Epidermal Growth Factor Receptor Immunoreactivity Research Articles

Factors for Differential Outcome Across Cancers in Clinical Molecule-Targeted Fluorescence Imaging.

Clinical imaging performance using a fluorescent antibody was compared across 3 cancers to elucidate physical and biologic factors contributing to differential translation of epidermal growth factor receptor (EGFR) expression to macroscopic fluorescence in tumors. Methods: Thirty-one patients with high-grade glioma (HGG, n = 5), head-and-neck squamous cell carcinoma (HNSCC, n = 23), or lung adenocarcinoma (LAC, n = 3) were systemically infused with 50 mg of panitumumab-IRDye800 1-3 d before surgery. Intraoperative open-field fluorescent images of the surgical field were acquired, with imaging device settings and operating room lighting conditions being tested on tissue-mimicking phantoms. Fluorescence contrast and margin size were measured on resected specimen surfaces. Antibody distribution and EGFR immunoreactivity were characterized in macroscopic and microscopic histologic structures. The integrity of the blood-brain barrier was examined via tight junction protein (Claudin-5) expression with immunohistochemistry. Stepwise multivariate linear regression of biologic variables was performed to identify independent predictors of panitumumab-IRDye800 concentration in tissue. Results: Optimally acquired at the lowest gain for tumor detection with ambient light, intraoperative fluorescence imaging enhanced tissue-size dependent tumor contrast by 5.2-fold, 3.4-fold, and 1.4-fold in HGG, HNSCC, and LAC, respectively. Tissue surface fluorescence target-to-background ratio correlated with margin size and identified 78%-97% of at-risk resection margins ex vivo. In 4-μm-thick tissue sections, fluorescence detected tumor with 0.85-0.89 areas under the receiver-operating-characteristic curves. Preferential breakdown of blood-brain barrier in HGG improved tumor specificity of intratumoral antibody distribution relative to that of EGFR (96% vs. 80%) despite its reduced concentration (3.9 ng/mg of tissue) compared with HNSCC (8.1 ng/mg) and LAC (6.3 ng/mg). Cellular EGFR expression, tumor cell density, plasma antibody concentration, and delivery barrier were independently associated with local intratumoral panitumumab-IRDye800 concentration, with 0.62 goodness of fit of prediction. Conclusion: In multicancer clinical imaging of a receptor-ligand-based molecular probe, plasma antibody concentration, delivery barrier, and intratumoral EGFR expression driven by cellular biomarker expression and tumor cell density led to heterogeneous intratumoral antibody accumulation and spatial distribution whereas tumor size, resection margin, and intraoperative imaging settings substantially influenced macroscopic tumor contrast.

Biphasic changes in airway epithelial cell EGF receptor binding and phosphorylation induced by components of hogbarn dust

Purpose of the study: Workers in enclosed hogbarns experience an increased incidence of airway inflammation and obstructive lung disease, and an aqueous hogbarn dust extract (HDE) induces multiple inflammation-related responses in cultured airway epithelial cells. Epidermal growth factor receptor (EGFR) phosphorylation and activation has been identified as one important mediator of inflammatory cytokine release from these cells. The studies here investigated both early and late phase adaptive changes in EGFR binding properties and subcellular localization induced by exposure of cells to HDE. Materials and Methods: Cell surface EGFRs were quantified as binding to intact cells on ice. EGFR phosphorylation, expression, and localization were assessed with anti-EGFR antibodies and either blotting or confocal microscopy. Results: In BEAS-2B and primary human bronchial epithelial cells, HDE induced decreases in cell surface EGFR binding following both 15-min and 18-h exposures. In contrast, H292 cells exhibited only the 15-min decrease, with binding near the control level at 18 hr. Confocal microscopy showed that the 15-min decrease in binding is due to EGFR endocytosis. Although total EGFR immunoreactivity decreased markedly at 18 hr in confocal microscopy with BEAS-2B cells, immunoblots showed no loss of EGFR protein. HDE stimulated EGFR phosphorylation at both 15 min and 18 hr in BEAS-2B cells and primary cells, but only at 15 min in H292 cells, indicating that the different EGFR binding changes among these cell types is likely related to their different time-dependent changes in phosphorylation. Conclusions: These studies extend the evidence for EGFRs as important cellular targets for components of HDE and they reveal novel patterns of EGFR phosphorylation and binding changes that vary among airway epithelial cell types. The results provide both impetus and convenient assays for identifying the EGFR-activating components and pathways that likely contribute to hogbarn dust-induced lung disease in agricultural workers.

