High Levels Of Epidermal Growth Factor Receptor Expression Research Articles

Abstract 1985: Activation of EGFR/IGF1R - beta catenin - CD44 pathway in periampullary cancer

Abstract Background: Periampullary cancer (PC) is a global medical burden, although infrequent but less than 5% of patients experience an overall survival of five years or more. The study aims to analyze the importance of Epidermal Growth Factor Receptor (EGFR)/Insulin-Like Growth Factor 1 Receptor (IGF1R) - Beta-catenin (β-catenin)- CD44 pathway in the development of periampullary cancer of Indian patients. Materials and Methods: Expression profile of EGFR, IGF1R, β-catenin, and CD44 was verified by immunohistochemical analysis in primary tumor samples (N=14) and respective adjacent normal tissues. Results: In periampullary carcinoma patients, a high level of EGFR expression was seen in 64% of the samples along with co-expression of IGF1-R in 77.8% samples. Furthermore, the high expression of EGFR and IGF1R significantly correlated with the increased expression of β-catenin along with CD44 expression of the tumors. Conclusion: The EGFR/IGF1R - β-catenin- CD44 pathway seems to be important in the development of PC with clinical importance. Key-words: Periampullary cancer; EGFR; IGF1R; β-catenin; CD44 Key Messages: Interplay of EGFR, IGF1R, β-Catenin and CD44 expressions is important phenomena in the development of PC. Citation Format: Suvendu Maji, Biswabandhu Bankura, Chinmay Kumar Panda, Neyaz Alam. Activation of EGFR/IGF1R - beta catenin - CD44 pathway in periampullary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1985.

Biodistribution of fluorescence-labelled EGF protein from slow release NanoZolid depots in mouse

AimThe study was designed to evaluate the ability of the calcium sulfate based NanoZolid® drug delivery technology to locally release the epidermal growth factor (EGF) protein while maintaining its biological activity. MethodsNanoZolid-formulated EGF protein labelled with a near infrared dye (EGF-NIR) depots or EGF-NIR dissolved in PBS were injected subcutaneously into mice bearing EGF receptor (EGFR) positive human A549 lung cancer tumors inoculated subcutaneously. The release and biodistribution of the EGF-NIR were investigated in vivo longitudinally up to 96 h post administration, utilizing whole body fluorescence imaging. In order to confirm the in vivo findings, histological analysis of tumor cryosections was performed to investigate EGF-NIR fluorescent signal and EGFR expression level by immunofluorescence labelling. ResultsThe in vivo fluorescence imaging showed a controlled release profile of the EGF-NIR loaded in the NanoZolid depots compared to free EGF-NIR. Histological analysis of the tumors further demonstrated a prevailing distribution of EGF-NIR in regions with high levels of EGFR expression. ConclusionCalcium sulfate based depots can be used to formulate EGF while maintaining its biological activity, e.g. receptor binding capability. This may have a good clinical potential for local delivery of biomolecules to enhance treatment efficacy and minimize systemic adverse effects.

Activation of epidermal growth factor receptor/insulin-like growth factor 1 receptor-β-Catenin-CD44 pathway in periampullary cancer

Background: Periampullary cancer (PC) is a global medical burden. Less than 5% of patients experience an overall survival of 5 years or more. The study aims to analyze the importance of epidermal growth factor receptor (EGFR)/insulin-like growth factor 1 receptor (IGF1R)–beta-catenin (β-catenin)–CD44 pathway in the development of periampullary cancer of Indian patients. Subjects and Methods: Expression profile of EGFR, IGF1R, β-catenin, and CD44 was verified by immunohistochemical analysis in primary tumor samples (N = 14) and respective adjacent normal tissues. Results: In periampullary carcinoma patients, a high level of EGFR expression was seen in 64% of the samples along with co-expression of IGF1R in 77.8% of samples. Furthermore, the high expression of EGFR and IGF1R significantly correlated with the increased expression of β-catenin along with CD44 expression of the tumors. Conclusion: The EGFR/IGF1R–β-catenin–CD44 pathway seems to be important in the development of PC with clinical importance.

Expression of EGFR in Pituitary Corticotroph Adenomas and Its Relationship With Tumor Behavior.

