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.     

Evaluation of HER-2/neu in serum and tissue of primary and metastatic breast cancer patients using an automated enzyme immunoassay

Serum HER-2/neu concentrations were evaluated in 172 healthy subjects, 176 primary and 55 metastatic breast cancer patients, employing a new automated assay (Bayer Immuno 1 serum HER-2/neu). Using 13 ng/mL as the cutoff, abnormal HER-2/neu serum levels were found in 8% (14/176) of primary and 50.9% (28/55) of metastatic breast cancer patients. Both in primary and metastatic breast cancer a significant relationship was found with the stage of the disease when serum HER-2/neu was considered as a categorized variable (p=0.0003 and p=0.02, respectively), but not when it was taken as a continuous variable (p=0.247 and p=0.146, respectively). Moreover, we evaluated the correlation between Immuno 1 HER-2/neu and Oncogene Research Products ELISA assay in 53 normal subjects, 46 primary and 34 metastatic breast cancer patients. The correlation was relatively good (p<0.0001), although substantial differences could be found in single cases. The Immuno 1 assay was also evaluated for the first time in breast cancer tissue. The method, which showed good performance both in terms of imprecision and linearity, was used to measure HER-2/neu protein in 140 cytosol samples from primary breast cancer tissue and in homogenates from 40 matched cases. The correlation between the two matrixes was very close (p<0.0001). By contrast, no correlation was found between serum and matched cytosol (p=0.101) or homogenate samples (p=0.511).     

HER-2/neu evaluation by fluorescence in situ hybridization on destained cytologic smears from primary and metastatic breast cancer

OBJECTIVE: To evaluate HER-2/neu amplification by fluorescence in situ hybridization (FISH) (HER-2/neu by FISH) on archival cytologic smears stained with May-Grünwald-Giemsa (MGG) stain. STUDY DESIGN: Cytologic specimens from 69 breast cancer lesions (48 primary and 21 metastatic), stained with MGG stain for routine diagnostic cytology, were destained and subjected to HER-2/neu by FISH. Fifteen of the 69 samples were also evaluated by FISH on paired fresh smears. RESULTS: HER-2/neu by FISH was successfully assayed in 25 of the 48 primary tumors and in 15 of the 21 metastatic lesions, corresponding to an overall feasibility of 58%. These cases had been archived between 1 month and 10 years prior to FISH analysis. Eight of the 25 primary and 5 of the 15 metastatic tumors were amplified. In 15 of the 40 evaluable cases, HER-2/neu was also assessed on the corresponding fresh smears: 8 tumors were amplified and 7 unamplified on both destained MGG and fresh smears. CONCLUSION: HER-2/neu can be detected by FISH on routinely MGG-stained cytologic slides. This approach allows HER-2/neu evaluation whenever histologic sections or fresh cytologic material are not available. In these cases, HER-2/neu assessment on destained cytologic smears plays a role in the selection of targeted therapy.     

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 phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses HER2/neu

A phase I study of escalating doses of humanized bispecific antibody (bsAb) MDX-H210 with granulocyte-colony-stimulating factor (G-CSF) was conducted in patients with metastatic breast cancer that overexpressed HER2/neu. The main objectives of the study were to define the maximal tolerated dose (MTD) of MDX-H210 when combined with G-CSF, to measure the pharmacokinetics of MDX-H210 when administered with G-CSF, and to determine the toxicity, biological effects and possible therapeutic effect of MDX-H210 with G-CSF. MDX-H210 is a F(ab)′ × F(ab)′ humanized bispecific murine antibody that binds to both HER2/neu and the FcγR1 receptor (CD64), and was administered intravenously weekly for three doses followed by a 2-week break and then three more weekly doses. A total of 23 patients were treated, and doses were escalated from 1 mg/m² to 40 mg/m² with no MTD reached. The toxicity of the bsAb + G-CSF combination was modest, with no dose-limiting toxicity noted: 19 patients had fevers, 7 patients had diarrhea, and 3 patients had allergic reactions that did not limit therapy. The β-elimination half-life varied from 4 h to 8 h at doses up to 20 mg/m². Significant release of cytokines interleukin-6, G-CSF, and tumor necrosis factor α was observed after administration of bsAb. Circulating monocytes disappeared within 1 h of bsAb infusion, which correlated with binding of bsAb, noted by flow-cytometric analysis. Significant levels of human anti-(bispecific antibody) were measured in the plasma of most patients by the third infusion. No objective clinical responses were seen in this group of heavily pre-treated patients. Received: 23 July 1998 / Accepted: 6 November 1998     