Immunohistochemical and genetic evaluations of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma

ObjectivesTo study the role of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma (OSCC), protein expression, gene amplification, and mutations were analyzed in OSCC as well as leukoplakia. Immunoreactivity of Ki-67 and K-Ras gene mutations were simultaneously evaluated. Material and methodsEighty-two OSCC and 10 leukoplakia specimens were immunohistochemically examined with antibodies against EGFR and Ki-67. Amplification of EGFR was evaluated by chromogenic in situ hybridization (CISH). In 14 OSCC samples, EGFR exons 19, 21 and K-Ras exon 2 were analyzed by direct DNA sequencing. ResultsImmunohistochemical reactivity for EGFR and Ki-67 was detected in epithelial cells of leukoplakia and in carcinoma cells of OSCC. EGFR and Ki-67 immunoreactivity was significantly higher in OSCC than in leukoplakia. Leukoplakia showed no EGFR amplification, whereas OSCC exhibited low-level amplification in 19 cases and high-level amplification in 3 cases, respectively. OSCC cases with EGFR amplification showed slightly higher EGFR and Ki-67 immunoreactivity than those without amplification. In OSCC, EGFR immunoreactivity significantly correlated with the mode of invasion, invasion depth, as well as EGFR amplification significantly correlated with the degree of differentiation, mode of invasion, recurrence, and postoperative metastasis. Direct DNA sequencing of EGFR and K-Ras did not show any gene alterations in OSCC. ConclusionEGFR aberrations might contribute to carcinogenesis, invasion, metastasis as well as poor outcomes in OSCC. EGFR-targeting therapy with anti-EGFR monoclonal antibody agents may be beneficial in OSCC patients.

Concurrent Expression of Cyclo-oxygenase-2 and Epidermal Growth Factor Receptor in Canine Malignant Mammary Tumours

Canine mammary tumours (CMTs) are reported to express cyclo-oxygenase (COX)-2 and epidermal growth factor receptor (EGFR); however, no studies have evaluated concurrent expression of these proteins. In this study, 43 malignant CMTs were evaluated immunohistochemically for concurrent expression of COX-2 and EGFR and expression was correlated with malignancy. High COX-2 expression was associated with tumour size (P = 0.033), mitotic index (P = 0.040), nuclear grade (P = 0.021), histological grade of malignancy (P = 0.020) and lymph node metastasis (P = 0.029). High EGFR immunoreactivity was associated with tumour size (P = 0.001), necrosis (P = 0.001), mitotic index (P = 0.022), histological grade of malignancy (P = 0.041) and lymph node metastasis (P = 0.005). Simultaneous high-expression of COX-2 and EGFR was associated with high-nuclear grade (P = 0.049), high-histological grade of malignancy (P = 0.031) and the presence of lymph node metastasis (P = 0.025). A positive correlation between COX-2 and EGFR expression (r = 0.474; P = 0.001) was also observed. These results suggest that combined use of selective inhibitors of COX-2 and EGFR may be a useful approach to the treatment of malignant CMTs.

Epidermal growth factor receptor (EGFR) status and prognosis in gallbladder cancer.

e14657 Background: Studies from USA, Asia and Australia showed EGFR over-expression in gallbladder cancer (GC), and recent case reports suggest a role for EGFR targeted therapies in this disease. Methods: We studied the prognostic role of EGFR expression and its association with clinicopathologic features in forty-two GC patients surgically treated at our center, from 1995 to 2006. None received targeted cancer therapeutics. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic biliary epithelia were obtained and assessed for immunohistochemical (IH) expression of EGFR, cyclo-oxygenase-2 (COX2), E-cadherin and p53 protein. Nonparametric correlations were assessed by Spearman’s rho; survival analysis was performed using the Kaplan-Meier method and Cox regression; and p-values were computed for two-tailed tests. Results: EGFR IH scores 0, +1, +2, and +3 were observed in 48.7%, 23.1%, 17.9%, and 10.3%, respectively. They were correlated with p53 staining (rho=0.48; p=0.002); T-stage (rho=0.38; p=0.016); serum CA-19.9 (rho=0.48; p=0.016); and serum CEA (rho=0.56; p=0.004). Median overall survival (OS) was 8.0 months (95% C.I.: 5.6 to 10.3); 1-year and 2-year survival rates were 43.7% and 23.5%, respectively. OS were 18.0 months, 14.0 months, and 4.0 months for tumors with 0, 1+ and 2+ to 3+ EGFR staining, respectively. Univariate analysis suggested a worse prognostic significance for low serum albumin (p=0.001); elevated CEA (p=0.007) and CA-19.9 (p=0.007) levels; positive surgical margins (p=0.001); gallbladder fundus tumors (p=0.007); histologic grade 3 (p=0.003); positive nodes (p=0.001); lymphovascular invasion (p=0.001); tumoral necrosis (p=0.004); perineural invasion (p=0.002); and EGFR expression (p=0.004). In the multivariate analysis, low serum albumin (p=0.006), tumoral necrosis (p<0.001) and EGFR expression (p<0.001) predicted poor OS. Conclusions: We found tumor immunoreactivity for EGFR in nearly half of the cases and it was independently associated with poor survival in GC patients.

