Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer

PubMed Central

Normanno, Nicola; Denis, Marc G.; Thress, Kenneth S.; Ratcliffe, Marianne; Reck, Martin

2017-01-01

Cancer treatment is evolving towards therapies targeted at specific molecular abnormalities that drive tumor growth. Consequently, to determine which patients are eligible, accurate assessment of molecular aberrations within tumors is required. Obtaining sufficient tumor tissue for molecular testing can present challenges; therefore, circulating free tumor-derived DNA (ctDNA) found in blood plasma has been proposed as an alternative source of tumor DNA. The diagnostic utility of ctDNA for the detection of epidermal growth factor receptor (EGFR) mutations harbored in tumors of patients with advanced non-small-cell lung cancer (NSCLC) is supported by the results of several large studies/meta-analyses. However, recent real-world studies suggest that the performance of ctDNA testing varies between geographic regions/laboratories, demonstrating the need for standardized guidance. In this review, we outline recommendations for obtaining an accurate result using ctDNA, relating to pre-analytical plasma processing, ctDNA extraction, and appropriate EGFR mutation detection methods, based on clinical trial results. We conclude that there are several advantages associated with ctDNA, including the potential for repeated sampling particularly following progression after first-line tyrosine kinase inhibitor (TKI) therapy, as TKIs targeting resistance mutations (eg T790M) are now approved for use in the USA/EU/Japan (at time of writing). However, evidence suggests that ctDNA does not allow detection of EGFR mutations in all patients with known mutation-positive NSCLC. Therefore, although tumor tissue should be the first sample choice for EGFR testing at diagnosis, ctDNA is a promising alternative diagnostic approach. PMID:27980215

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heravi, Mitra; Department of Radiation Oncology, McGill University, Montreal; Segal Cancer Center, Jewish General Hospital, Montreal

2015-06-01

Purpose: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. Methods and Materials: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Westernmore » blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Results: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Conclusions: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.« less

Dynamics of EGFR Mutation Load in Plasma for Prediction of Treatment Response and Disease Progression in Patients With EGFR-Mutant Lung Adenocarcinoma.

PubMed

Taus, Álvaro; Camacho, Laura; Rocha, Pedro; Hardy-Werbin, Max; Pijuan, Lara; Piquer, Gabriel; López, Eva; Dalmases, Alba; Longarón, Raquel; Clavé, Sergi; Salido, Marta; Albanell, Joan; Bellosillo, Beatriz; Arriola, Edurne

2018-03-23

The assessment of epidermal growth factor receptor (EGFR) mutations is crucial for the management of patients with lung adenocarcinoma. Circulating tumor DNA (ctDNA)-based assessment offers advantages over tumor as a minimally invasive method able to capture tumor heterogeneity. Consecutive patients diagnosed with EGFR-mutant lung adenocarcinoma in tumor biopsy were included in this study. Plasma samples were obtained at different time points during the course of the disease. EGFR mutations in plasma were quantified using BEAMing (beads, emulsions, amplification, and magnetics) or digital PCR and were correlated with mutations in tumor and with radiologic response and progression. Two hundred twenty-one plasma samples from 33 patients were analyzed. EGFR mutations in plasma were detected in 83% of all patients and 100% of those with extrathoracic metastases. The dynamics of the EGFR mutation load predicted response in 93% and progression in 89% of cases well in advance of radiologic evaluation. Progression-free survival for patients in whom ctDNA was not detected in plasma during treatment was significantly longer than for those in whom ctDNA remained detectable (295 vs. 55 days; hazard ratio, 17.1; P < .001). The detection of EGFR mutations in ctDNA showed good correlation with that in tumor biopsy and predicted tumor response and progression in most patients. The liquid biopsy for ctDNA-based assessment of EGFR mutations is a reliable technique for diagnosis and follow-up in patients with EGFR-mutant lung adenocarcinoma in routine clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics.

PubMed

Jeuken, Judith; Sijben, Angelique; Alenda, Cristina; Rijntjes, Jos; Dekkers, Marieke; Boots-Sprenger, Sandra; McLendon, Roger; Wesseling, Pieter

2009-10-01

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients

PubMed Central

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-01-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients. PMID:29067441

Cell-Free Plasma DNA-Guided Treatment With Osimertinib in Patients With Advanced EGFR-Mutated NSCLC.

PubMed

Buder, Anna; Hochmair, Maximilian J; Schwab, Sophia; Bundalo, Tatjana; Schenk, Peter; Errhalt, Peter; Mikes, Romana E; Absenger, Gudrun; Patocka, Kurt; Baumgartner, Bernhard; Setinek, Ulrike; Burghuber, Otto C; Prosch, Helmut; Pirker, Robert; Filipits, Martin

2018-03-02

Osimertinib is standard treatment for patients with advanced EGFR T790M-mutated non-small-cell lung cancer who have been pre-treated with EGFR-tyrosine kinase inhibitors (TKIs). We studied whether cell-free plasma DNA for T790M detection can be used to select patients for osimertinib treatment in the clinical routine. From April 2015 to November 2016, we included 119 patients with advanced EGFR-mutated non-small-cell lung cancer who had progressed under treatment with an EGFR-TKI. The T790M mutation status was assessed in cell-free plasma DNA by droplet digital polymerase chain reaction in all patients and by tissue analyses in selected patients. T790M mutations were detected in 85 (93%) patients by analyses of cell-free plasma DNA and in 6 (7%) plasma-negative patients by tumor re-biopsy. Eighty-nine of 91 T790M-positive patients received osimertinib. Median progression-free survival (PFS) was 10.1 months (95% confidence interval [CI]: 8.1-12.1). Median survival was not reached and the 1-year survival was 64%. The response rate was 70% in T790M-positive patients (n = 91) in the intention-to-treat population. PFS trended to be shorter in patients with high T790M copy number (≥10 copies/mL) compared to those with low T790M copy number (<10 copies/mL) (hazard ratio for PFS = 1.72, 95% CI: 0.92-3.2, p = 0.09). A comparable trend was observed for overall survival (hazard ratio for overall survival = 2.16, 95% CI: 0.89-5.25, p = 0.09). No difference in response rate was observed based on T790M copy numbers. Plasma genotyping using digital polymerase chain reaction is clinically useful for the selection of patients who had progressed during first-line EGFR-TKI therapy for treatment with osimertinib. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291.

PubMed

Thress, Kenneth S; Brant, Roz; Carr, T Hedley; Dearden, Simon; Jenkins, Suzanne; Brown, Helen; Hammett, Tracey; Cantarini, Mireille; Barrett, J Carl

2015-12-01

To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients. A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas(®) EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR]). Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78-100%) and specificity (93-100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas(®) EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82-87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas(®) EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma. The cobas(®) EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may

Cis-oriented solvent-front EGFR G796S mutation in tissue and ctDNA in a patient progressing on osimertinib: a case report and review of the literature.

PubMed

Klempner, Samuel J; Mehta, Pareen; Schrock, Alexa B; Ali, Siraj M; Ou, Sai-Hong Ignatius

2017-01-01

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) is a universal event and limits clinical efficacy. The third-generation EGFR inhibitor osimertinib is active in EGFR-mutant/T790M positive non-small-cell lung cancer. Mechanisms of acquired resistance are emerging, and here we describe a cis -oriented solvent-front EGFR G796S mutation as the resistance mechanism observed in a progression biopsy and circulating tumor DNA (ctDNA) from a patient with initial response followed by progression on osimertinib. This is one of the earliest reports of a sole solvent-front tertiary EGFR mutation as a resistance mechanism to osimertinib. Our case suggests a monoclonal resistance mechanism. We review the importance of the solvent-front residues across TKIs and describe known osimertinib resistance mechanisms. We observe that nearly all clinical osimertinib-resistant tertiary EGFR mutations are oriented in cis with EGFR T790M. This case highlights the importance of mutations affecting EGFR kinase domains and supports the feasibility of broad panel ctDNA assays for detection of novel acquired resistance and tumor heterogeneity in routine clinical care.

Comparison of plasma ctDNA and tissue/cytology-based techniques for the detection of EGFR mutation status in advanced NSCLC: Spanish data subset from ASSESS.

PubMed

Arriola, E; Paredes-Lario, A; García-Gomez, R; Diz-Tain, P; Constenla, M; García-Girón, C; Márquez, G; Reck, M; López-Vivanco, G

2018-04-05

The analysis of epidermal growth factor receptor (EGFR) mutations in many patients with advanced non-small-cell lung cancer (aNSCLC) has provided the opportunity for successful treatment with specific, targeted EGFR tyrosine kinase inhibitors. However, this therapeutic decision may be challenging when insufficient tumor tissue is available for EGFR mutation testing. Therefore, blood surrogate samples for EGFR mutation analysis have been suggested. Data were collected from the Spanish cohort of patients in the large, non-interventional, diagnostic ASSESS study (NCT01785888) evaluating the utility of circulating free tumor-derived DNA from plasma for EGFR mutation testing. The incidence of EGFR mutation in Spain and the level of concordance between matched tissue/cytology and plasma samples were evaluated. In a cohort of 154 eligible patients, EGFR mutations were identified in 15.1 and 11.0% of tumor and plasma samples, respectively. The most commonly used EGFR mutation testing method for the tumor tissue samples was the QIAGEN Therascreen ® EGFR RGQ PCR kit (52.1%). Fragment Length Analysis + PNA LNA Clamp was used for the plasma samples. The concordance rate for EGFR mutation status between the tissue/cytology and plasma samples was 88.8%; the sensitivity was 45.5%, and the specificity was 96.7%. The high concordance between the different DNA sources for EGFR mutation testing supports the use of plasma samples when tumor tissue is unavailable.

Diagnostic accuracy of droplet digital PCR for detection of EGFR T790M mutation in circulating tumor DNA

PubMed Central

Tong, Xiang; Wang, Ye; Wang, Chengdi; Jin, Jing; Tian, Panwen; Li, Weimin

2018-01-01

Objectives Although different methods have been established to detect epidermal growth factor receptor (EGFR) T790M mutation in circulating tumor DNA (ctDNA), a wide range of diagnostic accuracy values were reported in previous studies. The aim of this meta-analysis was to provide pooled diagnostic accuracy measures for droplet digital PCR (ddPCR) in the diagnosis of EGFR T790M mutation based on ctDNA. Materials and methods A systematic review and meta-analysis were carried out based on resources from Pubmed, Web of Science, Embase and Cochrane Library up to October 11, 2017. Data were extracted to assess the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio (NLR), diagnostic OR (DOR), and areas under the summary receiver-operating characteristic curve (SROC). Results Eleven of 311 studies identified have met the including criteria. The sensitivity and specificity of ddPCR for the detection of T790M mutation in ctDNA ranged from 0.0% to 100.0% and 63.2% to 100.0%, respectively. For the pooled analysis, ddPCR had a performance of 70.1% (95% CI, 62.7%–76.7%) sensitivity, 86.9 % (95% CI, 80.6%–91.7%) specificity, 3.67 (95% CI, 2.33–5.79) PLR, 0.41 (95% CI, 0.32–0.55) NLR, and 10.83 (95% CI, 5.86–20.03) DOR, with the area under the SROC curve being 0.82. Conclusion The ddPCR harbored a good performance for detection of EGFR T790M mutation in ctDNA. PMID:29844700

Association of mutant EGFR L858R and exon 19 concentration in circulating cell-free DNA using droplet digital PCR with response to EGFR-TKIs in NSCLC

PubMed Central

Zhu, Yan-Juan; Zhang, Hai-Bo; Liu, Yi-Hong; Zhu, Ya-Zhen; Chen, Jun; Li, Yong; Bai, Jian-Ping; Liu, Li-Rong; Qu, Yan-Chun; Qu, Xin; Chen, Xian; Zheng, Guang-Juan

2017-01-01

The present study aimed to determine the diagnostic concordance of plasma epidermal growth factor receptor (EGFR) mutation using droplet digital polymerase chain reaction (ddPCR) with tumor tissue samples and the predictive clinical significance of plasma EGFR mutation concentration. Plasma DNA samples from patients with non-small cell lung cancer (NSCLC) were analyzed for EGFR exon 21 codon 858 (L858R) mutation, deletion of exon 19 (ex19del) and exon 20 codon 790 (T790M) mutation using ddPCR. Firstly, the mutations in the plasma samples were compared with the matched tumor samples to determine the concordance. Secondly, image examination follow-ups were analyzed to assess the association between plasma EGFR mutation concentration and patients' response to EGFR-tyrosine kinase inhibitors (TKIs). A total of 51 patients with NSCLC were enrolled, including 48 newly diagnosed patients. Compared with tumor tissue samples, the sensitivity and specificity of ddPCR were 76.19% (16/21) and 96.55% (28/29) for mutant L858R, and 88.89% (8/9) and 100% (41/41) for ex19del, respectively. No patient exhibited the T790M mutation in the tumor tissue or plasma samples. Furthermore, 5 patients with the L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples had been followed up with image examination for ≥3 months following EGFR-TKI treatment. The baseline mutant EGFR concentrations were positively correlated with a reduction in tumor burden (Spearman's r=0.7000, P=0.0358). When analyzed separately, ex19del concentrations (Spearman's r=1.0000, P<0.0001) were also positively correlated with the reduction, while mutant L858R concentrations were not (Spearman's r=0.7000, P=0.1881). In the present study, detection of plasma EGFR mutations using ddPCR exhibited sufficient concordance with tumor tissue sample results. Baseline plasma mutant EGFR and ex19del concentrations were significantly and positively correlated with response to EGFR-TKIs. PMID:28789464

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients.

PubMed

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-08-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell‑free DNA (cfDNA) extracted from the plasma of advanced non‑small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) ‑sensitive (19DEL, L858R) and TKI‑resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR‑TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra‑sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR‑TKI decision‑making for advanced NSCLC patients.

DNA methylation down-regulates EGFR expression in chicken

USDA-ARS?s Scientific Manuscript database

The epidermal growth factor receptor (EGFR), a growth-factor-receptor tyrosine kinase, was found up-regulated in numerous tumors, which provides a good target for cancer therapy. Although it was documented that oncoviruses are responsible for the activation of EGFR in tumors, the impact of Marek’s d...

Emergence of EGFR G724S mutation in EGFR-mutant lung adenocarcinoma post progression on osimertinib.

PubMed

Oztan, A; Fischer, S; Schrock, A B; Erlich, R L; Lovly, C M; Stephens, P J; Ross, J S; Miller, V; Ali, S M; Ou, S-H I; Raez, L E

2017-09-01

Mutations in the epidermal growth factor receptor (EGFR) are drivers for a subset of lung cancers. Osimertinib is a third-generation tyrosine kinase inhibitor (TKI) recently approved for the treatment of T790M-positive non-small cell lung cancer (NSCLC); however, acquired resistance to osimertinib is evident and resistance mechanisms remain incompletely defined. The EGFR G724S mutation was detected using hybrid-capture based comprehensive genomic profiling (CGP) and a hybrid-capture based circulating tumor DNA (ctDNA) assays in two cases of EGFR-driven lung adenocarcinoma in patients who had progressed on osimertinib treatment. This study demonstrates the importance of both tissue and blood based hybrid-capture based genomic profiling at disease progression to identifying novel resistance mechanisms in the clinic. Copyright © 2017 Elsevier B.V. All rights reserved.

Metastatic EML4-ALK fusion detected by circulating DNA genotyping in an EGFR-mutated NSCLC patient and successful management by adding ALK inhibitors: a case report.

PubMed

Liang, Wenhua; He, Qihua; Chen, Ying; Chuai, Shaokun; Yin, Weiqiang; Wang, Wei; Peng, Guilin; Zhou, Caicun; He, Jianxing

2016-02-05

Rebiopsy is highly recommended to identify the mechanism of acquired resistance to EGFR-TKIs in advanced lung cancer. Recent advances in multiplex genotyping based on circulating tumor DNA (ctDNA) provide a strong and non-invasive alternative for detection of the resistance mechanism. Here we report a multiple metastatic NSCLC patient who was detected to have pure EGFR 19 exon deletion (negative for EML4-ALK and ROS1 in both IHC-based and sequencing assay) in the primary lesion and responded to first-line and second-line EGFR-TKI treatments (erlotinib then HY-15772). At 8 months, most lesions remained well controlled except for the liver metastases which presented dramatic progression. Considering the high risk of bleeding in rebiopsy of hepatic lesions, we conducted a multiplex genomic profiling with ctDNA. Results reported coexistence of EGFR mutation and EML4-ALK gene translocation in plasma which heavily indicated that ALK was the primary reason for progression of the liver lesions. This deduction was supported by the repeated response to ALK inhibitors (crizotinib then AP26113) of the hepatic metastases. This is the first report of the existence of ALK rearrangement in metastatic lesions in an EGFR mutated patient. It highlighted the feasibility and advantages of using ctDNA multiplex genotyping in identifying the heterogeneity across lesions and the resistance mechanism of targeted treatments.

Detection of EGFR Gene Mutation by Mutation-oriented LAMP Method.

PubMed

Matsumoto, Naoyuki; Kumasaka, Akira; Ando, Tomohiro; Komiyama, Kazuo

2018-04-01

Epidermal growth factor receptor (EGFR) is a target of molecular therapeutics for non-small cell lung cancer. EGFR gene mutations at codons 746-753 promote constitutive EGFR activation and result in worst prognosis. However, these mutations augment the therapeutic effect of EGFR-tyrosine kinase inhibitor. Therefore, the detection of EGFR gene mutations is important for determining treatment planning. The aim of the study was to establish a method to detect EGFR gene mutations at codons 746-753. EGFR gene mutation at codons 746-753 in six cancer cell lines were investigated. A loop-mediated isothermal amplification (LAMP)-based procedure was developed, that employed peptide nucleic acid to suppress amplification of the wild-type allele. This mutation-oriented LAMP can amplify the DNA fragment of the EGFR gene with codons 746-753 mutations within 30 min. Moreover, boiled cells can work as template resources. Mutation oriented-LAMP assay for EGFR gene mutation is sensitive on extracted DNA. This procedure would be capable of detecting EGFR gene mutation in sputum, pleural effusion, broncho-alveolar lavage fluid or trans-bronchial lung biopsy by chair side. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

PubMed Central

2018-01-01

Liquid biopsies to genotype the epidermal growth factor receptor (EGFR) for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA) scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA) in normal plasma. The limit of detection (LOD) of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems) was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94%) of results obtained using the cobas assay and next-generation sequencing (NGS) were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion), 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion). Deep sequencing of materials (>100,000X depth of coverage) resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell-Free DNA from Patients with Advanced Non-Small Cell Lung Cancer.

PubMed

Zhu, Guanshan; Ye, Xin; Dong, Zhengwei; Lu, Ya Chao; Sun, Yun; Liu, Yi; McCormack, Rose; Gu, Yi; Liu, Xiaoqing

2015-05-01

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Immunostaining with EGFR mutation-specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples.

PubMed

Fan, Xiangshan; Liu, Biao; Xu, Haodong; Yu, Bo; Shi, Shanshan; Zhang, Jin; Wang, Xuan; Wang, Jiandong; Lu, Zhenfeng; Ma, Henghui; Zhou, Xiaojun

2013-08-01

Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas. Copyright © 2013 Elsevier Inc. All rights reserved.

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

PubMed

Powrózek, Tomasz; Krawczyk, Paweł; Pankowski, Juliusz; Reszka, Katarzyna; Jakubiak, Magdalena; Obrochta, Anna; Wojas-Krawczyk, Kamila; Buczkowski, Jarosław; Milanowski, Janusz

2015-11-14

Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

EGFR-TKIs plus chemotherapy demonstrated superior efficacy than EGFR-TKIs alone as first-line setting in advanced NSCLC patients with EGFR mutation and BIM deletion polymorphism.

PubMed

Liu, Sangtian; He, Yayi; Jiang, Tao; Ren, Shengxiang; Zhou, Fei; Zhao, Chao; Li, Xuefei; Zhang, Jie; Su, Chunxia; Chen, Xiaoxia; Cai, Weijing; Gao, Guanghui; Li, Wei; Wu, Fengying; Li, Jiayu; Zhao, Jing; Hu, Qiong; Zhao, Mingchuan; Zhou, Caicun; Hirsch, Fred R

2018-06-01

Non-small-cell lung cancer (NSCLC) patients with both epidermal growth factor receptor (EGFR) positive mutation and B-cell chronic lymphocytic leukemia/lymphoma-like 11 (BIM) deletion polymorphism had a poor clinical response to EGFR-tyrosine kinase inhibitors (TKIs). The current study aimed to investigate the clinical efficacy and tolerability of EGFR-TKIs plus chemotherapy versus EGFR-TKIs alone as first-line treatment in advanced NSCLC patients with EGFR mutations and BIM deletion polymorphism. A retrospective, non-randomized analysis was conducted. BIM deletion polymorphism was detected using polymerase chain reaction (PCR) analysis and direct sequencing of DNA from peripheral blood cells. Clinical characteristics, overall survival (OS), progress-free-survival (PFS), objective response rate (ORR) and treatment-related adverse events were compared between EGFR-TKIs alone versus EGFR-TKIs plus chemotherapy group. 65 patients were enrolled. 36 of them received EGFR-TKIs and 29 received EGFR-TKIs plus chemotherapy. EGFR-TKIs plus chemotherapy had significantly higher ORR than TKIs alone (65.5% vs. 38.9%, P = 0.046). Median PFS was significantly longer in EGFR-TKIs plus chemotherapy group than in TKIs group (7.2 vs 4.7 m; P = 0.008). Median OS was numerically longer in EGFR-TKIs plus chemotherapy group than in TKIs alone (18.5 vs 14.2 m; P = 0.107). EGFR-TKIs plus chemotherapy was associated with more grade 3 or 4 hematological toxic effects than EGFR-TKIs alone. EGFR-TKIs plus chemotherapy conferred a significantly higher ORR, prolonged PFS and numerically longer OS in advanced NSCLC patients with EGFR mutation and BIM deletion polymorphism. Further prospective studies are needed to validate these findings. Copyright © 2018 Elsevier B.V. All rights reserved.

Improved therapeutic effectiveness by combining recombinant p14(ARF) with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma.

PubMed

Liu, Feng; Du, JinTao; Xian, Junming; Liu, Yafeng; Liu, Shixi; Lin, Yan

2015-01-01

The tumor suppressor p14(ARF) and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14(ARF) with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. After human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14(ARF) and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14(ARF) and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. Ad-p14(ARF) combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14(ARF) and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14(ARF) protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14(ARF) was combined with Ad-antisense EGFR. Besides, Ad-p14(ARF) plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14(ARF) or Ad-antisense EGFR alone. Combination Ad-p14(ARF) with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided. Copyright © 2015 Elsevier Inc. All rights reserved.

A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non-Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial.

PubMed

Yanagita, Masahiko; Redig, Amanda J; Paweletz, Cloud P; Dahlberg, Suzanne E; O'Connell, Allison; Feeney, Nora; Taibi, Myriam; Boucher, David; Oxnard, Geoffrey R; Johnson, Bruce E; Costa, Daniel B; Jackman, David M; Jänne, Pasi A

2016-12-15

Genotype-directed therapy is the standard of care for advanced non-small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression. EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression. Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival. cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010-20. ©2016 AACR. ©2016 American Association for Cancer Research.

Longitudinal monitoring of EGFR mutations in plasma predicts outcomes of NSCLC patients treated with EGFR TKIs: Korean Lung Cancer Consortium (KLCC-12-02).

PubMed

Lee, Ji Yun; Qing, Xu; Xiumin, Wei; Yali, Bai; Chi, Sangah; Bak, So Hyeon; Lee, Ho Yun; Sun, Jong-Mu; Lee, Se-Hoon; Ahn, Jin Seok; Cho, Eun Kyung; Kim, Dong-Wan; Kim, Hye Ryun; Min, Young Joo; Jung, Sin-Ho; Park, Keunchil; Mao, Mao; Ahn, Myung-Ju

2016-02-09

We hypothesized that plasma-based EGFR mutation analysis for NSCLC may be feasible for monitoring treatment response to EGFR TKIs and also predict drug resistance.Clinically relevant mutations including exon 19 deletion (ex19del), L858R and T790M were analyzed using droplet digital PCR (ddPCR) in longitudinally collected plasma samples (n = 367) from 81 NSCLC patients treated with EGFR TKI. Of a total 58 baseline cell-free DNA (cfDNA) samples available for ddPCR analysis, 43 (74.1%) had the same mutation in the matched tumors (clinical sensitivity: 70.8% [17/24] for L858R and 76.5% [26/34] for ex19del). The concordance rates of plasma with tissue-based results of EGFR mutations were 87.9% for L858R and 86.2% for ex19del. All 40 patients who were detected EGFR mutations at baseline showed a dramatic decrease of mutant copies (>50%) in plasma during the first two months after treatment. Median progression-free survival (PFS) was 10.1 months for patients with undetectable EGFR v 6.3 months for detectable EGFR mutations in blood after two-month treatment (HR 3.88, 95% CI 1.48-10.19, P = 0.006). We observed emerging resistance with early detection of T790M as a secondary mutation in 14 (28.6%) of 49 patients. Plasma-based EGFR mutation analysis using ddPCR can monitor treatment response to EGFR TKIs and can lead to early detection of EGFR TKIs resistance. Further studies confirming clinical implications of EGFR mutation in plasma are warranted to guide optimal therapeutic strategies upon knowledge of treatment response and resistance.

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the

Xenogeneic Acellular Conjunctiva Matrix as a Scaffold of Tissue-Engineered Corneal Epithelium

PubMed Central

Zhao, Haifeng; Qu, Mingli; Wang, Yao; Wang, Zhenyu; Shi, Weiyun

2014-01-01

Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface. PMID:25375996

Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

PubMed Central

Ishii, Hidenobu; Azuma, Koichi; Sakai, Kazuko; Kawahara, Akihiko; Yamada, Kazuhiko; Tokito, Takaaki; Okamoto, Isamu; Nishio, Kazuto; Hoshino, Tomoaki

2015-01-01

As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs. PMID:26334838

Unique genetic profiles from cerebrospinal fluid cell-free DNA in leptomeningeal metastases of EGFR-mutant non-small-cell lung cancer: a new medium of liquid biopsy.

PubMed

Li, Y S; Jiang, B Y; Yang, J J; Zhang, X C; Zhang, Z; Ye, J Y; Zhong, W Z; Tu, H Y; Chen, H J; Wang, Z; Xu, C R; Wang, B C; Du, H J; Chuai, S; Han-Zhang, H; Su, J; Zhou, Q; Yang, X N; Guo, W B; Yan, H H; Liu, Y H; Yan, L X; Huang, B; Zheng, M M; Wu, Y L

2018-04-01

Leptomeningeal metastases (LM) are more frequent in non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations. Due to limited access to leptomeningeal lesions, the purpose of this study was to explore the potential role of cerebrospinal fluid (CSF) as a source of liquid biopsy in patients with LM. Primary tumor, CSF, and plasma in NSCLC with LM were tested by next-generation sequencing. In total, 45 patients with suspected LM underwent lumbar puncture, and those with EGFR mutations diagnosed with LM were enrolled. A total of 28 patients were enrolled in this cohort; CSF and plasma were available in 26 patients, respectively. Driver genes were detected in 100% (26/26), 84.6% (22/26), and 73.1% (19/26) of samples comprising CSF cell-free DNA (cfDNA), CSF precipitates, and plasma, respectively; 92.3% (24/26) of patients had much higher allele fractions in CSF cfDNA than the other two media. Unique genetic profiles were captured in CSF cfDNA compared with those in plasma and primary tissue. Multiple copy number variations (CNVs) were mainly identified in CSF cfDNA, and MET copy number gain identified in 47.8% (11/23) of patients was the most frequent one, while other CNVs included ERBB2, KRAS, ALK, and MYC. Moreover, loss of heterozygosity (LOH) of TP53 was identified in 73.1% (19/26) CSF cfDNA, which was much higher than that in plasma (2/26, 7.7%; P < 0.001). There was a trend towards a higher frequency of concomitant resistance mutations in patients with TP53 LOH than those without (70.6% versus 33.3%; P = 0.162). EGFR T790M was identified in CSF cfDNA of 30.4% (7/23) of patients who experienced TKI progression. CSF cfDNA could reveal the unique genetic profiles of LM and should be considered as the most representative liquid biopsy medium for LM in EGFR-mutant NSCLC.

Stress-specific p38 MAPK activation is sufficient to drive EGFR endocytosis but not its nuclear translocation.

PubMed

Tomas, Alejandra; Jones, Sylwia; Vaughan, Simon O; Hochhauser, Daniel; Futter, Clare E

2017-08-01

EGF receptor (EGFR) endocytosis is induced by stress in a manner dependent on the p38 MAPK family. Ligand and stresses such as X-rays, reportedly promote nuclear trafficking of endocytosed EGFR for regulation of gene transcription and DNA repair. We fail to detect EGFR endocytosis or nuclear transport following X-ray treatment of HeLa or head and neck cancer cells, despite extensive DNA damage induction. Apparent nuclear staining with EGFR extracellular domain antibody remained present despite reduced/absent EGFR expression, and so did not represent nuclear EGFR. UVB and UVC, but not X-ray or UVA, treatment induced p38 activation and EGFR endocytosis, although all of these stresses induced DNA damage, indicating that DNA damage alone is not sufficient to induce EGFR endocytosis. Increased reactive oxygen species (ROS) levels following UVB treatment, compared to that seen with X-rays, do not alone explain differences in p38 activation. UVB, like UVC, induced EGFR accumulation predominantly in perinuclear endosomes, rather than in the nucleus. Our morphological techniques identifying major changes in receptor distribution do not exclude the possibility that small but biologically relevant amounts of EGFR enter the nucleus. This study highlights the importance and limitations of morphological analyses of receptor distribution in understanding signaling outcome. © 2017. Published by The Company of Biologists Ltd.

Detection of EGFR Variants in Plasma: A Multilaboratory Comparison of the cobas EGFR Mutation Test v2 in Europe.

PubMed

Keppens, Cleo; Palma, John F; Das, Partha M; Scudder, Sidney; Wen, Wei; Normanno, Nicola; Van Krieken, J Han; Sacco, Alessandra; Fenizia, Francesca; de Castro, David Gonzalez; Hönigschnabl, Selma; Kern, Izidor; Lopez-Rios, Fernando; Lozano, Maria D; Marchetti, Antonio; Halfon, Philippe; Schuuring, Ed; Setinek, Ulrike; Sorensen, Boe; Taniere, Phillipe; Tiemann, Markus; Vosmikova, Hana; Dequeker, Elisabeth M C

2018-04-25

Molecular testing of EGFR is required to predict the response likelihood to targeted therapy in non-small-cell lung cancer. Analysis of circulating tumor DNA in plasma may complement limitations of tumor tissue. This study evaluated the interlaboratory performance and reproducibility of the cobas EGFR Mutation Test v2 to detect EGFR variants in plasma. Fourteen laboratories received two identical panels of 27 single-blinded plasma samples. Samples were wild-type or spiked with plasmid DNA to contain seven common EGFR variants at six predefined concentrations from 50 to 5000 copies per mL. The circulating tumor DNA was extracted by the cobas cfDNA Sample Preparation kit, followed by duplicate analysis with the EGFRv2 kit (Roche Molecular Systems, Pleasanton, CA). Lowest sensitivities were obtained for the c.2156G>C p.(Gly719Ala) and c.2573T>G p.(Leu858Arg) variants for the lowest target copies. For all other variants, sensitivities varied between 96.3% and 100.0%. Specificities were all 98.8% to 100.0%. Coefficients of variation indicated good intra and interlaboratory repeatability and reproducibility, but increased for decreasing concentrations. Prediction models revealed a significant correlation for all variants between the pre-defined copy number and the observed semiquantitative index values which reflects the samples' plasma mutation load. This study demonstrates an overall robust performance of the EGFRv2 kit in plasma. Prediction models may be applied to estimate the plasma mutation load for diagnostic or research purposes. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients.

PubMed

Siravegna, Giulia; Mussolin, Benedetta; Buscarino, Michela; Corti, Giorgio; Cassingena, Andrea; Crisafulli, Giovanni; Ponzetti, Agostino; Cremolini, Chiara; Amatu, Alessio; Lauricella, Calogero; Lamba, Simona; Hobor, Sebastijan; Avallone, Antonio; Valtorta, Emanuele; Rospo, Giuseppe; Medico, Enzo; Motta, Valentina; Antoniotti, Carlotta; Tatangelo, Fabiana; Bellosillo, Beatriz; Veronese, Silvio; Budillon, Alfredo; Montagut, Clara; Racca, Patrizia; Marsoni, Silvia; Falcone, Alfredo; Corcoran, Ryan B; Di Nicolantonio, Federica; Loupakis, Fotios; Siena, Salvatore; Sartore-Bianchi, Andrea; Bardelli, Alberto

2015-07-01

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

Loss of Activating EGFR Mutant Gene Contributes to Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in Lung Cancer Cells

PubMed Central

Kubo, Takuya; Murakami, Yuichi; Kawahara, Akihiko; Azuma, Koichi; Abe, Hideyuki; Kage, Masayoshi; Yoshinaga, Aki; Tahira, Tomoko; Hayashi, Kenshi; Arao, Tokuzo; Nishio, Kazuto; Rosell, Rafael; Kuwano, Michihiko; Ono, Mayumi

2012-01-01

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11–18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11–18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11–18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance. PMID:22815900

Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

PubMed

Wei, Hong; Zhu, Zijie; Lu, Longtao

2017-01-01

Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

Brk/PTK6 Sustains Activated EGFR Signaling through Inhibiting EGFR Degradation and Transactivating EGFR

PubMed Central

Li, X; Lu, Y; Liang, K; Hsu, J -M.; Albarracin, C; Mills, G B; Hung, M-C; Fan, Z

2011-01-01

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from Cbl-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy. PMID:22231447

Xenogeneic Bio-Root Prompts the Constructive Process Characterized by Macrophage Phenotype Polarization in Rodents and Nonhuman Primates.

PubMed

Li, Hui; Sun, Jingjing; Li, Jie; Yang, Hefeng; Luo, Xiangyou; Chen, Jinlong; Xie, Li; Huo, Fangjun; Zhu, Tian; Guo, Weihua; Tian, Weidong

2017-03-01

Tissue or organ regeneration using xenogeneic matrices is a promising approach to address the shortage of donor matrices for allotransplantation. Success of such approach has been demonstrated to correlate with macrophage-mediated fibrotic homeostasis and tissue remodeling. The previous studies have demonstrated that treated dentin matrix (TDM) could be a suitable bioactive substrate for allogeneic tooth root regeneration. This study constructed xenogeneic bioengineered tooth root (bio-root) via a combination of porcine TDM (pTDM) with allogeneic dental follicle cells (DFCs). Macrophage phenotypes are used to evaluate the remodeling process of xenogeneic bio-roots in vitro and in vivo. pTDM can facilitate odontoblast differentiation of human derived DFCs. Xenogeneic bio-roots in rat subcutaneous tissue prompt constructive response via M1 macrophage infiltration during early postimplantation stages and increase restorative M2 phenotype at later stages. After implantation of bio-roots into jaws of rhesus monkeys for six months, periodontal ligament-like fibers accompanied by macrophage polarization are observed, which are positive for COL-1, Periostin, βIII-tubulin and display such structures as fibroblasts and blood vessels. The reconstructed bio-root possesses biomechanical properties for the dissipation of masticatory forces. These results support that xenogeneic bio-root could maintain fibrotic homeostasis during remodeling process and highlight the potential application of xenogeneic matrices in regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

WHSC1L1-mediated EGFR mono-methylation enhances the cytoplasmic and nuclear oncogenic activity of EGFR in head and neck cancer

PubMed Central

Saloura, Vassiliki; Vougiouklakis, Theodore; Zewde, Makda; Deng, Xiaolan; Kiyotani, Kazuma; Park, Jae-Hyun; Matsuo, Yo; Lingen, Mark; Suzuki, Takehiro; Dohmae, Naoshi; Hamamoto, Ryuji; Nakamura, Yusuke

2017-01-01

While multiple post-translational modifications have been reported to regulate the function of epidermal growth factor receptor (EGFR), the effect of protein methylation on its function has not been well characterized. In this study, we show that WHSC1L1 mono-methylates lysine 721 in the tyrosine kinase domain of EGFR, and that this methylation leads to enhanced activation of its downstream ERK cascade without EGF stimulation. We also show that EGFR K721 mono-methylation not only affects the function of cytoplasmic EGFR, but also that of nuclear EGFR. WHSC1L1-mediated methylation of EGFR in the nucleus enhanced its interaction with PCNA in squamous cell carcinoma of the head and neck (SCCHN) cells and resulted in enhanced DNA synthesis and cell cycle progression. Overall, our study demonstrates the multifaceted oncogenic function of the protein lysine methyltransferase WHSC1L1 in SCCHN, which is mediated through direct non-histone methylation of the EGFR protein with effects both in its cytoplasmic and nuclear functions. PMID:28102297

Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations.

