Epidermal Growth Factor Receptor Gene Alterations Research Articles

Inhibition of epidermal growth factor receptor suppresses parathyroid hormone-related protein expression in tumours and ameliorates cancer-associated cachexia.

BackgroundLung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour‐derived parathyroid hormone‐related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia.MethodsGlobal gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT‐PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour‐bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA‐seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression.ResultsExpression of EGFR ligands, EGFR itself, and PTHrP co‐clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8‐fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65–75% (P < 0.0005) in tumours upon treatment of LLC tumour‐bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour‐bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8‐fold, P < 0.05) and HNSC (17.5‐fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P < 0.0001 and R = 0.51, P < 0.0001; HNSC: n = 545, R = 0.34, P < 0.001 and R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log‐rank test, P = 0.0451).ConclusionsEpidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour‐bearing mice.

Epidermal Growth Factor Receptor Mutations in Resectable Non-Small Cell Lung Cancer Patients and their Potential Role in the Immune Landscape

BackgroundThe epidermal growth factor receptor (EGFR) is a therapeutic target for non-small cell lung cancer (NSCLC), but knowledge on gene mutations that contribute to NSCLC development and persistence is lacking. In this study, we investigated genetic variations in EGFR and their association with the clinical and pathological factors of NSCLC.Material/MethodsClinical cases (331 patients) and The Cancer Genome Atlas (TCGA) cases (1040 patients) were selected and analyzed using the refractory mutation systems cBioPortal and the Tumor Immune Estimation Resource (TIMER).ResultsEGFR mutation frequencies were 54.4% (180 of 331 patients) and 8.0% (83 of 1040 patients) in the clinical and TCGA cohorts, respectively. EGFR mutations were strongly associated with smoking and pathology (P≤0.05) in the clinical cohort, and with gender, smoking, and pathology (P=0.001, P<0.001, and P<0.001, respectively) in TCGA cohort. In cases of lung squamous carcinoma (LUSC), EGFR was overexpressed as a result of DNA amplification, but this amplified expression showed no association with the overall survival (OS) or progression-free survival of LUSC patients. EGFR gene alterations were, however, associated with worse OS in lung adenocarcinoma (LUAD) patients. Immune cell infiltrates from LUAD and LUSC tumors differed according to EGFR expression. EGFR mutations resulted in a decline of immune infiltration or a lack of infiltrating immune cells in the NSCLC microenvironment.ConclusionsMutational profiles of the EGFR in NSCLC patients provide useful information for the use of tyrosine kinase inhibitors for adjuvant or neoadjuvant therapy and immunotherapy.

Significant Effect of Anti-tyrosine Kinase Inhibitor (Gefitinib) on Overall Survival of the Glioblastoma Multiforme Patients in the Backdrop of Mutational Status of Epidermal Growth Factor Receptor and PTEN Genes.

Introduction:We aimed to assess the effect of anti-tyrosine kinase inhibitors (TKIs) (gefitinib) in overall survival (OS) of the glioblastoma multiforme (GBM) patients in the backdrop of mutational status of epidermal growth factor receptor (EGFR) and PTEN genes.Materials and Methods:All the patients subjected to resection or biopsies were put on gefitinib, and radiotherapy was delivered as per the hospital protocol. EGFR and PTEN mutational spectrum was performed by single-strand conformation polymorphism followed by DNA sequencing.Results:In total, 50% GBM tumors had mutation either in EGFR or PTEN. Median progression-free survival (PFS) and OS observed in patients with EGFR +ve/PTEN −ve were significantly favorable (P < 0.05) which aggregated to 9(7, 11) months and 20 (16, 24) months, respectively, than 6 (4, 8) months and 13 (7, 19) months in patients with PTEN +ve/EGFR −ve. Patients positive for both EGFR/PTEN had lower disease-free survival and OS of 6 and 9 months as compared to 6 (5, 7) and 14 (12, 24) months for those negative for both EGFR/PTEN.Conclusions:We conclude that EGFR gene alterations with wild-type PTEN are associated with significantly better PFS and OS in patients treated with anti-TKIs (gefitinib). Combined EGFR and PTEN gene mutation is associated with significantly poor response to gefitinib in terms of median OS.