Su1251 Overexpression of EGFR in the Exocrine Pancreas Induces a Chronic Pancreatitis

Background and Aim: Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) frequently overexpress epidermal growth factor receptor (EGFR) and EGF-family ligands. Although chronic pancreatitis is a risk factor for PDA, the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis in development of PDA is the lack of appropriate animal models for chronic pancreatitis with similar clinical courses as in human patients. Serine protease inhibitor Kazal type 1 (SPINK1: mouse homolog Spink3), which is also known as tumor associated trypsin inhibitor (TATI), is mainly produced in the pancreatic acinar cells. Recently, we showed that SPINK1 binds to EGFR to activate its downstream signaling. The present study aims to generate chronic pancreatitis model mice and assess the molecular events from chronic pancreatitis to PDA. Methods: 1) C57BL/6 female mice were repeatedly induced acute pancreatitis by using choline-deficient with ethionine (CDE) diet for 54 weeks. 2) To assess the molecular events caused by long term CDE diet treatment using Western blotting. 3) To further analyze the clinical importance of EGFR and SPINK1 in the chronic pancreatitis and PDA, we examined the expression of SPINK1 and EGFR. Immunohistochemical analyses were performed using human chronic pancreatitis and PDA samples. Results: 1) Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 wk of CDE-diet treatment. In spite of the severe pancreatic degeneration, neoplastic changes were not observed. 2) Expression of EGFR and its ligands (Spink3 and TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. In addition, cyclooxygenase-2 which induced by stimuli involved in inflammatory responses was also overexpressed. 3) In the human chronic pancreatitis samples, TCs were positive EGFR immunostaining. Furthermore, PDA cases were positive for both SPINK1 and EGFR immunoreactivity. Conclusion: We have successfully developed a chronic pancreatitis model with CDE diet. We also showed expression of EGFR and its potent ligands, phosphorylation of EGFR, and activation of K-Ras as the step of chronic inflammation. It was suggested that EGFR signaling cascades were important for the development from chronic pancreatitis to PDA. Howevere, chronic pancreatitis itself is not sufficient for the development of PDA. Further combinations of CDE-diet treatment with the administration of other promotion insults or genetic alterations may result in PDA development.

Su1252 Factors Predictive of Relapse, Cancer and Cyst Formation in Autoimmune Pancreatitis Patients

Background and Aim: Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) frequently overexpress epidermal growth factor receptor (EGFR) and EGF-family ligands. Although chronic pancreatitis is a risk factor for PDA, the relationship between chronic pancreatitis and PDA remains obscure. A critical obstacle to understanding the role of chronic pancreatitis in development of PDA is the lack of appropriate animal models for chronic pancreatitis with similar clinical courses as in human patients. Serine protease inhibitor Kazal type 1 (SPINK1: mouse homolog Spink3), which is also known as tumor associated trypsin inhibitor (TATI), is mainly produced in the pancreatic acinar cells. Recently, we showed that SPINK1 binds to EGFR to activate its downstream signaling. The present study aims to generate chronic pancreatitis model mice and assess the molecular events from chronic pancreatitis to PDA. Methods: 1) C57BL/6 female mice were repeatedly induced acute pancreatitis by using choline-deficient with ethionine (CDE) diet for 54 weeks. 2) To assess the molecular events caused by long term CDE diet treatment using Western blotting. 3) To further analyze the clinical importance of EGFR and SPINK1 in the chronic pancreatitis and PDA, we examined the expression of SPINK1 and EGFR. Immunohistochemical analyses were performed using human chronic pancreatitis and PDA samples. Results: 1) Histological evaluation revealed that chronic pancreatitis, characterized by acinar atrophy, fibrosis and well-developed tubular complexes (TCs), was observed after 24 wk of CDE-diet treatment. In spite of the severe pancreatic degeneration, neoplastic changes were not observed. 2) Expression of EGFR and its ligands (Spink3 and TGF α) and activation of K-Ras (GTP-Ras formation), which are frequently observed in human PDA, were indeed observed in parallel with TCs formation. In addition, cyclooxygenase-2 which induced by stimuli involved in inflammatory responses was also overexpressed. 3) In the human chronic pancreatitis samples, TCs were positive EGFR immunostaining. Furthermore, PDA cases were positive for both SPINK1 and EGFR immunoreactivity. Conclusion: We have successfully developed a chronic pancreatitis model with CDE diet. We also showed expression of EGFR and its potent ligands, phosphorylation of EGFR, and activation of K-Ras as the step of chronic inflammation. It was suggested that EGFR signaling cascades were important for the development from chronic pancreatitis to PDA. Howevere, chronic pancreatitis itself is not sufficient for the development of PDA. Further combinations of CDE-diet treatment with the administration of other promotion insults or genetic alterations may result in PDA development.