PubMed

Lou, Na-Na; Zhang, Xu-Chao; Chen, Hua-Jun; Zhou, Qing; Yan, Li-Xu; Xie, Zhi; Su, Jian; Chen, Zhi-Hong; Tu, Hai-Yan; Yan, Hong-Hong; Wang, Zhen; Xu, Chong-Rui; Jiang, Ben-Yuan; Wang, Bin-Chao; Bai, Xiao-Yan; Zhong, Wen-Zhao; Wu, Yi-Long; Yang, Jin-Ji

2016-10-04

The co-occurrence of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements constitutes a rare molecular subtype of non-small-cell lung cancer (NSCLC). Herein, we assessed the clinical outcomes and incidence of acquired resistance to tyrosine kinase inhibitors (TKIs) in this subtype. So we enrolled 118 advanced NSCLC treated with TKIs. EGFR mutations and ALK rearrangements were detected by DNA sequencing or Scorpion amplification refractory mutation system and fluorescence in situ hybridization respectively. Immunohistochemistry was used to evaluate the activation of associated proteins. We found that nine in ten patients with EGFR/ALK co-alterations had good response with first-line EGFR TKI, and the objective response rate (ORR) of EGFR TKIs was 80% (8/10) for EGFR/ALK co-altered and 65.5% (55/84) for EGFR-mutant (P = 0.57), with a median progression-free survival (PFS) of 11.2 and 13.2 months, (hazard ratio [HR]=0.95, 95% [CI], 0.49-1.84, P= 0.87). ORR of crizotinib was 40% (2/5) for EGFR/ALK co-altered and 73.9% (17/23) for ALK-rearranged (P= 0.29), with a median PFS of 1.9 and 6.9 months (hazard ratio [HR], 0.40; 95% [CI] 0.15-1.10, P = 0.08). The median overall survival (OS) was 21.3, 23.7, and 18.5 months in EGFR-mutant, ALK-rearranged, and EGFR/ALK co-altered (P= 0.06), and there existed a statistically significant difference in OS between ALK-rearranged and EGFR/ALK co-altered (P=0.03). Taken together, the first-line EGFR-TKI might be the reasonable care for advanced NSCLC harbouring EGFR/ALK co-alterations, whether or nor to use sequential crizotinib should be guided by the status of ALK rearrangement and the relative level of phospho-EGFR and phospho-ALK.

Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dittmann, Klaus H.; Mayer, Claus; Ohneseit, Petra A.

2008-01-01

Purpose: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. Methods and Materials: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by {gamma}H{sub 2}AX foci assay. Results: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observedmore » radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual {gamma}H2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. Conclusions: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.« less

EGFR-TKI-induced HSP70 degradation and BER suppression facilitate the occurrence of the EGFR T790 M resistant mutation in lung cancer cells.

PubMed

Cao, Xiang; Zhou, Yi; Sun, Hongfang; Xu, Miao; Bi, Xiaowen; Zhao, Zhihui; Shen, Binghui; Wan, Fengyi; Hong, Zhuan; Lan, Lei; Luo, Lan; Guo, Zhigang; Yin, Zhimin

2018-06-28

Non-small cell lung cancer (NSCLC) patients harboring EGFR-activating mutations initially respond to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and have shown favorable outcomes. However, acquired drug resistance to EGFR-TKIs develops in almost all patients mainly due to the EGFR T790 M mutation. Here, we show that treatment with low-dose EGFR-TKI results in the emergence of the EGFR T790 M mutation and in the reduction of HSP70 protein levels in HCC827 cells. Erlotinib treatment inhibits HSP70 phosphorylation at tyrosine 41 and increases HSP70 ubiquitination, resulting in HSP70 degradation. We show that EGFR-TKI treatment causes increased DNA damage and enhanced gene mutation rates, which are secondary to the EGFR-TKI-induced reduction of HSP70 protein. Importantly, HSP70 overexpression delays the occurrence of Erlotinib-induced EGFR T790 M mutation. We further demonstrate that HSP70 interacts with multiple enzymes in the base excision repair (BER) pathway and promotes not only the efficiency but also the fidelity of BER. Collectively, our findings show that EGFR-TKI treatment facilitates gene mutation and the emergence of EGFR T790 M secondary mutation by the attenuation of BER via induction of HSP70 protein degradation. Copyright © 2018. Published by Elsevier B.V.

Comparison of cross-platform technologies for EGFR T790M testing in patients with non-small cell lung cancer

PubMed Central

Li, Xuefei; Zhou, Caicun

2017-01-01

Somatic mutations in the gene encoding epidermal growth factor receptor (EGFR) play an important role in determining targeted treatment modalities in non-small cell lung cancer (NSCLC). The EGFR T790M mutation emerges in approximately 50% of cases who acquire resistance to tyrosine kinase inhibitors. Detecting EGFR T790M mutation in tumor tissue is challenging due to heterogeneity of the tumor, low abundance of the mutation and difficulty for re-biopsy in patients with advanced disease. Alternatively, circulating tumor DNA (ctDNA) has been proposed as a non-invasive method for mutational analysis. The presence of EGFR mutations in ctDNA predicts response to the EGFR TKIs in the first-line setting. Molecular testing is now considered a standard care for NSCLC. The advent of standard commercially available kits and targeted mutational analysis has revolutionized the accuracy of mutation detection platforms for detection of EGFR mutations. Our review provides an overview of various commonly used platforms for detecting EGFR T790M mutation in tumor tissue and plasma. PMID:29246024

EGFR Mutation Testing Practices within the Asia Pacific Region

PubMed Central

Kerr, Keith M.; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma. Luisa D.; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

2015-01-01

Introduction: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non–small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. Methods: A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Results: Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%–32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. Conclusions: In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia. PMID:25376513

Informed consent: cultural and religious issues associated with the use of allogeneic and xenogeneic mesh products.

PubMed

Jenkins, Eric D; Yip, Michael; Melman, Lora; Frisella, Margaret M; Matthews, Brent D

2010-04-01

Our aim was to investigate the views of major religions and cultural groups regarding the use of allogeneic and xenogeneic mesh for soft tissue repair. We contacted representatives from Judaism, Islam, Buddhism, Hinduism, Scientology, and Christianity (Baptists, Methodists, Seventh-Day Adventists, Catholics, Lutherans, Church of Jesus Christ of Latter-Day Saints, Evangelical, and Jehovah's Witnesses). We also contacted American Vegan and People for the Ethical Treatment of Animals (PETA). Standardized questionnaires were distributed to the religious and cultural authorities. Questions solicited views on the consumption of beef and pork products and the acceptability of human-, bovine-, or porcine-derived acellular grafts. Dietary restrictions among Jews and Muslims do not translate to tissue implantation restriction. Approximately 50% of Seventh-day Adventists and 40% of Buddhists practice vegetarianism, which may translate into a refusal of the use of xenogeneic tissue. Some Hindus categorically prohibit the use of human tissue and animal products; others allow the donation and receipt of human organs and tissues. PETA is opposed to all uses of animals, but not to human acellular grafts or organ transplantation. Some vegans prefer allogeneic to xenogeneic tissue. Allogeneic and xenogeneic acellular grafts are acceptable among Scientologists, Baptists, Lutherans, Evangelicals, and Catholics. Methodists, Jehovah's Witnesses, and The Church of Jesus Christ of Latter-Day Saints leave the decision up to the individual. Knowledge of religious and cultural preferences regarding biologic mesh assists the surgeon in obtaining a culturally sensitive informed consent for procedures involving acellular allogeneic or xenogeneic grafts. Copyright (c) 2010 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

Evaluation of pre-analytical conditions and comparison of the performance of several digital PCR assays for the detection of major EGFR mutations in circulating DNA from non-small cell lung cancers: the CIRCAN_0 study

PubMed Central

Garcia, Jessica; Dusserre, Eric; Cheynet, Valérie; Bringuier, Pierre Paul; Brengle-Pesce, Karen; Wozny, Anne-Sophie; Rodriguez-Lafrasse, Claire; Freyer, Gilles; Brevet, Marie; Payen, Léa; Couraud, Sébastien

2017-01-01

Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression. PMID:29152135

Spectrum of EGFR gene mutations in Vietnamese patients with non-small cell lung cancer.

PubMed

Vu, Hoang Anh; Xinh, Phan Thi; Ha, Hua Thi Ngoc; Hanh, Ngo Thi Tuyet; Bach, Nguyen Duc; Thao, Doan Thi Phuong; Dat, Ngo Quoc; Trung, Nguyen Sao

2016-03-01

Epidermal growth factor receptor (EGFR) mutational status is a crucial biomarker for prediction of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Although these mutations have been well characterized in other countries, little is known about the frequency or spectrum of EGFR mutations in Vietnamese NSCLC patients. Using Sanger DNA sequencing, we investigated mutations in EGFR exons 18-21 from 332 patients diagnosed with NSCLC at University of Medicine and Pharmacy, Ho Chi Minh City, Vietnam. DNA was extracted from formalin-fixed, paraffin-embedded tissues, followed by PCR amplification and sequencing. EGFR mutations were detected in 135 samples (40.7%), of which eight samples carried double mutations. In total, 46 different types of EGFR mutations were found, including six novel mutations (p.K713E, p.K714R, p.P794S, p.R803W, p.P848S, and p.K867E). Among the four exons investigated, exon 19 was most frequently mutated (63 out of 332 patients, 19%), with the p.E746_A750del appearing in 43 samples. Exon 21 was mutated in 56 samples (16.9%), of which 47 were p.L858R. Each of exons 18 and 20 was mutated in 12 samples (3.6%). The frequency of EGFR mutations was higher in females than in males (48.9% vs 35%, P = 0.012), but not statistically different between adenocarcinomas and other histological types of NSCLC (41.3% vs 34.5%, P = 0.478). DNA sequencing detected EGFR mutations with high frequency and revealed a broad spectrum of mutation type in Vietnamese patients with NSCLC. © 2015 Wiley Publishing Asia Pty Ltd.

EGFR-TKIs resistance via EGFR-independent signaling pathways.

PubMed

Liu, Qian; Yu, Shengnan; Zhao, Weiheng; Qin, Shuang; Chu, Qian; Wu, Kongming

2018-02-19

Tyrosine kinase inhibitors (TKIs)-treatments bring significant benefit for patients harboring epidermal growth factor receptor (EGFR) mutations, especially for those with lung cancer. Unfortunately, the majority of these patients ultimately develop to the acquired resistance after a period of treatment. Two central mechanisms are involved in the resistant process: EGFR secondary mutations and bypass signaling activations. In an EGFR-dependent manner, acquired mutations, such as T790 M, interferes the interaction between TKIs and the kinase domain of EGFR. While in an EGFR-independent manner, dysregulation of other receptor tyrosine kinases (RTKs) or abnormal activation of downstream compounds both have compensatory functions against the inhibition of EGFR through triggering phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling axes. Nowadays, many clinical trials aiming to overcome and prevent TKIs resistance in various cancers are ongoing or completed. EGFR-TKIs in accompany with the targeted agents for resistance-related factors afford a promising first-line strategy to further clinical application.

Mutational status of EGFR and KIT in thymoma and thymic carcinoma.

PubMed

Yoh, Kiyotaka; Nishiwaki, Yutaka; Ishii, Genichiro; Goto, Koichi; Kubota, Kaoru; Ohmatsu, Hironobu; Niho, Seiji; Nagai, Kanji; Saijo, Nagahiro

2008-12-01

This study was conducted to evaluate the prevalence of EGFR and KIT mutations in thymomas and thymic carcinomas as a means of exploring the potential for molecularly targeted therapy with tyrosine kinase inhibitors. Genomic DNA was isolated from 41 paraffin-embedded tumor samples obtained from 24 thymomas and 17 thymic carcinomas. EGFR exons 18, 19, and 21, and KIT exons 9, 11, 13, and 17, were analyzed for mutations by PCR and direct sequencing. Protein expression of EGFR and KIT was evaluated immunohistochemically. EGFR mutations were detected in 2 of 20 thymomas, but not in any of the thymic carcinomas. All of the EGFR mutations detected were missense mutations (L858R and G863D) in exon 21. EGFR protein was expressed in 71% of the thymomas and 53% of the thymic carcinomas. The mutational analysis of KIT revealed only a missense mutation (L576P) in exon 11 of one thymic carcinoma. KIT protein was expressed in 88% of the thymic carcinomas and 0% of the thymomas. The results of this study indicate that EGFR and KIT mutations in thymomas and thymic carcinomas are rare, but that many of the tumors express EGFR or KIT protein.

The combi-targeting concept: a novel 3,3-disubstituted nitrosourea with EGFR tyrosine kinase inhibitory properties.

PubMed

Qiu, Qiyu; Dudouit, Fabienne; Matheson, Stephanie L; Brahimi, Fouad; Banerjee, Ranjita; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

To study the dual mechanism of action of FD137, a 3,3-disubstituted nitrosourea designed to block signaling mediated by the epidermal growth factor receptor (EGFR) on its own and to be hydrolyzed to an inhibitor of EGFR plus a DNA-damaging species. HPLC was used to determine the half-life (t(1/2)) of FD137 and to characterize its derived metabolite FD110. The dual mechanisms of DNA damaging and EGFR tyrosine kinase (TK) targeting were ascertained by the comet assay for DNA damage and by inmunodetection of phosphotyrosine in an ELISA and a whole-cell assay for EGFR-mediated signaling. The antiproliferative effects of the different drugs and their combinations were determined by the sulforhodamine B (SRB) assay. In contrast to BCNU, FD137 significantly blocked EGF-induced EGFR autophosphorylation (IC(50) 4 micro M) in the human solid tumor cell line A431. DNA damage induced by FD137 could only be observed after 24 h exposure, but the level of DNA damage remained 3.6-fold lower than that induced by BCNU. This difference was rationalized by the 160-fold greater stability of FD137 when compared with BCNU in serum-containing medium. Further, degradation of FD137 was accompanied by the slow release of FD110, an extremely potent inhibitor of EGFR TK [IC(50) (EGFR autophosphorylation) <0.3 micro M]. The complex properties of FD137 translated into a 55-fold greater antiproliferative activity than BCNU against the EGFR-overexpressing A431 cells that coexpresses the O(6)-alkylguanine transferase (AGT). Depletion of AGT in these cells by the use of O(6)-benzylguanine (O(6)-BG) enhanced their sensitivity to BCNU by 8-fold, but only by 3-fold to FD137. The results overall suggest that the superior antiproliferative activity of FD137 when compared with BCNU may be associated with its ability to behave as a combination of many species with different mechanisms of action. However, the enhancement of its potency by O(6)-BG suggests that its antiproliferative effect was at least

Effect of verteporfin-PDT on epithelial growth factor receptor (EGFR) signaling pathway in cholangiocarcinoma cell lines

NASA Astrophysics Data System (ADS)

Andreola, Fausto; Cerec, Virginie; Pereira, Stephen P.

2009-06-01

EGFR, a member of the ERBB family, plays a pivotal role in carcinogenesis. EGFR overexpression is implicated in DNA repair and synergistic interactions between EGFR-targeting drugs and conventional chemo/radiotherapy have been reported in preclinical studies for different cancers but not cholangiocarcinoma (CCA). To date there are no in vitro data available on the cellular response and effect of either photodynamic therapy (PDT) or EGFR-targeting drugs on CCA. Therefore, we aimed to study the: (i) response to Verteporfin PDT and to EGFR-targeting drugs, as single agents; (ii) effect of PDT on ERBBs expression, phosporylation status and activation of its signaling pathways; (iii) response to combination of PDT and EGFR-targeting agents. We showed that two cholangiocarcinoma cell lines (HuCCT1 and TFK1 cells, intra- and extrahepatic, respectively) differentially respond to verteporfin-PDT treatment and are resistant to EGFR-targeting agents. A constitutive activation of EGFR in both cell lines was also observed, which could partly account for the observed resistance to EGFR-targeting drugs. In addition, verteporfin-PDT induced further phosphorylation of both EGFR and other Receptor Tyrosine Kinases. Mitochondria-independent apoptosis was induced by PDT in both CCA cell lines; in particular, PDT modulated the expression of members of the Inhibitor of Apoptosis (IAP) family of proteins. Interestingly, there was a PDT-induced EGFR nuclear translocation in both cell lines; co-treatment with either an EGFR-inhibitor (Cetuximab) or a nuclear import blocking agent (Wheat Germ Agglutinin) had an additive effect on PDT cell killing, thus implying a role of EGFR in repairing the potential PDT-induced DNA damage.

Association of EGFR mutation or ALK rearrangement with expression of DNA repair and synthesis genes in never-smoker women with pulmonary adenocarcinoma.

PubMed

Ren, Shengxiang; Chen, Xiaoxia; Kuang, Peng; Zheng, Limou; Su, Chunxia; Li, Jiayu; Li, Bing; Wang, Yongshen; Liu, Lu; Hu, Qiong; Zhang, Jie; Tang, Liang; Li, Xuefei; Zhou, Caicun; Schmid-Bindert, Gerald

2012-11-15

Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement may predict the outcome of targeted drug therapy and also are associated with the efficacy of chemotherapy in patients with nonsmall cell lung cancer (NSCLC). The authors of this report investigated the relation of EGFR mutation or ALK rearrangement status and the expression of DNA repair or synthesis genes, including excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), thymidylate synthetase (TS), and breast cancer-early onset (BRCA1), as a potential explanation for these observations. In total, 104 resected lung adenocarcinomas from women who were nonsmokers were analyzed concurrently for EGFR mutations, ALK rearrangements, and mRNA expression of the ERCC1, RRM1, TS, and BRCA1 genes. EGFR mutations were detected with a proprietary detection kit, ALK rearrangements were detected by polymerase chain reaction analysis, and genetic mRNA expression was detected by real-time polymerase chain reaction analysis. Of 104 patients, 73 (70.2%) had EGFR mutations, and 10 (9.6%) had ALK rearrangements. ERCC1 mRNA levels in patients who had EGFR mutations were 3.44 ± 1.94 × 10(-3) , which were significantly lower than the levels in patients who were positive for ALK rearrangements and in patients who were negative for both biomarkers (4.60 ± 1.95 × 10(-3) and 4.95 ± 2.33 × 10(-3) , respectively; P = .010). However, TS mRNA levels were significantly lower in patients who had EGFR mutations (1.15 ± 1.38 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .006) or ALK rearrangements (1.21 ± 0.78 × 10(-3) vs 2.69 ± 3.97 × 10(-3) ; P = .020) than in patients who were negative for both biomarkers. NSCLC specimens that harbored activating EGFR mutations were more likely to express low ERCC1 and TS mRNA levels, whereas patients with NSCLC who had ALK rearrangement were more likely to express low TS mRNA levels. Copyright © 2012 American Cancer

Sequential liquid biopsies reveal dynamic alterations of EGFR driver mutations and indicate EGFR amplification as a new mechanism of resistance to osimertinib in NSCLC.

PubMed

Knebel, Franciele H; Bettoni, Fabiana; Shimada, Andrea K; Cruz, Manoel; Alessi, João Victor; Negrão, Marcelo V; Reis, Luiz Fernando L; Katz, Artur; Camargo, Anamaria A

2017-06-01

Osimertinib is an EGFR-T790M-specific TKI, which has demonstrated impressive response rates in NSCLC, after failure to first-line anti-EGFR TKIs. However, acquired resistance to osimertinib is also observed and the molecular mechanisms of resistance are not yet fully understood. Monitoring and managing NSCLC patients who progressed on osimertinib is, therefore, emerging as an important clinical challenge. Sequential liquid biopsies were used to monitor a patient with EGFR-exon19del positive NSCLC, who received erlotinib and progressed through the acquisition of the EGFR-T790M mutation. Erlotinib was discontinued and osimertinib was initiated. Blood samples were collected at erlotinib progression and during osimertinib treatment for the detection of the activating (EGFR-exon19del) and resistance mutations (EGFR-T790M, EGFR-C797S, BRAF-V600E, METamp and ERBB2amp) in the plasma DNA using digital droplet PCR. Plasma levels of the activating EGFR-exon19del accurately paralleled the clinical and radiological progression of disease and allowed early detection of AR to osimertinib. Resistance to osimertinib coincided with the emergence of a small tumor cell subpopulation carrying the known EGFR-C797S resistance mutation and an additional subpopulation carrying amplified copies of EGFR-exon19del. Given the existence of multiple AR mechanisms, quantification of the original EGFR activation mutation, instead of the resistance mutations, can be efficiently used to monitor response to osimertinib, allowing early detection of AR. Absolute quantification of both activation and resistance mutations can provide important information on tumor clonal evolution upon progression to osimertinib. Selective amplification of the EGFR-exon19del allele may represent a novel resistance mechanism to osimertinib. Copyright © 2017 Elsevier B.V. All rights reserved.

EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

PubMed

Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

2017-10-01

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

De novo activating epidermal growth factor mutations (EGFR) in small-cell lung cancer.

PubMed

Thai, Alesha; Chia, Puey L; Russell, Prudence A; Do, Hongdo; Dobrovic, Alex; Mitchell, Paul; John, Thomas

2017-09-01

In Australia, mutations in epidermal growth factor mutations (EGFR) occur in 15% of patients diagnosed with non-small-cell lung cancer and are found with higher frequency in female, non-smokers of Asian ethnicity. Activating mutations in the EGFR gene are rarely described in SCLC. We present two cases of de novo EGFR mutations in patients with SCLC detected in tissue and in plasma cell free DNA, both of whom were of Asian ethnicity and never-smokers. These two cases add to the growing body of evidence suggesting that screening for EGFR mutations in SCLC should be considered in patients with specific clinical features. © 2017 Royal Australasian College of Physicians.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Wenjun; Ercan, Dalia; Chen, Liang

2010-01-12

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potentmore » against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.« less

Light of DNA-alkylating agents in castration-resistant prostate cancer cells: a novel mixed EGFR/DNA targeting combi-molecule.

PubMed

Liang, Guan-Can; Zheng, Hao-Feng; Chen, Yan-Xiong; Li, Teng-Cheng; Liu, Wei; Fang, You-Qiang

2017-01-01

The mechanism underlying the therapeutic effects of combi-molecule JDF12 on prostate cancer (PCa) DU145 cells remains still unclear. This study aimed to investigate the proteomic profile after JDF12 treatment in DU145 cells by comparing with that in Iressa treated cells and untreated cells. MTT was used to evaluate drug cytotoxicity, DAPI staining was done to assess apoptosis of cells, and flow cytometry was used to analyze cell cycle. iTRAQ and qPCR were employed to obtain the proteomic profiles of JDF12 treated, Iressa treated, and untreated DU145 cells, and validate the expression of selected differentially expressed proteins, respectively. JDF12 could significantly inhibit the proliferation and increase the apoptosis of DU145 cells when compared with Iressa or blank group. In total, 5071 proteins were obtained, out of which, 42, including 21 up-regulated and 21 down-regulated proteins, were differentially expressed in JDF12 group when compared with Iressa and blank groups. The up-regulated proteins were mainly involved in DNA damage/repair and energy metabolism; while the down-regulated proteins were mainly associated with cell apoptosis. qPCR confirmed the expression of several biologically important proteins in DU145 cells after JDF12 treatment. The molecular mechanisms of DNA alkylating agents on PCa therapy that with the assistant of EGFR-blocker were revealed on proteomic level, which may increase the possible applications of DNA alkylating agents and JDF12 on PCa therapy.

Establishing an EGFR mutation screening service for non-small cell lung cancer - sample quality criteria and candidate histological predictors.

PubMed

Leary, Alexandra F; Castro, David Gonzalez de; Nicholson, Andrew G; Ashley, Sue; Wotherspoon, Andrew; O'Brien, Mary E R; Popat, Sanjay

2012-01-01

EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (<2 ng/μL) had more test failures (30% versus 3.9% for [DNA]>2.2 ng/μL), the mutation rate was 9.2%. Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

PubMed Central

Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

2015-01-01

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

EGFR Targeted Therapies and Radiation: Optimizing Efficacy by Appropriate Drug Scheduling and Patient Selection

PubMed Central

Cuneo, Kyle C.; Nyati, Mukesh K.; Ray, Dipankar; Lawrence, Theodore S.

2015-01-01

The epidermal growth factor receptor (EGFR) plays an important role in tumor progression and treatment resistance for many types of malignancies including head and neck, colorectal, and nonsmall cell lung cancer. Several EGFR targeted therapies are efficacious as single agents or in combination with chemotherapy. Given the toxicity associated with chemoradiation and poor outcomes seen in several types of cancers, combinations of EGFR targeted agents with or without chemotherapy have been tested in patients receiving radiation. To date, the only FDA approved use of an anti-EGFR therapy in combination with radiation therapy is for locally advanced head and neck cancer. Given the important role EGFR plays in lung and colorectal cancer and the benefit of EGFR inhibition combined with chemotherapy in these disease sites, it is perplexing why EGFR targeted therapies in combination with radiation or chemoradiation have not been more successful. In this review we summarize the clinical findings of EGFR targeted therapies combined with radiation and chemoradiation regimens. We then discuss the interaction between EGFR and radiation including radiation induced EGFR signaling, the effect of EGFR on DNA damage repair, and potential mechanisms of radiosensitization. Finally, we examine the potential pitfalls with scheduling EGFR targeted therapies with chemoradiation and the use of predictive biomarkers to improve patient selection. PMID:26205191

Selective gene amplification to detect the T790M mutation in plasma from patients with advanced non-small cell lung cancer (NSCLC) who have developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance.

PubMed

Nishikawa, Shingo; Kimura, Hideharu; Koba, Hayato; Yoneda, Taro; Watanabe, Satoshi; Sakai, Tamami; Hara, Johsuke; Sone, Takashi; Kasahara, Kazuo; Nakao, Shinji

2018-03-01

The epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, tissues for the genotyping of the EGFR T790M mutation can be difficult to obtain in a clinical setting. The aims of this study were to evaluate a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients using the PointMan™ EGFR DNA enrichment kit, which is a novel method for the selective amplification of specific genotype sequences. Blood samples were collected from NSCLC patients who had activating EGFR mutations and who were resistant to EGFR-TKI treatment. Using cell-free DNA (cfDNA) from plasma, EGFR T790M mutations were amplified using the PointMan™ enrichment kit, and all the reaction products were confirmed using direct sequencing. The concentrations of plasma DNA were then determined using quantitative real-time PCR. Nineteen patients were enrolled, and 12 patients (63.2%) were found to contain EGFR T790M mutations in their cfDNA, as detected by the kit. T790M mutations were detected in tumor tissues in 12 cases, and 11 of these cases (91.7%) also exhibited the T790M mutation in cfDNA samples. The concentrations of cfDNA were similar between patients with the T790M mutation and those without the mutation. The PointMan™ kit provides a useful method for determining the EGFR T790M mutation status in cfDNA.

Profile of the Roche cobas® EGFR mutation test v2 for non-small cell lung cancer.

PubMed

Malapelle, Umberto; Sirera, Rafael; Jantus-Lewintre, Eloísa; Reclusa, Pablo; Calabuig-Fariñas, Silvia; Blasco, Ana; Pisapia, Pasquale; Rolfo, Christian; Camps, Carlos

2017-03-01

The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories' expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required. Areas covered: The EGFR mutation testing landscape is manifold and includes both screening and targeted methods, each with their own pros and cons. Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its liquid-biopsy applications. Expert commentary: The Roche cobas® EGFR mutation test v2, based on real time RT-PCR, is a reliable option for testing EGFR mutations in clinical practice, either using tissue-derived DNA or plasma-derived cfDNA. This application will be valuable for laboratories with whose purpose is purely diagnostic and lacking high-throughput technologies.

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

PubMed Central

Kriegs, Malte; Kasten-Pisula, Ulla; Riepen, Britta; Hoffer, Konstantin; Struve, Nina; Myllynen, Laura; Braig, Friederike; Binder, Mascha; Rieckmann, Thorsten; Grénman, Reidar; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai

2016-01-01

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells. PMID:27281611

Urine circulating-tumor DNA (ctDNA) detection of acquired EGFR T790M mutation in non-small-cell lung cancer: An outcomes and total cost-of-care analysis.

PubMed

Sands, Jacob; Li, Qianyi; Hornberger, John

2017-08-01

Third-generation tyrosine kinase inhibitors (TKIs) have proven effective in patients with the acquired EGFR T790M resistance mutation who progress on prior EGFR TKI therapy. Median progression-free survival (PFS) on a 3rd-gen TKI was 9-10 months for T790M+ patients compared to 2.8 months for T790M- patients. PFS is similar regardless of the specimen used to assess T790M, such as tissue, plasma, or urine ctDNA. This study aimed to assess the total cost of care of a urine-testing strategy (UTS) versus a tissue-testing strategy (TTS) for T790M detection, in patients with EGFR-mutation positive lung adenocarcinoma and progression on prior TKI therapy. Long-term outcomes and economic implications were assessed from a US payer perspective. Endpoints were PFS, overall survival (OS), medical resource use and related costs. We included published randomized drug trials and Medicare fee schedules. A state-transition analysis and Markov model tracked patients from stable disease to progression and death. Univariate and multivariate sensitivity analyses were performed to assess the robustness of findings and identify factors that most influenced outcomes and costs. UTS increased the rate of detection of patients with T790M mutation eligible for treatment with 3rd generation TKI by 7% compared with TTS; urine ctDNA testing detected T790M mutation in some patients for whom biopsy could not be performed or when tissue testing yielded indeterminate results. Due to enhanced targeting of TKI therapy, UTS increased PFS and OS by 0.44 and 0.35 months, respectively. UTS yields a savings of $1243-$1680 per patient due to avoidance of biopsy, potential biopsy-associated complications, and tissue-based molecular testing in approximately 55.6% of patients. Probability of T790M detection by tissue and cost of biopsy procedure were the most influential factors. UTS prolonged PFS/OS due to increased detection of T790M mutation and decreased biopsies and complication-related costs. Copyright �

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers

PubMed Central

Blakely, Collin M.; Watkins, Thomas B.K.; Wu, Wei; Gini, Beatrice; Chabon, Jacob J.; McCoach, Caroline E.; McGranahan, Nicholas; Wilson, Gareth A.; Birkbak, Nicolai J.; Olivas, Victor R.; Rotow, Julia; Maynard, Ashley; Wang, Victoria; Gubens, Matthew A.; Banks, Kimberly C.; Lanman, Richard B.; Caulin, Aleah F.; John, John St.; Cordero, Anibal R.; Giannikopoulos, Petros; Simmons, Andrew D.; Mack, Philip C.; Gandara, David R.; Husain, Hatim; Doebele, Robert C.; Riess, Jonathan W.; Diehn, Maximilian; Swanton, Charles; Bivona, Trever G.

2017-01-01

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant protein product (e.g. EGFR inhibitor treatment in EGFR-mutant lung cancers). However, genetically-driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1122 EGFR-mutant lung cancer cell-free DNA samples and whole exome analysis of seven longitudinally collected tumor samples from an EGFR-mutant lung cancer patient, we identify critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We define new pathways limiting EGFR inhibitor response, including WNT/β-catenin and cell cycle gene (e.g. CDK4, CDK6) alterations. Tumor genomic complexity increases with EGFR inhibitor treatment and co-occurring alterations in CTNNB1, and PIK3CA exhibit non-redundant functions that cooperatively promote tumor metastasis or limit EGFR inhibitor response. This study challenges the prevailing single-gene driver oncogene view and links clinical outcomes to co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancer patients. PMID:29106415

Are allogenic or xenogenic screws and plates a reasonable alternative to alloplastic material for osteosynthesis--a histomorphological analysis in a dynamic system.

PubMed

Jacobsen, C; Obwegeser, J A

2010-12-01

Despite invention of titanium and resorbable screws and plates, still, one of the main challenges in bone fixation is the search for an ideal osteosynthetic material. Biomechanical properties, biocompatibility, and also cost effectiveness and clinical practicability are factors for the selection of a particular material. A promising alternative seems to be screws and plates made of bone. Recently, xenogenic bone pins and screws have been invented for use in joint surgery. In this study, screws made of allogenic sheep and xenogenic human bone were analyzed in a vital and dynamic sheep-model and compared to conventional titanium screws over a standard period of bone healing of 56 days with a constant applied extrusion force. Biomechanical analysis and histomorphological evaluation were performed. After 56 days of insertion xenogenic screws made of human bone showed significantly larger distance of extrusion of on average 173.8 μm compared to allogenic screws made of sheep bone of on average 27.8 and 29.95 μm of the titanium control group. Severe resorption processes with connective tissue interposition were found in the histomorphological analysis of the xenogenic screws in contrast to new bone formation and centripetal vascularization of the allogenic bone screw, as well as in processes of incorporation of the titanium control group. The study showed allogenic cortical bone screws as a substantial alternative to titanium screws with good biomechanical properties. In contrast to other reports a different result was shown for the xenogenic bone screws. They showed insufficient holding strength with confirmative histomorphological signs of degradation and insufficient osseointegration. Before common clinical use of xenogenic osteosynthetic material, further evaluation should be performed. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison of EGFR signaling pathway somatic DNA mutations derived from peripheral blood and corresponding tumor tissue of patients with advanced non-small-cell lung cancer using liquidchip technology.

PubMed

Zhang, Hui; Liu, Deruo; Li, Shanqing; Zheng, Yongqing; Yang, Xinjie; Li, Xi; Zhang, Quan; Qin, Na; Lu, Jialin; Ren-Heidenreich, Lifen; Yang, Huiyi; Wu, Yuhua; Zhang, Xinyong; Nong, Jingying; Sun, Yifen; Zhang, Shucai

2013-11-01

Somatic DNA mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway are known to predict responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. We evaluated a sensitive liquidchip platform for detecting EGFR, KRAS (alias Ki-ras), proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations in plasma samples, which were highly correlated with matched tumor tissues from 86 patients with advanced non-small-cell lung cancers. Either EGFR exon 19 or 21 mutations were detected in 36 patients: 23 of whom had identical mutations in both their blood and tissue samples; whereas mutations in the remaining 13 were found only in their tumor samples. These EGFR mutations occurred at a significantly higher frequency in females, never-smokers, and in patients with adenocarcinomas (P ≤ 0.001). The EGFR exon 20 T790M mutation was detected in only one of the paired samples [100% (95% CI, 96% to 100%) agreement]. For KRAS, proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations, the overall agreements were 97% (95% CI, 90% to 99%), 98% (95% CI, 92% to 99%), and 97% (95% CI, 90% to 99%), respectively, and these were not associated with age, sex, smoking history, or histopathologic type. In conclusion, mutations detected in plasma correlated strongly with mutation profiles in each respective tumor sample, suggesting that this liquidchip platform may offer a rapid and noninvasive method for predicting tumor responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Deng-Liang; Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou; Song, Yan-Ling

2014-10-31

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the idealmore » antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.« less

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. | Office of Cancer Genomics

Cancer.gov

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration.

PubMed

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A

2017-05-09

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.

EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration

PubMed Central

Xiang, Jinyi; Bandura, Jennifer; Zhang, Peng; Jin, Yinhua; Reuter, Hanna; Edgar, Bruce A.

2017-01-01

Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. Here we report that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. We illustrate how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair. PMID:28485389

Bone regeneration in the presence of a synthetic hydroxyapatite/silica oxide-based and a xenogenic hydroxyapatite-based bone substitute material.

