Amplification and expression of c-erbB2 oncogene in normal, hyperplastic, and malignant endometria

OBJECTIVE: To study the correlation of development and survival with amplification of c-erbB2 oncogene in endometrial carcinoma. METHODS: The amplification and expression of c-erbB2 oncogene were determined from formalin-fixed, paraffin-embedded tissues of 25 normal, 31 hyperplastic, and 72 malignant samples of the endometrium in 128 patients, using differential polymerase chain reaction (DPCR) and an immunohistochemical technique. RESULTS: Amplified c-erbB2 (2 to 12 copies) were found in two of 25 (8.0%) normal, 15 of 31 (48.4%) hyperplastic, and 45 of 72 (62.5%) malignant samples. When the results of DPCR were compared with those of the immunohistochemical method, the negative findings concide well with one another, i.e., nonamplification was associated with the absence of immunoreactivity. It was noted that the amplified c-erbB2 was found more significantly in complex and a typical hyperplasias than in simple hyperplasias. The high-level c-erbB2 amplification (at least five copies) was significantly correlated with the histological grade of endometrial carcinoma and vascular or lymphatic invasion. No correlation was seen between c-erbB2 amplification and overall survival in the present group of patients. CONCLUSIONS: The amplified c-erbB2 may play a potential role in the early development of some endometrial carcinomas. The high-level c-erbB2 amplification may identify a subset of aggressive endometrial carcinoma that involves vascular or lymphatic invasiveness and poor cell differentiation playing the role of a marker for clinical prognosis.     

Amplification and expression of the c-erbB-2 oncogene in normal, hyperplastic, and malignant endometria

The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing < 0.5 mM glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 mumol min-1 per 10(6) cells in transformants compared with parental levels of 0.25 mumol min-1 per 10(6) cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.     

The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy

To examine whether and how local somatothermal stimulation inhibits the function of the sphincter of Oddi (SO) in humans and in animals with different types of SO, we measured the activity of SO in anesthetized cats and rabbits by using continuously perfused open-tip manometric methods. Local somatothermal stimulation was achieved by applying an electroheating rod 0.5 cm away from the skin area near the right subcostal region. A heating pad was applied to the corresponding area in patients undergoing endoscopic retrograde cholangiopancreatography and biliary manometry. The motility of the biliary tract in cats, in terms of gall bladder pressure, tonic and phasic contraction pressure and frequency of SO before and during local heat were significantly different, respectively. The local heat-induced SO relaxation was not inhibited by pretreatment with atropine, propranolol, phentolamine or anti-cholecystokinin-octapeptide, but was almost completely blocked by infiltration of local anesthetics. Pretreatment with a nitric oxide synthesis inhibitor also blocked the relaxation, which was reversed by pretreatment with L-arginine, but not by D-arginine. The inhibition of SO motility by local heat in rabbits was also blocked by pretreatment with L-NAME, and this blockade was reversed by L-arginine. Application of local heat on patients demonstrated obvious inhibitory SO responses. We conclude that local somatothermal stimulation inhibits the SO motility in animals with different types of SO through the activation of heat-sensitive neural release of nitric oxide. This procedure may represent a simplified approach for the treatment of diseases with hypofunction of the L-arginine/NO pathway.     

Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas

PURPOSE: To test whether p34cdc2 overexpression, CD44s downregulation, and HER-2/neu amplification correlate with disease recurrence after radical prostatectomy, and to evaluate a possible biologic association between p34cdc2 and HER-2/neu expression. MATERIALS AND METHODS: Immunohistochemical (IHC) detection of both p34cdc2 cyclin-dependent kinase (CDK) and CD44s expression and fluorescence in situ hybridization (FISH)-based analysis of HER-2/neu gene status were performed on formalin-fixed, paraffin-embedded sections of 106 prostatic adenocarcinomas (PACs). Findings were correlated with Gleason grade, pathologic stage, DNA ploidy, and postsurgical biochemical disease recurrence. RESULTS: CDK overexpression correlated with tumor grade (P = .001), DNA ploidy (P = .001), pathologic stage (P = .04), and disease recurrence (P = .01). CD44s downregulation correlated with grade (P = .03), ploidy (P = .01), and recurrence (P = .02). HER-2/neu amplification correlated with grade (P = .001), ploidy (P = .001), and recurrence (P = .01). On multivariate analysis, CDK overexpression independently predicted recurrence (P = .001) after prostatectomy. CDK expression correlated with HER-2/neu status with 32 of 65 (49%) tumors that overexpressed CDK and showed concomitant HER-2/neu amplification (P = .04). CONCLUSION: This study showed that p34cdc2, CD44s, and HER-2/neu are variably expressed or amplified in prostatic carcinoma and that such alteration may affect tumor behavior. In addition, CDK overexpression and HER-2/neu amplification may be biologically related.     

