Fluorescence in situ hybridization and immunohistochemical assays for HER-2/neu status determination: application to node-negative breast cancer

BACKGROUND: HER-2/neu (ERBB2) gene amplification and/or overexpression is a major event in human breast tumorigenesis. HER-2/neu gene alterations have been the most frequently assessed prognostic factors during the last 10 years in breast cancer and have recently emerged as a management decision tool and a therapeutic target. There is still controversy over the best method to determine whether a tumor is HER-2/neu positive. Because of the increasing demand for HER-2/neu gene status determination in clinical practice, we compared HER-2/neu gene alterations at the DNA level (gene amplification) and the protein level (overexpression) in a panel of patients with lymph node-negative breast cancer who had received local radiotherapy alone, with no adjuvant therapy. METHODS: We tested 100 excised lymph node-negative breast tumors, using fluorescence in situ hybridization (FISH) with a biotinylated HER-2/neu DNA probe and immunohistochemical assays (IHC) with 2 different antibodies. RESULTS: The FISH frequency of HER-2/neu gene amplification was 15%, and the IHC frequency of overexpression was 21%. CONCLUSION: Although HER-2/neu amplification by FISH and HER-2/neu overexpression by IHC correlated well in this panel of lymph node-negative breast carcinomas, there were a number of discordant cases, pointing to the important need for determining HER-2/neu alteration for the future management of HER-2/neu-based clinical applications.     

HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.

We evaluated 750 consecutive invasive breast carcinomas for HER-2/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2/neu positive and 0 and 1+ IHC reactions were considered HER-2/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH analysis was performed on the positive and negative IHC tests. One hundred thirty-eight cases (18.4%) were HER-2/neu positive by IHC and/or FISH. One hundred twenty-three of the positive cases (89%) were 3+ IHC reactions and 14 positive cases were inconclusive by IHC and amplified by FISH. There was concordance with FISH in 77 of 78 (98.7%) of the positive or negative IHC cases that were tested (95% confidence interval [CI] = 93.1 to 100%). A single IHC-negative case showed HER-2/neu amplification by FISH. Thirty-nine cases were 2+ IHC (5.2%); 14 (36%) were amplified, 24 (62%) were not amplified, and one was not interpretable. HER-2/neu positivity was observed in 34% of grade 3 ductal carcinomas, 11.4% of grade 2 ductal carcinomas, 3.2% of grade 1 ductal carcinomas, and 3.2% of lobular carcinomas. Occasional cases with discordant IHC expression of HER-2/neu within the in situ and invasive carcinoma elements were also identified. IHC reliably characterized HER-2/neu in approximately 95% of the cases studied (95% CI = 93.0 to 96.2%) and was effective as a primary method for evaluating HER-2/neu status. In this study, 2+ IHC reactions were a heterogeneous group best regarded as indeterminate or inconclusive; in this series, only 36% were amplified by FISH analysis. Our findings suggest that a combination of IHC and FISH testing with FISH analysis performed reflexly on all 2+ IHC cases can optimize HER-2/neu testing.     

Quantitative evaluation of HER-2/neu status in breast cancer by fluorescence in situ hybridization and by immunohistochemistry with image analysis

We correlated quantitative results obtained in 40 invasive breast cancer cases for HER-2 gene amplification by fluorescence in situ hybridization with protein expression by immunohistochemical studies with computer-assisted image analysis. Fluorescence in situ hybridization (FISH) results were quantified as the mean number of fluorescent signals per nucleus, and immunohistochemical slides were read by semiquantitatively assessing membranous immunostaining intensity in tumor cells vs nonneoplastic breast tissue or quantitatively evaluated by image analysis. We found high correlation between immunohistochemical results by semiquantitative scoring and by image analysis. FISH results correlated with immunohistochemical results moderately when the staining intensity of only tumor cells was assessed and significantly better when the difference in staining intensity between tumor cells and nonneoplastic breast tissue was assessed. The correlation with FISH results was further improved when immunohistochemical study was combined with heat-induced epitope retrieval (HIER). Although FISH and immunohistochemical studies assess different aspects of the HER-2/neu gene (amplification vs overexpression), we found good correlation between the diagnostic techniques. The correlation was best when immunohistochemical studies were combined with HIER and assessed as the difference between tumor cells and nonneoplastic breast tissue.     

Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer

Determination of HER-2/neu oncogene amplification has become clinically important in the present management of breast cancer and may have important applications in other areas of clinical oncology and scientific research. In situ hybridization is an extremely accurate and sensitive technique for assessing amplification of HER-2/neu. A new method using a chromogen-labeled probe offers numerous advantages, including the ability to view the morphologic features of the cells of interest using a light microscope, which can be found in every laboratory. We used both techniques to assay 31 cases of infiltrating breast carcinoma; assays were performed in laboratories at 2 institutions. Identical results for both methods were found in 26 cases (10 amplified, 16 nonamplified). One case was misinterpreted as overexpressed by chromogenic in situ hybridization (CISH) because of background precipitate. In 4 cases, CISH suggested low-level amplification. Three of these cases subsequently were found to have chromosome 17 polysomy. In the remaining case, the initial section chosen was suboptimal, showing weak signals by both methods. If a probe for chromosome 17 (now available for CISH) is used in cases of questionable HER-2/neu amplification, CISH seems to be as accurate and more practical than FISH.     

HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in situ hybridization and immunocytochemical analysis

We evaluated HER-2 receptor status by immunocytochemical and immunohistochemical analyses and fluorescence in situ hybridization (FISH) in 51 fine-needle aspiration (FNA) specimens together with the corresponding formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from surgically resected breast cancers. Three fixation methods were compared: ethanol, formalin, and CytoLyt-ThinPrep (Cytyc, Boxborough, MA). HER-2 was overexpressed and amplified in 8 (16%) of 51 FFPE specimens. Of the 8 cases, gene amplification was observed in 8 FNA specimens (100%) and overexpression in 2 (25%) ethanol-, 4 (50%) CytoLyt-, and 5 (63%) formalin-fixed FNA specimens. Strong pairwise kappa association between FISH results performed on FNA specimens and FFPE tissue samples (ethanol fixation, kappa = 0.848; ThinPrep, kappa = 0.918) and moderate (ThinPrep, kappa = 0.692; formalin fixation, kappa = 0.667) to poor (ethanol, kappa = 0.300) pairwise kappa agreement between tissue immunohistochemical and FNA immunocytochemical results was demonstrated. We conclude that HER-2 protein expression on cytologic preparations was insufficiently reliable for clinical use, whereas HER-2 gene amplification determined by FISH demonstrated strong and consistent correlation with HER-2 status of FFPE tissue samples.     

Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples

Determination of HER-2/neu oncogene amplification has become necessary for selection of breast cancer patients for trastuzumab (Herceptin) therapy. Fluorescence in situ hybridization (FISH) is currently regarded as a gold standard method for detecting HER-2/neu amplification, but it is not very practical for routine histopathological laboratories. We evaluated a new modification of in situ hybridization, the chromogenic in situ hybridization (CISH), which enables detection of HER-2/neu gene copies with conventional peroxidase reaction. Archival formalin-fixed paraffin-embedded tumor tissue sections were pretreated (by heating in a microwave oven and using enzyme digestion) and hybridized with a digoxigenin-labeled DNA probe. The probe was detected with anti-digoxigenin fluorescein, anti-fluorescein peroxidase, and diaminobenzidine. Gene copies visualized by CISH could be easily distinguished with a x40 objective in hematoxylin-stained tissue sections. HER-2/neu amplification typically appeared as large peroxidase-positive intranuclear gene copy clusters. CISH and FISH (according to Vysis, made from frozen pulverized tumor samples) correlated well in a series of 157 breast cancers (kappa coefficient, 0.81). The few different classifications were mostly because of low-level amplifications by FISH that were negative by CISH and immunohistochemistry with monoclonal antibody CB-11. We conclude that CISH, using conventional bright-field microscopy in evaluation, is a useful alternative for determination of HER-2/neu amplification in paraffin-embedded tumor samples, especially for confirming the immunohistochemical staining results.     

