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1.
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. 相似文献
2.
Her2 amplification: correlation of chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization. 总被引:4,自引:0,他引:4
Christopher R Wixom Elizabeth A Albers Noel Weidner 《Applied immunohistochemistry & molecular morphology》2004,12(3):248-251
Detecting Her2 gene amplification has become routine in predicting therapeutic responsiveness in patients with breast carcinoma. Fluorescence in situ hybridization (FISH) is a common technique for detecting Her2 amplification, yet dark field fluorescence microscopy remains problematic for many pathologists. Thus, a technique such as chromogenic in situ hybridization (CISH), in which the more familiar light microscopy can be used, is appealing. Paraffin-embedded sections from 61 breast carcinomas were tested for Her2 amplification by immunohistochemistry (IHC) and CISH. FISH was used to confirm CISH results. Excellent correlation was found between IHC and CISH except in cases considered negative (1+ on the DAKO scale) by IHC. CISH detected low-level Her2 amplification in 4 of 9 of these cases. Amplification was subsequently confirmed by FISH in all but 1 case. When compared with FISH, CISH was more sensitive than IHC for detecting low levels of Her2 gene amplification. Moreover, excellent concordance was found between FISH and CISH, supporting the conclusion that the CISH assay for Her2 gene amplification provides an accurate, effective, and practical alternative to FISH. 相似文献
3.
We studied HER-2/neu (HER-2) and topoisomerase IIa (topo2a) amplification (using chromogenic in situ hybridization) and overexpression (immunohistochemical analysis) in 113 invasive breast carcinomas. A gene copy number/chromosome 17 copy number ratio of 2.0 or higher indicated amplification. A topo2a/chromosome 17 ratio of less than 0.8 indicated gene deletion. HER-2 overexpression was scored according to standard HercepTest guidelines (DAKO, Carpinteria, CA). Overexpression of topo2a was identified when nuclear staining was found in more than 5% of tumor cells. Of 113 tumors, 104 were analyzed successfully for HER-2 and topo2a amplification. Of the 104, 64 showed HER-2 amplification; 25 of these (39%) also showed topo2a amplification. No amplification was found in 40 tumors. Deletion of topo2a was seen in 7 (11%) of 64 HER-2-amplified tumors and 2 (5%) of 40 nonamplified tumors. Of 25 tumors with topo2a amplification, 18 (72%) overexpressed topo2a. Only 3 (4%) of 79 tumors without topo2a amplification overexpressed topo2a. Amplification of topo2a is associated with HER-2 amplification but not vice versa. Amplification of topo2a resulted in protein overexpression in 72% of tumors, but topo2a overexpression rarely occurred without gene amplification. Identification of topo2a and HER-2 status might have therapeutic and prognostic implications. 相似文献
4.
Fluorescence in situ hybridization study of HER-2/neu oncogene amplification in prostate cancer. 总被引:4,自引:0,他引:4
H F Mark D Feldman S Das H Kye S Mark C L Sun M Samy 《Experimental and molecular pathology》1999,66(2):170-178
Prostate cancer is a serious disease affecting men worldwide and treatment compromises the quality of life of prostate cancer patients. We conducted a study of 88 cases of prostate cancer in an attempt to identify prognostic biomarkers that can distinguish aggressive cases that must be treated immediately. HER-2/neu oncogene amplification was initially studied because amplification of this gene has been reported in many other cancers, including those studied in this laboratory. Fluorescence in situ hybridization (FISH) using a HER-2/neu gene probe with a chromosome 17 centromere control probe was performed on formalin-fixed, paraffin-embedded tissues. Of a total of 86 cases successfully analyzed, only 8 (9.3%) were found to be amplified. This frequency was lower than the frequency of amplification found in other cancers studied. Furthermore, no case was found where the level of amplification can be considered high. Only one case was found to have moderate amplification. The rest of the positive cases can all be classified as low amplification. Thus, while we have demonstrated that FISH is a sensitive technique for detecting oncogene amplification, the frequency and level of HER-2/neu amplification detected in prostate cancer seem to be lower than those in most cancers that we studied. In view of the fact that HER-2/neu amplification does not seem to play as significant a role, exploration of other biomarkers in prostate cancer is warranted. 相似文献
5.
