Epidermal Growth Factor Receptor Protein Expression Research Articles

Curcumin and (−)-epigallocatechin-3-gallate attenuate acrylamide-induced proliferation in HepG2 cells

Acrylamide, a proven rodent carcinogen, is present in carbohydrate-rich food heated at high temperatures. It can be metabolized into glycidamide mainly by cytochrome P450 2E1 (CYP2E1). The fact that acrylamide is a potential carcinogen to human-beings draws public attention recently. This study aimed to elucidate the effect of acrylamide at low doses on proliferation of HepG2 cells, and to test whether the two well-studied chemopreventive agents, curcumin and (−)-epigallocatechin-3-gallate (EGCG), would have antagonistic effects against acrylamide. The results showed that lower concentration of acrylamide (⩽100μM) significantly increased the proliferation of HepG2 cells, but not of the other cancer cells (MDA-231, HeLa, A549, and PC-3). Only in HepG2 cells, low concentration of acrylamide was able to induce CYP2E1 expression significantly. Knockdown of CYP2E1 restrained acrylamide to increase viability of HepG2 cells. In addition, acrylamide raised expression of epidermal growth factor receptor (EGFR), cyclin D1 and nuclear factor-κB (NF-κB), which contributed to cell proliferation. Both curcumin and EGCG effectively reduced acrylamide-induced proliferation, as well as protein expression of CYP2E1, EGFR, cyclin D1 and NF-κB. All these results suggest that low concentration of acrylamide may contribute to progression of hepatocellular carcinoma (HCC). Curcumin or EGCG could prevent acrylamide triggering this effect.

EGFR expression in vulvar cancer: clinical implications and tumor heterogeneity.

Epidermal growth factor receptor (EGFR) protein expression was assessed by immunohistochemistry (IHC) in 150 cases of invasive vulvar squamous cell carcinoma. In addition, gene copy number status by fluorescence in situ hybridization was performed in a smaller set of samples. Results were correlated with patient's clinical data and prognostic factors. EGFR overexpression (2+ and 3+) was observed on the membrane in 24.66% and 21.33% of all cases, respectively. Higher EGFR expression was associated with depth of invasion (P = .0409) and disease recurrence (P = .0401). Cytoplasm staining was found in 21.33% of the cases and was associated with absence of nodal metastasis (P = .0061) and better survival (P = .0199). Intratumor heterogeneity of EGFR IHC staining was frequently observed (55.33%) and was associated with the presence of nodal metastasis (P = .0207) and tumor invasion (P = .0161). Worse survival outcomes have been demonstrated in tumors with EGFR heterogeneity (P = .0434). EGFR gene status evaluated by fluorescence in situ hybridization did not correlate with protein expression evaluated by IHC. In conclusion, EGFR cytoplasm staining has no link with poorer outcome; still, this pattern of staining is even more related to better prognosis. EGFR heterogeneity of staining correlated with more aggressive tumors, and presented to be an important marker of poor prognosis in vulvar squamous cell carcinoma. The usage of small biopsies or even tissue microarrays for vulvar cancer evaluation should be carefully reconsidered for the assessment of EGFR as the results may be misleading. Protein overexpression may be independent on gene amplification, showing that other molecular mechanisms than copy number variation may regulate protein expression of EGFR in vulvar cancer.

Canine pulmonary adenocarcinoma tyrosine kinase receptor expression and phosphorylation

BackgroundThis study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18–21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence.ResultsThe pro-angiogenic growth factor receptor, PDGFRα, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the corresponding normal lung tissue. The EGFR mRNA, protein expression and phosphorylation were not increased compared to the normal lung and no activating mutations were identified in exons 18–21.ConclusionsCanine pulmonary adenocarcinoma TKRs are detected at both the mRNA and protein levels and are activated. Further investigation into the contribution of TKR activation in cpAC tumorigenesis is warranted.

