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.     

Determination of HER2 amplification in primary breast cancer using dual‐colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens

García‐Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J
(2010) Histopathology 56, 472–480
Determination of HER2 amplification in primary breast cancer using dual‐colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual‐colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual‐colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual‐colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual‐colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual‐colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual‐colour CISH and FISH was highly significant with an overall correlation coefficient (ρ) of 0.96. Conclusions: We conclude that dual‐colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.     

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.     

Prognostic value of HER2 gene amplification detected by chromogenic in situ hybridization (CISH) in metastatic breast cancer

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.     

Dual-colour chromogenic in-situ hybridization is a potential alternative to fluorescence in-situ hybridization in HER2 testing

Hwang C‐C, Pintye M, Chang L‐C, Chen H‐Y, Yeh K‐Y, Chein H‐P, Lee N & Chen J‐R
(2011) Histopathology 59 , 984–992 Dual‐colour chromogenic in‐situ hybridization is a potential alternative to fluorescence in‐situ hybridization in HER2 testing Aim: Dual‐colour chromogenic in‐situ hybridization (dc‐CISH) is an emerging methodology for characterizing genomic alterations. This study was aimed at evaluating the performance of a dc‐CISH kit (ZytoVision) in determining human epidermal growth factor receptor 2 (HER2) status in breast cancer. Methods and results: Two hundred and twenty‐eight invasive breast carcinomas arranged in tissue microarrays were analysed in parallel with dc‐CISH, fluorescence in‐situ hybridization (FISH), and immunohistochemistry. Of 227 tumours with available FISH and dc‐CISH results, HER2 amplification and non‐amplification were detected in 49 (21.6%) and 178 (78.4%) tumours, respectively, by both assays. The concordance between dc‐CISH and FISH results showed 100% agreement (κ‐coefficient = 1.00). Immunohistochemically, 162 (71%), 25 (11.0%) and 41 (18%) tumours were scored 0/1+, 2+, and 3+, respectively. The corresponding results with both FISH and dc‐CISH demonstrated HER2 amplification in two (3.2%), nine (36%) and 38 (93%) tumours, respectively. Complete consensus among these three methods was observed in 197 cases, representing 98% of all 3+ and 0/1+ tumours (κ‐coefficient = 0.92). Confirmatory testing of 25 2+ tumours showed complete consensus between FISH and dc‐CISH. Conclusions: dc‐CISH is a promising alternative to FISH in HER2 testing, and the single‐institute incidence of HER2 amplification in breast cancer in Taiwan is 21.2%.     

乳腺癌组织HER2基因扩增的检测方法

目的:评价高分辨率熔体聚合酶链反应(high-resolution melt polymerase chain reaction,HRM-PCR)检测HER2基因扩增的有效性及其与荧光原位杂交(fluorescence in situ hybridization,FISH)和免疫组织化学(immunohistochemistry,IHC)检测法的一致性.方法:采用HRM-PCR法检测HER2阴性及阳性细胞株,评估检测方法的有效性;检测98例已行FISH和/或IHC的临床样本,并与FISH和IHC结果进行比较.结果:HRM-PCR检测可以有效区分HER2阴性及阳性细胞株(P<0.05),具有较好的可重复性;检测98例临床样本显示,阴性和阳性样本检测结果差异有统计学意义(0.18±0.14 vs 1.42±0.88,P<0.01),与IHC的一致性为80.33%(kappa=0.6,P<0.01),与FISH的一致性为87.88%(kappa=0.7,P<0.01),结论:HRM-PCR是一种可靠有效的检测HER2基因扩增的方法,与FISH和IHC均有较好的一致性.     

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.     

Detection of c-erbB-2 (HER-2/neu) amplification in breast carcinoma by fluorescence in situ hybridization on tissue sections and imprinted cells

Abnormalities in c-erbB-2 have attracted a great deal of attention. Treatment using an antibody against the c-erbB-2 gene product is effective against breast cancers with amplification and/or overexpression of c-erbB-2. There is an urgent need to establish methodology for selecting patients who would benefit from this therapy. A total of 235 breast carcinomas were examined for c-erbB-2 protein overexpression by immunohistochemistry. Tissue sections with discernible immunostaining from 52 tumors, 70 negative tumors and smear imprints from 35 patients were examined by dual-color fluorescence in situ hybridization (FISH) using probes specific for c-erbB-2 and the centromeric region of chromosome 17. The concordance between gene amplification and protein overexpression was 95.7%. When the findings of the two FISH preparation techniques were compared, no discrepancies were found in 24 of the tumors. However, differences were seen in eight cases. In six of these cases the differences did not affect the presence or absence of amplification, but in the other two cases, considered to show low-level amplification on paraffin sections, polysomy 17 was detected instead. It was concluded that FISH is an excellent tool to detect gene amplification in particular, and FISH on touch imprints is a useful adjunct to differentiate between low-level amplification and polysomy 17.     

