Epidermal Growth Factor Receptor Staining Research Articles

Insulin-Like Growth Factor-1 Receptor Expression in Thymic Malignancies

Thymic epithelial tumors are rare mediastinal malignancies that can be invasive and difficult to treat. Insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane receptor implicated in the regulation of cell metabolism, growth, and survival. As higher levels of IGF-1R protein expression may be associated with relative sensitivity to anti-IGF-1R antibody treatment, we investigated IGF-1R expression in thymic malignancies. Sixty-three thymic tumors (56 thymomas and seven thymic carcinomas) were analyzed for total IGF-1R expression using immunohistochemistry with a specific antibody (clone G11, Roche-Ventana, Tucson, AZ). Expression levels were correlated with relevant clinical and pathologic variables, including epidermal growth factor receptor and KIT expression, and patient outcome. IGF-1R staining was negative in 13 (21%) cases, low (1+) in 20 (32%) cases, moderate (2+) in 20 (32%) cases, and high (3+) in 10 (16%) cases. Moderate to high IGF-1R staining was observed in 6 of 7 (86%) thymic carcinomas and in 24 of 56 (43%) thymomas (p = 0.039). Moderate to high IGF-1R staining was associated with high epidermal growth factor receptor staining (p = 0.015). By multivariate analysis, only tumor stage and histologic type were significant prognostic factors on time to progression (hazard ratio [HR] = 4.12, 95% confidence interval [CI]: 1.98-14.23; p = 0.010 and HR = 2.79, 95% CI: 1.62-12.50; p = 0.018, respectively). There was no association between IGF-1R expression and time to progression (HR = 3.07, 95% CI: 0.38-24.59; p = 0.291). A majority of thymic malignancies display moderate to high expression of IGF-1R. The lack of preclinical models prevented us to further study the functional consequences of anti-IGF-1R therapy in this setting. However, given correlations in other cancers, these data support the evaluation of anti-IGF-1R inhibitors in thymic tumors.

Anti-Epidermal Growth Factor Receptor Antibodies in the Treatment of Metastatic Colorectal Cancer

Targeting the epidermal growth factor receptor (EGFR), an important component in carcinogenesis, is an attractive therapeutic option for selective anticancer therapy. Several EGFR inhibitors, mostly monoclonal antibodies and tyrosine kinase inhibitors are under investigation in the treatment of colorectal cancer. Although there has been some progress in the treatment of colorectal cancer with combination chemotherapy, the repertoire of active agents is still limited. More recently the anti-EGFR drugs cetuximab and panitumumab have made an impact in the treatment of metastatic disease but with variable response rates. Although the appearance of a skin rash confers a higher response rate, immunohistochemical staining of EGFR at baseline does not. Several studies have now focused on identifying predictive biological markers at a molecular level. Exciting data has demonstrated KRAS mutation status to be the first predictive marker of response to anti-EGFR monoclonal antibodies (mAbs) and has led a new era in the development of targeted therapies in colorectal malignant disease. The aim of this review is to evaluate the impact of anti-EGFR therapies in the treatment of metastatic colorectal cancer; and to present the current data on predictive markers including KRAS status, PTEN expression and germinal gene polymorphisms. The relevant patents are discussed.

Randomized phase III study of panitumumab (pmab) with FOLFIRI versus FOLFIRI alone as second-line treatment (tx) in patients (pts) with metastatic colorectal cancer (mCRC): Analysis by tumor epidermal growth factor receptor (EGFR) staining.

