Tyrosine kinase activation in breast carcinoma with correlation to HER-2/neu gene amplification and receptor overexpression

The HER-2/neu oncogene encodes a transmembrane receptor with intrinsic tyrosine kinase activity. A pilot study was performed to investigate downstream effects of HER-2/neu (or related growth factor receptor) activation by identifying phosphorylated tyrosine. Fifty-four breast carcinomas were evaluated for HER-2/neu overexpression by the HercepTest (Dako, Carpinteria, CA) and the monoclonal CB11 antibody (Ventana, Tucson, AZ). Phosphotyrosine (an indication of tyrosine kinase activity) was detected by an antiphosphotyrosine mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). The gene amplification status was evaluated in 50 of the 54 cases by fluorescence in situ hybridization (FISH) using the Ventana gene probe. The HER-2/neu oncogene amplification was detected in 28% (14 of 50) of cases. Of the 14 cases showing oncogene amplification, tyrosine kinase activity was detected in 9 (64.2%) cases. There was moderate agreement between HER-2/neu gene amplification and tyrosine kinase activity (κ = 0.43). Immunohistochemical staining of 3+ (with both HercepTest and CB11) showed better agreement with HER-2/neu oncogene amplification and increased tyrosine kinase activity than 2+ immunohistochemical staining. Overall, oncogene amplification and overexpression correlated with increased tyrosine kinase activity, supporting the mechanism of tyrosine kinase activation by HER-2/neu amplification and overexpression. However, 7 cases showing increased tyrosine kinase activity did not show gene amplification or 3+ receptor expression (by either HercepTest or CB11), raising the possibility of other growth factor receptors operating via the tyrosine kinase pathway. There was no apparent correlation between tyrosine kinase activity and hormone receptor status (estrogen or progesterone). Increased tyrosine kinase activity is more commonly associated with higher-grade tumors and thus may correlate with aggressive biologic behavior in breast carcinoma. The results of this pilot study suggest that a larger-scale investigation into downstream activation of tyrosine kinase and correlation to clinical outcome or response to Herceptin therapy may identify subsets of patients whose clinical response or outcome may be predicted by tyrosine kinase activation. H P 32:1344-1350. Copyright © 2001 by W.B. Saunders Company     

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.     

Preferential HER-2/neu overexpression and/or amplification in aggressive histological subtypes of invasive breast cancer

AIMS: To investigate whether alterations of the HER2 gene occur more frequently in histologically unfavourable subtypes of invasive breast cancer. METHODS: The study was composed of nine invasive apocrine, six lipid-rich, 12 glycogen-rich, 11 micropapillary and 33 pleomorphic lobular breast carcinomas. Lymph node involvement was represented in all subgroups. HER2 status was confirmed in all cases by using immunohistochemistry (CB11, Herceptest) and fluorescent in-situ hybridization (FISH) analysis (Vysis). RESULTS: Micropapillary and apocrine carcinomas showed the highest rate of protein overexpression (72% and 66%) and gene amplification (45% and 44%). Protein overexpression was common in poorly differentiated pleomorphic lobular carcinomas (56%); however, this subgroup failed to show an increased number of gene copies by FISH (31%). The incidence of HER2 overexpression (33% and 50%, respectively) and gene amplification (25% and 33%, respectively) among glycogen-rich and lipid-rich carcinomas was not higher than that observed in breast cancer generally. CONCLUSION: Our data suggest that preferential involvement of the HER2 gene in micropapillary and apocrine breast carcinomas may contribute to their aggressive behaviour.     

Prognostic significance of p34cdc2 cyclin-dependent kinase and MIB1 overexpression, and HER-2/neu gene amplification detected by fluorescence in situ hybridization in breast cancer.

