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.     

Dual-colour chromogenic in situ hybridization for testing of HER-2 oncogene amplification in archival breast tumours

Chromogenic in situ hybridization (CISH), which uses an enzymatic reaction to detect the hybridized DNA probe, is a new alternative to fluorescence in situ hybridization (FISH) for the assessment of HER-2 oncogene amplification status in breast cancer. The main advantage of CISH over FISH is the use of bright-field microscopy, which is rapid and allows the histopathological evaluation of tumour tissue sections. The main disadvantage of CISH has been the use of a single probe, thereby making it necessary to hybridize the control probe (chromosome 17 centromere) on an adjacent tissue section. The present paper presents an efficient protocol for dual-colour CISH (dc-CISH) based on the co-hybridization of probes to the HER-2 oncogene and chromosome 17 centromere. The probes were detected sequentially with antibodies to digoxigenin and biotin and with secondary antibody polymers labelled with horseradish peroxidase and alkaline phosphatase. The peroxidase reaction was visualized with tetramethyl benzidine (green reaction product) and the alkaline phosphatase reaction with New Fuchsin (red reaction product). The accuracy of the method was verified by comparing the results for four cell lines and 40 tumour samples with those obtained using FISH (Vysis Inc.). The results of FISH and dc-CISH showed high concordance (91%, Kappa coefficient = 0.82). It is concluded that dual-colour CISH, which is a new alternative to FISH enables the assessment of copy number ratio (HER-2/17 centromere) in conjunction with proper histopathological evaluation and the ease of bright-field microscopy.     

Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma.

PURPOSE: To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer. MATERIALS AND METHODS: HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest. RESULTS: HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively. CONCLUSION: By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.     

Chromogenic in situ hybridization for Her‐2/neu‐oncogene in breast cancer: comparison of a new dual‐colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization

Aims: Her‐2/neu testing is used as a marker for Herceptin^® therapy. The aim was to investigate new dual‐colour chromogenic in situ hybridization (CISH), in a large number of breast carcinomas (n = 205) with DNA‐specific dual‐colour probes (ZytoVision, Bremerhaven, Germany) and to compare the results with immunohistochemistry (n = 205) and fluorescence in situ hybridization (FISH) (n = 129). Methods and results: Paraffin‐embedded tissue of 205 patients was used. After immunohistochemistry with a focus on immunohistochemically uncertain cases, Her‐2/neu amplification using dual‐colour CISH (ZytoVision^®) was analysed. Validation by FISH was performed. The results were: immunohistochemistry, 27.8% with strong expression, 53.7% with uncertain overexpression and 18.5% with no expression; FISH, 25.6% amplified and 74.4% negative; CISH, 35.6% amplified, 62.9% negative and 1.5% not evaluable. Comparison of immunohistochemistry with CISH: CISH negative in 100% with immunohistochemistry 0/1+, amplified in 82.5% with immunohistochemistry 3+; 5.9% contradictory results: 4.4% immunohistochemistry 3+ and negative by CISH, 1.5% negative in immunohistochemistry but amplified by CISH; FISH (129 cases), 8.5% contradictory results to immunohistochemistry, 6.2% immunohistochemistry 3+ and negative by FISH, 2.3% negative by immunohistochemistry and amplified by FISH; comparison of CISH and FISH, 94.6% same results, 3.9% different ones, 1.6% CISH not analysable. Conclusions: CISH, using dual‐colour probes (ZytoVision^®) is as good as FISH for Her‐2/neu analysis. The few discrepant results are likely to be caused by polysomy or tumour heterogeneity.     

Chromogenic in-situ hybridization: a viable alternative to fluorescence in-situ hybridization in the HER2 testing algorithm.

