Pathobiologic findings in DCIS of the breast: morphologic features, angiogenesis, HER-2/neu and hormone receptors.

With the increasing incidence of ductal carcinoma in situ (DCIS) of the breast and its relationship to invasive breast carcinoma, it is important to understand the biology of this entity. We report on a hospital-based survey of 219 case subjects with DCIS of the breast without associated invasive carcinoma diagnosed between 1982 and 1994. The cases of DCIS were analyzed for architectural type, size, nuclear grade, necrosis, calcification, periductal fibrosis, neovascularization, estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu expression. Periductal neovascularization was associated with tumor size, microcalcifications, periductal fibrosis, and HER-2/neu overexpression. Expression of ER and PR was observed in 60 and 62% of the cases, respectively, and HER-2/neu overexpression in 28% of the cases. ER and PR expression were both inversely associated with comedo histology and nuclear grade. HER-2/neu overexpression was positively associated with comedo histology, high nuclear grade, and periductal neovascularization and was inversely correlated with both ER and PR expression. High nuclear grade was positively associated with comedocarcinoma, necrosis, microcalcification, and periductal fibrosis. The role of these molecular/pathologic markers in the biology of DCIS and their potential clinical implications are discussed.     

Immunohistochemical characterization of molecular subtypes of invasive breast cancer: a study from North India

In recent years breast cancer has been classified on the basis of its molecular characteristics by gene expression profiling. A similar classification using immunohistochemistry has been identified so that it has a wider application. This study was designed to define the precise prevalence of molecular subtypes of invasive breast carcinoma using immunohistochemistry in patients from north India and to correlate it with known clinical and histological prognostic factors. Based on ER/PR/Her2/neu expression, 100 cases of invasive breast cancer were categorized into: ER+ and ? or PR+ and Her2/neu? (47%), ER+ and?or PR+ and Her2/neu+ (15%), ER? and ? or PR? and Her2/neu+ (Her2/neu overexpressing, 21%), ER?, PR? and Her2/neu? (Triple negative, 17%). All cases demonstrated positivity for the luminal Cytokeratins 8/18. In addition, 10% of these tumours showed expression of the basal markers (CK4/14, CK5/6). Among the 17 triple negative cases, eight cases were positive for one of the basal markers and two cases with basal marker expression were Her2/neu overexpressing. The basal markers showed significant correlations only with histological grade and ER negative status. On the basis of hormone receptor, Her‐2/neu and cytokeratin expressions, distinct subclasses of breast cancer have been identified which show significant differences in relation to histological grade and ER status. Expression of basal markers is needed to define basal‐like breast cancer.     

Predominance of the basal type and HER-2/neu type in brain metastasis from breast cancer.

Although breast cancer is the second most common cause of central nervous system (CNS) metastases with a notable increase of incidence, only few studies on brain-metastasizing breast cancer are available. In this immunohistochemical and fluorescence in situ hybridization (FISH) study, metastases to the CNS (n=85) and primary breast cancers, with known involvement of the CNS (n=44) including paired primary and metastasized tumours (n=23), were investigated retrospectively for the expression of oestrogen- (ER) and progesterone- (PR) hormone receptors, Her-2/neu, epidermal growth factor receptor (EGFR), Ki-67, and cytokeratins (CKs) 5/14. The majority of brain metastases were steroid hormone receptor negative (ER 66%, PR 82%) corresponding to the findings in primary tumours with known involvement of the CNS (68% ER-negative, 75% PR-negative). The frequency of HER-2/neu-overexpressing or -amplified cancers was increased in both groups (34 and 32%, respectively). EGFR expression was more frequent in metastases (41%) than in primary tumours (16%). The proportions of cases with a basal phenotype were 26 and 30%, respectively. In paired primary tumours and metastases to the CNS, constancy of Her-2/neu status was observed in 87% of cases with only one sample turning Her-2/neu-negative and two samples acquiring overexpression/amplification in brain metastases. In contrast, steroid hormone receptors exhibited more frequently a loss of expression (17%) than a gain (9%) with 74% revealing a constant phenotype. We conclude that brain-metastasizing breast cancer belongs predominantly to the basal type or Her-2/neu type. Primary and metastatic tumours differ from each other only in a minority of cases, leading rather to a loss of steroid hormone receptors and to a gain of EGFR and Her-2/neu.     