Akishi Ooi Takuo Takehana Xiaoling Li Shioto Suzuki Kazuyoshi Kunitomo Hiroshi Iino Hideki Fujii Yasuhisa Takeda Yoh Dobashi 《Modern pathology》2004,17(8):895-904
Overexpression of HER-2 and the epidermal growth factor receptor (EGFR) has been observed in many cancers, sometimes accompanied by gene amplification. To assess whether novel chemotherapies targeting these overexpressed proteins may be effective for the treatment of colorectal cancers, we examined the exact frequency of HER-2 and EGFR overexpression, the relationship between gene amplification and protein expression, and the heterogeneity of gene amplification within and between primary and metastatic tumors. We evaluated 244 colorectal cancers immunohistochemically. All tumors found to overexpress HER-2 or EGFR were further analyzed for gene amplification by fluorescent in situ DNA hybridization. Overexpression of HER-2 and EGFR was found in 8 (3%) and 19 (8%) of the 244 colorectal carcinomas, respectively. Gene amplification was observed in 100 and 58% of the tumors exhibiting HER-2 and EGFR overexpression, respectively. HER-2 amplification in cancer cells was characterized by clusters of hybridization signals, suggesting amplicons in homogeneously staining regions that were predominant in most primary and metastatic tumors. EGFR amplification, observed as scattered signals reminiscent of amplicons in double minute chromosomes, or coamplification of EGFR with the centromeric regions was observed as a minor population within primary tumors, and found in variety of populations in metastatic tumors. Overexpression of HER-2 and EGFR were observed in only a small fraction of colorectal carcinomas, but were frequently accompanied by gene amplification. 相似文献
6.
Accurate determination of HER-2 status is important in the management of patients with breast cancer, especially in determining their eligibility for trastuzumab therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting HER-2 gene amplification. Recently, chromogenic in situ hybridization (CISH), in which HER-2 is detected by a peroxidase reaction and the gene copies are determined by regular bright-field microscopy, has emerged as a potential alternative to FISH. However, this method requires validation before it can be adopted into clinical practice. In this study, we evaluated 80 cases of invasive breast carcinoma by CISH, compared the results with those obtained by FISH, and assessed interobserver reproducibility among three observers. We found that agreement among the three pathologists on the CISH-determined HER-2 status was achieved in 73 cases (91%), all of which had results matching the corresponding FISH results: 54 nonamplified and 19 amplified. Of the 19 amplified cases, 13 were scored unanimously as high-level amplification; six had a minor scoring discrepancy (ie, low-level vs high-level amplification). A major scoring discrepancy (ie, nonamplification vs amplification) was found in the remaining seven cases, three of which were amplified and four of which were nonamplified by FISH. Two of the latter cases had a polysomy of chromosome 17. The cases that caused scoring difficulty were those with an equivocal or borderline signal number against a high background. Overall, there was nearly perfect agreement between the CISH and corresponding FISH results, and interpretation of CISH results were highly reproducible among the three pathologists. We conclude that, in general, HER-2 status can be reliably assessed by CISH. Confirmatory FISH is recommended in cases with equivocal or borderline CISH copy numbers. 相似文献
7.
Jinru Shia David S Klimstra Allan R Li Jing Qin Leonard Saltz Julie Teruya-Feldstein Muzaffar Akram Ki Young Chung Davis Yao Philip B Paty William Gerald Beiyun Chen 《Modern pathology》2005,18(10):1350-1356
Recent data suggest that detection of epidermal growth factor receptor protein by immunohistochemistry (IHC) does not predict response to the antiepidermal growth factor receptor drug, cetuximab, in patients with colorectal carcinoma. In searching for foundation for further investigation to optimize patient selection for cetuximab therapy, this study sought to exploit the tissue microarray and chromogenic in situ hybridization techniques to evaluate the status of epidermal growth factor receptor gene amplification in colorectal cancer and its relationship with protein expression by IHC. The study included 158 primary or metastatic colorectal adenocarcinomas. Immunohistochemical results were scored as 0-3+ based on the intensity of membrane staining. The in situ hybridization signals were counted in 30 nuclei per tissue core. Overall, the rate of tissue loss was 7%, yielding 147 analyzable cases: 123 primary, 24 metastatic. Positive immunohistochemical staining of any intensity was detected in 85% (105/123) of primary and 79% (19/24) of metastatic tumors, whereas gene amplification (>5 gene copies/nucleus) was only seen in 12% (15/123) of primary and 8% (2/24) of metastatic tumors. Only 2/15 primary and 1/2 metastatic tumors that showed gene amplification were amplified at a high level (>10 gene copies/nucleus). Although a positive correlation was detected between the intensity of protein expression and the likelihood of gene amplification in both the primary (P = 0.01) and the metastatic (P = 0.05) tumors, IHC had a low specificity (17% in primary, 23% in metastatic) in predicting gene amplification. Conversely, all tumors that did not express the protein by IHC lacked gene amplification. Thus, this study shows that only a small fraction of epidermal growth factor receptor- positive colorectal carcinomas detected by IHC are associated with gene amplification. Additional studies are needed to determine whether epidermal growth factor receptor gene amplification bears any informative value in predicting response to cetuximab-based therapy. 相似文献
8.