3565^ Background: Pmab, a fully human, anti-EGFR monoclonal antibody, plus FOLFIRI vs FOLFIRI was evaluated as 2nd-line tx for mCRC by tumor KRAS status; efficacy analyses by EGFR staining were also performed. Methods: Pts who received 1 prior chemotherapy tx for mCRC were randomized 1:1 to pmab 6.0 mg/kg Q2W+FOLFIRI (Arm 1) or FOLFIRI (Arm 2). Independently tested coprimary endpoints progression-free survival (PFS) and overall survival (OS) were prospectively analyzed by KRAS status. EGFR+ staining was not required for entry. Tumor tissue must have been sectioned within 2 months prior to testing using the Dako EGFR pharmDx immunohistochemistry kit. Results: 1,186 pts were randomized. Demographics and baseline characteristics were generally balanced. 1,083 pts (91%) had KRAS results and 719 (66%) pts had EGFR results. For WT KRAS, median PFS was 5.9 mos for Arm 1 and 3.9 mos for Arm 2; median OS was 14.5 mos for Arm 1 and 12.5 mos for Arm 2. 391 pts with WT KRAS also had EGFR results; 295 (75%) were EGFR+. The most common reason for no EGFR result was tissue section ≥2 months old. The analysis of treatment effect within the subpopulations by EGFR staining are shown (Table). No treatment effect by EGFR staining was observed in a stratified multivariate Cox model evaluating the predictive effect of EGFR parameters on PFS (p = 0.80) and OS (p = 0.81) in KRAS WT pts. In general, adverse event rates were comparable across arms except for known toxicities associated with anti-EGFR tx. Conclusions: Pmab significantly improves PFS and is well tolerated when added to FOLFIRI for 2nd-line tx in pts with WT KRAS mCRC; a consistent benefit in PFS and OS was observed among EGFR+ as well as EGFR- subsets of patients who received pmab. Further results by EGFR staining parameters will be presented. Efficacy by EGFR membrane staining—WT KRAS WT KRAS N = 597 EGFR evaluable WT KRAS N = 390 HR (95% CI) p value HR (95% CI) EGFR+ HR (95% CI) EGFR− PFS*Pmab FOLFIRI : FOLFIRI 0.73 0.78 0.89 Pmab FOLFIRI : FOLFIRI (0.59-0.90) 0.004 (0.61-1.01) (0.55-1.43) OS 0.85 0.88 0.97 Pmab FOLFIRI : FOLFIRI (0.70-1.04) 0.12 (0.66-1.17) (0.59-1.61) * By blinded central review. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen, Lilly, Merck, sanofiaventis Amgen Amgen, Lilly, Merck, Merck Serono, Roche, sanofi-aventis Amgen Amgen Amgen, Lilly, Merck, sanofiaventis In compliance with the guidelines established by the ASCO Conflict of Interest Policy (J Clin Oncol. 2006 Jan 20;24[3]:519-521) and the Accreditation Council for Continuing Medical Education (ACCME), ASCO strives to promote balance, independence, objectivity, and scientific rigor through disclosure of financial and other interests, and identification and management of potential conflicts. According to the ASCO Conflict of Interest Policy, the following financial and other relationships must be disclosed: employment or leadership position, consultant or advisory role, stock ownership, honoraria, research funding, expert testimony, and other remuneration (J Clin Oncol. 2006 Jan 20;24[3]:520). The ASCO Conflict of Interest Policy disclosure requirements apply to all authors who submit abstracts to the Annual Meeting. For clinical trials that began accrual on or after April 29, 2004, ASCO's Policy places some restrictions on the financial relationships of principal investigators (J Clin Oncol. 2006 Jan 20;24[3]:521). If a principal investigator holds any restricted relationships, his or her abstract will be ineligible for placement in the 2010 Annual Meeting unless the ASCO Ethics Committee grants an exception. Among the circumstances that might justify an exception are that the principal investigator (1) is a widely acknowledged expert in a particular therapeutic area; (2) is the inventor of a unique technology or treatment being evaluated in the clinical trial; or (3) is involved in international clinical oncology research and has acted consistently with recognized international standards of ethics in the conduct of clinical research. NIH-sponsored trials are exempt from the Policy restrictions. Abstracts for which authors requested and have been granted an exception in accordance with ASCO's Policy are designated with a caret symbol (^) in the Annual Meeting Proceedings. For more information about the ASCO Conflict of Interest Policy and the exceptions process, please visit www.asco.org/conflictofinterest.

Randomized phase III study of panitumumab (pmab) with FOLFOX4 compared to FOLFOX4 alone as first-line treatment (tx) for metastatic colorectal cancer (mCRC): PRIME trial analysis by epidermal growth factor receptor (EGFR) tumor staining.

