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.     

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)     

Discordant results obtained for different methods of HER-2/neu testing in breast cancer--a question of standardization, automation and timing

BACKGROUND: HER-2/neu positivity is required for the selection of stage IV breast cancer patients for trastuzumab therapy. We compared the results of the recommended immunohistochemistry (IHC) evaluation with the automated ACIS IHC system and with fluorescence in situ hybridization (FISH). These HER-2/neu tissue results were correlated with the serum HER-2/neu (sHER-2/neu) levels at the time of metastatic spread. PATIENTS AND METHODS: A total of 61 IHC slides from 30 patients were stained using the HercepTest. HER-2/neu gene amplification was determined using the Ventana FISH assay. sHER-2/neu levels were measured with the Oncogene Science" ELISA kit. The concordance of HER-2/neu results was determined using the concordance index Kappa (kappa). RESULTS: The best concordance between any IHC and FISH was found for the automated ACIS system (88.5%, kappa=0.68, category "good"). The comparison between the manual interpretations and the automated IHC was categorized as "very good" (95.1%, kappa=0.85). The median sHER-2/neu level of FISH positive patients was significantly higher (67 ng/mL) than that of FISH negative patients (17 ng/mL, p=0.018). The increase in HER-2/neu positivity comparing tissue to stage IV serum was statistically significant (p=0.001). CONCLUSIONS: The concordance between conventional IHC and computerized analysis was very good. The number of patients with stage IV breast cancer with an elevated sHER-2/neu level was much higher than HER-2/neu positivity in tissue. This discrepancy is only partially explained by the influence of tumor load. Patients with an elevated sHER-2/neu level and no tissue overexpression should be considered for retesting of tissue or a new biopsy.     

The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis

Because HER-2/neu overexpression is important in cancer development, we looked for a method of suppressing the cell transformation mediated by HER-2/neu overexpression. We have identified that the DNA-binding protein PEA3, which is encoded by a previously isolated gene of the ets family, specifically targeted a DNA sequence on the HER-2/neu promoter and downregulated the promoter activity. Expression of PEA3 resulted in preferential inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells. This is a new approach to targeting HER-2/neu overexpression and also provides a rationale to the design for repressors of diseases caused by overexpression of pathogenic genes.     

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.     

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intradermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient's age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing melanoma from melanocytic nevi, if we consider c-Kit expression in intraepidermal proliferating cells. The c-Kit expression in proliferating melanocytes in the dermis could help in the differential diagnosis between a superficial spreading melanoma (with dermis invasion) and a compound nevus or an intradermal nevus. Finally, c-Kit could be a good diagnostic tool for distinguishing benign compound nevi from malignant melanocytic lesions with dermis invasion and to differentiate metastatic melanoma from primary melanoma.     

HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization

The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.     

HER-2/neu oncogene expression, DNA ploidy and proliferation index in breast cancer.

HER-2/neu gene expression, DNA ploidy and proliferation index were studied in 250 cases of breast cancer. Expression of HER-2/neu was determined by using an antibody to the HER-2/neu receptor. Ki-67 antibody was used to determine the proliferation index of the breast cancers, and the Feulgen method was used to assess DNA amounts in the tumor cells. Histochemical staining was quantitated by image analysis. Of the cancers studied, 72 were positive for overexpression of HER-2/neu protein; of these, 62 (86%) possessed near-tetraploid DNA content, and 47 (65%) had more than one G0G1 stem line (polyploid) of DNA distribution. Cells from the cases negative for HER/2-neu overexpression contained DNA amounts that ranged from diploid to varying degrees of aneuploid. A significant difference in the amounts of cellular proliferation in HER-2/neu overexpressing cancers was found between those that expressed the HER-2/neu receptor on their membranes and those that exhibited mainly cytoplasmic receptors.     