Targeting the epidermal growth factor receptor for head and neck cancer chemoprevention

The epidermal growth factor receptor (EGFR) has been implicated in head and neck squamous cell carcinoma (HNSCC) carcinogenesis. It is currently the only molecular target in head and neck cancers for which there are pharmacologic therapeutic interventions approved by regulatory agencies worldwide to treat advanced disease. Oral pre-malignant lesions have increased EGFR protein expression and increased egfr gene copy number compared to normal mucosa. Oral pre-malignant lesions with overexpression of EGFR or egfr gene copy number gain are at higher risk for malignant transformation. Inhibition of EGFR in pre-clinical models of oral pre-malignancies validates this approach as an effective way to reduce the incidence of oral cancer, and supports investigation of this strategy in the clinic. Clinical trials with EGFR targeted agents, including cetuximab, erlotinib, and vandetanib, are currently under way, some with promising preliminary results. If ultimately shown to reduce the risk of oral cancer, chemoprevention with EGFR inhibitors may significantly reduce morbidity and possibly mortality from HNSCC.

Revisiting epidermal growth factor receptor in glioblastoma multiforme: does it play a role in response to therapy?

Glioblastoma multiforme (GBM) are the most aggressive class of cancer of central nervous system with hallmark characteristics that include rampant proliferation, necrosis, and endothelial proliferation. Epidermal growth factor receptor (EGFR) has been implicated as the primary contributor to glioblastoma initiation and succession. The present study was designed to evaluate EGFR protein expression in GBM as predictor of response to therapy and survival. Epidermal growth factor receptor was assessed by immunohistochemistry as a percentage of positive tumor cells in hot spots (10 high-power fields). The study group comprised of 35 cases of GBM. All cases underwent surgical resection and subsequently underwent radiotherapy (n = 17) or radiotherapy with adjuvant temozolomide chemotherapy (n = 18). Immediate response to therapy was assessed at 3 months using World Health Organization response evaluation criteria in solid tumors criteria and cases followed up for survival. Twenty-four cases (68.6%) expressed EGFR while 11/35 (31.4%) cases were negative. Response to therapy was evident in 21/35 cases (60.0%) and 14/35 were (40.0%) nonresponders. Mean EGFR protein expression in responders was 37.23 ± 33.70 and in nonresponders was 59.5 ± 39.46 (P = 0.542). The percentage of responders which were EGFR negative was 72.7% and while response in EGFR positive cases was observed in 54.2%. Mean survival in EGFR positive and negative GBM was 394.37 ± 189.11 and 420.54 ± 191.23 days, respectively. The EGFR negative cases appear to respond better to therapy, however, the difference is not statistically significant (P = 0.298). Further, EGFR protein expression does not play a definitive role in predicting survival. This is an original study evaluating EGFR in terms of therapeutic response.

Effects of Okadaic Acid, Retinoic Acid, and Phorbol Myristate Acetate Tumor Promoter on Oncogene Expression

The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.

Novel EGFR mutation-specific antibodies for lung adenocarcinoma: Highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry

ObjectivesActivating mutations in the epidermal growth factor receptor (EGFR) kinase domain are correlated with dramatic clinical responses in non-small cell lung cancer patients treated with EGFR-tyrosine kinase inhibitors (TKIs). The two most common EGFR mutations, representing 85–90% of EGFR mutations, are the E746_A750 deletion in exon 19 and the L858R point mutation in exon 21. We conducted this study to evaluate the suitability of mutation-specific antibodies that can detect E746_A750 deletion and L858R mutant EGFR proteins by immunohistochemistry (IHC). Materials and methodsIn a cohort of consecutive patients with surgically resected lung adenocarcinomas (n=240), mutant EGFR protein expression was assessed by IHC using specific antibodies (clone SP111 and SP125) to the 2 major forms of EGFR mutations. Immunoreactivity was scored as 0–3, and the results were compared with the EGFR-mutational status. ResultsWith a cutoff value of IHC 2+ for SP 111 (anti-EGFR E746_A750 del antibody) and SP 125 (anti-EGFR L858R antibody), both antibodies showed high specificity (99.0% and 89.7%, respectively) and sensitivity (70.6% and 80.4%, respectively). While cases with IHC scores of 3 using these 2 antibodies positively correlated with the EGFR-mutational status, cases with IHC scores lower than 3+ showed variable results regarding EGFR-mutational status. ConclusionAlthough each antibody showed relatively high specificity, some EGFR-mutant cases were not detected by the mutation-specific antibodies. Various forms of exon 19 deletions, except E746_A750, were rarely detected by the mutant-specific antibody. Therefore, IHC-negative cases require further molecular analysis to confirm the presence of EGFR mutations.