3566^ Background: This study evaluated the efficacy and safety of pmab + FOLFOX4 versus FOLFOX4 alone as first-line tx for mCRC by tumor KRAS status; analyses by EGFR staining were also performed. Methods: Pts who received no prior chemotherapy for mCRC and no prior oxaliplatin were randomized 1:1 to pmab 6.0 mg/kg Q2W+FOLFOX4 (Arm 1) or FOLFOX4 alone (Arm 2). The primary endpoint was progression-free survival (PFS); overall survival (OS) was secondary. PFS and OS were prospectively analyzed according to KRAS status. Using the Dako EGFR pharmDx immunohistochemistry kit, tumor tissue sectioned within 2 months prior to testing was evaluated for EGFR staining. EGFR results were not required for study entry. Results: 1,183 pts were randomized. Demographics and baseline characteristics were generally balanced. 1,096 pts (93%) had KRAS results. For WT KRAS, median PFS was 9.6 mos for Arm 1 and 8.0 mos for Arm 2; median OS was 23.9 mos for Arm 1 and 19.7 mos for Arm 2. 813 (69%) pts had EGFR results. The most common reason for no EGFR result was tissue section ≥2 months old. Of 479 WT KRAS pts with EGFR results, 326 (68%) were EGFR+. The Table shows the treatment effect within the subpopulations by EGFR staining. No treatment effect by EGFR staining was observed in a stratified multivariate Cox model evaluating the predictive effect of EGFR parameters on PFS (p = 0.89) and OS (p = 0.43) in KRAS WT pts. Except for known toxicities associated with anti-EGFR tx adverse event rates were generally comparable across arms. Conclusions: When added to FOLFOX for first-line mCRC tx, pmab significantly improves PFS and is well tolerated in pts with WT KRAS. A consistent effect of pmab on PFS and OS was observed in both EGFR+ and EGFR- subsets. Further results by EGFR staining parameters will be presented. Efficacy by EGFR membrane staining-WT KRAS WT KRAS N = 656 EGFR evaluable WT KRAS N = 479 HR (95% CI) p value HR (95% CI) EGFR+ HR (95% CI) EGFR- PFS* 0.80 0.77 0.80 Pmab FOLFOX : FOLFOX (0.66-0.97) 0.02 (0.58-1.03) (0.53-1.21) OS 0.83 0.77 0.64 Pmab FOLFOX : FOLFOX (0.67- 1.02) 0.07 (0.52-1.13) (0.34-1.18) * By blinded central review. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen Amgen, AstraZeneca, Celgene, Fresenius Biotech, Merck Serono, Roche, sanofi-aventis Amgen Amgen, Merck AG, Merck Serono, sanofiaventis Amgen, Merck AG, Merck Serono, Pfizer Amgen In compliance with the guidelines established by the ASCO Conflict of Interest Policy (J Clin Oncol. 2006 Jan 20;24[3]:519-521) and the Accreditation Council for Continuing Medical Education (ACCME), ASCO strives to promote balance, independence, objectivity, and scientific rigor through disclosure of financial and other interests, and identification and management of potential conflicts. According to the ASCO Conflict of Interest Policy, the following financial and other relationships must be disclosed: employment or leadership position, consultant or advisory role, stock ownership, honoraria, research funding, expert testimony, and other remuneration (J Clin Oncol. 2006 Jan 20;24[3]:520). The ASCO Conflict of Interest Policy disclosure requirements apply to all authors who submit abstracts to the Annual Meeting. For clinical trials that began accrual on or after April 29, 2004, ASCO's Policy places some restrictions on the financial relationships of principal investigators (J Clin Oncol. 2006 Jan 20;24[3]:521). If a principal investigator holds any restricted relationships, his or her abstract will be ineligible for placement in the 2010 Annual Meeting unless the ASCO Ethics Committee grants an exception. Among the circumstances that might justify an exception are that the principal investigator (1) is a widely acknowledged expert in a particular therapeutic area; (2) is the inventor of a unique technology or treatment being evaluated in the clinical trial; or (3) is involved in international clinical oncology research and has acted consistently with recognized international standards of ethics in the conduct of clinical research. NIH-sponsored trials are exempt from the Policy restrictions. Abstracts for which authors requested and have been granted an exception in accordance with ASCO's Policy are designated with a caret symbol (^) in the Annual Meeting Proceedings. For more information about the ASCO Conflict of Interest Policy and the exceptions process, please visit www.asco.org/conflictofinterest.

Epidermal growth factor receptor (EGFR), cyclooxygenase-2 (COX-2), and cyclin D1 expression in squamous cell carcinoma of the anal canal.