Association between hormone receptors and HER-2/neu is age-related

Purpose: To investigate the association between hormone receptors and HER-2/neu in different age groups of women with breast cancers. Methods: A total of 1036 women with breast cancers were recruited. All the patients were divided into nine groups. The expression of hormone receptors and HER-2/neu was studied by IHC, while FISH test was used to determine HER-2/neu status in cases scored IHC 2+. The association between hormone receptors and HER-2/neu in different age groups was evaluated using the χ² test. Multivariate analysis was used to find out the independent factors predicting HER-2/neu amplification. Significant findings: The expression of ER and PR was inversely correlated with HER-2/neu status in women aged >40 years. By multivariate analysis, as far as the overall groups were concerned, PR, lymph node status and tumor grade were independently associated with HER-2/neu; Considering the younger age group (≤40), the only predictor for HER-2/neu was the tumor grade; Considering the older age group (>40), tumor grade, PR status, tumor size and lymph node status were associated with HER-2/neu overexpression. Conclusions: Our data suggest that the association between ER, PR and HER-2/neu is age-related. The negative relationship is only applied for women aged >40 years.     

Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas

We studied the inverse relationship between HER-2 and estrogen (ER) and progesterone (PR) receptors using HER-2 testing and correlated HER-2 status with histologic features in 3,655 unselected invasive breast carcinomas. Immunohistochemical analysis for ER, PR, and HER-2 and fluorescence in situ hybridization for HER-2 were performed. ER and PR expression were decreased significantly in HER-2+ tumors compared with HER-2- tumors (ER, 49.1% vs 78.17%; PR, 24.3% vs 53.13%). Even among HER-2+ tumors, the rate of ER or PR expression in high-grade tumors was significantly decreased compared with intermediate-grade tumors. HER-2 was positive in 10.87% of grade 2 and 27.84% of grade 3 ductal carcinomas and negative in all grade 1 ductal carcinomas. HER-2 overexpression or amplification essentially was limited to grades 2 and 3 ductal carcinomas and correlated inversely with ER or PR expression. Although ER or PR expression is decreased in HER-2+ tumors, a substantial proportion of them still express ER or PR.     

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.     

Change of HER-2/neu status in a subset of distant metastases from breast carcinomas

It is assumed that HER-2/neu status remains consistent in breast carcinoma during metastatic spread, but in most previous studies primary tumours were compared with concurrent regional lymph node metastases. The present study investigated 31 breast carcinomas and their corresponding lymph node and distant metastases for HER-2/neu status by immunohistochemistry (HercepTest) and fluorescence in situ hybridization (FISH) (PathVysion). In 14 cases, serum HER-2/neu (SHER-2) was measured sequentially using the Bayer Immuno 1 HER-2/neu assay. Comparing HER-2/neu immunohistochemistry of primary tumours and distant metastases case by case, increased HER-2/neu expression was found in the distant metastases in 15 cases (48.4%), three (9.7%) of which showed an increase from score 0 to score 3+. In contrast, lymph node metastases showed the same HER-2/neu expression as the primary tumours, confirmed by FISH. Two cases, which showed HER-2/neu score 3+ and HER-2/neu amplification in the primary tumours, revealed increased SHER-2 levels above 50 ng/ml at the first measurement. Five of the 14 cases (36%) showed an increase of SHER-2 above 50 ng/ml towards the end of the patients' life. On the basis of these results, there is evidence that in a subset of breast carcinomas, HER-2/neu amplification and overexpression occur de novo in distant metastases at a late disease stage.     