Expression of Phosphorylated Histone H2AX (γ-H2AX) in Normal and Neoplastic Squamous Epithelia of the Uterine Cervix

The histone γ-H2AX is a marker of activated DNA damage and is overexpressed in different cancers and their precursor lesions, indicating a role in oncogenic transformation. Epidermal growth factor receptor (EGFR) is overexpressed in many kinds of epithelial neoplasms. This study aimed to determine whether immunohistochemical expression of γ-H2AX is involved in the progression of the morphological spectrum from normal squamous cervical epithelia (NE, n=33) to cervical intraepithelial neoplasia (CIN; CIN1, n=9; CIN2/3, n=33) and cervical invasive squamous cell carcinoma (ISCC, n=33), whether γ-H2AX expression follows the pattern of proliferation of atypical squamous cells as shown by EGFR immunoreactivity, and whether it is correlated with clinicopathologic variables in ISCC. Immunostaining for both the factors was scored semiquantitatively for moderate and strong intensities. Gamma-H2AX and EGFR expression, respectively, increased from NE and CIN1 to CIN2/3 and ISCCs significantly (P=0.0001, respectively). Gamma-H2AX reactivity was found in the nuclei of the cells of the upper epithelial levels and the cells of basal/parabasal type in variable quantities in NE and CIN; expression of γ-H2AX was seen in the nuclei of ISCC including keratinizing cells in horn pearls. EGFR staining was mainly membranous and noted in basal/parabasal cells in NE and atypically proliferating keratinocytes in CIN and nonkeratinizing cells of ISCC. In addition, immature squamous metaplasias were decorated by the antibodies used. Immunoscores for γ-H2AX and EGFR, respectively, did not differ between International Federation of Gynecology and Obstetrics stages I and II, keratinizing and nonkeratinizing ISCC, and CIN2/3 and ISCC. However, expression patterns were different for both the factors, suggesting their involvement in different biological mechanisms, with regard to γ-H2AX apart from proliferation. Overexpression of γ-H2AX in CIN2/3 and ISCC of the uterine cervix reflects the neoplastic transformation of cervical squamous epithelia in reaction to increased DNA-damage and supports the classification of dysplasia into low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions.

Fenofibrate exhibits a high potential to suppress the formation of squamous cell carcinoma in an oral-specific 4-nitroquinoline 1-oxide/arecoline mouse model

The excessive use of areca nut and/or tobacco may induce the production of free radicals and reactive oxygen species, which affect the lipid contents of the cell membrane and are possibly involved in tumorigenic processes in the oral cavity. The aim of this study was to investigate the therapeutic efficacy of fenofibrate (0.1% or 0.3%, w/w), a ligand of the peroxisome proliferator-activated receptor alpha (PPARα), in a 4-nitroquinoline 1-oxide (4-NQO)/arecoline-induced oral cancer mouse model. The carcinogen, 4-NQO/arecoline, was administrated to C57BL/6JNarl mice for 8weeks followed by fenofibrate treatment for 12 or 20weeks. After 28weeks, changes in serum lipids, the multiplicity of tumor lesions, and tumor sizes were determined together with changes in the immunohistochemical expressions of PPARα, acetyl-coenzyme A carboxylase (ACC), the epidermal growth factor receptor (EGFR), and cyclooxygenase-2 (COX2). The results showed that when compared to the 4-NQO/arecoline only group, 0.3% fenofibrate treatment increased serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. 0.3% fenofibrate treatment suppressed the incidence rate of tongue lesions, reduced the multiplicity of squamous cell carcinoma (SCC), decreased the tumor size, and increased the immunoreactivity of EGFR and COX2 in oral dysplasia but decreased EGFR and COX2 expressions in SCC. These findings indicated that fenofibrate reduced the tumor incidence rate and suppressed the tumor progression into SCC and that these molecular events might be linked to the EGFR and COX2 regulatory pathways. We suggest that fenofibrate provides a new strategy for preventing oral tumor progression.