PubMed

Kruse, A; Jung, R E; Nicholls, F; Zwahlen, R A; Hämmerle, C H F; Weber, F E

2011-05-01

A comparison of synthetic hydroxyapatite/silica oxide, xenogenic hydroxyapatite-based bone substitute materials with empty control sites in terms of bone regeneration enhancement in a rabbit calvarial four non-critical-sized defect model. In each of six rabbits, four bicortical calvarial bone defects were generated. The following four treatment modalities were randomly allocated: (1) empty control site, (2) synthetic hydroxyapatite/silica oxide-based (HA/SiO) test granules, (3) xenogenic hydroxyapatite -based granules, (4) synthetic hydroxyapatite/silica oxide -based (HA/SiO) test two granules. The results of the latter granules have not been reported due to their size being three times bigger than the other two granule types. After 4 weeks, the animals were sacrificed and un-decalcified sections were obtained for histological analyses. For statistical analysis, the Kruskal-Wallis test was applied (P<0.05). Histomorphometric analysis showed an average area fraction of newly formed bone of 12.32±10.36% for the empty control, 17.47±6.42% for the xenogenic hydroxyapatite -based granules group, and 21.2±5.32% for the group treated with synthetic hydroxyapatite/silica oxide -based granules. Based on the middle section, newly formed bone bridged the defect to 38.33±37.55% in the empty control group, 54.33±22.12% in the xenogenic hydroxyapatite -based granules group, and to 79±13.31% in the synthetic hydroxyapatite/silica oxide -based granules group. The bone-to-bone substitute contact was 46.38±18.98% for the xenogenic and 59.86±14.92% for the synthetic hydroxyapatite/silica oxide-based granules group. No significant difference in terms of bone formation and defect bridging could be detected between the two bone substitute materials or the empty defect. There is evidence that the synthetic hydroxyapatite/silica oxide granules provide comparable results with a standard xenogenic bovine mineral in terms of bone formation and defect bridging in non-critical size

Distribution of putative xenogeneic silencers in prokaryote genomes.

PubMed

Perez-Rueda, Ernesto; Ibarra, J Antonio

2015-10-01

Gene silencing is an important function as it keeps newly acquired foreign DNA repressed, thereby avoiding possible deleterious effects in the host organism. Known transcriptional regulators associated with this process are called xenogeneic silencers (XS) and belong to either the H-NS, Lsr2, MvaT or Rok families. In the work described here we looked for XS-like regulators and their distribution in prokaryotic organisms was evaluated. Our analysis showed that putative XS regulators similar to H-NS, Lsr2, MvaT or Rok are present only in bacteria (31.7%). This does not exclude the existence of alternative XS in the rest of the organisms analyzed. Additionally, of the four XS groups evaluated in this work, those from the H-NS family have diversified more than the other groups. In order to compare the distribution of these putative XS regulators we also searched for other nucleoid-associated proteins (NAPs) not included in this group such as Fis, EbfC/YbaB, HU/IHF and Alba. Results showed that NAPs from the Fis, EbfC/YbaB, HU/IHF and Alba families are widely (94%) distributed among prokaryotes. These NAPs were found in multiple combinations with or without XS-like proteins. In regard with XS regulators, results showed that only XS proteins from one family were found in those organisms containing them. This suggests specificity for this type of regulators and their corresponding genomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

EGFR-SGLT1 interaction does not respond to EGFR modulators, but inhibition of SGLT1 sensitizes prostate cancer cells to EGFR tyrosine kinase inhibitors.

PubMed

Ren, Jiangong; Bollu, Lakshmi R; Su, Fei; Gao, Guang; Xu, Lei; Huang, Wei-Chien; Hung, Mien-Chie; Weihua, Zhang

2013-09-01

Overexpression of epidermal growth factor receptor (EGFR) is associated with poor prognosis in malignant tumors. Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that is overexpressed in many cancers including prostate cancer. Previously, we found that EGFR interacts with and stabilizes SGLT1 in cancer cells. In this study, we determined the micro-domain of EGFR that is required for its interaction with SGLT1 and the effects of activation/inactivation of EGFR on EGFR-SGLT1 interaction, measured the expression of EGFR and SGLT1 in prostate cancer tissues, and tested the effect of inhibition of SGLT1 on the sensitivity of prostate cancer cells to EGFR tyrosine inhibitors. We found that the autophosphorylation region (978-1210 amino acids) of EGFR was required for its sufficient interaction with SGLT1 and that this interaction was independent of EGFR's tyrosine kinase activity. Most importantly, the EGFR-SGLT1 interaction does not respond to EGFR tyrosine kinase modulators (EGF and tyrosine kinase inhibitors). EGFR and SGLT1 co-localized in prostate cancer tissues, and inhibition of SGLT1 by a SGLT1 inhibitor (Phlorizin) sensitized prostate cancer cells to EGFR inhibitors (Gefitinib and Erlotinib). These data suggest that EGFR in cancer cells can exist as either a tyrosine kinase modulator responsive status or an irresponsive status. SGLT1 is a protein involved in EGFR's functions that are irresponsive to EGFR tyrosine kinase inhibitors and, therefore, the EGFR-SGLT1 interaction might be a novel target for prostate cancer therapy. © 2013 Wiley Periodicals, Inc. This article is a U.S. Government work and is in the public domain in the USA.

Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line

PubMed Central

Sullivan, Ivana; Planchard, David

2017-01-01

Tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (EGFR) are now standard treatment in the clinic for patients with advanced EGFR mutant non-small-cell lung cancer (NSCLC). First-generation EGFR TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR tyrosine kinase domain, have resulted in a significant improvement in outcome for NSCLC patients with activating EGFR mutations (L858R and Del19). However, after a median duration of response of ~12 months, all patients develop tumor resistance, and in over half of these patients this is due to the emergence of the EGFR T790M resistance mutation. The second-generation EGFR/HER TKIs were developed to treat resistant disease, targeting not only T790M but EGFR-activating mutations and wild-type EGFR. Although they exhibited promising anti-T790M activity in the laboratory, their clinical activity among T790M+ NSCLC was poor mainly because of dose-limiting toxicity due to simultaneous inhibition of wild-type EGFR. The third-generation EGFR TKIs selectively and irreversibly target EGFR T790M and activating EGFR mutations, showing promising efficacy in NSCLC resistant to the first- and second-generation EGFR TKIs. They also appear to have lower incidences of toxicity due to the limited inhibitory effect on wild-type EGFR. Currently, the first-generation gefitinib and erlotinib and second-generation afatinib have been approved for first-line treatment of metastatic NSCLC with activating EGFR mutations. Among the third-generation EGFR TKIs, osimertinib is today the only drug approved by the Food and Drug Administration and the European Medicines Agency to treat metastatic EGFR T790M NSCLC patients who have progressed on or after EGFR TKI therapy. In this review, we summarize the available post-progression therapies including third-generation EGFR inhibitors and combination treatment strategies for treating patients with NSCLC harboring EGFR mutations and address the

[Autogenous platelet-rich plasma gel with acellular xenogeneic dermal matrix for treatment of deep II degree burns].

PubMed

Hao, Tianzhi; Zhu, Jingmin; Hu, Wenbo; Zhang, Hua; Gao, Zhenhui; Wen, Xuehui; Zhou, Zhi; Lu, Gang; Liu, Jingjie; Li, Wen

2010-06-01

To investigate the effectiveness of autogenous platelet-rich plasma (PRP) gel with acellular xenogeneic dermal matrix in the treatment of deep II degree burns. From January 2007 to December 2009, 30 cases of deep II degree burns were treated. There were 19 males and 11 females with an average age of 42.5 years (range, 32-57 years). The burn area was 10% to 48% of total body surface area. The time from burn to hospitalization was 30 minutes to 8 hours. All patients were treated with tangential excision surgery, one side of the wounds were covered with autogenous PRP gel and acellular xenogeneic dermal matrix (PRP group), the other side of the wounds were covered with acellular xenogeneic dermal matrix only (control group). The healing rate, healing time, infection condition, and scar formation were observed. At 7 days after operation, the infection rate in PRP group (6.7%, 2/30) was significantly lower than that in control group (16.7%, 5/30, P < 0.05). The healing times were (18 +/- 4) days and (22 +/- 4) days respectively in PRP group and control group, showing significant difference (P < 0.05). The healing rates at 14 days and 21 days were 75% +/- 7% and 88% +/- 5% in PRP group, were 62% +/- 15% and 73% +/- 7% in control group, showing significant difference (P < 0.05). RPR group was superior to control group in elasticity, color, appearance, softness, scar formation, and healing quality. Autogenous PRP gel with acellular xenogeneic dermal matrix can accelerate the wound healing of deep II degree burns as well as alleviate the scar proliferation.

Evidence-based best practices for EGFR T790M testing in lung cancer in Canada.

PubMed

Stockley, T; Souza, C A; Cheema, P K; Melosky, B; Kamel-Reid, S; Tsao, M S; Spatz, A; Karsan, A

2018-04-01

Epidermal growth factor receptor (egfr) tyrosine kinase inhibitors (tkis) are recommended as first-line systemic therapy for patients with non-small-cell lung cancer (nsclc) having mutations in the EGFR gene. Resistance to tkis eventually occurs in all nsclc patients treated with such drugs. In patients with resistance to tkis caused by the EGFR T790M mutation, the third-generation tki osimertinib is now the standard of care. For optimal patient management, accurate EGFR T790M testing is required. A multidisciplinary working group of pathologists, laboratory medicine specialists, medical oncologists, a respirologist, and a thoracic radiologist from across Canada was convened to discuss best practices for EGFR T790M mutation testing in Canada. The group made recommendations in the areas of the testing algorithm and the pre-analytic, analytic, and post-analytic aspects of clinical testing for both tissue testing and liquid biopsy circulating tumour dna testing. The recommendations aim to improve EGFR T790M testing in Canada and to thereby improve patient care.

EGFR mutation testing practices within the Asia Pacific region: results of a multicenter diagnostic survey.

PubMed

Yatabe, Yasushi; Kerr, Keith M; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma Luisa D; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

2015-03-01

The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%-32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia.

Combined EGFR/MEK Inhibition Prevents the Emergence of Resistance in EGFR mutant Lung Cancer

PubMed Central

Uddin, Sharmeen; Capelletti, Marzia; Ercan, Dalia; Ogino, Atsuko; Pratilas, Christine A.; Rosen, Neal; Gray, Nathanael S.; Wong, Kwok-Kin; Jänne, Pasi A.

2016-01-01

Irreversible pyrimidine based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR activating and EGFR inhibitor resistant T790M mutations more potently than wild type EGFR. While this class of mutant selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFR T790M, prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002 induced apoptosis and inhibits the emergence of resistance in WZ4002 sensitive models known to acquire resistance via both T790M dependent and independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial co-targeting of EGFR and MEK could significantly impede the development of acquired resistance in mutant EGFR lung cancer. PMID:26036643

Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer.

PubMed

Lin, Chien-Chung; Huang, Wei-Lun; Wei, Fang; Su, Wu-Chou; Wong, David T

2015-01-01

Advances in target therapies for lung cancer have enabled detection of gene mutations, specifically those of EGFR. Assays largely depend on the acquisition of tumor tissue biopsy, which is invasive and may not reflect the genomic profile of the tumor at treatment due to tumor heterogeneity or changes that occur during treatment through acquired resistance. Liquid biopsy, a blood test that detects evidence of cancer cells or tumor DNA, has generated considerable interest for its ability to detect EGFR mutations. However, its clinical application is limited by complicated collection methods and the need for technique-dependent platforms. Recently, simpler techniques for EGFR mutant detection in urine or saliva samples have been developed. This review focuses on advances in liquid biopsy and discusses its potential for clinical implementation in lung cancer.

Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer

PubMed Central

Wong; Lin, David T; Huang, Chien-Chung; Wei, Wei-Lun; Su, Fang; Wu-Chou

2016-01-01

Summary Advances in target therapies for lung cancer have enabled detection of gene mutations, specifically those of EGFR. Assays largely depend on the acquisition of tumor tissue biopsy, which is invasive and may not reflect the genomic profile of the tumor at treatment due to tumor heterogeneity or changes that occur during treatment through acquired resistance. Liquid biopsy, a blood test that detects evidence of cancer cells or tumor DNA, has generated considerable interest for its ability to detect EGFR mutations, however, its clinical application is limited by complicated collection methods and the need for technique-dependent platforms. Recently, simpler techniques for EGFR mutant detection in in urine or saliva samples have been developed. This review focuses on advances in liquid biopsy and discusses its potential for clinical implementation in lung cancer. PMID:26420338

Inhibition of DYRK1A destabilizes EGFR and reduces EGFR-dependent glioblastoma growth

PubMed Central

Pozo, Natividad; Zahonero, Cristina; Fernández, Paloma; Liñares, Jose M.; Ayuso, Angel; Hagiwara, Masatoshi; Pérez, Angel; Ricoy, Jose R.; Hernández-Laín, Aurelio; Sepúlveda, Juan M.; Sánchez-Gómez, Pilar

2013-01-01

Glioblastomas (GBMs) are very aggressive tumors that are resistant to conventional chemo- and radiotherapy. New molecular therapeutic strategies are required to effectively eliminate the subpopulation of GBM tumor–initiating cells that are responsible for relapse. Since EGFR is altered in 50% of GBMs, it represents one of the most promising targets; however, EGFR kinase inhibitors have produced poor results in clinical assays, with no clear explanation for the observed resistance. We uncovered a fundamental role for the dual-specificity tyrosine phosphorylation–regulated kinase, DYRK1A, in regulating EGFR in GBMs. We found that DYRK1A was highly expressed in these tumors and that its expression was correlated with that of EGFR. Moreover, DYRK1A inhibition promoted EGFR degradation in primary GBM cell lines and neural progenitor cells, sharply reducing the self-renewal capacity of normal and tumorigenic cells. Most importantly, our data suggest that a subset of GBMs depends on high surface EGFR levels, as DYRK1A inhibition compromised their survival and produced a profound decrease in tumor burden. We propose that the recovery of EGFR stability is a key oncogenic event in a large proportion of gliomas and that pharmacological inhibition of DYRK1A could represent a promising therapeutic intervention for EGFR-dependent GBMs. PMID:23635774

Fatty acid synthase mediates EGFR palmitoylation in EGFR mutated non-small cell lung cancer.

PubMed

Ali, Azhar; Levantini, Elena; Teo, Jun Ting; Goggi, Julian; Clohessy, John G; Wu, Chan Shuo; Chen, Leilei; Yang, Henry; Krishnan, Indira; Kocher, Olivier; Zhang, Junyan; Soo, Ross A; Bhakoo, Kishore; Chin, Tan Min; Tenen, Daniel G

2018-02-15

Metabolic reprogramming is widely known as a hallmark of cancer cells to allow adaptation of cells to sustain survival signals. In this report, we describe a novel oncogenic signaling pathway exclusively acting in mutated epidermal growth factor receptor (EGFR) non-small cell lung cancer (NSCLC) with acquired tyrosine kinase inhibitor (TKI) resistance. Mutated EGFR mediates TKI resistance through regulation of the fatty acid synthase (FASN), which produces 16-C saturated fatty acid palmitate. Our work shows that the persistent signaling by mutated EGFR in TKI-resistant tumor cells relies on EGFR palmitoylation and can be targeted by Orlistat, an FDA-approved anti-obesity drug. Inhibition of FASN with Orlistat induces EGFR ubiquitination and abrogates EGFR mutant signaling, and reduces tumor growths both in culture systems and in vivo Together, our data provide compelling evidence on the functional interrelationship between mutated EGFR and FASN and that the fatty acid metabolism pathway is a candidate target for acquired TKI-resistant EGFR mutant NSCLC patients. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays.

PubMed

Wang, Jun; Ramakrishnan, Ramesh; Tang, Zhe; Fan, Weiwen; Kluge, Amy; Dowlati, Afshin; Jones, Robert C; Ma, Patrick C

2010-04-01

The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable. We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array. The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors. The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.

The "COLD-PCR approach" for early and cost-effective detection of tyrosine kinase inhibitor resistance mutations in EGFR-positive non-small cell lung cancer.

PubMed

Mairinger, Fabian D; Vollbrecht, Claudia; Streubel, Anna; Roth, Andreas; Landt, Olfert; Walter, Henry F R; Kollmeier, Jens; Mairinger, Thomas

2014-01-01

Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies. Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing. COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible. COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Bisphosphonates enhance EGFR-TKIs efficacy in advanced NSCLC patients with EGFR activating mutation: A retrospective study

PubMed Central

Cai, Xiao-Hong; Yao, Wen-Xiu; Xu, Yong; Liu, Xiao-Ke; Zhu, Wen-Jiang; Wang, Yan; Zhou, Jin; Lu, You; Wang, Yong-Sheng

2016-01-01

Background Bisphosphonates have exhibited anti-tumor activity in non-small cell lung cancer (NSCLC). We aimed to evaluate whether the combination of bisphosphonates with tyrosine kinase inhibitors of EGFR (EGFR-TKIs) could obtain a synergistic effect on advanced NSCLC patients with EGFR mutations. Methods Between January 2008 and October 2013, 114 advanced EGFR mutations NSCLC patients who received EGFR-TKIs as first-line therapy were recruited from two cancer centers. Patients were separated into EGFR-TKIs alone or EGFR-TKIs plus bisphosphonates (combination) group. Median progression free survival (mPFS), median overall survival (mOS) distributions and survival curves were analyzed. Results Among the 114 patients, 62 had bone metastases (19 patients treated with EGFR-TKIs, 43 patients treated with EGFR-TKIs + bisphosphonates). Median PFS and OS were significantly improved in combination group compared with EGFR-TKIs group (mPFS: 15.0 vs 7.3 months, P = 0.0017; mOS: 25.2 vs 10.4 months, P = 0.0015) in patients with bone metastases. Among the 71 patients (19 patients with bone metastases) treated with EGFR-TKIs alone, patients with bone metastases had poor survival prognosis (mPFS:7.3 vs 12.1 months, P = 0.0434; mOS:10.4 vs 22.0 months, P = 0.0036). The survival of patients with bone metastases who received EGFR-TKIs plus bisphosphonates therapy was non-inferior to patients without bone metastases treated with EGFR-TKIs alone (mPFS: 15.0 vs 12.1 months, p = 0.1871; mOS: 25.2 vs 22.0 months, p = 0.9798). Conclusions Concomitant use of bisphosphonates and EGFR-TKIs improves therapeutic efficacy and brings survival benefits to NSCLC patients with EGFR mutation and bone metastases. PMID:26624882

Gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancers by accelerating EGFR turnover.

PubMed

Nam, Boas; Rho, Jin Kyung; Shin, Dong-Myung; Son, Jaekyoung

2016-10-01

Gallic acid is a common botanic phenolic compound, which is present in plants and foods worldwide. Gallic acid is implicated in various biological processes such as cell growth and apoptosis. Indeed, gallic acid has been shown to induce apoptosis in many cancer types. However, the molecular mechanisms of gallic acid-induced apoptosis in cancer, particularly lung cancer, are still unclear. Here, we report that gallic acid induces apoptosis in EGFR-mutant non-small cell lung cancer (NSCLC) cells, but not in EGFR-WT NSCLC cells. Treatment with gallic acid resulted in a significant reduction in proliferation and induction of apoptosis, only in EGFR-mutant NSCLC cells. Interestingly, treatment with gallic acid led to a robust decrease in EGFR levels, which is critical for NSCLC survival. Treatment with gallic acid had no significant effect on transcription, but induced EGFR turnover. Indeed, treatment with a proteasome inhibitor dramatically reversed gallic acid-induced EGFR downregulation. Moreover, treatment with gallic acid induced EGFR turnover leading to apoptosis in EGFR-TKI (tyrosine kinase inhibitor)-resistant cell lines, which are dependent on EGFR signaling for survival. Thus, these studies suggest that gallic acid can induce apoptosis in EGFR-dependent lung cancers that are dependent on EGFR for growth and survival via acceleration of EGFR turnover. Copyright © 2016 Elsevier Ltd. All rights reserved.

EGFR gene copy number alterations are not a useful screening tool for predicting EGFR mutation status in lung adenocarcinoma.

PubMed

Russell, Prudence A; Yu, Yong; Do, Hongdo; Clay, Timothy D; Moore, Melissa M; Wright, Gavin M; Conron, Matthew; Wainer, Zoe; Dobrovic, Alexander; McLachlan, Sue-Anne

2014-01-01

We investigated if gene copy number (GCN) alterations of the epidermal growth factor receptor (EGFR), as detected by silver enhanced in situ hybridisation (SISH), could be used to select patients for EGFR mutation testing. Resected lung adenocarcinoma specimens with adequate tumour were identified. EGFR SISH was performed using the Ventana Benchmark Ultra platform. EGFR GCN was classified according to the Colorado Classification System. EGFR mutations were scanned by high resolution melting and confirmed by Sanger sequencing. Thirty-four of 96 tumours were EGFR SISH positive (35%), and 31 of 96 tumours harboured one or more EGFR mutations (32%). Of 31 EGFR-mutant tumours, 18 were EGFR SISH positive (58%). There was a statistically significant relationship between the presence of an EGFR mutation and EGFR GCN (p = 0.003). Thirteen of 31 EGFR-mutant tumours were EGFR SISH negative (42%), and 16 of 65 EGFR-wild type tumours were EGFR SISH positive (24%). The sensitivity, specificity, positive predictive value and negative predictive value were 58%, 75%, 52.9% and 79%, respectively. Despite a significant relationship between EGFR GCN alterations and EGFR mutations, our results indicate that EGFR GCN as detected by SISH is not a suitable way to select patients for EGFR mutation testing.

Osteoprotegerin inhibits bone resorption and prevents tumor development in a xenogenic model of Ewing's sarcoma by inhibiting RANKL

PubMed Central

Picarda, Gaëlle; Matous, Etienne; Amiaud, Jérôme; Charrier, Céline; Lamoureux, François; Heymann, Marie-Françoise; Tirode, Franck; Pitard, Bruno; Trichet, Valérie; Heymann, Dominique; Redini, Françoise

2013-01-01

Ewing's sarcoma (ES) associated with high osyeolytic lesions typically arises in the bones of children and adolescents. The development of multi-disciplinary therapy has increased current long-term survival rates to greater than 50% but only 20% for high risk group patients (relapse, metastases, etc.). Among new therapeutic approaches, osteoprotegerin (OPG), an anti-bone resorption molecule may represent a promising candidate to inhibit RANKL-mediated osteolytic component of ES and consequently to limit the tumor development. Xenogenic orthotopic models of Ewing's sarcoma were induced by intra-osseous injection of human TC-71 ES cells. OPG was administered in vivo by non-viral gene transfer using an amphiphilic non ionic block copolymer. ES bearing mice were assigned to controls (no treatment, synthetic vector alone or F68/empty pcDNA3.1 plasmid) and hOPG treated groups. A substantial but not significant inhibition of tumor development was observed in the hOPG group as compared to control groups. Marked bone lesions were revealed by micro-computed tomography analyses in control groups whereas a normal bone micro-architecture was preserved in the hOPG treated group. RANKL over-expressed in ES animal model was expressed by tumor cells rather than by host cells. However, TRAIL present in the tumor microenvironment may interfere with OPG effect on tumor development and bone remodeling via RANKL inhibition. In conclusion, the use of a xenogenic model of Ewing's sarcoma allowed discriminating between the tumor and host cells responsible for the elevation of RANKL production observed in this tumor and demonstrated the relevance of blocking RANKL by OPG as a promising therapy in ES. PMID:26909278

Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.

PubMed

Chang, Shing-Jyh; Liao, En-Chi; Yeo, Hsin-Yueh; Kuo, Wen-Hung; Chen, Hsin-Yi; Tsai, Yi-Ting; Wei, Yu-Shan; Chen, Ying-Jen; Wang, Yi-Shiuan; Li, Ji-Min; Shih, Chuan-Chi; Chan, Chia-Hao; Lai, Zih-Yin; Chou, Hsiu-Chuan; Chuang, Yung-Jen; Chan, Hong-Lin

2018-06-01

With the concept of precision medicine, combining multiple molecular-targeting therapies has brought new approaches to current cancer treatments. Malfunction of the tumor suppressor protein, p53 is a universal hallmark in human cancers. Under normal conditions, p53 is degraded through an ubiquitin-proteosome pathway regulated by its negative regulator, MDM2. In contrast, cellular stress such as DNA damage will activate p53 to carry out DNA repair, cell cycle arrest, and apoptosis. In this study, we focused on ovarian carcinoma with high EGFR and MDM2 overexpression rate. We assessed the effects of combined inhibition by MDM2 (JNJ-26854165) and EGFR (gefitinib) inhibitors on various ovarian cell lines to determine the importance of these two molecular targets on cell proliferation. We then used a proteomic strategy to investigate the relationship between MDM2 and EGFR inhibition to explore the underlying mechanisms of how their combined signaling blockades work together to exert cooperative inhibition. Our results demonstrated that all four cell lines were sensitive to both individual and combined, MDM2 and EGFR inhibition. The proteomic analysis also showed that gefitinib/JNJ-treated CAOV3 cells exhibited downregulation of proteins involved in nucleotide biosynthesis such as nucleoside diphosphate kinase B (NME2). In conclusion, our study showed that the combined treatment with JNJ and gefitinib exerted synergistic inhibition on cell proliferation, thereby suggesting the potential application of combining MDM2 inhibitors with EGFR inhibitors for enhancing efficacy in ovarian cancer treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

NASA Astrophysics Data System (ADS)

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-03-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR-TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure-activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

In vivo xenogeneic scaffold fate is determined by residual antigenicity and extracellular matrix preservation.

PubMed

Wong, Maelene L; Wong, Janelle L; Vapniarsky, Natalia; Griffiths, Leigh G

2016-06-01

The immunological potential of animal-derived tissues and organs is the critical hurdle to increasing their clinical implementation. Glutaraldehyde-fixation cross-links proteins in xenogeneic tissues (e.g., bovine pericardium) to delay immune rejection, but also compromises the regenerative potential of the resultant biomaterial. Unfixed xenogeneic biomaterials in which xenoantigenicity has been ameliorated and native extracellular matrix (ECM) architecture has been maintained have the potential to overcome limitations of current clinically utilized glutaraldehyde-fixed biomaterials. The objective of this work was to determine how residual antigenicity and ECM architecture preservation modulate recipient immune and regenerative responses towards unfixed bovine pericardium (BP) ECM scaffolds. Disruption of ECM architecture during scaffold generation, with either SDS-decellularization or glutaraldehyde-fixation, stimulated recipient foreign body response and resultant fibrotic encapsulation following leporine subpannicular implantation. Conversely, BP scaffolds subjected to stepwise removal of hydrophilic and lipophilic antigens using amidosulfobetaine-14 (ASB-14) maintained native ECM architecture and thereby avoided fibrotic encapsulation. Removal of hydrophilic and lipophilic antigens significantly decreased local and systemic graft-specific, adaptive immune responses and subsequent calcification of BP scaffolds compared to scaffolds undergoing hydrophile removal only. Critically, removal of antigenic components and preservation of ECM architecture with ASB-14 promoted full-thickness recipient non-immune cellular repopulation of the BP scaffold. Further, unlike clinically utilized fixed BP, ASB-14-treated scaffolds fostered rapid intimal and medial vessel wall regeneration in a porcine carotid patch angioplasty model. This work highlights the importance of residual antigenicity and ECM architecture preservation in modulating recipient immune and regenerative

Acquired Resistance Mechanisms to Combination Met-TKI/EGFR-TKI Exposure in Met-Amplified EGFR-TKI-Resistant Lung Adenocarcinoma Harboring an Activating EGFR Mutation.

PubMed

Yamaoka, Toshimitsu; Ohmori, Tohru; Ohba, Motoi; Arata, Satoru; Kishino, Yasunari; Murata, Yasunori; Kusumoto, Sojiro; Ishida, Hiroo; Shirai, Takao; Hirose, Takashi; Ohnishi, Tsukasa; Sasaki, Yasutsuna

2016-12-01

Met-amplified EGFR-tyrosine kinase inhibitor (TKI)-resistant non-small cell lung cancer (NSCLC) harboring an activating EGFR mutation is responsive to concurrent EGFR-TKI and Met-TKI treatment in a preclinical model. Here, we determined that Met-amplified gefitinib-resistant cells acquire dual resistance to inhibition of EGFR and Met tyrosine kinase activities. PC-9 lung adenocarcinoma cells harboring 15-bp deletions (Del E746_A750) in EGFR exon 19 were treated with increasing concentrations of the Met-TKI PHA665752 and 1 μmol/L gefitinib for 1 year; three resistant clones were established via Met amplification. The three dual-resistance cell lines (PC-9DR2, PC-9DR4, and PC-9DR6, designated as DR2, DR4, and DR6, respectively) exhibited different mechanisms for evading both EGFR and Met inhibition. None of the clones harbored a secondary mutation of EGFR T790M or a Met mutation. Insulin-like growth factor (IGF)/IGF1 receptor activation in DR2 and DR4 cells acted as a bypass signaling pathway. Met expression was attenuated to a greater extent in DR2 than in PC-9 cells, but was maintained in DR4 cells by overexpression of IGF-binding protein 3. In DR6 cells, Met was further amplified by association with HSP90, which protected Met from degradation and induced SET and MYND domain-containing 3 (SMYD3)-mediated Met transcription. This is the first report describing the acquisition of dual resistance mechanisms in NSCLC harboring an activating EGFR mutation to Met-TKI and EGFR-TKI following previous EGFR-TKI treatment. These results might inform the development of more effective therapeutic strategies for NSCLC treatment. Mol Cancer Ther; 15(12); 3040-54. ©2016 AACR. ©2016 American Association for Cancer Research.

Gefitinib targets EGFR dimerization and ERK1/2 phosphorylation to inhibit pleural mesothelioma cell proliferation.

PubMed

Favoni, Roberto E; Pattarozzi, Alessandra; Lo Casto, Michele; Barbieri, Federica; Gatti, Monica; Paleari, Laura; Bajetto, Adriana; Porcile, Carola; Gaudino, Giovanni; Mutti, Luciano; Corte, Giorgio; Florio, Tullio

2010-03-01

Altered EGFR activity is a causal factor for human tumor development, including malignant pleural mesotheliomas. The aim of the present study was the evaluation of the effects of Gefitinib on EGF-induced mesothelioma cell proliferation and the intracellular mechanisms involved. Cell proliferation, DNA synthesis and apoptosis were measured by MTT, thymidine incorporation and FACS analysis; EGFR, ERK1/2 and Akt expression and phosphorylation by Western blot, whereas receptor sites were analyzed by binding studies. Gefitinib inhibited EGF-induced proliferation in two mesothelioma cell lines, derived from pleural effusion (IST-Mes2) or tumor biopsy (ZL55). The treatment with Gefitinib induced cell cycle arrest in both cell lines, while apoptosis was observed only for high concentrations and prolonged drug exposure. EGF-dependent mesothelioma cell proliferation was mediated by EGFR and ERK1/2 phosphorylation, while Akt was not affected. Gefitinib inhibited both EGFR and ERK1/2 activation, being maximal at drug concentrations that induce cytostatic effects, suggesting that the proapoptotic activity of Gefitinib is independent from EGFR inhibition. Gefitinib treatment increased EGFR Bmax, possibly through membrane stabilization of inactive receptor dimers that we show to be induced by the drug also in the absence of EGF. EGFR activation of ERK1/2 represents a key pathway for pleural mesothelioma cell proliferation. Low concentrations of Gefitinib cause mesothelioma cell cycle arrest through the blockade of EGFR activity while high concentrations induce apoptosis. Finally, we propose that the formation of inactive EGFR dimers may contribute to the antitumoral activity of Gefitinib.

Highly expressed EGFR in pearl sac may facilitate the pearl formation in the pearl oyster, Pinctada fucata.

PubMed

Zhu, Wenjie; Fan, Sigang; Huang, Guiju; Zhang, Dongling; Liu, Baosuo; Bi, Xiaomin; Yu, Dahui

2015-07-25

Epidermal growth factor receptor (EGFR) plays an important role in cell growth, proliferation, differentiation and migration. Yet whether it functions in pearl formation or not is not reported. In this study, EGFR was cloned from the pearl oyster Pinctada fucata (named as Pf-EGFR) and its expression profiles were investigated. The cDNA was 2156bp long with an ORF of 1017bp encoding 338 amino acid residues. The deduced polypeptide contained an L domain and a cysteine-rich domain, consistent with the characteristics of ErbB family. The sequence of Pf-EGFR shared 22.78-56.71% identity with other EGFRs. The genomic sequence of Pf-EGFR consisted of six exons and five introns, being 5190bp in total length, and expressed in all the tested tissues with a higher expression level in the pearl sac (P<0.05). In situ hybridization showed that Pf-EGFR was specifically expressed on both the inner side of the outer fold and the outer side of the inner fold of the mantle, as well as on the whole pearl sac including the connective tissues. In addition, Pf-EGFR was markedly increased at larval metamorphosis, significantly higher than other development periods (P<0.05). These results indicated that the Pf-EGFR may facilitate pearl formation as well as larval metamorphosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

PubMed Central

2010-01-01

Background Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients. PMID:21167064

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials.

PubMed

Wang, Raymond M; Johnson, Todd D; He, Jingjin; Rong, Zhili; Wong, Michelle; Nigam, Vishal; Behfar, Atta; Xu, Yang; Christman, Karen L

2017-06-01

Current assessment of biomaterial biocompatibility is typically implemented in wild type rodent models. Unfortunately, different characteristics of the immune systems in rodents versus humans limit the capability of these models to mimic the human immune response to naturally derived biomaterials. Here we investigated the utility of humanized mice as an improved model for testing naturally derived biomaterials. Two injectable hydrogels derived from decellularized porcine or human cadaveric myocardium were compared. Three days and one week after subcutaneous injection, the hydrogels were analyzed for early and mid-phase immune responses, respectively. Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials. The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4 + T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice. T-helper cells, in response to the allogeneic hydrogel material, were also less polarized towards a pro-remodeling Th2 phenotype compared to xenogeneic extracellular matrix hydrogels in humanized mice. In both models, both biomaterials induced the infiltration of macrophages polarized towards a M2 phenotype and T-helper cells polarized towards a Th2 phenotype. In conclusion, these studies showed the importance of testing naturally derived biomaterials in immune competent animals and the potential of utilizing this humanized mouse model for further studying human immune cell responses to biomaterials in an in vivo environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intrinsic resistance to EGFR tyrosine kinase inhibitors in advanced non-small-cell lung cancer with activating EGFR mutations

PubMed Central

Wang, Jun; Wang, Baocheng; Chu, Huili; Yao, Yunfeng

2016-01-01

Identifying activating EGFR mutations is a useful predictive strategy that helps select a population of advanced non-small-cell lung cancer (NSCLC) patients for treatment with EGFR tyrosine kinase inhibitors (TKIs). Patients with sensitizing EGFR mutations (predominantly an in-frame deletion in exon 19 and an L858R substitution) are highly responsive to first-generation EGFR TKIs, such as gefitinib and erlotinib, and show improved progression-free survival without serious side effects. However, all patients with activating EGFR mutations who are initially responsive to EGFR TKIs eventually develop acquired resistance after a median progression-free survival of 10–16 months, followed by disease progression. Moreover, ~20%–30% of NSCLC patients have no objective tumor regression on initial EGFR TKI treatment, although they harbor an activating EGFR mutation. These patients represent an NSCLC subgroup that is defined as having intrinsic or primary resistance to EGFR TKIs. Different mechanisms of acquired EGFR TKI resistance have been identified, and several novel compounds have been developed to reverse acquired resistance, but little is known about EGFR TKI intrinsic resistance. In this review, we summarize the latest findings involving mechanisms of intrinsic resistance to EGFR TKIs in advanced NSCLC with activating EGFR mutations and present possible therapeutic strategies to overcome this resistance. PMID:27382309

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

PubMed

Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-10-31

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

EGFR TKI as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer

PubMed Central

Nan, Xueli; Xie, Chao; Yu, Xueyan; Liu, Jie

2017-01-01

After the discovery of activating mutations in EGFR, EGFR tyrosine kinase inhibitors (TKIs) have been introduced into the first-line treatment of non-small-cell lung cancer (NSCLC). A series of studies have shown that EGFR TKI monotherapy as first-line treatment can benefit NSCLC patients harbouring EGFR mutations. Besides, combination strategies based on EGFR TKIs in the first line treatment have also been proved to delay the occurrence of resistance. In this review, we summarize the scientific literature and evidence of EGFR TKIs as first-line therapy from the first-generation EGFR TKIs to conceptually proposed fourth-generation EGFR TKI, and also recommend the application of monotherapy and combination therapies of the EGFR-based targeted therapy with other agents such as chemotherapy, anti-angiogenic drugs and immunecheckpoint inhibitors. PMID:29088904

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

PubMed

Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

2013-09-01

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Osimertinib - effective treatment of NSCLC with activating EGFR mutations after progression on EGFR tyrosine kinase inhibitors.