Evaluation of oestrogen and progesterone receptor status in HER-2 positive breast carcinomas and correlation with outcome

AIM: HER-2/neu amplification occurs in 15-25% of breast carcinomas. This oncogene, also referred to as c-erbB-2, encodes a transmembrane tyrosine kinase receptor belonging to the epidermal growth factor receptor family. HER-2 over-expression is reported to be associated with a poor prognosis in breast carcinoma patients and in some studies is associated with a poorer response to anti-oestrogen therapy. These patients are less likely to benefit from CMF (cyclophosphamide, methotrexate, fluorouracil)-based chemotherapy compared with anthracycline-based chemotherapy. The aim of this study was to evaluate breast carcinomas to determine hormone receptor status and if there is a difference in breast cancer specific survival for HER-2 positive patients. METHODS: A total of 591 breast carcinomas were evaluated using immunohistochemistry (IHC) for oestrogen receptor (ERp), progesterone receptor (PRp) and three different HER-2 antibodies (CB11, A0485 and TAB250). Percentage of tumour cells and intensity of staining for ERp were evaluated using a semiquantitative method. RESULTS: Of the 591 tumours, 91 (15.4%) showed 3+ membrane staining for HER-2 with one or more antibodies. Of these 91 tumours, 41 (45.1%) were ERp+/PRp+, seven (7.7%) were ERp+/PR-, six (6.6%) were ERp-/PRp+ and 37 (40.7%) were ERp-/PR-. Of HER-2 positive tumours, 5.5% showed >80% 3+ staining for ERp compared with 31.8% of 0-2+ HER-2 tumours; 24.2% of HER-2-positive tumours showed 60% or more cells with 2+ or 3+ staining for ERp. Treatment data were available for 209 patients and no difference was observed in breast cancer specific survival (BCSS) with HER-2 status and tamoxifen. CONCLUSION: Oestrogen receptor status cannot be used to select tumours for evaluation of HER-2 status, and oestrogen and progesterone receptor positivity does not preclude a positive HER-2 status. There is a higher proportion of ERp negative tumours associated with HER-2 positivity, however, more than 20% of HER-2 positive tumours show moderate or strong staining for ERp. HER-2 positive patients in this study did not show an adverse BCSS with tamoxifen treatment unlike some previous studies.     

Prognostic implications of HER-2 status in steroid receptor-positive, lymph node-negative breast carcinoma

Patients with lymph node-negative breast carcinoma (LNNBC) and positive hormone receptor (HR) status during estrogen-based endocrine therapy have different prognoses. The contribution of HER-2 (human epidermal growth factor receptor-2) has not been extensively evaluated. We stained 230 LNNBCs for estrogen and progesterone receptors (ER and PR) and HER-2. HER-2 gene status was studied in 150 randomly selected tumors by chromogenic in situ hybridization and cases with discordant or nondefinitive results by fluorescence in situ hybridization. Patients with ER+ and/or PR+ tumors were treated with tamoxifen. We found positive expression of ER, PR, and HER-2 in 73.7%, 67%, and 27.8%, respectively, and HER-2 amplification in 18.0%. Poorer outcome was seen for patients with ER+ and/or PR+/HER-2 overexpressing tumors and as a trend for patients with HER-2 amplification. ER/PR and HER-2 expression showed an independent prognostic value. In LNNBCs with positive HR status, HER-2 overexpression and/or amplification confer an aggressive tumor phenotype, and this might be related to tamoxifen unresponsiveness.     

Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays

The amplification and/or overexpression of the HER-2/neu oncogene and its encoded receptor protein are increasingly used for prognostication and prediction of therapeutic response to Herceptin in breast cancer. However, large-scale examination of archival tumor blocks by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) is prohibitively laborious and technically challenging. The tissue microarray (TMA) technique enables hundreds of tumors to be studied simultaneously in a single experiment. To evaluate the HER-2/neu status of a selection of the breast tumors in our tumor bank, we constructed a TMA from 97 breast tumors, with a single 0.6-mm core per specimen. HER-2/neu gene amplification by FISH was found in 20 of the 87 interpretable cases (23%): in 14 of 14 IHC 3+ cases (100%), 5 of 8 IHC 2+ cases (62.5%) and 1 of 65 IHC 0/1+ cases (1.5%). Three of the 67 cases with no evidence of HER-2/neu gene amplification by FISH were moderately positive (2+) by IHC. A close relationship was observed between these 2 assays as applied to the TMA (95.4% concordance: 95% CI, -2.2% to 6.8%; P <0.0001), and both HER-2/neu gene amplification and protein overexpression were strongly associated with tumor grade, estrogen receptor status, and progesterone receptor status. Gene amplification was found in most of the tumors with high-level overexpression (IHC 3+) and not in the unequivocal IHC-negative cases. Complementary analysis by IHC and FISH are, however, recommended for tumors graded as 2+ by IHC, the group with the most result discrepancy. Hum Pathol 34:362-368.     