Fluorescence in situ hybridization (FISH) is the most widely used technique to detect HER-2/neu gene amplification; however, it is only available in some institutions. In contrast, chromogenic in situ hybridization (CISH) can be evaluated by routine light microscopy. In endometrial carcinoma there are few data concerning HER-2/neu status and prognosis. Therefore, we determined HER-2/neu gene status by CISH using a digoxigenin-labelled probe on 60 formalin-fixed paraffin-embedded endometrial carcinomas. The data were compared with the immunohistochemistry of HER-2/neu (A0485, TAB250), p53, Ki-67, clinicopathological factors, and survival. By conventional light microscopy, HER-2/neu amplification (>/=6 copies >50% cancer cells) was detected in 14% (8/59) tumours, HER-2/neu overexpression (>10% cells moderate/strong complete membrane staining) in 22% (13/60) for A0485, and 18% (11/60) for TAB250, p53 (>10% +cells) in 61% (36/59), and Ki-67 (>50% +cells) in 50% (30/60). Discordant cases for CISH and immunohistochemistry, as well as all (2+) were further analysed by FISH (Vysis). Among 10 cases (2+) and not amplified by CISH, two showed low-level amplification by FISH. Significant correlation was found between amplification and protein overexpression (P=0.001), and a trend with nonendometrioid type, higher grade, and older age. A better outcome (Kaplan-Meier) was observed for patients with nonamplified (1-5 copies per nucleus) or low-level (6-10 copies) amplification tumours, low Ki-67 expression, age <50 years, endometrioid type, low FIGO (International Federation of Obstetrics and Gynaecology) grade and stage, superficial myometrial infiltration, and no lymph-vascular invasion (P=0.036), but only as a trend for HER-2/neu protein negative (P=0.13). Cox analysis revealed age, FIGO grade and stage, myometrial infiltration, and lymph-vascular invasion to be independent prognostic factors (P=0.05), and a trend for HER-2/neu gene copy number (0.18). In endometrial carcinoma, HER-2/neu gene status can be readily assessed by CISH in routine clinical practice, and it gives more prognostic information than HER-2/neu by immunohistochemistry. FISH analysis in (2+) cases but negative by CISH may detect additional tumours with low-level amplification. 相似文献
9.
Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma. 总被引:12,自引:0,他引:12
Jianxin Zhao Rina Wu Alfred Au Abbey Marquez Yibing Yu Zuorong Shi 《Modern pathology》2002,15(6):657-665
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. 相似文献
10.
Determining of HER-2/neu oncogene amplification has become clinically important for managing breast cancer. Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently regarded as the standard methods. Chromogenic in situ hybridization (CISH) was investigated as a new modification with an accurate, sensitive technique. From 1998 to 2002, using CISH and IHC, the amplification and protein expression of the HER-2/neu oncogene were examined using paraffin sections in 130 breast carcinomas and to determine the prognostic role of HER-2/neu for outcome after a follow-up of 24- 64 months. Amplifications by CISH and overexpression by IHC were observed in 28 (22%) and 27 cases (20.8%), respectively. Of the 104 patients, 20 patients (19.2%) with amplification had a shorter disease-free interval (34.9 months vs. 38.0 months in controls) (p=0.372). 15 patients (14.4%) had a disease recurrence, but there is no significant difference between 3 patients amplifying the oncogene and 12 patients without oncogene (20.6 months vs. 19.6 months) (p=0.862). 6 patients (5.8%) of these died. CISH is a useful alternative, particularly for confirming the IHC results. There is no relationship between the early recurrence and the HER-2/neu positive group, but lymph node status was statistically significant. 相似文献
11.
Comparison between immunohistochemistry and chromogenic in situ hybridization in assessing HER-2 status in breast cancer 总被引:2,自引:0,他引:2
Todorović-Raković N Jovanović D Nesković-Konstantinović Z Nikolić-Vukosavljević D 《Pathology international》2005,55(6):318-323
Human epidermal growth factor receptor-2 (HER-2) is usually determined as a potential target for breast cancer therapy. The purpose of the present study was to compare chromogenic in situ hybridization (CISH) with immunohistochemistry (IHC) in determination of HER-2 status, in metastatic breast cancer patients screened for the clinical study of chemotherapy +/- herceptin. It was possible to assess both CISH and IHC in 56 cases, using CISH Detection Kit (Zymed) and HercepTest (DakoCytomation), respectively. HER-2 was amplified by CISH in 32 cases (57%) while 33 (59%) were HER-2-positive by IHC. A concordance between HER-2 status determined by CISH and IHC was noted in 43 of 56 cases (77%; P = 0.00008). Gene amplification was observed in 6/16 cases (37.5%) in IHC-negative subgroup (1+), while no amplification was observed in 5/10 cases (50%) in the IHC-positive subgroup (2+). These results suggest that there was a greater heterogeneity on the genetic level and that simple IHC classification was not sufficient. It is suggested that CISH could be considered as a useful additional method to IHC in determining HER-2 status in breast cancer patients, with a recommendation for testing not only the 2+ but also the 1+ subgroup of patients. 相似文献
12.