PubMed

Skrzypski, Marcin; Szymanowska-Narloch, Amelia; Dziadziuszko, Rafał

2017-01-01

Non-small cell lung cancer (NSCLC) driven by activating mutations in epidermal growth factor receptor (EGFR) constitutes up to 10% of NSCLC cases. According to the NCCN recommendations, all patients (with the exception of smoking patients with squamous cell lung cancer) should be screened for the presence of activating EGFR mutations, i.e. deletion in exon 19 or point mutation L858R in exon 21, in order to select the group that benefits from EGFR tyrosine kinase inhibitors (EGFR TKIs) treatment. Among approved agents there are the 1 st generation reversible EGFR TKIs, erlotinib and gefitinib, and the 2 nd generation irreversible EGFR TKI, afatinib. The objective response rates to these drugs in randomised clinical trials were in the range of 56-74%, and median time to progression 9-13 months. The most common determinant of resistance to these drugs is the clonal expansion of cancer cells with T790M mutation (Thr790Met) in exon 20 of EGFR. Osimertinib (Tagrisso™), a 3 rd generation, irreversible EGFR tyrosine kinase inhibitor, constitutes a novel, highly efficacious treatment for NSCLC patients progressing on EGFR TKIs with T790M mutation confirmed as the resistance mechanism. Resistance mutation can be determined in tissue or liquid biopsy obtained after progression on EGFR TKIs. Osimertinib has a favourable toxicity profile, with mild rash and diarrhoea being the most common. In this article, we present three cases that were successfully treated with osimertinib after progression on 1st and 2nd generation EGFR TKIs.

Intratumoral heterogeneity in EGFR mutant NSCLC results in divergent resistance mechanisms in response to EGFR tyrosine kinase inhibition

PubMed Central

Soucheray, Margaret; Capelletti, Marzia; Pulido, Inés; Kuang, Yanan; Paweletz, Cloud P.; Becker, Jeffrey H.; Kikuchi, Eiki; Xu, Chunxiao; Patel, Tarun B.; Al-shahrour, Fatima; Carretero, Julián; Wong, Kwok-Kin; Jänne, Pasi A.; Shapiro, Geoffrey I.; Shimamura, Takeshi

2015-01-01

Non-small cell lung cancers (NSCLC) that have developed resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, are clinically linked to an epithelial-to-mesenchymal transition (EMT) phenotype. Here we examined whether modulating EMT maintains the responsiveness of EGFR-mutated NSCLCs to EGFR TKI therapy. Using human NSCLC cell lines harboring mutated-EGFR and a transgenic mouse model of lung cancer driven by mutant EGFR (EGFR-Del19-T790M), we demonstrate that EGFR inhibition induces TGFβ secretion followed by SMAD pathway activation, an event that promotes EMT. Chronic exposure of EGFR-mutated NSCLC cells to TGFβ was sufficient to induce EMT and resistance to EGFR TKI treatment. Furthermore, NSCLC HCC4006 cells with acquired resistance to gefitinib were characterized by a mesenchymal phenotype and displayed a higher prevalence of the EGFR T790M mutated allele. Notably, combined inhibition of EGFR and the TGFβ receptor in HCC4006 cells prevented EMT, but was not sufficient to prevent acquired gefitinib resistance because of an increased emergence of the EGFR T790M allele compared to cells treated with gefitinib alone. Conversely, another independent NSCLC cell line, PC9, reproducibly develops EGFR T790M mutations as the primary mechanism underlying EGFR TKI resistance, even though the prevalence of the mutant allele is lower than that in HCC4006 cells. Thus, our findings underscore heterogeneity within NSCLC cells lines harboring EGFR kinase domain mutations that give rise to divergent resistance mechanisms in response to treatment and anticipate the complexity of EMT suppression as a therapeutic strategy. PMID:26282169

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

PubMed

Figueroa, Javier M; Skog, Johan; Akers, Johnny; Li, Hongying; Komotar, Ricardo; Jensen, Randy; Ringel, Florian; Yang, Isaac; Kalkanis, Steven; Thompson, Reid; LoGuidice, Lori; Berghoff, Emily; Parsa, Andrew; Liau, Linda; Curry, William; Cahill, Daniel; Bettegowda, Chetan; Lang, Frederick F; Chiocca, E Antonio; Henson, John; Kim, Ryan; Breakefield, Xandra; Chen, Clark; Messer, Karen; Hochberg, Fred; Carter, Bob S

2017-10-19

RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

PubMed

Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

2018-06-01

Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization

NASA Astrophysics Data System (ADS)

Wang, Yujia; Bao, Ji; Wu, Xiujuan; Wu, Qiong; Li, Yi; Zhou, Yongjie; Li, Li; Bu, Hong

2016-04-01

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host’s immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.

Dynamics of multiple resistance mechanisms in plasma DNA during EGFR-targeted therapies in non-small cell lung cancer.

PubMed

Tsui, Dana Wai Yi; Murtaza, Muhammed; Wong, Alvin Seng Cheong; Rueda, Oscar M; Smith, Christopher G; Chandrananda, Dineika; Soo, Ross A; Lim, Hong Liang; Goh, Boon Cher; Caldas, Carlos; Forshew, Tim; Gale, Davina; Liu, Wei; Morris, James; Marass, Francesco; Eisen, Tim; Chin, Tan Min; Rosenfeld, Nitzan

2018-06-01

Tumour heterogeneity leads to the development of multiple resistance mechanisms during targeted therapies. Identifying the dominant driver(s) is critical for treatment decision. We studied the relative dynamics of multiple oncogenic drivers in longitudinal plasma of 50 EGFR -mutant non-small-cell lung cancer patients receiving gefitinib and hydroxychloroquine. We performed digital PCR and targeted sequencing on samples from all patients and shallow whole-genome sequencing on samples from three patients who underwent histological transformation to small-cell lung cancer. In 43 patients with known EGFR mutations from tumour, we identified them accurately in plasma of 41 patients (95%, 41/43). We also found additional mutations, including EGFR T790M (31/50, 62%), TP53 (23/50, 46%), PIK3CA (7/50, 14%) and PTEN (4/50, 8%). Patients with both TP53 and EGFR mutations before treatment had worse overall survival than those with only EGFR Patients who progressed without T790M had worse PFS during TKI continuation and developed alternative alterations, including small-cell lung cancer-associated copy number changes and TP53 mutations, that tracked subsequent treatment responses. Longitudinal plasma analysis can help identify dominant resistance mechanisms, including non-druggable genetic information that may guide clinical management. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

PubMed Central

Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

2002-01-01

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

PubMed

Yamazaki, Shota; Higuchi, Youichi; Ishibashi, Masayuki; Hashimoto, Hiroko; Yasunaga, Masahiro; Matsumura, Yasuhiro; Tsuchihara, Katsuya; Tsuboi, Masahiro; Goto, Koichi; Ochiai, Atsushi; Ishii, Genichiro

2018-06-01

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

PubMed Central

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-01-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR. PMID:28287083

Gender-related survival differences associated with EGFR polymorphisms in metastatic colon cancer.

PubMed

Press, Oliver A; Zhang, Wu; Gordon, Michael A; Yang, Dongyun; Lurje, Georg; Iqbal, Syma; El-Khoueiry, Anthony; Lenz, Heinz-Josef

2008-04-15

Evidence is accumulating supporting gender-related differences in the development of colonic carcinomas. Sex steroid hormone receptors are expressed in the colon and interact with epidermal growth factor receptor (EGFR), a gene widely expressed in colonic tissue. Increased EGFR expression is linked with poor prognosis in colon cancer. Within the EGFR gene there are two functional polymorphisms of interest: a polymorphism located at codon 497 (HER-1 R497K) and a dinucleotide (CA)(n) repeat polymorphism located within intron 1. These germ-line polymorphisms of EGFR were analyzed in genomic DNA from 318 metastatic colon cancer patients, 177 males and 141 females, collected from 1992 to 2003. Gender-related survival differences were associated with the HER-1 R497K polymorphism (P(interaction) = 0.003). Females with the HER-1 497 Arg/Arg variant had better overall survival (OS) when compared with the Lys/Lys and/or Lys/Arg variants. In males the opposite was true. The EGFR dinucleotide (CA)(n) repeat also trended with a gender-related OS difference (P(interaction) = 0.11). Females with both short <20 (CA)(n) repeat alleles had better OS than those with any long >or=20 (CA)(n) repeats. In males the opposite was true. Combination analysis of the two polymorphisms taken together also revealed the same gender-related survival difference (P(interaction) = 0.002). These associations were observed using multivariable analysis. The two polymorphisms were not in linkage disequilibrium and are independent of one another. This study supports the role of functional EGFR polymorphisms as independent prognostic markers in metastatic colon cancer. As a prognostic factor, these variants had opposite prognostic implications based on gender.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

Emergence of novel and dominant acquired EGFR solvent-front mutations at Gly796 (G796S/R) together with C797S/R and L792F/H mutations in one EGFR (L858R/T790M) NSCLC patient who progressed on osimertinib.

PubMed

Ou, Sai-Hong Ignatius; Cui, Jean; Schrock, Alexa B; Goldberg, Michael E; Zhu, Viola W; Albacker, Lee; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M

2017-06-01

Acquired epidermal growth factor receptor (EGFR) resistance mutations to osimertinib are common, including the EGFR C797S that abolishes the covalent binding of osimertinib to EGFR. Here we report the emergence of novel EGFR solvent front mutations at Gly796 (G796S/R) in addition to a hinge pocket L792F/H mutations, and C797S/G all in cis with T790M in a single patient on progression on osimertinib as detected by plasma circulating tumor DNA (ctDNA) assay in the course of clinical care. A 69-year-old Caucasian female former light-smoker presented with stage IV EGFR L858R positive adenocarcinoma who developed EGFR T790M mutation after 8 month treatment of erlotinib. The patient was initiated on osimertinib with disease shrinkage after 2 months, but tumor regrowth was observed after 5 months of osimertinib treatment. Assay of plasma ctDNA at this time revealed these different secondary resistance mutations all in trans with each other including distinct mutations at the same codon producing different amino acid changes: G796S/R (mutant allele frequency [MAF]; 14.4%), C797S/G (MAF: 2.26%), L792F/H (MAF: 0.36%), and V802F (MAF: 0.40%), in addition to the pre-existing L858R (MAF:17.9%) and T790M (MAF:18.2%) but all in cis with T790M. The G796S/R mutations are homologous with known reported solvent front mutations in ALK G1202R, ROS1 G2032R, TrkA G595R and TrkC G623R, all of which are associated with acquired resistance to type I TKIs. In silico modeling revealed mutation at G796 interferes with osimertinib binding to the EGFR kinase domain at the phenyl aromatic ring position as this residue forms a narrow "hydrophobic sandwich" with L718, while L792F/H mutation interferes with osimertinib binding at the methoxyl group on the phenyl ring. Multiple resistance mutations at differing allele frequencies including novel EGFR solvent front mutations can emerge in a single patient with progression on osimertinib potentially due to tumor hetereogeneity and definitely present a

EGFR Amplification as a Target in Gastroesophageal Adenocarcinoma: Do Anti-EGFR Therapies Deserve a Second Chance?

PubMed

Strickler, John H

2018-06-01

Anti-EGFR therapies have failed to improve survival for unselected patients with metastatic gastroesophageal cancer, but in a subset of patients, EGFR amplification may predict treatment benefit. Maron and colleagues report the clinical activity of anti-EGFR therapies in a cohort of patients with EGFR -amplified metastatic gastroesophageal cancer and utilize serial blood and tumor tissue collection to identify molecular drivers of treatment sensitivity and resistance. Their insights offer a path to overcome technical limitations associated with EGFR amplification and facilitate molecularly targeted therapeutic strategies. Cancer Discov; 8(6); 679-81. ©2018 AACR See related article by Maron et al., p. 696 . ©2018 American Association for Cancer Research.

Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

PubMed Central

Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

2013-01-01

Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

PubMed Central

Kerner, Gerald S. M. A.; Schuuring, Ed; Sietsma, Johanna; Hiltermann, Thijo J. N.; Pieterman, Remge M.; de Leede, Gerard P. J.; van Putten, John W. G.; Liesker, Jeroen; Renkema, Tineke E. J.; van Hengel, Peter; Platteel, Inge; Timens, Wim; Groen, Harry J. M.

2013-01-01

Introduction In randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI. Patient and Methods Clinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis. Results Tumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively. Conclusion One out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival. PMID:23922984

Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes.

PubMed

Ogawa, T; Dobrinski, I; Avarbock, M R; Brinster, R L

1999-02-01

It was recently demonstrated that rat spermatogenesis can occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat. In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis would result. The hamster diverged at least 16 million years ago from the mouse and produces spermatozoa that are larger than, and have a shape distinctly different from, those of the mouse. In four separate experiments with a total of 13 recipient mice, hamster spermatogenesis was identified in the testes of each mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved hamster testis cells generated spermatogenesis in recipients. However, abnormalities were noted in hamster spermatids and acrosomes in seminiferous tubules of recipient mice. Hamster spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and heads and tails were separated. Thus, defects in spermiogenesis occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell. The generation of spermatogenesis from frozen hamster cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding has immediate application to valuable animals of many species, because the cells could be stored until suitable recipients are identified or culture techniques devised to expand the stem cell population.

Cancer stem cell-like population is preferentially suppressed by EGFR-TKIs in EGFR-mutated PC-9 tumor models.

PubMed

Yang, Fan; Li, Yang; Liu, Bin; You, Jiacong; Zhou, Qinghua

2018-01-01

Although the epidermal growth factor receptor (EGFR) and Wnt/β-catenin signaling systems synergistically regulate many essential developmental and regenerative processes in lung cancer, the mechanisms of their crosstalk remain poorly defined. Our study aimed to investigate an interaction between EGFR and the β-catenin signal. In this study, we described a potent activation of β-catenin by EGFR, which is dependent of the PtdIns3K/AKT pathway. We found EGF activated β-catenin signaling via phosphorylation of EGFR and AKT in EGFR-mutated PC-9 lung cancer cells. Meanwhile, EGFR tyrosine kinase inhibitors (EGFR-TKIs) regulated cancer stem-like cells (CSCs) by inhibiting autophosphorylation of EGFR and downstream signaling proteins, as well as β-catenin. Further, β-catenin depletion by RNA interference virtually eliminated cancer stem cell-like population in PC-9 cells in vitro. The nude mice transplantation model was also performed to confirm EGFR-TKIs strongly inhibited the β-catenin signal and decreased CSCs. Importantly, the reduction of CSCs that sorted out by side population (SP) cells significantly reduced the migration capability. Thus, our results improved the understanding of this process to provide insights into mechanisms of responding to EGFR-TKIs. Our discoveries raise an intriguing question of the role of β-catenin in EGFR-TKIs-treated cancer stem cell-like population(s) and its potential as a new therapeutic target for NSCLC in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg; Zhu Congju; Wong Yinling

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival,more » {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

PubMed

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem

Functional mutation analysis of EGFR family genes and corresponding lymph node metastases in head and neck squamous cell carcinoma.

PubMed

Hama, Takanori; Yuza, Yuki; Suda, Toshihito; Saito, Yoshimichi; Norizoe, Chihiro; Kato, Takakuni; Moriyama, Hiroshi; Urashima, Mitsuyoshi

2012-01-01

Tumors with certain mutations in the epidermal growth factor receptor (EGFR) family genes dramatically respond to EGFR inhibitors. Therefore, these mutations are important factors that influence disease progression and patient survival. We previously studied the mutation status of EGFR in patients with head and neck squamous cell carcinoma (HNSCC). However, the mutation status of lymph node metastases and the frequency of mutations in EGFR family genes have not been extensively studied. In this study, we sequenced the catalytic domains of the three other members of the EGFR family, HER2, HER3, and HER4 in 92 clinical samples of HNSCC. We identified a HER2 mutation (K716E) in one sample but no mutations were found in HER3 or HER4. Next to investigate the relationship between EGFR mutations and tumor metastasis, we compared the DNA sequences of the EGFR gene between the primary tumor and the lymph node metastasis in 31 clinical samples. Only one of the patients with an EGFR mutation in the primary HNSCC carried the same mutation (L858R) in the lymph node metastasis. Finally, we explored the tumorigenic potential of the EGFR mutations that we had previously identified and their sensitivity to two different EGFR tyrosine kinase inhibitors (CL-387785, OSI-420). Ba/F3 cells transformed with mutant EGFR genes were sensitive to treatment with lower concentrations of CL-387785 than of OSI-420. These results contribute to our understanding of the genetic basis of drug sensitivity and will help design drugs that specifically target different subtypes of HNSCC.

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells.

PubMed

Sathya, Shanmugaraj; Sudhagar, Selvaraj; Sarathkumar, Baskaran; Lakshmi, Baddireddi Subhadra

2014-01-24

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro. Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes. We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation. Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Purification processes of xenogeneic bone substitutes and their impact on tissue reactions and regeneration.

PubMed

Perić Kačarević, Zeljka; Kavehei, Faraz; Houshmand, Alireza; Franke, Jörg; Smeets, Ralf; Rimashevskiy, Denis; Wenisch, Sabine; Schnettler, Reinhard; Jung, Ole; Barbeck, Mike

2018-04-01

Xenogeneic bone substitute materials are widely used in oral implantology. Prior to their clinical use, purification of the former bone tissue has to be conducted to ensure the removal of immunogenic components and pathogens. Different physicochemical methods are applied for purification of the donor tissue, and temperature treatment is one of these methods. Differences in these methods and especially the application of different temperatures for purification may lead to different material characteristics, which may influence the tissue reactions to these materials and the related (bone) healing process. However, little is known about the different material characteristics and their influences on the healing process. Thus, the aim of this mini-review is to summarize the preparation processes and the related material characteristics, safety aspects, tissue reactions, resorbability and preclinical and clinical data of two widely used xenogeneic bone substitutes that mainly differ in the temperature treatment: sintered (cerabone ® ) and non-sintered (Bio-Oss ® ) bovine-bone materials. Based on the summarized data from the literature, a connection between the material-induced tissue reactions and the consequences for the healing processes are presented with the aim of translation into their clinical application.

A marked response to icotinib in a patient with large cell neuroendocrine carcinoma harboring an EGFR mutation: A case report.

PubMed

Wang, Yuehong; Shen, Yi Hong; Ma, Shanni; Zhou, Jianying

2015-09-01

The present study reports the case of an 84-year-old male with primary pulmonary large cell neuroendocrine carcinoma (LCNEC) harboring an epidermal growth factor receptor (EGFR) gene mutation that exhibited a long-lasting response to the EGFR-tyrosine kinase inhibitor (EGFR-TKI) icotinib. The patient had an extensive smoking history, a poor performance status, and presented with an irregular mass in the middle lobe of the right lung on computed tomography (CT) and an enlarged left supraclavicular lymph node on physical examination. Right middle lobe bronchial brushing during fiberoptic bronchoscopy identified poorly-differentiated cancer cells. The left supraclavicular lymph node was biopsied and a diagnosis of metastatic LCNEC was determined. Furthermore, an EGFR exon 19 deletion was identified by DNA sequencing. Following diagnosis, icotinib was administered at a dose of 125 mg three times a day. Chest CT scans were performed after 1 month of treatment, which indicated that the tumor was in partial remission. This marked response to icotinib lasted for 8 months. Thus, the present case illustrates the possibility of identifying EGFR mutations in LCNEC and indicates that EGFR-tyrosine kinase inhibitors may be an alternative treatment strategy for patients with LCNEC harboring activating EGFR mutations.

Sulforaphane attenuates EGFR signaling in NSCLC cells.

PubMed

Chen, Chi-Yuan; Yu, Zhu-Yun; Chuang, Yen-Shu; Huang, Rui-Mei; Wang, Tzu-Chien V

2015-06-03

EGFR, a receptor tyrosine kinase (RTK), is frequently overexpressed and mutated in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been widely used in the treatment of many cancers, including NSCLC. However, intrinsic and acquired resistance to TKI remains a common obstacle. One strategy that may help overcome EGFR-TKI resistance is to target EGFR for degradation. As EGFR is a client protein of heat-shock protein 90 (HSP90) and sulforaphane is known to functionally regulate HSP90, we hypothesized that sulforaphane could attenuate EGFR-related signaling and potentially be used to treat NSCLC. Our study revealed that sulforaphane displayed antitumor activity against NSCLC cells both in vitro and in vivo. The sensitivity of NSCLC cells to sulforaphane appeared to positively correlate with the inhibition of EGFR-related signaling, which was attributed to the increased proteasomal degradation of EGFR. Combined treatment of NSCLC cells with sulforaphane plus another HSP90 inhibitor (17-AAG) enhanced the inhibition of EGFR-related signaling both in vitro and in vivo. We have shown that sulforaphane is a novel inhibitory modulator of EGFR expression and is effective in inhibiting the tumor growth of EGFR-TKI-resistant NSCLC cells. Our findings suggest that sulforaphane should be further explored for its potential clinical applications against NSCLC.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode

PubMed Central

Chen, Cheng; Yu, Kailin; Zou, Fengming; Wang, Wenchao; Wang, Wei; Wu, Jiaxin; Liu, Juan; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shanchun; Yun, Cai-Hong; Liu, Jing; Liu, Qingsong

2017-01-01

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose. PMID:28407693

Antitumor Activity of Osimertinib, an Irreversible Mutant-Selective EGFR Tyrosine Kinase Inhibitor, in NSCLC Harboring EGFR Exon 20 Insertions.

PubMed

Floc'h, Nicolas; Martin, Matthew J; Riess, Jonathan W; Orme, Jonathan P; Staniszewska, Anna D; Ménard, Ludovic; Cuomo, Maria Emanuela; O'Neill, Daniel J; Ward, Richard A; Finlay, M Raymond V; McKerrecher, Darren; Cheng, Mingshan; Vang, Daniel P; Burich, Rebekah A; Keck, James G; Gandara, David R; Mack, Philip C; Cross, Darren A E

2018-05-01

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non-small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885-96. ©2018 AACR . ©2018 American Association for Cancer Research.

Clinical outcomes of EGFR-TKI treatment and genetic heterogeneity in lung adenocarcinoma patients with EGFR mutations on exons 19 and 21.

PubMed

Yu, Jiang-Yong; Yu, Si-Fan; Wang, Shu-Hang; Bai, Hua; Zhao, Jun; An, Tong-Tong; Duan, Jian-Chun; Wang, Jie

2016-03-21

Epidermal growth factor receptor (EGFR) mutations, including a known exon 19 deletion (19 del) and exon 21 L858R point mutation (L858R mutation), are strong predictors of the response to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment in lung adenocarcinoma. However, whether patients carrying EGFR 19 del and L858R mutations exhibit different responsiveness to EGFR-TKIs and what are the potential mechanism for this difference remain controversial. This study aimed to investigate the clinical outcomes of EGFR-TKI treatment in patients with EGFR 19 del and L858R mutations and explore the genetic heterogeneity of tumors with the two mutation subtypes. Of 1127 patients with advanced lung adenocarcinoma harboring EGFR 19 del or L858R mutations, 532 received EGFR-TKI treatment and were included in this study. EGFR 19 del and L858R mutations were detected by using denaturing high-performance liquid chromatography (DHPLC). T790M mutation, which is a common resistant mutation on exon 20 of EGFR, was detected by amplification refractory mutation system (ARMS). Next-generation sequencing (NGS) was used to explore the genetic heterogeneity of tumors with EGFR 19 del and L858R mutations. Of the 532 patients, 319 (60.0%) had EGFR 19 del, and 213 (40.0%) had L858R mutations. The patients with EGFR 19 del presented a significantly higher overall response rate (ORR) for EGFR-TKI treatment (55.2% vs. 43.7%, P = 0.017) and had a longer progression-free survival (PFS) after first-line EGFR-TKI treatment (14.4 vs. 11.4 months, P = 0.034) compared with those with L858R mutations. However, no statistically significant difference in overall survival (OS) was observed between the two groups of patients. T790M mutation status was analyzed in 88 patients before EGFR-TKI treatment and 134 after EGFR-TKI treatment, and there was no significant difference in the co-existence of T790M mutation with EGFR 19 del and L858R mutations before EGFR-TKI treatment (5.6% vs. 8.8%, P = 0.554) or after

Simultaneous Inhibition of EGFR and PI3K Enhances Radiosensitivity in Human Breast Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Ping; Zhang Qing; Torossian, Artour

2012-07-01

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells. Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used tomore » investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect. Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF{sub SF2}) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF{sub SF2} at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts. Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may

Apatinib enhances antitumour activity of EGFR-TKIs in non-small cell lung cancer with EGFR-TKI resistance.

PubMed

Li, Fang; Zhu, Tengjiao; Cao, Baoshan; Wang, Jiadong; Liang, Li

2017-10-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs)-rechallenged therapy for EGFR-mutant non-small cell lung cancer (NSCLC) patients who acquired resistance showed moderate efficacy. Considering the high interrelation between EGFR and vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR) pathways, we firstly evaluated EGFR-TKI combined with apatinib (a highly selective VEGFR2 inhibitor) in EGFR-TKI-resistant model and patients. Effects of apatinib, gefitinib and gefitinib plus apatinib were assessed on four NSCLC cell lines (A549 with wild-type EGFR, H1975 harbouring L858R and T790M, H1650 and HCC827 harbouring E746_A750 deletion) and xenograft model of acquired resistance that was established by injecting H1975 cells. Furthermore, we retrospectively evaluated EGFR-TKI rechallenge with apatinib in 16 patients. Gefitinib plus apatinib strengthened the effect of gefitinib and apatinib alone on the four NSCLC cell lines, and H1975 was the most susceptible one. Co-administration delayed the tumour growth than mono-therapy in the xenograft model and had better effect on inhibiting the activation of EGFR and VEGFR2 and expression of CD31 (an angiogenesis marker) and vascular endothelial growth factor A (an important pro-angiogenesis factor in the tumour microenvironment). Changes in protein expression of protein kinase B/mammalian target of rapamycin and extracellular signal-regulated kinase pathways demonstrated the potent inhibitory effect on the pro-survival signalling pathways by combined therapy. EGFR-TKI rechallenge with apatinib achieved a median progression-free survival of 4.60 months (95% confidence interval, 2.23-12.52 months) in the patients. Apatinib significantly potentiated the antitumour effect of gefitinib in NSCLC with T790M-related EGFR-TKI resistance both in vivo and vitro. EGFR-TKI rechallenge with apatinib might represent a new option for NSCLC with T790M or unknown resistance mechanism

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

PubMed

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-04-06

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients

PubMed Central

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-01-01

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection. PMID:29719623

Molecular alterations of EGFR and PIK3CA in uterine serous carcinoma.

PubMed

Hayes, Monica Prasad; Douglas, Wayne; Ellenson, Lora Hedrick

2009-06-01

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. 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. 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. 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

Are EGFR tyrosine kinase inhibitors effective in elderly patients with EGFR-mutated non-small cell lung cancer?

PubMed

Roviello, Giandomenico; Zanotti, Laura; Cappelletti, Maria Rosa; Gobbi, Angela; Dester, Martina; Paganini, Giovanni; Pacifico, Chiara; Generali, Daniele; Roudi, Raheleh

2018-02-01

EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib, gefitinib, and afatinib changed dramatically the history of metastatic non-small cell lung cancer (NSCLC) harbouring EGFR mutations. However, not enough data are available on the efficacy of these targeted drugs in elderly patients. The aim of this study is to analyse the available clinical data evaluating the efficacy of anti-EGFR therapies in elderly patients with advanced NSCLC carrying EGFR mutations. A literature-based meta-analysis of the results of randomized clinical trials was undertaken. Relevant publications from PubMed, the Cochrane Library, and abstracts from American Society of Clinical Oncology meetings were searched. Progression-free survival (PFS), as a measure of the efficacy of treatment, was the primary outcome investigated. The pooled analysis revealed an overall significant improvement in PFS (HR = 0.44, 95% CI 0.28-0.69; p = 0.0004) with the use of EGFR TKIs in EGFR-mutated NSCLC. The data stratification per age subgroups showed that EGFR TKIs were more effective in prolonging PFS in elderly patients, with HR 0.39 (p = 0.008), in comparison with young patients (HR = 0.48; p = 0.04). The results of this study suggest that EGFR TKIs have a significant effect in slowing down diseases progression in elderly patients with advanced NSCLC, therefore representing a valid therapeutic option in this age group.

Feasibility of cell-free circulating tumor DNA testing for lung cancer.

PubMed

Santarpia, Mariacarmela; Karachaliou, Niki; González-Cao, Maria; Altavilla, Giuseppe; Giovannetti, Elisa; Rosell, Rafael

2016-01-01

Tumor tissue genotyping is used routinely for lung cancer to identify specific targetable oncogenic alterations, including EGFR mutations and ALK rearrangements. However, tumor tissue from a single biopsy is often insufficient for molecular testing, may offer a limited evaluation because of tumor heterogeneity and can be difficult to obtain. Cell-free circulating tumor DNA has been widely investigated as a potential surrogate for tissue biopsy for noninvasive assessment of tumor-related genomic alterations. New techniques have improved EGFR mutations detection in ctDNA, thus supporting the use of this liquid biopsy for predicting response to EGFR tyrosine kinase inhibitors (TKIs) and monitoring the emergence of resistance. The serial evaluation of ctDNA during treatment is feasible and can be used to track tumor changes in real time and for a wide range of clinically useful applications.

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer

PubMed Central

2011-01-01

Background Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Methods Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. Results EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Conclusions Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series. PMID:21266046

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

PubMed

Peraldo-Neia, Caterina; Migliardi, Giorgia; Mello-Grand, Maurizia; Montemurro, Filippo; Segir, Raffaella; Pignochino, Ymera; Cavalloni, Giuliana; Torchio, Bruno; Mosso, Luciano; Chiorino, Giovanna; Aglietta, Massimo

2011-01-25

Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer

PubMed Central

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo, erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity. PMID:29312548

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

PubMed

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-12-12

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo , erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity.

Autophagosome-mediated EGFR down-regulation induced by the CK2 inhibitor enhances the efficacy of EGFR-TKI on EGFR-mutant lung cancer cells with resistance by T790M.

PubMed

So, Kwang Sup; Kim, Cheol Hyeon; Rho, Jin Kyung; Kim, Sun Ye; Choi, Yun Jung; Song, Joon Seon; Kim, Woo Sung; Choi, Chang Min; Chun, Young Jin; Lee, Jae Cheol

2014-01-01

Protein kinase CK2 has diverse functions promoting and maintaining cancer phenotypes. We investigated the effect of CK2 inhibition in lung cancer cells with T790M-mediated resistance to the EGFR-TK inhibitor. Resistant sublines of PC-9 to gefitinib (PC-9/GR) and erlotinib (PC-9/ER) were established by previous study, and T790M secondary mutation was found in both resistant sublines. A decrease of EGFR by siRNA treatment effectively controlled the growth of resistant cells, thus suggesting that they still have EGFR-dependency. CX-4945, a potent and selective CK2 inhibitor, induced autophagy in PC-9/GR and PC-9/ER, and which was supported by the induction of autophagic vacuoles and microtubule-associated protein 1 light chain 3 (LC3) expression, and the increase of punctate fluorescent signals in resistant cells pre-transfected with green fluorescent protein (GFP)-tagged LC3. However, the withdrawal of CX-4945 led to the recovery of cancer cells with autophagy. We found that the induction of autophagy by CX-4945 in both resistant cells was CK2 dependent by using small interfering RNA against CK2. The treatment with CX-4945 alone induced a minimal growth inhibition in resistant cells. However, combined treatment of CX-4945 and EGFR-TKI effectively inhibited cancer-cell proliferation and induced apoptosis. CX-4945 increased the translocation of EGFR from the cell surface into the autophagosome, subsequently leading to the decrease of EGFR while inhibition of autophagy by 3MA or Atg7-targeted siRNA pretreatment reduced the decrease of EGFR by CX-4945. Accordingly, apoptosis by a combination of CX-4945 and EGFR-TKI was suppressed by 3MA or Atg7-targeted siRNA pretreatment, thus suggesting that autophagosome-mediated EGFR down-regulation would have an important role regarding apoptotic cell death by EGFR-TKI. Combined treatment of the CK2 inhibitor and EGFR-TKI may be a promising strategy for overcoming T790M-mediated resistance.

PGE2/EP3/SRC signaling induces EGFR nuclear translocation and growth through EGFR ligands release in lung adenocarcinoma cells

PubMed Central

Bazzani, Lorenzo; Donnini, Sandra; Finetti, Federica; Christofori, Gerhard; Ziche, Marina

2017-01-01

Prostaglandin E2 (PGE2) interacts with tyrosine kinases receptor signaling in both tumor and stromal cells supporting tumor progression. Here we demonstrate that in non-small cell lung carcinoma (NSCLC) cells, A549 and GLC82, PGE2 promotes nuclear translocation of epidermal growth factor receptor (nEGFR), affects gene expression and induces cell growth. Indeed, cyclin D1, COX-2, iNOS and c-Myc mRNA levels are upregulated following PGE2 treatment. The nuclear localization sequence (NLS) of EGFR as well as its tyrosine kinase activity are required for the effect of PGE2 on nEGFR and downstream signaling activities. PGE2 binds its bona fide receptor EP3 which by activating SRC family kinases, induces ADAMs activation which, in turn, releases EGFR-ligands from the cell membrane and promotes nEGFR. Amphiregulin (AREG) and Epiregulin (EREG) appear to be involved in nEGFR promoted by the PGE2/EP3-SRC axis. Pharmacological inhibition or silencing of the PGE2/EP3/SRC-ADAMs signaling axis or EGFR ligands i.e. AREG and EREG expression abolishes nEGFR induced by PGE2. In conclusion, PGE2 induces NSCLC cell proliferation by EP3 receptor, SRC-ADAMs activation, EGFR ligands shedding and finally, phosphorylation and nEGFR. Since nuclear EGFR is a hallmark of cancer aggressiveness, our findings reveal a novel mechanism for the contribution of PGE2 to tumor progression. PMID:28415726

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

PubMed Central

Yasuda, Hiroyuki; Hamamoto, Junko; Oashi, Ayano; Ishioka, Kota; Arai, Daisuke; Nukaga, Shigenari; Miyawaki, Masayoshi; Kawada, Ichiro; Naoki, Katsuhiko; Costa, Daniel B.; Kobayashi, Susumu S.; Betsuyaku, Tomoko; Soejima, Kenzo

2015-01-01

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations. PMID:26515464

δ-Catenin Increases the Stability of EGFR by Decreasing c-Cbl Interaction and Enhances EGFR/Erk1/2 Signaling in Prostate Cancer.

PubMed

Shrestha, Nensi; Shrestha, Hridaya; Ryu, Taeyong; Kim, Hangun; Simkhada, Shishli; Cho, Young-Chang; Park, So-Yeon; Cho, Sayeon; Lee, Kwang-Youl; Lee, Jae-Hyuk; Kim, Kwonseop

2018-04-30

δ-Catenin, a member of the p120-catenin subfamily of armadillo proteins, reportedly increases during the late stage of prostate cancer. Our previous study demonstrates that δ-catenin increases the stability of EGFR in prostate cancer cell lines. However, the molecular mechanism behind δ-catenin-mediated enhanced stability of EGFR was not explored. In this study, we hypothesized that δ-catenin enhances the protein stability of EGFR by inhibiting its lysosomal degradation that is mediated by c-casitas b-lineage lymphoma (c-Cbl), a RING domain E3 ligase. c-Cbl monoubiquitinates EGFR and thus facilitates its internalization, followed by lysosomal degradation. We observed that δ-catenin plays a key role in EGFR stability and downstream signaling. δ-Catenin competes with c-Cbl for EGFR binding, which results in a reduction of binding between c-Cbl and EGFR and thus decreases the ubiquitination of EGFR. This in turn increases the expression of membrane bound EGFR and enhances EGFR/Erk1/2 signaling. Our findings add a new perspective on the role of δ-catenin in enhancing EGFR/Erk1/2 signaling-mediated prostate cancer.

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

PubMed

Johnson, Laura A; Scholler, John; Ohkuri, Takayuki; Kosaka, Akemi; Patel, Prachi R; McGettigan, Shannon E; Nace, Arben K; Dentchev, Tzvete; Thekkat, Pramod; Loew, Andreas; Boesteanu, Alina C; Cogdill, Alexandria P; Chen, Taylor; Fraietta, Joseph A; Kloss, Christopher C; Posey, Avery D; Engels, Boris; Singh, Reshma; Ezell, Tucker; Idamakanti, Neeraja; Ramones, Melissa H; Li, Na; Zhou, Li; Plesa, Gabriela; Seykora, John T; Okada, Hideho; June, Carl H; Brogdon, Jennifer L; Maus, Marcela V

2015-02-18

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376). Copyright © 2015, American Association for the Advancement of Science.

