Cases 207 90 41 79 100 116 127 52 52 189 FISH Method Vysis 100 Ventana 78 Ventana 100 and Vysis Vysis 100 Ventana 87 Ventana 97 and Vysis Vysis 63-84 Ventana Vysis Vysis 93 93 99 3+ Only 100 100 90 95 91 91 98 69 96 87 79 91 70 95-98 68 66 66 Specificity 3+ Only 90 100 100 100 89 89 94 40 86 83 65 62 59 54 50 65 PPV 3+ Only 72 100 100 100 93-100 77 77 92 100 93 100 100 97 98 93-97 (0); 53-87 (1+) 96 96 99 NPV 3+ Only 100 100 96 98 96 96 99 73 91 92 85 90 78 75 73 79 Efficiency 3+ Only 92 100 97 99 90 90 96

FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells. * For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. Data for 2 FISH methods not separately specified. Three observers, using a modified statistical method outlined in the article. Two FISH methods performed on all cases. || Manual method only.

study by Thomson et al,13 3 observers, including an experienced breast pathologist, a general surgical pathologist, and a pathology resident, independently interpreted slides in a blinded manner. Negative staining and strong staining (0, 3+) showed the greatest degree of agreement in scoring, whereas intermediate degrees of staining (1+ and 2+) showed significant variations in scoring. Hoang et al14 also compared interobserver reproducibility and found interobserver agreement in 85% of cases among 4 observers, with agreement in negative and strong positive cases and variability in weak positive cases. This disparity in scoring by observers may well be a good part of the reason for reported differences in results when the same antibody is used in different laboratories Table 2, Table 3, Table 4, and Table 5.13-29
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Fluorescent In Situ Hybridization

FISH quantifies the number of HER-2 gene copies that are located on chromosome 17 Image 2. Normal cells contain 2 copies of the HER-2 gene, but when a cell is undergoing division, the cell may contain 4 copies of the gene. Gene-amplified cells contain more than 4 copies or have a HER-2/chromosome 17 gene copies ratio greater than 2. The most compelling reason to use FISH analysis is that comparison of the clinical trial assay with FISH, which was used during the trastuzumab trials, showed that prediction of response was superior using FISH.10 These data were presented to the FDA and led to the recent approval (December 2001) of the Pathvysion FISH assay
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Table 4 CB11 vs FISH*

Sensitivity Authors Bnkfalvi et al15 Birner et al16 Couturier et al25 Gancberg et al26 Jimenez et al21 Lebeau et al18 Thomson et al13 Tubbs et al27 No. of Cases 40 207 100 160 41 85 127 144 FISH Method Ventana 86 Vysis 80 Ventana 92 Vysis 43 Ventana and Vysis 100 Vysis 91 Vysis 63-65 Vysis 73 3+ Only 83 76 89 100 85 68 100 96 99 100 93 96 97-98 86 Specificity 3+ Only 100 98 99 100 100 93 100 80 92 100 89 91 57 PPV 3+ Only 100 88 89 100 100 95-96 85 97 96 99 84 NPV 3+ Only 97 96 99 97 93 98 87 96 95 83 Efficiency 3+ Only 97 95 99 100 97 91

100 100 96 97 86-93 (0); 30-33 (1+) 93 93

FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells. * For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. If <10% cells stained, the result was regarded as negative (B data from the article are presented). Three observers, using a modified statistical method outlined in the article.

Table 5 TAB250 vs FISH*

Sensitivity Authors Farabegoli et al28 Gancberg et al26 Jimenez et al21 Lebeau et al18 Thomson et al13 Vang et al29|| No. of Cases 79 157 41 85 127 32 FISH Method OConnel and White 95 Vysis 53 Ventana and Vysis 100 Vysis 91 Vysis 82-100 Ventana 90 3+ Only Specificity 3+ Only PPV 3+ Only NPV 3+ Only Efficiency 3+ Only