e21095 Background: Identification of proteins overexpressed in squamous cell carcinoma of the anal canal will increase our understanding of the biology of these uncommon tumors, and may help to identify potential new therapeutic targets. Cetuximab, a monoclonal antibody that targets EGFR, is used in the treatment of head and neck squamous cell tumors, which frequently express this receptor. COX-2 activity and Cyclin D1 amplification have also been seen in other squamous cell carcinomas, but their role in anal cancers has not been well studied. The purpose of this study was to examine the expression of these proteins in squamous cell tumors of the anal canal. Methods: Twenty cases of invasive squamous cell carcinoma of the anal canal were examined immunohistochemically for expression of COX-2, EGFR and Cyclin D1. Tumor cells were graded based on the intensity of staining, and expression was considered positive when > 10% of cells were stained (1-3+). Protein expression was correlated with tumor histology and other clinical variables. Results: The majority of tumors (19/20; 95%) stained positive for COX-2 and EGFR. Moderate to intense staining (2-3+) was observed in 75% and 45% of tumor tissues for COX-2 and EGFR, respectively. Fifty percent of these tumors were also positive for Cyclin D1, with the majority exhibiting a moderate staining pattern. Interestingly, the most intense staining for each of these markers was observed in the more actively proliferating basal layer of cells, especially in the keratinized sub-type. In addition, keratinized tumors exhibited more intense COX-2 expression (10/11; 90.9%) than non-keratinized tumors (5/9; 55.6%). No associations were found between tumor grade or Cyclin D1 expression and other variables. However, all four patients identified with positive lymph node status exhibited moderate to intense staining for both EGFR and COX-2. Conclusions: Since EGFR and COX-2 are expressed in the majority of squamous cell anal cancers, the role of EGFR-targeted therapies and COX-2 inhibitors in the treatment of these tumors should be further investigated. No significant financial relationships to disclose.

Prognostic Impact of p16, p53, Epidermal Growth Factor Receptor, and Human Papillomavirus in Oropharyngeal Cancer in a Betel Nut–Chewing Area

To evaluate the prevalence of human papillomavirus (HPV) and the prognostic significance of epidermal growth factor receptor (EGFR), p53, and p16 among patients with oropharyngeal carcinoma. Retrospective study. Academic Institute of Otolaryngology, Kaohsiung, Taiwan. Two hundred seventy-four patients who were diagnosed as having oropharyngeal carcinoma underwent testing for the presence of the HPV genome in the nuclei of their tumor cells from January 1, 1992, through March 31, 2008. The HPV genome was detected by performing polymerase chain reaction-based assays and in situ hybridization on tumor tissue from paraffin blocks. Immunohistochemistry staining for p16, p53, and EGFR was also performed. We used the Fisher exact test to evaluate the correlation between the clinicopathological variables and the presence of HPV in tumor cells. Survival analysis was based on the Kaplan-Meier method. We detected HPV in 45 of the 274 patients (16.4%); of these, HPV-16 and -18 were identified in 42 (93.3%) of the HPV-positive tumors. The HPV-positive oropharyngeal cancers were more likely to occur in females, nonsmoking individuals, and those who did not chew betel quid. The HPV-positive tumors significantly expressed p16 and were inversely associated with EGFR and p53 expression (all, P < .001). In addition, patients with tumor tissue that was positive for HPV (P = .008) and had negative expression of EGFR (P = .01), low expression of p53 (P = .01), and high expression of p16 (P = .04) had a better prognosis. Our results suggest that HPV, EGFR, p53, and p16 are useful biomarkers in predicting the clinical outcomes of oropharyngeal cancer.

Abstract 3667: ERβ and PR expression show opposite effects on survival in NSCLC with no sex differences

Abstract Sex steroids may be targets for lung cancer intervention. Estrogen has been shown to stimulate, while progesterone has been shown to inhibit, lung cancer growth. Cross-talk between the estrogen and the epidermal growth factor (EGF) signaling pathways in lung cancer has been reported. To further clarify the roles of estrogen, progesterone and EGF receptors, we analyzed primary lung tumors and normal lung tissue from 183 patients (primarily non-small cell lung cancer histologies) for expression and localization of estrogen receptor α (ERα), estrogen receptor β (ERβ), aromatase, progesterone receptor (PR), and EGF receptor (EGFR) using immunohistochemistry. No differences were observed in any marker expression analyzed by sex or histology. ERα expression was positive only when using an antibody that also detects ERα deletion variants, consistent with mRNA profiles of lung tumor cell lines showing multiple ERα variants with no full-length mRNA. ERα expression showed no effect on survival. Tumor tissues were found to express significantly higher levels (p&amp;lt;.001) of ERβ (both nuclear and cytoplasmic) and variant ERα compared to matched normal lung tissues, suggesting up-regulation of these proteins in lung cancer. Conversely, PR was expressed at significantly higher levels (p=.007) in normal tissue compared to tumor. Positive correlations were observed between ER subtypes and the other markers, suggesting hormonal pathways are often expressed together. Cytoplasmic ERβ expression was identified as a negative prognostic predictor of overall survival in all patients, males and females (p=.021). However, elevated ERβ cytoplasmic expression was predictive of survival in stage III/IV patients (p=.012) but not in stage I/II patients (p=.353), suggesting ERβ signaling is a more important survival determinant in advanced disease. Patients with high PR expression had longer time to progression (TTP) than patients with low PR expression; this was significant in stage I/II patients (p=0.008) but not in stage III/IV patients (p=.834). PR signaling may protect against recurrence in early stage patients. Survival advantage in all patients was more pronounced when degree of EGFR staining was combined with PR staining (p=0.004). When degree of ERβ was included, patients with low ERβ, low EGFR and high PR had longer TTP than the opposite (p=0.021) suggesting that these signaling pathways interact and cooperate within the tumor to affect prognosis. Low aromatase was protective only when considering other markers. Patients with high ERβ expression and advanced disease may be good candidates for anti-estrogen therapy while patients with low PR expression and early stage disease may be candidates for progesterone supplementation. Phenotyping all interacting targets may select patients for combining hormonal modulation with anti-EGFR therapy. Supported by NCI P50 CA090440 (JMS) and Flight Attendant Medical Research Institute (LPS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3667.