Inhibition of oncogenic epidermal growth factor receptor kinase triggers release of exosome-like extracellular vesicles and impacts their phosphoprotein and DNA content.

PubMed

Montermini, Laura; Meehan, Brian; Garnier, Delphine; Lee, Wan Jin; Lee, Tae Hoon; Guha, Abhijit; Al-Nedawi, Khalid; Rak, Janusz

2015-10-02

Cancer cells emit extracellular vesicles (EVs) containing unique molecular signatures. Here, we report that the oncogenic EGF receptor (EGFR) and its inhibitors reprogram phosphoproteomes and cargo of tumor cell-derived EVs. Thus, phosphorylated EGFR (P-EGFR) and several other receptor tyrosine kinases can be detected in EVs purified from plasma of tumor-bearing mice and from conditioned media of cultured cancer cells. Treatment of EGFR-driven tumor cells with second generation EGFR kinase inhibitors (EKIs), including CI-1033 and PF-00299804 but not with anti-EGFR antibody (Cetuximab) or etoposide, triggers a burst in emission of exosome-like EVs containing EGFR, P-EGFR, and genomic DNA (exo-gDNA). The EV release can be attenuated by treatment with inhibitors of exosome biogenesis (GW4869) and caspase pathways (ZVAD). The content of P-EGFR isoforms (Tyr-845, Tyr-1068, and Tyr-1173), ERK, and AKT varies between cells and their corresponding EVs and as a function of EKI treatment. Immunocapture experiments reveal the presence of EGFR and exo-gDNA within the same EV population following EKI treatment. These findings suggest that targeted agents may induce cancer cells to change the EV emission profiles reflective of drug-related therapeutic stress. We suggest that EV-based assays may serve as companion diagnostics for targeted anticancer agents. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Wenqing; Weng, Shuqiang; Zhang, Si

2013-05-10

Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits {sup 125}I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFRmore » in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using {sup 125}I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited {sup 125}I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation.« less

Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

PubMed Central

Liu, Bing; Li, Lei; Huang, Lixia; Li, Shaoli; Rao, Guanhua; Yu, Yang; Zhou, Yanbin

2017-01-01

Emerging evidence has indicated that circulating tumor DNA (ctDNA) from plasma could be used to analyze EGFR mutation status for NSCLC patients; however, due to the low level of ctDNA in plasma, highly sensitive approaches are required to detect low frequency mutations. In addition, the cutoff for the mutation abundance that can be detected in tumor tissue but cannot be detected in matched ctDNA is still unknown. To assess a highly sensitive method, we evaluated the use of digital PCR in the detection of EGFR mutations in tumor tissue from 47 advanced lung adenocarcinoma patients through comparison with NGS and ARMS. We determined the degree of concordance between tumor tissue DNA and paired ctDNA and analyzed the mutation abundance relationship between them. Digital PCR and Proton had a high sensitivity (96.00% vs. 100%) compared with that of ARMS in the detection of mutations in tumor tissue. Digital PCR outperformed Proton in identifying more low abundance mutations. The ctDNA detection rate of digital PCR was 87.50% in paired tumor tissue with a mutation abundance above 5% and 7.59% in paired tumor tissue with a mutation abundance below 5%. When the DNA mutation abundance of tumor tissue was above 3.81%, it could identify mutations in paired ctDNA with a high sensitivity. Digital PCR will help identify alternative methods for detecting low abundance mutations in tumor tissue DNA and plasma ctDNA. PMID:28978074

Triple Inhibition of EGFR, Met, and VEGF Suppresses Regrowth of HGF-Triggered, Erlotinib-Resistant Lung Cancer Harboring an EGFR Mutation

PubMed Central

Nakade, Junya; Takeuchi, Shinji; Nakagawa, Takayuki; Ishikawa, Daisuke; Sano, Takako; Nanjo, Shigeki; Yamada, Tadaaki; Ebi, Hiromichi; Zhao, Lu; Yasumoto, Kazuo; Matsumoto, Kunio; Yonekura, Kazuhiko

2014-01-01

Introduction: Met activation by gene amplification and its ligand, hepatocyte growth factor (HGF), imparts resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant lung cancer. We recently reported that Met activation by HGF stimulates the production of vascular endothelial growth factor (VEGF) and facilitates angiogenesis, which indicates that HGF induces EGFR-TKI resistance and angiogenesis. This study aimed to determine the effect of triple inhibition of EGFR, Met, and angiogenesis on HGF-triggered EGFR-TKI resistance in EGFR-mutant lung cancer. Methods: Three clinically approved drugs, erlotinib (an EGFR inhibitor), crizotinib (an inhibitor of anaplastic lymphoma kinase and Met), and bevacizumab (anti-VEGF antibody), and TAS-115, a novel dual TKI for Met and VEGF receptor 2, were used in this study. EGFR-mutant lung cancer cell lines PC-9, HCC827, and HGF-gene–transfected PC-9 (PC-9/HGF) cells were examined. Results: Crizotinib and TAS-115 inhibited Met phosphorylation and reversed erlotinib resistance and VEGF production triggered by HGF in PC-9 and HCC827 cells in vitro. Bevacizumab and TAS-115 inhibited angiogenesis in PC-9/HGF tumors in vivo. Moreover, the triplet erlotinib, crizotinib, and bevacizumab, or the doublet erlotinib and TAS-115 successfully inhibited PC-9/HGF tumor growth and delayed tumor regrowth associated with sustained tumor vasculature inhibition even after cessation of the treatment. Conclusion: These results suggest that triple inhibition of EGFR, HGF/Met, and VEGF/VEGF receptor 2, by either a triplet of clinical drugs or TAS-115 combined with erlotinib, may be useful for controlling progression of EGFR-mutant lung cancer by reversing EGFR-TKI resistance and for inhibiting angiogenesis. PMID:24828661

Protein Phosphorylation Profiling Using an In Situ Proximity Ligation Assay: Phosphorylation of AURKA-Elicited EGFR-Thr654 and EGFR-Ser1046 in Lung Cancer Cells

PubMed Central

Chen, Tzu-Chi; Liu, Yu-Wen; Huang, Yei-Hsuan; Yeh, Yi-Chen; Chou, Teh-Ying; Wu, Yu-Chung; Wu, Chun-Chi; Chen, Yi-Rong; Cheng, Hui-Chuan; Lu, Pei-Jung; Lai, Jin-Mei; Huang, Chi-Ying F.

2013-01-01

The epidermal growth factor receptor (EGFR), which is up-regulated in lung cancer, involves the activation of mitogenic signals and triggers multiple signaling cascades. To dissect these EGFR cascades, we used 14 different phospho-EGFR antibodies to quantify protein phosphorylation using an in situ proximity ligation assay (in situ PLA). Phosphorylation at EGFR-Thr654 and -Ser1046 was EGF-dependent in the wild-type (WT) receptor but EGF-independent in a cell line carrying the EGFR-L858R mutation. Using a ProtoAarray™ containing ∼5000 recombinant proteins on the protein chip, we found that AURKA interacted with the EGFR-L861Q mutant. Moreover, overexpression of EGFR could form a complex with AURKA, and the inhibitors of AURKA and EGFR decreased EGFR-Thr654 and -Ser1046 phosphorylation. Immunohistochemical staining of stage I lung adenocarcinoma tissues demonstrated a positive correlation between AURKA expression and phosphorylation of EGFR at Thr654 and Ser1046 in EGFR-mutant specimens, but not in EGFR-WT specimens. The interplay between EGFR and AURKA provides an explanation for the difference in EGF dependency between EGFR-WT and EGFR-mutant cells and may provide a new therapeutic strategy for lung cancer patients carrying EGFR mutations. PMID:23520446

Berberine inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors in gastric cancer.

PubMed

Wang, Junxiong; Yang, Shuo; Cai, Xiqiang; Dong, Jiaqiang; Chen, Zhangqian; Wang, Rui; Zhang, Song; Cao, Haichao; Lu, Di; Jin, Tong; Nie, Yongzhan; Hao, Jianyu; Fan, Daiming

2016-11-15

Cetuximab plus chemotherapy for advanced gastric cancer (GC) shows an active result in phase 2 trials. Unfortunately, Combination of cetuximab does not provide enough benefit to chemotherapy alone in phase 3 trials. Studies have demonstrated that berberine can suppress the activation of EGFR in tumors. In this study, we evaluated whether berberine could enhance the effects of EGFR-TKIs in GC cell lines and xenograft models. Our data suggest that berberine could effectively enhance the activity of erlotinib and cetuximab in vitro and in vivo. Berberine was found to inhibit growth in GC cell lines and to induce apoptosis. These effects were linked to inhibition of EGFR signaling activation, including the phosphorylation of STAT3. The expressions of Bcl-xL and Cyclind1 proteins were decreased, whereas the levels of cleavage of poly-ADP ribose polymerase (PARP) were considerably increased in the cell lines in response to berberine treatment. These results suggest a potential role for berberine in the treatment of GC, particularly in combination with EGFR-TKIs therapy. Berberine may be a competent therapeutic agent in GC where it can enhance the effects of EGFR inhibitors.

Berberine inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors in gastric cancer

PubMed Central

Wang, Junxiong; Yang, Shuo; Cai, Xiqiang; Dong, Jiaqiang; Chen, Zhangqian; Wang, Rui; Zhang, Song; Cao, Haichao; Lu, Di; Jin, Tong; Nie, Yongzhan; Hao, Jianyu; Fan, Daiming

2016-01-01

Cetuximab plus chemotherapy for advanced gastric cancer (GC) shows an active result in phase 2 trials. Unfortunately, Combination of cetuximab does not provide enough benefit to chemotherapy alone in phase 3 trials. Studies have demonstrated that berberine can suppress the activation of EGFR in tumors. In this study, we evaluated whether berberine could enhance the effects of EGFR-TKIs in GC cell lines and xenograft models. Our data suggest that berberine could effectively enhance the activity of erlotinib and cetuximab in vitro and in vivo. Berberine was found to inhibit growth in GC cell lines and to induce apoptosis. These effects were linked to inhibition of EGFR signaling activation, including the phosphorylation of STAT3. The expressions of Bcl-xL and Cyclind1 proteins were decreased, whereas the levels of cleavage of poly-ADP ribose polymerase (PARP) were considerably increased in the cell lines in response to berberine treatment. These results suggest a potential role for berberine in the treatment of GC, particularly in combination with EGFR-TKIs therapy. Berberine may be a competent therapeutic agent in GC where it can enhance the effects of EGFR inhibitors. PMID:27738318

Osimertinib As First-Line Treatment of EGFR Mutation-Positive Advanced Non-Small-Cell Lung Cancer.

PubMed

Ramalingam, Suresh S; Yang, James C-H; Lee, Chee Khoon; Kurata, Takayasu; Kim, Dong-Wan; John, Thomas; Nogami, Naoyuki; Ohe, Yuichiro; Mann, Helen; Rukazenkov, Yuri; Ghiorghiu, Serban; Stetson, Daniel; Markovets, Aleksandra; Barrett, J Carl; Thress, Kenneth S; Jänne, Pasi A

2018-03-20

Purpose The AURA study ( ClinicalTrials.gov identifier: NCT01802632) included two cohorts of treatment-naïve patients to examine clinical activity and safety of osimertinib (an epidermal growth factor receptor [EGFR] -tyrosine kinase inhibitor selective for EGFR-tyrosine kinase inhibitor sensitizing [ EGFRm] and EGFR T790M resistance mutations) as first-line treatment of EGFR-mutated advanced non-small-cell lung cancer (NSCLC). Patients and Methods Sixty treatment-naïve patients with locally advanced or metastatic EGFRm NSCLC received osimertinib 80 or 160 mg once daily (30 patients per cohort). End points included investigator-assessed objective response rate (ORR), progression-free survival (PFS), and safety evaluation. Plasma samples were collected at or after patients experienced disease progression, as defined by Response Evaluation Criteria in Solid Tumors (RECIST), to investigate osimertinib resistance mechanisms. Results At data cutoff (November 1, 2016), median follow-up was 19.1 months. Overall ORR was 67% (95% CI, 47% to 83%) in the 80-mg group, 87% (95% CI, 69% to 96%) in the 160-mg group, and 77% (95% CI, 64% to 87%) across doses. Median PFS time was 22.1 months (95% CI, 13.7 to 30.2 months) in the 80-mg group, 19.3 months (95% CI, 13.7 to 26.0 months) in the 160-mg group, and 20.5 months (95% CI, 15.0 to 26.1 months) across doses. Of 38 patients with postprogression plasma samples, 50% had no detectable circulating tumor DNA. Nine of 19 patients had putative resistance mechanisms, including amplification of MET (n = 1); amplification of EGFR and KRAS (n = 1); MEK1, KRAS, or PIK3CA mutation (n = 1 each); EGFR C797S mutation (n = 2); JAK2 mutation (n = 1); and HER2 exon 20 insertion (n = 1). Acquired EGFR T790M was not detected. Conclusion Osimertinib demonstrated a robust ORR and prolonged PFS in treatment-naïve patients with EGFRm advanced NSCLC. There was no evidence of acquired EGFR T790M mutation in postprogression plasma samples.

Emerging functions of the EGFR in cancer.

PubMed

Sigismund, Sara; Avanzato, Daniele; Lanzetti, Letizia

2018-01-01

The physiological function of the epidermal growth factor receptor (EGFR) is to regulate epithelial tissue development and homeostasis. In pathological settings, mostly in lung and breast cancer and in glioblastoma, the EGFR is a driver of tumorigenesis. Inappropriate activation of the EGFR in cancer mainly results from amplification and point mutations at the genomic locus, but transcriptional upregulation or ligand overproduction due to autocrine/paracrine mechanisms has also been described. Moreover, the EGFR is increasingly recognized as a biomarker of resistance in tumors, as its amplification or secondary mutations have been found to arise under drug pressure. This evidence, in addition to the prominent function that this receptor plays in normal epithelia, has prompted intense investigations into the role of the EGFR both at physiological and at pathological level. Despite the large body of knowledge obtained over the last two decades, previously unrecognized (herein defined as 'noncanonical') functions of the EGFR are currently emerging. Here, we will initially review the canonical ligand-induced EGFR signaling pathway, with particular emphasis to its regulation by endocytosis and subversion in human tumors. We will then focus on the most recent advances in uncovering noncanonical EGFR functions in stress-induced trafficking, autophagy, and energy metabolism, with a perspective on future therapeutic applications. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

PubMed

Strioga, Marius M; Darinskas, Adas; Pasukoniene, Vita; Mlynska, Agata; Ostapenko, Valerijus; Schijns, Virgil

2014-07-07

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens. Copyright © 2014. Published by Elsevier Ltd.

Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

PubMed Central

Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

2016-01-01

Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

Association between BIM deletion polymorphism and clinical outcome of EGFR-mutated NSCLC patient with EGFR-TKI therapy: A meta-analysis.

PubMed

Ma, Ji-Yong; Yan, Hai-Jun; Gu, Wei

2015-01-01

BIM deletion polymorphism was deemed to be associated with downregulation of BIM, resulting in a decreased apoptosis induced by epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutation-positive non-small cell lung cancer (NSCLC). However, accumulating evidences concerning the association between BIM deletion polymorphism and efficacy of EGFR-TKI and survival in EGFR-mutation-driven NSCLC patient reported contradictory results. A meta-analysis was conducted by combing six original eligible studies including 871 NSCLC patients. Our study showed that BIM deletion polymorphism was significantly associated with poor response to EGFR-TKI therapy in mutant EGFRNSCLC patients (P(h) = 0.309, P(z) = 0.001, OR = 0.39, 95% confidence interval (CI) = 0.23-0.67). Disease control rate (DCR) in mutant EGFRNSCLC patient with treatment of EGFR-TKI was significantly decreased in patients with BIM deletion polymorphism comparing to patients harbored BIM wild variant (P(h) = 0.583, P(Z) = 0.007, OR = 0.46, 95%CI = 0.25-0.85). EGFR mutation-derived NSCLC patient carrying BIM deletion polymorphism had a shorter progression-free survival (PFS; P(h) < 0.001, P(z) < 0.001, hazard ratio (HR) = 1.37, 95%CI = 1.09-1.71) and overall survival (OS; P(h) = 0.90, P(z) = 0.003, HR = 1.25, 95%CI = 1.08-1.45), than those harbored BIM wild variant. These results suggested that BIM deletion polymorphism might be a cause that contributes to primary EGFR-TKI resistance, and it could be used as a genetic predictor for EGFR-TKI outcome and an independent prognostic factor of EGFR mutation-driven NSCLC patient.

Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

PubMed

Takeuchi, Shinji; Wang, Wei; Li, Qi; Yamada, Tadaaki; Kita, Kenji; Donev, Ivan S; Nakamura, Takahiro; Matsumoto, Kunio; Shimizu, Eiji; Nishioka, Yasuhiko; Sone, Saburo; Nakagawa, Takayuki; Uenaka, Toshimitsu; Yano, Seiji

2012-09-01

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Vertical ridge augmentation using xenogenous bone blocks: a comparison between the flap and tunneling procedures.

PubMed

Xuan, Feng; Lee, Chun-Ui; Son, Jeong-Seog; Fang, Yiqin; Jeong, Seung-Mi; Choi, Byung-Ho

2014-09-01

Previous studies have shown that the subperiosteal tunneling procedure in vertical ridge augmentation accelerates healing after grafting and prevents graft exposure, with minor postoperative complications. It is conceivable that new bone formation would be greater with the tunneling procedure than with the flap procedure, because the former is minimally invasive. This hypothesis was tested in this study by comparing new bone formation between the flap and tunneling procedures after vertical ridge augmentation using xenogenous bone blocks in a canine mandible model. Two Bio-Oss blocks were placed on the edentulous ridge in each side of the mandibles of 6 mongrel dogs. The blocks in each side were randomly assigned to grafting with a flap procedure (flap group) or grafting with a tunneling procedure (tunneling group). The mean percentage of newly formed bone within the block was 15.3 ± 6.6% in the flap group and 46.6 ± 23.4% in the tunneling group. Based on data presented in this study, when a tunneling procedure is used to place xenogenous bone blocks for vertical ridge augmentation, bone formation in the graft sites is significantly greater than when a flap procedure is used. Copyright © 2014 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Graf regulates hematopoiesis through GEEC endocytosis of EGFR.

PubMed

Kim, Sungdae; Nahm, Minyeop; Kim, Najin; Kwon, Yumi; Kim, Joohyung; Choi, Sukwoo; Choi, Eun Young; Shim, Jiwon; Lee, Cheolju; Lee, Seungbok

2017-11-15

GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. We show that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. We also find that loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on our findings, we propose a model in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation. © 2017. Published by The Company of Biologists Ltd.

EGFR-targeted therapies in the post-genomic era.

PubMed

Xu, Mary Jue; Johnson, Daniel E; Grandis, Jennifer R

2017-09-01

Over 90% of head and neck cancers overexpress the epidermal growth factor receptor (EGFR). In diverse tumor types, EGFR overexpression has been associated with poorer prognosis and outcomes. Therapies targeting EGFR include monoclonal antibodies, tyrosine kinase inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, and antisense gene therapy. Few EGFR-targeted therapeutics are approved for clinical use. The monoclonal antibody cetuximab is a Food and Drug Administration (FDA)-approved EGFR-targeted therapy, yet has exhibited modest benefit in clinical trials. The humanized monoclonal antibody nimotuzumab is also approved for head and neck cancers in Cuba, Argentina, Colombia, Peru, India, Ukraine, Ivory Coast, and Gabon in addition to nasopharyngeal cancers in China. Few other EGFR-targeted therapeutics for head and neck cancers have led to as significant responses as seen in lung carcinomas, for instance. Recent genome sequencing of head and neck tumors has helped identify patient subgroups with improved response to EGFR inhibitors, for example, cetuximab in patients with the KRAS-variant and the tyrosine kinase inhibitor erlotinib for tumors harboring MAPK1 E322K mutations. Genome sequencing has furthermore broadened our understanding of dysregulated pathways, holding the potential to enhance the benefit derived from therapies targeting EGFR.

Identification of somatic mutations in EGFR/KRAS/ALK-negative lung adenocarcinoma in never-smokers

PubMed Central

2014-01-01

Background Lung adenocarcinoma is a highly heterogeneous disease with various etiologies, prognoses, and responses to therapy. Although genome-scale characterization of lung adenocarcinoma has been performed, a comprehensive somatic mutation analysis of EGFR/KRAS/ALK-negative lung adenocarcinoma in never-smokers has not been conducted. Methods We analyzed whole exome sequencing data from 16 EGFR/KRAS/ALK-negative lung adenocarcinomas and additional 54 tumors in two expansion cohort sets. Candidate loci were validated by target capture and Sanger sequencing. Gene set analysis was performed using Ingenuity Pathway Analysis. Results We identified 27 genes potentially implicated in the pathogenesis of lung adenocarcinoma. These included targetable genes involved in PI3K/mTOR signaling (TSC1, PIK3CA, AKT2) and receptor tyrosine kinase signaling (ERBB4) and genes not previously highlighted in lung adenocarcinomas, such as SETD2 and PBRM1 (chromatin remodeling), CHEK2 and CDC27 (cell cycle), CUL3 and SOD2 (oxidative stress), and CSMD3 and TFG (immune response). In the expansion cohort (N = 70), TP53 was the most frequently altered gene (11%), followed by SETD2 (6%), CSMD3 (6%), ERBB2 (6%), and CDH10 (4%). In pathway analysis, the majority of altered genes were involved in cell cycle/DNA repair (P <0.001) and cAMP-dependent protein kinase signaling (P <0.001). Conclusions The genomic makeup of EGFR/KRAS/ALK-negative lung adenocarcinomas in never-smokers is remarkably diverse. Genes involved in cell cycle regulation/DNA repair are implicated in tumorigenesis and represent potential therapeutic targets. PMID:24576404

[Application of the xenogenic acellular dermal matrix membrane application used in the postoperative tissue shortage repair].

PubMed

Bai, Yanxia; Yan, Liying; Zhang, Shaoqiang; Shao, Yuan; Yao, Xiaobao; Li, Honghui; Zhao, Ruimin; Zhao, Qian; Zhang, Pengfei; Yang, Qi

2014-09-01

To observe the short-term and long-term curative effect of the xenogenic acellular dermal matrix membrane (or joint muscle flap transfer) application used in the 82 cases postoperative tissue shortage repair that after the head neck carcinoma resection. To held the 82 cases head neck carcinoma postoperative mucosa shortage repaired after resection by the xenogenic acellular dermal matrix membrane (or joint muscle flap transfer), 65 cases mucosa shortage wound be directly covered by the repair membrane and the other 17 cases mucosa shortage wound be repaired by the tranfered muscle tissue flap with the repair membrane covered; 53 cases underwent additional postoperative radiotherapy between 2-4 weeks and follow-up in 1, 3, 6, 12, 18, 24, 30, 36, 48, 60 months and observed the operation site repair process through the electronic laryngoscope, observed the patients respiration, swallow, phonation function. Seventy-seven cases patients operation incision reached I phase healing standard, another 5 cases patients operation incision reached II phase healing standard because of the wound infection and fully-recovered through the local wound drainage,dressing process. All the patients tracheal cannula,the stomach tube be extubated successfully and without the local cicatricial constriction occurred. Seventy-eight cases follow up period reached 1 year including 53 cases who underwent postoperative radiotherapy, 49 cases follow up period reached 3 years including 32 cases who underwent postoperative radiotherapy, 14 cases follow up period reached 5 years including 12 cases who underwent postoperative radiotherapy. The patients with static local lesions discovered no reaction such as exclusion, allergy. The application of xenogenic acellular dermal matrix membrane (or joint muscle flap transfer used in in the postoperative tissue shortage repair that after the head neck carcinoma resection have several advantage such as comparatively easily implementation, operation safety

Usefulness of circulating free DNA for monitoring epidermal growth factor receptor mutations in advanced non-small cell lung cancer patients: a case report

PubMed Central

Gonzalez-Cao, Maria; Ramirez, Santiago Viteri; Ariza, Nuria Jordana; Balada, Ariadna; Garzón, Mónica; Teixidó, Cristina; Karachaliou, Niki; Morales-Espinosa, Daniela; Molina-Vila, Miguel Ángel; Rosell, Rafael

2016-01-01

Genomic analysis of circulating tumor DNA (ctDNA) released from cancer cells into the bloodstream has been proposed as a useful method to capture dynamic changes during the course of the disease. In particular, the ability to monitor epidermal growth factor receptor (EGFR) mutation status in cell-free circulating DNA (cfDNA) isolated from advanced non-small cell lung cancer (NSCLC) patients EGFR can help to the correct management of the disease and overcome the challenges associated with tumor heterogeneity and insufficient biopsied material to perform key molecular diagnosis. Here, we report a case of long term monitorization of EGFR mutation status in cfDNA from peripheral blood in an NSCLC patient in, with excellent correlation with clinical evolution. PMID:27826535

Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

PubMed Central

2015-01-01

Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

Gallic acid abolishes the EGFR/Src/Akt/Erk-mediated expression of matrix metalloproteinase-9 in MCF-7 breast cancer cells.

PubMed

Chen, Ying-Jung; Lin, Ku-Nan; Jhang, Li-Mei; Huang, Chia-Hui; Lee, Yuan-Chin; Chang, Long-Sen

2016-05-25

Several studies have revealed that natural compounds are valuable resources to develop novel agents against dysregulation of the EGF/EGFR-mediated matrix metalloproteinase-9 (MMP-9) expression in cancer cells. In view of the findings that EGF/EGFR-mediated MMP-9 expression is closely related to invasion and metastasis of breast cancer. To determine the beneficial effects of gallic acid on the suppression of breast cancer metastasis, we explored the effect of gallic acid on MMP-9 expression in EGF-treated MCF-7 breast cancer cells. Treatment with EGF up-regulated MMP-9 mRNA and protein levels in MCF-7 cells. EGF treatment induced phosphorylation of EGFR and elicited Src activation, subsequently promoting Akt/NFκB (p65) and ERK/c-Jun phosphorylation in MCF-7 cells. Activation of Akt/p65 and ERK/c-Jun was responsible for the MMP-9 up-regulation in EGF-treated cells. Gallic acid repressed the EGF-induced activation of EGFR and Src; furthermore, inactivation of Akt/p65 and ERK/c-Jun was a result of the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. Over-expression of constitutively active Akt and MEK1 or over-expression of constitutively active Src eradicated the inhibitory effect of gallic acid on the EGF-induced MMP-9 up-regulation. A chromosome conformation capture assay showed that EGF induced a chromosomal loop formation in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun activation. Treatment with gallic acid, EGFR inhibitor, or Src inhibitor reduced DNA looping. Taken together, our data suggest that gallic acid inhibits the activation of EGFR/Src-mediated Akt and ERK, leading to reduced levels of p65/c-Jun-mediated DNA looping and thus inhibiting MMP-9 expression in EGF-treated MCF-7 cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

PubMed

Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

2016-06-01

Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.

DIFFERENTIAL EFFECTS OF SPECIFIC ANTISERA ON THE REJECTION OF ALLOGENIC AND XENOGENIC SKIN GRAFTS BY SUBLETHALLY X-IRRADIATED MICE

DTIC Science & Technology

an allogenic skin graft can be significantly inhibited, in sublethally irrsdiated mice, by specific antisera, while the first-set response to a...xenogenic skin graft remains resistant to similar treatient. Specific antisera had no effect upon a pre-existing second-set response. The significance of these data is discussed.

First-line therapy for advanced non-small cell lung cancer with activating EGFR mutation: is combined EGFR-TKIs and chemotherapy a better choice?

PubMed

Wang, Shuyun; Gao, Aiqin; Liu, Jie; Sun, Yuping

2018-03-01

As the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation, EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have significantly improved the median progression-free survival (PFS) up to 18.9 months. However, almost all patients eventually develop acquired resistance to EGFR-TKIs, which limits the first-line PFS. To overcome the resistance and improve overall survival, researchers have tried to identify the resistance mechanisms and develop new treatment strategies, among which a combination of EGFR-TKIs and cytotoxic chemotherapy is one of the hotspots. The data from preclinical and clinical studies on combined EGFR-TKIs and chemotherapy have shown very interesting results. Here, we reviewed the available preclinical and clinical studies on first-line EGFR-TKIs-chemotherapy combination in patients with advanced NSCLC harboring activating EGFR mutation, aiming to provide evidences for more potential choices and shed light on clinical treatment.

Sialylation of EGFR by the ST6Gal-I sialyltransferase promotes EGFR activation and resistance to gefitinib-mediated cell death.

PubMed

Britain, Colleen M; Holdbrooks, Andrew T; Anderson, Joshua C; Willey, Christopher D; Bellis, Susan L

2018-02-05

The ST6Gal-I sialyltransferase is upregulated in numerous cancers, and high expression of this enzyme correlates with poor patient prognosis in various malignancies, including ovarian cancer. Through its sialylation of a select cohort of cell surface receptors, ST6Gal-I modulates cell signaling to promote tumor cell survival. The goal of the present study was to investigate the influence of ST6Gal-I on another important receptor that controls cancer cell behavior, EGFR. Additionally, the effect of ST6Gal-I on cancer cells treated with the common EGFR inhibitor, gefitinib, was evaluated. Using the OV4 ovarian cancer cell line, which lacks endogenous ST6Gal-I expression, a kinomics assay revealed that cells with forced overexpression of ST6Gal-I exhibited increased global tyrosine kinase activity, a finding confirmed by immunoblotting whole cell lysates with an anti-phosphotyrosine antibody. Interestingly, the kinomics assay suggested that one of the most highly activated tyrosine kinases in ST6Gal-I-overexpressing OV4 cells was EGFR. Based on these findings, additional analyses were performed to investigate the effect of ST6Gal-I on EGFR activation. To this end, we utilized, in addition to OV4 cells, the SKOV3 ovarian cancer cell line, engineered with both ST6Gal-I overexpression and knockdown, as well as the BxPC3 pancreatic cancer cell line with knockdown of ST6Gal-I. In all three cell lines, we determined that EGFR is a substrate of ST6Gal-I, and that the sialylation status of EGFR directly correlates with ST6Gal-I expression. Cells with differential ST6Gal-I expression were subsequently evaluated for EGFR tyrosine phosphorylation. Cells with high ST6Gal-I expression were found to have elevated levels of basal and EGF-induced EGFR activation. Conversely, knockdown of ST6Gal-I greatly attenuated EGFR activation, both basally and post EGF treatment. Finally, to illustrate the functional importance of ST6Gal-I in regulating EGFR-dependent survival, cells were

EGFR TKIs plus WBRT Demonstrated No Survival Benefit Other Than That of TKIs Alone in Patients with NSCLC and EGFR Mutation and Brain Metastases.

PubMed

Jiang, Tao; Su, Chunxia; Li, Xuefei; Zhao, Chao; Zhou, Fei; Ren, Shengxiang; Zhou, Caicun; Zhang, Jun

2016-10-01

Whether EGFR tyrosine kinase inhibitors (TKIs) plus whole brain radiation therapy (WBRT) provide a better survival benefit than EGFR TKIs alone remains undetermined in patients with NSCLC with EGFR mutation and brain metastases (BMs). A total of 230 patients with NSCLC with EGFR mutation and BM were identified. Within this group, 116 patients received EGFR TKIs alone (as first-line therapy in 91 cases) and 51 patients received EGFR TKIs plus WBRT therapy (as first-line treatment in 30 cases). Compared with TKIs alone, EGFR TKIs plus WBRT had no superior intracranial progression-free survival (PFS) (6.9 versus 7.4 months [p = 0.232]) and systemic PFS (7.5 versus 7.9 months [p = 0.546]) but were associated with worse overall survival (OS) (21.6 versus 26.4 months [p = 0.049]) in NSCLC with EGFR mutation and BM. Chemotherapy plus WBRT was shown to have an intracranial PFS (5.2 versus 5.9 months [p = 0.339]) and OS (10.5 versus 11.0 months [p = 0.977]) similar to those with chemotherapy alone in patients with EGFR of unknown or wild-type status. In multivariate analysis, EGFR mutation was found to be an independent risk factor for BM (hazard ratio = 1.476, p = 0.039) and also a significant independent prognostic factor for OS in patients with NSCLC with BM (hazard ratio = 0.601, p = 0.028). The addition of WBRT to EGFR TKIs did not appear to have survival benefit superior to that of EGFR TKIs alone in with EGFR-mutant NSCLC with BM. WBRT also did not bring additional benefit to chemotherapy in patients with BM and EGFR of wild-type or unknown status. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Zhiwei; Cui, Binbin; Jin, Yinghu

2011-08-12

Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study,more » using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.« less

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT

PubMed Central

Choudhary, Kumari Sonal; Rohatgi, Neha; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

2016-01-01

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend. PMID:27253373

EGFR Signal-Network Reconstruction Demonstrates Metabolic Crosstalk in EMT.

PubMed

Choudhary, Kumari Sonal; Rohatgi, Neha; Halldorsson, Skarphedinn; Briem, Eirikur; Gudjonsson, Thorarinn; Gudmundsson, Steinn; Rolfsson, Ottar

2016-06-01

Epithelial to mesenchymal transition (EMT) is an important event during development and cancer metastasis. There is limited understanding of the metabolic alterations that give rise to and take place during EMT. Dysregulation of signalling pathways that impact metabolism, including epidermal growth factor receptor (EGFR), are however a hallmark of EMT and metastasis. In this study, we report the investigation into EGFR signalling and metabolic crosstalk of EMT through constraint-based modelling and analysis of the breast epithelial EMT cell model D492 and its mesenchymal counterpart D492M. We built an EGFR signalling network for EMT based on stoichiometric coefficients and constrained the network with gene expression data to build epithelial (EGFR_E) and mesenchymal (EGFR_M) networks. Metabolic alterations arising from differential expression of EGFR genes was derived from a literature review of AKT regulated metabolic genes. Signaling flux differences between EGFR_E and EGFR_M models subsequently allowed metabolism in D492 and D492M cells to be assessed. Higher flux within AKT pathway in the D492 cells compared to D492M suggested higher glycolytic activity in D492 that we confirmed experimentally through measurements of glucose uptake and lactate secretion rates. The signaling genes from the AKT, RAS/MAPK and CaM pathways were predicted to revert D492M to D492 phenotype. Follow-up analysis of EGFR signaling metabolic crosstalk in three additional breast epithelial cell lines highlighted variability in in vitro cell models of EMT. This study shows that the metabolic phenotype may be predicted by in silico analyses of gene expression data of EGFR signaling genes, but this phenomenon is cell-specific and does not follow a simple trend.

Third generation EGFR TKIs: current data and future directions.

PubMed

Tan, Chee-Seng; Kumarakulasinghe, Nesaretnam Barr; Huang, Yi-Qing; Ang, Yvonne Li En; Choo, Joan Rou-En; Goh, Boon-Cher; Soo, Ross A

2018-02-19

Acquired T790 M mutation is the commonest cause of resistance for advanced non-small cell lung cancer (NSCLC) epidermal growth factor receptor (EGFR) mutant patients who had progressed after first line EGFR TKI (tyrosine kinase inhibitor). Several third generation EGFR TKIs which are EGFR mutant selective and wild-type (WT) sparing were developed to treat these patients with T790 M acquired resistant mutation. Osimertinib is one of the third generation EGFR TKIs and is currently the most advanced in clinical development. Unfortunately, despite good initial response, patients who was treated with third generation EGFR TKI would develop acquired resistance and several mechanisms had been identified and the commonest being C797S mutation at exon 20. Several novel treatment options were being developed for patients who had progressed on third generation EGFR TKI but they are still in the early phase of development. Osimertinib under FLAURA study had been shown to have better progression-free survival over first generation EGFR TKI in the first line setting and likely will become the new standard of care.

Mutation analysis of the EGFR pathway genes, EGFR, RAS, PIK3CA, BRAF, and AKT1, in salivary gland adenoid cystic carcinoma.