Objective: Epidermal growth factor receptor (EGFR) has been found to localize in several human neoplasms and has been shown to have a significant correlation with adenomaigenesis and patient prognosis. EGFR is also overexpressed in pituitary corticotroph adenomas. However, its clinical significance and relationship with tumor behavior, especially tumor recurrence status, remain obscure. The purpose of the present study was to identify the expression patterns of EGFR and its downstream signaling pathway molecules in pituitary corticotroph adenomas and to investigate the association of EGFR with clinicopathological characteristics and tumor recurrence.Methods: Fifty-two sporadic pituitary adenoma specimens and six normal pituitary glands were collected. The expression levels of EGFR and its downstream signaling molecules in each sample were evaluated and quantified using immunohistochemistry and Western blot. The relationships of EGFR expression with clinicopathological characteristics and tumor recurrence status were analyzed.Results: EGFR was overexpressed in 55.8% of the pituitary corticotroph adenomas and in 1 of 6 of the normal adenohypophysial tissues. The expression degree was significantly higher in pituitary corticotroph adenomas than in normal adenohypophysial tissues. In EGFR-overexpressing adenomas, the downstream pathway phosphorylated Erk (p-Erk) was also significantly activated. Moreover, the expression levels of EGFR were positively correlated with the adrenocorticotropic hormone (ACTH) and cortisol levels but were not correlated with age, sex or symptom duration. The expression levels of EGFR, phosphorylated EGFR (p-EGFR) and p-Erk were significantly up-regulated in the recurrent adenoma group compared with those in the non-recurrent adenoma group (all p < 0.05). The expression levels of EGFR were strongly correlated with the recurrence-free interval (p = 0.005, CC = −0.31).Conclusion: The expression levels of EGFR and its downstream pathway components were significantly increased in pituitary corticotroph adenomas compared to the levels in normal adenohypophysial tissues. EGFR expression levels were positively associated with the ACTH and cortisol levels and with tumor recurrence status. Pituitary corticotroph adenomas with high EGFR expression levels were correlated with an increased recurrence rate and a decreased recurrence-free interval. EGFR could be used as a promising biomarker for predicting pituitary corticotroph tumor recurrence.

Nuclear Delivery of Radiosensitizing EGFR-Targeted Nanoparticles Is Abrogated in Isogenic Cervical Cancer Cell Lines Following Mutagenesis of the Ligand Binding Domain

The development of targeted treatment regimens for cervical cancer remains a critically unexplored area of research. One such possible target is epidermal growth factor receptor (EGFR). Our laboratory has previously synthesized radiosensitizing nanoparticles (NPs) that specifically target cells that express EGFR. Here, we test the hypothesis that nuclear localization following treatment of EGFR-targeted NPs is preferentially achieved in cervical cancer cell lines with high levels of EGFR expression and is dependent upon an intact ligand binding domain. A panel of cervical cancer cell lines (HeLa, SiHa, CaSki, ME180, and C33A) with varying levels of endogenous and genetically manipulated EGFR expression were cultured and treated with Fe3O4@TiO2 NPs conjugated to EGF “mimic” (B-loop) peptide (EGFR-targeted NPs) or its scrambled version (scrambled non-targeted NPs). Transient overexpression and knockdown of EGFR was achieved with transfection of EGFR DNA or small interfering RNA (siRNA). Isogenic cell lines were expanded from single cell clones of the ME-180 cervical cancer cell line with heterozygous or homozygous mutations, introduced by CRISPR-Cas9 technology, of the critical amino acids Leucine-14 (Leu-14) and Tyrosine-45 (Tyr-45) located within the ligand binding domain of the EGFR protein. Immunocytochemistry with confocal microscopy and flow cytometry was utilized to assess EGF-binding ability of isogenic cell lines as well ascellular delivery, subcellular distribution, and receptor binding ability of EGFR-targeted NPs and scrambled non-targeted NPs in this panel of cell lines. All tested cervical cancer cell lines endogenously produced both full-length EGFR mRNA variant 1 (v1), as well as shorter mRNA variants 3 and 4 which encode soluble isoforms of EGFR, while expression of full-length EGFR (v1) varied dramatically across cell lines. Cell lines with high levels of endogenous or genetically upregulated EGFR expression experienced increased nuclear uptake of EGFR-targeted NPs compared to cell lines with low levels of endogenous EGFR expression or following siRNA-mediated knockdown, while uptake of scrambled non-targeted NPs was similar across all cell lines. Isogenic clones of the ME-180 cervical cancer cell line demonstrated impaired EGF-binding as well as nuclear localization of EGFR-targeted NPs but scrambled non-targeted NPs. Nuclear accumulation of EGFR-targeted radiosensitizing NPs, but not scrambled non-targeted NPs, occurred preferentially in cervical cancer cell lines with high expression of EGFR and was abrogated following mutation of the ligand binding domain. Further studies are required to demonstrate in vivo radiosensitization of cervical cancer following treatment with EGFR-targeted NPs.