Breast Cancer Molecular Class ERBB2: Preponderance of Tumors With Apocrine Differentiation and Expression of Basal Phenotype Markers CK5, CK5/6, and EGFR

This is a study of 205 consecutive invasive breast carcinomas. The principal aim was to identify morphologic and immunohistochemical features of tumors that belong to the molecular class ERBB2. The invasive breast carcinomas were classified using semiquantitative immunohistochemical results for estrogen receptor (ER), progesterone receptor, (PR), and HER2 into following classes: Luminal A (strong ER+, HER2 negative), Luminal B (weak to moderate ER/PR+, HER2 negative), Triple Negative (TN; ER/PR negative, HER2 negative), ERBB2 (ER/PR negative, HER2 positive), Luminal A-HER2 Hybrid (strong ER+, HER2 positive), Luminal B-HER2 Hybrid (weak to moderate ER/PR+, HER2 positive). Of the 205 tumors, 113 (55%) were classified as Luminal A, 34 (17%) as Luminal B, 32 (15%) as TN, 8 (4%) as ERBB2, 10 (5%) as Luminal A-HER2 Hybrid, and 8 (4%) as Luminal B-HER2 Hybrid. Majority of the ERBB2 tumors were high grade as expected, with average Nottingham score of 8. Moderate lymphoid infiltrate (constituting 25% to 50% of the tumor) was seen in 5 of 8 (63%) cases and necrosis in 3 of 8 (38%) cases. The most striking morphologic feature associated with ERBB2 tumors was the presence of apocrine differentiation seen in 7 of 8 (88%) cases. CK5 immunoreactivity was seen in 5 of 8 cases (63%). Epidermal growth factor receptor staining with 2+ or 3+ score was also seen in 5 cases (63%). Due to low prevalence of ERBB2 tumors, additional data set of 191 cases enriched in ERBB2 and TN tumors was used for confirmation of morphologic findings. We conclude that tumors with apocrine differentiation are most often of ERBB2 type. ERBB2 tumors demonstrate some features classically ascribed to TN basal-like tumors. Epidermal growth factor receptor overexpression in ERBB2 tumors may have additional predictive value.

Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis

The epidermal growth factor receptor (EGFR)-family and cyclin-D1 have been extensively studied in breast cancer; however systematic studies that examine protein expression and gene status in the same cohort of patients are lacking. Also emerging evidences suggest existence of a direct EGFR-signaling pathway, which involves cellular transport of EGFR from cell membrane to the nucleus, and transcriptional regulation of the target genes. Thus, we examined the protein expression of membrane EGFR, nuclear EGFR, cyclin-D1 and the corresponding gene status in 113 breast carcinomas by immunohistochemistry and fluorescence in situ hybridization using tissue microarrays. Membrane EGFR overexpression and EGFR gene amplification were detected in 2% cases, while nuclear EGFR was detected in 40% of cases, with 12% having high nuclear EGFR staining. Nuclear EGFR correlated with tumor size (P=0.0005), lymph node metastasis (P=0.0288), Nottingham prognostic index (P=0.0011) and estrogen receptor (ER) expression (P=0.0258) but the letter correlation was observed only in premenopausal group of patients. Strong cyclin-D1 expression and cyclin-D1 gene (CCND1) amplification were found in 64 and 13% of the cases, respectively. Cyclin-D1 expression showed positive correlation with ER (P=0.0113) and inverse correlation with Nottingham prognostic index (P=0.0309) and membrane EGFR (P=0.0201). CCND1 amplification also showed inverse correlation with membrane EGFR (P=0.0420). A strong correlation between membrane EGFR expression and gene amplification (P=0.0035), as well as cyclin-D1 overexpression and gene amplification (P=0.0362), was demonstrated. On univariate analysis cyclin-D1 expression showed a correlation with longer overall survival in the premenopausal group and nuclear EGFR correlated with shorter overall survival in whole cohort as well in the premenopausal group of patients. Multivariate analysis revealed nuclear EGFR to be an independent prognostic factor and showed 3.4 times greater mortality risk for nuclear EGFR+++ patients as compared with nuclear EGFR negative patients (hazard ratio =3.402; P=0.0026).