EGFR as a clinical marker in glioblastomas and other gliomas

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein and a member of the tyrosine kinase superfamily receptor. Gliomas are tumors originating from glial cells, which show a range of aggressiveness depending on grade and stage. Many EGFR gene alterations have been identified in gliomas, especially glioblastomas, including amplifications, deletions and single nucleotide polymorphisms (SNPs). Glioblastomas are discussed as a separate entity due to their high correlation with EGFR mutants and the reported association of the latter with survival and response to treatment in this glioma subgroup. This review is a comprehensive report of EGFR gene alterations and their relations with several clinical factors in glioblastomas and other gliomas. It covers all EGFR gene alterations including point mutations, SNPs, methylations, copy number variations and amplifications, assessed with regard to different clinical variables, including response to therapy and survival. This review also discusses the current prognostic status of EGFR in glioblastomas and other gliomas, and highlights gaps in previous studies. This serves as an update for the medical community about the role of EGFR gene alterations in gliomas and specifically glioblastomas, as a means for targeted treatment and prognosis.

Epidermal growth factor receptor mutations in nonsmall cell lung carcinoma patients in Kuwait.

Context:Nonsmall cell lung carcinoma (NSCLC) is the most frequently diagnosed form of lung cancer in Kuwait. NSCLC samples from Kuwait have never been screened for epidermal growth factor receptor (EGFR) gene aberration, which is known to affect treatment options.Aims:This study investigated the feasibility of using fine-needle aspiration (FNA) material for mutational screening, and whether common EGFR mutations are present in NSCLC samples from Kuwait.Settings and Design:Eighteen NSCLC samples from five Kuwaitis and 13 non-Kuwaitis were included in this study.Materials and Methods:DNA was extracted from FNA cell blocks and screened for EGFR gene mutations using peptide nucleic acid (PNA)-clamp assay, and EGFR gene amplification using fluorescent in situ hybridization (EGFR-FISH). EGFR protein expression was assessed using immunohistochemistry.Results:Five EGFR mutations were detected in five non-Kuwaiti NSCLC patients (27.8%). EGFR gene amplification was evident in 10 samples (55.5%) by direct amplification or under the influence of chromosomal polysomy. Four samples had EGFR mutations and EGFR gene amplification, out of which only one sample had coexisting EGFR overexpression.Conclusions:Given the evidence of EGFR gene alterations occurring in NSCLC patients in Kuwait, there is a need to incorporate EGFR gene mutational screen for NSCLC patients to implement its consequent use in patient treatment.

EGFR, KRAS, BRAF and ALK gene alterations in lung adenocarcinomas: patient outcome, interplay with morphology and immunophenotype

Numerous studies have been published on single aspects of pulmonary adenocarcinoma (ADC). To comprehensively link clinically relevant ADC characteristics, we evaluated established morphological, diagnostic and predictive biomarkers in 425 resected ADCs. Morphology was reclassified. Cytokeratin-7, thyroid transcription factor (TTF)1, napsin A, thymidylate synthase and excision repair cross-complementing rodent repair deficiency complementation group-1 expression, anaplastic lymphoma kinase rearrangements as well as epidermal growth factor receptor (EGFR), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (KRAS) and v-Raf murine sarcoma viral oncogene homologue B1 (BRAF) mutations were analysed. All characteristics were correlated with clinical and survival parameters. Morphological ADC subtypes were significantly associated with smoking history and distinct patterns of diagnostic biomarkers. KRAS mutations were prevalent in male smokers, while EGFR mutations were associated with female sex, nonsmoking and lepidic as well as micropapillary growth patterns. TTF1 expression (hazard ratio (HR) for overall survival 0.61, p=0.021) and BRAF mutations (HR for disease-free survival 2.0, p=0.046) were found to be morphology- and stage-independent predictors of survival in multivariate analysis. Adjuvant radio-/chemotherapy, in some instances, strongly impacted on the prognostic effect of both diagnostic and predictive biomarkers. Our data draw a comprehensive picture of the prevalence and interplay of established histological and molecular ADC characteristics. These data will help to develop time- and cost-effective diagnostic and treatment algorithms for ADC.

MUC5AC, cytokeratin 20 and HER2 expression and K‐RAS mutations within mucinogenic growth in congenital pulmonary airway malformations

To analyse the expression of several mucins (MUC1, MUC2, MUC3, MUC5AC and MUC6), epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2 (HER2), thyroid transcription factor-1 (TTF-1), caudal type homeobox 2 (CDX2) and cytokeratin 20 (CK20), and the presence of mutations of EGFR, KRAS and HER2 in congenital pulmonary airway malformations (CPAM). Forty-one cases of CPAM and six pulmonary sequestrations were included. TTF-1 expression was observed in all cases but was not seen in mucinogenic growths in CPAM. CDX2 expression was completely negative. MUC1 expression was noted in 12 (29%) CPAM and in 33% sequestrations. MUC5AC was noted in only five cases (26%) by immunohistochemistry and was found in the mucinogenic proliferations of type 1 CPAM. No immunolabelling was noted for the other mucins. EGFR was expressed variably in almost all cases, while HER2 and CK20 was seen exclusively in the mucinogenic proliferations. All mucinous growths were characterized by KRAS mutations. No EGFR and HER2 gene alterations were identified. KRAS mutations and MUC5AC, CK20 and HER2 expression was seen in all mucinogenic proliferations, supporting the neoplastic nature of these cytologically bland growths. These findings emphasize the importance of complete surgical resection of such lesions.