Clinical implications of HER-2/neu overexpression and proteolytic activity imbalance in breast cancer

The aggressive biological behavior of invasive and metastatic cancer is considered to be the most insidious and life-threatening aspect for breast cancer patients. It is mostly the result of changes in many molecular characteristics of tumor cells, including alterations in gene expression and the balance of proteolytic activity. The objective of this study was to determine the level of MMP-2, its natural inhibitor TIMP-2, their ratio and HER-2/neu as diagnostic and prognostic factors. Markers were analyzed in 240 tissue samples categorized into 96 benign breast disease and 144 breast cancer patients. Enzyme linked immunosorbent assay procedure was used to evaluate the level of MMP-2 and TIMP-2 in the cell lysate, HER-2/neu in the membrane fraction, and steroid hormone receptors (ER and PgR) in the cytosol fraction. Breast cancer patients were followed-up for three years. Receiver operating characteristic curves were used to determine the cutoff points for the investigated factors. Positive values for all investigated factors were significantly increased in breast cancer patients compared to benign ones. Mean levels for all investigated factors were significantly correlated with lymph node and hormone receptor status, while MMP-2 and TIMP-2 were correlated with tumor grade (P < 0.05). In Univariate analysis, positive MMP-2, MMP-2/TIMP-2, HER-2/neu overexpression, higher tumor grade, late clinical stages and positive lymph nodes status were significantly associated with relapse. By multivariate analysis, all aforementioned factors apart from tumor grade were independent variables. Thus, the investigated markers are constructive for biologic aggressiveness of breast cancer and MMP-2/TIMP-2 ratio might be a new significant marker in early diagnosis and estimate prognosis in breast cancer.     

HER-2/neu gene in primary and local metastatic axillary lymph nodes in human breast tumors.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.     

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.     

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.     

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.     

Interobserver reproducibility of immunohistochemical HER-2/neu evaluation in human breast cancer: the real-world experience

In spite of the large number of studies on technical problems affecting the interlaboratory reproducibility of IHC HER-2/neu determination, only little is known about factors limiting the intra- and interobserver reproducibility in the actual practice of HER-2/neu expression analysis. The aim of the present INQAT study was to evaluate the intra- and inter-observer reproducibility of IHC HER-2 analysis among pathologists routinely working in Italian laboratories. Twenty immunostained slides were distributed to 12 pathologists who had to report, for each slide, the semiquantitative analysis of the percentage of immunopositive cells and the qualitative evaluation of the intensity of membrane staining. The intra- and interobserver reproducibility as well as the reproducibility between each laboratory and the reference values were quantified adopting an approach based on computation of the weighted kappa statistic (Kw). Additionally, in order to evaluate the contribution of each category to the overall agreement, the kappa category-specific statistics (Kcs) were estimated for both classification criteria by jointly considering all the participating laboratories. The intraobserver analyses showed a satisfactory level of reproducibility for both the percentage of positive cells (median Kw, 0.94; range: 0.80-0.96) and membrane staining (median Kw, 0.86; range: 0.78-0.96). Similarly, a fairly good level of reproducibility for the percentage of cells (median Kw, 0.89; range, 0.73-0.96) and the intensity of membrane staining (median Kw, 0.84; range, 0.72-0.92) were observed from comparisons with reference values. When all possible pairwise comparisons were performed, a satisfactory level of interobserver reproducibility was found for most laboratories. Kw values varied between 0.51 and 0.98 (median Kw, 0.80) and between 0.61 and 0.94 (median Kw, 0.81) for semiquantitative and qualitative measurements, respectively. Analysis of the contribution of the extreme categories to the overall agreement showed a substantial or almost perfect agreement for both classification criteria. Conversely, the contribution of intermediate categories appeared to be scarce or slight for the percentage of immunostained cells and slight or fair for the intensity of membrane staining. We conclude that, overall, the interobserver reproducibility in IHC analysis of HER-2/neu expression is satisfactory, although classification of the intermediate categories is problematic, both with regard to the percentage of immunostained cells and the intensity of membrane staining.     