Expression of epidermal growth factor receptor in adult human airway epithelium--application of AMeX method

To elucidate the role of epidermal growth factor (EGF) in the maintenance and repair of airway epithelial cells in adult humans, we investigated immunohistochemically whether EGF receptor (EGFR) is expressed in these cells. In the present study, we employed the AMeX method (Sato et al., 1986) for tissue preparation. We first examined the expression of EGFR in peripheral lung tissue and bronchial tissue obtained at surgery from thirteen adult patients with lung carcinoma. The results showed that there was no positive staining for EGFR in the bronchial surface epithelium, bronchial glandular cells, bronchiolar epithelium or alveolar lining cells. There was also no expression of EGFR in cells comprising areas of basal cell hyperplasia and epidermoid metaplasia in the bronchus, or alveolar cell hyperplasia and columnar cell metaplasia in fibrotic lung tissue. Second, we examined the expression of EGFR in regenerating distal airway epithelial cells, which were experimentally produced by xenotransplantation of human lung tissue into the subcutaneous tissue of nude mice. There was also no expression of EGFR at any stage of regeneration from one week through six weeks after the transplantation. It is concluded that EGFR is not expressed, at least at an immunohistochemically detectable level, by airway epithelial cells of adult humans, not only in the quiescent state but also under conditions in which epithelial cells are stimulated to replicate. Thus, it appears unlikely that the EGF/EGFR system plays an important role in either maintenance or repair of airway epithelial cells in adult humans.

153. COMBINATION METHOTREXATE AND EPIDERMAL GROWTH FACTOR RECEPTOR INHIBITION AS A NOVEL MEDICATION-BASED CURE OF ECTOPIC PREGNANCIES

Ectopic pregnancies are serious gynaecological emergencies that can cause fatal haemorrhage. Most are treated surgically. Given the placenta is heavily reliant on Epidermal Growth Factor Receptor (EGFR) signaling, we set out to develop a medication-based treatment of ectopics using combination gefitinib (EGFR inhibitor) and methotrexate (folate antagonist). Both drugs are well tolerated and available in tablet form. We used in vitro and in vivo approaches to test the ability of gefitinib and methotrexate to regress placental tissue, and to explore molecular mechanisms. In vitro assays included immunohistochemistry (EGFR staining) western blot (EGFR phosphorylation), cell viability assays (Cell-Titre Blue, LDH cytotoxicity assay, xCELLigence system), apoptosis assays (PCR, FACS of M30 antibody) and The Bioplex Platform (phosphorylation of the EGFR pathway). JEG-3 xenografts were used to assess regression of placental tissue in vivo, where serum hCG was also measured (ELISA). EGFR was highly expressed in placentae from ectopic pregnancies. Combination treatment was supra-additive in inducing cell death of placental tissue in vitro, with &gt;70% cell death by 48 hours (Syncytialised BeWos, JEG-3 and 1st trimester trophoblast). This supra-additive effect was demonstrated in both end point assays (cell viability) and regular monitoring using The xCELLigence system. Gefitinib potently blocks EGFR phosphorylation in placental tissues in vitro (Western blot). Both drugs may be converging to inhibit Akt phosphorylation (Bioplex analysis). Combination treatment increases apoptosis (FACS of M30 antibody). In vivo, Gefitinib or MTX as single agents induced significant decrease in xenograft tumour volume in a dose dependent manner (n ≤ 5 mice per treatment). However, combining these drugs was supra-additive in decreasing xenograph tumour volume and weight. Serum hCG in mice was lowest with combination treatment. Combination gefitinib and methotrexate potently regresses placental tissue. It may be a novel therapeutic approach to cure ectopic pregnancies, potentially replacing surgery with tablets.