Dual inhibition of EGFR and mTOR pathways in small cell lung cancer

Background:In this report we investigated the combination of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) pathway inhibition as a possible new therapeutic strategy for small cell lung cancer (SCLC).Methods:EGFR, p-AKT, p-ERK, p-mTOR and p-p70s6K protein expressions were studied by immunohistochemistry in 107 small cell lung carcinomas and correlated with clinicopathological parameters. Cells of SCLC were treated with erlotinib±RAD001 and analysed for cell viability, proliferation, autophagy, and pathway regulation.Results:Epidermal growth factor receptor, p-AKT, p-ERK, p-mTOR, and p-p70s6K were expressed in 37, 24, 13, 55 and 91% of the tumour specimens of all SCLC patients, respectively, and were not associated with disease-free or overall survival. The expression of EGFR was lower in neoadjuvant-treated patients (P=0.038); mTOR pathway activation was higher in the early stages of disease (P=0.048). Coexpression of EGFR/p-mTOR/p-p70s6K was observed in 28% of all patients . EGFR immunoreactivity was associated with p-ERK and p-mTOR expression (P=0.02 and P=0.0001); p-mTOR immunoreactivity was associated with p-p70s6K expression (P=0.001). Tumour cells comprised a functional EGFR, no activating mutations in exons 18–21, and resistance to RAD001 monotherapy. We found synergistic effects of erlotinib and RAD001 combination therapy on the molecular level, cell viability, proliferation and autophagy.Conclusions:The combined inhibition of EGFR/mTOR pathways could be a promising approach to treat SCLC.

Immunohistochemical expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis

ObjectivesSquamous cell carcinoma (SCC) is by far the most common malignant neoplasm of the oral cavity. A number of etiologic factors have been implicated in its development. During the past few decades, a particular focus has been placed on the investigation of valid biomarkers predictive of cancer behavior and cervical lymph node metastasis in head and neck Squamous cell carcinoma (HNSCC).The present study was designed to investigate the expression of epidermal growth factor in these tumors in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis.Materials and methodsImmunohistochemical (IHC) evaluation of epidermal growth factor receptor (EGFR) expression in 40 retrospective OSCC specimens and its correlation with proliferating cell nuclear antigen (PCNA), antiapoptotic antibody (P53), vascular endothelial growth factor (VEGF), and D2-40 monoclonal antibodies (Mab), in relation to the clinicopathological parameters.ResultsData revealed positive EGFR immunoreactivity in 35(87.5%) cases. There was a statistically significant correlation regarding EGFR extent score with respect to intratumoral lymphatic vessel density (ILVD) (r = 0.35) as well as EGFR intensity score with respect to ILVD and peritumoral lymphatic vessel density (PLVD) (r = 0.33, r = 0.36 respectively). EGFR expression was not correlated with the clinicopathological parameters. Conclusions: EGFR is expressed by most of the cases. EGFR correlation with D2- 40 positive lymphatic vessels suggests a higher tendency of OSCC for lymphatic dissemination. Lack of correlation among the studied markers suggests their independent effect on tumor behavior.

Epidermal Growth Factor Receptor, C-kit, and Her2/neu Immunostaining in Advanced or Recurrent Thymic Epithelial Neoplasms Staged According to the 2004 World Health Organization in Patients Treated with Octreotide and Prednisone: An Eastern Cooperative Oncology Group Study

Advanced or recurrent nonresectable thymic epithelial tumors show only a modest response to standard chemotherapy. A recent study using octreotide and prednisone in thymic tumors, Eastern Cooperative Oncology Group study E1C97, was conducted to verify the activity of octreotide for thymic tumors. The aim of this study was to determine whether epidermal growth factor receptor (EGFR) immunoreactivity correlated with outcomes and to identify new biologic markers for potential targeted therapy. Three markers, EGFR, C-kit, and Her2/neu, were selected for evaluation in patients with advanced thymic epithelial tumors treated on E1C97. Of the 42 patients entered onto E1C97, 34 patients (World Health Organization [WHO] categories: type A = 1, type AB = 1, type B1 = 10, type B2 = 11 type B3 = 8, and type C = 3) had sufficient tissue available for immunohistologic study. Each tumor was assessed to have 0, 1+, 2+, or 3+ immunoreactivity in the cytoplasm or membranes of the neoplastic cells for Her2/neu and EGFR and for the presence or absence of C-kit immunoreactivity. EGFR immunoreactivity of 2+ or 3+ was associated with more aggressive thymic tumors (WHO types B2 and B3). However, strong EGFR immunoreactivity was not consistently seen with thymic carcinoma. The presence of EGFR within cells was associated with a significantly improved progression-free survival (PFS) and a trend for overall survival (OS). Twelve patients demonstrated C-kit immunoreactivity; the lack of C-kit immunoreactivity was significantly associated with superior PFS but not OS. Her2/neu immunoreactivity was uniformly negative for all tumors evaluated. There was no association between response and biomarker status. High EGFR immunoreactivity is seen in more aggressive thymic neoplasms as classified according to the 2004 WHO, but regardless of classification, the presence of EGFR in tumor cells (1+, 2+, and 3+) is associated with improved performance free survival (PFS) and a trend for better OS. In contrast, the absence of C-kit immunoreactivity was associated with improved PFS. These data suggest that EGFR and C-kit may be prognostic, and further studies of these markers in subcategories of thymic malignancies is warranted.