Increased natural killer cell activity correlates with low or negative expression of the HER-2/neu oncogene in patients with breast cancer

BACKGROUND: Increased expression of the HER-2/neu oncogene in breast cancer correlates with decreased estrogen receptor concentration and seems to be an important prognostic factor. The authors investigated whether there is a correlation between HER-2/neu expression and immunologic parameters representing tumor defense in patients with breast cancer. METHOD: A Western blot analysis was used to investigate HER-2/neu expression, whereas a chromium-release assay using the K562 cell line as target was used to measure natural killer (NK) cell activity. RESULTS: In patients with breast cancer, NK cell activity was significantly higher compared with patients with benign tumors (P = 0.006) or healthy control subjects (P = 0.002). Moreover, 23.3% of patients with breast cancer showed an overexpression of HER-2/neu protein. Within this group of patients, NK cell activity was significantly lower (45.6 +/- 16.1%) compared with the group with no HER-2/neu overexpression (57.3 +/- 11.0%). NK cell activity did not increase in patients with HER-2/neu overexpression. Thus, there was a statistically significant correlation of cytolytic effector cell function with HER-2/neu expression of the tumor (P = 0.003), and HER-2/neu overexpression correlated with a negative estrogen receptor status (P = 0.005). CONCLUSION: These data add further evidence to previous observations from the authors' laboratory that certain tumor characteristics may be associated with reactions of the host with breast cancer.     

Estrogen receptor, progesterone receptor, and HER-2/neu protein in breast cancers from pregnant patients

BACKGROUND: Because the occurrence of breast cancer during pregnancy is uncommon and because the high levels of estrogens and progestins associated with pregnancy could cause false-negative results from ligand binding assays (LBA), the actual incidence of steroid hormone receptor positivity in tumors from this subset of women is unclear. METHODS: Estrogen receptor (ER) and progesterone receptor (PgR) were determined using LBA methods in 15 tumors from 15 pregnant patients with breast cancer. In addition, immunohistochemistry was done for ER, PgR, pS2, heat shock protein 27 (hsp27), and HER-2/neu on 12 of the 15 tumors. RESULTS: Five of 15 (33%) tumors were positive for ER by LBA, compared with 52% of tumors from age-matched nonpregnant patients. Six of 12 (50%) were ER-positive by immunohistochemistry. For PgR, 7 of 15 (47%) tumors were positive by LBA, compared with 42% of tumors from nonpregnant patients. Ten of 12 (83%) stained positive for PgR. By LBA, 67% of tumors studied were positive for ER or PgR or both, as opposed to 57% of tumors from the nonpregnant comparison group. Two other estrogen receptor-mediated proteins, pS2 and hsp27, were present by staining in 8 of 12 (67%) and 10 of 12 (83%) of tumors, respectively. Seven of 12 tumors (58%) had positive staining for HER-2/neu, whereas only 16% of age-matched nonpregnant patients had positive-staining tumors. CONCLUSION: By LBA, the incidence of ER and PgR in breast tumors from pregnant women was not significantly different from that of tumors from nonpregnant age-matched patients. Some ER-negative tumors were PgR, pS2, or hsp27 positive, indicating that an intact estrogen response system was operative although ER was not detectable by standard LBA.     

Prognostic significance of HER-2/neu overexpression in stage I adenocarcinoma of lung

BACKGROUND: Even with early diagnosis and adequate resection, the 5-year survival rate for stage I lung cancer patients is around 60% to 70%. Overexpression of HER-2/neu protein is associated with poor prognosis in lung cancers. In this study, we evaluated the expression of HER-2/neu in cancer cells of lung and assessed their clinicopathologic and prognostic significance. METHODS: From 1986 to 1995, clinical data on 42 consecutive patients who underwent complete surgical resection for stage I lung adenocarcinoma were collected. Expression of HER-2/neu in paraffin-embedded tumor samples was determined by immunohistochemistry and scored with a semiquantitative method. RESULTS: Twenty-one of 42 patients were positive for HER-2/neu overexpression in tumor. Compared with patients with low HER-2/neu expression, patients with HER-2/neu overexpression had a significantly higher incidence of early tumor recurrence (p = 0.014). Survival was also significantly better in patients without HER-2/neu overexpression than in those with HER-2/neu overexpression (p = 0.0047). By univariate analysis, HER-2/neu overexpression and poor cell differentiation are two important factors correlated with poor prognosis. CONCLUSIONS: Expression of HER-2/neu oncoprotein in stage I lung adenocarcinoma can predict the tumor's aggressiveness. Early tumor recurrence was frequently detected in patients with HER-2/neu overexpression. We recommend an individualized therapeutic strategy based on the level of HER-2/neu oncoprotein in the tumor cells.     

