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.     

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.     

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.     

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。     

ADVIA Centaur HER-2/neu shows value in monitoring patients with metastatic breast cancer

INTRODUCTION:The proteolytic breakdown product corresponding to the extracellular domain (ECD) of the HER-2/neu oncoprotein p185 is found in the circulation of healthy individuals and patients having cancers of epithelial origin. For the current evaluation we sought to determine the analytical performance as well as the clinical utility of the newly developed ADVIA Centaur HER-2/neu assay (Bayer HealthCare LLC, Diagnostics Division, Tarrytown, NY, USA) in monitoring patients with metastatic breast cancer during the course of disease and treatment and to compare the obtained results with those of CA 15-3. METHODS: The analytical performance (including precision, normal range, interfering substances, minimum detectable concentration, dilution recovery, spiking recovery and high-dose hook effect) were determined. HER-2/neu and CA 15-3 values were measured in retrospective samples obtained from 59 patients with metastatic breast cancer undergoing treatment over a 6-12 month period. Serial changes in serum HER-2/neu and CA 15-3 were correlated with changes in clinical status on a visit-to-visit basis. For each pair of serial measurements, changes of equal to or greater than, or less than 15% for HER-2/neu and 21% for CA 15-3 were considered to indicate progression or lack of progression, respectively. RESULTS: The ADVIA Centaur HER-2/neu assay demonstrated within-run imprecision and total imprecision ranging from 3.0-5.6% and from 3.2-5.7%, respectively. The upper limit of normal was 15.2 ng/mL (90% CI: 14.2-17.0 ng/mL). No significant interference (<5%) was seen with bilirubins, hemoglobin, triglycerides and cholesterol or therapeutic drugs commonly present in the sera of breast cancer patients. The minimum detectable concentration (analytical sensitivity) was found to be 0.5 ng/mL. The patient population in the clinical study included breast cancer patients who responded to therapy (stable, partial or complete response) or had disease progression. HER-2/neu levels showed a concordance of 78.1% (82/105 restaging time points) with the clinical course of disease, whereas CA 15-3 levels showed a concordance of 76.2% (80/105 restaging time points). The concordance with clinical status increased to 85.7% (90/105 restaging time points) when both results were used in combination as a series test. CONCLUSIONS: The ADVIA Centaur HER-2/neu assay provides excellent analytical performance for serial testing of serum HER-2/neu levels. The clinical data demonstrate the usefulness of serum HER-2/neu in monitoring metastatic breast cancer patients during treatment. Furthermore, the results indicate that serum HER-2/neu and CA 15-3 may be useful in identifying disease progression or therapeutic response in different subgroups of women with metastatic breast cancer.     

Comparison of in situ hybridization methods for the assessment of HER-2/neu gene amplification status in breast cancer using a tissue microarray

BackgroundThis project compared HER-2/neu gene status in breast cancers, as demonstrated by FISH (fluorescent in situ hybridization) and CISH (chromogenic in situ hybridization) and using a tissue microarray (TMA). The study also aimed to show whether the TMA technique could be used in clinical diagnostics, rather than remain a scientific tool.Materials and methodsA TMA was constructed using 121 breast cancer specimens, 6 cores from each specimen. Demonstration and assessment of HER-2/neu gene status was by FISH (Vysis Path) and CISH (DAKO Duo CISH).ResultsThe 121 breast cancer specimens were divided into 3 groups by HER-2 status, as determined by immunohistochemistry. In the HER-2 negative group no amplification was observed in 36 out of 40 cases. 3 cases showed amplification by both methods and one by CISH alone. The equivocal HER-2 group showed no amplification in 30 out of 41 cases and amplification in 9 cases. One case was FISH negative CISH positive and one was discarded. In the HER-2 positive group, amplification was confirmed in 37 of the 40 cases by both methods. 3 cases were unsuitable for assessment.ConclusionsThis study indicated that CISH is a sensitive alternative to FISH in detecting HER2 gene amplification and may replace FISH in HER2 testing. Good agreement was observed between methods (98.5% – 119 out of 121 cases).Furthermore, as only 4 out of 121 cases were unsuitable for assessment (no signal or missing TMA cores) – it may be feasible to use TMA in diagnostics.     

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 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.     