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.     

Putting the colours into chromogenic in situ hybridization (CISH)

Recurrent genomic alterations are the hallmarks of particular cancers. Application of molecular cytogenetic technologies to tumour material in order to detect these alterations has become important for molecular diagnostics and research. A dual-colour chromogenic in situ hybridization (dc-CISH) method described recently in the Journal of Pathology has the advantage of visualizing two probes simultaneously with the ability to discern morphological features. In addition, the bright field microscopy required is readily accessible to many laboratories. The approach has been validated by comparison of results with standard analyses for HER-2 amplification status in formalin-fixed, paraffin-embedded breast cancers and is applicable to the analysis of other clinically relevant genomic aberrations as well as of use in research investigations.     

Comparison between immunohistochemistry and chromogenic in situ hybridization in assessing HER-2 status in breast cancer

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.     

Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer

The determination of HER2/neu status in breast carcinomas has become essential for the selection of breast cancer patients for Herceptin therapy. Herceptin treatment is used in patients with metastatic breast carcinoma with HER2/neu protein overexpression detected by immunohistochemistry (IHC) or gene amplification analysed by fluorescence in situ hybridization (FISH). A multiparametric fluorescent approach based on the simultaneous detection of HER2/neu gene amplification and protein expression was established to increase the accuracy, and to improve the reproducibility, of HER2/neu diagnostics. Based on four paraffin-embedded breast cancer cell lines, a combined fluorescent immunostaining (FIHC) and FISH method was developed by using the PathVysion HER2 DNA Probe Kit (VYSIS) and the polyclonal antibody from the HercepTest (DAKO). Diagnostic applicability was documented on 215 formalin-fixed primary breast carcinomas. Criteria for immunofluorescence quantification were chosen by analogy with the FDA-approved HercepTest scoring, ranging from 0 to 3+. There was 97.7% concordance between conventional IHC and fluorescence IHC. The FISH data resulting from the multiparametric approach did not differ from conventional FISH. Breast carcinomas with HER2/neu protein overexpression and simultaneous gene amplification were detected with 100% sensitivity. In addition, five of the 215 cases (2.3%) had HER2/neu gene amplification without protein overexpression. The main advantage of this novel approach is that polysomy, aneuploidy, gene amplification, and protein content can be analysed simultaneously in the same cell.     

Comparison of Dako fluorescence in situ hybridization assays (FISH and IQFISH) in the assessment of HER2 amplification in breast cancer

Abstract

Determining human epidermal growth factor receptor 2 (HER2) status in breast cancer patients can be attained by fluorescent in situ hybridization (FISH) analysis, which prompts targeted treatment options. This study was performed to compare the staining results of Dako’s manual overnight HER2 FISH pharmDx^? assay with the manual (4 hours) HER2 IQFISH pharmDx^? assay and the Dako Omnis automated (4 hours) HER2 IQFISH assay, for assessment of HER2 amplification.

Thirty-four formalin-fixed paraffin-embedded (FFPE) tissue blocks from patients diagnosed with invasive breast cancer were used to compare three FISH assays in three experiments. All assays included Texas Red-labeled HER2 and fluorescein-labeled centromere 17 (CEN-17) probes. First, 20 blocks with known HER2 immunohistochemistry (IHC) and FISH results from a reference laboratory were used in the validation of HER2 FISH pharmDx^? assay. Secondly, 19 of the same blocks were used in a comparison of HER2 FISH pharmDx^? assay and HER2 Instant Quality FISH pharmDx (IQFISH pharmDx^?) assays. The third experiment, which included 23 blocks, compared the HER2 FISH pharmDx^? assay with the Dako Omnis IQFISH assay (stained on a Beta version of Dako’s new Omnis platform). Stained slides were counted following American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines (2007 version), with three categories of labeling: <1·8 (negative), 1·8–2·2 (equivocal), and >2·2 (positive) calculated HER2/CEN-17 ratio. The results showed high agreement between all assays employed. Initial validation of HER2 FISH pharmDx^? assay to the results provided by a reference laboratory had a concordance of 95%. The concordance between HER2 FISH pharmDx^? assay and HER2 IQFISH pharmDx^? assay was 100%. There was also 100% concordance between manual and Omnis IQFISH staining. A high level of concordance was obtained in all three experiments, indicating that manual and automated IQFISH are comparable to HER2 FISH pharmDx^?. IQFISH has a faster turn-around-time (TAT) and showed comparable results in quality and quantitation.     