Influence of leucine-rich repeats and immunoglobulin-like domains 3 antisense oligodeoxynucleotide on proliferation in nude mice transplanted tumor of human cervical cancer

Objective Leucine-rich repeats and Immunoglobulin-like domains (LRIGs) gene family is a group of recently discovered tumor-associated genes,which acts in a negatingve feedback loop of epidermal growth factor receptor (EGFR) and inhibits the deterioration and proliferation of cancer.The present study investigated the effect of LRIG3 silencing on tumor growth and EGFR expression of the human cervical carcinoma xenografts in nude mice.Methods The Hela229 cells were inoculated subcutaneously into nude mice.The mice were divided into three groups after intratumoral injection of saline,LRIG3 antisense oligodeoxynucleotide (ASODN),and LRIG3 MSODN,respectively.Results Compared to MSODN and saline groups,the tumors in ASOND group grew faster and showed larger size,and demonstrated more deeply stained nucleolus by HE staining.LRIG3 protein expression in the tumors of saline,MSODN and ASODN group were 0.2302 ± 0.0108,0.2203 ± 0.0183 and 0.1532 ± 0.0132,respectively,the expression was significantly down-regulated in ASODN group compared to the other two groups (P ＜ 0.05).EGFR protein expression in the tumors in ASODN group was 1.2730 ± 1.1116,and significantly elevated compared to that in saline and MSODN group,which was 0.9212 ±0.0796 and 0.9904 ±0.0830,respectively (P ＜ 0.05).Conclusion Inhibition of LRIG3 gene expression can accelerate human cervical carcinoma xenograft growth and elevate EGFR expression in xenograft tumors. Key words: Leucine-rich repeats and Immunoglobulin-like domains 3 ; Epidermal growth factor receptor; Nude

Phosphatidylinositol 3-Kinase dependent upregulation of the epidermal growth factor receptor upon Flotillin-1 depletion in breast cancer cells.

BackgroundFlotillin-1 and flotillin-2 are two homologous and ubiquitously expressed proteins that are involved in signal transduction and membrane trafficking. Recent studies have reported that flotillins promote breast cancer progression, thus making them interesting targets for breast cancer treatment. In the present study, we have investigated the underlying molecular mechanisms of flotillins in breast cancer.MethodsHuman adenocarcinoma MCF7 breast cancer cells were stably depleted of flotillins by means of lentivirus mediated short hairpin RNAs. Western blotting, immunofluorescence and quantitative real-time PCR were used to analyze the expression of proteins of the epidermal growth factor receptor (EGFR) family. Western blotting was used to investigate the effect of EGFR stimulation or inhibition as well as phosphatidylinositol 3-kinase (PI3K) inhibition on mitogen activated protein kinase (MAPK) signaling. Rescue experiments were performed by stable transfection of RNA intereference resistant flotillin proteins.ResultsWe here show that stable knockdown of flotillin-1 in MCF7 cells resulted in upregulation of EGFR mRNA and protein expression and hyperactivation of MAPK signaling, whereas ErbB2 and ErbB3 expression were not affected. Treatment of the flotillin knockdown cells with an EGFR inhibitor reduced the MAPK signaling, demonstrating that the increased EGFR expression and activity is the cause of the increased signaling. Stable ectopic expression of flotillins in the knockdown cells reduced the increased EGFR expression, demonstrating a direct causal relationship between flotillin-1 expression and EGFR amount. Furthermore, the upregulation of EGFR was dependent on the PI3K signaling pathway which is constitutively active in MCF7 cells, and PI3K inhibition resulted in reduced EGFR expression.ConclusionsThis study demonstrates that flotillins may not be suitable as cancer therapy targets in cells that carry certain other oncogenic mutations such as PI3K activating mutations, as unexpected effects are prone to emerge upon flotillin knockdown which may even facilitate cancer cell growth and proliferation.