Tissue and serum EGFR as prognostic factors in malignant pleural mesothelioma

Background Malignant pleural mesothelioma (MPM) is an asbestos related aggressive tumor. Asbestos causes genetic modifications and cell signaling events that favor resistance to chemotherapy. A variety of receptor tyrosine kinases have been identified to play a central role in various aspects of tumorigenesis. Epidermal growth factor receptor ( EGFR) is overexpressed in a variety of epithelial malignancies including lung cancer in which EGFR aberrations not only predict response to EGFR tyrosine kinase inhibitors but also indicate tumor progression. However in MPM, the role of EGFR is less clear. This study was designed to identify serum and tissue EGFR levels in patients with MPM and to evaluate the relationship between serum and tissue EGFR levels and clinicao-pathological prognostic factors and survival. Methods We investigated 71 cases of MPM for EGFR expression in tissue. Serum EGFR was assessed in 40 out of those 71 cases and 20 healthy subjects as a control. Pre-treatment serum EGFR levels were measured using quantitative enzyme-linked immunosorbent assay. Tissue EGFR protein overexpression was assessed by immunohistochemistry and gene amplification was assessed by the chromogen in situ hybridization (CISH) technique. Results were correlated with the clinical–pathological factors of the patients and overall survival (OS). Results Out of the 71 patients included in the study, 19 had undergone extrapleural pneumonectomy. As for the rest of the patients, 46 received chemotherapy while 6 had only best supportive care. EGFR immuno-reactivity was detected in 74.6% of the cases, 37 (52.1%) cases were positive for EGFR gene amplification by CISH, 31 of them revealed moderate to high (++, +++) EGFR immuno-reactivity. Elevated serum EGFR >2.5 ng/ml (the median concentration of EGFR in MPM) was reported in 45% of the cases. The overall response rate (RR) for the 46 treated patients who received chemotherapy was 24.1%. After a median follow up of 29 months, the median overall survival (OS) was 10 months. Elevated serum and tissue EGFR is significantly associated with advanced disease stage. However neither EGFR overexpression in tissues nor high serum levels were associated with survival rates. Conclusions EGFR expression is a common feature in MPM patients. High pre-treatment levels of serum EGFR are associated with advanced stage but not with reduced OS. Detailed mutational analysis of EGFR on a larger number of patients is still needed to clarify the exact role of EGFR in MPM patients.

Correlation between tumor volume response to radiotherapy and expression of biological markers in patients with cervical squamous cell carcinoma

To determine the factors associated with tumor volume response to radiotherapy (RT) in cervical cancer patients, and the relationship between the tumor volume response and alteration of the expression of biological markers during RT. Twenty consecutive patients with cervical squamous cell carcinoma who received definitive RT were enrolled. Tumor volumes were calculated by MRI examinations performed at the start of RT (pre-RT), at the fourth week of RT (mid-RT), and 1 month after RT completion (post-RT). Two serial punch biopsies were performed at pre- and mid-RT, and immunohistochemical staining was performed for cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR). For the pre-RT evaluation, fourteen (70%) and eleven (55%) patients showed positive immunoreactivity for COX-2 and EGFR, respectively. Among the seven patients whose median percentage residual tumor at mid-RT (V2R) was greater than 0.5, seven (100%, p=0.0515) and five (71.4%, p=0.3742) patients showed positive immunoreactivity for COX-2 and EGFR, respectively. The logistic regression analysis showed that positive immunoreactivity for both COX-2 and EGFR at pre-RT were associated with V2R (p=0.0782). For the mid-RT evaluation, eight cases showed an interval increase in the distribution of immunoreactivity for COX-2, and six out of the eight patients had a V2R greater than 0.5 (p=0.2222). The poor mid-RT tumor response was associated with the coexpression of COX-2 and EGFR.