PubMed

Saida, Kosuke; Murase, Takayuki; Ito, Mayuko; Fujii, Kana; Takino, Hisashi; Masaki, Ayako; Kawakita, Daisuke; Ijichi, Kei; Tada, Yuichiro; Kusafuka, Kimihide; Iida, Yoshiyuki; Onitsuka, Tetsuro; Yatabe, Yasushi; Hanai, Nobuhiro; Hasegawa, Yasuhisa; Shinomiya, Hitomi; Nibu, Ken-Ichi; Shimozato, Kazuo; Inagaki, Hiroshi

2018-03-30

Adenoid cystic carcinoma (AdCC), one of the most common salivary gland carcinomas, usually has a fatal outcome. Epidermal growth factor receptor (EGFR) pathway gene mutations are important in predicting a patient's prognosis and estimating the efficacy of molecular therapy targeting the EGFR pathway. In this study of salivary gland AdCC (SAdCC), we looked for gene mutations in EGFR, RAS family ( KRAS, HRAS, and NRAS ), PIK3CA, BRAF, and AKT1 , using a highly sensitive single-base extension multiplex assay, SNaPshot. Out of 70 cases, EGFR pathway missense mutations were found in 13 (18.6%): RAS mutations in 10 (14.3%), EGFR in one (1.4%), and PIK3CA in 5 (7.1%). None of the cases showed an EGFR deletion by direct sequencing. Concurrent gene mutations were found in three cases (4.3%). EGFR pathway mutations were significantly associated with a shorter disease-free ( p = 0.011) and overall survival ( p = 0.049) and RAS mutations were as well; ( p = 0.010) and ( p = 0.024), respectively. The gene fusion status as determined by a FISH assay had no significant association with mutations of the genes involved in the EGFR pathway. In conclusion, EGFR pathway mutations, especially RAS mutations, may be frequent in SAdCC, and associated with a poor prognosis for the patient.

Stress-induced EGFR trafficking: mechanisms, functions, and therapeutic implications

PubMed Central

Tan, Xiaojun; Lambert, Paul F.; Rapraeger, Alan C.; Anderson, Richard A.

2016-01-01

Epidermal growth factor receptor (EGFR) has fundamental roles in normal physiology and in cancer, making it a rational target for cancer therapy. Surprisingly, however, inhibitors that target canonical, ligand-stimulated EGFR signaling have proven to be largely ineffective in treating many EGFR-dependent cancers. Recent evidence indicates that both intrinsic and therapy-induced cellular stress triggers robust, non-canonical pathways of ligand-independent EGFR trafficking and signaling, which provides cancer cells with a survival advantage and resistance to therapeutics. Here we review the mechanistic regulation of non-canonical EGFR trafficking and signaling, the pathological and therapeutic stresses that activate it, and discuss the implications of this pathway in clinical treatment of EGFR-overexpressing cancers. PMID:26827089

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR.

PubMed

Watanabe, Masaru; Kawaguchi, Tomoya; Isa, Shun-Ichi; Ando, Masahiko; Tamiya, Akihiro; Kubo, Akihito; Saka, Hideo; Takeo, Sadanori; Adachi, Hirofumi; Tagawa, Tsutomu; Kawashima, Osamu; Yamashita, Motohiro; Kataoka, Kazuhiko; Ichinose, Yukito; Takeuchi, Yukiyasu; Watanabe, Katsuya; Matsumura, Akihide; Koh, Yasuhiro

2017-07-01

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R 2 ) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M). Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Surmounting the resistance against EGFR inhibitors through the development of thieno[2,3-d]pyrimidine-based dual EGFR/HER2 inhibitors.

PubMed

Milik, Sandra N; Abdel-Aziz, Amal Kamal; Lasheen, Deena S; Serya, Rabah A T; Minucci, Saverio; Abouzid, Khaled A M

2018-06-06

In light of the emergence of resistance against the currently available EGFR inhibitors, our study focuses on tackling this problem through the development of dual EGFR/HER2 inhibitors with improved enzymatic affinities. Guided by the binding mode of the marketed dual EGFR/HER2 inhibitor, Lapatinib, we proposed the design of dual EGFR/HER2 inhibitors based on the 6-phenylthieno[2,3-d]pyrimidine as a core scaffold and hinge binder. After two cycles of screening aiming to identify the optimum aniline headgroup and solubilizing group, we eventually identified 27b as a dual EGFR/HER2 inhibitor with IC 50 values of 91.7 nM and 1.2 μM, respectively. Notably, 27b dramatically reduced the viability of various patient-derived cancer cells preferentially overexpressing EGFR/HER2 (A431, MDA-MBA-361 and SKBr3 with IC 50 values of 1.45, 3.5 and 4.83 μM, respectively). Additionally, 27b efficiently thwarted the proliferation of lapatinib-resistant human non-small lung carcinoma (NCI-H1975) cells, harboring T790 M mutation, with IC 50 of 4.2 μM. Consistently, 27b significantly blocked EGF-induced EGFR activation and inactivated its downstream AKT/mTOR/S6 signalling pathway triggering apoptotic cell death in NCI-H1975 cells. The present study presents a promising candidate for further design and development of novel EGFR/HER2 inhibitors capable of overcoming EGFR TKIs resistance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Epiregulin and EGFR interactions are involved in pain processing

PubMed Central

Martin, Loren J.; Smith, Shad B.; Khoutorsky, Arkady; Magnussen, Claire A.; Samoshkin, Alexander; Sorge, Robert E.; Cho, Chulmin; Yosefpour, Noosha; Sivaselvachandran, Sivaani; Tohyama, Sarasa; Cole, Tiffany; Khuong, Thang M.; Mir, Ellen; Gibson, Dustin G.; Wieskopf, Jeffrey S.; Sotocinal, Susana G.; Austin, Jean Sebastien; Meloto, Carolina B.; Gitt, Joseph H.; Sonenberg, Nahum; Greenspan, Joel D.; Fillingim, Roger B.; Slade, Gary D.; Knott, Charles; Dubner, Ronald; Nackley, Andrea G.; Ribeiro-da-Silva, Alfredo; Neely, G. Gregory; Maixner, William; Zaykin, Dmitri V.; Mogil, Jeffrey S.; Diatchenko, Luda

2017-01-01

The EGFR belongs to the well-studied ErbB family of receptor tyrosine kinases. EGFR is activated by numerous endogenous ligands that promote cellular growth, proliferation, and tissue regeneration. In the present study, we have demonstrated a role for EGFR and its natural ligand, epiregulin (EREG), in pain processing. We show that inhibition of EGFR with clinically available compounds strongly reduced nocifensive behavior in mouse models of inflammatory and chronic pain. EREG-mediated activation of EGFR enhanced nociception through a mechanism involving the PI3K/AKT/mTOR pathway and matrix metalloproteinase-9. Moreover, EREG application potentiated capsaicin-induced calcium influx in a subset of sensory neurons. Both the EGFR and EREG genes displayed a genetic association with the development of chronic pain in several clinical cohorts of temporomandibular disorder. Thus, EGFR and EREG may be suitable therapeutic targets for persistent pain conditions. PMID:28783046

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

PubMed Central

McFadden, David G.; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K.; Song, Xiaoling; Pirun, Mono; Santiago, Philip M.; Kim-Kiselak, Caroline; Platt, James T.; Lee, Emily; Hodges, Emily; Rosebrock, Adam P.; Bronson, Roderick T.; Socci, Nicholas D.; Hannon, Gregory J.; Jacks, Tyler; Varmus, Harold

2016-01-01

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity. PMID:27702896

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma.

PubMed

McFadden, David G; Politi, Katerina; Bhutkar, Arjun; Chen, Frances K; Song, Xiaoling; Pirun, Mono; Santiago, Philip M; Kim-Kiselak, Caroline; Platt, James T; Lee, Emily; Hodges, Emily; Rosebrock, Adam P; Bronson, Roderick T; Socci, Nicholas D; Hannon, Gregory J; Jacks, Tyler; Varmus, Harold

2016-10-18

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

The impact of common and rare EGFR mutations in response to EGFR tyrosine kinase inhibitors and platinum-based chemotherapy in patients with non-small cell lung cancer.

PubMed

Arrieta, Oscar; Cardona, Andrés Felipe; Corrales, Luis; Campos-Parra, Alma Delia; Sánchez-Reyes, Roberto; Amieva-Rivera, Eduardo; Rodríguez, July; Vargas, Carlos; Carranza, Hernán; Otero, Jorge; Karachaliou, Nikki; Astudillo, Horacio; Rosell, Rafael

2015-02-01

In non-small cell lung cancer (NSCLC), the association between common EGFR mutations (Del EX19/L858R) with EGFR tyrosine kinase inhibitors (EGFR-TKIs) has been well established. However, this has not been investigated for rare EGFR mutations or their impact on treatment response and outcome to EGFR TKIs (primary objective) and chemotherapy (secondary objective). In an observational prospective cohort, we analyzed 188 NSCLC patients from Mexico, Colombia and Costa Rica with EGFR mutations. As a first line of treatment, 66.5% received platinum-based chemotherapy. All patients received TKIs in first-line treatment or after progression to chemotherapy. The clinical-pathological characteristics as well as the f of common and rare EGFR mutations associated with treatment response were analyzed. Of all patients, 79.5% had common and 20.5% had rare EGFR mutations. Lepidic and acinar adenocarcinomas were associated with common EGFR mutations (p=0.010). Patients with common EGFR mutations had higher response rates to EGFR-TKIs than those who had rare EGFR mutations (63.8 vs 32.4%, p<0.001). Women had increased progression-free survival (PFS) to EGFR-TKIs than men (16.4 vs 9.5 months, p=0.02). The median PFS and overall survival (OS) were better in patients with common EGFR mutations (15.5 vs 3.9 months, p<0.001; and 37.3 vs 17.4 months, p<0.001) respectively. Our findings suggested that only patients with rare EGFR mutations could receive platinum-based chemotherapy as a first-line treatment, due to their low response rates and short PFS in response to EGFR-TKIs. Consequently, EGFR-TKIs could be reserved as a second- or third-line treatment. In patients with EGFR mutations, women have better PFS to EGFR-TKIs than men, and rare EGFR mutations are more frequent in high grade adenocarcinomas than in low grade tumors. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Processed xenogenic cartilage as innovative biomatrix for cartilage tissue engineering: effects on chondrocyte differentiation and function.

PubMed

Schwarz, Silke; Elsaesser, Alexander F; Koerber, Ludwig; Goldberg-Bockhorn, Eva; Seitz, Andreas M; Bermueller, Christian; Dürselen, Lutz; Ignatius, Anita; Breiter, Roman; Rotter, Nicole

2015-12-01

One key point in the development of new bioimplant matrices for the reconstruction and replacement of cartilage defects is to provide an adequate microenvironment to ensure chondrocyte migration and de novo synthesis of cartilage-specific extracellular matrix (ECM). A recently developed decellularization and sterilization process maintains the three-dimensional (3D) collagen structure of native septal cartilage while increasing matrix porosity, which is considered to be crucial for cartilage tissue engineering. Human primary nasal septal chondrocytes were amplified in monolayer culture and 3D-cultured on processed porcine nasal septal cartilage scaffolds. The influence of chondrogenic growth factors on neosynthesis of ECM proteins was examined at the protein and gene expression levels. Seeding experiments demonstrated that processed xenogenic cartilage matrices provide excellent environmental properties for human nasal septal chondrocytes with respect to cell adhesion, migration into the matrix and neosynthesis of cartilage-specific ECM proteins, such as collagen type II and aggrecan. Matrix biomechanical stability indicated that the constructs retrieve full stability and function during 3D culture for up to 42 days, proportional to collagen type II and GAG production. Thus, processed xenogenic cartilage offers a suitable environment for human nasal chondrocytes and has promising potential for cartilage tissue engineering in the head and neck region. Copyright © 2012 John Wiley & Sons, Ltd.

A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients.

PubMed

Feng, Qin; Gai, Fei; Sang, Yaxiong; Zhang, Jie; Wang, Ping; Wang, Yue; Liu, Bing; Lin, Dongmei; Yu, Yang; Fang, Jian

2018-01-01

The AURA3 clinical trial has shown that advanced non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations in circulating tumor DNA (ctDNA) could benefit from osimertinib. The aim of this study was to assess the usefulness of QuantStudio™ 3D Digital PCR System platform for the detection of plasma EGFR T790M mutations in NSCLC patients, and compare the performances of 3D Digital PCR and ARMS-PCR. A total of 119 Chinese patients were enrolled in this study. Mutant allele frequency of plasma EGFR T790M was detected by 3D Digital PCR, then 25 selected samples were verified by ARMS-PCR and four of them were verified by next generation sequencing (NGS). In total, 52.94% (69/119) had EGFR T790M mutations detected by 3D Digital PCR. In 69 positive samples, the median mutant allele frequency (AF) was 1.09% and three cases presented low concentration (AF <0.1%). Limited by the amount of plasma DNA, 17 samples (AF <2.5%) and eight samples (T790M-) were selected for verification by ARMS-PCR. Four of those samples were verified by NGS as a third verification method. Among the selected 17 positive cases, ten samples presented mutant allele frequency <0.5%, and seven samples presented intermediate mutant allele frequency (0.5% AF 2.5%). However, only three samples (3/17) were identified as positive by ARMS-PCR, namely, P6 (AF =1.09%), P7 (AF =2.09%), and P8 (AF =2.21%). It is worth mentioning that sample P9 (AF =2.05%, analyzed by 3D Digital PCR) was identified as T790M- by ARMS-PCR. Four samples were identified as T790M+ by both NGS and 3D Digital PCR, and typically three samples (3/4) presented at a low ratio (AF <0.5%). Our study demonstrated that 3D Digital PCR is a novel method with high sensitivity and specificity to detect EGFR T790M mutation in plasma.

Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients.

PubMed

Yang, Zhe; Yang, Nong; Ou, Qiuxiang; Xiang, Yi; Jiang, Tao; Wu, Xue; Bao, Hua; Tong, Xiaoling; Wang, Xiaonan; Shao, Yang W; Liu, Yunpeng; Wang, Yan; Zhou, Caicun

2018-03-05

Background: The third-generation EGFR tyrosine kinase inhibitor osimertinib is approved to treat patients with EGFR T790M-positive non-small cell lung cancer (NSCLC) who have developed resistance to earlier-generation drugs. Acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in some patients. However, the remaining resistance mechanisms are largely unknown. Methods: We performed mutation profiling using targeted next-generation sequencing (NGS) for 416 cancer-relevant genes on 93 osimertinib-resistant lung cancer patients' samples, mainly cell-free DNAs (cfDNAs), and matched pretreatment samples of 12 patients. In vitro experiments were conducted to functionally study the secondary EGFR mutations identified. Results: EGFR G796/C797, L792, and L718/G719 mutations were identified in 24.7%, 10.8%, and 9.7% of the cases, respectively, with certain mutations coexisting in one patient with different prevalence. L792 and L718 mutants markedly increased the half inhibitory concentration (IC 50 ) of osimertinib in vitro , among which the L718Q mutation conferred the greatest resistance to osimertinib, as well as gefitinib resistance when not coexisting with T790M. Further analysis of the 12 matched pretreatment samples confirmed that these EGFR mutations were acquired during osimertinib treatment. Alterations in parallel or downstream oncogenes such as MET, KRAS , and PIK3CA were also discovered, potentially contributing to the osimertinib-resistance in patients without EGFR secondary mutations. Conclusions: We present comprehensive mutation profiles of a large cohort of osimertinib-resistance lung cancer patients using mainly cfDNA. Besides C797 mutations, novel secondary mutations of EGFR L718 and L792 residues confer osimertinib resistance, both in vitro and in vivo , and are of great clinical and pharmaceutical relevance. Clin Cancer Res; 1-11. ©2018 AACR. ©2018 American Association for Cancer Research.

Gefitinib and EGFR Gene Copy Number Aberrations in Esophageal Cancer.

PubMed

Petty, Russell D; Dahle-Smith, Asa; Stevenson, David A J; Osborne, Aileen; Massie, Doreen; Clark, Caroline; Murray, Graeme I; Dutton, Susan J; Roberts, Corran; Chong, Irene Y; Mansoor, Wasat; Thompson, Joyce; Harrison, Mark; Chatterjee, Anirban; Falk, Stephen J; Elyan, Sean; Garcia-Alonso, Angel; Fyfe, David Walter; Wadsley, Jonathan; Chau, Ian; Ferry, David R; Miedzybrodzka, Zosia

2017-07-10

Purpose The Cancer Esophagus Gefitinib trial demonstrated improved progression-free survival with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib relative to placebo in patients with advanced esophageal cancer who had disease progression after chemotherapy. Rapid and durable responses were observed in a minority of patients. We hypothesized that genetic aberration of the EGFR pathway would identify patients benefitting from gefitinib. Methods A prespecified, blinded molecular analysis of Cancer Esophagus Gefitinib trial tumors was conducted to compare efficacy of gefitinib with that of placebo according to EGFR copy number gain (CNG) and EGFR, KRAS, BRAF, and PIK3CA mutation status. EGFR CNG was determined by fluorescent in situ hybridization (FISH) using prespecified criteria and EGFR FISH-positive status was defined as high polysomy or amplification. Results Biomarker data were available for 340 patients. In EGFR FISH-positive tumors (20.2%), overall survival was improved with gefitinib compared with placebo (hazard ratio [HR] for death, 0.59; 95% CI, 0.35 to 1.00; P = .05). In EGFR FISH-negative tumors, there was no difference in overall survival with gefitinib compared with placebo (HR for death, 0.90; 95% CI, 0.69 to 1.18; P = .46). Patients with EGFR amplification (7.2%) gained greatest benefit from gefitinib (HR for death, 0.21; 95% CI, 0.07 to 0.64; P = .006). There was no difference in overall survival for gefitinib versus placebo for patients with EGFR, KRAS, BRAF, and PIK3CA mutations, or for any mutation versus none. Conclusion EGFR CNG assessed by FISH appears to identify a subgroup of patients with esophageal cancer who may benefit from gefitinib as a second-line treatment. Results of this study suggest that anti-EGFR therapies should be investigated in prospective clinical trials in different settings in EGFR FISH-positive and, in particular, EGFR-amplified esophageal cancer.

In-silico evidences for binding of Glucokinase activators to EGFR C797S to overcome EGFR resistance obstacle with mutant-selective allosteric inhibition.

PubMed

Patel, Harun; Pawara, Rahul; Surana, Sanjay

2018-03-29

The tyrosine kinase inhibitors (TKI) against epidermal growth factor receptor (EGFR) are generally utilized as a part of patients with non-small cell lung carcinoma (NSCLC). However, EGFR T790M mutation results in resistance to most clinically available EGFR TKIs. Third-generation EGFR TKIs against the T790M mutation has been in active clinical development to triumph the resistance problem; they covalently bind with conserved Cys797 inside the EGFR active site, offering both potency and kinase-selectivity. Third generation drugs target C797, which makes the C797S resistance mutation more subtle. EGFR C797S mutation was accounted to be a main mechanism of resistance to the third-generation inhibitors. The C797S mutation gives off an impression of being an ideal target for conquering the acquired resistance to the third generation inhibitors. We have performed structure based-virtual screening strategies for binding of glucokinase activator to EGFR C797S, which can overcome EGFR resistance impediment with mutant-selective allosteric inhibition towards all kinds of mutant EGFR (T790M, L858R, TMLR) and WT EGFR. The final filter of Lipinski's Rule of Five, Jargan's Rule of Three and in silico ADME predictions gave 23 hits, which conform to Lipinski's rule and Jorgensen's rule and all their pharmacokinetic parameters are inside the appropriate range characterized for human use, in this manner demonstrating their potential as a drug-like molecule. Copyright © 2018 Elsevier Ltd. All rights reserved.

Propofol enhances the cisplatin-induced apoptosis on cervical cancer cells via EGFR/JAK2/STAT3 pathway.

PubMed

Li, Haoran; Lu, Yan; Pang, Yangyang; Li, Mengjiao; Cheng, Xi; Chen, Jiawei

2017-02-01

The main purpose of this study was to evaluate propofol and its combined effect with cisplatin on apoptosis of cervical cancer cells and molecular mechanisms of this phenomenon. The effects of propofol and cisplatin on cell viability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, colony formation assay and flow cytometry assay. Besides, protein expression of EGFR/JAK2/STAT3 pathway was determined by western blot. STAT3 was over-expressed in cervical cancer cells by STAT3 cDNA. Expression of EGFR and STAT3 protein of human tissues was evaluated by immunohistochemistry (IHC) assay. In this study, we found that not only propofol alone could inhibit cervical cancer cells viability but also could increase the inhibitory effect of cisplatin on cervical cancer cells growth. Meanwhile, propofol sensitized cervical cancer cells to cisplatin-induced apoptosis but not affected normal cervical cells. In genetic level, propofol could enhance the anti-tumor effect of cisplatin through EGFR/JAK2/STAT3 pathway. Further studies indicated that overexpression of EGFR and STAT3 is related to poor prognoses in cervical cancer patients, which contributed to confirm the clinical role of combined application of propofol and cisplatin. Propofol enhances the cisplatin-induced cell apoptosis cervical cancer cells via EGFR/JAK2/STAT3 pathway and may be developed as a potential therapeutic agent to treat cervical cancer. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Osimertinib and Necitumumab in Treating Patients With EGFR-Mutant Stage IV or Recurrent Non-small Cell Lung Cancer Who Have Progressed on a Previous EGFR Tyrosine Kinase Inhibitor

ClinicalTrials.gov

2018-03-07

EGFR Exon 19 Deletion Mutation; EGFR Exon 20 Insertion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR NP_005219.2:p.T790M; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage IV Non-Small Cell Lung Cancer AJCC v7

Sapanisertib and Osimertinib in Treating Patients With Stage IV EGFR Mutation Positive Non-small Cell Lung Cancer After Progression on a Previous EGFR Tyrosine Kinase Inhibitor

ClinicalTrials.gov

2018-04-25

EGFR Activating Mutation; EGFR Exon 19 Deletion Mutation; EGFR NP_005219.2:p.G719X; EGFR NP_005219.2:p.L858R; EGFR NP_005219.2:p.L861Q; EGFR T790M Mutation Negative; Recurrent Non-Small Cell Lung Carcinoma; Stage III Non-Small Cell Lung Cancer AJCC v7; Stage IIIA Non-Small Cell Lung Cancer AJCC v7; Stage IIIB Non-Small Cell Lung Cancer AJCC v7; Stage IV Non-Small Cell Lung Cancer AJCC v7

Detection of EGFR and KRAS mutations in fine-needle aspirates stored on Whatman FTA cards: is this the tool for biobanking cytological samples in the molecular era?

PubMed

da Cunha Santos, Gilda; Liu, Ni; Tsao, Ming-Sound; Kamel-Reid, Suzanne; Chin, Kayu; Geddie, William R

2010-12-25

The aims of this study were to compare the quality of DNA recovered from fine-needle aspirates (FNAs) stored on Whatman FTA cards with that retrieved from corresponding cell blocks and to determine whether the DNA extracted from the cards is suitable for multiple mutation analyses. FNAs collected from 18 resected lung tumors and cell suspensions from 4 lung cancer cell lines were placed on FTA Indicating Micro Cards and further processed to produce paired formalin-fixed paraffin-embedded (FFPE) cell blocks. Fragment analysis was used for the detection of EGFR exon 19 deletion, and direct sequencing for detection of EGFR exon 21 L858R mutation and exon 2 deletion of KRAS. Corresponding FFPE tissue sections from 2 resection specimens were also tested. Analyses were successful with all FNAs and lung cancer-derived cell lines collected on cards. Polymerase chain reaction failed in 2 cell blocks. For FNAs collected on cards, 5 cases showed EGFR and 3 showed KRAS mutations. Eleven cases were wild type. With cell blocks, 4 cases were found to harbor KRAS and 4 harbored EGFR mutations. All lung cancer-derived cell lines tested positive for their respective mutations, and there was complete agreement between card and cell block FNA samples for EGFR exon 21. For EGFR exon 19, 1 of 18 cases showed discordant results between the card and cell block, and for KRAS 1 of 17. The two resection specimens tested gave concordant results with the FTA card. Storage of cytologic material on FTA cards can maximize and simplify sample procurement for multiple mutational analyses with results similar to those from cell blocks.

The APPLE Trial: Feasibility and Activity of AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients. EORTC 1613.

PubMed

Remon, Jordi; Menis, Jessica; Hasan, Baktiar; Peric, Aleksandra; De Maio, Eleonora; Novello, Silvia; Reck, Martin; Berghmans, Thierry; Wasag, Bartosz; Besse, Benjamin; Dziadziuszko, Rafal

2017-09-01

The AZD9291 (Osimertinib) Treatment on Positive PLasma T790M in EGFR-mutant NSCLC Patients (APPLE) trial is a randomized, open-label, multicenter, 3-arm, phase II study in advanced, epidermal growth factor receptor (EGFR)-mutant and EGFR tyrosine kinase inhibitor (TKI)-naive non-small-cell lung cancer (NSCLC) patients, to evaluate the best strategy for sequencing gefitinib and osimertinib treatment. Advanced EGFR-mutant NSCLC patients, with World Health Organization performance status 0-2 who are EGFR TKI treatment-naive and eligible to receive first-line treatment with EGFR TKI will be randomized to: In all arms, a plasmatic ctDNA T790M test will be performed by a central laboratory at the Medical University of Gdansk (Poland) but will be applied as a predictive marker for making treatment decisions only in arm B. The primary objective is to evaluate the best strategy for sequencing of treatment with gefitinib and osimertinib in advanced NSCLC patients with common EGFR mutations, and to understand the value of liquid biopsy for the decision-making process. The progression-free survival rate at 18 months is the primary end point of the trial. The activity of osimertinib versus gefitinib to prevent brain metastases will be evaluated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas

PubMed Central

Goyal, Amit; Gonda, David; Akers, Johnny; Adhikari, Bandita; Patel, Kunal; Vandenberg, Scott; Yan, Wei; Bao, Zhaoshi; Carter, Bob S.; Wang, Renzhi; Mao, Ying; Jiang, Tao; Chen, Clark C.

2014-01-01

The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf−/− EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients. PMID:25277177

Epigenetic suppression of EGFR signaling in G-CIMP+ glioblastomas.

PubMed

Li, Jie; Taich, Zachary J; Goyal, Amit; Gonda, David; Akers, Johnny; Adhikari, Bandita; Patel, Kunal; Vandenberg, Scott; Yan, Wei; Bao, Zhaoshi; Carter, Bob S; Wang, Renzhi; Mao, Ying; Jiang, Tao; Chen, Clark C

2014-09-15

The intrinsic signaling cascades and cell states associated with the Glioma CpG Island Methylator Phenotype (G-CIMP) remain poorly understood. Using published mRNA signatures associated with EGFR activation, we demonstrate that G-CIMP+ tumors harbor decreased EGFR signaling using three independent datasets, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), and The Cancer Genome Atlas (TCGA; n=406). Additionally, an independent collection of 25 fresh-frozen glioblastomas confirmed lowered pERK levels in G-CIMP+ specimens (p<0.001), indicating suppressed EGFR signaling. Analysis of TCGA glioblastomas revealed that G-CIMP+ glioblastomas harbored lowered mRNA levels for EGFR and H-Ras. Induction of G-CIMP+ state by exogenous expression of a mutated isocitrate dehydrogenase 1, IDH1-R132H, suppressed EGFR and H-Ras protein expression as well as pERK accumulation in independent glioblastoma models. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. The IDH1-R132H expression-induced pERK suppression can be reversed by exogenous expression of H-RasG12V. Finally, the G-CIMP+ Ink4a-Arf-/- EGFRvIII glioblastoma line was more resistant to the EGFR inhibitor, Gefitinib, relative to its isogenic G-CIMP- counterpart. These results suggest that G-CIMP epigenetically regulates EGFR signaling and serves as a predictive biomarker for EGFR inhibitors in glioblastoma patients.

Cigarette smoke enhances oncogene addiction to c-MET and desensitizes EGFR-expressing non-small cell lung cancer to EGFR TKIs.

PubMed

Tu, Chih-Yen; Cheng, Fang-Ju; Chen, Chuan-Mu; Wang, Shu-Ling; Hsiao, Yu-Chun; Chen, Chia-Hung; Hsia, Te-Chun; He, Yu-Hao; Wang, Bo-Wei; Hsieh, I-Shan; Yeh, Yi-Lun; Tang, Chih-Hsin; Chen, Yun-Ju; Huang, Wei-Chien

2018-05-01

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke-derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine-derived nitrosamine ketone (NNK), reduced the sensitivity of wild-type EGFR-expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P-treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c-MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c-MET reduces B[α]P-induced Akt activation. The CSE-treated NSCLC cells are sensitive to the c-MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c-MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

[Xenogeneic cell therapeutics: Treatment of type 1 diabetes using porcine pancreatic islets and islet cells].

PubMed

Godehardt, Antonia W; Schilling-Leiß, Dagmar; Sanzenbacher, Ralf; Tönjes, Ralf R

2015-11-01

In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

Role of Epidermal Growth Factor Receptor (EGFR) Inhibitors and Radiation in the Management of Brain Metastases from EGFR Mutant Lung Cancers.

PubMed

Khandekar, Melin J; Piotrowska, Zofia; Willers, Henning; Sequist, Lecia V

2018-04-27

The growth of genotype-directed targeted therapies, such as inhibitors of the epidermal growth factor receptor (EGFR), has revolutionized treatment for some patients with oncogene-addicted lung cancer. However, as systemic control for these patients has improved, brain metastases remain an important source of morbidity and mortality. Traditional treatment for brain metastases has been radiotherapy, either whole-brain radiation or stereotactic radiosurgery. The growing availability of drugs that can cross the blood-brain barrier and have activity in the central nervous system (CNS) has led to many studies investigating whether targeted therapy can be used in combination with or in lieu of radiation. In this review, we summarize the key literature about the incidence and nature of EGFR-mutant brain metastases (EGFR BMs), the data about the activity of EGFR inhibitors in the CNS, and whether they can be used as front-line therapy for brain metastases. Although initial use of tyrosine kinase inhibitors for EGFR BMs can often be an effective treatment strategy, multidisciplinary evaluation is critical, and prospective studies are needed to clarify which patients may benefit from early radiotherapy. Management of brain metastases in epidermal growth factor receptor (EGFR) mutant lung cancer is a common clinical problem. The question of whether to start initial therapy with an EGFR inhibitor or radiotherapy (either whole-brain radiotherapy or stereotactic radiosurgery) is controversial. The development of novel EGFR inhibitors with enhanced central nervous system (CNS) penetration is an important advance in the treatment of CNS disease. Multidisciplinary evaluation and evaluation of extracranial disease status are critical to choosing the best treatment option for each patient. © AlphaMed Press 2018.

Antibodies Specifically Targeting a Locally Misfolded Region of Tumor Associated EGFR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garrett, T.; Burgess, A; Gan, H

2009-01-01

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR{sub 287-302} complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR.more » However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR{sub C271A/C283A} mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR{sub C271A/C283A}. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.« less

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

PubMed

Mehrabi, Masomeh; Mahdiuni, Hamid; Rasouli, Hassan; Mansouri, Kamran; Shahlaei, Mohsen; Khodarahmi, Reza

2018-04-14

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln 43 , Tyr 44 , Arg 45 and Asp 46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast,more » amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.« less

Silibinin preferentially radiosensitizes prostate cancer by inhibiting DNA repair signaling

PubMed Central

Nambiar, Dhanya K.; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K.; Agarwal, Rajesh; Singh, Rana P.

2015-01-01

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer (PCa). The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant PCa cell lines by clonogenic, cell cycle, cell death and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μM) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P<0.001) of colony formation selectively in PCa cells, and prolonged and enhanced IR-caused G2/M arrest, apoptosis and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of anti-apoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P<0.01) with higher apoptotic response (10-fold, P<0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced pro-survival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Since silibinin is already in phase II clinical trial for PCa patients, the present finding has translational relevance for radioresistant PCa. PMID:26516160

Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

PubMed

Nambiar, Dhanya K; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K; Agarwal, Rajesh; Singh, Rana P

2015-12-01

Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. ©2015 American Association for Cancer Research.

Bioinformatics-driven discovery of rational combination for overcoming EGFR-mutant lung cancer resistance to EGFR therapy.

PubMed

Kim, Jihye; Vasu, Vihas T; Mishra, Rangnath; Singleton, Katherine R; Yoo, Minjae; Leach, Sonia M; Farias-Hesson, Eveline; Mason, Robert J; Kang, Jaewoo; Ramamoorthy, Preveen; Kern, Jeffrey A; Heasley, Lynn E; Finigan, James H; Tan, Aik Choon

2014-09-01

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Targeted tyrosine kinase inhibitors (TKIs) directed against the epidermal growth factor receptor (EGFR) have been widely and successfully used in treating NSCLC patients with activating EGFR mutations. Unfortunately, the duration of response is short-lived, and all patients eventually relapse by acquiring resistance mechanisms. We performed an integrative systems biology approach to determine essential kinases that drive EGFR-TKI resistance in cancer cell lines. We used a series of bioinformatics methods to analyze and integrate the functional genetics screen and RNA-seq data to identify a set of kinases that are critical in survival and proliferation in these TKI-resistant lines. By connecting the essential kinases to compounds using a novel kinase connectivity map (K-Map), we identified and validated bosutinib as an effective compound that could inhibit proliferation and induce apoptosis in TKI-resistant lines. A rational combination of bosutinib and gefitinib showed additive and synergistic effects in cancer cell lines resistant to EGFR TKI alone. We have demonstrated a bioinformatics-driven discovery roadmap for drug repurposing and development in overcoming resistance in EGFR-mutant NSCLC, which could be generalized to other cancer types in the era of personalized medicine. K-Map can be accessible at: http://tanlab.ucdenver.edu/kMap. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

β-catenin contributes to lung tumor development induced by EGFR mutations

PubMed Central

Nakayama, Sohei; Sng, Natasha; Carretero, Julian; Welner, Robert; Hayashi, Yuichiro; Yamamoto, Mihoko; Tan, Alistair J.; Yamaguchi, Norihiro; Yasuda, Hiroyuki; Li, Danan; Soejima, Kenzo; Soo, Ross A.; Costa, Daniel B.; Wong, Kwok-Kin; Kobayashi, Susumu S.

2014-01-01

The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and development of EGFR tyrosine kinase inhibitors (TKIs) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. β-catenin was upregulated and activated in EGFR mutated cells. Mutant EGFR preferentially bound to and tyrosine-phosphorylated β-catenin, leading to increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacological inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs. PMID:25164010

Understanding resistance to EGFR inhibitors—impact on future treatment strategies

PubMed Central

Wheeler, Deric L.; Dunn, Emily F.; Harari, Paul M.

2010-01-01

EGFR is a tyrosine kinase that participates in the regulation of cellular homeostasis. Following ligand binding, EGFR stimulates downstream cell signaling cascades that influence cell proliferation, apoptosis, migration, survival and complex processes, including angiogenesis and tumorigenesis. EGFR has been strongly implicated in the biology of human epithelial malignancies, with therapeutic applications in cancers of the colon, head and neck, lung, and pancreas. Accordingly, targeting EGFR has been intensely pursued, with the development of a series of promising molecular inhibitors for use in clinical oncology. As is common in cancer therapy, challenges with respect to treatment resistance emerge over time. This situation is certainly true of EGFR inhibitor therapies, where intrinsic and acquired resistance is now well recognized. In this Review, we provide a brief overview regarding the biology of EGFR biology, preclinical and clinical development of EGFR inhibitors, and molecular mechanisms that underlie the development of treatment resistance. A greater understanding of the mechanisms that lead to EGFR resistance may provide valuable insights to help design new strategies that will enhance the impact of this promising class of inhibitors for the treatment of cancer. PMID:20551942

Synchronous occurrence of squamous-cell carcinoma "transformation" and EGFR exon 20 S768I mutation as a novel mechanism of resistance in EGFR-mutated lung adenocarcinoma.

PubMed

Longo, Lucia; Mengoli, Maria Cecilia; Bertolini, Federica; Bettelli, Stefania; Manfredini, Samantha; Rossi, Giulio

2017-01-01

The occurrence of secondary EGFR mutation T790M in exon 20 and histologic "transformation" are common mechanisms underlying resistance to EGFR first- or second-generation tyrosine kinase inhibitors (TKI). We describe here on a hitherto unreported mechanism of EGFR TKI resistance synchronously combining squamous-cell carcinoma change and occurrence of the EGFR exon 20 S768I secondary mutation in a 43 year-old woman with stage IV adenocarcinoma harbouring EGFR exon 21 L858R mutation. After 8 months of response to gefitinib, the patient experienced EGFR TKI resistance and died of leptomeningeal neoplastic dissemination. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DIFFERENTIAL RESPONSE TO ALLOGENIC AND XENOGENIC SKIN GRAFTS BY SUBLETHALLY IRRADIATED (670 RAD) AND NON-IRRADIATED MICE SENSITIZED BY VARIOUS MEANS

DTIC Science & Technology

consecutive BALB/c or rat skin tail grafts. One week following the last injection or the rejection of the second, skin graft , the mice either were grafted...resulted in prolonged survival of subsequent allogenic skin grafts in sublethally irradiated mice. The second-set response to a xenogenic skin graft was

New Insights from Drosophila into the Regulation of EGFR Signaling.