100 90

85 98 80 98 94-95 100

100 100

67 90 77 97 100

100 100 92-95

98 88 100 95 80-100 (0); 100 (1+) 96

100 97

89 88 88 96 97

100 97

FISH, fluorescence in situ hybridization; NPV, negative predictive value; PPV, positive predictive value; 3+, strong complete membranous staining in more than 10% of cells. * For the methods used to calculate sensitivity, specificity, PPV, NPV, and efficiency and for proprietary information, see the text. The probes were gifts of OConnel and White, Howard Hughes Medical Institute, Salt Lake City, UT. If <10% cells stained, the result was regarded as negative (B data from the article are presented). Three observers, using a modified statistical method outlined in the article. || Unclear from the article whether all cases were tested by TAB250.

for the selection of patients for trastuzumab therapy. In addition, studies of HER-2 measured by FISH are more consistently predictive of prognosis than are immunohistochemical studies.30 Slamon31 argues for the use of FISH as the primary testing method for these reasons: (1) FISH data will separate curves of patient prognosis, even at low levels of gene amplification; (2) when immunohistochemical analysis is done as the primary assay, then FISH is superimposed, there is a significant change in the outcome data, but the opposite is not true. This implies that immunohistochemical analysis is missing some cases (false-negative results), although the theoretical ideal immunohistochemical analysis would be more sensitive than FISH.31
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FISH is less subject to the problems of fixation, storage, antigen preservation, and interobserver variability than is immunohistochemical analysis. DNA is relatively stable and not subject to problems of preservation and storage. Formalin fixation, used in the vast majority of pathology laboratories for breast tissue, has no significant detrimental effect on the preservation of DNA. Interobserver variability is not usually much of a problem in a quantitative test, but the fluorescent signals are not always clearly defined; clumping of signals occurs, and some cases are undecipherable by FISH for this reason. Cases may be indeterminate by FISH assay because of heterogeneity in the gene number among cells.21 It sometimes is difficult to get an exact count of gene copy number because the examined tissue section is
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Image 2 A, Breast cancer, fluorescent in situ hybridization (FISH) for the HER-2 gene (orange dots). This tumor is gene amplified for HER-2. B, Breast cancer, FISH with probe for the centromeric region of chromosome 17 (green dots). Note that some nuclei overlap and cells in division appear to have more than 2 copies.

only 5 m thick, whereas the tumor nucleus may be several times this size, thereby necessarily missing some signals. Counting multiple cells (40 for INFORM and 60 for Pathvysion) and using the presence of 2 chromosome 17 centromeric signals (Pathvysion) helps ensure a more accurate count. Measuring chromosome 17 and giving a ratio of gene copy number of HER-2 to chromosome 17 excludes cases with chromosome 17 polysomy from being scored as positive, although the data are not completely clear whether cases with polysomy chromosome 17 respond to trastuzumab treatment. Compared with immunohistochemical analysis, FISH is more expensive, more time-consuming, and technically more difficult to perform. FISH reagent costs are typically at least 3 times the costs of immunohistochemical analysis. Technical assay performance takes approximately 16 hours for FISH compared with 3 hours for immunohistochemical analysis, and technicians need to be specially trained for FISH. Technologists may be delegated the timeconsuming task of counting cells, but the differentiation of tumor cells vs normal cells and in situ vs invasive carcinoma cells needs to be performed by a trained pathologist. Additional negative aspects of FISH testing include missing some overexpressed cases, the need for special equipment, and the lack of permanent slides for review. FISH detects only gene amplification and will not detect protein overexpression, which occurs in 3% to 10% of breast cancer cases.32 The additional equipment required for FISH (fluorescent microscope) is often available in
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standard hospital laboratories for other purposes, such as for evaluating kidney biopsy specimens, but most pathologists do not routinely perform fluorescent microscopy. Special filters are required, as well as photography equipment, if permanent records are desired. Fluorescent signals will quench, and although slides may be viewed more than once, they cannot be retained for years, as immunostained slides can be. The use of photography may overcome the permanent record problem, but of course photos permit the viewing of only small areas of the original slide. On a high-power image, one may not be sure whether the area being viewed is part of an in situ or an invasive carcinoma. This is a crucial point in determining a positive assay, as in situ areas more often are positive for HER-2 than are invasive areas of tumor, but it is the invasive area that should be analyzed for determination of HER-2 status.

