Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.
ABSTRACT Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis.
A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy.
Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4.
Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.
- SourceAvailable from: ascpjournals.orgAmerican Journal of Clinical Pathology 03/2000; 113(2):171-5. · 2.88 Impact Factor
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ABSTRACT: Her-2/neu, a proto-oncogene located on chromosome 17, is an important biomarker in breast carcinoma. Immunohistochemistry (IHC) is currently the most widely used method for assessing Her-2/neu status. Some IHC-positive cases do not show Her-2/neu gene amplification by fluorescence in situ hybridization (FISH). It has been suggested that some of these IHC "false positive" results may in part be due to increased copy number of chromosome 17 resulting in increased Her-2/neu protein expression. We analyzed IHC and FISH data from 561 cases of invasive breast carcinoma to test this hypothesis. IHC and FISH for Her-2/neu were performed on formalin-fixed, paraffin-embedded sections of 561 invasive breast carcinomas. The IHC results were interpreted as 0, 1+, 2+, or 3+ according to the manufacturer's recommended criteria. The FISH results were expressed as a ratio of Her-2/neu/chromosome 17 and were interpreted as positive (> = 2.0) or negative (<2.0) for gene amplification according to the manufacturer's recommended scoring system. We found that in IHC 3+/FISH-negative cases (n = 15) both the average chromosome 17 copy number and the average Her-2/neu copy number were significantly higher than that in IHC (0 to 2+)/FISH-negative cases (n = 411) (2.45 vs. 1.68; P < 0.0001, and 3.19 vs. 1.95; P < 0.0001, respectively). In contrast, the IHC 2+/FISH-negative cases did not exhibit a significantly increased number of chromosome 17 compared to IHC 0 to 1+ cases. In addition, the average copy number of chromosome 17 in FISH-positive cases (n = 135) was significantly higher than that in FISH-negative cases (n = 426) (2.27 vs. 1.70; P < 0.0001), indicating a general association of increased chromosome 17 copy number with Her-2/neu gene amplification. Thus, our data suggest that IHC 3+ immunostaining without scorable gene amplification may indeed be, at least in some cases, the result of increased Her-2/neu protein expression secondary to an increased copy number of chromosome 17, associated with an increased total number of Her-2/neu gene copies per tumor cell.Journal of Molecular Diagnostics 08/2003; 5(3):155-9. · 3.95 Impact Factor
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ABSTRACT: 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. 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. 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. 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.Journal of Clinical Pathology 06/2000; 53(5):374-81. · 2.44 Impact Factor