Immunocytochemistry and fluorescence in situ hybridization in HER-2/neu status in cell block preparations

OBJECTIVE: Recent studies have validated the use of cytologic materials to determine HER-2/neu status. Good concordance has been shown between results obtained by immunocytochemistry (ICC), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) on cytologic and surgical specimens. However, the utility of cytologic cell block material in determining HER-2/neu status has not been reported and is the subject of this study. STUDY DESIGN: HER-2/neu status was determined in 25 cases of primary or metastatic breast carcinoma by IHC and FISH. All cases were formalin-fixed, paraffin-embedded (FPPE) cell block preparations. ICC was performed using monoclonal antibodies TAB250 (Zymed) and CB11 (Novacastra Laboratories). FISH analysis was performed using the PathVysion HER-2 probe kit (Vysis, Inc.). Results of ICC and FISH were compared in each case. RESULTS: Of 25 cases studied, 17 showed no protein overexpression or amplification. Five cases showed protein overexpression and amplification. The remaining 3 cases showed 2+ staining intensity by ICC in 10, 20, and 50% of carcinoma cells, respectively, and all demonstrated lack of amplification. CONCLUSION: Immunocytochemistry performed on FFPE cell block material is a reliable method for determining HER-2/neu status in cytologic specimens. We recommend routine preparation of FFPE cell block material in instances of suspected primary of metastatic breast carcinoma.     

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.     

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.     

HER-2/neu raises SHP-2, stops IFN-gamma anti-proliferation in bladder cancer

Gene amplification or HER-2/neu protein overexpression signals a poor outcome for bladder cancer patients. We investigated the anti-proliferative effect of IFN-gamma in HER-2/neu-transfected human bladder cancer cells (TCC-N5 and TCC-N10). The cells continued growing after IFN-gamma stimulation but did not activate the Janus kinase (Jak)/Stat pathway. We found Jak/Stat protein phosphatase in TCC-N5 and TCC-N10 cells with upregulated Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2). After the cells had been treated with AG825, a HER-2/neu-specific inhibitor, SHP-2 expression declined, and Jak2/Stat1 reactivated. Similar results were reported in a mouse bladder cancer cell line, MBT2, with constitutive HER-2/neu overexpression. Further, AG825 pretreatment restored the anti-proliferation activity of IFN-gamma in TCC-N5 and TCC-N10 cells. Therefore, the suppression of IFN-gamma signaling in HER-2/neu-overexpressing bladder cancer cells might be due to SHP-2 upregulation. The regulation of SHP-2 by HER-2/neu provides a new target for blocking the HER-2/neu oncogenic pathway.     

p53 and HER2/neu expression in relation to chemotherapy response in patients with non-small cell lung cancer

The aim of the study was to investigate a relation between p53 and HER2/neu expression in resected lung tumors and the response of those tumors to neoadjuvant chemotherapy. The study population included 67 consecutive patients with non-small cell lung cancer (NSCLC) in stage II or III who were operated on at the Institute of Tuberculosis, Warsaw, Poland, between 20 April 2001 and 10 March 2003. All patients received two cycles of chemotherapy consisting of cisplatin and vinorelbine prior to the operation. The response to therapy was assessed as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD), on the basis of CT scans performed before and after neoadjuvant chemotherapy. p53 and HER2/neu protein expression were evaluated by immunohistochemistry (IHC) using antibodies against p53 (clone PAb 1801, Novocastra) and against HER2/neu (Dako) in paraffin-embedded specimens of tumors. A response to therapy (CR+PR) was observed in 27 patients, while 40 patients (SD+PD) were regarded as resistant to therapy. Resistance was observed significantly more often in tumors above 3 cm in diameter. p53 expression was found in 16 tumors (23.9%) and HER2/neu in 26 tumors (38.8%). We observed a nonsignificant tendency to chemoresistance in tumors with HER-2/neu overexpression and also in tumors with p53 overexpression. If we consider HER-2/neu and p53 together, chemoresistance was observed statistically significantly more often when one or both markers were positive (p<0.05). This significance was independent of tumor size.     

