scholarly journals Expression of HER2 and the Coamplified Genes GRB7 and MLN64 in Human Breast Cancer: Quantitative Real-time Reverse Transcription-PCR as a Diagnostic Alternative to Immunohistochemistry and Fluorescence In situ Hybridization

2005 ◽  
Vol 11 (23) ◽  
pp. 8348-8357 ◽  
Author(s):  
Ursula Vinatzer ◽  
Brigitta Dampier ◽  
Berthold Streubel ◽  
Margit Pacher ◽  
Michael J. Seewald ◽  
...  
Keyword(s):  
Breast Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Reverse Transcription ◽  
Human Breast Cancer ◽  
Human Breast ◽  
Reverse Transcription Pcr

Assessment of Methods for Tissue-Based Detection of the HER-2/neu Alteration in Human Breast Cancer: A Direct Comparison of Fluorescence In Situ Hybridization and Immunohistochemistry

2000 ◽  
Vol 18 (21) ◽  
pp. 3651-3664 ◽  
Author(s):  
Giovanni Pauletti ◽  
Suganda Dandekar ◽  
HongMei Rong ◽  
Lilllian Ramos ◽  
HongJun Peng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Gene Amplification ◽  
Survival Probability ◽  
Human Breast Cancer ◽  
Patient Survival ◽  
Human Breast ◽  
Her 2

PURPOSE: To compare the efficacy of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in detecting the HER-2/neu alteration in human breast cancer. PATIENTS AND METHODS: Unselected stage I, II, and III breast cancer patients (N = 900) were tested for HER-2/neu gene amplification by FISH in paraffin-embedded, formalin-fixed archival material. Of these samples, 856 were tested for HER-2/neu overexpression by non–antigen-retrieval IHC with the polyclonal antibody R60, the sensitivity and specificity of which was preliminarily compared with the United States Food and Drug Administration–approved HercepTest (Dako Corp, Carpinteria, CA). Patient survival was analyzed in relation to the presence of the HER-2/neu alteration as determined by these two methodologies. RESULTS: A total of 189 (21%) of 900 patients were positive by FISH and 147 (17.2%) of 856 were positive by IHC. This discrepancy is consistent with expected loss of IHC sensitivity associated with tissue fixation/embedding. The HercepTest did not improve sensitivity and introduced false positives. Comparison of R60-based IHC with FISH demonstrates that patient survival is associated progressively to gene amplification level as determined by FISH, whereas for IHC an association is found only in the highest (3+) immunostaining group. Among FISH-negative tumors, 45 (6.6%) of 678 were IHC-positive, with a survival probability similar to that of FISH-negative/IHC-negative cases; FISH-positive/IHC-negative patients have a survival probability similar to that of FISH-positive/IHC-positive cases. CONCLUSION: IHC does not consistently discriminate patients likely to have a poor prognosis, whereas FISH provides superior prognostic information in segregating high-risk from lower-risk beast cancers. HER-2/neu protein overexpression in the absence of gene amplification occurs infrequently in breast cancer, in which case, patient outcome is similar to that of patients without the alteration.

Copy Number Analysis of c-erb-B2 (HER-2/neu) and Topoisomerase IIα Genes in Breast Carcinoma by Quantitative Real-Time Polymerase Chain Reaction Using Hybridization Probes and Fluorescence In Situ Hybridization

2005 ◽  
Vol 129 (1) ◽  
pp. 39-46
Author(s):  
Sabita K. Murthy ◽  
Anthony M. Magliocco ◽  
Douglas J. Demetrick
Keyword(s):  
Breast Cancer ◽  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Hybridization Probes ◽  
Fresh Frozen