Study of Her-2/neu oncogene in relation to prognosis of human breast cancer

A follow-up study of 143 cases of human breast cancer for over 5 years proved that Her-2/neu oncogene overexpression is much more common in the high risk group (patients died within 5 years) in comparison with the low risk group (patients survived over 5 years). The difference between these 2 groups was statistically significant. The Her-2/neu oncogene positive rate in infiltrative ductal carcinoma was 33.3%, the lower the differentiation, the higher the positive rate. Histological typing is also related to the positive rate, comedocarcinoma (intraductal carcinoma) expresses the highest positive rate while lobular carcinoma the lowest. Selection of fixation fluid and the mastering of diagnostic criteria are also important. In the author's opinion, only membrane staining in monoclonal antibody C-erbB-2 can be recognized as truly positive. In conclusion, Her-2/neu oncogene expression can be used as a supplemental marker when considering prognosis in breast cancer.     

Quantitation of HER-2/neu and c-myc gene amplification in breast carcinoma using fluorescence in situ hybridization

HER-2/neu and c-myc amplification or overexpression have been reported to be associated with poor prognosis in breast carcinoma. The prognostic significance, however, remains somewhat controversial, partly because of discrepancies among different methodologies used for detection of the oncogene amplification or overexpression. Fluorescence in situ hybridization (FISH) has recently been shown to be a useful technique for analyzing genetic alterations in interphase nuclei in various tumors. In this study, FISH was used to quantitate HER-2/ neu and c-myc gene amplification in touch preparations of frozen tissue from 100 node-negative breast carcinomas. HER-2/neu amplification was found to be associated with an abnormal DNA index (P < .001) and tumor size (P < .04). Amplification of c-myc was associated with S phase (P < .0003), abnormal DNA index (P < .003), and a negative estrogen receptor status (P < .01). The coamplification of both oncogenes was strongly associated with an abnormal DNA index (P < .0001) and with tumor size (P < .009). The use of FISH for detection of HER-2/neu gene amplification was 92% concordant with immunocytochemistry (ICC) used for detection of overexpression of HER-2/neu protein. Fifteen of the 100 cases were both amplified for HER-2/neu by FISH and positive by ICC analysis. Seven cases without HER-2/neu gene amplification demonstrated HER-2/neu protein overexpression by ICC. One HER-2/neu-amplified case was negative by ICC. Repeat analysis of a subset of cases showed FISH to be a more reproducible method than ICC in the analysis of HER-2/neu in touch preparations of breast carcinoma. FISH is a rapid and reproducible method that allows the accurate measurement of the level of oncogene amplification within interphase nuclei. The use of FISH should provide a more accurate assessment of the prognostic significance of oncogene amplification in breast carcinoma.     

High-titer HER-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer

PURPOSE: To evaluate HER-2/neu-specific antibody immunity in patients with breast cancer, to determine the rate of occurrence of serum antibodies to HER-2/neu in patients with breast cancer, and to relate the presence of specific immunity to overexpression of HER-2/neu protein in primary tumor. METHODS: The antibody response to HER-2/neu protein was analyzed in 107 newly diagnosed breast cancer patients. Sera was analyzed for the presence of HER-2/neu-specific antibodies with a capture enzyme-linked immunosorbent assay (ELISA) and verified by Western blot. Sera from 200 volunteer blood donors was used as a control population. RESULTS: The presence of antibodies to HER-2/neu correlated with the presence of breast cancer. HER-2/neu antibodies at titers of > or = 1:100 were detected in 12 of 107 (11%) breast cancer patients versus none of 200 (0%) normal controls (P < .01). The presence of antibodies to HER-2/neu also correlated to overexpression of HER-2/neu protein in the patient's primary tumor. Nine of 44 (20%) patients with HER-2/neu-positive tumors had HER-2/neu-specific antibodies, whereas three of 63 (5%) patients with HER-2/neu-negative tumors had antibodies (P = .03). The antibody responses could be substantial. Titers of greater than 1:5,000 were detected in five of 107 (5%). CONCLUSION: The presence of HER-2/neu antibodies in breast cancer patients and the correlation with HER-2/neu-positive cancer implies that immunity to HER-2/neu develops as a result of exposure of patients to HER-2/neu protein expressed by their own cancer. These findings should stimulate further studies to develop the detection of immunity to oncogenic proteins as tumor markers, as well as the development and testing of vaccine strategies to induce and augment immunity to HER-2/neu for the treatment of breast cancer or prevention of recurrent disease.     