Epidermal growth factor receptor and HER-2/neu status by immunohistochemistry and fluorescence in situ hybridization in adenocarcinomas of the biliary tree and gallbladder

Adenocarcinomas of the biliary tract and gallbladder are aggressive tumors with a poor prognosis. Standard chemotherapy often offers minimal benefit. Because epidermal growth factor receptor and HER-2/neu antagonists have been successfully used in adenocarcinomas from other sites, their use in cholangiocarcinoma can be potentially beneficial. This study examines the epidermal growth factor receptor and HER-2/neu expression and the epidermal growth factor receptor gene copy number in biliary tract adenocarcinomas. Fifty-one formalin-fixed, paraffin-embedded cases of adenocarcinomas (26 intrahepatic, 19 extrahepatic, 6 gallbladder) were stained with monoclonal antibodies against epidermal growth factor receptor and HER-2/neu. Fluorescence in situ hybridization analysis was performed in 37 cases using probes directed against epidermal growth factor receptor and centromeric region of chromosome 7. Epidermal growth factor receptor expression was present in 41 (80%) cases, with moderate or strong epidermal growth factor receptor staining in 30 (59%) cases. HER-2/neu was positive in 2 (4%) cases. Fluorescence in situ hybridization analysis showed gain in epidermal growth factor receptor gene copy number in 17 (46%) tumors. Of the latter, 1 showed gene amplification, whereas all others showed gain in chromosome 7, indicating balanced polysomy. Epidermal growth factor receptor overexpression by immunohistochemistry correlated significantly with epidermal growth factor receptor copy number by fluorescence in situ hybridization (P = .02). HER2/neu expression is uncommon in these tumors.

Expression of Epidermal Growth Factor Receptor (EGFR) and Activated EGFR Predict Poor Response to (Chemo)radiation and Survival in Cervical Cancer

Activation of the epidermal growth factor receptor (EGFR) signaling pathway has been reported to induce resistance to (chemo)radiation in cancers, such as head and neck cancer, whereas EGFR-targeted agents in combination with (chemo)radiation seem to improve treatment efficacy. The aim of this study was to determine the relation between proteins involved in the EGFR pathway and response to (chemo)radiation and survival in a large, well-documented series of cervical cancer patients. Pretreatment tissue samples of 375 consecutive International Federation of Gynecologists and Obstetricians stage Ib to IVa cervical cancer patients treated with (chemo)radiation between January 1980 and December 2006 were collected. Clinicopathologic and follow-up data were prospectively obtained during standard treatment and follow-up. Protein expression of EGFR, phosphorylated EGFR (pEGFR), PTEN, phosphorylated AKT, and phosphorylated extracellular signal-regulated kinase (pERK) was assessed by immunohistochemistry on tissue microarrays. EGFR staining was present in 35.3%, pEGFR in 19.7%, PTEN in 34.1%, phosphorylated AKT in 4.1%, and pERK in 29.2% of tumors. pEGFR staining was related to PTEN (P = 0.001) and pERK staining (P = 0.004). EGFR staining was inversely related to PTEN (P = 0.011). In multivariate analysis, membranous staining of EGFR [hazard ratio (HR), 1.84; 95% confidence interval (95% CI), 1.20-2.82; P = 0.005] and cytoplasmic staining of pEGFR (HR, 1.71; 95% CI, 1.11-2.66; P = 0.016) were independent predictors of poor response to (chemo)radiation. Membranous EGFR staining also was an independent prognostic factor for poor disease-specific survival (HR, 1.54; 95% CI, 1.09-2.17; P = 0.014). EGFR and pEGFR immunostainings are frequently observed and independently associated with poor response to therapy and disease-specific survival in cervical cancer patients primarily treated by (chemo)radiation. Our data present the EGFR pathway as a promising therapeutic target in already ongoing clinical trials.

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches ( Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.