Assessment of human epidermal growth factor receptor-2 status is standard practice in women with breast cancer. Most laboratories use immunohistochemistry as a screening test, with equivocal results confirmed by fluorescence in-situ hybridization (FISH). Chromogenic in-situ hybridization (CISH) is a relatively new method for detection of gene amplification using a peroxidase reaction, which can be viewed using a standard light microscope. This study was undertaken to validate CISH as a method for assessing human epidermal growth factor receptor-2 gene amplification. The gene amplification status of human epidermal growth factor receptor-2 immunohistochemistry negative (0/1+, n = 69; Group 1), immunohistochemistry positive (3+, n = 50; Group 2) and equivocal tumor samples (2+, n = 135; Group 3) was evaluated by FISH and CISH, and the concordance between FISH and CISH results calculated. In Group 1, 67/69 cases did not show amplification by CISH and 69/69 showed no amplification by FISH. Two cases were discordant; therefore, fluorescence/CISH concordance was 97%. In Group 2, 46/50 cases were amplified by FISH and 47/50 cases were amplified by CISH; three cases were not amplified by either method (immunohistochemistry false-positives). Only one case showed discordant FISH and CISH results, making the fluorescence/CISH concordance 98%. In Group 3, 89/135 cases were not amplified and 37/135 were amplified by both methods. Nine cases were discordant, giving a fluorescence/CISH concordance of 93%. The discordant cases were those with very low or borderline amplification with FISH. The high level of concordance between FISH and CISH seen in this study suggests that CISH may be a viable alternative to FISH for use in the human epidermal growth factor receptor-2 testing algorithm.     

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.     

Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer

Determination of HER-2/neu oncogene amplification has become clinically important in the present management of breast cancer and may have important applications in other areas of clinical oncology and scientific research. In situ hybridization is an extremely accurate and sensitive technique for assessing amplification of HER-2/neu. A new method using a chromogen-labeled probe offers numerous advantages, including the ability to view the morphologic features of the cells of interest using a light microscope, which can be found in every laboratory. We used both techniques to assay 31 cases of infiltrating breast carcinoma; assays were performed in laboratories at 2 institutions. Identical results for both methods were found in 26 cases (10 amplified, 16 nonamplified). One case was misinterpreted as overexpressed by chromogenic in situ hybridization (CISH) because of background precipitate. In 4 cases, CISH suggested low-level amplification. Three of these cases subsequently were found to have chromosome 17 polysomy. In the remaining case, the initial section chosen was suboptimal, showing weak signals by both methods. If a probe for chromosome 17 (now available for CISH) is used in cases of questionable HER-2/neu amplification, CISH seems to be as accurate and more practical than FISH.     

Comparing fluorescence in situ hybridization and chromogenic in situ hybridization methods to determine the HER2/neu status in primary breast carcinoma using tissue microarray.

Identification of HER2/neu status is important for predicting response to specific chemotherapy in breast carcinoma. Chromogenic in situ hybridization was performed using tissue microarray technology on 188 primary breast carcinomas. To validate the reliability of novel chromogenic in situ hybridization technology, the results of chromogenic in situ hybridization were correlated with the results of two-color fluorescence in situ hybridization done with the same tumors. On tissue microarray panels containing 188 breast carcinoma tissues, fluorescence in situ hybridization and chromogenic in situ hybridization were conducted simultaneously. HER2/neu amplification was detected in 46 tumors (24.5%) by fluorescence in situ hybridization and in 43 tumors (22.9%) by chromogenic in situ hybridization. Results of each method agreed with each other in 177 tumors (concordance: 94.1%). HER2/neu amplification by fluorescence in situ hybridization was associated with nuclear pleomorphism (P =.021), and HER2/neu amplification by chromogenic in situ hybridization was associated with poor nuclear grade (P =.037). High concordance between fluorescence in situ hybridization and chromogenic in situ hybridization indicated that chromogenic in situ hybridization can be a tempting alternative to fluorescence in situ hybridization for the detection of HER2/neu amplification in breast carcinoma because of its accuracy and relative low cost. HER2/neu appeared to have a prognostic implication because its amplification was associated with aggressive biologic features of the breast carcinoma. Integration of tissue microarray technology enabled high-throughput determination of HER2/neu amplification profile with rapidity and accuracy in large cohorts of the breast carcinoma.     

Amplification of c-myc oncogene by chromogenic and fluorescence in situ hybridization in archival breast cancer tissue array samples.