The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells

INTRODUCTION: Determination of the optimal electrode configuration during implantable cardioverter defibrillator (ICD) implantation remains largely an empirical process. This study investigated the feasibility of using a finite element model of the thorax to predict clinical defibrillation metrics for internal defibrillation in humans. Computed defibrillation metrics from simulations of three common electrode configurations with a monophasic waveform were compared to pooled metrics for similar electrode and waveform configurations reported in humans. METHODS AND RESULTS: A three-dimensional finite element model was constructed from CT cross-sections of a human thorax. Myocardial current density distributions for three electrode configurations (epicardial patches, right ventricular [RV] coil/superior vena cava [SVC] coil, RV coil/SVC coil/subcutaneous patch) and a truncated monophasic pulse with a 65% tilt were simulated. Assuming an inexcitability threshold of 25 mA/cm2 (10 V/cm) and a 75% critical mass criterion for successful defibrillation, defibrillation metrics (interelectrode impedance, defibrillation threshold current, voltage, and energy) were calculated for each electrode simulation. Values of these metrics were within 1 SD of sample-size weighted means for the corresponding metrics determined for similar electrode configurations and waveforms reported in human clinical studies. Simulated myocardial current density distributions suggest that variations in current distribution and uniformity partially explain differences in defibrillation energy requirements between electrode configurations. CONCLUSION: Anatomically realistic three-dimensional finite element modeling can closely simulate internal defibrillation in humans. This may prove useful for characterizing patient-specific factors that influence clinically relevant properties of current density distributions and defibrillation energy requirements of various ICD electrode configurations.     

HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas

PURPOSE: The HER-2/neu gene codes for a membrane receptor protein that is homologous, but distinct from the epidermal growth factor receptor. This investigation was performed to validate fluorescence in situ hybridization (FISH) as a sensitive and specific method for assessing HER-2/neu gene amplification in archival tissue and to test whether this alteration is associated with poor prognosis. MATERIALS AND METHODS: HER-2/neu gene amplification was determined by FISH in 140 archival breast cancers, previously characterized for gene amplification by Southern hybridization or dot-blot hybridization, and for gene expression by Northern hybridization, Western immunoblot, or immunohistochemistry. A separate cohort of 324 node-negative breast cancers was assessed for amplification by FISH to determine the utility of HER-2/neu gene amplification. RESULTS: Relative to solid-matrix blotting procedures, FISH analysis of HER-2/neu gene amplification showed a sensitivity of 98% and a specificity of 100% in 140 breast cancers. Among patients treated by surgery only, the relative risks (relative hazard) of early recurrence (recurrent disease within 24 months of diagnosis), recurrent disease (at any time), and disease-related death were statistically significantly associated with amplification. The prognostic information contributed by HER-2/neu amplification was independent of the other markers studied. CONCLUSION: FISH was an alternative technique for determining gene amplification and had some distinct advantages over Southern hybridization. Our results demonstrate that HER-2/neu gene amplification in the absence of adjuvant therapy is an independent predictor of poor clinical outcome and is a stronger discriminant than tumor size. Women with small tumors that had gene amplification were at increased risk of recurrence and disease-related death.     

NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer

We identified an NH2-terminally truncated HER-2/neu product of M(r) 95,000 with in vitro kinase activity by Western blotting and immunoprecipitations using domain-specific antibodies. p95 levels correlated with the extracellular domain (ECD) shed from different cells under varied conditions. Both ECD and p95 were at approximately 20-fold lower levels in SKOV3 ovarian carcinoma cells, as compared to BT474 breast carcinoma cells. Both were stimulated by treatment of cells with the phorbol ester tumor promoter phorbol 12-myristate 13-acetate and the lysosomotrophic agent chloroquine. The hydroxamate inhibitor of metalloproteases, TAPI, suppressed both p95 and ECD in a dose-dependent fashion, with maximal inhibition at < or = 10 microM in BT474 cells. Cancer tissues were analyzed by Western blotting and scored for p95HER-2/neu and for p185HER-2/neu expression. Breast and ovarian cancer tissues were both found to express p95HER-2/neu in addition to p185HER-2/neu. Of 161 breast cancer tissues, 22.4% expressed p95, 21.7% overexpressed p185, and 14.3% were p95 positive and overexpressed p185. A higher proportion of node-positive patients (23 of 78) than node-negative patients (9 of 63) expressed p95 in all tumors combined (P = 0.032). In the group that overexpressed p185, those that contained p95 were associated with node-positive patients (15 of 21), whereas those that were p95 negative were associated with node-negative patients (8 of 11; P = 0.017). Neither p95- nor p185-rich patients significantly correlated with tumor size or with hormone receptor status in this study. Our findings show that breast cancers, which express the HER-2/neu oncogene, are heterogeneous with respect to HER-2/neu protein products. p95HER-2/neu appears to distinguish tumors that have metastasized to the lymph nodes from those in node-negative patients.     