Analysis of ErbB Receptors in Pulmonary Carcinoid Tumors

This study aimed to investigate the expression of the ErbB family of receptor tyrosine kinases in pulmonary typical carcinoid and atypical carcinoid tumors and to understand the role of epidermal growth factor receptor (EGFR) signaling in pulmonary carcinoid tumor proliferation. Surgically resected typical carcinoid (n = 24) and atypical carcinoid (n = 7) tumor tissues were analyzed by immunohistochemical staining for EGFR, ErbB2, ErbB3, and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene and the KRAS gene was carried out. Biochemical analysis of lung carcinoid cell lines was used to investigate EGFR signal transduction and response to erlotinib inhibition. The analysis showed that 45.8% of typical carcinoid and 28.6% of atypical carcinoid tumors express EGFR, 100% of the tumors lack expression of ErbB2, and 100% have moderate to intense staining for ErbB3 and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene revealed the absence of tyrosine kinase domain mutations in these tumors. Instead, 80.6% tumors harbored a synonymous single nucleotide polymorphism in exon 20. Because EGFR and KRAS mutations tend not to be present at the same time, we sequenced the KRAS gene from pulmonary carcinoid tumor DNA and found that 100% were wild-type. Using a lung carcinoid cell line that expresses EGFR, we found that erlotinib reduced proliferation by inhibiting EGFR signal transduction. Our findings suggest clinical potential for the use of EGFR inhibitors in the treatment of patients with pulmonary carcinoid tumors, particularly for patients with EGFR-positive pulmonary carcinoid tumors not amenable to surgical resection.

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (⩾four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression ( P = 0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly ( P = 0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers ( P = 0.001, P = 0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

Expression modes and clinical manifestations of latent membrane protein 1, Ki‐67, cyclin‐B1, and epidermal growth factor receptor in nonendemic nasopharyngeal carcinoma

We aimed to identify clinical significance of latent membrane protein 1 (LMP1), Ki-67, cyclin-B1, and epidermal growth factor receptor (EGFR), in nonendemic nasopharyngeal carcinoma (NPC). The relation between expression of the markers in 45 NPC specimens and clinicopathological and survival variables was statistically analyzed. LMP1 was present in 33% of the tumors, and its presence was associated with advanced nodal and disease stages. Overexpression was defined as labeling index > or = median value for Ki-67, > or = 15% for cyclin-B1, and > or =50% for EGFR, and it was displayed in 50%, 55%, and 80% of the specimens, respectively. Strong EGFR staining intensity and not overexpression of the 3 markers was the variable with statistically significant impact on treatment outcomes in terms of worse local and locoregional tumor control rates. Our results suggest that the evaluation of EGFR staining intensity in patients with NPC may identify a subgroup of patients with poor prognosis.

Molecular alterations of EGFR and PIK3CA in uterine serous carcinoma

Objectives Uterine serous carcinoma (USC) is an aggressive endometrial cancer associated with poor prognosis despite comprehensive surgical staging and adjuvant chemotherapy and radiation therapy. Biologic targets have yet to be fully explored in this disease and research on such targets could lead to clinical trials utilizing a new class of therapeutics. This study sought to evaluate primary USC tumors for molecular alterations in epidermal growth factor receptor ( EGFR) and the recently characterized oncogene PIK3CA, which encodes the catalytic p110-alpha subunit of phosphatidylinositol 3-kinase (PI3K) and thus activates the AKT–mTOR oncogenic pathway. Methods Paraffin-embedded archival tissue of 45 primary USC tumors was utilized in this study. Immunohistochemical analysis of EGFR was performed and cases given a score of 0 to 12 calculated as the product of staining intensity (0 to 3+) and the percentage of positively stained cells (0–4), with 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%. For mutational analysis, neoplastic tissue was microdissected and DNA was extracted with phenol–chloroform. Exons 18 through 21 of EGFR and exons 9 and 20 of PIK3CA, the most commonly mutated exons of these genes, were amplified and directly sequenced. Results When EGFR was evaluated, moderate or strong EGFR membranous staining was observed in 25/45 (56%) USC cases. Thus, a mutational analysis was performed on 35 cases, including all cases with moderate and strong EGFR staining. No mutations were identified in EGFR. In contrast, PIK3CA mutations were confirmed in 5/34 (15%) of USC cases. Four cases were mutated in exon 20 and one case was mutated in exon 9. Conclusions Since optimal treatment of uterine serous carcinoma remains unknown, novel therapeutic approaches need to be actively pursued. In the current study of primary USC tumors, oncogenic mutations of the PIK3CA gene were seen in 15% of USC cases. This represents the first report of this gene mutation in USC. In addition, EGFR stained positively in the majority of cases, suggesting a possible target protein. These findings warrant further investigation and suggest a potential role for therapeutic agents targeting the PI3K–AKT–mTOR pathway, such as rapamycin, as well as possible targets of EGFR in the treatment of uterine serous carcinoma.

Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain

Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.