THE ASSOCIATION BETWEEN EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTOR WITH PRIMARY TUMOR VOLUME OF NASOPHARYNGEAL CARCINOMA

Objective: Tumor development was triggered by the excess population of the Epidermal Growth Factor Receptor (EGFR). Expression of EGFR with primary tumor volume of in patients with NPC. Expression of EGFR has a role in the increasing of metastasis, self-renewal, drug resistance and anti-apoptosis. EGFR expression associated with tumors size, positively nodules and advanced tumor stage in NPC patients. High level of EGFR expression by immunohistochemical examination associated with the development, progression and differentiation cell of tumors.&#x0D; Methods: Using Cross-sectional as study design. Formalin-fixed paraffin-embedded biopsy specimens were obtained. The expression of EGFR was studied with immunohistochemistry using EGFR polyclonal antibody (Bioss, USA). Assessment of the staining was performed by pathologist consultant used histoscore. The Pearson’s correlation test was used to determine the correlation between expression of EGFR and primary tumor volume of nasopharyngeal carcinoma. Statistical significance was defined as p &lt;0.05.&#x0D; Result: Total samples are 19 patients. The result of EGFR expression in NPC patients with a primary tumor volume of 1-50 ml is 5 samples with a weak positive expression and 4 samples with moderate positive expression. NPC with a primary tumor volume of 51-100 ml is 2 samples of negative expression, 1 sample of weak positive expression, 3 samples with moderate positive expression and 2 samples with strong positive expression. NPC with primary tumor volume &gt;100 ml is 1 sample weak positive expression and 1 sample strong positive expression. Statistical analysis using Pearson’s exact test was obtained p=0.047 with a correlation coefficient 0.461. EGFR expression from all of the patients there was 2 samples (10.53%) negatives, 7 samples (36.84%) weak positive, moderate positive was 7 samples (36.84%), and strong positive 3 samples (15.79%). &#x0D; Conclusion: There was an association between the expression of EGFR with the primary tumor volume of nasopharyngeal carcinoma.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.

Induction of autophagy enhances apoptotic cell death via epidermal growth factor receptor inhibition by canertinib in cervical cancer cells

BackgroundIt has been known epidermal growth factor receptor (EGFR) frequently overexpressed in cervical cancer. High levels of EGFR expression in their tumors leads to a poor prognosis and inhibition frequently induces autophagy in cancer cells. This study aimed to investigate whether EGFR inhibition by canertinib induces autophagy and this induction influence the effect of Palladium (Pd) (II) complex and 5-fluorouracil (5-FU) especially in nontoxic doses. MethodsCytotoxicity was evaluated by using SRB assay. Apoptosis, autophagy, and EGFR key markers were determined by flow cytometry, fluorescence staining, and immunoblotting. Colony formation, invasion, and wound healing assays were performed to investigate cell proliferation, invasion, and migration, respectively. ResultsBlocking EGFR by the pan-ErbB tyrosine kinase inhibitor canertinib inhibited cell growth of HeLa cervical cancer cells in combination with Pd(II) complex and 5-FU. Combination of canertinib and Pd(II) complex promotes autophagy and apoptosis of HeLa cancer cells via blockade of the PI3K/AKT and MAPK/ERK pathway, which leads to cervical cancer cell death. ROS accumulation and DNA damage were increased after combinatorial treatment which causes depolarization of the mitochondrial inner membrane and leads to apoptotic cell death. Canertinib combined with Pd(II) complex leads to inhibition of migration and invasion. ConclusionInhibition of EGFR signaling by canertinib in combination with Pd(II) complex promotes apoptosis and autophagy via blockade of the PI3K/AKT and MAPK/ERK. General significanceThe cytotoxic activity of Pd(II) complex and 5-FU on HeLa cells is mediated by EGFR inhibition and autophagy induction, leading to activation of mitochondrial apoptotic cell death.