The HER-2/neu oncogene, localized to chromosome 17q, shares substantial homology with the epidermal growth factor receptor. HER-2/neu gene amplification and protein overexpression have been associated with poor prognosis in breast cancer. Formalin-fixed paraffin-embedded primary invasive breast cancer tissues from 135 women were tested for HER-2/neu gene amplification by automated fluorescence in situ hybridization (FISH) using a sequence probe. The tumors also were evaluated by immunohistochemistry for proliferation markers Ki 67 (MIB1) and p34cdc2 cyclin-dependent kinase. Patients were followed up for a mean of 61 months. There were 70 node-negative and 65 node-positive cases. Ki 67 and p34cdc2 proliferation marker overexpression, HER-2/neu oncogene amplification, large tumor size, high tumor grade, advanced tumor stage, positive lymph node status, and distant metastasis at the time of diagnosis predicted disease-related death in combined node-negative and node-positive breast cancer. HER-2/neu gene amplification, tumor stage, lymph node metastasis, tumor grade, and distant metastasis at the time of diagnosis independently predicted disease outcome. HER-2/neu amplification detected by FISH also predicted disease-related death independent of lymph node status, tumor grade, and distant metastasis in breast cancer patients who received adjuvant therapy.     

Overexpression/amplification of HER-2/neu is uncommon in hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent fatal cancers in the world. Despite advances in early diagnosis and improvements in surgical techniques, the survival of patients with HCC even after resection is poor because of the high incidence of recurrences. Therefore, the identification of prognostic factors may be helpful in the development of new treatment protocols. AIMS: To investigate HER-2/neu status in HCC by immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), and to explore the possibility of using trastuzumab in the treatment of HCC. METHODS: Eight hundred and sixty eight surgical samples from patients with primary HCC were examined for their HER-2/neu status. IHC for HER-2/neu was performed with the HercepTest kit; FISH analysis was performed with the PathVysion HER-2 DNA probe kit. The correlations between HER-2/neu overexpression and clinicopathological characteristics were analysed statistically. RESULTS: HER-2/neu overexpression was detected in 21 (2.42%) of the 868 primary HCCs. Only one specimen showed HER-2/neu gene amplification by FISH. No significant associations were found between HER-2/neu overexpression and the clinicopathological parameters. CONCLUSIONS: There is a low frequency of HER-2/neu overexpression/amplification in HCC. There appears to be no role for HER-2/neu as a prognostic marker and no benefit of anti-HER-2/neu trastuzumab treatment in patients with HCC.     

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.     

Immunohistochemical COX-2 overexpression correlates with HER-2/neu overexpression in invasive breast carcinomas: a pilot study

Cyclooxygenase-2 (COX-2) is a prostaglandin synthase that catalyzes the synthesis of prostaglandin G2 and H2. It has been shown that COX-2 plays an important role in tumorigenesis of different tumor types and it is thought to take part in breast carcinogenesis. In the present study, we aimed to investigate the relationship of immunohistochemical COX-2 expression with clinicopathological parameters, including HER-2/neu overexpression in invasive breast carcinoma (IBC).Our study population comprised 10 normal breasts, 25 ductal carcinomas in situ (DCIS), and 51 invasive breast carcinomas. Immunohistochemical overexpressions of COX-2 and HER-2/neu were investigated in sections of formalin-fixed, paraffin-embedded blocks by 3 observers.In normal breast, DCIS and IBC, the COX-2 overexpression rate was 0%, 84%, and 58.8%, respectively. In IBC, COX-2 overexpression had a significant relationship with HER-2/neu overexpression (p = 0.026) and a high histological grade (p = 0.026). COX-2 expression in both DCIS (n = 25) and IBC (n = 51) was significantly higher than in normal breast tissue (p < 0.0001). In addition, the COX-2 expression rate was significantly higher in DCIS than in IBC (p = 0.042).Our results indicated that COX-2 overexpression correlates with aggressive phenotypic features, such as HER-2/neu overexpression and high histological grade in IBC. Increased expression of COX-2 in both DCIS and IBC in comparison to normal breast could indicate a role in breast carcinogenesis. COX-2 overexpression may provide a clinically useful biomarker for estimating tumor aggressiveness.     

Review: significance of, and optimal screening for, HER-2 gene amplification and protein overexpression in breast carcinoma.

