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      Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

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          Abstract

          García-Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J (2010) Histopathology 56, 472–480 Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

          Aims:

          Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results.

          Methods and results:

          We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96.

          Conclusions:

          We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.

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          Most cited references 22

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          A phase III randomized comparison of lapatinib plus capecitabine versus capecitabine alone in women with advanced breast cancer that has progressed on trastuzumab: updated efficacy and biomarker analyses.

          Lapatinib is a small molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor type 2 (HER2). Initial results of a phase III trial demonstrated that lapatinib plus capecitabine is superior to capecitabine alone in women with HER2-positive advanced breast cancer that progressed following prior therapy including trastuzumab. Updated efficacy and initial biomarker results from this trial are reported. Women with HER2-positive, locally advanced or metastatic breast cancer previously treated with anthracycline-, taxane-, and trastuzumab-containing regimens were randomized to lapatinib 1,250 mg/day continuously plus capecitabine 2,000 mg/m(2) days 1-14 of a 21-day cycle or capecitabine 2,500 mg/m(2) on the same schedule. The primary endpoint was time to progression (TTP) as determined by an independent review panel. Relationship between progression-free survival (PFS) and tumor HER2 expression and serum levels of HER2 extracellular domain (ECD) were assessed. 399 women were randomized. The addition of lapatinib prolonged TTP with a hazard ratio (HR) of 0.57 (95% CI, 0.43-0.77; P < 0.001) and provided a trend toward improved overall survival (HR: 0.78, 95% CI: 0.55-1.12, P = 0.177), and fewer cases with CNS involvement at first progression (4 vs. 13, P = 0.045). Baseline serum HER2 ECD did not predict for benefit from lapatinib. The addition of lapatinib to capecitabine provides superior efficacy for women with HER2-positive, advanced breast cancer progressing after treatment with anthracycline-, taxane-, and trastuzumab-based therapy. Biomarker studies could not identify a subgroup of patients who failed to benefit from the addition of lapatinib to capecitabine.
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            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.
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              A general methodology for the analysis of experiments with repeated measurement of categorical data.

              This paper is concerned with the analysis of multivariate categorical data which are obtained from repeated measurement experiments. An expository discussion of pertinent hypotheses for such situations is given, and appropriate test statistics are developed through the application of weighted least squares methods. Special consideration is given to computational problems associated with the manipulation of large tables including the treatment of empty cells. Three applications of the methodology are provided.
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                Author and article information

                Journal
                Histopathology
                his
                Histopathology
                Blackwell Publishing Ltd
                0309-0167
                1365-2559
                March 2010
                : 56
                : 4
                : 472-480
                Affiliations
                Department of Morphological Sciences, School of Medicine-University Clinical Hospital, University of Santiago de Compostela Santiago de Compostela, Spain
                [1 ]Division of Pathology and Cytology, University Hospital of Lund Lund, Sweden
                [2 ]Division of Pathology, School of Molecular Medical Sciences, University of Nottingham Nottingham
                [3 ]University Hospital Birmingham NHS Foundation Trust Birmingham, UK
                [4 ]Institute of Pathology, Johannes Gutenberg University Hospital Mainz, Germany
                [5 ]Dako Denmark A/S Glostrup, Denmark
                Author notes
                J Mollerup, Dako Denmark A/S, Produktionsvej 42, 2600 Glostrup, Denmark. e-mail: jmollerup@ 123456dako.com
                T.G-C., D.G., A.R.G., J.G. and A.S. contributed equally to this work.

                Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html

                Article
                10.1111/j.1365-2559.2010.03503.x
                2855864
                20459554
                0298be09-7161-4145-896a-7438db15d1d3
                Journal compilation © 2010 Blackwell Publishing Ltd

                Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

                Categories
                Original Articles

                Pathology

                cen-17, breast cancer, her2, her2 amplification, fish, cish, in situ hybridization

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