HER-2 status in breast cancer: correlation of gene amplification by FISH with immunohistochemistry expression using advanced cellular imaging system.

It has become important to accurately evaluate the status of HER-2/neu in invasive breast cancer, especially when one is considering the use of anti-HER-2 monoclonal antibody therapy (Trastuzumab). Almost one third of invasive breast carcinomas overexpress the HER-2/neu protein, so the use of the anti-HER-2/neu monoclonal antibody Herceptin (trastuzumab) to block the protein has become important in the management of and in prolonging the survival for patients with metastatic breast cancer. The effectiveness of this therapy is dependent on accurately evaluating the HER-2 status in these tumors, which can be done either by studying the expression of HER-2 protein by immunohistochemistry (IHC) or by evaluating HER-2 gene amplification by fluorescent in situ hybridization (FISH). Since interobserver variability may occur in manually grading HER-2 protein expression by IHC, the aim of this study was to compare the HER-2/neu expression by IHC using a computer-based image analysis system with that of the gene amplification by FISH. Formalin-fixed paraffin-embedded archival tissue from 108 primary infiltrating ductal carcinomas were immunostained using the HercepTest (DAKO). To reduce interobserver variability, membrane staining was evaluated using the Automated Cellular Imaging System (ACIS) by ChromaVision, and the cases were divided into four groups: group 1 (n=23) with HER-2/neu expression ACIS score less than or equal to 1.5; group 2 (n=17) with a score ranging from 1.6 to 1.9; group 3 (n=46) with a score 2.0 to 2.5; and group 4 (n=22) with a score greater than or equal to 2.6. FISH was performed on all of the 108 cases using the PathVysion HER-2/neu DNA probe kit from Vysis Inc. All cases were also manually reviewed and graded as negative, 1+, 2+, and 3+ according to the DAKO HercepTest grading scheme. Cases with negative and 1+immunostaining were considered as HER-2 not overexpressed, and cases with 2+ and 3+ staining were classified as showing HER-2 overexpression. In group 1, 1 of 23 (4%), in group 2, 2 of 17 (12%), in group 3, 5 of 46 (11%), and in group 4, 19 of 22 (86%) cases showed gene amplification by FISH. Furthermore, in group 4 all 15 (100%) cases with an ACIS score of 3 or greater were FISH positive. Correlation with manual IHC score and FISH showed that 2 of the 23 (9%) IHC negative (0 and 1+) cases and 25 of the 85 (29%) IHC positive (2+ and 3+) cases showed gene amplification by FISH. This study shows that the amplification of the HER-2/neu gene correlates better with overexpression of the HER-2/neu protein by IHC when the score is either less than 1.5 or greater than 2.6 by ACIS. Therefore, FISH may be useful to better evaluate HER-2/neu status in breast cancer in cases where the ACIS score by immunohistochemistry is 1.6 to 2.5, and since the correlation is so good, FISH may not be needed for HER-2 evaluation in cases with ACIS scores less than 1.5 and greater than 2.6.     

Human milk fat globule antigen III D 5, steroid receptors and histopathologic parameters in breast cancer

Immunohistochemical reactivity of mammary carcinomas with the monoclonal human milk fat globule (HMFG) antibody III D 5, and the estrogen receptor (ER) and progesterone receptor (PR) status were compared with the histopathology of primary breast cancer. The reactivity with III D 5 has earlier been shown to be associated with the estrogen receptor status of tumours and with a favourable prognosis. The reactivity of tumours with III D 5, as well as the presence of ER and PR correlated significantly with the histological features of differentiation; histological grade, nuclear grade, tumour necrosis and lymphoid infiltration. Reactivity with III D 5 correlated with all these parameters, while the presence of ER did not correlate with the nuclear grade and that of PR correlated only with the nuclear grade and the lymphoid infiltration of tumours. Reactivity of III D 5 may thus have prognostic and therapeutic implications in the management of breast cancer.     

