Weak Epidermal Growth Factor Receptor Research Articles

Synthesis and in vitro evaluation of a novel thienopyrimidine with phototoxicity towards rat glioma F98 cells

Glioblastoma multiforme is one of the most aggressive cancer forms in humans, and has low recovery rates after surgery, ionizing radiation and chemotherapy. Therefore, there is a high interest in the development of new treatment methods, as for instance photodynamic therapy (PDT). It is here presented results of the cytotoxic properties of the novel compound N1-(4-(4-(benzyl(methyl)amino)thieno[2,3-d]pyrimidin-6-yl)benzyl)-N2,N2-dimethylethane-1,2-diamine (1), which was found ten-fold more active on the rat glioma cell model F98 than the reference drug Temozolomide (TMZ). Further cell survival studies showed a profound increase in F98 cell death on UVA-radiation (330 nm, 0.5 mW/cm2). Photochemical internalization induced delivery of compound 1, but in contrast to the cytostatic drug Bleomycin, a higher cytotoxicity was not observed. Localization studies using fluorescence microscopy revealed that compound 1 readily internalized into the cytosol but did not enter the cell nucleus. The compound was shown to be a relatively weak epidermal growth factor receptor inhibitor, which is not likely to explain its cytotoxicity. However, the quantum efficiency for generation of singlet oxygen was 23%, suggesting generation of reactive oxygen species as one possible mechanism. Although more studies are needed to reveal detailed mode of action, compound 1 is a promising photosensitizer candidate for further development in tests of animal models.

Amphiregulin in intestinal acute graft-versus-host disease: a possible diagnostic and prognostic aid

Amphiregulin, a weak epidermal growth factor receptor agonist, is elevated, while epidermal growth factor, a strong epidermal growth factor receptor agonist, is low in the blood of patients with severe acute graft-versus-host disease. However, the tissue expression and function of these epidermal growth factor receptor ligands in acute graft-versus-host disease target organs is unknown. We compared by immunohistochemistry expression of amphiregulin and epidermal growth factor in archived, formalin-fixed, paraffin-embedded intestinal tissues of 48 patients with biopsy-proven gastrointestinal acute graft-versus-host disease to 3 groups: (1) 10 non-hematopoietic cell transplant normal controls, (2) 11 patients with newly diagnosed ulcerative colitis (ulcerative colitis), (3) 8 patients with a clinical diagnosis of acute graft-versus-host disease despite pathologically non-diagnostic biopsies, (4) and 10 cases of cytomegalovirus colitis. We used a semi-quantitative score of 0 (absent) through 3 (strong) to describe the intensity of immunohistochemical staining. We correlated serum and tissue amphiregulin and epidermal growth factor in patients with acute graft-versus-host disease. Gastrointestinal amphiregulin was significantly lower in acute graft-versus-host disease biopsies (median score 1), ulcerative colitis (median score 1.5), and cytomegalovirus colitis (median score 1) than in normal colon (median score 2, p = 0.004, p = 0.03, p = 0.009 respectively). Amphiregulin expression in was low in 74% of acute graft-versus-host disease cases with or without significant apoptosis. Patients with acute graft-versus-host disease exhibiting the pattern of high gastrointestinal amphiregulin but low serum amphiregulin (n = 14) had the best 1-year survival at 71%, but patients with high serum amphiregulin had poorer survival (<30%) regardless of gastrointestinal amphiregulin expression. Overall, our results lead to the hypothesis that amphiregulin is released into the circulation from damaged intestinal epithelium and stroma, although contributions from other cellular sources are likely. Low gastrointestinal amphiregulin expression by immunohistochemistry may be further studied for its utility in the pathologic acute graft-versus-host disease diagnosis without classic apoptotic changes.