RETRACTED ARTICLE: Genomic amplification and high expression of EGFR are key targetable oncogenic events in malignant peripheral nerve sheath tumor

BackgroundThe dismal outcome of malignant peripheral nerve sheath tumor (MPNST) highlights the necessity of finding new therapeutic methods to benefit patients with this aggressive sarcoma. Our purpose was to investigate epidermal growth factor receptor (EGFR) as a potential therapeutic target in MPNSTs.Patients and methodsWe performed a microarray based-comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center (MD Anderson) and 26 patients from Tianjin Medical University Cancer Institute & Hospital (TMUCIH). Fluorescence in situ hybridization (FISH) method was used to validate the gene amplification detected by aCGH analysis. Another independent cohort of 56 formalin fixed paraffin embedded (FFPE) MPNST samples was obtained to explore EGFR protein expression by immunohistochemical analysis. Cell biology detection and validation were performed on human MPNST cell lines ST88-14 and STS26T.ResultsaCGH and pathway analysis of the 51 MPNSTs identified significant gene amplification events in EGFR pathway, including frequent amplifications of EGFR gene itself, which was subsequently validated by FISH assay. High expression of EGFR protein was associated with poor disease-free and overall survival of human MPNST patients. In human MPNST cell lines ST88-14 and STS26T, inhibition of EGFR by siRNA or Gefitinib led to decreased cell proliferation, migration, and invasion accompanied by attenuation of PI3K/AKT and MAPK pathways.ConclusionThese results suggest that EGFR is a potential therapeutic target for MPNST.

Prognostic value of survivin and EGFR protein expression in triple-negative breast cancer (TNBC) patients

Triple-negative breast cancer (TNBC) is a particular type of breast cancer which is characterized by its biological aggressiveness, worse prognosis, and lack of prognostic markers or therapeutic targets in contrast with hormonal receptor-positive and human epidermal growth factor receptor 2-positive (HER2+) breast cancers. We aimed to evaluate survivin and epidermal growth factor receptor (EGFR) expression and their prognostic value and determine their relationships with the clinicopathological parameters of TNBC. A total of 136 patients who had undergone a resection of primary TNBC were enrolled at the Third Affiliated Hospital of Harbin Medical University from March 2003 to September 2005. Expression of ER, PR, HER2, EGFR, and survivin was assessed by immunohistochemistry. The association of TNBC and other clinicopathological variables and the prognostic value of survivin and EGFR expression were evaluated. Survivin was expressed in 62 (45.6 %) cases and EGFR was expressed in 82 (60.3 %) cases. Survivin expression was associated with menopausal status (P = 0.011), tumor size (P = 0.037), and lymph node status (P = 0.001). EGFR expression was associated with menopausal status (P = 0.029), lymph node status (P = 0.004), P53 expression (P = 0.001), Ki-67 expression (P = 0.028), and lymphatic vascular invasion (P = 0.037). A multivariate analysis demonstrated that tumor size (hazard ratio (HR) 1.587, 95 % confidence interval (CI) 1.081–2.330, P = 0.018 for disease-free survival (DFS); HR 1.606, 95%CI 1.096–2.354, P = 0.015 for overall survival (OS)), lymph node status (HR 2.873, 95%CI 1.544–5.344, P = 0.001 for DFS; HR 2.915, 95%CI 1.553–5.471, P = 0.001 for OS), tumor grade (HR 1.914, 95%CI 1.218–3.007, P = 0.005 for DFS; HR 1.983, 95%CI 1.228–3.203, P = 0.005 for OS), EGFR (HR 3.008, 95%CI 1.331–6.792, P = 0.008 for DFS; HR 3.151, 95%CI 1.374–7.226, P = 0.007 for OS), and survivin (HR 1.573, 95%CI 1.087–2.277, P = 0.016 for DFS; HR 1.607, 95%CI 1.088–2.374, P = 0.017 for OS) were of prognostic significance for disease-free and overall survival. We draw a conclusion from the present study that survivin and EGFR expression are useful prognostic markers of TNBC and might be useful for molecular targeting therapy of TNBC treatment.