Subcutaneous and orthotopic xenograft models of human bladder carcinoma in nude mice for epidermal growth factor receptor-targeted treatment

Introduction.Approaches based on the principles of a targeted therapy are considered a promising strategy that is capable to improve the effectiveness of treatment for bladder cancer (BC) patients.The purposeof the study was to establish an orthotopic xenograft model of human BC in mice and to prove its suitability for experimental examination of drugs targeting the epidermal growth factor receptor (EGFR).Materials and methods.The objects of the study were ectopic subcutaneous and orthotopic human BC xenografts established using EJ and 5637 human BC cell lines. The growth of orthotopic xenografts in vivo was assessed by magnetic resonance imaging. Tumor tissues were investigated using histological and immunohistochemical techniques.Results.It was shown that EJ and 5637 xenografts exhibit a good reproducibility, a sufficient blood supply of the tumor tissues, a high level of EGFR expression, and different pattern of a subcellular receptor localization. Implantation and subsequent proliferation of human EJ or 5637 cells in the murine bladder mucosa presumably results in muscle-non-invasive tumor formation.Conclusions.The EJ and 5637 xenograft models can be useful for investigation of the efficacy of EGFR-targeted biotherapeutic treatments.

P‐cadherin Overexpression Enhances Survival Signaling in Dysplastic Oral Epithelia via the Epidermal Growth Factor Receptor

P‐cadherin is a calcium‐dependent transmembrane protein that mediates cell‐cell adhesion within oral epithelial tissue. Previous histological analyses have suggested that P‐cadherin protein levels increase in oral dysplasia and subsequently decrease in oral squamous cell carcinoma (OSCC). The epidermal growth factor receptor (EGFR) gene has also been shown to undergo amplification in the dysplastic stage of oral tumor development. In comparative studies, we found that levels of P‐cadherin were increased two‐fold in dysplastic oral keratinocytes (DOK) compared to primary human oral keratinocytes (HOK) obtained from normal oral mucosa. We hypothesized that in dysplastic oral epithelia, upregulated P‐cadherin cooperates with amplified EGFR to enhance survival signaling and facilitate tumor progression. To further explore the interaction of P‐cadherin and EGFR, DOK cells were retrovirally infected to generate stably‐selected control and P‐cadherin‐overexpressing cell lines. We have previously demonstrated that the DOK cell line exhibits comparatively high levels of EGFR expression. We found that P‐cadherin overexpression increased basal, as well as EGF‐stimulated EGFR phosphorylation levels. Furthermore, this was accompanied by increased phosphorylation of a downstream effector of EGFR, signal transducer and activator of transcription (STAT3). This increased phosphorylation was not dependent on an increase in total STAT3 protein level. STAT3 phosphorylation suggested activation of a pro‐survival mechanism, a finding that was further supported by P‐cadherin‐dependent increases in the anti‐apoptotic protein Bcl‐xL. When exposed to chemotherapeutic agents such as paclitaxel and carboplatin, P‐cadherin overexpression increased cell survival compared to control cells. P‐cadherin‐overexpressing cells treated with paclitaxel also exhibited decreased apoptosis. Further analysis confirmed that the difference in cell death was not due to altered proliferation of the two cell lines. We also explored the possibility that EGFR was activated by P‐cadherin‐mediated adherens junction formation. In contrast to ligand‐dependent signaling, we observed that calcium‐dependent cadherin engagement suppressed the phosphorylation of both EGFR and STAT3. These studies suggest that the upregulation of P‐cadherin, together with increased EGFR expression, promotes tumor progression in dysplastic oral epithelia through the activation of prosurvival signaling pathways.Support or Funding InformationMidwestern University Glendale College of Osteopathic Medicine, College of Health Sciences, College of Veterinary MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High expression of EGFR predicts poor survival in patients with resected T3 stage gastric adenocarcinoma and promotes cancer cell survival.