Comparison of immunohistochemical and fluorescence in situ hybridization assessment of HER-2 status in routine practice

Because HER-2 expression in invasive carcinoma of the breast has well-documented ramifications for treatment and prognosis, accurate assessment of HER-2 status is critical. Comparative studies have shown high concordance rates between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in cases with immunohistochemical scores of 0 or 1+ (negative) and 3+ (strongly positive) and low concordance rates among cases with immunohistochemical scores of 2+. The present study was performed to determine concordance rates in a setting more representative of routine clinical practice, in which multiple pathologists submit specimens to a single cytogenetics referral laboratory. We found a higher rate of discordance between immunohistochemical analysis and FISH (approximately 92%) in the groups with immunohistochemical scores of 2+ than reported in other studies. These results strongly support the practice of performing FISH in all cases with immunohistochemical scores of 2+, particularly in routine practice, in which interobserver variability in immunohistochemical scoring among multiple pathologists is likely to be high.     

Reliability of chromogenic in situ hybridization for detecting HER-2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility.

Accurate determination of HER-2 status is important in the management of patients with breast cancer, especially in determining their eligibility for trastuzumab therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting HER-2 gene amplification. Recently, chromogenic in situ hybridization (CISH), in which HER-2 is detected by a peroxidase reaction and the gene copies are determined by regular bright-field microscopy, has emerged as a potential alternative to FISH. However, this method requires validation before it can be adopted into clinical practice. In this study, we evaluated 80 cases of invasive breast carcinoma by CISH, compared the results with those obtained by FISH, and assessed interobserver reproducibility among three observers. We found that agreement among the three pathologists on the CISH-determined HER-2 status was achieved in 73 cases (91%), all of which had results matching the corresponding FISH results: 54 nonamplified and 19 amplified. Of the 19 amplified cases, 13 were scored unanimously as high-level amplification; six had a minor scoring discrepancy (ie, low-level vs high-level amplification). A major scoring discrepancy (ie, nonamplification vs amplification) was found in the remaining seven cases, three of which were amplified and four of which were nonamplified by FISH. Two of the latter cases had a polysomy of chromosome 17. The cases that caused scoring difficulty were those with an equivocal or borderline signal number against a high background. Overall, there was nearly perfect agreement between the CISH and corresponding FISH results, and interpretation of CISH results were highly reproducible among the three pathologists. We conclude that, in general, HER-2 status can be reliably assessed by CISH. Confirmatory FISH is recommended in cases with equivocal or borderline CISH copy numbers.     

HER-2/neu gene amplification in breast imprint cytology analyzed by fluorescence in situ hybridization: direct comparison with companion tissue sections

Fluorescence in situ hybridization (FISH) is a validated method for detection of HER-2/neu gene amplification and was recently approved by the FDA for diagnostic use in paraffin-embedded tissue. Its use in cytologic specimens, however, has not been investigated. To see whether HER-2/neu gene amplification is detectable in cytologic specimens, we examined touch imprints and corresponding tissue sections of 27 breast carcinomas (20 invasive and 7 in situ) and 3 atypical epithelial hyperplastic lesions, using the FISH technique with the HER-2/neu DNA probe kit (Vysis, Inc., Downers Grove, IL). HER-2/neu gene amplification was determined, using the ratio of HER-2/neu:CEP 17 signal counts; a ratio of 2.0 or greater was considered amplified. Successful hybridization occurred in 55/60 (92%) slides. In all cases, at least one of the paired slides was adequate for evaluation. Whole-cell imprint and tissue section slides yielded comparable HER-2/neu:CEP 17 signal counts and ratios, including one case of low-level HER-2/neu gene copy numbers where the ratio was 2.0. Our findings indicate that whole-cell imprint cytology preparations are a reliable medium for HER-2/neu gene quantification by FISH, and may substitute for or complement tissue section analysis.     

A formalin-fixed, paraffin-processed cell line standard for quality control of immunohistochemical assay of HER-2/neu expression in breast cancer.

To ensure the accuracy and reproducibility of immunohistochemical assays for determining HER-2/neu status of patients with breast cancer, a reliable standard for monitoring assay sensitivity is necessary. We optimally fixed and paraffin processed human ovarian and breast carcinoma cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 in quantities sufficient to meet the needs of a laboratory for the foreseeable future. The material was tested, alongside HercepTest kit cell lines (DAKO, Carpinteria, CA), by 7 breast cancer centers in the United Kingdom and France with different immunohistochemical assays and markers. The cell lines also were analyzed by fluorescence in situ hybridization (FISH) by 2 centers using HER-2/neu kits. FISH produced 100% agreement between the 2 centers: SKOV-3 and MDA-MB-453 showed HER-2/neu amplification and BT-20 and MCF-7 did not. Immunohistochemical analysis and a common evaluation method produced 100% agreement that SKOV-3 and MCF-7 showed 3+ and zero HER-2/neu overexpression, respectively. For MDA-MB-453, there was 71% (5/7) concordance of 2+ immunohistochemical staining and 86% (6/7) concordance of zero or 1 + staining for BT-20. The cell lines provide a valuable standard for gauging HER-2/neu assay sensitivity irrespective of the antibody, antigen retrieval system, detection system, or method of evaluation used.     

