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.     

Histological tumor grade correlates with HER2/c-erB-2 status in invasive breast cancer: a comparative analysis between immunohistochemical (CB11 clone and Herceptest), FISH and differential PCR procedures

There is a growing clinical demand for analysis of the HER2/c-erbB-2 (HER2) status of breast cancer specimens because it provides valuable prognostic, predictive and therapeutic information. In this sense, a variety of methods is available for detection of HER2 status, although to date a reliable and sensitive test does not exist. In order to choose the most suitable procedure to assess HER2 status, we analyzed 102 invasive breast cancers for HER2 overexpression by means of immunohistochemistry (IHC), with the CB11 Mo-Ab and the Hercep Test kit, and for HER2 gene amplification by fluorescence in situ hyubridization (FISH) and differential PCR (dPCR). HER2 overexpression, determined by CB11 (group C) and HercepTest (2+ and 3+), was observed in 19 samples (18.6%) whereas genetic amplification was detected in 31 (30.4%) and 14 (13.7%) cases by FISH and dPCR, respectively. The majority of overexpressed/amplified specimens corresponded to high grade tumors. We found concordances of 78-80% and 93-95% between IHC vs FISH and IHC vs dPCR, respectively. Considering FISH procedure as a gold standard, we found a sensitivity and specificity of 48.4% and 94.3% for CB11 antibody, of 45.2% and 92.9% for HercepTest, and of 45.2% and for 100% for the dPCR. Thus, considering the sensitivity, specificity and the high grade of concordance between IHC and dPCR, we suggest the use of IHC for assessing HER2 status. However, due that sensitivity of IHC test is lower than FISH, we also suggest to carry out FISH on those cass in which IHC results are not definitive for its clinical evaluation.     

HER2 amplification status in breast cancer: a comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques

AIMS: To compare the results of breast cancer sections with HercepTesttrade mark immunohistochemistry (IHC) scores ranging from 0 to 3+ with fluorescence in situ hybridisation (FISH) for HER2 amplification. The HER2 digital scoring application of the Micrometastasis Detection System (MDS) was used, together with manual scoring of FISH and HercepTest, to determine whether this system provides an accurate alternative. METHODS: Paraffin wax embedded sections were stained using HercepTest and analysed by eye and automated quantitative image analysis. FISH was performed using the PathVysion fluorescent probe and scored by eye and automated quantitative image analysis using MDS. RESULTS: Of 114 cases, 26% were amplified by FISH, whereas only 18% scored 3+; 32% of IHC 2+ cases were amplified by FISH, and one showed borderline amplification. Six percent of IHC negative cases (0 or 1+) were amplified by FISH, and one showed borderline amplification. Of IHC 3+ cases, 10% were non-amplified by FISH. Classification discrepancies were seen in 18% of HercepTest cases scored by eye and using the MDS system. MDS was consistent with visual FISH scoring and correctly differentiated most ambiguous visual IHC scores. CONCLUSIONS: FISH provides a more accurate and consistent scoring system for determining HER2 amplification than HercepTest. The MDS system provides a reliable, consistent alternative to visual IHC and FISH scoring. IHC is still a valuable technique to aid in identification of isolated or heterogeneous tumour populations for subsequent FISH analysis, and a combined FISH and HercepTest approach to all breast cancer cases may be the most efficient strategy.     

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.     

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 hybridisation testing for HER2 gene amplification in breast cancer produces highly reproducible results concordant with fluorescence in situ hybridisation and immunohistochemistry

AIMS: The objective of this study was to evaluate the accuracy, ease of use and reproducibility of chromogenic in situ hybridisation (CISH) for HER2 testing by studying its inter-laboratory concordance in five Australian pathology laboratories. METHODS: The HER2 status of 49 breast cancers was determined by CISH twice in two different laboratories. Each sample had previously been tested by immunohistochemistry (IHC; 2+ and 3+ cases selected) and fluorescence in situ hybridisation (FISH). Participating laboratories were blinded to these test results. Oestrogen receptor (ER) status was also evaluated for each cancer. RESULTS: High correlation was observed between FISH and CISH results. No cases showing high gene amplification by FISH were scored as non-amplified by CISH (kappa coefficient = 1). High correlation was observed between IHC and CISH, all IHC 3+ samples showing amplification by CISH. Inter-laboratory CISH concordance was also good (kappa coefficient = 0.67). Fifty-six per cent of HER2-amplified samples tested ER positive, while 42% of ER-positive cases showed HER2 gene amplification, confirming that HER2 testing should not be confined to ER-negative breast cancers. CONCLUSIONS: These findings demonstrate that CISH is a robust test to assess HER2 status in breast cancer and therefore is an important addition to the HER2 testing algorithm.     

Evaluating HER2 amplification and overexpression in breast cancer.