Chromogenic in situ hybridization analysis of HER-2/neu status in cytological samples of breast carcinoma

The current use of humanized monoclonal antibody trastuzumab for the treatment of patients with metastatic breast cancer has made evaluation of HER-2/neu status an important clinical issue. Chromogenic in situ hybridization (CISH), in which the DNA probe is detected with an immunohistochemistry (IHC)-like peroxidase reaction, has been recently developed for the assessment of HER-2/neu status in formalin-fixed breast cancer specimens. We have applied the technique of dual-colour CISH using HER-2/neu and chromosome 17 centromere probes in 27 cytological smears, and these cytological samples were obtained from scrapings of fresh breast tumours. We also investigated HER-2/neu amplification and protein overexpression in the corresponding surgical tissues by CISH and IHC using the monoclonal antibody CB11. Of the 27 cytological cases, HER-2/neu gene amplification was observed in nine cases that were positive cases (2+ and 3+) for IHC. Among the 13 IHC positive cases (2+ and 3+), four of them showed no gene amplification. Identical results for the CISH technique were obtained in the matched surgical samples. The scrape samples from fresh breast tumour offer a monolayer cell population that is especially suitable for CISH. This study has shown that the cytological smear might be a good alternative for the CISH test.     

Immunohistochemical evaluation of HER-2/neu expression in infiltrating breast carcinoma: a study of reproducibility

OBJECTIVE: To determine interobserver and intraobserver reproducibility in the assessment of the HercepTest- and TAB250-immunostained slides. STUDY DESIGN: Three independent expert pathologists (two with and one without training in HercepTest assessment) evaluated the HercepTest and TAB250-immunostained slides of 108 infiltrating breast carcinomas with a triple-blind method. The evaluation was repeated, with the same method and sequence of view, after 60 days. RESULTS: Expert pathologists, after adequate training in HercepTest evaluation, could reach excellent interobserver (K=.911, P<.001) and intraobserver reproducibility (K of .863-.926; P <.001 for all). The percentage of disagreement in intraobserver reproducibility ranged from 0.9% to 3.7%. Interobserver and intraobserver reproducibility in the evaluation of TAB250-immunostained slides was good (K = .658, P < .001) and from good to excellent (K of .600-.895, P < .001 for all), respectively. CONCLUSION: Optimization of the level of accuracy in HercepTest evaluation is mandatory because the decision to initiate therapy with Herceptin depends on the result. Moreover, considering that the percentage of disagreement in intraobserver reproducibility ranges from 0.9% to 3.7%, it is advisable that two expert pathologists evaluate all HercepTest slides with a double-blind method. If there are discordant results, they must be discussed by the same pathologists.     

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.     

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.     

Fluorescence in situ hybridization for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy specimens

OBJECTIVE: To assess the usefulness of fluorescence in situ hybridization (FISH) for HER-2/neu amplification of breast carcinoma in archival fine needle aspiration biopsy (FNAB) specimens. STUDY DESIGN: All FISH performed on formalin-fixed, paraffin-embedded surgical specimens during January 2003-August 2003 at the University of California Irvine Medical Center were selected. Prior FNABs were retrieved. One cytologic slide was destained in each case. The results were compared with those obtained on histologic specimens using the paired t test. RESULTS: FISH was performed on 41 surgical specimens of breast carcinoma. Thirteen patients had prior FNABs that were positive for adenocarcinoma. After hybridization on destained fine needle aspiration slides, no cells were found in 2 cases, and the results were not readable in 2 cases. In the remaining 9 cases, the results, expressed as the ratio of copies of the HER-2/neu gene to copies of the chromosome 17 centromere, were 5.10, 1.14, 1.21, 1.12, 0.74, 1.11, 1.21, 9.87 and 2.4. Results on the corresponding histologic specimens were 5.25, 1.05, 1.13, 1.22, 1.13, 1.12, 1.21, 9.35 and 2.61, respectively. No significant difference was found (p = 0.23). CONCLUSION: HER-2/neu amplification status by FISH can be accurately and reliably evaluated in existing archival cytologic slides.     

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.     

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.     

Application of a gene vaccine targeting HER-2/neu in immunocontraception

HER-2/neu was widely used as a target for tumor prevention and therapy because of its overexpression in many tumors. However, it also plays an important role in proliferation of endometrium, embryo implantation, and development. Here, HER-2/neu was used in immunocontraception. A gene vaccine encoding the extracellular domain of human HER-2/neu was constructed. After immunization, it especially elicited both humoral and cellular responses in mice. Embryo implantation was interfered by intravenous and intraluminal injection of anti-HER-2/neu serum or lymphocytes. Lower fertility was induced after vaccination when compared with the control groups, while injuries to the uterus and ovary were not observed. Our results suggested a new and impactful target for contraceptive vaccines development.