Comparative investigations of the determination of the HER-2 status in breast cancer by fluorescence and chromogenic in situ hybridization

Forty invasive breast ductal carcinoma biopsy specimens were investigated. The techniques of fluorescence and chromogenic in situ hybridization were used to study HER-2/neu gene amplification and the concordance of these techniques was examined. The advantages and disadvantages of both techniques are shown. The optimum ranges of using these techniques in the determination of the herzept status of breast cancer are given.     

Comparison between immunohistochemistry and chromogenic in situ hybridization in assessing HER-2 status in breast cancer

Human epidermal growth factor receptor-2 (HER-2) is usually determined as a potential target for breast cancer therapy. The purpose of the present study was to compare chromogenic in situ hybridization (CISH) with immunohistochemistry (IHC) in determination of HER-2 status, in metastatic breast cancer patients screened for the clinical study of chemotherapy +/- herceptin. It was possible to assess both CISH and IHC in 56 cases, using CISH Detection Kit (Zymed) and HercepTest (DakoCytomation), respectively. HER-2 was amplified by CISH in 32 cases (57%) while 33 (59%) were HER-2-positive by IHC. A concordance between HER-2 status determined by CISH and IHC was noted in 43 of 56 cases (77%; P = 0.00008). Gene amplification was observed in 6/16 cases (37.5%) in IHC-negative subgroup (1+), while no amplification was observed in 5/10 cases (50%) in the IHC-positive subgroup (2+). These results suggest that there was a greater heterogeneity on the genetic level and that simple IHC classification was not sufficient. It is suggested that CISH could be considered as a useful additional method to IHC in determining HER-2 status in breast cancer patients, with a recommendation for testing not only the 2+ but also the 1+ subgroup of patients.     

HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring

We analyzed concordance between immunohistochemical analysis and fluorescence in situ hybridization (FISH) in HER-2 status and studied the effect of dual-color (D-FISH) vs single-color FISH (S-FISH) scoring on the assignment of tumors to amplified or nonamplified categories. The assays were performed on formalin-fixed, paraffin-embedded sections of 2,279 invasive breast carcinomas. Immunohistochemical results were interpreted as negative (0, 1+) or positive (2+, 3+). For FISH analyses, a ratio for HER-2/chromosome 17 of 2.0 or more (D-FISH) or an absolute HER-2 copy number per nucleus of more than 4.0 (S-FISH) were interpreted as positive gene amplification. We found 547 (24.0%) cases positive immunohistochemically, 326 (14.3%) by D-FISH, and 351 (15.4%) by S-FISH. Overall concordance in HER-2 status with immunohistochemical analysis was 87% for D-FISH and 86% for S-FISH. Excellent concordance was found among groups scored immunohistochemically as 0, 1+, and 3+ (with D-FISH, 97%; with S-FISH, 96%). The most discordant category was the group scored 2+ immunohistochemically, in which only a quarter of the 2+ tumors were FISH(+). D-FISH and S-FISH scoring results were discordant in 89 tumors (4%), of which 8 (9%) had 3+ immunohistochemical staining and none showed high-level HER-2 amplification. Among all FISH(+) tumors, 10% were negative by immunohistochemical analysis, and notably almost half (47%) showed borderline to low HER-2 amplification (D-FISH score, 2.0-3.9); the clinical significance of these findings warrants further investigation.     

erb-b2 amplification by fluorescence in situ hybridization in breast cancer specimens read as 2+ in immunohistochemical analysis