The development of Herceptin (Trazumatab) makes testing for HER2 status important for choosing optimal therapy in breast cancer. This study addresses the precision, accuracy, and reproducibility of HER2 assays. HER2 was assessed retrospectively by immunohistochemistry (IHC) with Dako 'Herceptest', by IHC with the monoclonal antibody CB11, and by fluorescence in situ hybridization (FISH, PathVysion), in a series of 216 formalin-fixed breast carcinomas including 191 for which quantitative HER2 data from radioimmunohistochemistry (Q-IHC) were available. All tests were scored independently by two observers. Positivity rates varied between Herceptest (12.6%), FISH (19.4%), and CB11 IHC (28.5%). Kappa values showed that IHC-based tests were more susceptible to inter-observer variation (kappa=0.67 and 0.74 for Herceptest and CB11, respectively) than FISH (kappa=0.973). Overall test accuracy (see the Materials and methods section) for CB11 IHC (83.8%) was lower than Herceptest (87.4%) or FISH (93.2%). FISH predicted p185 HER2 overexpression (determined by Q-IHC) better (concordance index C.Ind. 0.90) than CB11 IHC (C.Ind.=0.85) or Herceptest (C.Ind.=0.81). Of 42 cases with gene amplification by FISH, 67% were positive in the Herceptest (2+ or 3+) vs. 83% with CB11. Of 174 cases negative by FISH, 96% were negative in the Herceptest and 68% with CB11. In conclusion, FISH is the most accurate, reproducible, and precise predictor of HER2 overexpression in routine diagnostic laboratories.     

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.     

Amplification of the HER2 gene in breast cancers testing 2+ weak positive by HercepTest immunohistochemistry: false-positive or false-negative immunohistochemistry?

BACKGROUND: The majority of cases of breast cancer scoring HER2 weak positive (2+) on immunohistochemistry (IHC) using the HercepTest are not associated with amplification of the HER2/neu gene. AIM: To examine the reproducibility of IHC in cases scoring 2+ subsequently shown to have gene amplification by fluorescence in-situ hybridisation (FISH). METHODS: A retrospective analysis of 153 cases referred for FISH confirmation of a weak positive HercepTest (2+) result was performed. Repeat IHC was undertaken in cases with weak positive (2+) referral IHC and amplification of the HER2 gene by FISH. RESULTS: Amplification of the HER2 gene was confirmed in 29/153 cases (19%) scoring 2+ on IHC. Repeat IHC was carried out on 25 IHC 2+ cases: 7 (28%) scored 2+ on repeat IHC, 18 (72%) scored 3+ and were reclassified as strong positive. A heterogeneous expression pattern was present in 3/17 cases scoring 3+. CONCLUSIONS: The majority of HercepTest 2+ results are not accompanied by gene amplification and represent "false positive" IHC in terms of prognostic or therapeutic relevance. A small proportion of HercepTest 2+ scores represent true 2+ IHC positive cases accompanied by gene amplification: a category probably biologically related to 3+ IHC cases. The remainder of cases of HercepTest 2+ accompanied by gene amplification represent a category of referral IHC 2+ weak positive, FISH amplified, repeat IHC 3+ strong positive, best described as "false negative 2+ IHC". This has implications for selection of cases for FISH analysis where weak positive (2+) IHC score is used as a triage for FISH testing, and for testing strategies in referral laboratories undertaking FISH analysis.     

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。     

Role of chromogenic in situ hybridization (CISH) in the evaluation of HER2 status in breast carcinoma: comparison with immunohistochemistry and FISH

We report our experience with Chromogenic in Situ Hybridization (CISH) for the evaluation of HER2 amplification on 55 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of different histology. All the results were corrected for chromosome 17 aneusomy and compared with immunohistochemistry (IHC); a subset of cases was compared to FISH. Thirty-one of 32 cases in which FISH and CISH were performed yielded the same results. CISH and IHC showed a good concordance in the 0/1+ and 3+ category, while a poor agreement with weakly protein overexpression was confirmed. Chromosome 17 analysis was necessary in cases with a low number of HER2 gene copies. CISH is a useful tool to evaluate breast cancer HER2 status that can be easily implemented in a laboratory of surgical pathology.     

Selecting antibodies to detect HER2 overexpression by immunohistochemistry in invasive mammary carcinomas.