Androgen receptor expression in estrogen receptor-negative breast cancer. Immunohistochemical, clinical, and prognostic associations

We sought to determine the prevalence of androgen receptor (AR) expression in a predominantly estrogen receptor (ER)-negative subset of breast cancers and delineate the immunohistochemical and clinical associations, including whether AR expression has prognostic significance in ER-negative tumors. We identified 69 ER-negative and 19 ER-positive breast cancer cases with concurrent immunohistochemical prognostic panels (ER, PR, HER-2/neu, Ki-67, and p53); immunohistochemical analysis was performed for AR using standard techniques. Clinical data were extracted from medical records. chi 2 tests were used to assess associations between variables. AR was found in 49% (34/69) of ER-negative and 89% (17/19) of ER-positive cases. In ER-negative tumors, AR was associated with increased age (P = .02), postmenopausal status (P < .001), tumor grade (P = .03), tumor size (P = .03), and HER-2/neu overexpression (P = .003). In ER-positive tumors, AR was associated with progesterone receptor expression (P < .03). In univariate analysis of ER-negative tumors, patients with AR-positive tumors had significantly better disease-free survival (P = .049). AR is expressed in a significant number of ER-negative cases and shows significant associations with important clinical and pathologic prognostic factors.     

Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in invasive breast cancer in women

Introduction

Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression are crucial in the biology of breast carcinoma. HER-2/neu gene is amplified and overexpressed in 15-30% of invasive breast cancers. HER-2-positive breast cancers have worse prognosis than HER-2 negative tumors and possess distinctive clinical features. The aim of this study was to assess the expression of HER2 in cancer tissue of patients with invasive breast cancer in correlation with tumor type, histological grade, tumor size, lymph node status, and expression of estrogen receptor and progesterone receptor.

Material and methods

Formalin-fixed, paraffin-embedded tissues from 40 patients with invasive HER-2-positive breast cancer and from 191 patients with HER-2-negative breast cancer were used in this study. HER2 expression was determined using the test HerceptTest™ DAKO.

Results

Among 231 cases of breast cancer, 18 invasive lobular carcinomas and 213 invasive ductal carcinomas were diagnosed. Sixty percent of HER-2-positive breast cancers were ER-positive compared with 77% in the HER-2-negative group (p = 0.002). The expression of PR was observed in 43% of HER-2-positive breast cancers and in 72% of HER2-negative tumors (p = 0.003). Excessive expression of HER2 protein was detected in 60% of patients positive for estrogen receptors, which may worsen prognosis in these patients.

Conclusions

Determination of HER2 overexpression in breast cancer patients, allows for a determination of a group of patients with a worse prognosis.     

Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression of in situ to invasive breast cancer.

Using permanent-section immunohistochemistry, we investigated the role of HER-2/neu in the development and progression of human breast cancer by measuring its overexpression in a series of hyperplastic (n = 30), dysplastic (n = 15), and malignant neoplastic (n = 708) lesions of ductal epithelium and by evaluating the relationships between overexpression and clinicopathologic features known to have prognostic significance in these lesions. The neoplasms included pure ductal carcinoma in situ (DCIS; n = 59) and infiltrating ductal carcinoma (IDC; n = 649). The latter were all node negative and stratified into IDC combined (n = 237) or not combined (n = 412) with a "significant amount" of DCIS (defined as DCIS greater than or equal to 10% of total tumor cellularity). Overexpression of HER-2/neu was not observed in any of the hyperplastic or dysplastic lesions. In contrast, it was present in 56% of pure DCIS and in 77% of the comedo subtype of this group. Only 15% of IDC overexpressed HER-2/neu. However, the rate of overexpression was significantly higher in the subset of IDC combined with DCIS compared with the subset of IDC not combined with DCIS (22% v 11%, respectively; P less than .0001). These results are consistent with the hypothesis that HER-2/neu plays a more important role in initiation than in progression of ductal carcinomas. They also suggest that overexpression decreases within individual tumors as they evolve from in situ to increasingly invasive lesions or, alternatively, that many invasive carcinomas arise de novo (ie, without progressing through a significant in situ stage) by mechanisms not involving HER-2/neu. In addition, overexpression of HER-2/neu was associated with several poor prognostic features (younger patient age, premenopause, negative estrogen receptor status, negative progesterone receptor status, and high nuclear grade) in the subset of IDC combined with DCIS. With one exception (negative estrogen receptor status) these associations were lost in IDC not combined with DCIS, also suggesting that the role of HER-2/neu changes during the progression of human breast cancer.     