Analysis of epidermal growth factor receptor expression and gene expression status in tissue microarray of cervical squamous cell carcinoma

To elucidate the protein expression and gene expression status and the relationship between epidermal growth factor receptor (EGFR) protein expression and EGFR gene status. Tissue microarray containing 72 cervical squamous cell carcinoma tissues was constructed, and EGFR protein expression and gene status were evaluated by immunohistochemical and fluorescence in situ hybridization (FISH) techniques. Protein expression of EGFR: 69 of 72 cervical squamous cell carcinomas were observed. The results demonstrated it was significant association with invasion depth, lymph node metastasis and lymph-vessel invasion (χ(2) = 4.998, P < 0.05; χ(2) = 4.299, P < 0.05; χ(2) = 4.686, P < 0.05) in cervical squamous cell carcinomas. For FISH assessing EGFR gene, 64 of 72 carcinomas were observed; 7 of 64 cases showed EGFR gene amplification, and 25 disomy, 23 trisomy and 9 polysomy were detected. There were high levels of protein expression in all the EGFR gene amplification cases, and there were significant association between EGFR protein expression and the gene copy number (χ(2) = 13.564, P < 0.05). EGFR may participate in the occurrence, progression and metastasis of cervical squamous cell carcinoma. Overexpression of EGFR protein may result from gene amplification and gene copy number increases, which showed that EGFR gene expression status may be a more effective biological indicator of cervical squamous cell carcinoma targeted therapy.

Detecting EGFR alterations in clinical specimens—pitfalls and necessities

We investigated the epidermal growth factor receptor (EGFR) status in early stage lung cancer in Southern Sweden, a population for which there are no previous reports on the EGFR mutation frequency. Three hundred fifty small cell lung cancers, adenocarcinomas (AC), squamous cell carcinomas (SqCC), and large cell carcinomas were analyzed using a combination of techniques for the analysis of protein expression, gene copy numbers, and mutations. Immunohistochemical (IHC) staining with antibodies for the EGFR mutations L858R and del E746-A750 revealed intratumoral heterogeneity and several discrepant cases when compared to mutation-specific polymerase chain reaction (PCR)-based analysis. The frequencies of these two mutations, when considering IHC staining with mutation-specific antibodies in a cohort of 298 cases and subsequent confirmation by PCR, were 10 % in AC and <2 % in SqCC. Furthermore, screening by sequencing of EGFR in a cohort of 52 lung AC and squamous carcinomas demonstrated a more diverse mutation spectrum, not covered by the mutation-specific antibodies. High expression of total EGFR protein was correlated to high gene copy numbers but did not reflect the mutational status of the tumors. We believe that the mutation spectra in a Southern Swedish population is too diverse to be covered by the mutation-specific antibodies, and we also raise some other issues regarding the use of the mutation-specific antibodies, for example concerning heterogeneous expression of the mutated protein, optimal antibody dilution, and discrepancies between staining results and PCR.

Abstract C37: Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors

Abstract The role of annexin A6 (AnxA6), a calcium-dependent phospholipid binding protein, in breast carcinogenesis remains poorly understood. Some reports suggest that AnxA6 inhibits the activation and down-stream signaling of epidermal growth factor receptor (EGFR). On the contrary, other studies have shown that AnxA6 not only stabilizes activated receptors on the cell surface but also promotes the expression of differentiation markers in chondrocytes. Together with our recent study showing that AnxA6 expression is reduced in breast cancer, we investigated whether AnxA6 expression status influences breast cancer outcome in patients and whether this may be attributed to its modulation of EGFR expression. We show that low AnxA6 expression correlates with reduced distant metastasis-free and overall survival, but is associated with a higher probability for recurrence-free survivalof patients with basal-like breast cancer. To determine whether these effects are mediated via EGFR, a predictive marker of basal-like breast cancer, we examined the effects of altered expression of AnxA6 in AnxA6-high invasive triple negative breast cancer cells. Interestingly, depletion of AnxA6 in invasive BT-549 cells was accompanied by decreased expression of EGFR protein and mRNA. We also show that in invasive breast cancer cells, AnxA6 expression is required for sustained membrane localization of activated EGFR and persistent downstream signaling. Consequently, AnxA6-depleted cells exhibited decreased cell motility, and increased sensitivity to the tyrosine kinase inhibitors (TKIs), lapatinib and PD153035. On the contrary, over-expression of AnxA6 in AnxA6-low HCC1806 cells did not affect their motility but as expected, inhibited their growth in 3D matrigel cultures. These data suggest that AnxA6-modulation of EGFR expression and activity underlies at least in part, the changes in proliferation, metastatic potentials and sensitivity to EGFR-targeted TKIs of invasive breast cancer cells. Our data also suggest that AnxA6 expression status may predict the survival and likelihood to respond to EGFR-targeted therapies of patients with basal-like breast cancer. Citation Format: Rainelli B. Koumangoye, Josiah Ochieng, Amos M. Sakwe. Reduced annexin A6 expression enhances EGFR degradation and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C37.