Breast carcinoma is a common disease, with an estimated 183,000 new cases expected in the USA during 2000. Whereas early stage patients have high likelihood of cure, only 20-40% of patients with metastatic breast carcinoma respond to presently available chemotherapy. A need exists to identify the underlying biological subsets of morphologically similar carcinomas in order to develop customized therapies for patients who require chemotherapy. The HER-2 receptor tyrosine kinase is overexpressed in 15-30% of breast carcinomas, and is associated with a worse prognosis stage-for-stage. Humanized monoclonal antibody therapy (Herceptin; Genentech Co.) appears to benefit this subset of patients by improving their response rate and survival following anthracycline- or taxane-based chemotherapeutic regimens. Both HER-2 gene amplification and protein overexpression correlate with clinical outcomes, and screening for HER-2 gene amplification appears to be the more informative test. This article reviews data on the HER-2 gene and protein, discusses their association with clinical outcomes, and proposes a strategy for screening for HER-2 excess in formalin-fixed specimens of breast carcinoma.     

HER-2/neu protein-receptor-positive breast carcinoma: an immunologic perspective

Immunotherapy using a monoclonal antibody against the human epidermal growth factor receptor 2 protein, HER-2/neu, has proven to be clinically efficacious in about one-half of breast cancer patients who exhibit strong (3+) plasmalemmal immunoreactivity for this protein. The tumoricidal effect of this antibody relies in part upon antibody-dependent cell-mediated cytotoxicity. This report provides observations on certain factors or circumstances which could have an impact on this aspect of the therapeutic approach. These include: (1) concurrent medications; (2) the composition (immunophenotype) of peritumoral lymphocytes and the generally limited numbers of intratumoral T-lymphocytes/natural killer (NK) cells, monocytes, and neutrophilic granulocytes; (3) the presence of circulating HER-2/neu antigens which might bind the exogenous antibody and lead to immune complex formation; (4) the variable co-expression in the tumor of cytokines known to downregulate NK cell function (ie, transforming growth factor-beta1 [TGF-beta1] and platelet-derived growth factor [PDGF]-AB); and (5) the tumoral and/or stromal immunoreactivity for angiotensin-converting enzyme, which forms a part of one of the pathways for the activation of latent TGF-beta1 and for the biosynthesis of PDGF-AB. These observations provide an immunologic perspective for the use of monoclonal antibody therapy in HER-2/neu protein-receptor-positive breast carcinoma and suggest a role for the clinical laboratory in identifying potential avenues for additional manipulations of the immune system in individual cases in order to enhance the therapeutic response.     

The role of HER2/neu overexpression/amplification in the progression of ductal carcinoma in situ to invasive carcinoma of the breast.

HER2/neu overexpression/amplification is seen more frequently in ductal carcinoma in situ, particularly high-grade ductal carcinoma in situ (50-60%), than in invasive ductal carcinoma of the breast (25-30%). To date, however, the role of HER2/neu in the progression of in situ to invasive disease has not been clarified. Two hundred fifty-one breast tumors were retrieved from the pathology files at Mount Sinai Hospital. These included 91 cases of ductal carcinoma in situ, 136 cases of invasive ductal carcinomas with associated ductal carcinoma in situ, and 24 cases of pure invasive carcinomas. All cases were reviewed and stained with two monoclonal antibodies to HER2/neu (CB11 and TAB250). Immunohistochemical staining was recorded using a semiquantitative scoring system (1). Representative cases were also investigated using fluorescence in situ hybridization. HER2/neu protein overexpression (defined as immunohistochemical staining with score of >or=5) was seen in 34% of cases of pure ductal carcinoma in situ, 17% of invasive carcinomas with associated ductal carcinoma in situ, and 12.5% of pure invasive carcinomas (P =.01). Sixty percent of cases of high-grade ductal carcinoma in situ showed HER2/neu protein overexpression, versus 29% of high-grade invasive carcinomas with associated ductal carcinoma in situ and 22% of high-grade pure invasive ductal carcinomas (P =.02). The concordance between the immunohistochemical staining in the in situ and invasive components of individual tumors was 90%. Thirty-three cases were also evaluated by fluorescence in situ hybridization and showed concordance between the immunohistochemical results and the degree of gene amplification in 91% of cases, whereas 3 of 33 cases showed HER2/neu gene amplification (HER2/CEP17 = 2.3-3.7) by fluorescence in situ hybridization in the absence of positive immunohistochemical staining. One case showed HER2/neu gene amplification in the associated ductal carcinoma in situ (HER2/CEP17 ratio = 6.5), with no evidence of gene amplification in the invasive tumor (HER2/CEP17 ratio = 1.14). Multiple genetic events are required for the development of an invasive phenotype. The findings from this study suggest that the genetic event of HER2/neu gene amplification/protein overexpression may not play a key role in the progression of ductal carcinoma in situ to invasive carcinoma and that other molecular alterations may be more important in the initiation of invasion in ductal carcinoma of the breast.     