Immunohistochemical expression of epidermal growth factor receptor (EGFR) in South Asian head and neck squamous cell carcinoma: association with various risk factors and clinico-pathologic and prognostic parameters

BackgroundIn this study, we intend to determine the immunohistochemical expression of EGFR in cases of head and neck squamous cell carcinoma and its association with prognostic clinico-pathologic features.MethodsA total of 115 cases of head and neck squamous cell carcinoma treated at Liaquat National Hospital, Karachi, Pakistan, were included in the study. Clinico-pathologic features, risk factors, and recurrence status of cases were evaluated, and EGFR immunohistochemistry was performed.ResultsIn our study, 52 cases (45.2%) of head and neck SCC were positive and 10 cases (8.7%) were focal positive for EGFR expression, while 53 cases (46.1%) were negative for EGFR expression. High EGFR expression (> 70%) was noted in 6.1% (7 cases), while 12.2% (14 cases) and 26.1% (30 cases) revealed 51–70% and 11–50% EGFR expression respectively. On the basis of intensity, strong EGFR expression was noted in 13.9% (16 cases) while 16.5% (19 cases) and 23.5% (27 cases) revealed intermediate and weak EGFR expression respectively. Significant association of EGFR expression was noted with tumor stage and disease-free survival.ConclusionWe found a significant association of EGFR expression with tumor stage and disease-free survivals, which are the most important prognostic factors in head and neck squamous cell carcinoma; therefore, EGFR expression can help as a prognostic biomarker in head and neck squamous cell carcinoma. On the other hand, we suggest that molecular studies should be performed in squamous cell carcinoma of head and neck in our setup to identify patients that can avail response from anti-EGFR therapy.

Expression of epidermal growth factor receptor in patients with insulin resistance and seborrheic keratomas

to study the expression of epidermal growth factor receptor (EGFR) in patients with seborrheic keratomas (SK) and insulin resistance (type 2 diabetes mellitus (DM2)) and in those without concomitant carbohydrate metabolic disturbances. 80 patients with SK were examined. According to the presence or absence of DM2, the patients were divided into 2 groups: 1) 40 patients with concomitant DM2; 2) 40 people without carbohydrate metabolic disturbances. DM2 was diagnosed on the basis of laboratory studies and an endocrinologist's consultation. Histological and immunohistochemical (IHC) examinations using anti-EGFR antibodies were performed; two intact skin sections from the patients with DM2 and two intact skin sections from those without carbohydrate metabolic disturbances were used as a control. The ICH examination using anti-EGFR monoclonal antibodies revealed that Group 1 showed intense diffuse membrane staining of more than 50% of the cells in 32 (80%) patients, moderate (30-50% cells) and weak (10-30% cells) staining in 6 (15%) and 2 (5%) patients, respectively. Marked EGFR expression was also noted in two intact skin biopsy specimens taken from patients with DM2. In Group 2, the staining intensity was weak in more than 10% but less than 30% of the SK cells in 28 (70%) patients; moderate EGFR expression was observed in 9 (22.5%) and diffuse, pronounced, staining in more than 50% of the SK cells was in 3 (7.5%) patients. The intact skin biopsy specimens taken from 2 patients without carbohydrate metabolic disturbances displayed a weak EGFR expression in the basal cell layer of the epidermis. EGFR overexpression in SK may be a result of metabolic disorders rather than a diagnostic sign of malignant neoplasms of the internal organs. The increased EGFR expression revealed in patients with SK and concomitant DM2 is caused by insulin resistance and hyperinsulinemia, in which the dysregulation of insulin signal transmission into the cell leads to changes in EGF synthesis and signaling pathway that regulates cell proliferation and growth.

Epidermal growth factor receptor expression in carcinoma gallbladder: A prospective study in Indian scenario.