Tissue microarrays (TMAs) have been commonly used to study protein expression by immunohistochemistry (IHC). However, limited data exist on the validity of using TMAs to study gene amplification. In this study, we evaluated the feasibility of using breast carcinoma TMAs to study HER-2 gene amplification by fluorescence in situ hybridization (FISH). In addition, hormonal receptor status (ER and PR) and HER-2 protein overexpression by IHC were also studied, and results were compared with whole tissue sections. FISH for HER-2 was performed on formalin-fixed paraffin-embedded tissue from 114 invasive breast carcinomas both on whole tissue sections and on TMAs containing the same tumors. The TMA was created using 0.6-mm tissue cores with four sampled cores per tumor from the same tissue block used for whole section FISH. The PathVysion HER-2 probe kit was used for the FISH analysis. A ratio of HER-2:Chromosome17 > or =2.0 was interpreted as positive for gene amplification. The ER or PR was interpreted as positive when nuclear staining was detected in more than 10% of tumor cells. The HER-2 IHC (HercepTest; DAKO Corp, Carpinteria, CA) results were interpreted as 0, 1+, 2+, and 3+ according to standard criteria. The FISH results in the TMA and whole sections were concordant in 99 out of 101 successfully analyzed cases (99%). The FISH scores were consistent among the two to four cores in the majority of the cases. ER and PR results were concordant between whole sections and TMA cores in 97% (107/110) and 89% (97/109) cases, respectively. The overall concordance for HER-2 status by IHC between whole sections and TMA cores was 86% (94 out of 109 cases). TMAs are a reliable approach to study HER-2 gene amplification in a high throughput manner. 相似文献
13.
Rodriguez-Pinilla SM Jones RL Lambros MB Arriola E Savage K James M Pinder SE Reis-Filho JS 《Journal of clinical pathology》2007,60(9):1017-1023
AIMS: To analyse the correlation between MYC amplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based chemotherapy. Given the high prevalence of MYC amplification in tumours of BRCA1 mutation carriers and the similarities between these and sporadic "basal-like" carcinomas, the prevalence of MYC amplification in "basal-like" breast carcinomas was investigated. METHODS: MYC gene copy number was assessed on tissue microarrays containing duplicate cores of 245 invasive breast carcinomas by means of chromogenic in situ hybridisation using SpotLight C-MYC amplification probe and chromosome 8 centromeric probe (CEP8). Signals were evaluated at 400x magnification; 30 morphologically unequivocal neoplastic cells in each core were counted for the presence of the gene and CEP8 probes. RESULTS: Amplification was defined as a MYC:CEP8 ratio >2. Signals for both MYC and CEP8 were assessable in 196/245 (80%) tumours. MYC amplification was found in 19/196 cases (9.7%) and was not associated with tumour size, histological grade, positivity for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor, cytokeratins 14, 5/6 and 17, MIB1 or p53. Only 4% of basal-like carcinomas showed MYC amplification, compared to 8.75% and 10.7% of luminal and HER2 tumours respectively. On univariate analysis, MYC amplification displayed a significant association with shorter metastasis-free and overall survival and proved to be an independent prognostic factor on multivariate survival analysis. CONCLUSION: MYC amplification is not associated with "basal-like" phenotype and proved to be an independent prognostic factor for breast cancer patients treated with anthracycline-based chemotherapy. 相似文献
14.
15.