PubMed

Harden, Nicholas

2017-01-01

Genetic analysis of Egfr signaling in Drosophila has a long-standing track record of making important contributions to our understanding of the Egfr pathway. While the central Ras/MAPK pathway has long been well defined, there is much to learn with regard to its cross talk with other pathways and how it is regulated. A better understanding of the regulation of Egfr signaling is of particular interest with regard to the participation of misregulated Egfr signaling in tumorigenesis. Recent studies in the fly have led to some surprising results, identifying regulators of Egfr acting in unexpected ways.

Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation.

PubMed

Osoegawa, Atsushi; Hashimoto, Takafumi; Takumi, Yohei; Abe, Miyuki; Yamada, Tomonori; Kobayashi, Ryoji; Miyawaki, Michiyo; Takeuchi, Hideya; Okamoto, Tatsuro; Sugio, Kenji

2018-03-28

Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.

A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients

PubMed Central

Sang, Yaxiong; Zhang, Jie; Wang, Ping; Wang, Yue; Liu, Bing; Lin, Dongmei; Yu, Yang; Fang, Jian

2018-01-01

Background The AURA3 clinical trial has shown that advanced non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations in circulating tumor DNA (ctDNA) could benefit from osimertinib. Purpose The aim of this study was to assess the usefulness of QuantStudio™ 3D Digital PCR System platform for the detection of plasma EGFR T790M mutations in NSCLC patients, and compare the performances of 3D Digital PCR and ARMS-PCR. Patients and methods A total of 119 Chinese patients were enrolled in this study. Mutant allele frequency of plasma EGFR T790M was detected by 3D Digital PCR, then 25 selected samples were verified by ARMS-PCR and four of them were verified by next generation sequencing (NGS). Results In total, 52.94% (69/119) had EGFR T790M mutations detected by 3D Digital PCR. In 69 positive samples, the median mutant allele frequency (AF) was 1.09% and three cases presented low concentration (AF <0.1%). Limited by the amount of plasma DNA, 17 samples (AF <2.5%) and eight samples (T790M-) were selected for verification by ARMS-PCR. Four of those samples were verified by NGS as a third verification method. Among the selected 17 positive cases, ten samples presented mutant allele frequency <0.5%, and seven samples presented intermediate mutant allele frequency (0.5% AF 2.5%). However, only three samples (3/17) were identified as positive by ARMS-PCR, namely, P6 (AF =1.09%), P7 (AF =2.09%), and P8 (AF =2.21%). It is worth mentioning that sample P9 (AF =2.05%, analyzed by 3D Digital PCR) was identified as T790M- by ARMS-PCR. Four samples were identified as T790M+ by both NGS and 3D Digital PCR, and typically three samples (3/4) presented at a low ratio (AF <0.5%). Conclusion Our study demonstrated that 3D Digital PCR is a novel method with high sensitivity and specificity to detect EGFR T790M mutation in plasma. PMID:29403309

JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

PubMed Central

Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.

2016-01-01

Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877

Efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with non-small-cell lung cancer harboring sensitive EGFR mutations.

PubMed

Imai, Hisao; Minemura, Hiroyuki; Sugiyama, Tomohide; Yamada, Yutaka; Kaira, Kyoichi; Kanazawa, Kenya; Kasai, Takashi; Kaburagi, Takayuki; Minato, Koichi

2018-05-08

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is effective as first-line chemotherapy for patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive EGFR mutations. However, whether the efficacy of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment is similar to that of first-line cytotoxic drug chemotherapy in elderly patients aged ≥ 75 years harboring sensitive EGFR mutations is unclear. Therefore, we aimed to investigate the efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations. We retrospectively evaluated the clinical effects and safety profiles of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations (exon 19 deletion/exon 21 L858R mutation). Between April 2008 and December 2015, 78 elderly patients with advanced NSCLC harboring sensitive EGFR mutations received first-line EGFR-TKI at four Japanese institutions. Baseline characteristics, regimens, responses to first- and second-line treatments, whether or not patients received subsequent treatment, and if not, the reasons for non-administration were recorded. Overall, 20 patients with a median age of 79.5 years (range 75-85 years) were included in our analysis. The overall response, disease control, median progression-free survival, and overall survival rates were 15.0, 60.0%, 2.4, and 13.2 months, respectively. Common adverse events included leukopenia, neutropenia, anemia, thrombocytopenia, malaise, and anorexia. Major grade 3 or 4 toxicities included leukopenia (25.0%) and neutropenia (45.0%). No treatment-related deaths were noted. Second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment among elderly patients with NSCLC harboring sensitive EGFR mutations was effective and safe and showed equivalent outcomes to first

Anti-EGFR Agents: Current Status, Forecasts and Future Directions.

PubMed

Kwapiszewski, Radoslaw; Pawlak, Sebastian D; Adamkiewicz, Karolina

2016-12-01

The epidermal growth factor receptor (EGFR) is one of the most important and attractive targets for specific anticancer therapies. It is a robust regulator of pathways involved in cancer pathogenesis and progression. Thus far, clinical trials have demonstrated the benefits of monoclonal antibodies and synthetic tyrosine kinase inhibitors in targeting this receptor; however, novel strategies are still being developed. This article reviews the current state of efforts in targeting the EGFR in cancer therapy. Following a brief characterization of EGFR, we will present a complete list of anti-EGFR agents that are already approved, and available in clinical practice. Aside from the indications, we will present the sales forecasts and expiry dates of product patents for the selected agents. Finally, we discuss the novel anti-EGFR strategies that are currently in preclinical development.

[Regulation on EGFR function via its interacting proteins and its potential application].

PubMed

Zheng, Jun-Fang; Chen, Hui-Min; He, Jun-Qi

2013-12-01

Epidermal growth factor receptor (EGFR) is imptortant for cell activities, oncogenesis and cell migration, and EGFR inhibitor can treat cancer efficiently, but its side effects, for example, in skin, limited its usage. On the other hand, EGFR interacting proteins may also lead to oncogenesis and its interacting protein as drug targets can avoid cutaneous side effect, which implies possibly a better outcome and life quality of cancer patients. For the multiple EGFR interaction proteins, B1R enhances Erk/MAPK signaling, while PTPN12, Kek1, CEACAM1 and NHERF repress Erk/MAPK signaling. CaM may alter charge of EGFR juxamembrane domain and regulate activation of PI3K/Akt and PLC-gamma/PKC. STAT1, STAT5b are widely thought to be activated by EGFR, while there is unexpectedly inhibiting sequence within EGFR to repress the activity of STATs. LRIG1 and ACK1 enhance the internalization and degration of EGFR, while NHERF and HIP1 repress it. In this article, proteins interacting with EGFR, their interacting sites and their regulation on EGFR signal transduction will be reviewed.

A view on EGFR-targeted therapies from the oncogene-addiction perspective.

PubMed

Perez, Rolando; Crombet, Tania; de Leon, Joel; Moreno, Ernesto

2013-01-01

Tumor cell growth and survival can often be impaired by inactivating a single oncogen- a phenomenon that has been called as "oncogene addiction." It is in such scenarios that molecular targeted therapies may succeed. among known oncogenes, the epidermal growth factor receptor (EGFR) has become the target of different cancer therapies. So far, however, the clinical benefit from EGFR-targeted therapies has been rather limited. a critical review of the large amount of clinical data obtained with anti-EGFR agents, carried out from the perspective of the oncogene addiction concept, may help to understand the causes of the unsatisfactory results. In this article we intend to do such an exercise taking as basis for the analysis a few case studies of anti-EGFR agents that are currently in the clinic. There, the "EGFR addiction" phenomenon becomes apparent in high-responder patients. We further discuss how the concept of oncogene addiction needs to be interpreted on the light of emerging experimental evidences and ideas; in particular, that EGFR addiction may reflect the interconnection of several cellular pathways. In this regard we set forth several hypotheses; namely, that requirement of higher glucose uptake by hypoxic tumor cells may reinforce EGFR addiction; and that chronic use of EGFR-targeted antibodies in EGFR-addicted tumors would induce stable disease by reversing the malignant phenotype of cancer stem cells and also by sustaining an anti-tumor T cell response. Finally, we discuss possible reasons for the failure of certain combinatorial therapies involving anti-EGFR agents, arguing that some of these agents might produce either a negative or a positive trans-modulation effect on other oncogenes. It becomes evident that we need operational definitions of EGFR addiction in order to determine which patient populations may benefit from treatment with anti-EGFR drugs, and to improve the design of these therapies.

Xenogeneic Collagen Matrix Versus Connective Tissue Graft: Case Series of Various Gingival Recession Treatments.

PubMed

Chevalier, Grégoire; Cherkaoui, Selma; Kruk, Hanna; Bensaïd, Xavier; Danan, Marc

A xenogeneic collagen matrix recently has been suggested as an alternative to connective tissue graft for the treatment of gingival recession. The matrix avoids the second surgical site, and as a consequence could decrease surgical morbidity. This new matrix was used in various clinical situations and compared to connective tissue graft (CTG) in a split-mouth design case series. A total of 17 recessions were treated with a coronally advanced flap, 9 with CTG, and 8 with the matrix. Mean recession reduction was 2.00 mm with the CTG and 2.00 mm with the matrix. No significant statistical differences between the techniques were observed in this case report.

Xenogeneic Collagen Matrix Versus Connective Tissue Graft: Case Series of Various Gingival Recession Treatments.

PubMed

Chevalier, Grégoire; Cherkaoui, Selma; Kruk, Hanna; Bensaïd, Xavier; Danan, Marc

2016-08-24

A xenogeneic collagen matrix recently has been suggested as an alternative to connective tissue graft for the treatment of gingival recession. The matrix avoids the second surgical site, and as a consequence could decrease surgical morbidity. This new matrix was used in various clinical situations and compared to connective tissue graft (CTG) in a split-mouth design case series. A total of 17 recessions were treated with a coronally advanced flap, 9 with CTG, and 8 with the matrix. Mean recession reduction was 2.00 mm with the CTG and 2.00 mm with the matrix. No significant statistical differences between the techniques were observed in this case report.

EGFR and Ras regulate DDX59 during lung cancer development.

PubMed

Yang, Lin; Zhang, Hanyin; Chen, Dan; Ding, Peikun; Yuan, Yunchang; Zhang, Yandong

2018-02-05

Oncogenes EGFR and ras are frequently mutated and activated in human lung cancers. In this report, we found that both EGFR and Ras signaling can upregulate RNA helicase DDX59 in lung cancer cells. DDX59 can be induced through the mitogen activated protein kinase (MAPK) pathway after EGFR or Ras activation. Inhibitors for Ras/Raf/MAP pathway significantly decreased DDX59 expression at both protein and mRNA levels. Through immunohistochemistry, we found that DDX59 protein expression correlated with Ras and EGFR mutation status in human lung adenocarcinoma. Finally, through a xenograft nude mice model, we demonstrated that DDX59 is pivotal for EGFR mutated lung cancer cell growth in vivo. Our study identified a novel protein downstream of Ras and EGFR, which may serve as a potential therapeutic drug target for lung cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Past Decline Versus Current eGFR and Subsequent ESRD Risk.

PubMed

Kovesdy, Csaba P; Coresh, Josef; Ballew, Shoshana H; Woodward, Mark; Levin, Adeera; Naimark, David M J; Nally, Joseph; Rothenbacher, Dietrich; Stengel, Benedicte; Iseki, Kunitoshi; Matsushita, Kunihiro; Levey, Andrew S

2016-08-01

eGFR is a robust predictor of ESRD risk. However, the prognostic information gained from the past trajectory (slope) beyond that of the current eGFR is unclear. We examined 22 cohorts to determine the association of past slopes and current eGFR level with subsequent ESRD. We modeled hazard ratios as a spline function of slopes, adjusting for demographic variables, eGFR, and comorbidities. We used random effects meta-analyses to combine results across studies stratified by cohort type. We calculated the absolute risk of ESRD at 5 years after the last eGFR using the weighted average baseline risk. Overall, 1,080,223 participants experienced 5163 ESRD events during a mean follow-up of 2.0 years. In CKD cohorts, a slope of -6 versus 0 ml/min per 1.73 m(2) per year over the previous 3 years (a decline of 18 ml/min per 1.73 m(2) versus no decline) associated with an adjusted hazard ratio of ESRD of 2.28 (95% confidence interval, 1.88 to 2.76). In contrast, a current eGFR of 30 versus 50 ml/min per 1.73 m(2) (a difference of 20 ml/min per 1.73 m(2)) associated with an adjusted hazard ratio of 19.9 (95% confidence interval, 13.6 to 29.1). Past decline contributed more to the absolute risk of ESRD at lower than higher levels of current eGFR. In conclusion, during a follow-up of 2 years, current eGFR associates more strongly with future ESRD risk than the magnitude of past eGFR decline, but both contribute substantially to the risk of ESRD, especially at eGFR<30 ml/min per 1.73 m(2). Copyright © 2016 by the American Society of Nephrology.

Osimertinib in Untreated EGFR-Mutated Advanced Non-Small-Cell Lung Cancer.

PubMed

Soria, Jean-Charles; Ohe, Yuichiro; Vansteenkiste, Johan; Reungwetwattana, Thanyanan; Chewaskulyong, Busyamas; Lee, Ki Hyeong; Dechaphunkul, Arunee; Imamura, Fumio; Nogami, Naoyuki; Kurata, Takayasu; Okamoto, Isamu; Zhou, Caicun; Cho, Byoung Chul; Cheng, Ying; Cho, Eun Kyung; Voon, Pei Jye; Planchard, David; Su, Wu-Chou; Gray, Jhanelle E; Lee, Siow-Ming; Hodge, Rachel; Marotti, Marcelo; Rukazenkov, Yuri; Ramalingam, Suresh S

2018-01-11

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and EGFR T790M resistance mutations. We compared osimertinib with standard EGFR-TKIs in patients with previously untreated, EGFR mutation-positive advanced non-small-cell lung cancer (NSCLC). In this double-blind, phase 3 trial, we randomly assigned 556 patients with previously untreated, EGFR mutation-positive (exon 19 deletion or L858R) advanced NSCLC in a 1:1 ratio to receive either osimertinib (at a dose of 80 mg once daily) or a standard EGFR-TKI (gefitinib at a dose of 250 mg once daily or erlotinib at a dose of 150 mg once daily). The primary end point was investigator-assessed progression-free survival. The median progression-free survival was significantly longer with osimertinib than with standard EGFR-TKIs (18.9 months vs. 10.2 months; hazard ratio for disease progression or death, 0.46; 95% confidence interval [CI], 0.37 to 0.57; P<0.001). The objective response rate was similar in the two groups: 80% with osimertinib and 76% with standard EGFR-TKIs (odds ratio, 1.27; 95% CI, 0.85 to 1.90; P=0.24). The median duration of response was 17.2 months (95% CI, 13.8 to 22.0) with osimertinib versus 8.5 months (95% CI, 7.3 to 9.8) with standard EGFR-TKIs. Data on overall survival were immature at the interim analysis (25% maturity). The survival rate at 18 months was 83% (95% CI, 78 to 87) with osimertinib and 71% (95% CI, 65 to 76) with standard EGFR-TKIs (hazard ratio for death, 0.63; 95% CI, 0.45 to 0.88; P=0.007 [nonsignificant in the interim analysis]). Adverse events of grade 3 or higher were less frequent with osimertinib than with standard EGFR-TKIs (34% vs. 45%). Osimertinib showed efficacy superior to that of standard EGFR-TKIs in the first-line treatment of EGFR mutation-positive advanced NSCLC, with a similar safety profile and lower rates of serious adverse events. (Funded

High resolution melting analysis for epidermal growth factor receptor mutations in formalin-fixed paraffin-embedded tissue and plasma free DNA from non-small cell lung cancer patients.

PubMed

Jing, Chang-Wen; Wang, Zhuo; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Zhong

2014-01-01

The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

Turning EGFR mutation-positive non-small-cell lung cancer into a chronic disease: optimal sequential therapy with EGFR tyrosine kinase inhibitors

PubMed Central

Hirsh, Vera

2018-01-01

Four epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), erlotinib, gefitinib, afatinib and osimertinib, are currently available for the management of EGFR mutation-positive non-small-cell lung cancer (NSCLC), with others in development. Although tumors are exquisitely sensitive to these agents, acquired resistance is inevitable. Furthermore, emerging data indicate that first- (erlotinib and gefitinib), second- (afatinib) and third-generation (osimertinib) EGFR TKIs differ in terms of efficacy and tolerability profiles. Therefore, there is a strong imperative to optimize the sequence of TKIs in order to maximize their clinical benefit. Osimertinib has demonstrated striking efficacy as a second-line treatment option in patients with T790M-positive tumors, and also confers efficacy and tolerability advantages over first-generation TKIs in the first-line setting. However, while accrual of T790M is the most predominant mechanism of resistance to erlotinib, gefitinib and afatinib, resistance mechanisms to osimertinib have not been clearly elucidated, meaning that possible therapy options after osimertinib failure are not clear. At present, few data comparing sequential regimens in patients with EGFR mutation-positive NSCLC are available and prospective clinical trials are required. This article reviews the similarities and differences between EGFR TKIs, and discusses key considerations when assessing optimal sequential therapy with these agents for the treatment of EGFR mutation-positive NSCLC. PMID:29383041

Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

PubMed

Wang, Aoli; Yan, Xiao-E; Wu, Hong; Wang, Wenchao; Hu, Chen; Chen, Cheng; Zhao, Zheng; Zhao, Peng; Li, Xixiang; Wang, Li; Wang, Beilei; Ye, Zi; Wang, Jinhua; Wang, Chu; Zhang, Wei; Gray, Nathanael S; Weisberg, Ellen L; Chen, Liang; Liu, Jing; Yun, Cai-Hong; Liu, Qingsong

2016-10-25

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 μM), Ibrutinib exhibited less efficient binding (Kd: 0.18 μM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

PubMed

Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

2014-10-01

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

The clinical features of squamous cell lung carcinoma with sensitive EGFR mutations.

PubMed

Taniguchi, Yuri; Matsumoto, Yoko; Furukawa, Ryutaro; Ohara, Sayaka; Usui, Kazuhiro

2018-06-01

The process of selecting patients on the basis of epidermal growth factor receptor (EGFR) mutations would likely result in a patient population with greater sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, EGFR mutation status is not routinely examined in patients with squamous cell lung cancer (Sq) because of the low incidence of EGFR mutations and the poor clinical response to EGFR-TKIs. We retrospectively reviewed the clinical features of patients at our hospital with Sq who carried EGFR-TKI-sensitive EGFR mutations and assessed their responses to EGFR-TKIs. EGFR mutation status was tested in 23 of 441 patients with Sq (5.2%) admitted to our hospital during the study period. An EGFR mutation (exon 19 deletion 3, L858R 2) was identified in five of the 23 patients (21.7%), all of whom were female never-smokers. Of these five patients, four (4/9; 44.4%) were in the normal lung group, one (1/12; 8.3%) was in the emphysematous lung group, and none (0/2; 0%) in the fibrotic lung group. Two of these five patients with the EGFR mutation received gefitinib and two received afatinib. Although the two patients who were treated with gefitinib did not respond well to treatment (stable disease, 1 patient; progressive disease, 1 patient), the two patients who were treated with afatinib showed a good response (partial response, 2 patients). The administration of afatinib to Sq patients after selecting patients using the EGFR mutation test based on their underlying pulmonary disease and smoking status would likely result in a population with a greater sensitivity to afatinib.

EGFR transactivation: mechanisms, pathophysiology and potential therapies in cardiovascular system

PubMed Central

Forrester, Steven J.; Kawai, Tatsuo; Elliott, Katherine J.; O’Brien, Shannon; Thomas, Walter; Harris, Raymond C.; Eguchi, Satoru

2017-01-01

Accumulating studies suggest that the epidermal growth factor receptor (EGFR) activation is associated with the physiology and pathophysiology of the cardiovascular system, and inhibition of EGFR activity is emerging as a potential therapeutic strategy to treat diseases, including hypertension, cardiac hypertrophy, renal fibrosis and abdominal aortic aneurysm. The capacity of G protein-coupled receptor (GPCR) agonists, such as angiotensin II (AngII), to promote EGFR signaling is well described – a process termed EGFR “transactivation” – yet delineating the molecular processes and functional relevance of this crosstalk has been challenging. Moreover, these critical findings are dispersed among many different fields. The aim of our review is to highlight the recent advancement of the signaling cascades and downstream consequences of EGFR transactivation within the cardiovascular renal system in vitro and in vivo. We will also focus on linking EGFR transactivation to animal models of the disease as well as the potential therapeutic applications. PMID:26566153

EGFR and HER2 activate rigidity sensing only on rigid matrices

NASA Astrophysics Data System (ADS)

Saxena, Mayur; Liu, Shuaimin; Yang, Bo; Hajal, Cynthia; Changede, Rishita; Hu, Junqiang; Wolfenson, Haguy; Hone, James; Sheetz, Michael P.

2017-07-01

Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two different cell lines, that in serum- and EGF-free conditions, EGFR or HER2 activity increase spreading and rigidity-sensing contractions on rigid, but not soft, substrates. Contractions peak after 15-20 min, but diminish by tenfold after 4 h. Addition of EGF at that point increases spreading and contractions, but this can be blocked by myosin-II inhibition. We further show that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFK). On soft surfaces, neither EGFR inhibition nor EGF stimulation have any effect on cell motility. Thus, EGFR or HER2 can catalyse rigidity sensing after associating with nascent adhesions under rigidity-dependent tension downstream of SFK activity. This has broad implications for the roles of EGFR and HER2 in the absence of EGF both for normal and cancerous growth.

PD-L1 expression according to the EGFR status in primary lung adenocarcinoma.

PubMed

Takada, Kazuki; Toyokawa, Gouji; Tagawa, Tetsuzo; Kohashi, Kenichi; Shimokawa, Mototsugu; Akamine, Takaki; Takamori, Shinkichi; Hirai, Fumihiko; Shoji, Fumihiro; Okamoto, Tatsuro; Oda, Yoshinao; Maehara, Yoshihiko

2018-02-01

It was reported that programmed cell death-ligand 1 (PD-L1) expression is associated with smoking and wild-type epidermal growth factor receptor (EGFR) in lung adenocarcinoma. However, the association between PD-L1 expression and EGFR mutation site in EGFR mutation-positive lung adenocarcinoma is unclear. We retrospectively examined the relationship between PD-L1 expression and EGFR status in 441 surgically resected primary lung adenocarcinomas. Membrane PD-L1 expression on tumor cells was evaluated by immunohistochemical analysis using a PD-L1 antibody (clone SP142) and defined by tumor proportion scores (TPSs) of 0%, 1-4%, 5-49%, and ≥50%, respectively. Two hundred and eighteen (49.4%) patients had wild-type EGFR, and 223 (50.6%) had mutant EGFR-98 (44.0%) with exon 19 deletion, 116 (52.0%) with exon 21 L858R point mutation, and nine (4.0%) with another EGFR mutation. Overall, Fisher's exact test showed that PD-L1 positivity was associated with wild-type EGFR, and there was only one case with PD-L1 TPS ≥50% among the cases with mutant EGFR. The analysis of cases with mutant EGFR indicated no significant association between EGFR mutation site and PD-L1 expression. However, the prevalence of PD-L1 TPS 5-49% was higher among patients with EGFR exon 19 deletion than with EGFR exon 21 L858R point mutation. PD-L1 expression was significantly associated with wild-type EGFR, and PD-L1 TPS ≥50% seldom overlaps with presence of driver oncogene EGFR. There was no significant difference in PD-L1 expression among the EGFR mutation sites. Copyright © 2017 Elsevier B.V. All rights reserved.

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

PubMed

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma

PubMed Central

Cossu-Rocca, Paolo; Muroni, Maria R; Sanges, Francesca; Sotgiu, Giovanni; Asunis, Anna; Tanca, Luciana; Onnis, Daniela; Pira, Giovanna; Manca, Alessandra; Dore, Simone; Uras, Maria G; Ena, Sara; De Miglio, Maria R

2016-01-01

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms.

PubMed

Li, Jiannong; Bennett, Keiryn; Stukalov, Alexey; Fang, Bin; Zhang, Guolin; Yoshida, Takeshi; Okamoto, Isamu; Kim, Jae-Young; Song, Lanxi; Bai, Yun; Qian, Xiaoning; Rawal, Bhupendra; Schell, Michael; Grebien, Florian; Winter, Georg; Rix, Uwe; Eschrich, Steven; Colinge, Jacques; Koomen, John; Superti-Furga, Giulio; Haura, Eric B

2013-11-05

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.

Frequency of EGFR T790M mutation and multimutational profiles of rebiopsy samples from non-small cell lung cancer developing acquired resistance to EGFR tyrosine kinase inhibitors in Japanese patients.

PubMed

Ko, Ryo; Kenmotsu, Hirotsugu; Serizawa, Masakuni; Koh, Yasuhiro; Wakuda, Kazushige; Ono, Akira; Taira, Tetsuhiko; Naito, Tateaki; Murakami, Haruyasu; Isaka, Mitsuhiro; Endo, Masahiro; Nakajima, Takashi; Ohde, Yasuhisa; Yamamoto, Nobuyuki; Takahashi, Kazuhisa; Takahashi, Toshiaki

2016-11-08

The majority of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR tyrosine kinase inhibitors (TKIs). Minimal information exists regarding genetic alterations in rebiopsy samples from Asian NSCLC patients who develop acquired resistance to EGFR-TKIs. We retrospectively reviewed the medical records of patients with NSCLC harboring EGFR mutations who had undergone rebiopsies after developing acquired resistance to EGFR-TKIs. We analyzed 27 practicable samples using a tumor genotyping panel to assess 23 hot-spot sites of genetic alterations in nine genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, and HER2), gene copy number of EGFR, MET, PIK3CA, FGFR1, and FGFR2, and ALK, ROS1, and RET fusions. Additionally, 34 samples were analyzed by commercially available EGFR mutation tests. Sixty-one patients underwent rebiopsy. Twenty-seven samples were analyzed using our tumor genotyping panel, and 34 samples were analyzed for EGFR mutations only by commercial clinical laboratories. Twenty-one patients (34 %) had EGFR T790M mutation. Using our tumor genotyping panel, MET gene copy number gain was observed in two of 27 (7 %) samples. Twenty patients received continuous treatment with EGFR-TKIs even after disease progression, and 11 of these patients had T790M mutation in rebiopsy samples. In contrast, only 10 of 41 patients who finished EGFR-TKI treatment at disease progression had T790M mutation. The frequency of T790M mutation in patients who received continuous treatment with EGFR-TKIs after disease progression was significantly higher than that in patients who finished EGFR-TKI treatment at disease progression (55 % versus 24 %, p = 0.018). The frequency of T790M mutation in this study was lower than that in previous reports examining western patients. These results suggest that continuous treatment with EGFR-TKI after disease progression may enhance the frequency of EGFR T

Redox Regulation of EGFR Signaling Through Cysteine Oxidation1

PubMed Central

Truong, Thu H.; Carroll, Kate S.

2012-01-01

Epidermal growth factor receptor (EGFR) exemplifies the family of receptor tyrosine kinases that mediate numerous cellular processes including growth, proliferation and differentiation. Moreover, gene amplification and EGFR mutations have been identified in a number of human malignancies, making this receptor an important target for the development of anticancer drugs. In addition to ligand-dependent activation and concomitant tyrosine phosphorylation, EGFR stimulation results in the localized generation of H2O2 by NADPH-dependent oxidases. In turn, H2O2 functions as a secondary messenger to regulate intracellular signaling cascades, largely through the modification of specific cysteine residues within redox-sensitive protein targets, including Cys797 in the EGFR active site. In this review, we highlight recent advances in our understanding of the mechanisms that underlie redox regulation of EGFR signaling and how these discoveries may form the basis for development of new therapeutic strategies to target this and other H2O2-modulated pathways. PMID:23186290

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2015-09-01

AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina Politi...CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER 6...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Phenotypic changes have been observed in EGFR mutant lung cancers that become resistant to targeted

BIM Gene Polymorphism Lowers the Efficacy of EGFR-TKIs in Advanced Nonsmall Cell Lung Cancer With Sensitive EGFR Mutations: A Systematic Review and Meta-Analysis.

PubMed

Huang, Wu Feng; Liu, Ai Hua; Zhao, Hai Jin; Dong, Hang Ming; Liu, Lai Yu; Cai, Shao Xi

2015-08-01

The strong association between bcl-2-like 11 (BIM) triggered apoptosis and the presence of epidermal growth factor receptor (EGFR) mutations has been proven in nonsmall cell lung cancer (NSCLC). However, the relationship between EGFR-tyrosine kinase inhibitor's (TKI's) efficacy and BIM polymorphism in NSCLC EGFR is still unclear.Electronic databases were searched for eligible literatures. Data on objective response rates (ORRs), disease control rates (DCRs), and progression-free survival (PFS) stratified by BIM polymorphism status were extracted and synthesized based on random-effect model. Subgroup and sensitivity analyses were conducted.A total of 6 studies that involved a total of 773 EGFR mutant advanced NSCLC patients after EGFR-TKI treatment were included. In overall, non-BIM polymorphism patients were associated with significant prolonged PFS (hazard ratio 0.63, 0.47-0.83, P = 0.001) compared to patients with BIM polymorphism. However, only marginal improvements without statistical significance in ORR (odds ratio [OR] 1.71, 0.91-3.24, P = 0.097) and DCR (OR 1.56, 0.85-2.89, P = 0.153) were observed. Subgroup analyses showed that the benefits of PFS in non-BIM polymorphism group were predominantly presented in pooled results of studies involving chemotherapy-naive and the others, and retrospective studies. Additionally, we failed to observe any significant benefit from patients without BIM polymorphism in every subgroup for ORR and DCR.For advanced NSCLC EGFR mutant patients, non-BIM polymorphism ones are associated with longer PFS than those with BIM polymorphism after EGFR-TKIs treatment. BIM polymorphism status should be considered an essential factor in studies regarding EGFR-targeted agents toward EGFR mutant patients.

The International Association for the Study of Lung Cancer Consensus Statement on Optimizing Management of EGFR Mutation-Positive Non-Small Cell Lung Cancer: Status in 2016.

PubMed

Tan, Daniel S W; Yom, Sue S; Tsao, Ming S; Pass, Harvey I; Kelly, Karen; Peled, Nir; Yung, Rex C; Wistuba, Ignacio I; Yatabe, Yasushi; Unger, Michael; Mack, Philip C; Wynes, Murry W; Mitsudomi, Tetsuya; Weder, Walter; Yankelevitz, David; Herbst, Roy S; Gandara, David R; Carbone, David P; Bunn, Paul A; Mok, Tony S K; Hirsch, Fred R

2016-07-01

Mutations in the epidermal growth factor receptor gene (EGFR) represent one of the most frequent "actionable" alterations in non-small cell lung cancer (NSCLC). Typified by high response rates to targeted therapies, EGFR tyrosine kinase inhibitors (TKIs) are now established first-line treatment options and have transformed the treatment paradigm for NSCLC. With the recent breakthrough designation and approval of the third-generation EGFR TKI osimertinib, available systemic and local treatment options have expanded, requiring new clinical algorithms that take into account individual patient molecular and clinical profiles. In this International Association for the Study of Lung Cancer commissioned consensus statement, key pathologic, diagnostic, and therapeutic considerations, such as optimal choice of EGFR TKI and management of brain metastasis, are discussed. In addition, recommendations are made for clinical guidelines and research priorities, such as the role of repeat biopsies and use of circulating free DNA for molecular studies. With the rapid pace of progress in treating EGFR-mutant NSCLC, this statement provides a state-of-the-art review of the contemporary issues in managing this unique subgroup of patients. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Statistical analysis of EGFR structures' performance in virtual screening

NASA Astrophysics Data System (ADS)

Li, Yan; Li, Xiang; Dong, Zigang

2015-11-01

In this work the ability of EGFR structures to distinguish true inhibitors from decoys in docking and MM-PBSA is assessed by statistical procedures. The docking performance depends critically on the receptor conformation and bound state. The enrichment of known inhibitors is well correlated with the difference between EGFR structures rather than the bound-ligand property. The optimal structures for virtual screening can be selected based purely on the complex information. And the mixed combination of distinct EGFR conformations is recommended for ensemble docking. In MM-PBSA, a variety of EGFR structures have identically good performance in the scoring and ranking of known inhibitors, indicating that the choice of the receptor structure has little effect on the screening.

EGFR Gene Amplification and KRAS Mutation Predict Response to Combination Targeted Therapy in Metastatic Colorectal Cancer.

PubMed

Khan, Sajid A; Zeng, Zhaoshi; Shia, Jinru; Paty, Philip B

2017-07-01

Genetic variability in KRAS and EGFR predicts response to cetuximab in irinotecan refractory colorectal cancer. Whether these markers or others remain predictive in combination biologic therapies including bevacizumab is unknown. We identified predictive biomarkers from patients with irinotecan refractory metastatic colorectal cancer treated with cetuximab plus bevacizumab. Patients who received cetuximab plus bevacizumab for irinotecan refractory colorectal cancer in either of two Phase II trials conducted were identified. Tumor tissue was available for 33 patients. Genomic DNA was extracted and used for mutational analysis of KRAS, BRAF, and p53 genes. Fluorescence in situ hybridization was performed to assess EGFR copy number. The status of single genes and various combinations were tested for association with response. Seven of 33 patients responded to treatment. KRAS mutations were found in 14/33 cases, and 0 responded to treatment (p = 0.01). EGFR gene amplification was seen in 3/33 of tumors and in every case was associated with response to treatment (p < 0.001). TP53 and BRAF mutations were found in 18/33 and 0/33 tumors, respectively, and there were no associations with response to either gene. EGFR gene amplification and KRAS mutations are predictive markers for patients receiving combination biologic therapy of cetuximab plus bevacizumab for metastatic colorectal cancer. One marker or the other is present in the tumor of half of all patients allowing treatment response to be predicted with a high degree of certainty. The role for molecular markers in combination biologic therapy seems promising.

Development of the Third Generation EGFR Tyrosine Kinase Inhibitors for Anticancer Therapy.

PubMed

Cheng, Weiyan; Zhou, Jianhua; Tian, Xin; Zhang, Xiaojian

2016-01-01

Epidermal growth factor receptor (EGFR) is one of the most important targets in anticancer therapy. Till date, a large number of first and second generation EGFR tyrosine kinase inhibitors (TKIs) have been marketed or advanced into clinical studies. However, the occurrence of TKI-resistant mutations has led to the loss of efficacy of these inhibitors. In the purpose of overcoming resistant mutations and reducing side effects, lots of third generation EGFR inhibitors are explored with promising potencies against EGFR mutations while sparing wild-type EGFR. This review outlines the current landscape of the development of third generation EGFR inhibitors, mainly focusing on the biological properties, clinical status and structure-activity relationships.

Dimerization drives EGFR endocytosis through two sets of compatible endocytic codes.

PubMed

Wang, Qian; Chen, Xinmei; Wang, Zhixiang

2015-03-01

We have shown previously that epidermal growth factor (EGF) receptor (EGFR) endocytosis is controlled by EGFR dimerization. However, it is not clear how the dimerization drives receptor internalization. We propose that EGFR endocytosis is driven by dimerization, bringing two sets of endocytic codes, one contained in each receptor monomer, in close proximity. Here, we tested this hypothesis by generating specific homo- or hetero-dimers of various receptors and their mutants. We show that ErbB2 and ErbB3 homodimers are endocytosis deficient owing to the lack of endocytic codes. Interestingly, EGFR-ErbB2 or EGFR-ErbB3 heterodimers are also endocytosis deficient. Moreover, the heterodimer of EGFR and the endocytosis-deficient mutant EGFRΔ1005-1017 is also impaired in endocytosis. These results indicate that two sets of endocytic codes are required for receptor endocytosis. We found that an EGFR-PDGFRβ heterodimer is endocytosis deficient, although both EGFR and PDGFRβ homodimers are endocytosis-competent, indicating that two compatible sets of endocytic codes are required. Finally, we found that to mediate the endocytosis of the receptor dimer, the two sets of compatible endocytic codes, one contained in each receptor molecule, have to be spatially coordinated. © 2015. Published by The Company of Biologists Ltd.