Immunohistochemical determination of HER-2/neu expression in invasive breast carcinoma

Numerous methods exist for HER-2/neu assessment; however, technical and interpretive standardization is virtually absent. We evaluated 2 commercially available antibodies on routinely fixed paraffin-embedded tissue sections to establish our own guidelines. Thirty-three cases of infiltrating breast carcinoma were evaluated simultaneously with monoclonal and polyclonal antibodies. Only membranous staining, no matter how focal, was considered positive. An additional 32 tumors were studied subsequently using only the polyclonal antibody. Of all carcinomas, 13.0% showed immunohistochemical evidence of HER-2/neu overexpression. High-grade tumors were more often positive. There was no HER-2/neu gene expression in the benign epithelium that generally was present in the tissue section or in any of the well-differentiated tumors tested. The polyclonal antibody proved more sensitive than the monoclonal antibody. While true cytoplasmic staining was present occasionally, it did not create substantial difficulty in interpretation. The polyclonal antibody cost substantially less than the monoclonal antibody. Fluorescence in situ hybridization assay for HER-2/neu gene amplification performed on 32 of 65 cases showed concordant results in 31 cases. The immunohistochemical assay for HER-2/neu gene overexpression, using our methods, is accurate, economic, and easily integrated into the laboratory.     

Correlation of HER 2/neu gene amplification with immunohistochemistry and other prognostic factors in breast carcinoma

The purpose of this study was to determine relationship between HER-2/neu status and estrogen receptor, progesterone receptor, grade and age by comparing fluorescence in situ hybridization and immunohistochemistry. One hundred invasive breast carcinomas were reviewed and fluorescence in situ hybridization analysis was performed in all cases. Immunohistochemical scores showed a perfect concordance with fluorescence in situ hybridization amplification ratios (p < 0.0001). The results indicated a significant correlation between HER-2/neu and grade, but an inverse relationship between HER-2/neu and hormone receptors. In women aged ≤ 45 years, an inverse relationship between HER-2/neu and progesterone receptor was found and no association was noted between HER-2/neu and other factors. In women aged > 45 years, the results indicated a significant correlation between HER-2/neu and grade, and there was an inverse relationship between HER-2/neu and hormone receptors.     

HER-2/neu oncogene amplification in stage I and stage III ovarian papillary serous carcinoma.

Oncogene amplification has been implicated in the genesis and progression of many cancers. Overexpression of the HER-2/neu proto-oncogene occurs in 20-30% of ovarian epithelial cancers, in which it may be of prognostic significance. Oncogene overexpression is traditionally studied using immunohistochemistry. In this study we used fluorescent in situ hybridization (FISH) to determine HER-2/neu amplification in ovarian papillary serous carcinoma and compared the frequency of amplification in two stages of the disease. Archival tissues from 23 cases of papillary serous ovarian carcinoma (9 cases of stage I and 14 cases of stage III) were analyzed by FISH using a HER-2/neu probe and a chromosome 17 centromere control probe. Determination of the level of amplification was performed according to the standard protocols of the Cytogenetics Laboratory at Rhode Island Hospital. Of the 23 cases successfully analyzed, the frequency of amplification among stage I tumors was 22% (2/9) and the frequency of amplification among stage III tumors was 71% (10/14). These results are significant (P = 0.036). The frequency of stage I tumors among amplified cases was 17% (27/12) and the frequency of stage III tumors among amplified cases was 83% (10/12). This study not only confirms the presence of a subset of ovarian papillary serous carcinoma with HER-2/neu gene amplification, but it also indicates that HER-2/neu oncogene amplification is more likely to be associated with a more advanced stage. Thus, the present data are consistent with the hypothesis that HER-2/neu amplification, similar to HER-2/neu protein over expression, is a prognostic marker of poor outcome.     