Abstract Context.—The Topoisomerase IIα (TOP2A) protein is the target of the anthracycline class of chemotherapeutic agents. TOP2A is frequently coamplified with c-erb-B2 and consequently might be a prognostic and/or predictive factor for breast cancer patients when anthracycline-based chemotherapy is a consideration. A total of 20% to 35% of breast carcinomas show amplification of the erb-B2 gene, some of which also have coamplification of the TOP2A gene. Investigation of the prognostic or predictive significance of these gene amplifications requires a reliable and sensitive method for the measurement of gene copy number in clinical tumor samples. Objective.—To assess 2 different assay methods that might allow accurate, reproducible, quantitative, and high-throughput estimation of gene copy number in fresh, frozen, or paraffin-embedded breast cancer specimens. Design.—We developed an assay and analyzed the gene copy numbers of the erb-B2 and TOP2A genes in 8 breast cancer cell lines, 6 fresh frozen samples, and 38 paraffin-embedded breast tumor specimens by a novel real-time polymerase chain reaction (PCR) assay using hybridization probes. The results were compared with standard fluorescence in situ hybridization. Results.—We discovered a 100% concordance between assessment of gene copy number of erb-B2 and TOP2A between quantitative PCR and fluorescence in situ hybridization (FISH). Quantitative PCR also had the additional feature of uncovering an erb-B2 gene polymorphism. Finally, we observed that TOP2A amplification only occurred in conjunction with erb-B2 amplification in our paraffin-embedded cases of invasive breast carcinoma and that this event was present in 5 (42%) of 12 erb-B2 amplified cases. Conclusions.—We conclude that the potentially automatic, real-time PCR analysis using hybridization probes is an efficient method to perform copy number analysis, with results that appear identical to the FISH technique and with the benefit of identifying HER-2 polymorphisms.

scholarly journals Evaluation of the Quantitative Analytical Methods Real-Time PCR for HER-2 Gene Quantification and ELISA of Serum HER-2 Protein and Comparison with Fluorescence in Situ Hybridization and Immunohistochemistry for Determining HER-2 Status in Breast Cancer Patients

2005 ◽  
Vol 51 (7) ◽  
pp. 1093-1101 ◽  
Author(s):  
Chantal Tse ◽  
Didier Brault ◽  
Joseph Gligorov ◽  
Martine Antoine ◽  
Rainer Neumann ◽  
...  
Keyword(s):  
Breast Cancer ◽  
In Situ Hybridization ◽  
Metastatic Breast Cancer ◽  
Fluorescence In Situ Hybridization ◽  
Real Time ◽  
Metastatic Breast ◽  
Gene Copy ◽  
Primary Breast Tumor ◽  
Her 2

Abstract Background: HER-2 status is generally determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Both methods are only semiquantitative, require a tumor sample, and can be difficult to reproduce. We compared these methods with 2 quantitative approaches, one measuring HER-2 gene copy number in tissue by real-time quantitative PCR (qPCR), and the other measuring shed HER-2 protein in serum by ELISA in patients with metastatic disease. Methods: We analyzed 52 cases of metastatic breast cancer for which both serum collected at the diagnosis of metastasis and stored primary breast tumor specimens were available. The within- and between-run imprecision of real-time qPCR and ELISA were evaluated according to Clinical and Laboratory Standards Institute (formerly known as NCCLS) recommendations. Concordance among the 4 methods was assessed by calculating the κ statistic and its 95% confidence interval (95% CI). Results: The CVs for within- and between-run imprecision were both <10% with qPCR and ELISA. There was good agreement of results between qPCR and IHC (κ = 0.81; 95% CI, 0.64–0.99), qPCR and FISH (κ = 0.77; 95% CI, 0.58–0.96), ELISA and IHC (κ = 0.65; 95% CI, 0.41–0.89); and ELISA and FISH (κ = 0.69; 95% CI, 0.46–0.92). Conclusions: Measurements of HER-2 gene expression by qPCR and of serum HER-2 protein by ELISA are highly reproducible approaches for determining HER-2 status in metastatic breast cancer. In addition, ELISA eliminates the need for biopsy.

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