Todorović-Raković N Jovanović D Nesković-Konstantinović Z Nikolić-Vukosavljević D 《Experimental and molecular pathology》2007,82(3):262-268
After so many years of research, clinical value of HER2 (Human epidermal growth factor receptor 2) is unclear. Perhaps the main reason is variability of testing methods that produce controversial results. There is a lack of studies regarding prognostic value of CISH especially in metastatic breast cancer (MBC) when risk evaluation is based on different parameters than for primary breast cancer. Aim of this study was to compare prognostic relevance of HER2 status in MBC tested by two different methods i.e. immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).HER2 status of the same group of 107 MBC patients was determined by IHC (protein overexpression) and by CISH (gene amplification). HER2 results obtained by IHC and CISH showed significant correlation, beside the existence of discrepancies. Beside the significant correlation in two methods, there was a difference in prognostic values of compared methods during the course of metastatic disease. There was a significant difference in progression-free interval (PFI) between HER2 non-amplified and HER2 amplified cases determined by CISH, in postmenopausal subgroup and node-positive subgroup, but no significant difference for IHC stratified MBC patients. CISH seems to be accurate and more informative method than IHC regarding prognostic value of HER2 in metastatic breast cancer. 相似文献
16.
Rohit Bhargava Orit Oppenheimer William Gerald Suresh C Jhanwar Beiyun Chen 《Diagnostic molecular pathology》2005,14(2):72-76
Chromogenic in situ hybridization (CISH) is a recently developed technique, which utilizes the general principles of in situ hybridization and a detection system similar to immunohistochemistry. To assess the utility of CISH for analysis of MYCN gene amplification, we compared this assay with established diagnostic assays such as Southern blot analysis (SB) and fluorescent in situ hybridization (FISH). CISH was performed on 67 cases of neuroblastoma using tissue microarray (65 cases) and whole tissue sections (2 cases). Unequivocal, high-level amplification (> or =10 gene copies per tumor nucleus) was identified in 19 of 67 (28.4%) tumors. Two (3%) tumors showed low-level amplification (6-9 gene copies per tumor nucleus). No amplification was seen in 46 of 67 (68.6%) tumors. SB data were available in 44 tumors. Forty-one of the 44 tumors (93%) showed concordant results between CISH and SB. Three tumors showed MYCN amplification by CISH but no amplification by SB, most likely due to dilution effect of nonneoplastic tissue in the test samples. Two of these three tumors also showed MYCN amplification by FISH, and the third tumor was not analyzed by FISH. FISH data were available in total of 30 tumors. All 30 tumors showed concordant results between CISH and FISH for classifying a tumor as MYCN amplified or not amplified. We conclude that CISH is an accurate method for determining MYCN gene amplification, with added advantages that make it a more practically useful method. 相似文献
17.
Forty invasive breast ductal carcinoma biopsy specimens were investigated. The techniques of fluorescence and chromogenic in situ hybridization were used to study HER-2/neu gene amplification and the concordance of these techniques was examined. The advantages and disadvantages of both techniques are shown. The optimum ranges of using these techniques in the determination of the herzept status of breast cancer are given. 相似文献
18.
Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples 总被引:19,自引:0,他引:19
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Tanner M Gancberg D Di Leo A Larsimont D Rouas G Piccart MJ Isola J 《The American journal of pathology》2000,157(5):1467-1472
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. 相似文献
19.
Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer 总被引:5,自引:0,他引:5
Gupta D Middleton LP Whitaker MJ Abrams J 《American journal of clinical pathology》2003,119(3):381-387
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. 相似文献
20.
Persons DL Bui MM Lowery MC Mark HF Yung JF Birkmeier JM Wong EY Yang SJ Masood S 《Annals of clinical and laboratory science》2000,30(1):41-48
Amplification and/or overexpression of HER-2/neu has been shown to be both a prognostic and predictive marker in breast cancer. Recent studies have also confirmed the efficacy of Herceptin (trastuzumab) as adjuvant therapy for patients with overexpression of HER-2/neu. Therefore, it is critical that precise and reproducible assays be used in the clinical laboratory setting for determination of the HER-2/neu status in patients with breast cancer. The objective of this study was to determine the portability (reproducibility between different institutions) of the PathVysion HER-2 fluorescence in situ hybridization (FISH) assay used for detection of amplification of the HER-2/neu gene in formalin-fixed, paraffin-embedded tissue sections of invasive ductal carcinoma of the breast. Study specimens consisted of one breast tumor with a normal HER-2/neu copy number, two tumors with a low level, and one tumor with a high level of HER-2/neu amplification. The PathVysion HER-2 assay was shown to be highly reproducible on different assay days (n = 3) and between different institutions (n = 5) in the detection of amplification of the HER-2/neu gene in routinely processed clinical specimens of breast carcinoma. In addition, this study examined the feasibility of enumerating FISH signals in 20 nuclei in contrast to 60 nuclei per specimen. Although a modest increase in variation was observed when analyzing 20 compared to 60 nuclei, the mean ratios were similar. Therefore, analysis of as few as 20 nuclei with this FISH HER-2/neu assay may be sufficient for determining the amplification level of the HER-2/neu gene. 相似文献