Detection and quantitation of HER-2 gene amplification and protein expression in breast carcinoma

We compared fluorescence in situ hybridization (FISH), immunohistochemical analysis, immunocytochemical analysis, and relative quantification assays using polymerase chain reaction (PCR) as methods for estimating HER-2 gene amplification and protein overexpression infine-needle aspirate (FNA) specimens and paraffin-embedded tissue samples from 49 cases of breast cancer: FISH can be performed successfully on FNA smears. Immunohistochemical and immunocytochemical staining intensity of 3+ corresponds to a FISH ratio of more than 2.5. Immunohistochemical and immunocytochemical staining of 2+ and 1+ are not necessarily associated with gene amplification. Increased DNA PCR ratios might be seen without amplification, reflecting polysomy. HER-2 messenger RNA relative quantitation scores correlate well with HER-2 gene amplification. Owing to the ease with which it can be performed and interpreted, we conclude that FISH is the test of choice for HER-2 estimation and, when possible, should be performed on whole nuclei, which are readily available in FNA smears or imprint cytology. FISH may be used primarily or to confirm immunohistochemical, immunocytochemical, and PCR results.     

HER-2/neu oncogene expression and proliferation in breast cancers.

Amplification of the HER-2/neu proto-oncogene in breast cancer has been reported to correlate with poor patient prognosis. The proliferation, or growth fraction, of cells has also been shown to be of prognostic importance in breast cancer. A study was conducted to evaluate the correlation between HER-2/neu gene expression and proliferation in breast cancer. Quantitative immunohistochemical methods for the detection of the HER-2/neu protein expression and for assessing the proliferation fraction on frozen sections of tumor cells were used. The detection of epidermal growth factor receptor (EGFR) along with quantitative DNA ploidy analysis, also was performed on the same breast cancers. The results indicated two subgroups of invasive ductal carcinoma; 1) HER-2/neu overexpressing cases that were negative for EGFR expression and had low proliferation fraction, and a tetraploid DNA pattern (22 cases), and 2) other combinations of HER-2/neu expression and EGFR expression, with a high proliferation fraction and an aneuploid DNA pattern (38 cases). Eight cases of carcinoma in situ were positive for HER-2/neu overexpression and negative for EGFR expression, and had a high proliferation fraction and a tetraploid DNA pattern. Twenty-six cases of low-grade carcinoma exhibited low proliferation and a diploid DNA pattern.     

HER-2 amplification in tubular carcinoma of the breast

The prognostic and therapeutic implications of HER-2 gene amplification and estrogen and progesterone receptor status in breast cancer are well described. To address the relative paucity of information concerning HER-2 amplification for tubular carcinomas, we assessed the frequency of gene amplification in 55 tubular carcinomas of the breast from 54 patients, 5 of which had axillary node metastases. The HER-2 gene copy number was assessed by fluorescence in situ hybridization for the majority of tumors analyzed, whereas estrogen and progesterone receptor status was achieved by immunohistochemical analysis. HER-2 gene amplification was not observed in any of the tumors examined, and most were estrogen receptor-positive. This HER-2 gene amplification frequency was significantly lower than the frequency of gene amplification previously reported for all invasive ductal carcinoma of no special type (P < .01). HER-2 gene amplification likely occurs infrequently, or not at all, in tubular carcinomas of the breast, whereas most express estrogen receptors.