The correction of immunoendocrine disorders in patients with hyperplastic processes in the endometrium

The nervous systems of invertebrates and vertebrates consist of neuronal networks of varying complexity, and the elucidation of the organization of these networks is essential if we are to understand neural function. Up until the mid-19th Century gross dissection was the primary tool available to scientists to study the nervous system. The development of neurohistological techniques, electrical stimulation, and observation of neural function in humans and animals following injury added rapidly to our understanding of the nervous system during the following century. Over the last 3 decades investigators seeking to unravel the complexities of neural circuits have made use of analytical methods based upon the biological properties of neurons, including orthograde and retrograde axonal transport of tracer substances, the expression of particular genes and gene products that can be assessed with immunocytochemical or in situ methods, and the imaging of the utilization of oxygen or glucose by active populations of neurons. Advances in neuroscience have led to an enormous expansion in our knowledge of normal neural functioning and how that function is altered by injury or disease. Modern studies of neuronal organization have been at the center of our increased understanding of how the brain works.     

Expression of p53 protein in normal, dysplastic, and malignant gastric mucosa: an immunohistochemical study

Mutations in the p53 nuclear oncogene are the most frequent genetic abnormalities encountered in human malignancies. Using the polyclonal antibody CM-1, we have examined the expression of the p53 oncoprotein immunohistochemically in archival material of normal, dysplastic, and malignant gastric mucosa. Abnormal expression of this protein was not observed in biopsies of normal gastric tissue (n = 30) but was detected in 22 of the 36 gastric cancers analysed (61 per cent). Nuclear staining was diffuse in 15 of the positive cancer cases, the remaining seven showing a more varied heterogeneous staining pattern. Abnormal p53 protein was not detected in mild (n = 14) or moderate (n = 16) gastric dysplasia but was present in 3 out of 15 severe dysplasia cases. The results suggest that expression of the p53 oncoprotein is a common finding in gastric cancer and occurs as a late event in the malignant transformation process.     

Immunohistochemical localization of p21(WAF1/CIP1) in normal, hyperplastic, and neoplastic uterine tissues

Assuming that the chemical reactions used to synthesize a combinatorial library member are successful, then knowledge of the specific reaction sequence is equivalent to knowing the member's chemical identity. Because the determination of chemical identity is typically not automatable and requires a substantial amount of material, schemes that encode a member's reaction history onto the reaction platform are of value. The primary benefits of encoding are relational nomenclature (all methods) and automated handling (some methods). Encoding methods evaluated to date are spatial, graphical, chemical, spectrometric, electronic, and physical.     

Identification of HLA-A3-restricted cytotoxic T lymphocyte epitopes from carcinoembryonic antigen and HER-2/neu by primary in vitro immunization with peptide-pulsed dendritic cells

The human carcinoembryonic antigen (CEA) and HER-2/neu are potential target antigens for CTL specific immunotherapy for common malignancies such as breast, lung, colon, and gastric carcinomas. Several CTL epitopes restricted by HLA-A2, the most common human histocompatibility molecule, have been previously reported. However, to develop CTL-based immunotherapies for the general population, it is necessary to identify epitopes restricted by other common histocompatibility alleles. Here, we describe two HLA-A3-restricted CTL epitopes from the CEA and HER-2/neu antigens. HLA-A3 binding synthetic peptides from CEA and HER-2/neu were tested for immunogenicity by in vitro primary CTL induction protocol using peripheral blood mononuclear cells from normal healthy volunteers. One peptide from CEA (CEA[9(61)]: HLFGYSWYK) and one peptide from HER-2/neu (HER2[9(754)]: VLRENTSPK) were shown to induce CTL that was capable of killing a tumor cell line expressing HLA-A3 and the corresponding tumor-associated antigen. Additional MHC binding studies with the most common HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301, and -A*6801), demonstrated that CEA[9(61)] binds five of five A3 supertype molecules with high affinity, and the HER2[9(754)] epitope was able to bind to four of the same five alleles. These results indicate that these two new CTL epitopes should be immunogenic in individuals expressing either HLA-A3, or other members of the HLA-A3 superfamily.