Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.     

The expression of HER-2/neu (c-erbB2), survivin and cycline D1 in serous ovarian neoplasms: their correlation with clinicopathological variables

Ovarian cancer is the most common cause of death among all gynecologic malignancies and a result of complex interaction of multiple oncogenes and tumor suppressor genes. The aim of this study was to evaluate expression of HER-2/neu (c-erbB2), survivin and cycline D1 biomarkers in serous ovarian neoplasms and their correlations with clinicopathological variables in serous ovarian cancers. We analyzed pathological specimens of 62 patients with benign (n = 25), borderline (n = 14) and malignant (n = 23) serous ovarian neoplasms. Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded specimens. Significantly more immunoreactivity with HER-2/neu was detected in malignant tumors (100 %) compared to borderline (78.6 %) and benign tumors (48 %) (P < 0.01). Survivin expression was significantly higher in malignant tumors (91.3 %) than those found in borderline (71.4 %) and benign tumors (24 %) (P < 0.001). Similarly, higher cyclin D1 expression was observed in malignant tumors (95.6 %) compared to borderline (85.7 %) and benign tumors (48 %) (P < 0.001). Expression of all biomarkers analyzed significantly and gradually increased from benign to borderline and borderline to malignant serous tumors. In terms of clinicopathological variables, only tumor grade was associated with the expression of all biomarkers others exhibited different correlations in serous ovarian cancers. The expressions of HER-2/neu (c-erbB2), survivin and cycline D1 are positively correlated with the malignant potential of serous ovarian neoplasms.     

Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells.

Amplification and resulting overexpression of the HER-2/ neu proto-oncogene is found in approximately 30% of human breast and 20% of human ovarian cancers. To better understand the molecular events associated with overexpression of this gene in human breast cancer cells, differential hybridization was used to identify genes whose expression levels are altered in cells overexpressing this receptor. Of 16 000 clones screened from an overexpression cell cDNA library, a total of 19 non-redundant clones were isolated including seven whose expression decreases (C clones) and 12 which increase (H clones) in association with HER-2/ neu overexpression. Of these, five C clones and 11 H clones have been confirmed to be differentially expressed by northern blot analysis. This group includes nine genes of known function, three previously sequenced genes of relatively uncharacterized function and four novel genes without a match in GenBank. Examination of the previously characterized genes indicates that they represent sequences known to be frequently associated with the malignant phenotype, suggesting that the subtraction cloning strategy used identified appropriate target genes. In addition, differential expression of 12 of 16 (75%) cDNAs identified in the breast cancer cell lines are also seen in HER-2/ neu -overexpressing ovarian cancer cells, indicating that they represent generic associations with HER-2/ neu overexpression. Finally, up-regulation of two of the identified cDNAs, one novel and one identified but as yet uncharacterized gene, was confirmed in human breast cancer specimens in association with HER-2/ neu overexpression. Further characterization of these genes may yield insight into the fundamental biology and pathogenetic effects of HER-2/ neu overexpression in human breast and ovarian cancer cells.     

HER-2/neu oncogene expression and DNA ploidy in normal human kidney and renal cell carcinoma.

Using flow cytometry (FCM), we have investigated both the DNA content (stained with propidium iodide) and HER-2/neu oncogene expression (revealed by means of an anti-HER-2/neu monoclonal antibody) in neoplastic and non-neoplastic kidney samples from 20 patients with renal cell carcinoma. All the non-neoplastic samples and 15/20 (75%) renal cell cancers showed diploid modal DNA content while the remaining 5 neoplastic sample (25%) showed both diploid and hyperdiploid cell populations. In normal kidney the level of HER-2/neu oncoprotein was low (median fluorescence values in arbitrary units = 7.5 AU, range: 4-10 AU). In diploid renal cancers the level of HER-2/neu was slightly increased (median fluorescence values = 20 AU, range: 9.5-30 AU) (p < .005). The relationship of HER-2/neu expression to the cell cycle in these tumor samples is not clear since most of the cells express the antigen in all phases of the cell cycle. On the other hand, there is an association between HER-2/neu expression and abnormal DNA content suggesting that aneuploid pattern may be biologically related to overexpression of the HER-2/neu gene.     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

Timely DNA vaccine combined with systemic IL-12 prevents parotid carcinomas before a dominant-negative p53 makes their growth independent of HER-2/neu expression

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.     

