Chromogenic in situ hybridization analysis of HER-2/neu status in breast carcinoma: application in screening of patients for trastuzumab (Herceptin) therapy

Evaluation of HER-2/neu status is important in the management of patients with breast carcinoma, especially in determining the possible application of trastuzumab, a humanized anti-HER-2/neu monoclonal antibody. Chromogenic in situ hybridization (CISH) detection of the HER-2/neu oncogene is a newly developed in situ hybridization method that utilizes a robust and unique-sequence DNA probe labeled with digoxygenin, and sequential incubations with antidigoxygenin fluorescein, antifluorescein peroxidase, and diaminobenzidine. In this study, we examined 20 archival specimens of human breast carcinoma using CISH, and we correlated findings with immunohistochemical findings for HER-2/neu. HER-2/neu immunohistochemistry was carried out with HercepTest, a standardized immunohistochemical examination system for HER-2/neu overexpression in surgical pathology specimens. CISH analysis could be done in 18 out of 20 cases examined. Gene copy signals for HER-2/neu were recognized as intranuclear brown dots in both neoplastic and non-neoplastic cells. Seven carcinomas showed an increased number or size of signals and were interpreted as being positive for HER-2/neu amplification. Eight cases were positive with the HercepTest. Seven out of eight carcinoma cases found to overexpress immunoreactive HER-2/neu also demonstrated HER-2/neu gene amplification following CISH analysis. There was a significant correlation between immunohistochemical and CISH analyses (P < 0.001). We found that CISH was a specific, sensitive and easily applicable method for the detection of HER-2/neu gene amplification, which may be used together with immunohistochemical examination for the evaluation of patients with breast carcinoma.     

进展期乳腺浸润性导管癌HER-2/neu蛋白表达的稳定性分析

目的探讨HER-2/neu蛋白在进展期乳腺浸润性导管癌原发灶和同期腋窝淋巴结转移灶中的表达,指导临床检测标本的选择以及检测报告的分析与应用。方法收集进展期乳腺浸润性导管癌标本100例制备全信息组织芯片,采用免疫组化法检测HER-2/neu蛋白表达,FISH法检测HER-2/neu基因扩增状态,分析其特点、稳定性及其相互关系。结果 69%病例HER-2/neu蛋白呈均质性表达,与同期腋窝淋巴结转移灶相比,原发灶蛋白均质阴性病例一致性最强,均质阳性病例次之,均质不确定病例最差;三者相比差异有统计学意义(P0.000 1)。36例HER-2/neu蛋白不确定和异质性病例的FISH检测显示20例为基因均质扩增,13例为基因均质非扩增,3例为基因异质性,其中31.65%的蛋白阴性位点HER-2/neu基因扩增。结论乳腺浸润性导管癌HER-2/neu蛋白表达在肿瘤原发灶和同期腋窝淋巴结转移灶之间稳定性较高,尤以阴性病例最稳定;免疫组化与FISH同步检测及对比分析可能为乳腺癌患者的治疗及预后提供更为精准的信息;应用全信息组织芯片更能全面检测HER-2/neu蛋白表达及基因状态,并可用于深入分析乳腺癌基因分型及其它相关指标在肿瘤进展中的变化。     

胃癌中HER-2表达异质性的病理学分析

目的检测胃癌组织中HER-2蛋白表达水平和基因扩增情况,定量分析HER-2表达和分布特征的异质性,探讨其在判读中的影响。方法应用免疫组化检测并观察胃癌组织中HER-2蛋白表达水平及分布特征,应用荧光原位杂交(fluorescence in situ hybridization,FISH)技术检测HER-2基因扩增情况,并运用欧式距离等进行统计学处理。结果 373例胃癌手术切除标本中HER-2蛋白阳性率(3+)为12.33%,与基因扩增情况显著相关(P0.001)。98例有HER-2蛋白表达的标本总异质性、组内异质性及组间异质性大小差异均有统计学意义(P值分别为0.025、0.001、0.040);组内异质性大小明显低于组间异质性。根据标准判读为HER-2蛋白为2+及3+的病例异质性最大,1+病例最小。HER-2蛋白表达分布较均一的病例近乎为零,而同时具有阴阳脸及花斑样特征的病例最多。根据标准判读HER-2蛋白为0的病例中,有7例具有明显异质性。结论胃癌组织中HER-2蛋白表达的异质性较大且类型多样,判读时首先要增加观察视野,选择至少2个组织块进行HER-2蛋白检测;其次无论HER-2蛋白表达水平高低,均需同时检测基因扩增情况,以确保判读结果的准确性,更好的指导临床用药。     