EGFR Gene Alterations in a Norwegian Cohort of Lung Cancer Patients Selected for Surgery

Lung cancer is the leading cause of cancer-related deaths worldwide. New therapies targeting the epidermal growth factor receptor (EGFR) tyrosine kinase are promising and show high response rates in the subset of patients with activating mutations in EGFR. The frequency of these mutations is largely unknown in unselected Caucasian patients. Mutation analysis of EGFR exons 18-21 was performed on 240 lung cancer samples using the TheraScreen EGFR mutation kit and denaturing high-performance liquid chromatography in addition to sequencing. In a cohort of 240 Norwegian lung cancer patients selected for surgery, we identified 18 tumors with EGFR-activating mutations (7.5%, 14 women and 4 men), of which 14 were adenocarcinomas, 2 squamous cell carcinomas, and 2 bronchoalveolar carcinomas. Five of the mutations were found in patients with more than 20 pack-years of smoking history. The frequency of EGFR mutations is lower in our cohort than among Asian lung cancer patients and present in both men and women and smokers and never-smokers. However, the frequency is significantly higher among women and never-smokers and among patients with adenocarcinomas.

EGFR gene alterations as a prognostic biomarker in advanced esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) exhibits abnormalities in epidermal growth factor receptor (EGFR) gene. To identify a prognostic marker, the overexpression of EGFR protein, mutations in EGFR and p53 mutations were analyzed in pretreatment biopsy specimens removed from T3-4 and/or M1 LYM ESCC patients who received chemoradiotherapy. A silent mutation comprised of a single nucleotide polymorphism (SNP) at codon 787 of exon 20 of the EGFR gene was found in 19 patients (33%). In multivariate analysis, a significant difference was seen in the overall survival (odds ratio; 2.347, 95% confidence interval; 1.183-4.656, p = 0.015) between patients with and without the EGFR heterozygous genotype. Among the 57 eligible patients, 3-year survival rates was 21%, while in patients with EGFR heterozygous genotype the rate were 0%. However, neither overexpression of EGFR nor p53 mutations was associated with the overall survival. These results suggest that the EGFR SNP at codon 787 of exon 20 determined in pretreatment biopsy specimens may be a clinically useful biomarker for predicting the prognosis of ESCC patients.

Comparison of FISH, PCR, and Immunohistochemistry in Assessing EGFR Status in Lung Adenocarcinoma and Correlation With Clinicopathologic Features

The purpose of this study is to investigate the relationship between mutation status, copy number, and protein expression of epidermal growth factor receptor (EGFR) in lung adenocarcinoma, and to correlate the genetic status and clinicopathologic features. Forty-nine adenocarcinomas were tested by polymerase chain reaction (PCR) for EGFR gene mutations, by fluorescence in situ hybridization (FISH) for increased EGFR gene copy number, and by immunohistochemistry (IHC) for EGFR protein expression. Terminal respiratory unit (TRU) morphology, tumor grade, mitotic count, and pleural and lymphovascular invasion were also examined. Five of 49 tumors (10%) had EGFR mutation by PCR. Eighteen (36%) had high EGFR gene copy number by FISH, all of which were polysomies without gene amplification. Fifteen (31%) had EGFR protein overexpression by IHC, 13 (87%) of which were also positive by FISH. Of the PCR-positive cases, 4/5 (80%) were positive by FISH and 3/5 (60%) were positive by IHC. TRU morphology was observed in all PCR-positive cases and in about 50% of FISH-positive and IHC-positive cases. Pleural invasion was significantly more common in IHC and FISH-positive cases, and lymphovascular invasion was more often present in FISH-positive cases. No statistically significant differences in mitotic count, age, sex, smoking status, degree of differentiation, or stage were seen. Our findings suggest that a staged approach, with FISH testing as the first step, followed by PCR for the FISH-negative cases would be most effective in the identification of patients with EGFR gene alterations.