Clinical utility of determination of HER-2/neu and EGFR fragments in serum of patients with metastatic breast cancer

INTRODUCTION: The growth factor receptors EGFR and HER-2/neu are targets for new treatment strategies and are of potential use as prognostic and predictive factors. However, the optimal method of determination in order to obtain clinically relevant information remains a source of controversy. METHODS: HER-2/neu and EGFR expression was examined by immunohistochemistry in primary tumors of patients with breast cancer. In addition, serum was tested for the extracellular domains of HER-2/neu (HER-2/neu ECD) and EGFR (sEGFR) before initiation of therapy for metastatic disease (n=76). The course of disease from the time of metastasis with regard to these parameters was evaluated by univariate and multivariate analyses. RESULTS: HER-2/neu ECD levels at the time of metastatic disease were correlated with HER-2/neu expression determined by immunohistochemistry from primary tumors (p=0.001). No correlation was observed between expression of EGFR in primary tumors and sEGFR serum levels. HER-2/neu ECD and sEGFR levels at the onset of metastatic disease did not show a significant impact on overall survival. CONCLUSIONS: Determination of HER-2/neu ECD levels in the serum measured by ELISA at the onset of metastatic disease could offer an alternative to immunohistochemistry of the primary tumor since serum levels are correlated with protein expression in primary tumors. In contrast, no such correlation was observed for EGFR.     

Progesterone receptor inhibits aromatase and inflammatory response pathways in breast cancer cells via ligand-dependent and ligand-independent mechanisms

Aromatase (product of CYP19 gene), the critical enzyme in estrogen biosynthesis, is up-regulated in 70% of all breast cancers and is highly correlated with cyclooxygenase 2 (COX-2), the rate-determining enzyme in prostanoid biosynthesis. Expression of COX-2 also is correlated with the oncogene HER-2/neu. The efficacy of current endocrine therapies for breast cancer is predicted only if the tumor contains significant amounts of estrogen receptor. Because the progesterone receptor (PR) is an estrogen-induced target gene, it has been suggested that its presence may serve as an indicator of estrogen receptor functional capacity and the differentiation state of the tumor. In the present study, we tested the hypothesis that PR serves a crucial protective role by antagonizing inflammatory response pathways in the breast. We observed that progesterone antagonized the stimulatory effects of cAMP and IL-1beta on aromatase, COX-2, and HER-2/neu expression in T47D breast cancer cells. These actions of progesterone were associated with increased expression of the nuclear factor-kappaB inhibitor, IkappaBalpha. In 28 breast cancer cell lines, IkappaBalpha expression was positively correlated with PR mRNA levels; overexpression of a phosphorylation-defective mutant of IkappaBalpha inhibited expression of aromatase, COX-2, and HER-2/neu. Moreover, in breast cancer cell lines cultured in the absence of progesterone, up-regulation of endogenous PR caused decreased expression of aromatase, COX-2, and HER-2/neu expression, whereas down-regulation of endogenous PR resulted in a marked induction of aromatase and HER-2/neu mRNA. Collectively, these findings suggest that PR plays an important antiinflammatory role in breast cancer cells via ligand-dependent and ligand-independent mechanisms.     

High-level detection of gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer using FISH: a new approach for retrospective studies

A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested.     

Alterations of estrogen receptors, progesterone receptors and c-erbB2 oncogene protein expression in ductal carcinomas of the breast