Epidermal growth factor receptor (EGFR) is an essential regulator and biomarker of several types of cancer. However, the association between its expression and prognosis in patients with resected T3 stage gastric adenocarcinoma (RT3-GA) remains to be determined. In total, 683 patients with resectable T3-GA who underwent surgery were retrospectively included in the present study, and their immunohistochemical data for EGFR expression were collected. The associations between the patients' clinicopathologic characteristics and EGFR immunohistochemistry data were analyzed by multiple statistical methods. Annexin V apoptosis and MTT cell viability assays were performed to explore the effect of EGFR on AGS gastric adenocarcinoma cell survival. EGFR expression levels were categorized into two groups: low (406 cases) and high (277 cases). High EGFR was demonstrated to be significantly associated with distant metastasis (P=0.043) and severely decreased median overall survival time (MOST) and recurrence-free survival time (MRFST). MOST and MRFST in the low EGFR group were 39 and 37 months, respectively; whereas in the high EGFR group these values were only 18 and 13 months (P=3.10×10−9 and P=6.74×10−8, respectively). Multivariate analysis confirmed that high EGFR expression levels were associated with poor survival, which was associated with significantly increased recurrence risk and ~2-fold elevation in mortality risk [hazard ratio (HR), 1.73; 95% confidence interval (CI), 1.43–2.10; P=2.37×10−8 and HR, 1.80; 95% CI, 1.50–2.17; P=3.80×10−10]. Inhibiting EGFR with AG1478 suppressed its effect on promoting AGS cell survival. These results suggest that high EGFR expression indicates poor survival in patients with RT3-GA, which may be correlated with EGFR promoting GA cell survival.

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

BackgroundThough overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors.MethodsThe EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with 11C for in vivo PET studies. Kinetic scans with the 11C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions.ResultsFlow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-11C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-11C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels.ConclusionsSame-day in vivo dual tracer imaging enabled by the Sel-tag technology and 11C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0213-8) contains supplementary material, which is available to authorized users.

Novel EGFR-targeted strategy with hybrid peptide against oesophageal squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is a key molecule in the pathophysiology of oesophageal squamous cell carcinoma (OSCC). However, EGFR-targeted agents such as anti-EGFR antibody or tyrosine kinase inhibitors for OSCC have not demonstrated any clinical benefits. Recently, a novel chemotherapeutic agent, EGFR(2R)-lytic hybrid peptide, a composite of EGFR-binding peptide and lytic peptide fragments, has been shown to exhibit a potent anti-tumour effect against cancers that express high EGFR levels. In this study, we investigated the validity of employing EGFR(2R)-lytic hybrid peptide against OSCC cells both in vitro and in vivo. Additionally, the toxicity of this peptide was assessed in mice. We found high EGFR expression levels on the cell surface of OSCC cells, and the EGFR-binding peptide fragment showed high affinity for OSCC cells. A potent cytotoxic effect was induced within 30 minutes by the exposure of OSCC cells to EGFR(2R)-lytic hybrid peptide. Furthermore, EGFR(2R)-lytic hybrid peptide markedly suppressed the tumour growth of OSCC cells in a xenograft model. Moreover, it did not cause any identifiable adverse effects in mice. Taken together, EGFR(2R)-lytic hybrid peptide was shown to be a valid therapeutic agent against OSCC, providing a crucial rationale regarding novel EGFR-targeted therapies against OSCC.

High levels of EGFR expression in tumor stroma are associated with aggressive clinical features in epithelial ovarian cancer.

PurposeThe aim of this study was to investigate the clinical significance and biological function of epidermal growth factor receptor (EGFR) expressed in tumor stroma of epithelial ovarian cancer.MethodsImmunohistological staining of EGFR was evaluated in 242 patients with epithelial ovarian cancer. The correlations of EGFR expression in tumor stroma with clinicopathological features and with the expression level of Ki-67 were analyzed by SPSS software. Kaplan–Meier analysis and the Cox proportional hazard model were used to analyze the effect of EGFR expression in tumor stroma on the prognosis of patients with epithelial ovarian cancer. Meanwhile, the activities of proliferation and migration of tumor cells were detected when EGFR overexpressed in stroma cells.ResultsEGFR expression in tumor stroma correlated significantly with clinical stage (χ2=7.002, P=0.008) and distant metastases (χ2=16.59, P<0.001). Furthermore, there was a significantly positive correlation between the level of EGFR expressed in tumor stroma and the level of Ki-67 expressed in tumor cells (χ2=6.120, P=0.013). Patients with high EGFR expression level in tumor stroma showed poor survival (P=0.002). Multivariate analysis showed that high expression of EGFR in tumor stroma was an independent predictor for epithelial ovarian cancer patients (hazard ratio =1.703; 95% confidence interval 1.125–2.578, P=0.012). Furthermore, stroma cells overexpressing EGFR could promote the proliferation and migration of adjacent tumor cells.ConclusionHigh expression of EGFR in tumor stroma correlates with aggressive clinical features in epithelial ovarian cancer, and is an independent prognostic factor.

Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.

A new A 431/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening epidermal growth factor receptor antagonists from Radix sophorae flavescentis

The intracellular kinase domains of epidermal growth factor receptor (EGFR) in some tumor cells such as human epidermal squamous cells (A 431 cells) are an important target for drug discovery. We have developed a new A 431/cell membrane chromatography (A 431/CMC)-online–high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening EGFR antagonists from medicinal herbs such as traditional Chinese medicines (TCMs). In this study, A 431 cells with high EGFR expression levels were used to prepare cell membrane stationary phase (CMSP) in an A 431/CMC model. The retention fractions eluted from the CMSP column were enriched onto an ODS pre-column and then switched into an HPLC/MS system by combining a 10 port columns switching valve. The screening results found that oxymatrine and matrine from Radix sophorae flavescentis (RSF) were the targeted components which could act on EGFR in similar manner of gefitinib as a control drug. There was a good relationship of their inhibiting effects on EGFR secretion and A 431 cell growth in vitro. This new A 431/CMC-online-HPLC/MS method can be applied for screening EGFR antagonists from TCMs such as RSF. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.

Lack of Correlation between Epidermal Growth Factor Receptor Status and Response to Panitumumab Monotherapy in Metastatic Colorectal Cancer

Panitumumab, a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody, is approved as monotherapy for the treatment of metastatic colorectal cancer. We evaluated the association of tumor EGFR expression levels with outcomes in patients with chemorefractory metastatic colorectal cancer. Two phase II, multicenter, single-arm, open-label studies enrolled chemorefractory patients with tumors expressing low/negative (1-9%/<1%; Low/Negative EGFR study) or high (> or =10%; High EGFR study) levels of EGFR. Patients received panitumumab 6 mg/kg every two weeks until disease progression or intolerance. End points included objective response rate (per response evaluation criteria in solid tumors), progression-free survival (PFS), overall survival (OS), and safety. Exploratory analyses by tumor KRAS status were carried out. A total of 203 patients (Low/Negative EGFR) and 185 patients (High EGFR) enrolled in the studies. The overall response rate was 5.7% [95% confidence interval (95% CI), 2.6-10.5] in patients with low/negative EGFR and 4.2% (95% CI, 1.6-9.0) in patients with high EGFR; the response rate at week 16 was 4% in both studies (all partial responses). Median PFS times were 8.1 weeks (95% CI, 7.1-12.6), 8.1 weeks (95% CI, 7.4-11.1), and 7.3 weeks (95% CI, 7.1-7.6) in patients with negative, low, and high levels of EGFR expression, respectively. PFS and OS were longer in patients with wild-type KRAS than those with mutant KRAS. As expected, most adverse events were skin related. These studies confirm previous reports that tumor EGFR expression levels are not associated with efficacy with an anti-EGFR antibody and that anti-EGFR antibody therapy should be limited to those patients whose tumors express wild-type KRAS.

Epidermal growth factor receptor expression in and targeted therapy for metastatic squamous cell carcinoma of the penis