Predictive markers in breast cancer--the future

The published literature is awash with examples of new tissue biomarkers promising to predict responses to therapy in breast cancer patients. However, few, if any, of these progress from the laboratory to the clinic. In this review we discuss some of the reasons for this, illustrating our discussion with a selection of biomarkers which are in development and which may be candidates for clinical application within the next few years (topoisomerase IIα, epidermal growth factor receptor, AKT, phosphatase and tensin homologue). In particular, we explore how our ever increasing knowledge of molecular and pathway biology is facilitating hypothesis-driven biomarker discovery, and the statistical considerations which need to be addressed in order to validate new candidate biomarkers adequately.     

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%.     

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.     

Expression and amplification of Her2, EGFR and cyclin D1 in breast cancer: immunohistochemistry and chromogenic in situ hybridization

Determination of Her2, epidermal growth factor receptor (EGFR) and cyclin D1 status is now of major clinical importance due to the development of molecule-targeting drugs in anticancer therapy. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) are the most simple and convenient methods for evaluating gene alterations and their protein consequences. The purpose of the present study was to investigate the status of Her2, EGFR and cyclin D1 on both IHC and CISH in 95 primary breast carcinomas. There was substantial consistency between the IHC and CISH results of Her2 and EGFR, showing fair agreement between protein overexpression and gene amplification. However, cyclin D amplification was not related to protein overexpression. Moreover, there was no correlation between Her2, EGFR and cyclin D1. Her2 protein overexpression and amplification were positively associated with histological grade, nuclear grade and inversely correlated with the expression of estrogen receptor (ER) and progesterone receptor (PR). In ER-negative and postmenopausal patients, EGFR gene amplification was strongly associated with worse recurrence-free survival (P = 0.0087, P = 0.0149, respectively). Overall, the present findings suggest that EGFR gene amplification is important in predicting prognosis and this should be evaluated in breast carcinoma in addition to Her2 status in routine pathological practice.     

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one-third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray-based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SEC61G. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2.     

Assessment of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status in the fine needle aspirates of metastatic breast carcinomas

The assessment of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) status in the fine needle aspirates of metastatic breast carcinomas has prognostic and therapeutic implications. In this study, expression of ER, PR, and HER2 was assessed by immunohistochemical study in 70 cases of metastatic breast carcinomas and HER2 gene amplification was further evaluated by fluorescence in situ hybridization (FISH) in 38 (54%) cases. Positive expression of ER and PR was seen in 42 (60%) and 16 (23%) cases of metastatic breast carcinomas, respectively. HER2 immunoreactivity was scored as 0/1+ in 39 (56%), 2+ in 10 (14%), and 3+ in 21 (30%) cases. HER2 gene amplification was seen in 20% of HER2 2+ and 64% of HER2 3+ cases. ER, PR, and HER2 status in primary breast cancers were available to comparison in 31 cases (44%). The concordance rates between metastatic and primary breast carcinomas were 81% for ER, 65% for PR and 71% for HER2. Our study demonstrates that ER, PR, and HER2 status can be assessed in the fine needle aspirates of metastatic breast carcinomas and ER has a higher concordance rate between metastatic and primary breast carcinomas than PR and HER2. The addition of HER2 gene amplification FISH test helps in accurate assessment of HER2 status in metastatic breast carcinomas.     

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.     

Evaluation of HER2 gene amplification in invasive breast cancer using a dual‐color chromogenic in situ hybridization (dual CISH)

Fluorescence in situ hybridization (FISH) assay is considered the ‘gold standard’ for evaluation of HER2/neu (HER2) gene status, however, it is difficult to recognize morphologic features of tumors using fluorescence microscopy. Thus, chromogenic in situ hybridization (CISH) has been proposed as an alternative method to evaluate HER2 gene amplification. Here, we examined the dual color CISH (dual CISH) method which provides information regarding the copy number of the HER2 gene and chromosome 17 centromere from a single slide. We examined 40 cases of invasive ductal carcinomas of the breast that were resected surgically. HER2 gene status was assessed with FISH (Abbott) and dual CISH (Dako). HER2 gene amplification status was classified according to the guidelines of the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP). Comparison of the cut‐off values for HER2/chromosome 17 centromere copy number ratio obtained by dual CISH and FISH showed that there was almost perfect agreement between two methods (Kappa coefficient 0.96). The results of the two commercial products were almost consistent for evaluation of HER2 gene counts on the sections. The current study proved that dual CISH is comparable with FISH for evaluating HER2 gene status.     

Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization

Chromogenic in situ hybridization (CISH) is a molecular technique used to visualize specific genes. Both heat treatment and protease treatment play important roles for the success of CISH on formalin‐fixed paraffin‐embedded (FFPE) tissue sections. In contrast to heat treatment, the optimal condition of protease treatment may vary depending on each sample. Because trypsin has a substrate specificity to cleave lysine and arginine, we hypothesized that trypsin could effectively degrade histones rich in lysine and arginine and that the removal of histones from DNA following heat treatment could improve CISH results. We selected 21 patients with lung adenocarcinoma previously known to be positive or negative for anaplastic lymphoma kinase (ALK) gene rearrangement and used FFPE tissue sections collected from these patients. Then, we assessed histone degradation among the following protease treatments; trypsin, pepsin, and proteinase K, and compared the ALK CISH results with results obtained using commercially available kits and these protease treatments. The results showed that trypsin effectively degraded histones. Additionally, compared with the other treatments, ALK CISH with trypsin treatment showed the most evaluable cells and the smallest standard deviation. Our study suggests that the degradation of histones by trypsin subsequent to heat treatment might improve CISH results.     

Demonstration of oestrogen and progesterone receptors in freeze-dried, paraffin-embedded sections of breast cancer

Immunohistochemical evaluation of oestrogen and progesterone receptors is of importance in evaluating human breast tumours. Staining techniques can be performed on snap-frozen, cryostat-cut tissues or, as recently reported, on formalin-fixed, paraffin-embedded tissues. These methods are, however, limited by several drawbacks, including difficulties in retrospective studies and in storage of the material, and the relatively high frequency of false negative results for chemically fixed specimens. We therefore investigated the application of freeze-drying technology to assess the feasibility and reliability of this technique as an alternative method for diagnostic breast pathology. Morphological and immunohistochemical studies were performed on snap-frozen, freeze-dried and paraffin-embedded tissue obtained from 16 cases of benign and malignant breast neoplasms. Our results showed good preservation of tissue morphology, similar to standard formalin fixation, and excellent preservation of antigenic reactivity of nuclear receptors, comparable to that obtained with cryostat sections. We therefore suggest that freeze drying and paraffin embedding of frozen tissue blocks is equivalent or even preferable to formalin fixation for the demonstration of oestrogen and progesterone receptors, at least in the case of small tumours.     

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.     

Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archives

A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype-phenotype analysis of individual cells.     

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.     

Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization

Breast cancer patients with HER-2/neu oncogene amplification by fluorescence in situ hybridization (FISH) have been shown to have a better response to trastuzumab (Herceptin) therapy than those showing HER-2/neu protein overexpression only. Many centers currently perform FISH only on tumors showing 2+ HER-2/neu positivity by immunohistochemistry (IHC), with the assumption that 3+ positivity virtually equates with amplification. Results of FISH performed on 102 breast cancer cases over a 12-month period were correlated with HER-2/neu IHC results. FISH was performed using a ratio of HER-2/neu and chromosome 17 centromere signal counts (PathVysion; Vysis, Downers Grove, IL). Immunohistochemical expression of HER-2/neu was evaluated according to the published scoring guidelines of the HercepTest (Dako, Carpinteria, CA). Only 22 of 45 tumors with 3+ positivity (49%) showed amplification by FISH. Only 2 of 25 cases with 2+ staining by IHC (6%) showed gene amplification, and 1 of 25 cases with negative IHC staining (4%) showed weak amplification. Of the 25 cases showing oncogene amplification, 22 (88%) showed 3+ IHC positivity, 2 (8%) showed 2+ positivity, and 1 (4%) was negative by IHC. More than 50% of breast tumors showing strong 3+ HER-2/neu staining do not show oncogene amplification by FISH. Most tumors with 2+ and negative IHC also fail to amplify. In our experience, FISH studies should be performed on all 3+ and 2+ staining tumors to avoid inappropriate and toxic treatment. The decision to perform FISH on IHC-negative tumors should be guided by additional parameters, including tumor grade and estrogen receptor status.