We conducted this study to ascertain the prevalence of erb-b2 gene amplification in breast cancer specimens read as 2+ in immunohistochemical analysis. Slides from patients with metastatic or recurrent breast cancer were eligible for fluorescent in situ hybridization (FISH) study if they were read as 2+ immunohistochemically for erb-b2 by a certified pathologist. The PathVysion kit (Vysis, Downers Grove, IL) was used for FISH studies. Amplification of the erb-b2 gene was defined as an erb-b2/CEP17 (chromosome 17 centromere) ratio of 2 or more in 30 tumor cells counted. From May 2003 to June 2004, 221 slides were submitted from 24 hospitals around the island. Of 216 successful hybridizations, 96 (44.4%) were determined to be erb-b2 amplified. In addition, the topoisomerase IIa gene was coamplified in 11 (21%) of 53 and deleted in 8 (15%) of 53 erb-b2 amplified cases. The erb-b2 gene amplification rate was very high in cases determined to be 2+ by immunohistochemical analysis; therefore, determination of erb-b2 status by FISH in cases scored 2+ immunohistochemically is strongly recommended.     

Contribution of fluorescence in situ hybridization to immunohistochemistry for the evaluation of HER-2 in breast cancer

The main focus of the present study was to assess the efficacy of interphase cytogenetics using fluorescence in situ hybridization (FISH) as a valid alternative to immunohistochemistry (IHC) in paraffin-embedded tissue sections and/or the efficacy of the combination of the two methods, while, at the same time, aiming to provide additional information on the use of the two methods. For this study, selected breast cancer patients (n=66) were tested for HER-2 gene amplification by FISH. The probe contains DNA sequences specific for the HER-2 human gene locus and hybridizes to the 17q11.2 through q12 region of human chromosome 17. The same samples were tested previously for HER-2 overexpression by two monoclonal antibodies (300G9 and CB11), recognizing an extracellular and an internal domain of gp185(Her-2), respectively. HER-2 overexpression also was evaluated using the HerceptTest Kit (Dako, Milan, Italy). The HerceptTest was performed according to the manufacturer's standard procedures, and results were scored on a 0 to 3+ scale. A total of 34 (51%) of 66 breast tumors enrolled in this study were positive by FISH. Of the 34 cases amplified by FISH, 9 were negative by IHC using both monoclonal antibody (MoAb) 300G9 and MoAb CB11, with a concordance rate from 80.3% to 83.3%. A higher concordance was verified (92.4%) when we used the HerceptTest Kit. Of the 32 cases found negative with the HerceptTest, FISH analysis identified HER-2 gene amplification in more than 10%. Our results indicate that with the combined use of both methods, several amplified samples classified negative by IHC can be used thus improving therapeutic planning for specific therapy with the monoclonal antibody trastuzumab.     

Comparison of immunohistochemistry by automated cellular imaging system (ACIS) versus fluorescence in-situ hybridization in the evaluation of HER-2/neu expression in primary breast carcinoma

AIMS: Immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) are both commonly used assays for evaluation of HER-2/neu status in breast cancer. However, there is still no consensus on which method is most predictive of patient response to Herceptin. Recently, the automated cellular imaging system (ACIS) has been shown to improve the accuracy and reproducibility in scoring IHC. Our aim was to compare the results of HER-2/neu expression and gene amplification in the same patients by IHC using the ACIS system and by FISH. METHODS AND RESULTS: Two hundred and forty-seven breast cancer cases were studied. The concordance rate between IHC-ACIS (> or = 2.2) and FISH (> or = 2.0) was 94%. Fifteen patients were discordant; three had borderline FISH values and three had borderline IHC values. The other nine discordant cases consisted of five IHC-ACIS+, FISH- and six IHC-ACIS-, FISH+. HER-2/neu overexpression was more common in tumours that were high-grade, aneuploid, progesterone receptor and bcl-2 negative, with MIB-1 > 10%. CONCLUSION: HER-2/neu assessment by the ACIS is reliable, rapid and inexpensive, and correlates highly with results obtained by FISH.     

HER-2/neu and topoisomerase IIa gene amplification and protein expression in invasive breast carcinomas: chromogenic in situ hybridization and immunohistochemical analyses