Prognostic role of epidermal growth factor receptor in head and neck cancer: A meta-analysis

To evaluate the predicting value of the epidermal growth factor receptor (EGFR) for survival in patients with head and neck cancer (HNC). Data were collected from studies comparing overall survival (OS) or progression-free survival (PFS) in patients with higher or lower EGFR levels. Studies were pooled and combined hazard ratios (HRs) of EGFR for survival were calculated. A total of 68 studies involving 6,781 patients were included for meta-analysis. Either EGFR protein expression or gene copy number had prognostic value in HNC patients. EGFR overexpression could predict worse outcome, with HRs of 1.65 (95% CI: 1.45, 1.86) for OS and 1.27 (95% CI: 1.17, 1.37) for PFS. Increased EGFR copy number was also associated with reduced survival, with HRs of 1.5 (95% CI: 1.15, 1.96) for OS and 1.35 (95% CI: 1.14, 1.61) for PFS. Furthermore, EGFR overexpression could predict poorer OS in both eastern and western countries. Particularly, EGFR was considered a strong predictor in laryngeal squamous cell cancer (HR > 2). Elevated EGFR expression and gene copy number could predict poor survival in HNC patients.

Alterations in the EGFR pathway coincide in colorectal cancer and impact on prognosis

Alterations of the downstream effectors of the epidermal growth factor receptor (EGFR) are common events in colorectal cancer (CRC) carcinogenesis. Since EGFR serves as a target for therapy and some downstream effectors of EGFR have predictive and prognostic impact, reliable information on the frequency and concordance of alterations in the signaling pathway has become clinically significant. We, therefore, determined the frequency and coincidence of mutations in the EGFR pathway. We also analyzed the concordance of these alterations between primary tumor and distant metastases. Furthermore, we assessed their prognostic relevance for the development of metastasis. Mutations of KRAS exon 2, BRAF exons 11 and 15, AKT exon 3, and PIK3CA exons 9 and 20 were analyzed by pyrosequencing in 171 primary CRC samples as well as in 63 corresponding metastases. Furthermore, the expression of PTEN and EGFR was assessed by immunohistochemistry. Of the 171 tumors investigated, 60.2% showed mutations in one or more genes of pathways downstream of EGFR. KRAS exon 2 and BRAF exon 15 mutations were detected in 40.9 and 11.1% of cases, respectively, and were mutually exclusive. Mutations in exons 9 and 20 of the PIK3CA gene (18.7%) largely overlapped with exon 2 KRAS mutations (16 of 32 cases; 50.0%) and, to a lesser extent, with exon 15 mutations of BRAF (2 of 32 cases; 6.3%). Only one case had simultaneous mutations of AKT exon 3 (0.6%) and BRAF exon 15. Mutation analysis for KRAS exon 2, BRAF exon 15, PIK3CA exon 20, and AKT exon 3 in primary tumors and in their corresponding metastases revealed 100% concordance. In one case, a PIK3CA exon 9 mutation in the primary tumor could not be detected in the matched distant metastases (κ = 0.9). Three different scores were applied for the evaluation of EGFR immunohistochemistry, and the range of positive cases varied between 8.8 and 52.6%. Loss of PTEN expression was detected in 38.6%. Although the expression of both markers does coincide with KRAS exon 2, BRAF exon 15, AKT exon 3, and PIK3CA exons 9 and 20 mutations, high discordance rates were found. The presence of at least one alteration in downstream effectors of the EGFR pathway was associated with a higher rate of distant metastases (p = 0.002). PIK3CA exons 9 and 20 mutations overlap with KRAS exon 2 and BRAF exon 15 mutations, and BRAF exon 15 and AKT exon 3 mutations co-occur in a single tumor, whereas KRAS exon 2 and BRAF exon 15 mutations are mutually exclusive. This suggests that mutations in the PIK3CA/PTEN/AKT branch of the EGFR pathway are less important than those of the RAS/RAF/MAPK branch for the progression of CRC. We found no difference in the mutational status of KRAS exon 2, BRAF exon 15, and AKT exon 3 between primary tumor and distant metastasis, validating both for diagnostic purposes. PIK3CA exons 9 and 20 mutations can be discordant between primary tumor and distant metastasis, and therefore, the lesion which is targeted for therapy should be tested. Protein expression of PTEN and EGFR using current protocols yields highly discordant results, and better standardization is needed before these markers can be used for diagnostic purposes.