Flow cytometry: a new approach for the molecular profiling of breast cancer

The established method in prognosis of breast cancer includes detection of molecular markers, such as the estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu. These markers are routinely checked via immunohistochemistry (IHC). HER-2/neu is also detected by fluorescent in situ hybridization (FISH). Flow cytometric analysis has not yet been used for detection of such markers. Flow cytometry was performed on four established breast cancer cell lines: MCF7, T47D, BT474, MDA-MB-231, and on one normal breast epithelial cell line: MCF10A. Flow cytometric analysis was used for the detection of ER, PR, HER-2/neu, epidermal growth factor receptor (EGFR), and E-cadherin. Currently, EGFR and E-cadherin are not standard predictive factors in determining survival of breast cancer patients, but both may be beneficial to prognosis. Cells undergoing flow cytometric analysis lost marker expression with increasing passage number. The highest expression was found at cells passaged 0-1 times. MCF7, T47D, and BT474 all had similar marker expression patterns. E-cadherin demonstrated a strongly positive pattern with marker expression of 85-92% among the three cell lines. ER, PR, and HER-2/neu demonstrated a weakly positive expression pattern when compared with E-cadherin. Marker expression ranged from 15 to 61%. These three cell lines were almost negative for expression of EGFR where expression ranged from 0 to 6%. MDA-MB-231 had almost no expression of all 5 markers, with positive values ranging from 0 to 5%. MCF10A had weak positive to almost negative expression values of ER, PR, HER-2/neu, and E-cadherin, which ranged from 3 to 13%. EGFR, both surface and cytoplasmic markers, again were not expressed in MCF10A cells with an expression value of <1%. We found that ER, PR, and HER-2/neu marker expressions in 5 out of 5 cell lines were consistent with established expression patterns. EGFR and E-cadherin expression in 4 out of 5 cell lines were also consistent with established expression patterns. We have shown that flow cytometry provides quantitative data on expression patterns of important prognostic markers in breast cancer.     

Amplification and overexpression of HER-2/neu in parotid gland salivary duct carcinoma. Immunohistochemical study and fluorescence in situ hybridization

Salivary duct carcinoma (SDC) is highly malignant salivary gland tumour with aggressive clinical behaviour, characterised by its histological resemblance to invasive ductal carcinoma of the breast. Amplification of gene HER-2/neu and overexpression of its gene product have been shown to have both prognostic and treatment implications in breast cancer. The reports concerning the expression of c-erbB2/HER-2/neu in salivary gland tumours are few and controversial. Thus, eleven cases of SDC were evaluated for HER-2/neu status using immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). To the best of our knowledge, this is the first molecular genetic analysis of SDCs using FISH. HER-2/neu overexpression, identified as strong membrane staining, was observed in all but one case of SDC in majority of neoplastic cells while only four tumours, of nine cases analysed, revealed HER-2/neu gene amplification by means of FISH analysis. SDCs were associated with poor clinical outcome, 6 patients (55%) died of disseminated carcinoma within 4 to 44 months after therapy. There was no difference in outcome of patients with IHC positive-nonamplified and IHC positive-amplified tumours.     

A formalin-fixed, paraffin-processed cell line standard for quality control of immunohistochemical assay of HER-2/neu expression in breast cancer.

To ensure the accuracy and reproducibility of immunohistochemical assays for determining HER-2/neu status of patients with breast cancer, a reliable standard for monitoring assay sensitivity is necessary. We optimally fixed and paraffin processed human ovarian and breast carcinoma cell lines SKOV-3, MDA-MB-453, BT-20, and MCF-7 in quantities sufficient to meet the needs of a laboratory for the foreseeable future. The material was tested, alongside HercepTest kit cell lines (DAKO, Carpinteria, CA), by 7 breast cancer centers in the United Kingdom and France with different immunohistochemical assays and markers. The cell lines also were analyzed by fluorescence in situ hybridization (FISH) by 2 centers using HER-2/neu kits. FISH produced 100% agreement between the 2 centers: SKOV-3 and MDA-MB-453 showed HER-2/neu amplification and BT-20 and MCF-7 did not. Immunohistochemical analysis and a common evaluation method produced 100% agreement that SKOV-3 and MCF-7 showed 3+ and zero HER-2/neu overexpression, respectively. For MDA-MB-453, there was 71% (5/7) concordance of 2+ immunohistochemical staining and 86% (6/7) concordance of zero or 1 + staining for BT-20. The cell lines provide a valuable standard for gauging HER-2/neu assay sensitivity irrespective of the antibody, antigen retrieval system, detection system, or method of evaluation used.     

