Available Assays and Interpretation

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IHC staining (Figure 24-1, lower panel), which has been the predominant method utilized for clinical testing of HER2/NEU, can be significantly affected by technical issues, especially for archival fixed paraffin-embedded tissues. Advantages of IHC testing include its wide availability, relatively low cost, easy preservation of stained slides, and use of routine microscopy. Difficulties with the technical aspects of IHC include the effects of preanalytic issues (including duration and type of fixation, and block storage conditions), intensity of antigen retrieval, type of antibody (polyclonal versus monoclonal),nature of control samples, and, most important, lack of an objective slide scoring system. Two commercially available HER2/NEU IHC kits, the Dako Herceptest and the Ventana Pathway, are FDA approved for determining the eligibility of patients to receive the anti-HER2/NEU therapeutic antibody trastuzumab (Herceptin). Problems with standardization in slide scoring recently have been highlighted and raise questions regarding the best method for determining HER2/NEU status for prediction of response to Her-ceptin.15 Slide scoring can be improved by avoiding specimen edges, retraction artifacts, under- or overfixation, cases with significant staining of benign tissues, and tumor cells lacking a complete membranous staining pattern (the so-called chicken wire appearance). The use of a computerized image analysis system can reduce slide scoring variability among pathologists and improve the reproducibility of interpreting the IHC tests.16

The FISH technique (Figure 24-1, upper panel), which is morphology-driven and like IHC can be automated, has the advantages of an objective scoring system and the presence of a built-in internal control consisting of the two HER2/NEU gene signals present in all cells in the specimen. Disadvantages of FISH testing include the higher cost compared with IHC, longer time required for slide scoring, requirement of a fluorescence microscope, and inability to preserve the slide for storage and review. Two versions of the FISH assay are FDA approved: the Ventana Inform test, which measures only HER2/NEU gene copies, and the Abbott-Vysis Pathvysion test, which includes a chromosome 17 probe in a dual-color format. Published studies indicate that the two assays are highly correlative.17 The chromogenic in situ hybridization method, or CISH technique (Figure 24-1, middle panel), features the advantages of both IHC (routine microscopy, lower cost, familiarity) and FISH (built-in internal control, objective scoring, more robust DNA target), but is not, to date, FDA approved for determining patient eligibility for Herceptin treatment.18,19 The reverse transcription-polymerase chain reaction (RT-PCR) technique20,21 has predominantly been used to detect HER2/NEU messenger RNA (mRNA) from occult breast cancer cells found in peripheral blood and bone marrow samples. It has correlated more with gene amplification status than IHC levels,22 and failed to predict

Figure 24-1. HER2/NEU testing in breast cancer. Upper panel: HER2/NEUgene amplification detected by FISH (Abbott-Vysis Pathvysion System). Middle panel: HER2/NEU gene amplification detected by CISH (Zymed System). Lower panel: IHC using Herceptest System (Dako Corp.) with continuous membranous 3+ positive immunostaining for HER2/NEU protein. (Figure prepared in collaboration with Dr.Ken Bloom of US Labs Inc,Irvine,CA.)

Figure 24-1. HER2/NEU testing in breast cancer. Upper panel: HER2/NEUgene amplification detected by FISH (Abbott-Vysis Pathvysion System). Middle panel: HER2/NEU gene amplification detected by CISH (Zymed System). Lower panel: IHC using Herceptest System (Dako Corp.) with continuous membranous 3+ positive immunostaining for HER2/NEU protein. (Figure prepared in collaboration with Dr.Ken Bloom of US Labs Inc,Irvine,CA.)

survival,but did correlate with estrogen receptor and progesterone receptor (ER/PR) status and tumor grade in one breast cancer outcome study of 365 patients.23

The enzyme-linked immunosorbent assay (ELISA) technique avoids the potential antigen damage associated with fixation, embedding, and uncontrolled storage, when performed on tumor cytosols made from fresh tissue samples. However, the small size of breast cancers associated with expanded screening programs in the United States generally precludes tumor tissue ELISA methods because insufficient tumor tissue is available to produce a cytosol.

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