Metallographic in situ hybridization

Case Western Reserve University, Cleveland, Ohio, United States
Human Pathlogy (Impact Factor: 2.77). 09/2007; 38(8):1145-59. DOI: 10.1016/j.humpath.2007.05.004
Source: PubMed


Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins.

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    • "The recent development of dual-color probes for HER2 and CEP17 enables the identification of polysomy using CISH [9]. Silver-enhanced in situ hybridization (SISH) has been developed as an alternative method to FISH and CISH for HER2 determination [10-12]. SISH is a novel bright-field in situ hybridization technique similar to CISH. "
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    ABSTRACT: Valid determination of HER2 status is a prerequisite to establish an adequate treatment strategy for breast cancer patients, regardless of the disease stage. The goal of this study was to examine the feasibility of the newly developed silver-enhanced in situ hybridization (SISH) technique as an alternative to fluorescence in situ hybridization (FISH) for HER2 assay in primary invasive breast cancer. FISH and SISH for HER2 amplification were performed using tissue microarray. Both methods were used in 257 consecutive primary breast cancers. HER2 amplification was observed in 62 (23.1%) of a total of 257 breast cancers based on SISH. Of the 257 breast cancers measured using both methods, the results of the two methods were consistent in 248 (concordance, 96.5%; kappa=0.903). When we compared HER2 amplification in the primary tumor with the metastatic lymph nodes of the same patients, HER2 amplification was observed in nine cases (14.0%) out of 64 cases in which HER2 was not amplified in the primary tumors. In contrast, HER2 status was completely preserved in metastatic lymph nodes showing HER2 amplification in the primary tumor. These results indicate that SISH can be a feasible alternative to FISH in the clinical setting. In node-positive breast cancer, confirmation of the HER2 status of the metastatic lymph nodes appears to be mandatory, regardless of the HER2 status of the primary tumors.
    12/2011; 14(4):276-82. DOI:10.4048/jbc.2011.14.4.276
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    • "If HER2 status differed between primary tumor and metastases, or when either primary tumor or metastasis were IHC 2+ (see below), silver in situ hybridization (SISH) analysis [31] was performed with a fully automated technique (INFORM, Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer's guidelines. "
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    ABSTRACT: When breast cancer patients develop distant metastases, the choice of systemic treatment is usually based on tissue characteristics of the primary tumor as determined by immunohistochemistry (IHC) and/or molecular analysis. Several previous studies have shown that the immunophenotype of distant breast cancer metastases may be different from that of the primary tumor ("receptor conversion"), leading to inappropriate choice of systemic treatment. The studies published so far are however small and/or methodologically suboptimal. Therefore, definite conclusions that may change clinical practice could not yet be drawn. We therefore aimed to study receptor conversion for estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) in a large group of distant (non-bone) breast cancer metastases by re-staining all primary tumors and metastases with current optimal immunohistochemical and in situ hybridization methods on full sections. 233 distant breast cancer metastases from different sites (76 skin, 63 liver, 43 lung, 44 brain and 7 gastro-intestinal) were IHC stained for ERα, PR and HER2, and expression was compared to that of the primary tumor. HER2 in situ hybridization (ISH) was done in cases of IHC conversion or when primary tumors or metastases showed an IHC 2+ result. Using a 10% threshold, receptor conversion by IHC for ERα, PR occurred in 10.3%, 30.0% of patients, respectively. In 10.7% of patients, conversion from "ER+ or PR+" to ER-/PR- and in 3.4% from ER-/PR- to "ER+ or PR+" was found. Using a 1% threshold, ERα and PR conversion rates were 15.1% and 32.6%. In 12.4% of patients conversion from "ER+ or PR+" to ER-/PR-, and 8.2% from ER-/PR- to "ER+ or PR+" occurred. HER2 conversion occurred in 5.2%. Of the 12 cases that showed HER2 conversion by IHC, 5 showed also conversion by ISH. One further case showed conversion by ISH, but not by IHC. Conversion was mainly from positive in the primary tumor to negative in the metastases for ERα and PR, while HER2 conversion occurred equally both ways. PR conversion occurred significantly more often in liver, brain and gastro-intestinal metastases. Receptor conversion by immunohistochemistry in (non-bone) distant breast cancer metastases does occur, is relatively uncommon for ERα and HER2, and more frequent for PR, especially in brain, liver and gastro-intestinal metastases.
    Breast cancer research: BCR 09/2010; 12(5):R75. DOI:10.1186/bcr2645 · 5.49 Impact Factor
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    • "The reaction product can be seen as discrete black dots under a brightfield microscope. Advantages of SISH include the high sensitivity for detection of single gene copies, the high resolution for quantifying DNA targets, and the high contrast with tissue counterstaining for visual separation of the signal and tissue morphology [27]. "
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    ABSTRACT: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatase (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 - 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 - 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 - 1.0000). Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.
    Diagnostic Pathology 11/2008; 3(1):41. DOI:10.1186/1746-1596-3-41 · 2.60 Impact Factor
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