14045 Background: Penile carcinoma is highly fatal when associated with bulky regional or distant metastases, despite chemotherapy and surgery. Epidermal growth factor receptor (EGFR) is a potential target for systemic therapy, but little is known about its expression or significance in penile carcinoma. Methods: The results of EGFR immunohistochemistry in cases of metastatic squamous cell carcinoma of the penis performed in our Department of Pathology over a 2-year period were reviewed retrospectively. The antibody used in the assay was Zymed clone 31G7. To assess the biological significance of EGFR expression, we reviewed the charts of these patients for EGFR-targeted therapies and outcomes. Results: Thirteen cases were assayed for EGFR expression. The tissue was from a lymph node metastasis in 5 patients, inguinal or scrotal skin metastasis in 2 patients, and the primary tumor in six patients. Tumor cells in all cases were positive for EGFR, and 9 were scored as 3+ or strongly positive. The percentage of cells positive for EGFR was &gt;90% in six cases, 40–50% in one case, and not specified in six cases. All of the patients received one or more EGFR-targeted therapies including erlotinib (one patient), cetuximab (three patients), or cetuximab combined with one or more cytotoxic agents (nine patients). Six patients received a second or third EGFR-targeted therapy. Grade 3 or 4 adverse events were limited to cellulitis, thrombocytopenia, and tumor hemorrhage (one patient each). Two patients had disease progression during initial treatment with paclitaxel, ifosfamide, and cisplatin (TIP), then had partial responses to TIP plus cetuximab. Two patients had partial responses to cetuximab and cisplatin, also after having disease progression while receiving TIP. A patient with visceral metastases who had extensive prior chemotherapy received cetuximab and cisplatin as salvage treatment and experienced objective tumor regression lasting 3 months. Conclusions: High levels of EGFR expression are common in metastatic penile carcinoma. Five of 13 patients showed evidence of response to cetuximab in combination with cisplatin or TIP. These results indicate that EGFR plays a clinically important role in metastatic penile carcinoma and is a promising target for therapy. No significant financial relationships to disclose.

Epidermal growth factor receptor as a predictor of tumor downstaging in locally advanced rectal cancer patients treated with preoperative chemoradiotherapy

To examine retrospectively whether levels of epidermal growth factor receptor (EGFR) expression can predict tumor downstaging after preoperative chemoradiotherapy in patients with locally advanced rectal cancer. A total of 183 patients with rectal cancer (cT3-T4 or N+) were enrolled in this study. Preoperative chemoradiotherapy consisted of 50.4 Gy of pelvic radiation with concurrent 5-fluorouracil and leucovorin bolus intravenous chemotherapy in 94 patients or oral capecitabine and leucovorin in 89 patients. EGFR expression in pretreatment paraffin-embedded tumor biopsy specimens was assessed by immunohistochemistry. EGFR expression was determined from the intensity and extent of staining. Tumor downstaging was defined as a reduction of at least one T-stage level. Tumor downstaging occurred in 97 patients (53%), and the tumors showed a pathologic complete response in 27 patients (15%). Positive EGFR expression was observed in 140 (76%) of 183 patients. EGFR expression levels were low in 113 patients (62%) and high in 70 patients (38%). On logistic regression analysis, the significant predictive factor for increased tumor downstaging was a low level of EGFR expression and preoperative chemotherapy using oral capecitabine (odds ratio, 0.437; p = 0.012 vs. odds ratio, 3.235; p < 0.001, respectively). A high level of EGFR expression may be a significant predictive molecular marker for decreased tumor downstaging after preoperative chemoradiotherapy in locally advanced rectal cancer.

Epidermal growth factor receptor as a prognostic factor in locally advanced rectal-cancer patients treated with preoperative chemoradiation

We investigated the prognostic value of epidermal growth factor receptor (EGFR) expression in pretreatment biopsy specimens from patients with locally advanced rectal cancer treated with preoperative chemoradiation. Pretreatment biopsy specimens from 92 patients with locally advanced rectal cancer were examined for EGFR expression by immunohistochemistry. EGFR expression was assessed by immunoreactive score (IRS). The prognostic value of EGFR expression was evaluated according to the level of EGFR expression. Epidermal growth factor receptor expression was positive in 65 patients (71%). EGFR expression levels were low (IRS 0 to 5) in 83 patients (90%) and high (IRS 6 to 7) in 9 patients (10%). A high level of EGFR expression was statistically significant for shorter overall survival (p = 0.013), disease-free survival (p = 0.002), and distant metastasis-free survival (p = 0.003), as compared with a low level of expression in univariate analysis. Grouping based on positive or negative EGFR expression did not represent prognostic significance for survival. In multivariate analysis, high EGFR expression was an independent prognostic factor for decreased disease-free survival (relative risk 2.4, p = 0.041) and distant metastasis-free survival (relative risk 2.6, p = 0.04). Our results suggest that high level of EGFR expression in a pretreatment biopsy specimen may be a significant adverse prognostic factor for disease-free survival and distant metastasis-free survival.