Assessment of HER‐2/neu overexpression and/or gene amplification in breast carcinomas: should in situ hybridization be the method of choice?

AIMS: Since the release of Herceptin, pathology laboratories have been requested to test breast carcinomas for HER-2/neu overexpression and/or gene amplification. Standardized IHC and FISH are mandatory in order to get reliable results, but there are problems even with standardized procedures. We decided to evaluate the two methods to determine which, or possibly if both, should be the primary investigation method(s). METHODS AND RESULTS: The material consisted of 215 primary invasive breast carcinomas with complete clinical follow-up of 15 years. HER-2/neu protein expression was determined for all specimens, whereas FISH for assessing HER-2/neu gene signal number was done in 165 cases. IHC was double-checked with two or three different antibodies in 35 tumours, including all cases with discrepancies between IHC and FISH. Among these, there were discrepancies in a third. IHC overexpression of HER-2/neu was found in 13% and gene amplification in 18%. Discordance between IHC and FISH was found in 11 cases (8%). Five tumours were IHC+/FISH- and six were IHC-/FISH+. IHC and FISH positive cases as well as FISH only positive tumours had the same prognosis respecting survival. Tumours with >2 but 4 gene signals per nucleus. In contrast, cases with IHC overexpression without gene amplification had a prognosis similar to that of IHC-/FISH- tumours. CONCLUSIONS: From our data, it seems to be more important to assess HER-2/neu gene amplification than IHC overexpression. Failure to detect FISH-amplified (IHC-negative) cases would have an adverse effect on the survival of these patients. On the other hand, IHC overexpression tumours without gene amplification appear to belong to a better prognostic group, and failure to detect them would probably not have a negative effect on the survival of these women. Even though FISH is a more complex and expensive procedure, it should be considered the method of choice for primary assessment of HER-2/neu status in breast cancer patients.     

HER-2/neu and bcl-2 in ovarian carcinoma: clinicopathologic, immunohistochemical, and molecular study in patients with shorter and longer survival.

The bcl-2 protein is a membrane protein involved in prolonging cell survival by inhibiting apoptosis. The HER-2 oncogene, which is located on chromosome 17 and encodes for a tyrosine-kinase growth factor receptor, is amplified and HER-2/neu is overexpressed in 25% to 30% of breast carcinomas. The authors analyzed the bcl-2 expression and the bcl-2 gene and HER-2/neu overexpression and amplification in FIGO stage IIIC, serous, G3, ovarian carcinomas obtained from living patients who had no evident disease 5 years after primary treatment compared with ovarian carcinomas obtained from patients, matched for stage, grade of differentiation, and treatment, who had died of progression of disease no later than 2 years after primary treatment. bcl-2 overexpression was statistically correlated with progression of disease during first-line chemotherapy (P=0.021). The HER-2/neu status was found not to correlate with progression of disease during first-line chemotherapy. Both bcl-2 and HER-2/neu expression were not statistically associated with the clinical outcome of ovarian cancer patients. Gene amplification of the HER-2/neu chromosome 17 was found in all the HER-2/neu, 3+ score, positive-staining ovarian carcinomas. None of the analyzed samples revealed a translocation t(14;18)(q32;q21) in the bcl-2 gene. The knowledge of additional prognostic or even predictive factors, such as bcl-2 expression, in patients with advanced ovarian carcinoma before the primary chemotherapeutic treatment may help in the management of patients who require a more tailored treatment. In addition, the gene amplification of the HER-2/neu suggests that HER-2 is a potential target for treatment in ovarian cancer.