Gallbladder cancer (GBC) is an aggressive disease with poor prognosis and complete surgical resection offering the only cure. Increased epidermal growth factor receptor (EGFR) expression has been noted in various cancers including GBC. Several studies across the world have examined the expression of EGFR in GBC. This study has been done to see the EGFR expression in GBC in Indian context. Fifty cases of GBC were evaluated histopathologically using hematoxylin and eosin stained sections. Immunohistochemical assessment of EGFR expression was done, and scoring was done as per Kaufman et al. Data were collected, tabulated, and analyzed statistically by SPSS 16.0 version (Chicago, Inc., USA) software. Of 50 cases, 44 revealed EGFR over-expression while 6 were negative. Of the 44 cases, 10 had weak EGFR immunostaining intensity (1+), 26 had moderate (2+), and 8 showed strong EGFR immunostaining (3+). We found that most of the cases showing weak EGFR immunostaining intensity (1+) were well-differentiated tumor (70%) and cases with a strong EGFR immunostaining intensity (3+) were poorly differentiated cases of adenocarcinoma (75%). Moderately differentiated adenocarcinoma showed moderate EGFR immunostaining intensity (2+) in most of the cases (53.8%). EGFR is expressed in most of the cases of GBC. In well-differentiated adenocarcinoma, the EGFR expression is less compared to EGFR expression in poorly differentiated tumor, leading to the conclusion that the differentiation of the tumor and EGFR expression is inversely related. Thus, intensity of EGFR expression may correlate with aggressiveness of disease.

Epidermal growth factor receptor expression and gene copy number analysis in gastric carcinoma samples from Chinese patients.

Epidermal growth factor receptor (EGFR) expression and gene copy number have been observed to be associated with a positive clinical response to EGFR inhibitors. The present study aimed to evaluate EGFR expression and gene copy number in samples of gastric carcinoma (GC) from Chinese patients. EGFR expression and gene copy number were detected using immunohistochemistry and fluorescence in situ hybridization, in tissue array slides containing 150 individual samples of GC tissue. The association between EGFR status, clinicopathological features and overall patient survival was analyzed. Out of the 150 cases of GC evaluated, 63 (42.00%) demonstrated weak EGFR expression and 20 (13.33%) demonstrated EGFR overexpression. EGFR expression was observed to be associated with tumor location (P<0.05). Out of 104 cases of GC, which produced a clear FISH signal, 6 (5.77%) exhibited EGFR gene amplification and 5 (4.80%) exhibited balanced polysomy. Patients exhibiting GC, who demonstrated weak EGFR expression, EGFR overexpression or increased EGFR gene copy number, possessed an unfavorable prognosis. Multivariate analysis revealed that EGFR expression, tumor/node/metastasis stage and tumor location were potential independent unfavorable prognostic factors for GC patients. In conclusion, EGFR overexpression, gene amplification and polysomy were observed in GC patients and were associated with an unfavorable prognosis. Evaluation of EGFR status may therefore facilitate the identification of a subset of GC patients sensitive to treatment with EGFR-targeted therapies.

Abstract 2096: Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa .

Abstract Treatment of non-small cell lung cancer (NSCLC) with drugs targeting the epidermal growth factor receptor (EGFR), e.g., gefitinib and erlotinib, will eventually fail because of the development of secondary mutations such as T790M in EGFR. Strategies to overcome this resistance are therefore an urgent need. In this study, we synthesized a dozen of novel gefitinib analogues and evaluated their effects on L858R/T790M-EGFR harboring NSCLC cells, and reported that one of these gefitinib mimetics, N-(2-romo-5-(trifluoromethyl)phenyl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)uinazolin-4-amine (hereafter, V1801), triggered apoptosis of the NSCLC cells and overcame gefitinib-resistance in mice inoculated with NCI-H1975 cells. Though V1801 only moderately inhibited EGFR kinase activity, it markedly induced the expression of the BH3-only protein Noxa, and Noxa silencing significantly reduced V1801-induced apoptosis of NCI-H1975 cells. It is showed that V1801 interfered with the expression of the transcription factor c-Myc and the extracellular signal regulated kinase (Erk) pathway. V1801 in combination with proteasome inhibitor bortezomib exerted enhanced cytotoxicity in NCI-H1975 cells possibly due to potentiated induction of Noxa expression. These data indicate that gefinitib analogues with weak EGFR inhibitory activity may overcome drug-resistance via activation of BH-3 only pro-apoptotic proteins, and V1801 may have therapeutic potentials for NSCLC. Citation Format: Bo Zhang, Zhujun Yao, Guangbiao Zhou. Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2013-2096

Gefitinib analogue V1801 induces apoptosis of T790M EGFR-harboring lung cancer cells by up-regulation of the BH-3 only protein Noxa.