EGFR gene copy number heterogeneity in fine-needle aspiration cytology from breast carcinomas determined by chromogenic in situ hybridization

Most studies have shown epidermal growth factor receptor (EGFR) overexpression to be associated with poor prognostic factors in breast carcinomas. The relationship to EGFR gene copy number is unclear. The aim of our study was to investigate the heterogeneity of the EGFR gene copy number in breast carcinomas. The material consisted of air-dried smears from 29 breast carcinomas and 3 breast cancer cell lines (MCF-7, SKBR3, and T47D). Chromogenic in situ hybridization (CISH) was done using chromogenic detection. The mean signal numbers for EGFR gene and chromosome 7 as well as the EGFR gene/chromosome 7 centromere probe (CEP7) ratio were recorded. Immunohistochemical (IHC) staining was done on the corresponding paraffin sections.The copy number of the EGFR gene in each tumor/cell line ranged from 1.2 to 5.6. The EGFR gene/CEP7 ratio showed a biological continuum ranging from 0.59 to 1.94 with a mean of 1.04. EGFR gene copy loss was found in 16.6% of cases whereas copy gain was demonstrated in 19.4%. There was no relationship between IHC protein expression of EGFR and EGFR gene copy number or EGFR gene/CEP7 ratio.In conclusion, most breast carcinomas had a balanced EGFR gene/CEP7 copy number with a mean ratio of 1.04. Almost equal subpopulations revealed limited copy gain and copy loss. EGFR high dosage amplification, like in HER-2, was not demonstrated. Demonstration of EGFR gene copy loss might have a potential as a surrogate marker for EGFR gene mutation and/or deletion.     

Impact of polysomy 17 on HER2 testing of invasive breast cancer patients

Background: Current American Society of Clinical Oncology/College of American Pathologists guidelines define HER2-positive tumors as those with > 6 HER2 genes per nucleus or those with HER2/CEP17 (chromosome 17) ratio > 2.2. These guidelines are potentially contradictory in tumors with polysomy of chromosome 17. The current study was performed to determine the impact of polysomy 17 on the interpretation of HER2 testing of invasive breast carcinomas. Methods: Chromosome 17 copies and HER2 gene status were identified by fluorescent in situ hybridization in 384 cases with invasive breast cancer, and the corresponding HER2 expression was obtained by immunohistochemistry stain. Results: The average CEP17 copy number for the group was 2.1 (range, 1.0-12.4). Forty-eight cases (13.8%, 48/348) were identified as chromosome 17 polysomy with CEP 17 copy number ≥ 3. Ninety-two (26.4%) cases had > 6 copies of HER2 per nucleus, and 92 cases (26.4%) qualified as HER2 gene amplified using the HER2/CEP17 ratio (> 2.2) guideline. Polysomy 17 showed poorly positive correlations with both HER2 gene copy number and HER2 overexpression (P < 0.01, r = 0.338 and 0.271, respectively). The distribution of clinicopathologic parameters of Polysomy 17 tumors was more similar to HER2 negative than HER2 positive tumors. Conclusions: Polysomy 17 is a crucial cause of equivocal HER2 testing results by FISH, depending on which criterion (ratio vs. absolute number) is used for interpretation. Polysomy 17 cannot be an independent predictive factor for HER2 gene amplification or protein overexpression.     

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.     