An overview of the epidermal growth factor receptor fluorescence in situ hybridisation challenge in tumour pathology

Epidermal growth factor receptor (EGFR) is deregulated in a variety of solid malignant tumours. Due to the availability of specific targeted therapies, the request to evaluate EGFR in neoplastic tissues...

Analysis of epidermal growth factor receptor (EGFR) downstream proteins in metastatic colorectal cancer patients: Comparison between primary tumor and related metastatic sites

11075 Background: EGFR expression by immunohistochemistry (IHC) is not predictive of response to cetuximab in metastatic colorectal cancer (mCRC). EGFR gene status (fluorescent in situ hybridization, FISH) and alterations in EGFR downstream proteins (K-Ras, PTEN) represent independent predictive markers of cetuximab efficacy. These data have been obtained analysing primary tumors (PT) of mCRC patients. However, PT and metastatic lesion (M) might share different patterns. Methods: We analyzed PT and M in 32 consecutive mCRC patients. We evaluated EGFR gene status and protein expression by IHC and FISH, K-Ras mutations in codons 12 and 13, and PTEN expression by IHC. Results: EGFR expression in PT was classified as score 1+ in 5 cases, score 2+ in 4, and score 3 in 21. In PT and M we observed the same pattern of EGFR expression in 26/30. Three patients showed score 3+ at PT and score 1+ at M, and 1 score 1+ in PT and score 2+ in M. We found in PT chromosome (Chr) 7 loss in 1 case, Chr 7 disomy in 9, Chr 7 polysomy in 14, and EGFR gene amplification in 6. The same pattern between PT and M was observed in 19 cases. A trend in favour of deregulation was observed in 7 cases (from disomy to polysomy in 5 cases, and from polysomy to gene amplification in 2 cases), whereas 4 patients showed gene amplification or polysomy in PT and polysomy or disomy in related M. K-Ras mutations were observed in 14 PT (47%). Only one patient showed the GtT change. The same mutational pattern between PT and M was observed in 26 cases. In 3 cases the mutation was restricted to PT, in 1 case to M. Loss of PTEN expression was found in 7 cases. The same PTEN expression pattern between PT and M was observed in 29 cases. Three cases showed a loss of PTEN expression limited to M. Conclusions: 1) PT and paired M may exhibit different gene and protein profiles. 2) A trend in favour of gene or protein expression deregulation most likely reflects a malignant progression of metastatic cells. The loss of deregulation in M with respect to paired PT may be explained by not representative biopsies. 3) EGFR and K-Ras gene status, and PTEN expression analysis should be performed on PT and possibly on representative (multiple) biopsies of M. 4) We confirm that GtT K-Ras mutation confers an indolent phenotype. No significant financial relationships to disclose.

Short-Term Gefitinib Treatment Brought About a Long-Term Regression of Bronchioloalveolar Carcinoma Without EGFR Gene Alterations: A Case Report

The tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) gefitinib has beneficial effect in some patients with refractory advanced non-small cell lung cancer (NSCLC). However, the majority of responders eventually develop acquired resistance during the course of prolonged continuous treatment. Here we present a case of 76-year-old Japanese female, who had never smoked, with poor performance status from bronchioloalveolar carcinoma (BAC), in whom a brief initial 5-week administration of gefitinib resulted in dramatic antitumor effects that lasted approximately 8.5 months after cessation of the treatment. Furthermore, the relapsed tumor later regressed again by re-treatment with the TKI. She survived 26 months since she first took gefitinib. Unexpectedly, neither sensitizing mutations for EGFR-TKIs nor increased copy numbers were detected in EGFR gene of her BAC cells. This case suggests that, in some patients with NSCLC, even short-term administration of gefitinib may bring about clinical benefits and disease response comparable to the standard long-term daily dosing schedule. Short-term use of gefitinib will also be able to minimize the expensive medical cost of the TKI. The potential role of short-term or pulse-dose therapy with EGFR-TKIs should be clarified in further prospective studies. Moreover, it is urgent to develop better strategies by which we could distinguish responders to the TKIs from nonresponders among patients who do not have any EGFR gene alterations.