Estrogens are important for stimulating the growth of a large proportion of breast cancers. Progesterone plays critical roles in breast development and tumorigenesis. The c-erbB2 gene (HER-2/neu) is a proto-oncogene expressed in 10-34% of breast cancers. Its expression is associated with poor clinical outcome. The hypothesis that the progression of in situ ductal carcinoma of breast to invasive ductal carcinoma is associated with alterations of ER, PgR and HER-2/neu protein expression was tested. Of 100 mastectomy specimens examined, all contained both ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC) not otherwise specified (NOS). The status of ER, PgR and HER-2/neu proteins was examined by immunochemistry. ER and PgR protein expression was scored as the mean value of positively stained cells. HER-2/neu protein expression was evaluated on ts staining pattern (0, 1+, 2+ and 3+). We found variations between DCIS and IDC with significant decrease of the mean values of ER and PgR positively stained cells in high-grade (Grade 3) IDC (ER: 49.2+/-10.3 vs. 30.8+/-5.5 and PgR: 40.0+/-10.0 vs. 22.3+/-5.1 in DCIS and IDC, respectively, P<0.05). Invasive carcinomas with lymph node metastases or lymphovascular invasion or both had lower mean values of ER and PgR positively stained cells compared to those without these features. In IDC (Grade 3), HER-2/neu protein expression values (1.2+/-0.2) were significantly high compared to DCIS (0.7+/-0.3, P<0.05). In addition, HER-2/neu protein expression values were significantly higher (P<0.05) in IDC with lymph node metastases or lymphovascular invasion (1.5+/-0.3) than those without these features (0.8+/-0.2). A significantly high mean (P<0.05) of ER and PgR positively stained cells was observed in postmenopausal females compared to premenopausal women. In contrast, high HER-2/neu expression values were seen only in premenopausal females. A significant positive correlation was observed between ER and PgR receptor expression (r=0.81). A low degree inverse correlation (r=-0.24, P<0.012) was found between ER+/PgR+ tumors and HER-2/neu expression. These findings substantiate the notion that breast cancer progression is often associated with alterations of ER, PgR and HER-2/neu expression. The underlying mechanisms of these alterations are open for further investigation.     

Proteomic study reveals that proteins involved in metabolic and detoxification pathways are highly expressed in HER-2/neu-positive breast cancer

The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase (FASN)), stress-mediated chaperonage (heat shock protein 27 (Hsp27)), and antioxidant and detoxification pathways (haptoglobin, aldo-keto reductase (AKR), glyoxalase I (GLO), and prolyl-4-hydrolase beta-isoform (P4HB)) were found to be up-regulated in HER-2/neu-positive breast tumors. HER-2/neu-dependent differential expression of PGK1, FASN, Hsp27, and GLO was further validated in four breast cancer cell lines and 12 breast tumors by immunoblotting and confirmed by partially switching off the HER-2/neu signaling in the high HER-2/neu-expressing SKBr3 cell line with Herceptin treatment. Statistical correlations of these protein expressions with HER-2/neu status were further verified by immunohistochemistry on a tissue microarray comprising 97 breast tumors. Our findings suggest that HER-2/neu signaling may result, directly or indirectly, in enhanced activation of various metabolic, stress-responsive, antioxidative, and detoxification processes within the breast tumor microenvironment. We hypothesize that these identified changes in the cellular proteome are likely to drive cell proliferation and tissue invasion and that the key cell cycle modulators involved, when uncovered by future research, would serve as naturally useful targets for the development of therapeutic strategies to negate the metastatic potential of HER-2/neu-positive breast tumors.     

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer.

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-β(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ～5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.     

pcDNA3.1(−)-mediated ribozyme targeting of <Emphasis Type="Italic">HER-2</Emphasis> suppresses breast cancer tumor growth

The HER-2 proto-oncogene (also called c-erbB-2/neu) encodes the protein, p185, which is closely related to the growth and metastasis of adenocarcinoma, and is overexpressed in 25–30% of human breast cancers. In this study, we attempt to reverse the malignant phenotype of the breast cancer cell line, MCF-7, using a HER-2-specific hammerhead ribozyme. Two anti-HER-2 hammerhead ribozymes, RZ1 and RZ2, were synthesized, inserted separately into the nonviral eukaryotic expression vector, pcDNA3.1(−), and transfected into MCF-7 cells. Analyses showed that the HER-2 mRNA and p185, as well as oncogene k-ras were down-regulated remarkably in the ribozyme-transfected cells, while the onco-suppressor gene, p53, was up-regulated. Furthermore, the tumorigenicity of the RZ1-stably transfected MCF-7 cells was decreased dramatically in nude mice. These results demonstrate that the use of anti-HER-2 ribozymes may be a beneficial strategy for gene therapy of breast cancer.     

Targeted therapy in breast cancer: the HER-2/neu gene and protein

The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.     