FISH vs Immunohistochemical Analysis

The College of American Pathologists issued a consensus statement, as a result of a 1999 conference, regarding the measurement of HER-2 as a prognostic marker.33 Recommendations include specifying the method and reagent used. For cases that are indeterminate by immunohistochemical analysis, another method is recommended to confirm the results, such as FISH. At that time, specific reagents or methods for HER-2 testing were not recommended because of lack of standardization and comparability data. Since that time, a number of studies
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have been published directly comparing FISH and immunohistochemical analysis.7,13-29,32 Tables 2 through 5 show a summary of recent literature detailing the results of comparison of FISH with the HercepTest and with 3 commonly used antibodies, A0485, CB11, and TAB250. For purposes of this review, data presented in the articles were analyzed and the following parameters were calculated, where possible (TP, true positive; FP, false positive; TN, true negative; and FN, false negative): Sensitivity = TP/(TP + FN); Specificity = TN/(FP + TN); Positive Predictive Value = TP/(TP + FP); Negative Predictive Value = TN/(TN + FN); and efficiency = (TP + FP)/(TP + FP + TN + FN).34 There are variations in study populations, methods, and scoring, even when analyzing the same antibodies, which hinders the comparison of studies. The analyses were performed on invasive breast cancer cases, if specified, using standard scoring of 0 and 1+ as negative and 2+ and 3+ as positive for each antibody, with FISH used as the gold standard. The analyses then were repeated by excluding the 2+ cases (or weakly positive cases, if an alternative scoring method was used) and using the scores 0 and 1+ as negative and considering only the 3+ cases as positive (Tables 2-5). Note that some of the articles did not present data so that the second analysis could be performed from published material. In reviewing these data, the following observations are made: (1) There is substantial variation among laboratories in reported sensitivity, specificity, positive predictive value, and efficiency, but the negative predictive value is excellent across all antibodies. (2) The positive predictive value is increased by excluding 2+ cases. (3) There is no consistent pattern of an antibody having the best sensitivity, specificity, or other parameter, although the monoclonal antibodies seem to have better positive predictive values than the polyclonal, especially when considering both 2+ and 3+ cases as positive; results seem to depend on the laboratory performing the assay. An automated reading system for immunohistochemical analysis has been tested and may show increased sensitivity with improved correlation with FISH. 20 However, this method adds substantially to the cost of the procedure; sensitivity and specificity are improved more by simply excluding the 2+ cases, as shown in the present analysis. It seems clear that immunohistochemical results that are intermediate (2+) should not be regarded as positive, but rather as borderline, and that these specimens should undergo further testing. The 3+ cases by immunohistochemical analysis are excellent predictors of gene amplification, and negative (0 and 1+) immunohistochemical results predict negative gene amplification very well. A few false-negative results will occur using either immunohistochemical analysis or FISH as the primary testing method.
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Enzyme-Linked Immunosorbent Assay