Epidermal growth factor ligand/receptor loop and downstream signaling activation pattern in completely resected nonsmall cell lung cancer

In recent years, molecular insights shed light on the role of the epidermal growth factor receptor (EGFR) in nonsmall cell lung cancer (NSCLC), and new therapeutic agents, such as the EGFR tyrosine kinase inhibitors, were tested successfully, with responsiveness to those agents more likely in those patients with specific EGFR gene alterations. The objective of the current study was to investigate the protein profiles of EGFR, c-erb-B2, transforming growth factor alpha (TGF-alpha) (one of the EGFR ligands commonly expressed in NSCLC), and some downstream molecules, potentially to detect a subset of tumors with an activated autocrine loop that is responsible for higher intracellular signaling. One hundred twelve consecutive patients with resected NSCLC were analyzed by immunohistochemistry for EGFR, the c-erb-B2 receptor, TGF-alpha, and pivotal molecules downstream from EGFR activation. Statistical correlations between the investigated molecular expression profiles and clinicopathologic data were performed. EGFR, c-erb-B2, TGF-alpha and downstream molecule expression, per se, was not correlated significantly with any clinicopathologic variables, with the exception of a significant correlation between squamous histology and EGFR and between adenocarcinoma and TGF-alpha. However, nearly 30% of NSCLCs demonstrated coexpression of both TGF-alpha and EGFR, and this molecular status was associated positively with a statistically significant expression of phosphatidylinositol 3 kinase and an inversely with mitogen-activated protein kinase expression. The presence of a subgroup of NSCLCs with an activated autocrine loop may help to explain the mechanisms that lead to the relative ineffectiveness of the EGFR tyrosine kinase inhibitor and may support new clinical trials to define whether the subgroup of patients with these tumors reasonably may benefit from higher doses of such inhibitors or from the simultaneous inhibition of EGFR downstream signaling targets.

Epidermal Growth Factor Receptor Gene Amplification as a Prognostic Marker in Glioblastoma Multiforme: Results of a Meta-Analysis

Amplification of the epidermal growth factor receptor (EGFR) gene occurs in approximately 40% of cases of glioblastoma multiforme (GBM) and is considered a possible marker of poor prognosis. This report presents the results of a meta-analysis of the available data addressing this issue. Using a prospective protocol, a meta-analysis was designed to assess the possible prognostic importance of EGFR gene amplification in GBM. One-year survival data derived from seven published studies were analyzed using a general variance based method employing confidence intervals described by Greenland. The outcome of interest was a summary relative risk (RRs) reflecting the risk of death at 1 year from diagnosis associated with EGFR amplification-positive versus -negative disease. Prior to calculation of a RRs, an analysis for homogeneity (Q) showed Q to equal 9.21. With 6 df this yielded a P value of 0.12, indicating that the data were homogenous and could be combined in a meta-analysis. Pooling all available studies gave a RRs of 1.13 with a 95% confidence interval of 0.71-1.80, a nonstatistically significant result. The data suggest that the available studies are insufficient for determining whether EGFR gene amplification is of prognostic value in GBM. Important potential confounding factors are the influence of underlying EFGR gene mutation on patient survival and lack of control for important known clinical prognostic indicators in many studies. Future work must incorporate these parameters in multivariate analyses to determine whether EGFR gene alterations are truly associated with poor clinical outcome.

Reverse transcriptase polymerase chain reaction as a reliable method to detect epidermal growth factor receptor exon 2–7 gene deletion in human glioblastomas

Epidermal growth factor receptor (EGFR) gene amplification has been reported to occur in diverse carcinoma types such as lung, ovarian, and breast carcinomas and in glioblastomas. A 801-bp in-frame deletion close to the aminoterminus of the receptor protein has been found to occur more or less frequently within at least three of these tumor entities. We studied EGFR gene alterations using the polymerase chain reaction and EGFR gene expression of 65 astrocytic tumors (51 glioblastomas World Health Organization [WHO] IV, five anaplastic astrocytomas WHO III, and nine astrocytomas WHO II). EGFR gene amplification, as determined by Southern blotting using a full-length cDNA probe, was observed in 22 of 51 glioblastomas (43%) but in none of the grade II astrocytomas. Two of five anaplastic astrocytomas at WHO III showed a considerable degree of EGFR amplification but, according to the neuroradiological data, these two tumors had to be considered as glioblastomas. The most frequently found genetic alteration was the 801-bp deletion near the receptor aminoterminus comprising a complete loss of exon 2 to exon 7 (del 2–7). We showed that RT-PCR is superior to Southern blot analysis in detection of this type of deletion and can be assigned to 9 of 38 (24%) glioblastomas examined. Expression of a EGF receptor protein was enhanced in most of the tumors with gene amplification. However, 5 of 18 tumors that express a receptor protein in the absence of EGFR gene amplification also showed elevated levels of EGFR gene expression. In addition to the full-length receptor protein, a signal in the 140-kDa range was observed in 17 of 35 glioblastomas (49%). This fragment may correspond to the truncated del 2–7 receptor protein or might be due to proteolysis of the full-length receptor protein.