Fluorescence in situ hybridization (FISH) is currently considered to be the most specific and sensitive method for detection of oncogene amplifications in human tumor samples. However, FISH requires fluorescence microscopy, which is tedious and does not allow histopathologic evaluation of the cells and tissues examined. Here we compared FISH with the newly developed chromogenic in situ hybridization (CISH), which uses peroxidase enzyme for probe detection instead of fluorescent dyes. CISH was found to be highly concordant with FISH in a tissue array series of 177 archival breast cancer samples. This was true both when comparing CISH with single-color and two-color FISH, the latter including the chromosome 8 centromere probe as reference (the kappa coefficients were 0.67 and 0.76, respectively). Clinicopathologic correlations of c-myc amplification as detected by FISH and CISH were generally the same. By both methods, c-myc amplification was significantly associated with high histologic grade, negative progesterone receptor status, DNA aneuploidy, and high S-phase fraction. c-myc amplification was strongly associated with poor distant metastasis-free survival when amplification was detected by CISH (p = 0.0013), but this association was weaker when FISH was used (p = 0.16 for two-color FISH and p = 0.065 for single-color FISH). These data suggest that CISH is at least as sensitive and specific as FISH in the detection of oncogene amplification in human tumor samples. The possibility for concomitant tissue architecture evaluation using an ordinary transmitted light microscope may favor the use of CISH over FISH in oncogene amplification detection in large tumor series, and tissue arrays and, ultimately, in routine clinical diagnostics.     

Analysis of HER2 by chromogenic in situ hybridization and immunohistochemistry in lymph node-negative breast carcinoma: Prognostic relevance

In patients with lymph node-negative breast carcinoma (LNNBC), the prevalence of HER2 overexpression and gene amplification and their prognostic value have not been extensively evaluated. We examined 162 patients with LNNBC with complete follow-up. Immunohistochemistry (IHC) for HER2, Ki67, and p53 was performed. HER2 gene status was analyzed by chromogenic in situ hybridization (CISH) and discordant cases by fluorescence in situ hybridization. HER2 overexpression was seen in 24.7% of cases (40/162) and amplification by CISH in 17.6% (28/159). Agreement between IHC and CISH was achieved in 147 (92.5%) cases. Amplification was seen in 21 (100%) of 21 (3+), 6 (35.3%) of 17 (2+), and 1 (0.6%) of 121 (0-1+) tumors. Fluorescence in situ hybridization detected 3 (1.8%) additional cases. HER2 overexpression and amplification were present in tumors of high grade, with necrosis and lymph-vascular invasion (LVI) (all P < .027). In addition, amplified tumors showed Ki67 of more than 20% and p53 overexpression (P < .05). By univariate analysis, shorter disease-free survival (DFS) and overall survival (OS) were seen for patients with tumors showing HER2 amplification, LVI, and Ki67 of more than 20% (P < .05) (Kaplan-Meier). However, the multivariate analysis (Cox regression) demonstrated only Ki67 as an independent prognostic factor for both DFS (P = .017) and OS (P = .010), and as a trend for HER2 gene status (OS, P = .087) and LVI (DFS, P = .11; OS, P = .063). We conclude that IHC is a reliable method for detecting HER2 expression that can be complemented by CISH in nondefinitive cases (2+). Moreover, CISH is a valuable tool for the assessment of HER2 gene status with potential prognostic value and, therefore, in clinical decision making for treatment of high-risk LNNBC.     

HER2 positivity in breast carcinoma: a comparison of chromogenic in situ hybridization with fluorescence in situ hybridization in tissue microarrays, with targeted evaluation of intratumoral heterogeneity by in situ hybridization.

An accurate and reproducible assay method for determining HER2 status is crucial, as a positive HER2 gene status is an eligibility requirement for Herceptintrade mark therapy. Although immunohistochemical (IHC) assessment is both practical and inexpensive, a worrying trend of high false-positive rates has been reported. Fluorescence in situ hybridization (FISH) is the universally accepted gold standard for confirming IHC 2+ cases and ambiguous results but is costly and requires specialized equipment and technical expertise. Chromogenic in situ hybridization (CISH) amalgamates the practical advantages of IHC with the reproducibility of FISH, and high concordance between the CISH and FISH methods has been reported in conventional sections. Tissue microarrays (TMAs) allow high throughput of specimens, and HER2 status assessment in TMA cores using IHC and FISH has correlated well with scores in conventional sections. The authors used TMA technology to compare the efficacy of ZYMED(R) CISH with PathVysiontrade mark FISH in a cohort of 119 archival breast resection cases and investigated possible intratumoral heterogeneity in a "mini-array" of 21 HercepTest "equivocal"/2+ cases. Concordance between FISH and CISH in TMA sections was 99%. All prescored 2+ HercepTest cases were nonamplified. Four 3+ HercepTest cases were classed as potential false-positives. The authors suggest that confirmatory ISH should be performed on all positive HercepTest cases. CISH was easier to perform and quicker to enumerate than FISH. The authors conclude that CISH is a practical alternative to FISH as a confirmatory tool for HER2 gene amplification status. Intratumoral heterogeneity did not affect the patient's HER2 status.     