Treatment of non-small cell lung cancer (NSCLC) with drugs targeting the epidermal growth factor receptor (EGFR), e.g., gefitinib and erlotinib, will eventually fail because of the development of secondary mutations such as T790M in EGFR. Strategies to overcome this resistance are therefore an urgent need. In this study, we synthesized a dozen of novel gefitinib analogues and evaluated their effects on L858R/T790M-EGFR harboring NSCLC cells, and reported that one of these gefitinib mimetics, N-(2-bromo-5-(trifluoromethyl) phenyl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-amine (hereafter, V1801), triggered apoptosis of the NSCLC cells and overcame gefitinib-resistance in mice inoculated with NCI-H1975 cells. Though V1801 only moderately inhibited EGFR kinase activity, it markedly induced the expression of the BH3-only protein Noxa, and Noxa silencing significantly reduced V1801-induced apoptosis of NCI-H1975 cells. It is showed that V1801 interfered with the expression of the transcription factor c-Myc and the extracellular signal regulated kinase (Erk) pathway. V1801 in combination with proteasome inhibitor bortezomib exerted enhanced cytotoxicity in NCI-H1975 cells possibly due to potentiated induction of Noxa expression. These data indicate that gefinitib analogues with weak EGFR inhibitory activity may overcome drug-resistance via activation of BH-3 only pro-apoptotic proteins, and V1801 may have therapeutic potentials for NSCLC.

Heterogeneous Distribution of EGFR Mutations Is Extremely Rare in Lung Adenocarcinoma

Some studies have shown that epidermal growth factor receptor (EGFR) mutations can be heterogeneously distributed in individual tumors. In this study, we re-evaluated the distribution of EGFR mutations within tumors. We used multiple approaches, including an analysis of simultaneous dual hot spot mutations, a trans-sectional analysis of individual lung adenocarcinomas, and comparisons of the mutation patterns between primary and metastatic sites and between primary and recurrent tumors. None of the 862 tumors harboring an EGFR mutation showed simultaneous dual hot spot mutations, although identical EGFR mutations were found throughout individual tumors in a trans-sectional analysis involving 50 tumors divided into three parts and five lung adenocarcinomas divided into 100 parts. In addition, no discordant mutation patterns were detected among 77 paired primary and metastatic site samples or among 54 primary and recurrent tumor pairs. All of these results suggest that the heterogeneous distribution of EGFR mutations is extremely rare. However, it is possible that pseudoheterogeneity occurs because of a combination of mutant allele-specific imbalance and heterogeneously distributed EGFR amplification, especially when a less sensitive method is used for detection. Specifically, when EGFR amplification occurs, the mutant allele is amplified, and this amplification is involved in invasive growth. Accordingly, invasive growth area significantly over-represents the mutation signal. In contrast, weak EGFR mutation signals in the area without EGFR amplification may not reach the threshold of detection because of the mixture with normal cells. Such unbalanced mutation signals might lead to pseudoheterogeneity.