Epidermal Growth Factor Receptor Expression in Serous Ovarian Carcinoma: An Immunohistochemical Study With Galectin-3 and Cyclin D1 and Outcome

This study investigated the expression of epidermal growth factor receptor (EGFR), galectin-3 and cyclin D1 in a cohort of ovarian serous carcinomas with regard to outcome and clinicopathologic parameters. Formalin-fixed paraffin-embedded archival tissues of fifty ovarian serous carcinomas were stained with anti-bodies to EGFR, Gal-3, and cyclin D1 by automated immunohistochemistry. Additionally, 10 benign serous cystadenomas and 10 typical serous borderline ovarian tumors were included in the study. Immunostaining was scored with regard to quantity and intensity of positively stained nuclei. Staining patterns were recorded. The EGFR expression was scored negative in all serous cystadenomas and serous borderline ovarian tumors. Membranous and cytoplasmic EGFR immunoreactivity was determined in 64% of ovarian serous carcinomas; it was related to high grade (P=0.0005) and poor outcome (P=0.0137) but not with stage (P=0.5118). Galectin-3 and cyclin D1 immunostaining decreased from serous cystadenomas and serous borderline ovarian tumors to the carcinomas significantly (P=0.0022 and P=0.0083, respectively). Galectin-3 immunostaining of any pattern (nuclear and cytoplasmic as well as merely cytoplasmic taken together) was not related to grade or stage in cancers; mere cytoplasmic expression was associated with poor outcome (P=0.0097). Cyclin D1 immunoreactivity in predominantly nuclear pattern was increased in low-grade carcinomas (P=0.0378) but was not related to stage and outcome (P=0.6578 and P=0.0675, respectively). This study indicates that with regard to EGFR and cytoplasmic galectin-3 immunoexpression, multiple marker testing may be an adjunct in the identification of high-risk ovarian serous cancers.

EGFR and K-RAS gene status evaluation in anal canal squamous cell carcinoma

15569 Background: Mutations affecting the epidermal growth factor receptor (EGFR) gene and the K-RAS gene have been identified in a variety of human solid tumours and have been validated as promising therapeutic targets. Approximately 85% of non small-cell lung cancer patients, who responded to gefinitib or erlotinib, have somatic mutations in the EGFR gene (exons 18–21) and for metastatic colorectal cancer patients, wild-type K-RAS is required for responsiveness to panitumumab. In squamous cell carcinoma of the anal canal however, the EGFR and K-RAS gene status has not systematically been investigated. Patients and Methods: In this study, 30 primary squamous cell carcinomas of the anal canal were investigated. The panel consisted of twenty men and 10 women with a median age of 60.5 years (range: 34–86 years). The formalin-fixed paraffin embedded tissues were immunohistochemically (ICH) examined for EGFR expression (Zymed Laboratories) and analysed by fluorescence in situ hybridisation (FISH) for EGFR gene copy numbers. Furthermore, EGFR and K-RAS mutation analysis was performed on DNA isolated from formalin-fixed paraffin embedded tissues. EGFR mutation analysis was carried out using exons 18 through 21 specific primers. For K-RAS, exon 2 was analysed for the presence of mutations. Results: EGFR immunoreactivity was detected in 27 of 29 (93%) tumours. One tumour yielded no conclusive results. Nineteen (65%) cases exhibited a moderate (2+) or strong (3+) staining intensity. FISH results were not interpretable in 18 (60%) cases. Of the remaining 12 cases, none showed EGFR gene amplification. Mutation analysis was performed on 20 samples, but no mutations could be identified in the kinase domain of the EGFR gene nor in the K-RAS gene. Conclusion: In our panel of squamous cell carcinoma of the anal canal, all samples showed a strong EGFR protein expression as evaluated by IHC. However, no EGFR gene amplification nor EGFR and K-RAS mutation could be identified. No significant financial relationships to disclose.

Metastasizing breast carcinoma to the brain: a clinicopathologic and immunohistochemical study of 14 matched pairs.

Brain metastasizing breast cancer is the second most common cause of brain metastases. The frequency of brain metastasis ranges between 10‐40% of breast cancers. Hormonal status of the primary malignancy is thought to play a role in determining which tumors eventually will show brain metastasis. In this study, we examined 14 pairs of matched primary breast carcinomas and their respective brain metastases for various clinicopathologic parameters and performed immunohistochemical staining for estrogen receptor (ER), progesterone receptor (PR), Her‐2/neu, and epidermal growth factor receptor (EGFR). The mean age at diagnosis of breast primary and the subsequent brain metastases was 54.8years (36–74) and 57.7years (39–75), respectively. The mean interval between the diagnosis of breast carcinoma and the brain metastasis was 32 months (4 months–8 years). All 14 breast primary cancers were of the invasive ductal type and over half (8/14) were high grade (SBR III/III). Nine (64%) of the matched pairs were ER negative and 10 (71%) were PR negative but only 7 (50%) were both ER/PR negative. Three (21%) pairs were found to be Her2/neu positive by immunohistochemistry. Overall, 5 (36%) matched pairs were “triple negative”. Diffuse and strong EGFR immunoreactivity was observed in 4 primary breast cancers and only 2 brain metastases. Both EGFR positive brain metastases had EGFR positive primaries. EGFR‐ positive pairs were more frequently “triple negative” or in one case, ER/PR negative and Her2/neu positive. Her2/neu analysis by FISH is underway.