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

Aims:  To validate the use of the silver-enhanced in situ hybridization (SISH) technique in assessing HER2 status of breast carcinoma in excision biopsy specimens, and to assess its reliability in determining HER2 status in core biopsy specimens.
Methods and results:  Routinely processed paraffin sections of 65 excised breast carcinomas and 56 available preoperative core biopsy specimens from the same patients were selected from the archives for testing with the SISH technique using the automated Ventana Benchmark XT machine. For each case, two sections were used, one for the assessment of HER2 gene amplification and the other for assessment of chromosome 17. Of the 65 excision specimens tested, sections of 53 cases were also available for fluorescence in situ hybridization (FISH) examination. HER2 gene amplification was detected by SISH in 14 (21%) out of 65 excision specimens and in eight (14%) out of 56 core biopsy specimens. The results of SISH and FISH were identical in 50 (94%) out of the 53 excision cases examined by the two techniques. Two cases were SISH−, FISH+, and one case was the other way round. SISH results of core biopsy specimens and corresponding excision biopsy specimens were identical in 50 (89%) out of 56 cases. Four cases (7%) were SISH− in cores but positive in excision specimens, whereas two cases were the other way round.
Conclusions:  The results validate the use of the SISH technique for assessing HER2 status of excised breast carcinoma tissue sections. The results are comparable to those obtained with FISH, but SISH has the advantage of having a permanent end result that can be visualized by an ordinary light microscope. There is a reasonable 89% concordance between SISH results obtained in core and excision biopsy specimens. However, it may be prudent to postpone doing SISH, if possible, until sections of the resected specimen are available, as these seem to be more reliable.     

Comparison of chromogenic in situ hybridisation with fluorescence in situ hybridisation and immunohistochemistry for the assessment of her-2/neu oncogene in archival material of breast carcinoma

The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy.The aim of this study was to prove the consistency of chromogenic in situ hybridisation (CISH) technique after analyzing the overexpression of the Her-2/neu proto-oncogene in 100 invasive breast carcinomas and by comparing CISH results with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). Moreover, it was done to evaluate the possible correlation of estrogen (ERs) and progesterone receptors (PRs), the proliferation marker Ki67 and the tumour suppressor gene p53 with HER-2/neu status of these breast carcinomas.Of the 100 breast carcinomas that were analysed, 22 cases showed HER-2/neu amplification, 66 cases showed no amplification, whereas 12 cases were non-interpretable in both assays (FISH and CISH). Consequently, the overall concordance between FISH and CISH was 100%. Additionally, it was observed that when HER-2/neu gene was overexpressed, there was an association with negative PRs and ERs status, negative p53 protein expression and high Ki67 labelling index.It is concluded that patients with tumours scoring 2+ with the CBE356 antibody (borderline immunohistochemistry-tested cases) would also benefit from CISH as it is shown to be highly accurate, practical and can be easily integrated into routine testing in any histopathology laboratory. Finally, CISH represents an important addition to the HER2 testing algorithm.     

Detection of Her-2/neu oncogene in breast carcinoma by chromogenic in situ hybridization in cytologic specimens

Determination of Her-2/neu oncogene amplification is important in the current treatment of breast carcinoma. In addition to fluorescence in situ hybridization (FISH) and immunohistochemical stain (HercepTest), chromogenic in situ hybridization (CISH) has been shown to be a sensitive and specific method to determine the Her-2/neu status of surgical specimens. The effectiveness of CISH in detecting the Her-2/neu oncogene in cytologic specimens has not been well documented. Twenty-five cases of fine needle aspirate smears and touch imprints from infiltrating ductal carcinomas were examined. Both CISH and FISH were performed on each case using a digoxigenin-labeled Her-2 DNA probe for CISH (Zymed) and both Her-2 and chromosome 17 probes for FISH (Vysis). Sixty tumor cells were evaluated in each case. The scoring system and interpretation of CISH were as follows: (1) no amplification (<5 brown dots/nucleus), (2) amplification (>10 brown dots/nucleus), and (3) low-level amplification (5-9 brown dots/nucleus). Of the 25 cases analyzed, 23 (3 amplified and 20 nonamplified) showed similar results for both methods. Two cases were discordant. In these cases, low-level amplification was suggested by CISH but nonamplification by FISH. One of the cases can be explained by polysomy for chromosome 17 by FISH. In conclusion, our preliminary data suggest that CISH is a useful technique to determine Her-2/neu oncogene status in cytologic specimens. In a case of low-level amplification, a CISH chromosome 17 probe should be used, or FISH is recommended for confirmation.     