EGFR immunolabeling pattern may discriminate low-grade gliomas from gliosis

Overexpression of epidermal growth factor receptor (EGFR) is common in gliomas. Gliomas are infiltrating tumors in which neoplastic glial cells can be intermingled with reactive glial cells, particularly in diffuse low-grade gliomas. As overexpression of EGFR has also been described in gliosis, it can be difficult to evaluate EGFR immunolabeling in diffuse low-grade gliomas because of this cell mix. We compared EGFR immunolabeling between gliosis and low-grade gliomas in order to identify distinctive criteria. We studied EGFR expression in 28 cases of gliosis and 39 diffuse low-grade gliomas (23 astrocytomas and 16 oligodendrogliomas). EGFR immunohistochemistry staining was performed on paraffin-embedded sections with a mouse monoclonal antibody (clone 2-18C9; Dako). Co-expression of EGFR with Olig2, Mib-1, and p53 was assessed in seven cases of low-grade gliomas using double immunolabeling. Then, EGFR immunostaining was blindly tested on 22 small specimens of indeterminate glial lesions provided by a reference neuropathological center. Two pathologists of our local center were asked to classify the lesions into diffuse low-grade glioma or gliosis according to the pattern of EGFR expression. Weak expression of EGFR was commonly detected in gliosis (23/28 cases). Strongly-stained cells were absent. Positive cells had reactive glial cell morphology. EGFR expression in gliomas was characterized by constant strongly-stained cells (39/39 cases). All strongly-stained cells had a high nucleus-to-cytoplasm ratio, with minimal to moderate nuclear atypia. Most of the strongly EGFR-positive cells were Olig2-positive. All the cases displayed cells co-expressing EGFR and Mib-1. In three p53-positive tumors, many p53-positive cells were strongly EGFR-positive. On the basis of EGFR expression, 14 out of the 22 indeterminate cases were classified as gliomas and eight as gliosis by both pathologists. Concordance with the initial diagnosis established by the reference center and concordance between the pathologists were 100%. Our results confirm that weak EGFR expression can be detected by immunohistochemistry in gliosis. They show that strong EGFR expression may be specific for neoplastic glial cells. As all low-grade gliomas contained strongly-stained cells in our study, we believe that EGFR immunohistochemistry could be a useful tool for detection of neoplastic glial cells in case of indeterminate glial lesions.

Angiotensin II and epidermal growth factor receptor cross‐talk mediated by a disintegrin and metalloprotease accelerates tumor cell proliferation of hepatocellular carcinoma cell lines

The cross-talk pathway between angiotensin II (AngII) and the epidermal growth factor receptor (EGFR) mediated by epidermal growth factor (EGF)-like ligands cleaved by a disintegrin and metalloprotease (ADAM) has been elucidated in several cell types. Even though the liver is a representative angiotensinogen-producing organ, such cross-talk has never been elucidated in hepatocellular carcinomas (HCCs). We investigated whether AngII exerted a mitogenic effect on HCC cell lines through the AngII-EGFR cross-talk pathway. We determined the expression and/or phosphorylation status of AngII receptor type 1 (AGTR1), ADAM9, ADAM17, ERK1/2, STAT3, AKT and EGFR in five HCC cell lines using Western blotting. Proliferation and invasion activities were measured by ATP and Matrigel invasion assays, respectively. AGTR1 was expressed ubiquitously in HCC cell lines. EGFR expression in HepG2 was relatively weaker than that in the remaining HCC cell lines. The phosphorylation status of EGFR, ERK1/2, STAT3 and AKT was upregulated by AngII treatment in two EGFR-overexpressing cell lines (Huh7 and PLC/PRF/5), but not in HepG2 (showing weak EGFR expression). AngII stimulation significantly accelerated proliferation and invasion activities in Huh7 and PLC/PRF/5, and was inhibited by pretreatment with an ADAM inhibitor. A selective AGTR1 blocker significantly repressed proliferation activity in both cell lines, but did not significantly repress the invasion activity. Both chemical agents and neutralizing antibodies against ADAMs (ADAM9 and ADAM17) and EGF-like ligands suppressed EGFR transactivation and/or subsequent phosphorylation of ERK1/2, STAT3 and AKT. These results suggest that AngII-EGFR cross-talk signaling mediated by ADAMs is involved in the proliferation and invasion activities of several HCC cell lines.

Signal Strength Dictates Phosphoinositide 3-Kinase Contribution to Ras/Extracellular Signal-Regulated Kinase 1 and 2 Activation via Differential Gab1/Shp2 Recruitment: Consequences for Resistance to Epidermal Growth Factor Receptor Inhibition