There is an increasing clinical demand for HER2 analysis in breast cancer, especially since the release of trastuzumab. The authors assessed the ability of immunohistochemistry to detect HER2 overexpression in invasive mammary carcinomas (IMC) using five antibodies. Paraffin-embedded samples of 86 IMCs (T2N0) were used to compare the immunohistochemical overexpression of HER2 using two polyclonal antibodies (HercepTest [DAKO] and A0485 [DAKO]) and three monoclonal antibodies (CB11 from two different laboratories, Biogenex and Novocastra, and 4D5 [Genentech]). All immunostainings were scored according to the FDA-approved HercepTest recommendations. The HercepTest-positive cases were compared with gene amplification by FISH (Oncor Inform, Ventana). The HercepTest was positive in 31 of the 86 cases (36.1%). The DAKO antibody A0485 was positive in 25 of the 66 (37.8%). Monoclonal antibody 4D5 was positive in only 15 of the 86 cases (17.4%). There was almost total agreement in results between the two CB11 antibodies: 25 of the 86 positive cases (29.1%). All cases positive for CB11 or 4D5 were HercepTest positive. Most of the HercepTest 2+ cases were negative when using either monoclonal antibody. FISH was positive in 19 of the 20 HercepTest 3+ cases and negative in 5 HercepTest 2+ cases. Three CB11-2+ cases showed no amplification by FISH. In three FISH-positive cases the immunohistochemistry showed no overexpression by all antibodies used. These findings suggest that immunohistochemistry may be used reliably as a primary methodology for evaluating HER2; however, the use of polyclonal antibodies may not be adequate to assess HER2 overexpression. CB11, regardless of the manufacturer (Biogenex or Novocastra), showed better concordance with FISH (kappa=0.83) than did the polyclonal antibodies.     

HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas

BACKGROUND: Laboratory methods for HER2 assessment currently include immunohistochemical (IHC) methods (measuring protein overexpression) and fluorescence in situ hybridisation (FISH) (measuring gene amplification). The measure of HER2 protein by IHC is usually assessed by the mouse monoclonal antibody CB11, and polyclonal antibodies (Herceptest) directed against the internal portion of the receptor. Recently, chromogenic in situ hybridisation (CISH), in which HER2 is detected by a peroxidase reaction and the gene amplification can be determined by regular bright-field microscopy, has emerged as an alternative to FISH. AIMS: To evaluate the status of HER2 in tissue microarrays (TMAs) of invasive breast cancer using the novel rabbit monoclonal antibody SP3 directed against the external portion of HER2, and correlate the results with CB11 and CISH. METHODS: IHC was performed with two antibodies (CB11 and SP3) and CISH for HER2 in 10 TMA blocks with 190 formalin-fixed paraffin-embedded cases of invasive breast carcinomas. RESULTS: The correlation between SP3 and CB11 was significant (p<0.001) with an agreement rate of 86.9%. When the staining pattern of the two antibodies was compared, the majority of SP3 immunostainings were assessed more easily, with a strong complete membrane staining pattern without non-specific cytoplasmic staining. There was a good correlation between SP3 and CISH (p<0.001). 23/24 SP3 3+ cases showed gene amplification, 97.3% of the cases without gene amplification were SP3 negative, and 6/7 SP3 2+ were amplified. CONCLUSION: The high level of agreement between SP3, a monoclonal antibody that recognises the extracellular domain of the HER2 receptor, and CB11 and CISH, shows that this novel antibody is a reliable candidate to evaluate the expression of HER2 in breast cancer.     

Correlation of HER2 gene amplification with immunohistochemistry in breast cancer as determined by a novel monoplex polymerase chain reaction assay.

Amplification of the HER2 oncogene in breast cancer identifies patients who are likely to respond to anti-HER2 mAb therapy. Current clinical practice dictates that all breast cancers first undergo HER2 screening by IHC. Strongly positive (3+ on a 0-to-3+ scale) IHC cases are considered as HER2-amplified tumors and are not evaluated further because of the strong correlation between HER2 gene amplification as measured by FISH and 3+ IHC. This strong correlation has recently been questioned, and some data suggest that over 50% of 3+ IHC HER2 immunostains may not be due to HER2 gene amplification. To help resolve this discrepancy, the authors developed a quantitative PCR assay for HER2. Quantitative PCR was used to determine the amount of HER2 DNA relative to a control gene, IF2 (eukaryotic translation initiation factor, 2p11.1-q11.1). The PCR assay is performed on genomic DNA isolated from paraffin-embedded breast cancer tissue. The PCR assay developed is a monoplex assay in which the HER2 and IF2 PCRs are performed in separate cuvettes. Cases of HER2 FISH amplified breast cancer and HER2 FISH nonamplified breast cancer were chosen for study by monoplex HER2 PCR. HER2 overexpression was evaluated by IHC. Twenty-two cases of HER2-positive and 22 cases of HER2-negative breast cancer, as determined by FISH, were assayed for HER2 by PCR and IHC. Sixteen of the 44 cases were interpreted as 3+ IHC. All 16 showed HER2 amplification by PCR and 15 showed HER2 amplification by FISH. One FISH negative case was found to be HER2 amplified by PCR and showed 3+ IHC stain, suggesting the FISH result in this case was underinterpreted. Two FISH positive cases were found to be negative by PCR and negative in IHC as well, suggesting the FISH result in these cases was overinterpreted. The authors conclude that 3+ IHC membrane staining correctly identifies neoplasms showing HER2 gene amplification. Monoplex HER2 PCR may offer significant advantages over both IHC and FISH for HER2 testing in breast cancer.     