Significance of tyrosine kinase activity on malign transformation of ovarian tumors: A comparison between EGF-R and TGF- α

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF- α) are members of the polypeptide growth factor family. The epidermal growth factor-receptor (EGF-R) is a receptor tyrosine kinase of the ErbB family. Many types of cancer, including ovarian cancer, display enhanced EGF-R immunoreactivity on their cell surface membranes. Also, an increase in TGF- α synthesis and secretion usually occurs in human carcinoma cell lines. In this study, we compared the immunoreactivities of TGF- α and EGF-R in ovarian tumors and related immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumor and serous-mucinous adenocarcinoma of the ovary ( n=10 each) were stained with hematoxylin–eosin and labeled for binding of primary antibodies against TGF- α and EGF-R using an avidin–biotin–peroxidase method. A semi-quantitative grading system was used to compare immunohistochemical labeling intensities. Increased immunoreactivity of EGF-R and moderate immunoreactivity of TGF- α was detected in adenocarcinomas. There was no significant difference in the immunoreactivity of TGF- α among the histologic types of ovarian tumors. The results of this study support the hypothesis that EGF-R may be a more useful marker than TGF- α in epithelial ovarian tumors.

Terminal Respiratory Unit Type Lung Adenocarcinoma is Associated With Distinctive EGFR Immunoreactivity and EGFR Mutations

Approximately 10% to 20% of nonsmall cell lung cancer patients respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib. Responders are mostly nonsmokers and women with tumors displaying bronchioloalveolar features. Mutations of the tyrosine kinase domain of the EGFR gene have been associated with a clinical response to gefitinib. A recent study reported that the terminal respiratory unit (TRU)-type adenocarcinoma shares the clinical profile and EGFR mutations of gefitinib responders. EGFR immunoreactivity in this context has not been reported in the literature. We performed a detailed immunohistochemical analysis of EGFR expression on 124 consecutive lung resection specimens for malignancy, to survey the EGFR immunoreactivity in lung cancers in general and to correlate EGFR immunoreactivity with EGFR mutations and TRU-type histology. EGFR positivity was seen most frequently in squamous cell carcinomas (77%), followed by TRU-type adenocarcinomas (63%), large cell carcinomas (23%), and non-TRU-type adenocarcinomas (12%). A distinctive basally oriented cytoplasmic positivity was observed exclusively in TRU-type adenocarcinomas. EGFR mutation was identified in 6 of 54 cases studied and all 6 cases were TRU-type adenocarcinomas. Five of six cases with EGFR mutation were positive for EGFR immunostain with the basal cytoplasmic localization. In conclusion, EGFR immunoreactivity with basal cytoplasmic pattern was exclusively seen in TRU-type adenocarcinoma and a subset of these cases was seen with EGFR mutations in the responders to EGFR inhibitor therapy.

Epidermal Growth Factor Receptor is a Possible Predictor of Sensitivity to Chemoradiotherapy in the Primary Lesion of Esophageal Squamous Cell Carcinoma

Chemoradiotherapy (CRT) is currently performed for patients with esophageal squamous cell carcinoma (SCC). Some reports have revealed that patients who responded well to CRT had favorable outcomes, whereas poor responders conversely showed a worse prognosis. The aim of this study was to identify molecular markers predicting sensitivity to CRT. We reviewed 62 patients with T(3-4), N-any, and M-any esophageal SCC treated with definitive CRT. The regimen comprised protracted 5-fluorouracil infusion and a 2-h infusion of cisplatinum combined with radiation therapy (2 Gy/day) at a total radiation dose of 60 Gy. The expressions of epidermal growth factor receptor (EGFR), vascular endothelial growth factor, cyclin D1, and proliferating cell nuclear antigen were investigated immunohistochemically in biopsy specimens obtained before treatment from all 62 patients. The immunoreactivities were compared with responsiveness to CRT, as evaluated by endoscopy. The complete response rate of the primary tumor estimated by endoscopy was 62% (13/21) in patients in the EGFR-positive group. The difference in the CR rate between EGFR-positive and -negative groups was significant (p = 0.037). The immunoreactivities of the other molecular markers did not show a significant correlation with the responsiveness of the primary lesion to CRT. Multiple logistic regression analysis revealed that positive immunostaining for EGFR was significantly correlated with primary CR for CRT in esophageal SCC. Among 62 patients with esophageal SCC, differences in the responsiveness of primary lesions to CRT were correlated with EGFR immunoreactivity assessed in the biopsy specimens. These results suggest that EGFR may help to predict the response of primary sites to definitive CRT in esophageal SCC, although the results should be confirmed in a larger, more homogeneous series.