基质金属蛋白酶-2和p185HER-2/neu蛋白在卵巢癌中的研究进展

基质金属蛋白酶-2(Matrix Metalloproteinase-2,MMP-2)是基质金属蛋白酶家族的重要成员,能降解明胶蛋白和Ⅳ型、V型胶原,在细胞外基质的降解过程中起着关键作用,能够促进肿瘤细胞发生侵袭和转移。p185HER-2/neu蛋白是一种相对分子质量185×103的跨膜糖蛋白,由HER-2/neu基因编码,属于酪氨酸激酶受体家族,p185HER-2/neu蛋白在人类多种癌症中存在扩增及过量表达,并与肿瘤的侵袭性表型及生存期短密切相关。就基质金属蛋白酶-2和p185HER-2/neu蛋白的生物学特性,与卵巢癌侵袭转移和预后的关系及MMP-2和p185HER-2/neu蛋白的研究情况等予以综述。     

Quantitation of HER-2/neu oncoprotein overexpression in invasive breast cancer by image analysis: a study comparing fresh and paraffin-embedded material.

HER-2/neu oncoprotein overexpression was compared in fresh frozen and paraffin-embedded formalin-fixed invasive breast cancer material from the same patients. The HER-2/neu protein was detected by an immunohistochemical staining method, and the average amount of protein staining per cell was measured using the CAS-200 image analysis system and expressed relative to the amount of HER-2/neu protein of calibration cells of the SKBR3 cell line which are known to have amplification of the HER-2/neu gene and overexpression of the HER-2/neu protein. There was a significant correlation between degree of HER-2/neu protein overexpression and DNA-hyperdiploidy (P less than 0.01, chi 2 test). No significant correlation could be demonstrated between degree of HER-2/neu overexpression and tumor size, lymph node status, number of positive nodes or morphometric features. There was in general a good concordance (r = 0.83) in HER-2/neu expression values between fresh and paraffin-embedded material. Pairwise comparison of the two series (Wilcoxon signed ranks test) revealed no significant differences, indicating that there were no systematic differences between HER-2/neu assessments in fresh and paraffin material. When analysing the HER-2/neu expression values according to thresholds used earlier for overexpression, comparable results for fresh and paraffin material were obtained for most cases. In the fresh and paraffin material a different staining pattern was observed (more membrane staining in the fresh material in contrast to a more diffuse staining pattern in the paraffin material). It was concluded that both fresh-frozen and paraffin-embedded, formalin-fixed material is suitable for assessment of HER-2/neu protein overexpression by image analysis and provides comparable HER-2/neu expression values in most cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay

PURPOSE: The HER-2/neu protein (p185) has become a promising target for antibody therapy in breast cancer. We tested the feasibility of a quantitative approach for HER-2/neu testing based on the analysis of tumor tissue extracts by an enzyme-linked immunosorbent assay (ELISA). EXPERIMENTAL DESIGN: Tumor tissue extracts of primary human breast cancers (n=124) were prepared using a triton-based buffer. HER-2/neu concentration was quantified by ELISA. Paraffin-embedded tissue sections of the same tumors were analyzed by immunohistochemical staining applying the monoclonal HER-2/neu antibody TAB 250 (n=124) and by chromogenic in situ hybridization (CISH) (n=73). RESULTS: Concentrations of p185 in tissue extracts determined by ELISA varied from 1 to 927 ng per mg protein with a median of 25 ng/mg protein, whereas normal breast tissue showed values from 0.4 to 5.5 ng/mg with a median of 2.2 ng/mg (p<0.0001, Mann-Whitney U test). A significant correlation between p185 concentration and immunohistochemical staining was observed (p<0.0001, Kruskal-Wallis test). In addition, p185 concentration measured by ELISA was correlated with the degree of HER-2/neu gene amplification determined by CISH. HER-2/neu-amplified tumors had significantly higher p185 concentrations (median value 181 ng/mg protein) than non-amplified tumors (median value 20 ng/mg; p<0.0001, Mann-Whitney U test). CONCLUSIONS: ELISA-based measurement of HER-2/neu protein concentration in breast cancer tissue extracts is feasible and provides quantitative results for p185 protein concentrations that correlate closely with HER-2/neu immunoscore and gene amplification.