Computational model to explore the endocrine response to trastuzumab action in HER-2/neu positive breast cancer

Breast cancer is a very frequent type of cancer and much attention is paid to therapy with considerable efforts both in the pharmacological and clinical fields.The present work aims to create a non-linear dynamic model of action of the drug Trastuzumab against HER-2 + breast cancer, mainly considering its action of ADCP (antibody-dependent phagocytosis) killing of cancer cells. The model, while also considering the other therapeutic effects induced by Trastuzumab, shows how the action of this monoclonal antibody in the induction of ADCP through the action of macrophages, is strictly connected to the formation of a multi-complex “Trastuzumab -HER-2 - macrophage” that shows a prolonged action over time, responsible for the increase in the Overall Survivor (OS) parameter reported in various. The model shows the correlation between the various therapeutic effects and the killing action of cancer cells through the variation of the dynamic fluctuation of the representative ”c” parameter.     

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.     

HER-2/neu vaccine-primed autologous T-cell infusions for the treatment of advanced stage HER-2/neu expressing cancers

This phase I study evaluated the feasibility of expanding HER-2/neu (HER2) vaccine-primed peripheral blood T-cells ex vivo and assessed the safety of T-cell infusions. Eight patients with HER2⁺ treatment refractory metastatic cancers were enrolled. T-cells could be expanded to predefined parameters in seven patients (88 %). Ninety-two percent of adverse events were grade 1 or 2. Three of seven patients developed infusion-related inflammatory reactions at their disease sites. HER2-specific T-cells significantly increased in vivo compared to pre-infusion levels (p = 0.010) and persisted in 4/6 patients (66 %) over 70 days after the first infusion. Partial clinical responses were observed in 43 % of patients. Levels of T-regulatory cells in peripheral blood prior to infusion (p < 0.001), the level of HER2-specific T-cells in vivo (p = 0.030), and development of diverse clonal T-cell populations (p < 0.001) were associated with response. The generation of HER2 vaccine-primed autologous T-cells for therapeutic infusion is feasible and well tolerated. This approach provides a foundation for the application of T-cell therapy to additional solid tumor types.     

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.     

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。     

Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products

GVAX^® immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.     

Interobserver reproducibility of immunohistochemical HER-2/neu assessment in human breast cancer: an update from INQAT round III

The clinical interest in HER-2/neu is related to trastuzumab, a drug used to treat patients with invasive breast carcinoma overexpressing the HER-2/neu protein. It is very important to correctly identify those patients who may benefit from trastuzumab by accurate assessment of the HER-2/neu status. Of the various methods available, the Dako Herceptest for immunohistochemical assay is considered the most reliable to reach this goal. The aim of this study was to investigate within a group of Italian laboratories the reproducibility of the results of HER-2/neu assessment by means of the Dako scoring system on slides stained with the Herceptest kit. This study was also conceived as the continuation of one of our previous studies, which was similar in its aims but different in the classification criteria adopted. Our results show that, whereas the intra-observer reproducibility was generally satisfactory, the interobserver reproducibility was not. Moreover, our findings confirm that the two extreme classes (0 and 3+) are more easy to identify than the other two and that the Herceptest does not allow to discriminate optimally between scoring classes 2+ and 3+. These findings are relevant in clinical practice where the treatment choice is based on categories defined by this assay, suggesting the need of adopting educational programs and/or new reference materials to improve the assay performance.