HER-2/neu testing in breast carcinoma: a combined immunohistochemical and fluorescence in situ hybridization approach.

We evaluated 750 consecutive invasive breast carcinomas for HER-2/neu utilizing a combination of immunohistochemical (IHC) and fluorescence in situ hybridization (FISH) methodologies. IHC reactions of 3+ were considered HER-2/neu positive and 0 and 1+ IHC reactions were considered HER-2/neu negative. IHC reactions of 2+ were considered inconclusive and reflexed to FISH analysis. In addition, a 10% sampling and validation FISH analysis was performed on the positive and negative IHC tests. One hundred thirty-eight cases (18.4%) were HER-2/neu positive by IHC and/or FISH. One hundred twenty-three of the positive cases (89%) were 3+ IHC reactions and 14 positive cases were inconclusive by IHC and amplified by FISH. There was concordance with FISH in 77 of 78 (98.7%) of the positive or negative IHC cases that were tested (95% confidence interval [CI] = 93.1 to 100%). A single IHC-negative case showed HER-2/neu amplification by FISH. Thirty-nine cases were 2+ IHC (5.2%); 14 (36%) were amplified, 24 (62%) were not amplified, and one was not interpretable. HER-2/neu positivity was observed in 34% of grade 3 ductal carcinomas, 11.4% of grade 2 ductal carcinomas, 3.2% of grade 1 ductal carcinomas, and 3.2% of lobular carcinomas. Occasional cases with discordant IHC expression of HER-2/neu within the in situ and invasive carcinoma elements were also identified. IHC reliably characterized HER-2/neu in approximately 95% of the cases studied (95% CI = 93.0 to 96.2%) and was effective as a primary method for evaluating HER-2/neu status. In this study, 2+ IHC reactions were a heterogeneous group best regarded as indeterminate or inconclusive; in this series, only 36% were amplified by FISH analysis. Our findings suggest that a combination of IHC and FISH testing with FISH analysis performed reflexly on all 2+ IHC cases can optimize HER-2/neu testing.     

HER2 testing in gastric cancer: a practical approach

Trastuzumab in combination with capecitabine or 5-fluorouracil and cisplatin is approved by the European Medicines Agency for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive (immunohistochemistry 3+ or immunohistochemistry 2+/fluorescence in situ hybridization-positive or immunohistochemistry 2+/silver in situ hybridization-positive) metastatic adenocarcinoma of the stomach or gastro-esophageal junction. Approvals are underway in other countries, with recent approvals granted in the United States and Japan. Experience and data from trastuzumab use in breast cancer have highlighted the importance of quality HER2 testing and scoring to ensure accurate identification of patients eligible for treatment. HER2 testing in gastric cancer differs from testing in breast cancer due to inherent differences in tumor biology; gastric cancer more frequently shows HER2 heterogeneity (focal staining) and incomplete membrane staining. Consequently, gastric cancer-specific HER2 testing protocols have been developed and standardized and it is imperative that these recommendations be adhered to. Given the predictive value of HER2 protein levels with response in the trastuzumab for GAstric cancer study (ToGA), immunohistochemistry should be the initial testing methodology and fluorescence in situ hybridization or silver in situ hybridization should be used to retest immunohistochemistry 2+ samples. Wherever possible, bright-field methodologies should be used as these are considered to be superior to fluorescent methodologies at identifying heterogeneous staining. Specific training is required before embarking on HER2 testing in gastric cancer, irrespective of the experience of HER2 testing in breast cancer. This paper provides the most up-to-date practical guidance on HER2 testing and scoring in patients with gastric and gastro-esophageal junction cancer, as agreed by a panel of expert pathologists with extensive experience of HER2 testing particularly reflecting the European Medicines Agency-approved indication. It is anticipated that these recommendations should ensure accurate and consistent HER2 testing, which will allow appropriate selection of patients eligible for treatment with trastuzumab.     