We studied HER-2/neu (HER-2) and topoisomerase IIa (topo2a) amplification (using chromogenic in situ hybridization) and overexpression (immunohistochemical analysis) in 113 invasive breast carcinomas. A gene copy number/chromosome 17 copy number ratio of 2.0 or higher indicated amplification. A topo2a/chromosome 17 ratio of less than 0.8 indicated gene deletion. HER-2 overexpression was scored according to standard HercepTest guidelines (DAKO, Carpinteria, CA). Overexpression of topo2a was identified when nuclear staining was found in more than 5% of tumor cells. Of 113 tumors, 104 were analyzed successfully for HER-2 and topo2a amplification. Of the 104, 64 showed HER-2 amplification; 25 of these (39%) also showed topo2a amplification. No amplification was found in 40 tumors. Deletion of topo2a was seen in 7 (11%) of 64 HER-2-amplified tumors and 2 (5%) of 40 nonamplified tumors. Of 25 tumors with topo2a amplification, 18 (72%) overexpressed topo2a. Only 3 (4%) of 79 tumors without topo2a amplification overexpressed topo2a. Amplification of topo2a is associated with HER-2 amplification but not vice versa. Amplification of topo2a resulted in protein overexpression in 72% of tumors, but topo2a overexpression rarely occurred without gene amplification. Identification of topo2a and HER-2 status might have therapeutic and prognostic implications.     

Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER2/neu status in primary breast carcinoma using tissue microarray.

Identification of HER2/neu status is important for predicting response to specific chemotherapy in breast carcinoma. Chromogenic in situ hybridization was performed using tissue microarray technology on 188 primary breast carcinomas. To validate the reliability of novel chromogenic in situ hybridization technology, the results of chromogenic in situ hybridization were correlated with the results of two-color fluorescence in situ hybridization done with the same tumors. On tissue microarray panels containing 188 breast carcinoma tissues, fluorescence in situ hybridization and chromogenic in situ hybridization were conducted simultaneously. HER2/neu amplification was detected in 46 tumors (24.5%) by fluorescence in situ hybridization and in 43 tumors (22.9%) by chromogenic in situ hybridization. Results of each method agreed with each other in 177 tumors (concordance: 94.1%). HER2/neu amplification by fluorescence in situ hybridization was associated with nuclear pleomorphism (P =.021), and HER2/neu amplification by chromogenic in situ hybridization was associated with poor nuclear grade (P =.037). High concordance between fluorescence in situ hybridization and chromogenic in situ hybridization indicated that chromogenic in situ hybridization can be a tempting alternative to fluorescence in situ hybridization for the detection of HER2/neu amplification in breast carcinoma because of its accuracy and relative low cost. HER2/neu appeared to have a prognostic implication because its amplification was associated with aggressive biologic features of the breast carcinoma. Integration of tissue microarray technology enabled high-throughput determination of HER2/neu amplification profile with rapidity and accuracy in large cohorts of the breast carcinoma.     

Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays

AIM: To evaluate the clinical usefulness of three commercially available assays for Her-2/neu oncogene and protein measurements. The Her-2/neu protein is overexpressed, mostly as a result of gene amplification, in 20-30% of human breast cancers, and has been shown to have prognostic and predictive value for treatment with chemotherapy or the new monoclonal antibody, Herceptin. METHODS: An immunohistochemistry (IHC) assay using the Dako polyclonal antibody A0485, which measures the Her-2/neu protein, was compared with two new Food and Drug Administration (FDA) approved fluorescence in situ hybridisation (FISH) assays--INFORM and PathVysion, in a cohort of 52 formalin fixed, paraffin wax embedded breast tissues. These tissues were selected randomly from 84 consecutive infiltrating breast cancer specimens, which were first stratified according to the Her-2/neu protein levels as measured by IHC. RESULTS: The two FISH assays achieved a 98% concordance rate: 14 specimens (27%) showed Her-2/neu gene amplification and 37 specimens (71%) showed no Her-2/neu gene amplification. The PathVysion assay had certain advantages over the INFORM assay. In contrast, the IHC assay detected Her-2/neu overexpression in a high percentage of cases, including 13 high positive specimens (25%) and 13 medium positive specimens (25%). Although 10 of these 13 IHC high positive specimens showed gene amplification by FISH, nine of 13 IHC medium positive specimens showed no gene amplification. Statistical analyses showed that the differences between IHC and FISH assays were primarily in the specimens with medium positive IHC, but negative FISH results. CONCLUSIONS: Because of the increasing importance of the Her-2/neu oncogene and oncoprotein in the clinical management of patients with breast cancer, the accurate and consistent evaluation of Her-2/neu status is crucial. This study suggests that the best approach is to combine both IHC and FISH assays; that is, to use the IHC assay as a triage step, followed by the PathVysion FISH assay to analyse the IHC medium and high positive cases.