Evaluation of EGFR protein expression by immunohistochemistry using H-score and the magnification rule: Re-analysis of the SATURN study

IntroductionThe phase III SATURN study demonstrated that first-line maintenance erlotinib extended progression-free survival (PFS) and overall survival (OS) versus placebo in patients with advanced non-small cell lung cancer (NSCLC). Analysis of epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC) found no significant interaction between EGFR IHC status and PFS (p=0.63) or OS (p=0.52). The FLEX study of first-line cetuximab plus chemotherapy demonstrated that EGFR IHC expression was predictive of improved OS with cetuximab when assessed by H-score with a magnification rule. This novel method was used to reassess samples from SATURN. MethodsThe H-score method assigned a score of 0–300 to each patient, based on the percentage of cells stained at different intensities viewed at various magnifications. The discriminatory threshold was set at 200, per the FLEX study, and existing samples were re-read and classed as low (H-score<200) or high (≥200) EGFR expression. PFS and OS were re-analyzed based on these new classifications. ResultsIn the overall and EGFR wild-type populations, erlotinib provided a consistent survival benefit versus placebo. Hazard ratios (HRs) in the overall population were similar between EGFR IHC-positive and -negative patients for median PFS (HR 0.68 [95% confidence interval (CI) 0.53–0.86] and 0.76 [95% CI 0.62–0.93], respectively) and OS (HR 0.80 [95% CI 0.62–1.05] and 0.80 [95% CI 0.64–1.01] for IHC-positive and IHC-negative, respectively). In the EGFR wild-type population, HRs were again similar between EGFR IHC-positive and -negative subpopulations for PFS (HR 0.69 [95% CI 0.51–0.95] and 0.84 [95% CI 0.63–1.12], respectively) and OS (HR 0.78 [95% CI 0.55–1.10] and 0.76 [95% CI 0.55–1.05], respectively). ConclusionsThese data suggest that EGFR IHC does not have value as a marker to predict erlotinib benefit in the first-line maintenance setting for advanced NSCLC.

MicroRNA‐133a regulates the cell cycle and proliferation of breast cancer cells by targeting epidermal growth factor receptor through the EGFR/Akt signaling pathway

microRNAs are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition, and are widely involved in carcinogenesis and cancer development. In this study, the expression profile of microRNA-133a (miR-133a) was examined in breast cancer cells and breast cancer tissues. The results showed that expression of miR-133a in both breast cancer cells and breast cancer tissues was significantly down-regulated. Over-expression of miR-133a in tumor cells arrested the cell cycle by drastically decreasing the G2 /S phase and retarded the newly synthesized DNA, suggesting a regulatory role for miR-133a in proliferation of breast cancer cells. Bioinformatics prediction showed that epidermal growth factor receptor (EGFR) is a potential target for miR-133a. A dual luciferase reporter gene assay showed that miR-133a bound to the 3' UTR of EGFR but not a mutated 3' UTR, thereby down-regulating the protein expression level. Accordingly, we found that expression of EGFR protein decreased with increased expression of miR-133a in MCF-7 and MDA-MB-231 cells. Over-expression of miR-133a in breast cancer cells resulted in suppression of the level of phosphorylated Akt protein (p-Akt) and inhibition of p-Akt nuclear translocation. These results demonstrate that miR-133a, which may act as a tumor suppressor in breast cancer, regulates the cell cycle and proliferation in tumorigenesis by targeting EGFR through the downstream signal molecule Akt. Overall, these results show that miR-133a may be used as biomarker and/or therapeutic target for diagnosis and therapy of breast cancer.