荧光原位杂交检测乳腺癌HER2基因状态

目的 探讨荧光原位杂交（FISH）,与免疫组织化学（IHC）染色（3＋）和（2＋）检测HER2基因状态的结果的一致性、导致两者差异的可能原因及FISH检测HER2基因状态的必要性和可行性。方法 采用PathVysion^TM探针试剂盒,以FISH方法,对28例IHC EnVision法染色分别为（3＋）、（2＋）、（1＋）和阴性（-）的乳腺癌石蜡切片标本进行HER2基因状态的检测。结果 HER2表达（3＋）的12例标本中,10例HER2基L大1扩增,其中2例为17号染色体多体,另2例无扩增的病例均为多体;IHC为（2＋）的10例标本中,7例为HER2基因扩增,其中1例为多体,另3例无扩增病例中2例为多体;IHC为（1＋）的3例标本均无HER2基因扩增,其中1例为多体;IHC（-）的3例标本均无基因扩增,其中1例为多体。结论 IHC是初步筛查HER2状态的首选方法;因IHC（2＋）与FISH检测结果差异较大,所以这类患者应做FISH确诊;IHC（3＋）存在假阳性,主要原因可能是由于17号染色体非整倍体,必要时这类患者也应做FISH。     

Evaluation of analytical accuracy of HER2 status in patients with breast cancer

Human epidermal growth factor receptor 2 (HER2) gene status and overexpression, occurring in ~ 13.6% of primary breast cancers, is essential for identifying patients likely to benefit from biological treatment. In this method of evaluation study, we tested and compared the HER2 gene–protein assay (GPA) with routine HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The GPA was evaluated using 67 formalin-fixed paraffin-embedded (FFPE) HER2 equivoval IHC (2+) breast cancer tissue samples. Overall, agreement between GPA silver in situ hybridization (SISH) and FISH was 91.9% (57/62). Regression analysis revealed slightly higher, but non-significant difference in HER2/chromosome enumeration probe 17 (CEP17) ratio for GPA as compared to FISH (p = 0.074). Intraclass correlation coefficients (ICCs) of 0.94 and Spearman´s rank correlation coefficients of 0.93 (p < 0.0001) for FISH and GPA SISH suggested strong inter-observer association for methods with one observer counting on average 0.23 significant higher for GPA SISH (p = 0.014). Intra-observer IHC method reproducibility was 52.6% (κ = 0.3122, p = 0.004) and 79.7% (κ = 0.6428, p = 0.9197), suggesting fair significant and substantial non-significant difference between tests for reviewers. Inter-observer reproducibility for IHC methods was 53%. While inter-observer reproducibility for experienced IHC interpretation suggested significant differences (κ = 0.3636, p = 0.0332), unexperienced interpretation of IHC GPA suggested fair non-significant difference between reviewers (κ = 0.3101, p = 0.0747). Using FISH as reference, the diagnostic indices for GPA SISH were as follows: sensitivity 100%, specificity 95% and accuracy 92%. Inaccuracy between tests was in 80% of cases due to ISH categorization as equivocal by one of the methods. IHC results highlight that it may be beneficial with a method for simultaneously visualization of HER2 gene and protein status.     

Immunohistochemical over-expression of HER2 does not always match with gene amplification in invasive bladder cancer

BackgroundHER2 is a potential target of therapy in urothelial cancer (UC). Pathological case stratification according to HER2 gene amplification or HER2 protein overexpression was critical for patients’ selection in previous unsuccessful clinical trial with HER2 targeting agents.Study designWe evaluated the HER2 overexpression by immunohistochemistry (IHC) together with the amplification of the HER2 gene with chromogenic(CISH) and fluorescent (FISH) in situ hybridization in a cohort of 61 patients covering the whole spectrum of bladder UC variants, using a tissue microarray (TMA) approach.ResultsIHC was available in all the 61 cases while ISH in 37 and FISH in 42. At IHC, 2/61 cases (3%) were scored 3+; 2 (3%) scored 2+; 2 (3%) scored 1+; the remaining 55 (91%) scored 0. At CISH analysis 10/37 cases (27%) were amplified, 6 cases with HER2 amplification showed positive HER2 IHC (3+, 2+, 1+). Seven cases with IHC score 0 were amplified at CISH. FISH analysis revealed an amplification in 5/42 cases (12%). The total number of HER2amplified cases was different between chromogenic and fluorescent ISH with 5 cases amplified using FISH compared to 10 with CISH.ConclusionsIn clinical trials with HER2 targeting agents the candidate patients should be investigated not only by IHC but also by ISH, independently of the IHC results. Since also usual type UC can overexpress HER2 we recommend to extend the patients’ selection to all the histotypes of bladder cancer other than the micropapillary type.