JC virus genomes in progressive multifocal leukoencephalopathy: detection using a sensitive non-radioisotopic in situ hybridization method

JC virus genomes have been localized in formalin-fixed, paraffin-embedded brain tissues of two cases of known progressive multifocal leukoencephalopathy by in situ hybridization utilizing a biotinylated JC virus DNA probe. A three-stage immunoperoxidase system with gold-silver amplification of the diaminobenzidine substrate was used to visualize biotinylated nucleic acid hybrids. Dot-blot quantification of this visualization system indicates that subpicogramme amounts of biotinylated DNA can be detected. Optimal detection of the virus genomes in the brain tissues required a microwave irradiation step prior to hybridization. JC virus genomes were observed in the nuclei of enlarged oligodendrocytes and of some bizarre astrocytes. No other cell types were found to harbour the genomes.     

Comparison of dual-color dual-hapten brightfield in situ hybridization (DDISH) and fluorescence in situ hybridization in breast cancer HER2 assessment

The most optimal method for assessing HER2 status is still subject to controversy as far as the type of assay used, the optimal method to perform, and the costs of each assay are concerned. The current study was done as a validation study prior to setting up a clinical HER2 testing service using the new commercial dual-color dual-hapten brightfield in situ hybridization (DDISH), but it was felt that our experience may be of interest to other laboratories considering setting up HER2 diagnostic facilities.     

显色原位杂交和免疫组织化学检测乳腺癌HER2/neu基因状况和蛋白表达的对照性研究

目的探讨显色原位杂交（CISH）在检测乳腺癌中HER2／neu基因扩增上的作用。方法挑选乳腺浸润性导管癌患者组织石蜡蜡块（回顾性255例,前瞻性271例）,进行免疫组织化学（IHC）、CISH检测。15例回顾性标本送往德国HERA检测中心进行FISH检测。结果（1）回顾性病例中IHC阳性3＋者CISH基因扩增率为91．6％（120／131）,IHC2＋者CISH基因扩增率为56．5％（39／69）,IHC与CISH检测结果符合率为81．2％（207／255）,两者明显相关（P〈0．01）。（2）前瞻性病例中IHC蛋白过表达率31．7％．CISH基因扩增率27．3％。IHC3＋者CISH基因扩增率为91．4％（53／58）,IHC2＋者CISH基因扩增率为46．4％（13／28）,IHC与CISH检测结果符合率为89．7％（243／271）,两者明显相关（P〈0．01）。（3）经德国检测中心荧光原位杂交（FISH）检测的15例中14例和CISH结果完全一致,1例检测失败,而CISH为无扩增。（4）CISH检测基因扩增与雌激素受体（ER）、孕激素受体（PR）表达明显负相关（P值均〈0．01）。结论CISH检测HER2基因扩增结果与IHC检测蛋白过表达及FISH结果高度一致,CISH是检测HER2基因扩增的一项新技术。     

Interphase fluorescence in situ hybridization studies for the detection of 9q34 deletions in chronic myelogenous leukemia: a practical approach to clinical diagnosis

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome (Ph) in more than 90% of cases. Recent studies using fluorescence in situ hybridization (FISH) have shown that in a subset of patients with CML, deletions of 9q34 involving the argininosuccinate synthetase region occur at the time of the Philadelphia translocation and are associated with a poor prognosis. We performed interphase FISH studies in 152 cases of CML using a dual-color, dual-fusion probe system with a third probe directed at 9q34. Cytogenetic studies showed a simple (typical) Ph in 124/152 (82%), a cryptic Ph in 11/152 (7%), and a variant Ph chromosome with a complex translocation in 17/152 (11%) of cases. Interphase FISH studies showed single BCR/ABL fusion patterns in 48/152 (32%) of cases. Deletions of 9q34 were observed in 14% of all the cases and were present in 46% of cases with single BCR/ABL fusion pattern. All the 9q34 deletions occurred in cases with single BCR/ABL fusion signal. However, a single-fusion pattern is not specific for 9q34 deletions, and cases should be routinely screened for the presence of this prognostically significant abnormality by using a third probe directed specifically at 9q34.