Phosphoinositide 3-kinase (PI3K) participates in extracellular signal-regulated kinase 1 and 2 (ERK1-2) activation according to signal strength, through unknown mechanisms. We report herein that Gab1/Shp2 constitutes a PI3K-dependent checkpoint of ERK1-2 activation regulated according to signal intensity. Indeed, by up- and down-regulation of signal strength in different cell lines and through different methods, we observed that Gab1/Shp2 and Ras/ERK1-2 in concert become independent of PI3K upon strong epidermal growth factor receptor (EGFR) stimulation and dependent on PI3K upon limited EGFR activation. Using Gab1 mutants, we observed that this conditional role of PI3K is dictated by the EGFR capability of recruiting Gab1 through Grb2 or through the PI3K lipid product PIP(3), according to a high or weak level of receptor stimulation, respectively. In agreement, Grb2 siRNA generates, in cells with maximal EGFR stimulation, a strong dependence on PI3K for both Gab1/Shp2 and ERK1-2 activation. Therefore, Ras/ERK1-2 depends on PI3K only when PIP(3) is required to recruit Gab1/Shp2, which occurs only under weak EGFR mobilization. Finally, we show that, in glioblastoma cells displaying residual EGFR activation, this compensatory mechanism becomes necessary to efficiently activate ERK1-2, which could probably contribute to tumor resistance to EGFR inhibitors.

Effect of an epidermal growth factor receptor tyrosine kinase inhibitor on actin remodeling in anin vitrobladder cancer carcinogenesis model

Alteration of actin remodeling is a marker of malignant-associated field defect and a potential surrogate biomarker for chemoprevention trials. We tested erlotinib, a specific tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), on actin remodeling in a bladder carcinogenic model consisting of untransformed HUC-PC cells and transformed MC-T11 cells, both derived from the same normal human urothelial clone immortalized by SV40. Erlotinib had a selective growth inhibitory and actin remodeling effect on MC-T11 cells over HUC-PC cells, as examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and immunofluorescence labeling with laser scan cytometer analysis, respectively. The IC(50) of untransformed HUC-PC cells was significantly higher than that of transformed MC-T11 cells (P < 0.05, t test). The actin remodeling effect was more prominent at lower dosage levels (1/8-1/4 of IC(50)), which was accompanied by an increased cell adhesion and decreased motility. At higher dosage levels (1/2 of IC(50)), erlotinib induced a decreased adhesion and anoikis (detachment-associated apoptosis). The transformed MC-T11, but not HUC-PC, showed a weak constitutive EGFR phosphorylation activity, which was inhibited by erlotinib in a dose-response manner. However, on epidermal growth factor stimulation, both cell lines showed a similar dose-response inhibitory effect on phosphorylated EGFR and mitogen-activated protein kinase (MAPK; P44/P42) activities, and MAPK inhibitor PD98059 showed no specific effect on erlotinib-induced actin remodeling, suggesting that pathways other than MAPK (P44/P42) may be responsible for erlotinib-induced actin remodeling. The findings provide evidence to support erlotinib-based bladder cancer chemoprevention and using actin remodeling as a marker for erlotinib-based intervention trials.

Increased expression of EGFR and TGF-? in segmental renal dysplasia in duplex kidney

Renal dysplasia (RD) is a disorganised development of renal parenchyma that results in a deficit of functional renal tissue. It is known that the epidermal growth factor (EGF) and the transforming growth factor-alpha (TGF-alpha) enhance renal cell proliferation, migration and differentiation during kidney development through binding to the same EGF receptor (EGFR). The aim of the study was to analyse the expression of TGF-alpha and EGFR in the dysplastic kidney. The specimens of dysplastic upper poles duplex kidneys were surgically resected from 19 patients. Indirect immunohistochemistry was performed using the ABC method employing antibodies against EGFR and TGF-alpha, and gene expression using primers specific to the human genes. There was absent or weak EGFR and TGF-alpha immunoreactivity in normal kidney tissue. In dysplastic kidneys, there was strong TGF-alpha and EGFR immunoreactivity in the epithelium of primitive tubules and strong EGFR immunoreactivity in the connective tissue around the primitive tubules. Our findings of markedly increased local expression of EGFR and TGF-alpha in primitive tubules suggest that EGFR and TGF-alpha may play an important role in altering renal morphogenesis resulting in renal dysplasia.

Gene delivery using a receptor-mediated gene transfer system targeted to hepatocellular carcinoma cells.