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.     

Oncogenic signals of HER-2/neu in regulating the stability of the cyclin-dependent kinase inhibitor p27

Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neu in cancers has been associated with poor prognosis. Reduced expression of p27(Kip1), a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neu overlap with those affected by reduced p27 expression, we studied the link between HER-2/neu oncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neu overexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neu activity causes mislocation of p27 and Jun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neu signals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.     

HER-2/neu-derived peptide 884–899 is expressed by human breast, colorectal and pancreatic adenocarcinomas and is recognized by in-vitro-induced specific CD4+ T cell clones

HER-2/neu peptides recognized in the context of HLA-DR molecules by CD4(+) Th lymphocytes on antigen-presenting cells have been identified. In this report, we demonstrate for the first time that HER-2/neu helper epitopes are also expressed on the surface of metastatic breast, colorectal and pancreatic carcinomas. Peripheral blood mononuclear cells from an HLA-DR4 healthy donor were used to induce HER-2/neu peptide-specific CD4(+) T cell clones by in vitro immunization with HER-2/neu peptide (884-899)-pulsed autologous dendritic cells (DCs). Strong proliferation and significant levels of IFN-gamma were induced by the CD4(+) T cell clones in response to specific stimulation with autologous DCs loaded with HER-2(884-899). Furthermore, these clones also recognized HER-2/neu(+) tumor cell lines, and tumor cells from breast, colorectal and pancreatic adenocarcinomas induced to express HLA-DR4, but also the HLA-DR4(+) melanoma cell line FM3 transfected to express HER-2/neu. The recognition of tumor cells was strongly inhibited by an anti-HLA-DR mAb. Taken altogether, we provide novel information for the role of HER-2(884-899) as a naturally processed epitope expressed by breast, colorectal and pancreatic carcinomas and the capacity of HER-2/neu protein to follow the endogenous class II processing pathway. Our results suggest that HER-2(884-899) might be attractive for broadly applicable vaccines and may prove useful for adoptive immunotherapy designed for breast, colorectal and pancreatic carcinomas.     

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.     

A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase

Prostate cancer progresses from a hormone-sensitive, androgen-dependent stage to a hormone-refractory, androgen-independent tumor. The androgen receptor pathway functions in these androgen-independent tumors despite anti-androgen therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines expressed higher levels of the HER-2/neu receptor tyrosine kinase than their androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent prostate cancer cells allowed ligand-independent growth. HER-2/neu activated the androgen receptor pathway in the absence of ligand and synergized with low levels of androgen to 'superactivate' the pathway. By modulating the response to low doses of androgen, a tyrosine kinase receptor can restore androgen receptor function to prostate cancer cells, a finding directly related to the clinical progression of prostate cancer.     

Monitoring therapy by serum HER-2/neu

We have evaluated the performance of the Bayer Immuno 1 serum HER-2/neu assay. The precision is excellent and varied between 1.7% and 2.1% at values of HER-2/neu ranging from 16.8 ng/mL to 108.6 ng/mL. In normal women who were followed on a monthly basis the average deviation was 6%. The concentration (mean +/- SD) in normal women was 8.7 +/- 3.2 ng/mL. There was no difference between pre and postmenopausal women. The normal/abnormal cutoff was defined as greater than 15 ng/mL. In normal women and those with benign disease the specificity varied between 95% and 100%. In women with breast cancer the sensitivity was 1.7% in stage I disease, 3.0% in stage II, 16.7 in stage III and 35.5% in stage IV. There were 56 elevations (14.7%) in the total group of 285 women with breast cancer. There was an excellent correlation with a microtitre plate method (r=0.9944). Longitudinal studies showed clearly that women who expressed HER-2/neu in their tissue had elevated serum levels and these levels reflected the clinical course of the patient. More extensive control studies are required to establish the role of HER-2/neu assays in the management of women with breast cancer.