The external domain of the HER-2 receptor is the ligandbinding portion, also known as the extracellular domain (ECD). Proteolytic processes cause the external domain to be shed into the serum, possibly mediated by matrix metalloproteinases,35 and this shed ECD has been shown to be present in breast cancer patients. ELISA is a monoclonal assay that can be performed on either cell lysates from tissues or on fluids. Because immunohistochemical analysis and FISH permit histologic correlation on tissues, which is not possible on cell lysates, the marketing of ELISA has concentrated on its use in serum. ELISA can be performed on serum at any time during the disease and has the advantage of being a quantitative test. However, serum ELISA levels tend to vary with tumor burden, time, and factors such as the phase of menstrual cycle. HER-2 ECD levels may be detected in healthy patients, and studies comparing healthy patients and those with breast cancer are ongoing, with optimal levels for the cutoff value still being defined. ELISA is available on an automated platform (Bayer Diagnostics, Tarrytown, NY), so that reproducibility should be excellent. Circulating HER-2 antigen levels correspond to expression shown by immunohistochemical analysis and tumor bulk,36-38 but relatively little ECD is shed during early-stage disease. Only 25% to 50% of patients with HER-2positive breast cancer by tissue assays will have detectable serum HER-2 ECD. The clinical sensitivity of the assay is nearly 0 in stage I tumors and increases to about 40% for stage IV tumors.39,40 For this reason, the serum assay may be useful only in cases of metastatic disease. Approximately 17% to 20% of patients with breast cancer whose tumors initially test negatively for HER-2 may experience recurrence, with HER-2positive serum. Possible reasons for this may be tumor heterogeneity with selection for an HER-2positive clone with disease progression, a false-negative initial assay result, mutations after the initial assay, and up-regulation of HER-2 expression by estrogen deprivation. For disease monitoring, surprisingly, trastuzumab does not seem to interfere with the ELISA assay results, although this drug is a monoclonal antibody against HER-2. Nor does a high level of HER-2 ECD inhibit response to trastuzumab therapy. Trastuzumab seems to down-regulate HER-2 and prevent cleavage.

Immunohistochemical Analysis, FISH, and ELISA

For pathologists, immunohistochemical testing remains the favored initial testing method in typical hospital laboratories because most such laboratories are set up to perform
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Use of one assay method does not exclude measurement by alternative methods. A triage system of using immunohistochemical analysis as a screening method, FISH as a confirmatory test,23,27,41 and ELISA for disease monitoring has been proposed42 Figure 2. This triage system uses the relatively cost-effective immunohistochemical method for detecting high expressers on tissue and uses the more expensive FISH method for a limited number of borderline cases. ELISA ECD is most likely to be clinically useful in the early detection of recurrent disease in HER-2positive tumors and, possibly, to monitor results of trastuzumab therapy.

Future Directions
Figure 2 Proposed triage of HER-2 testing procedures. ELISA, enzyme-linked immunosorbent assay; FISH, fluorescent in situ hybridization; IHC, immunohistochemical analysis.

Image 3 Chromogenic in situ hybridization for HER-2 in breast cancer (brown dots).

CISH is a new method of analyzing HER-2 that combines the best characteristics of both immunohistochemical analysis and FISH, that is, being able to identify gene copy number on a permanent slide that can be visualized by a standard light microscope Image 3. CISH assays depend on a technique called subtractive hybridization,43,44 which uses a DNA probe, visualized by a peroxidase reaction. Compared with FISH, this method is easier to perform, easier to visualize, and results in a stable prepared slide that does not require special microscopes or filters to view. Compared with immunohistochemical analysis, one can actually identify gene amplification and at least semiquantitate the gene copy number, and reading the gene copy number will not depend on factors such as the degree of antigen retrieval. These positive aspects make this technique one that may become widely used. However, there are too few data on the clinical usefulness and pathologic correlations to make a recommendation.45,46 The need to measure HER-2 continues to gain importance in the management of patients with breast carcinoma, as well as patients with a variety of other carcinomas. Selection of optimal methods to measure HER-2 likely will depend on availability, ease of use, cost, and correlation with clinical characteristics.
From the Department of Pathology, University of Texas Health Science Center at San Antonio. Address reprint requests to Dr Yeh: Dept of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229. Acknowledgments: I thank Tonya Keys for help with manuscript preparation, technologists Oredius Pressley and Guadalupe Castillo for help in the laboratory, and Jaishree Jagirdar, MD, for reading and commenting on the manuscript.

immunohistochemical analysis, but not necessarily FISH. In addition, the costs for immunohistochemical analysis are relatively low, and it is less time-consuming to perform and interpret. FISH has become favored by many oncologists because of the relatively high false-positive rate (primarily in low-positive, 2+ cases) and occasional false-negative results seen with immunohistochemical analysis. The particular antibody used may not be as important as the laboratory that is performing the assay. In some hands, one antibody performs better than others, and it should be up to the individual laboratory to determine the best method for its own conditions. At this time, serum ELISA seems to be mainly useful for monitoring advanced-stage disease.
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References
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