For gene therapy to be effective in cancers, it is necessary to deliver therapeutic genes into cells with high specificity and efficiency. In this study, we examined the in vitro and in vivo gene delivery efficiency of a new, growth receptor-mediated gene transfer system in hepatocellular carcinoma (HCC). The effects of transfection of wild-type p53 using this system were also studied. The system consisted of a ligand oligopeptide for epidermal growth factor receptor (EGFR) recognition, a polypeptide for DNA binding, and an endosome-releasing oligopeptide for endosomolysis. Two human HCC cell lines and a normal liver cell line were used, and pCMV-beta-galactosidase (beta-gal) was used as a reporter gene. Both HCC cell lines had strong expression of EGFR and the in vitro transfer efficiency peaked at day 5 at about 50%. This finding was in contrast to the normal liver cell line, which had weak EGFR expression and less than 1% transfer efficiency throughout. For in vivo gene transfer in tumors produced by inoculating HCC cells in nude mice and with the vector-beta-gal gene complex injected peritumorally, beta-gal expression was detected within the tumors at 12 hr, peaked at day 5 involving about 50% of the tumor cells and persisted at 2 weeks. Using this vector system, transfection of wild-type p53 into Huh-7 cells that had mutated p53 resulted in significant growth inhibition of cancer cells accompanied by a decreased G2/M phase and increased p53 protein. In conclusion, this receptor-mediated gene transfer system appears to work specifically in HCC cells with high efficiency, and may be promising in delivering apoptotic and other genes into HCC cells.

Oncogene Expression and Amplification in Barrett Adenocarcinoma

Oncogene activation by amplification and/or overexpression has been implicated in the development and progression of a number of malignancies. To better define the role of oncogene activation in adenocarcinomas arising in the setting of Barrett esophagus, we examined 44 resected Barrett adenocarcinomas for c-erb B-2, epidermal growth factor receptor (EGFR), c-myc, and cyclin DI gene amplification as well as c-erb B-2 and EGFR expression. DNA was extracted from formalin-fixed paraffin-embedded tissue and analyzed by differential polymerase chain reaction, a technique that permits semiquantitative analysis of gene dosage on archival material. Formalinfixed paraffin-embedded sections were stained with monoclonal antibodies directed against either the internal domain of c-erb B-2 or the extracellular domain of EGFR with a labeled streptavidin biotin technique. C-erb B-2 amplification was identified in three (7%) cases. A plasma membrane pattern of c-erb B-2 immunoreactivity in &gt;50% of tumor cells correlated highly with c-erb B-2 amplification ( P=.00008). EGFR amplification was noted in 18% of cases and correlated highly with the intensity of EGFR immunoreactivity ( P=.0004). C-myc amplification was present in 18% of cases. No tumors showed cyclin DI amplification. Follow-up was available regarding 29 patients and showed decreased mean survival ( P=.029) in patients with strong EGFR immunoreactivity (5.8 months) versus those with weak or absent EGFR labeling (11.8 months), and a trend toward decreased one-year survival (P=.069) for patients with (17%) versus those without (52%) c-myc amplification. Our results indicate one or more selected oncogenes are amplified/overexpressed in some Barrett adenocarcinomas and that EGFR and c-erb B-2 overexpression correlates with amplification. Additionally, strong EGFR expression in tumor cells indicates a poorer prognosis.

Immunocytochemical localization of epidermal growth factor receptors in human testis from infertile subjects