Epidermal growth factor receptor expression and KRAS and BRAF mutations: study of 39 sinonasal intestinal-type adenocarcinomas

Sinonasal intestinal-type adenocarcinomas (ITACs) are uncommon tumors of poor prognosis defined by their similarities to colorectal adenocarcinomas. The involvement of the epidermal growth factor receptor (EGFR) pathway in colorectal adenocarcinoma oncogenesis is well established, and the same is expected to apply to ITACs. In a series of 39 ITACs, we investigated EGFR amplification and chromosome 7 polysomy by fluorescence in situ hybridization; EGFR, KRAS, and BRAF mutational status by polymerase chain reaction sequencing; EGFR variant messenger RNA expression by quantitative reverse transcriptase polymerase chain reaction; and EGFR protein expression by immunohistochemistry with antibodies targeting the extracellular domain, the intracellular domain, and the phosphorylated isoform. The findings were analyzed with respect to clinical data, histologic typing, and patient outcome. EGFR amplification was observed in 3 cases with a focal distribution. EGFR proteins were overexpressed in all these foci with both extracellular domain and intracellular domain antibodies, suggesting involvement of the whole receptor. Chromosome 7 polysomy was observed in 15 cases and was not associated with EGFR protein expression. EGFR, KRAS, or BRAF mutations were observed in 5 different cases. The EGFRvIII mutant was not detected. In all cases, EGFR variants were expressed. There was no association between these molecular features and patient survival. In conclusion, (1) our study revealed various EGFR expression patterns in ITACs, indicating tumor heterogeneity; (2) EGFR amplification should be distinguished from chromosome 7 polysomy; (3) fluorescence in situ hybridization analysis could be guided by immunohistochemistry; and (4) ITACs share common alterations of the EGFR pathway with colorectal adenocarcinomas, except for a lower frequency of KRAS and BRAF mutations.

Effect of leucine-rich repeats and immunoglobulin-like domains 1 on human heptoma cell line HepG2 and potential mechanism involved

Objective To investigate the effect of high mobility group leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) on the proliferative activity of human hepatoma cell line Hep-G2 in vivo and in vitro and its potential regulating mechanism.Methods The cultured Hep-G2 cells were treated with recombinant LRIG1.Cell proliferation was observed by cell counting kit-8 (CCK-8) analysis.Transwell invasion assay was used to monitor the changes of Hep-G2 cell malignant phenotypes.Changes of LRIG1 and epidermal growth factor receptor (EGFR) expression at mRNA and protein levels were measured by using real-time polymerase chain reaction (Real-time PCR) and Western blotting,respectivetly.Results At 24,48 and 72 h,the CCK-8 analysis results showed that the cell survival rate in pEGFP-N1LRIG1 group and pEGFP-N1 group was 92.4％,86.4％ and 80.3％,and 55.2％,50.9％ and 46.5％ respectively.As compared with pEGFP-N1 group,proliferation of Hep-G2 cells was obviously inhibited in pEGFP-N1-LRIG1 group (P ＜0.05).The percentage of invasive cells in pEGFP-N1-LRIG1 group,control group and pEGFP-N1 group was 33.24％,100％ and 92.85％ respectively.The invasive ability of Hep-G2 cells in pEGFP-N1-LRIG1 group was significantly inhibited as compared with pEGFP-N1 group and control group (P ＜ 0.05).LRIG1 obviously inhibited the expression of EGFR mRNA and protein in vitro (P ＜ 0.05).Conclusion LRIG1 could inhibit the priliferation of Hep-G2 cells probably by suppressing the expression of EGFR mRNA and protein. Key words: Carcinoma, hepatocellular; Leucine-rich repeats and immunoglobulin-like domains 1; Epidermal growth factor receptor; Phenotypes