HER-2/neu gene amplification compared with HER-2/neu protein overexpression and interobserver reproducibility in invasive breast carcinoma

We compared the detection of HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) with detection of HER-2/neu protein overexpression by immunohistochemistry using 2 antibodies on 100 archival invasive breast carcinomas. Protein overexpression for each marker was scored independently by 4 pathologists using standardized criteria, and consensus was compared with results obtained from gene amplification. The concordance rate between FISH and immunohistochemistry was 76% for e2-4001 and 91% for the HercepTest. Of the 37 cases positive by e2-4001, 21 demonstrated no gene amplification; 7 of 24 cases positive by the HercepTest demonstrated no gene amplification. However, 1 of 61 cases negative by e2-4001 showed gene amplification; none of the cases negative by the HercepTest showed amplification. The predictive values of gene amplification based on 0-1+, 2+, and 3+ immunohistochemical staining were best for cases scored as 3+ (75% for e2-4001 and 89% for the HercepTest). Complete agreement among observers for immunohistochemical scoring of e2-4001 and the HercepTest was achieved in 75 and 85 cases, respectively. The pairwise kappa agreement values were substantial for e2-4001 and substantial to almost perfect for the HercepTest. Immunohistochemical staining may be considered a useful screening test. While negative staining almost always correlated with a lack of gene amplification, positive membranous staining, especially 2+, did not predict gene amplification. The low interobserver reproducibility in separating 2+ from 3+ cases necessitates further confirmation by FISH before treatment decisions are made.     

Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays

AIM: To evaluate the clinical usefulness of three commercially available assays for Her-2/neu oncogene and protein measurements. The Her-2/neu protein is overexpressed, mostly as a result of gene amplification, in 20-30% of human breast cancers, and has been shown to have prognostic and predictive value for treatment with chemotherapy or the new monoclonal antibody, Herceptin. METHODS: An immunohistochemistry (IHC) assay using the Dako polyclonal antibody A0485, which measures the Her-2/neu protein, was compared with two new Food and Drug Administration (FDA) approved fluorescence in situ hybridisation (FISH) assays--INFORM and PathVysion, in a cohort of 52 formalin fixed, paraffin wax embedded breast tissues. These tissues were selected randomly from 84 consecutive infiltrating breast cancer specimens, which were first stratified according to the Her-2/neu protein levels as measured by IHC. RESULTS: The two FISH assays achieved a 98% concordance rate: 14 specimens (27%) showed Her-2/neu gene amplification and 37 specimens (71%) showed no Her-2/neu gene amplification. The PathVysion assay had certain advantages over the INFORM assay. In contrast, the IHC assay detected Her-2/neu overexpression in a high percentage of cases, including 13 high positive specimens (25%) and 13 medium positive specimens (25%). Although 10 of these 13 IHC high positive specimens showed gene amplification by FISH, nine of 13 IHC medium positive specimens showed no gene amplification. Statistical analyses showed that the differences between IHC and FISH assays were primarily in the specimens with medium positive IHC, but negative FISH results. CONCLUSIONS: Because of the increasing importance of the Her-2/neu oncogene and oncoprotein in the clinical management of patients with breast cancer, the accurate and consistent evaluation of Her-2/neu status is crucial. This study suggests that the best approach is to combine both IHC and FISH assays; that is, to use the IHC assay as a triage step, followed by the PathVysion FISH assay to analyse the IHC medium and high positive cases.     

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.     

Her2 amplification: correlation of chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

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.     

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.     

HER-2/neu expression in germ cell tumours

AIMS: To determine the rate of HER-2/neu positivity of germ cell tumours by immunohistochemistry (IHC) and by fluorescence in situ hybridisation (FISH). PATIENTS/METHODS: Ninety six archival, paraffin wax embedded pathology specimens were chosen from four groups of germ cell tumours. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the INFORM assay and confirmed with a centromere 17 probe. RESULTS: Twenty two of 96 specimens overexpressed the HER-2/neu protein when measured by IHC. Only three specimens showed HER-2/neu gene amplification by FISH. There was no correlation between the results obtained by IHC and FISH. CONCLUSIONS: The lack of concordance between IHC and FISH makes it unlikely that overexpression of the HER-2/neu protein in germ cell tumours is of prognostic or therapeutic relevance. Because of the low rate of HER-2/neu gene amplification in germ cell tumours, a clinical trial of trastuzumab treatment in patients with germ cell tumours is not warranted.