To investigate the immunolocalization of the epidermal growth factor receptor (EGFR) in normal and pathological human testis by immunocytochemical technique. Cytologic specimens were obtained by bilateral fine needle aspiration (FNA) of the testis and stained in May Grünwald-Giemsa for the cytologic analysis; immunolocalization of EGFR was analyzed on duplicate slides from each testis using two anti-EGFR monoclonal antibodies and peroxidase-antiperoxidase technique. Infertility center of an academic unit. A total of 42 infertile patients, affected by various testicular diseases. The control group was made up of 10 normal sperm patients with autoimmune infertility and cytologic picture of normal spermatogenesis. Exogenous FSH was administered 75 IU IM on alternate days for 3 months on 16 of the infertile patients who showed oligospermia and normal FSH plasma levels. Semen analysis and testicular FNA (and after cytologic and immunocytochemical studies) were repeated at 3 months of treatment. Luteinizing hormone and FSH plasma levels were determined by RIA methods; qualitative and quantitative parameters for the cytologic evaluation are reported in our previous works. The cytologic analysis permitted identification of seven classes of infertile subjects, characterized by different cytologic pictures. Epidermal growth factor receptor immunostaining evidenced weak positivity on Sertoli and germ cells (with the exception of spermatozoa) in the presence of normal germ line and normal FSH plasma levels and strongly intense positivity in the presence of serious hypospermatogenesis, spermatogonial or spermatocytic arrest, and Sertoli cell-only syndrome. These conditions were characterized by higher FSH plasma levels than normal controls. All of the subjects who received exogenous FSH, with moderate hypospermatogenesis or spermatidic arrest, showed on Sertoli and germ cells a weak EGFR immunostaining before the treatment and intense immunostaining after the treatment. These results confirm recent demonstrations of EGFRs in human testis and evidence different EGFR immunostaining in the presence of various degrees of testicular damage, suggesting a role of this growth factor in growth and differentiation of the germ cells throughout spermatogenesis. The observation that intense EGFR immunostaining was found in subjects showing high FSH plasma levels and in all of the patients who received exogenous FSH, supports a possible role of this gonadotropin in the modulation of the EGFR expression.

Expression of epidermal growth factor receptors by odontogenic jaw cysts.

The expression of epidermal growth factor receptor (EGFr) by odontogenic epithelium was studied in odontogenic cysts (n = 35), ameloblastoma (n = 6), and periapical granulomas containing proliferating epithelial rests of Malassez (n = 7) using a panel of monoclonal antibodies to EGFr (clone E30, F4 and C11) known to react with formalin-fixed, paraffin-embedded sections. Odontogenic epithelium in all specimens demonstrated immunoreactivity with all three antibodies. Clone E30 consistently gave the most intense, membrane located staining pattern of the three antibodies tested. Generally, staining of epithelial cells progressively diminished with movement away from the basal cell layers toward the most superficial layers of cystic lining or centre of epithelial rests and tumour islands. Developmental odontogenic cysts (odontogenic keratocysts, n = 13; dentigerous cysts, n = 11) and ameloblastoma (follicular type, n = 5; unicystic type, n = 1) expressed a higher level of EGFr staining than inflammatory cysts (radicular cysts, n = 11) and the proliferating epithelial rests in periapical granulomas. However, foci of weak EGFr staining of odontogenic keratocyst lining, similar to that seen in radicular cysts, were found in areas associated with inflammation. In addition, epithelial rests not associated with inflammatory cell infiltrates exhibited stronger reactivity for EGFr than proliferating rests within periapical granulomas. These results indicate that the level of EGFr expression by odontogenic cysts and rests is related to the presence of inflammation within adjacent connective tissue and that there is no detectable difference in receptor expression between developmental cysts and ameloblastoma.

Immunohistochemical study of epidermal growth factor receptor and estrogen receptor in human thyroid tumors

Expressions of epidermal growth factor receptor (EGFR) and estrogen receptor (ER) in human thyroid tumors were examined immunohistochemically in frozen sections. Forty-eight fresh surgical specimens of thyroid tissues, 19 carcinomas, 12 adenomas, 7 adenomatous goiters and 10 normal thyroid tissues were studied. All cases, including normal thyroid tissues, expressed the EGFR immunoreaction in the cytoplasm. Incidences of stronger than (+) staining intensity of EGFR were 84% (16/19) in carcinoma, 67% (8/12) in adenoma, 57% (4/7) in adenomatous goiter and 30% (3/10) in normal thyroid tissue. This result suggests that the staining intensity of EGFR expression is associated with malignant potential. The frequency of ER positive cases was 11% (2/19) in carcinoma, 17% (2/12) in adenoma and 0% in both adenomatous goiter and normal thyroid tissue. No relationship between EGFR and ER expression was recognized. However, a case of papillary carcinoma with strong ER immunoreactive intensity expressed weak EGFR intensity, suggesting an inverse relation in immunoreaction between EGFR and ER in papillary carcinoma.