Silver-enhanced In Situ Hybridization Research Articles

HER2-Positive Breast Carcinomas with Co-amplification or Gain of Chromosome 17 Centromere Locus: Report of Three Cases and an Impact on HER2 Testing

Recently we experienced three cases of human epidermal growth factor receptor 2 (HER2)-amplified invasive breast carcinomas associated with co-amplification or gain of chromosome 17 centromere (CEP17) in silver-enhanced in situ hybridization (SISH) analysis. These cases revealed 2+ or 3+ staining for HER2 immunohistochemistry and > 6 HER2 copies per cell on SISH analyses. However, the calculated HER2/CEP17 ratios were low ( 6 per cell vs HER2/CEP17 ratio> 2.2). We recommend reporting raw SISH or fluorescence in situ hybridization data, including number of cells counted, average numbers of HER2 and CEP17 signals, and the calculated HER2/CEP17 ratio to prevent underreporting of HER2 amplification.

Determination of the Her-2/neu Gene Amplification Status in Cytologic Breast Cancer Specimens Using Automated Silver-enhanced In-situ Hybridization (SISH)

Silver-enhanced in-situ hybridization (SISH) is an emerging tool for the determination of the Her-2/neu amplification status in breast cancer. SISH is technically comparable to fluorescence in-situ hybridization (FISH) but does not require a fluorescence microscope for its interpretation. Although recent studies on histologic evaluations of SISH are promising, we aimed to evaluate its performance on 71 cytologic breast cancer specimens with the new combined Her-2/Chr17 probe. Her-2/neu status as routinely determined by FISH was available for all patients. We found SISH signals in cytologic cell blocks and smear specimens easy to evaluate in most cases. Small numbers of tumor cells and difficulties in identifying tumor cells in lymphocyte-rich backgrounds were limiting factors. Her-2/neu status, as determined by Her-2/Chr17 SISH, was basically identical to the results of the corresponding FISH. The discrepancies were mainly owing to the heterogeneity of Her-2/neu amplification in the tumor tissue. Interobserver agreement for the SISH evaluation was high (kappa value: 0.972). We conclude that Her-2/Chr17 SISH is a useful and accurate method for the evaluation of the Her-2/neu gene amplification status in cytologic breast cancer specimens, particularly in metastatic breast cancer lesions. The advantages of signal permanency and bright-field microscopic result interpretation make this technique an attractive alternative to the current FISH-based gold standard.

Automated silver-enhanced in situ hybridization detection assay for human epidermal growth factor receptor 2 gene status determination in breast cancer

To evaluate the concordance of human epidermal growth factor receptor 2 (HER2) gene amplification in invasive breast cancer by the techniques of fluorescence in situ hybridization (FISH) and silver-enhanced in situ hybridization (SISH). A prospective study of 63 invasive breast cancer specimens excised between May 2009 and November 2009 was conducted by automated immunohistochemistry (IHC) staining and bright field automated SISH. HER2 gene status was also examined by FISH in 43 cases. An evaluation was performed according to the ASCO/CAP guidelines (2007). Among 63 breast invasive ductal carcinoma tested by automated SISH, 61 cases were successfully evaluated. Gene amplification was detected in 97% (29/30) HER2 protein expression positive (3+) cases and in 18% (2/11) equivocal (2+) cases. Two of 11 (18%) HER2 expression 2+ cases were equivocal for gene amplification. In 43 cases examined by FISH, HER2 gene amplification was detected in 95% (20/21) HER2 expression positive (3+) cases and in one equivocal (2+) cases. Three of 10 (30%) expression 2+ cases were equivocal for gene amplification. No gene amplification was detected in expression negative (1 +/0) cases by either SISH or FISH (0/20 and 0/12 respectively). The results of SISH and FISH were identical in 42 cases examined by the above two techniques, including 21 HER2 gene amplification positive, 2 equivocal and 19 negative cases. One case was FISH equivocal and SISH negative. And the overall concordance between FISH and SISH was 98%. There is high concordance rate between SISH and FISH results for assessing the HER2 gene amplification status of excised breast cancer specimens. However, SISH has more advantages and it is particularly suited for routine application in surgical pathology.

Silver In Situ Hybridization (SISH) For Determination of HER2 Gene Status in Breast Carcinoma

The importance of HER2 status in breast cancer management has focused attention on the ability of clinical assays to correctly assign HER2 amplification status. There is no consensus as to the best method for assessing HER2 status. Disadvantages of fluorescence in situ hybridization (FISH) testing include longer time required for staining and scoring slides, requirements for specialized training and fluorescence microscopy, and loss of the signal due to quenching of the fluorescent dye. Silver-enhanced in situ hybridization (SISH) is a rapid fully automated assay providing permanently stained slides that are interpreted by conventional bright field microscopy which enables pathologists to evaluate slides within the context of tissue morphology. This study evaluates the concordance between SISH and FISH assays in determining the status of HER2 gene amplification in a cohort of 298 primary invasive breast carcinomas. Furthermore, we assessed in detail the variables contributing to interobserver interpretive reproducibility of HER2 SISH among 10 pathologists. HER2 was quantified using the ratio of HER2 to CHR17 signals using the conventional historical interpretation scale and also by the American Society of Clinical Oncology/College of American Pathologists reporting scheme. For SISH status determined by consensus among 10 pathologists, overall concordance between SISH and FISH was identified in 288 of 298 cases (96.6%) using the conventional Food and Drug Administration approved criteria. Overall agreement was observed in 282 of 285 cases (98.9%) using the American Society of Clinical Oncology/College of American Pathologists result reporting scheme (with equivocal cases removed). In conclusion, SISH represents a novel approach for the determination of HER2 status in breast cancer. The overall concordance between SISH and FISH is excellent, and the interpretation of SISH results by pathologists is most reproducible using the HER2/CHR17 ratio.

Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens

Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens

HER2Status in Gastric Adenocarcinomas Assessed by Immunohistochemistry, Automated Silver-EnhancedIn SituHybridization and FluorescenceIn SituHybridization

<i>HER2</i>Status in Gastric Adenocarcinomas Assessed by Immunohistochemistry, Automated Silver-Enhanced<i>In Situ</i>Hybridization and Fluorescence<i>In Situ</i>Hybridization

Emerging Technologies for Assessing HER2 Amplification

Patients with human epidermal growth factor receptor-2 (HER2)+ breast cancer are eligible for trastuzumab treatment; therefore, accurate assessment of HER2 status is essential. Until recently, only 2 methods were validated for determining the HER2 status of breast tumors in the routine diagnostic setting: immunohistochemical analysis and fluorescence in situ hybridization (FISH). Recently, bright-field in situ hybridization techniques such as chromogenic in situ hybridization (CISH) and silver-enhanced in situ hybridization (SISH), which combine features of immunohistochemical analysis and FISH, have been introduced for the determination of HER2 status. These new techniques use a peroxidase enzyme-labeled probe with chromogenic detection, instead of a fluorescent-labeled probe, allowing results to be visualized by standard bright-field microscopy. Thus, the histologic features and HER2 status of a specimen can be evaluated in parallel. Moreover, signals do not decay over time. This review discusses recent publications regarding CISH and SISH testing, including results scoring and concordance between FISH and immunohistochemical analysis.

Bright-field In Situ Hybridization for HER2 Gene Amplification in Breast Cancer Using Tissue Microarrays

HER2 gene amplification or overexpression occurs in 15% to 25% of breast cancers and has implications for treatment and prognosis. The most commonly used methods for HER2 testing are fluorescence in situ hybridization (FISH) and immunohistochemistry. FISH is considered to be the reference standard and more accurately predicts response to trastuzumab, but is technically demanding, expensive, and requires specialized equipment. In situ hybridization is required to be eligible for adjuvant treatment with trastuzumab in Australia. Bright-field in situ hybridization is an alternative to FISH and uses a combination of in situ methodology and a peroxidase-mediated chromogenic substrate such as diaminobenzidine [chromogenic in situ hybridization (CISH)] or multimer technology coupled with enzyme metallography [silver-enhanced in situ hybridization (SISH)] to create a marker visible under bright-field microscopy. CISH was introduced into diagnostic testing in Australia in October 2006. SISH methodology is a more recent introduction into the testing repertoire. An evaluation of CISH and SISH performance to assess patient outcome were performed using tissue microarrays. Tissue microarrays were constructed in duplicate using material from 593 patients with invasive breast carcinoma and assessed using CISH and SISH. Gene amplification was assessed using the American Society of Clinical Oncology/College of American Pathologists guideline and Australian HER2 Advisory Board criteria (single probe: diploid, 1 to 2.5 copies/nucleus; polysomy >2.5 to 4 copies/nucleus; equivocal, >4 to 6 copies/nucleus; low-level amplification, >6 to 10 copies/nucleus and high-level amplification >10 copies/nucleus; dual probe HER2/CHR17 ratio: nonamplified <1.8, equivocal 1.8 to 2.2, amplified >2.2). Results were informative for 337 tissue cores comprising 230 patient samples. Concordance rates were 96% for HER2 single probe CISH and SISH and 95.5% for single probe CISH and dual probe HER2/CHR17 SISH. Both bright-field methods correlated with immunohistochemistry results and with breast cancer-specific survival. HER2 SISH testing combines the advantages of automation and bright-field microscopy to facilitate workflow within the laboratory, improves turnaround time, and correlates with patient outcome.

Comparison of Silver-Enhanced in situ Hybridization and Fluorescence in situ Hybridization for HER2 Gene Status in Breast Carcinomas

specified elements to reduce assay variation such as specPurpose: Silver-enhanced in situ hybridization (SISH) is a newly developed method to evaluate HER2 gene amplification in invasive breast carcinomas. Most laboratories widely use fluorescence in situ hybridization (FISH) to evaluate the HER2 gene amplification status because FISH is a very sensitive and accurate technique. However, this technique is not the best because it requires specialized equipment and interpretation skills. We compared a new technique of SISH with FISH for assessing HER2 gene amplification in invasive breast carcinomas. Methods: HER2 gene amplification was assessed in 165 cases of invasive breast carcinoma by FISH and SISH with constructing a tissue microarray. The tumors were assessed by the guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/ CAP). Positivity was defined as a HER2/Chromosome 17 ratio greater than 2.2. Negativity was defined if the ratio was less than 1.8. The tumor was considered as equivocal for HER2 gene amplification if the ratio was between 1.8 and 2.2. The HER2 protein status was assessed. Immunostaining for HER2 protein was performed in a Benchmark automatic immunostaining device with using whole tissue sections. Results: There was agreement of the HER2 gene amplification status by SISH and FISH in 162 of 165 cases, which is a concordance rate of 98.2% (κ=0.94). There were three discrepant cases, with two of them being FISH positive and SISH negative (one case was IHC negative and one case was IHC positive) and one case was FISH negative and SISH equivocal. Conclusion: The 98.2% concordance between FISH and SISH meets the ASCO/CAP requirements for test validation of >95% concordance. These results indicate that SISH can be used as an alternative to FISH for assessing the HER2 gene amplification status in breast carcinomas.