EGFR and HER2 Activate Rigidity Sensing Only on Rigid Matrices

PubMed Central

Saxena, Mayur; Liu, Shuaimin; Yang, Bo; Hajal, Cynthia; Changede, Rishita; Hu, Junqiang

2017-01-01

Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two different cell lines, that in serum- and EGF-free conditions, EGFR or HER2 activity increase spreading and rigidity-sensing contractions on rigid, but not soft, substrates. Contractions peak after 15–20 min, but diminish by 10-fold after 4 hours. Addition of EGF at that point increases spreading and contractions, but this can be blocked by myosin-II inhibition. We further show that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFK). On soft surfaces, neither EGFR inhibition nor EGF stimulation have any effect on cell motility. Thus, EGFR or HER2 can catalyse rigidity sensing after associating with nascent adhesions under rigidity-dependent tension downstream of SFK activity. This has broad implications for the roles of EGFR and HER2 in absence of EGF both for normal and cancerous growth. PMID:28459445

Stem cell-like ALDHbright cellular states in EGFR-mutant non-small cell lung cancer

PubMed Central

Corominas-Faja, Bruna; Oliveras-Ferraros, Cristina; Cuyàs, Elisabet; Segura-Carretero, Antonio; Joven, Jorge; Martin-Castillo, Begoña; Barrajón-Catalán, Enrique; Micol, Vicente; Bosch-Barrera, Joaquim; Menendez, Javier A

2013-01-01

The enrichment of cancer stem cell (CSC)-like cellular states has not previously been considered to be a causative mechanism in the generalized progression of EGFR-mutant non-small cell lung carcinomas (NSCLC) after an initial response to the EGFR tyrosine kinase inhibitor erlotinib. To explore this possibility, we utilized a pre-clinical model of acquired erlotinib resistance established by growing NSCLC cells containing a TKI-sensitizing EGFR exon 19 deletion (ΔE746-A750) in the continuous presence of high doses of erlotinib. Genome-wide analyses using Agilent 44K Whole Human Genome Arrays were evaluated via bioinformatics analyses through GSEA-based screening of the KEGG pathway database to identify the molecular circuitries that were over-represented in the transcriptomic signatures of erlotinib-refractory cells. The genomic spaces related to erlotinib resistance included a preponderance of cell cycle genes (E2F1, -2, CDC2, -6) and DNA replication-related genes (MCM4, -5, -6, -7), most of which are associated with early lung development and poor prognosis. In addition, metabolic genes such as ALDH1A3 (a candidate marker for lung cancer cells with CSC-like properties) were identified. Thus, we measured the proportion of erlotinib-resistant cells expressing very high levels of aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms. Using flow cytometry and the ALDEFLUOR® reagent, we confirmed that erlotinib-refractory cell populations contained drastically higher percentages (>4500%) of ALDHbright cells than the parental erlotinib-responsive cells. Notably, strong decreases in the percentages of ALDHbright cells were observed following incubation with silibinin, a bioactive flavonolignan that can circumvent erlotinib resistance in vivo. The number of lung cancer spheres was drastically suppressed by silibinin in a dose-dependent manner, thus confirming the ability of this agent to inhibit the self-renewal of erlotinib-refractory CSC-like cells

Bevacizumab plus chemotherapy for patients with advanced pulmonary adenocarcinoma harboring EGFR mutations.

PubMed

Chen, R-L; Chen, H-J; Jiang, B-Y; Zhang, X-C; Zhou, Q; Tu, H-Y; Zhong, W-Z; Wu, Y-L; Yang, J-J

2018-02-01

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and bevacizumab plus chemotherapy were effective for EGFR-mutant patients. However, the appropriated treatment orders remained controvertible. We investigated the efficacy of treatment orders between bevacizumab plus chemotherapy and EGFR-TKIs for EGFR-mutant patients with advanced pulmonary adenocarcinoma. This study involved 40 EGFR-mutant patients with advanced pulmonary adenocarcinoma who were treated with bevacizumab plus carboplatin and paclitaxel (Bev + CP) and EGFR-TKIs in different treatment orders or gemcitabine plus cisplatin (GP) in first-line setting. Seventeen patients were treated with Bev + CP and 10 cases with GP in first-line treatment. Thirteen patients received EGFR-TKIs after first-line Bev + CP regimen, while 13 patients were treated with first-line EGFR-TKIs. Progression-free survival (PFS), the response rate (ORR) and overall survival (OS) were evaluated. Median PFS of Bev + CP treatment was significantly longer in first-line than non-first-line settings (11.7 vs. 5.6 months, P = 0.003). Median OS was 37.8 months for EGFR-mutant patients with first-line Bev + CP followed by second-line EGFR-TKIs and 31.0 months for those with first-line EGFR-TKIs and non-first-line Bev + CP, respectively (P = 0.509). Median PFS was 11.7 (95% CI 10.6-12.8) months for Bev + CP group and 4.7 (95% CI 4.4-5.0) months for GP group with the hazard ratio of 0.17 (P = 0.001). ORR was 70.6 and 50.0% in the two groups, respectively (P = 0.415). However, there was no significant difference in median OS (33.7 vs 27.8 months, P = 0.293). First-line Bev + CP followed by EGFR-TKIs might possibly provide favorable prognosis for EGFR-mutant patients. Bev + CP regimen significantly prolonged PFS in first-line than non-first-line settings. These findings warrant further investigations.

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

PubMed Central

Needham, Sarah R.; Roberts, Selene K.; Arkhipov, Anton; Mysore, Venkatesh P.; Tynan, Christopher J.; Zanetti-Domingues, Laura C.; Kim, Eric T.; Losasso, Valeria; Korovesis, Dimitrios; Hirsch, Michael; Rolfe, Daniel J.; Clarke, David T.; Winn, Martyn D.; Lajevardipour, Alireza; Clayton, Andrew H. A.; Pike, Linda J.; Perani, Michela; Parker, Peter J.; Shan, Yibing; Shaw, David E.; Martin-Fernandez, Marisa L.

2016-01-01

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling. PMID:27796308

The BIM Deletion Polymorphism and its Clinical Implication in Patients with EGFR-Mutant Non-Small-Cell Lung Cancer Treated with EGFR Tyrosine Kinase Inhibitors.

PubMed

Lee, Ji Yun; Ku, Bo Mi; Lim, Sung Hee; Lee, Min-Young; Kim, Haesu; Kim, Moonjin; Kim, Sungmin; Jung, Hyun Ae; Sun, Jong-Mu; Ahn, Jin Seok; Park, Keunchil; Ahn, Myung-Ju

2015-06-01

A germline BIM deletion polymorphism has been proposed to predict poor treatment response to certain kinase inhibitors. The purpose of this study was to explore whether the BIM deletion polymorphism predicts treatment efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in Korean patients with EGFR-mutant non-small-cell lung cancer (NSCLC). Peripheral blood samples from a total of 205 patients with EGFR-mutant NSCLC who were treated with EGFR TKIs between July 2008 and April 2013 were included. The incidence of BIM deletions in these samples was detected by polymerase chain reaction. We compared the clinical outcomes in patients with and without the polymorphism after treatment with EGFR TKIs (gefitinib or erlotinib). The BIM deletion polymorphism was present in 15.6% (32 of 205) of patients. One patient was homozygous for the deletion, and the remaining 31 had heterozygous deletions. The majority of patients were younger than 65 years (74%), female (68%), never smokers (76%), and had stage IV NSCLC (67%). There were no associations between the BIM deletion polymorphism and clinicopathological features including gender, age, smoking status, histology, stage, and number of metastasis sites. Patients with and without the BIM deletion polymorphism had similar objective response rates (91 vs. 84%, p = 0.585). Progression-free survival and overall survival did not differ significantly between patients with and without the BIM deletion polymorphism (median progression-free survival 12 vs. 11 months, p = 0.160; median overall survival 31 vs. 30 months, p = 0.452). Multivariate analysis identified significantly predictive markers for clinical outcomes of EGFR TKIs including Eastern Cooperative Oncology Group performance status 0-1, adenocarcinoma histology, recurrent disease, and EGFR mutation type. The results were validated in an independent cohort of 69 NSCLC patients. It remains to be determined whether the BIM deletion polymorphism

Treating EGFR mutation resistance in non-small cell lung cancer - role of osimertinib.

PubMed

Mazza, Valentina; Cappuzzo, Federico

2017-01-01

The discovery of mutations in EGFR significantly changed the treatment paradigm of patients with EGFR -mutant non-small cell lung cancer (NSCLC), a particular group of patients with different clinical characteristics and outcome to EGFR -wild-type patients. In these patients, the treatment of choice as first-line therapy is first- or second-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, erlotinib, or afatinib. Inevitably, after the initial response, all patients become refractory to these drugs. The most common mechanism of acquired resistance to EGFR-TKIs is the development of a second mutation in exon 20 of EGFR ( T790M ). Osimertinib is a third-generation EGFR-TKI designed for overcoming T790M -mediated resistance. Based on the results of efficacy and tolerability of Phase II and Phase III studies, osimertinib has been approved for treatment of advanced EGFR T790M+ mutation NSCLC following progression on a prior EGFR-TKI. Occurrence of acquired resistance to osimertinib represents an urgent need for additional strategies including combination with other agents, such as other targeted therapies or checkpoint inhibitors, or development of new and more potent compounds.

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Biomarker-driven EGFR therapy improves outcomes in patients with metastatic colorectal cancer.

PubMed

Hendifar, Andrew; Tan, Carlyn-Rose; Annamalai, Anand; Tuli, Richard

2014-09-01

As new data from randomized studies comparing EGFR-targeting therapies with VEGF inhibitors emerge, the treatment landscape for metastatic colorectal cancer is expected to change. Although both the VEGF inhibitor bevacizumab and the anti-EGFR antibody cetuximab are approved in the first-line setting, they have not until recently been compared directly in randomized studies. Unlike targeted therapy in the EGFR pathway, there are no biomarkers guiding VEGF treatment. Recent data, discussed in this review, demonstrate that patients with KRAS/NRAS wild-type tumors benefit from anti-EGFR therapy in the first-line setting and that anti-EGFR therapy may be superior when compared with anti-VEGF approaches. This review focuses on the clinical utility of targeting EGFR by revisiting the biologic rationale for EGFR inhibition in metastatic colorectal cancer and providing new insight on the advancements in biomarker analyses with the potential to change practice.

The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer

PubMed Central

Wang, Shu; Yu, Bing; Ng, Chiu Chin; Mercorella, Belinda; Selinger, Christina I.; O’Toole, Sandra A.

2015-01-01

Background Patients with advanced non-small cell lung cancer (NSCLC) benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) when their tumor harbors an activating EGFR mutation. As the majority of NSCLC patients present with advanced disease, cytology and small biopsy specimens are frequently the only tissue available for mutation testing, but can pose challenges due to low tumor content. We aim to better define the suitability of these specimens for mutation testing. Methods NSCLC cases referred to our institution for mutation testing over a 15-month period were retrospectively reviewed. Specimens were tested for mutations including EGFR, KRAS, and BRAF, using a multiplex PCR assay (OncoCarta Panel v1.0) and analyzed on the Agena Bioscience MassARRAY platform. Results A total of 146 specimens were tested, comprising 53 (36.3%) resection specimens (including 28 lung resection specimens), 55 (37.7%) small biopsy specimens and 38 (26%) cytology specimens. Of 142 cases with sufficient DNA for mutation testing, EGFR mutations were detected in 31 specimens (21.8%), KRAS mutations in 31 specimens (21.8%) and BRAF mutations in three specimens (2.1%). There was no significant difference in the EGFR mutation rate between lung resection (10 of 28 cases; 35.7%), small biopsy (9 of 53 cases; 17%), and cytology specimens (8 of 36 cases; 22.2%). Conclusions Our results support the utility of small biopsy and cytology specimens for mutation testing. Careful evaluation of the adequacy of small specimens is required to minimize the risk of false negative or positive results. PMID:25870794

Ultrasensitive detection of EGFR gene based on surface plasmon resonance enhanced electrochemiluminescence of CuZnInS quantum dots.

PubMed

Chen, Xueqian; Gui, Wenying; Ma, Qiang

2018-06-07

In our work, a novel DNA electrochemiluminescence (ECL) sensor based on CuZnInS quantum dots (QDs) and gold-nanoparticles (Au NPs) is developed for highly sensitive detection of epidermal growth factor receptor (EGFR) Gene, which has a close relation with the lung cancer. The CuZnInS QDs work as a novel kind of ECL luminophore, whose defect state emission is suitable for ECL sensing. To enhance the sensitivity of the sensing system, Au NPs are utilized creatively to strengthen the ECL intensity of CuZnInS QD S according to the surface plasmon resonance (SPR) effect. An ultrasensitive and universal detecting platform is built based on the SPR effect between Au NPs and CuZnInS QD S . The effect of the capped stabilizer on the ECL signal of QDs is firstly investigated. Three different stabilizers are used to cap the CuZnInS QDs, including mercaptopropionic acid (MPA), l-glutathione (GSH) and cysteamine (CA). MPA capped CuZnInS QDs possess the strongest ECL intensity among the three kinds of the CuZnInS QDs. Under the optimum conditions, a good linear relationship between ECL intensity and the concentration of target DNA is obtained in the range from 0.05 nmol L -1 to 1 nmol L -1 . The detection limit is 0.0043 nmol L -1 . The proposed DNA sensor has been employed for the determination of target DNA EGFR in human serum samples with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

Prevalence of EGFR Mutations in Lung Cancer in Uruguayan Population

PubMed Central

Touya, Diego; Bertoni, Bernardo; Osinaga, Eduardo; Varangot, Mario

2017-01-01

Background Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR) gene into routine clinical practice represents a milestone for personalized therapy of the non-small-cell lung cancer (NSCLC). However, the genetic testing of EGFR mutations has not yet become a routine clinical practice in developing countries. In view of different prevalence of such mutations among different ethnicities and geographic regions, as well as the limited existing data from Latin America, our aim was to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients from Uruguay. Methods We examined EGFR mutations in exons 18 through 21 in 289 NSCLC Uruguayan patients by PCR-direct sequencing. Results EGFR mutations were detected in 53 of the 289 (18.3%) patients, more frequently in women (23.4%) than in men (14.5%). The distribution by exon was similar to that generally reported in the literature. Conclusions This first epidemiological study of EGFR mutations in Uruguay reveals a wide spectrum of mutations and an overall prevalence of 18.3%. The background ethnic structure of the Uruguayan population could play an important role in explaining our findings. PMID:28744312

Prognostic Significance of NSCLC and Response to EGFR-TKIs of EGFR-Mutated NSCLC Based on PD-L1 Expression.

PubMed

Kobayashi, Kenichi; Seike, Masahiro; Zou, Fenfei; Noro, Rintaro; Chiba, Mika; Ishikawa, Arimi; Kunugi, Shinobu; Kubota, Kaoru; Gemma, Akihiko

2018-02-01

Recent clinical trials have shown that immune checkpoint blockades that target either PD-1 or PD-L1 yield remarkable responses in a subgroup of patients with non-small cell lung cancer (NSCLC). We retrospectively examined, by immunohistochemical analysis, 211 NSCLC samples. Using 32 independent samples, we also evaluated PD-L1 expression in NSCLC patients with EGFR gene mutations treated by EGFR-TKIs. Overall survival of PD-L1-positive stages I-III NSCLC and stage I NSCLC and stages I-III squamous cell carcinoma (SQ) were significantly shorter than those of PD-L1-negative NSCLC (p<0.01 and p=0.02 and p=0.01, respectively). In stage I NSCLC and stages I-III SQ, PD-L1 expression was found to be independent predictor of death after multivariate analysis. Response to EGFR-TKIs was not significantly different between PD-L1-positive and PD-L1-negative NSCLC patients with EGFR mutations. PD-L1 expression was a significant independent predictor of poor outcome in NSCLC patients. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Novel agents that downregulate EGFR, HER2, and HER3 in parallel

PubMed Central

Ferreira, Renan Barroso; Law, Mary Elizabeth; Jahn, Stephan Christopher; Davis, Bradley John; Heldermon, Coy Don; Reinhard, Mary; Castellano, Ronald Keith; Law, Brian Keith

2015-01-01

EGFR, HER2, and HER3 contribute to the initiation and progression of human cancers, and are therapeutic targets for monoclonal antibodies and tyrosine kinase inhibitors. An important source of resistance to these agents arises from functional redundancy among EGFR, HER2, and HER3. EGFR family members contain conserved extracellular structures that are stabilized by disulfide bonds. Compounds that disrupt extracellular disulfide bonds could inactivate EGFR, HER2, and HER3 in unison. Here we describe the identification of compounds that kill breast cancer cells that overexpress EGFR or HER2. Cell death parallels downregulation of EGFR, HER2, and HER3. These compounds disrupt disulfide bonds and are termed Disulfide Bond Disrupting Agents (DDAs). DDA RBF3 exhibits anticancer efficacy in vivo at 40 mg/kg without evidence of toxicity. DDAs may complement existing EGFR-, HER2-, and HER3-targeted agents that function through alternate mechanisms of action, and combination regimens with these existing drugs may overcome therapeutic resistance. PMID:25865227

InsR/IGF1R pathway mediates resistance to EGFR inhibitors in glioblastoma

PubMed Central

Ma, Yufang; Tang, Nan; Thompson, Reid; Mobley, Bret C.; Clark, Steven W.; Sarkaria, Jann N.; Wang, Jialiang

2015-01-01

Purpose Aberrant activation of epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR inhibitors exhibit at best modest efficacy in glioblastoma. This is in sharp contrast to the observations in EGFR-mutant lung cancer. We examined whether activation of functionally redundant receptor tyrosine kinases (RTKs) conferred resistance to EGFR inhibitors in glioblastoma. Experimental Design We collected a panel of patient-derived glioblastoma xenograft (PDX) lines that maintained expression of wild type or mutant EGFR in serial xenotransplantation and tissue cultures. Using this physiologically relevant platform, we tested the abilities of several RTK ligands to protect glioblastoma cells against an EGFR inhibitor, gefitinib. Based on the screening results, we further developed a combination therapy co-targeting EGFR and insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF1R). Results Insulin and IGF1 induced significant protection against gefitinib in the majority of EGFR-dependent PDX lines with one exception that did not expression InsR or IGF1R. Blockade of the InsR/IGF1R pathway synergistically improved sensitivity to gefitinib or dacomitinib. Gefitinib alone effectively attenuated EGFR activities and the downstream MEK/ERK pathway. However, repression of AKT and induction of apoptosis required concurrent inhibition of both EGFR and InsR/IGF1R. A combination of gefitinib and OSI-906, a dual InsR/IGF1R inhibitor, was more effective than either agent alone to treat subcutaneous glioblastoma xenograft tumors. Conclusions Our results suggest that activation of the InsR/IGF1R pathway confers resistance to EGFR inhibitors in EGFR-dependent glioblastoma through AKT regulation. Concurrent blockade of these two pathways holds promise to treat EGFR-dependent glioblastoma. PMID:26561558

Identification of a novel fusion gene HMGA2-EGFR in glioblastoma.

PubMed

Komuro, Akiyoshi; Raja, Erna; Iwata, Caname; Soda, Manabu; Isogaya, Kazunobu; Yuki, Keiko; Ino, Yasushi; Morikawa, Masato; Todo, Tomoki; Aburatani, Hiroyuki; Suzuki, Hiromichi; Ranjit, Melissa; Natsume, Atsushi; Mukasa, Akitake; Saito, Nobuhito; Okada, Hitoshi; Mano, Hiroyuki; Miyazono, Kohei; Koinuma, Daizo

2018-04-15

Glioblastoma is one of the most malignant forms of cancer, for which no effective targeted therapy has been found. Although The Cancer Genome Atlas has provided a list of fusion genes in glioblastoma, their role in progression of glioblastoma remains largely unknown. To search for novel fusion genes, we obtained RNA-seq data from TGS-01 human glioma-initiating cells, and identified a novel fusion gene (HMGA2-EGFR), encoding a protein comprising the N-terminal region of the high-mobility group AT-hook protein 2 (HMGA2) fused to the C-terminal region of epidermal growth factor receptor (EGFR), which retained the transmembrane and kinase domains of the EGFR. This fusion gene product showed transforming potential and a high tumor-forming capacity in cell culture and in vivo. Mechanistically, HMGA2-EGFR constitutively induced a higher level of phosphorylated STAT5B than EGFRvIII, an in-frame exon deletion product of the EGFR gene that is commonly found in primary glioblastoma. Forced expression of HMGA2-EGFR enhanced orthotopic tumor formation of the U87MG human glioma cell line. Furthermore, the EGFR kinase inhibitor erlotinib blocked sphere formation of TGS-01 cells in culture and inhibited tumor formation in vivo. These findings suggest that, in addition to gene amplification and in-frame exon deletion, EGFR signaling can also be activated by gene fusion, suggesting a possible avenue for treatment of glioblastoma. © 2017 UICC.

STAT3 signaling mediates tumour resistance to EGFR targeted therapeutics.

PubMed

Zulkifli, Ahmad A; Tan, Fiona H; Putoczki, Tracy L; Stylli, Stanley S; Luwor, Rodney B

2017-08-15

Several EGFR inhibitors are currently undergoing clinical assessment or are approved for the clinical management of patients with varying tumour types. However, treatment often results in a lack of response in many patients. The majority of patients that initially respond eventually present with tumours that display acquired resistance to the original therapy. A large number of receptor tyrosine and intracellular kinases have been implicated in driving signaling that mediates this tumour resistance to anti-EGFR targeted therapy, and in a few cases these discoveries have led to overall changes in prospective tumour screening and clinical practice (K-RAS in mCRC and EGFR T790M in NSCLC). In this mini-review, we specifically focus on the role of the STAT3 signaling axis in providing both intrinsic and acquired resistance to inhibitors of the EGFR. We also focus on STAT3 pathway targeting in an attempt to overcome resistance to anti-EGFR therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

MLH1 V384D polymorphism associates with poor response to EGFR tyrosine kinase inhibitors in patients with EGFR L858R-positive lung adenocarcinoma.

PubMed

Chiu, Chao-Hua; Ho, Hsiang-Ling; Doong, Howard; Yeh, Yi-Chen; Chen, Mei-Yu; Chou, Teh-Ying; Tsai, Chun-Ming

2015-04-10

A significant fraction of patients with lung adenocarcinomas harboring activating epidermal growth factor receptor (EGFR) mutations do not experience clinical benefits from EGFR tyrosine kinase inhibitor (TKI) therapy. Using next-generation sequencing, we screened 739 mutation hotspots in 46 cancer-related genes in EGFR L858R-mutant lung adenocarcinomas from 29 patients who received EGFR-TKI therapy; 13 had short (< 3 months) and 16 had long (> 1 year) progression-free survival (PFS). We discovered MLH1 V384D as a genetic variant enriched in the group of patients with short PFS. Next, we investigated this genetic variation in 158 lung adenocarcinomas with the EGFR L858R mutation and found 14 (8.9%) patients had MLH1 V384D; available blood or non-tumor tissues from patients were also tested positive for MLH1 V384D. Patients with MLH1 V384D had a significantly shorter median PFS than those without (5.1 vs. 10.6 months; P= 0.001). Multivariate analysis showed that MLH1 V384D polymorphism was an independent predictor for a reduced PFS time (hazard ratio, 3.5; 95% confidence interval, 1.7 to 7.2; P= 0.001). In conclusion, MLH1 V384D polymorphism is associated with primary resistance to EGFR-TKIs in patients with EGFR L858R-positive lung adenocarcinoma and may potentially be a novel biomarker to guide treatment decisions.

Contribution of EGFR and ErbB-3 Heterodimerization to the EGFR Mutation-Induced Gefitinib- and Erlotinib-Resistance in Non-Small-Cell Lung Carcinoma Treatments.

PubMed

Wang, Debby D; Ma, Lichun; Wong, Maria P; Lee, Victor H F; Yan, Hong

2015-01-01

EGFR mutation-induced drug resistance has become a major threat to the treatment of non-small-cell lung carcinoma. Essentially, the resistance mechanism involves modifications of the intracellular signaling pathways. In our work, we separately investigated the EGFR and ErbB-3 heterodimerization, regarded as the origin of intracellular signaling pathways. On one hand, we combined the molecular interaction in EGFR heterodimerization with that between the EGFR tyrosine kinase and its inhibitor. For 168 clinical subjects, we characterized their corresponding EGFR mutations using molecular interactions, with three potential dimerization partners (ErbB-2, IGF-1R and c-Met) of EGFR and two of its small molecule inhibitors (gefitinib and erlotinib). Based on molecular dynamics simulations and structural analysis, we modeled these mutant-partner or mutant-inhibitor interactions using binding free energy and its components. As a consequence, the mutant-partner interactions are amplified for mutants L858R and L858R_T790M, compared to the wild type EGFR. Mutant delL747_P753insS represents the largest difference between the mutant-IGF-1R interaction and the mutant-inhibitor interaction, which explains the shorter progression-free survival of an inhibitor to this mutant type. Besides, feature sets including different energy components were constructed, and efficient regression trees were applied to map these features to the progression-free survival of an inhibitor. On the other hand, we comparably examined the interactions between ErbB-3 and its partners (EGFR mutants, IGF-1R, ErbB-2 and c-Met). Compared to others, c-Met shows a remarkably-strong binding with ErbB-3, implying its significant role in regulating ErbB-3 signaling. Moreover, EGFR mutants corresponding to poor clinical outcomes, such as L858R_T790M, possess lower binding affinities with ErbB-3 than c-Met does. This may promote the communication between ErbB-3 and c-Met in these cancer cells. The analysis verified

Inhibition of GRP78 abrogates radioresistance in oropharyngeal carcinoma cells after EGFR inhibition by cetuximab.

PubMed

Sun, Chaonan; Han, Chuyang; Jiang, Yuanjun; Han, Ning; Zhang, Miao; Li, Guang; Qiao, Qiao

2017-01-01

The EGFR-specific mAb cetuximab is one of the most effective treatments for oropharyngeal carcinoma, while patient responses to EGFR inhibitors given alone are modest. Combination treatment with radiation can improve the efficacy of treatment through increasing radiosensitivity, while resistance to radiation after administration of cetuximab limits its efficiency. Radiation and drugs can damage the endoplasmic reticulum (ER) homeostatic state and result in ER stress (ERS), subsequently causing resistance to radiation and drugs. Whether the ERS pathway is involved in radioresistance after administration of cetuximab has not been reported. Herein, we show that cetuximab could increase the radiosensitivity of FaDu cells but not Detroit562 cells. In addition, cetuximab inhibited the radiation-induced activation of the ERS signalling pathway IRE1α/ATF6-GRP78 in FaDu cells, while this effect was absent in Detroit562 cells. Silencing GRP78 increased the radiosensitivity of oropharyngeal carcinoma cells and inhibited radiation-induced DNA double-strand-break (DSB) repair and autophagy. More interestingly, silencing GRP78 abrogated resistance to cetuximab and radiation in Detroit562 cells and had a synergistic effect with cetuximab in increasing the radiosensitivity of FaDu cells. Immunohistochemistry showed that overexpression of both GRP78 and EGFR was associated with a poor prognosis in oropharyngeal carcinoma patients (P<0.05). Overall, the results of this study show that radioresistance after EGFR inhibition by cetuximab is mediated by the ERS signalling pathway IRE1α/ATF6-GRP78. This suppression was consequently unable to inhibit radiation-induced DSB repair and autophagy in oropharyngeal carcinoma cells, which conferred resistance to radiotherapy and cetuximab. These results suggest that the cooperative effects of radiotherapy and cetuximab could be further improved by inhibiting GRP78 in non-responsive oropharyngeal carcinoma patients.

EGFR-targeted nonviral NIS gene transfer for bioimaging and therapy of disseminated colon cancer metastases

PubMed Central

Urnauer, Sarah; Müller, Andrea M.; Schug, Christina; Schmohl, Kathrin A.; Tutter, Mariella; Schwenk, Nathalie; Rödl, Wolfgang; Morys, Stephan; Ingrisch, Michael; Bertram, Jens; Bartenstein, Peter; Clevert, Dirk-André; Wagner, Ernst; Spitzweg, Christine

2017-01-01

Liver metastases present a serious problem in the therapy of advanced colorectal cancer (CRC), as more than 20% of patients have distant metastases at the time of diagnosis with less than 5% being cured. Consequently, new therapeutic approaches are of major need together with high-resolution imaging methods that allow highly specific detection of small metastases. The unique combination of reporter and therapy gene function of the sodium iodide symporter (NIS) may represent a promising theranostic strategy for CRC liver metastases allowing non-invasive imaging of functional NIS expression and therapeutic application of 131I. For targeted NIS gene transfer polymers containing linear polyethylenimine (LPEI), polyethylene glycol (PEG) and the epidermal growth factor receptor (EGFR)-specific ligand GE11 were complexed with human NIS DNA (LPEI-PEG-GE11/NIS). Tumor specificity and transduction efficiency were examined in high EGFR-expressing LS174T metastases by non-invasive imaging using 18F-tetrafluoroborate (18F-TFB) as novel NIS PET tracer. Mice that were injected with LPEI-PEG-GE11/NIS 48 h before 18F-TFB application showed high tumoral levels (4.8±0.6% of injected dose) of NIS-mediated radionuclide uptake in comparison to low levels detected in mice that received untargeted control polyplexes. Three cycles of intravenous injection of EGFR-targeted NIS polyplexes followed by therapeutic application of 55.5 MBq 131I resulted in marked delay in metastases spread, which was associated with improved animal survival. In conclusion, these preclinical data confirm the enormous potential of EGFR-targeted synthetic polymers for systemic NIS gene delivery in an advanced multifocal CRC liver metastases model and open the exciting prospect of NIS-mediated radionuclide therapy in metastatic disease. PMID:29190908

Comparison of Thoracic Radiotherapy Efficacy Between Patients With and Without EGFR-mutated Lung Adenocarcinoma.

PubMed

Li, Ming-Hsien; Tsai, Jo-Ting; Ting, Lai-Lei; Lin, Jang-Chun; Liu, Yu-Chang

2018-01-01

To investigate the association between tumor response to thoracic radiotherapy and epidermal growth factor receptor (EGFR) mutation status in patients with lung adenocarcinoma, we collected 48 patients treated between January 2010 and December 2013. Of the 18 patients with EGFR mutation, 15 (83.3%) had a single mutation, and three (16.7%) had double mutation. Different EGFR mutation subtypes exhibited different responses to radiotherapy. The identified double EGFR mutations were associated with reduction of residual tumor burden (RTB) after radiotherapy. In univariate analysis, EGFR mutations in exon 18, 20, and 21 and double EGFR mutation were significant factors predicting RTB. In multivariate analysis, exon 20 mutation was the only significant factor. Patients with EGFR mutation seemed to have longer mean overall survival (OS) compared to the group with wild-type EGFR (31.1 vs. 26.6 months, p=0.49). The median and mean OS in patients with double EGFR mutation vs. wild-type EGFR were 20.1 vs. 16.9 months and 28.9 vs. 26.6 months, respectively. Further studies with larger sample size are warranted to clarify the association of EGFR mutation status with the lung tumor response after radiotherapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Afatinib and Cetuximab in Four Patients With EGFR Exon 20 Insertion-Positive Advanced NSCLC.

PubMed

van Veggel, Bianca; de Langen, Adrianus J; Hashemi, Sayed M S; Monkhorst, Kim; Heideman, Daniëlle A M; Thunnissen, Erik; Smit, Egbert F

2018-04-24

EGFR exon 20 insertions comprise 4% to 9% of EGFR mutated NSCLC. Despite being an oncogenic driver, they are associated with primary resistance to EGFR tyrosine kinase inhibitors (TKIs). We hypothesized that dual EGFR blockade with afatinib, an irreversible EGFR TKI, and cetuximab, a monoclonal antibody against EGFR, could induce tumor responses. Four patients with EGFR exon 20 insertion-positive NSCLC were treated with afatinib 40 mg once daily and cetuximab 250 mg/m 2 to 500 mg/m 2 every 2 weeks. All patients had stage IV adenocarcinoma of the lung harboring an EGFR exon 20 insertion mutation. Previous lines of treatment consisted of platinum doublet chemotherapy (n = 4) and EGFR TKI (n = 2). Three of four patients showed a partial response according to Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Median progression-free survival was 5.4 months (95% confidence interval: 0.0 - 14.2 months; range 2.7 months - 17.6 months). Toxicity was manageable with appropriate skin management and dose reduction being required in two patients. Dual EGFR blockade with afatinib and cetuximab may induce tumor responses in patients with EGFR exon 20 insertion-positive NSCLC. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC.

PubMed

Che, Ting-Fang; Lin, Ching-Wen; Wu, Yi-Ying; Chen, Yu-Ju; Han, Chia-Li; Chang, Yih-leong; Wu, Chen-Tu; Hsiao, Tzu-Hung; Hong, Tse-Ming; Yang, Pan-Chyr

2015-11-10

Dysfunction of the mitochondria is well-known for being associated with cancer progression. In the present study, we analyzed the mitochondria proteomics of lung cancer cell lines with different invasion abilities and found that EGFR is highly expressed in the mitochondria of highly invasive non-small-cell lung cancer (NSCLC) cells. EGF induces the mitochondrial translocation of EGFR; further, it leads to mitochondrial fission and redistribution in the lamellipodia, upregulates cellular ATP production, and enhances motility in vitro and in vivo. Moreover, EGFR can regulate mitochondrial dynamics by interacting with Mfn1 and disturbing Mfn1 polymerization. Overexpression of Mfn1 reverses the phenotypes resulting from EGFR mitochondrial translocation. We show that the mitochondrial EGFR expressions are higher in paired samples of the metastatic lymph node as compared with primary lung tumor and are inversely correlated with the overall survival in NSCLC patients. Therefore, our results demonstrate that besides the canonical role of EGFR as a receptor tyrosine, the mitochondrial translocation of EGFR may enhance cancer invasion and metastasis through regulating mitochondria dynamics.

EGFR is involved in dermatofibrosarcoma protuberans progression to high grade sarcoma.

PubMed

Osio, Amélie; Xu, Shuo; El Bouchtaoui, Morad; Leboeuf, Christophe; Gapihan, Guillaume; Lemaignan, Christine; Bousquet, Guilhem; Lebbé, Céleste; Janin, Anne; Battistella, Maxime

2018-02-02

Dermatofibrosarcoma protuberans (DFSP), amounting to 6% of all soft tissue sarcomas, has a slow growth rate, contrasting with a likelihood for local recurrence and a 10-20% evolution to higher-grade sarcoma, or "transformed DFSP" (DFSP-T). At molecular level, the characteristic COL1A1-PDGFB rearrangement, leading to sustained PDGFR signaling, is not linked to the evolutive potential. Here, we studied EGFR, another tyrosine kinase receptor, using laser-microdissection to select the different histologic components of DFSP (DFSP center, DFSP infiltrative periphery, DFSP-T higher-grade sarcoma), in 22 patients followed over 3 to 156 months. EGFR protein and mRNA were expressed in 13/22 patients with DFSP or DFSP-T, and increased with tumor progression, both in microdissected areas of higher-grade sarcomas and in microdissected areas of local extension. No cancer-associated EGFR gene mutation or copy-number variation, nor any KRAS, BRAF, NRAS hotspot mutations were found in any microdissected area. Among epithelial-mesenchymal transition factors tested, SNAIL 1/2 had the same expression pattern as EGFR while ZEB1/2 or TWIST1/2 did not. Using a proteome profiler phospho-kinase array on 3 DFSP and 3 DFSP-T cryopreserved tissue samples, EGFR phosphorylation was detected in each case. Among EGFR downstream pathways, we found positive correlations between phosphorylation levels of EGFR and STAT5a/b (r = 0.87, p < 0.05) and TOR (r = 0.95, p < 0.01), but not ERK in the MAPK pathway (r = -0.18, p > 0.70). We thus demonstrated that in DFSP evolution to high grade sarcoma, EGFR and SNAIL were involved, with EGFR activation and signaling through TOR and STAT5a/b downstream effectors, which could lead on to new therapies for advanced DFSP.

2D-QSAR and 3D-QSAR Analyses for EGFR Inhibitors

PubMed Central

Zhao, Manman; Zheng, Linfeng; Qiu, Chun

2017-01-01

Epidermal growth factor receptor (EGFR) is an important target for cancer therapy. In this study, EGFR inhibitors were investigated to build a two-dimensional quantitative structure-activity relationship (2D-QSAR) model and a three-dimensional quantitative structure-activity relationship (3D-QSAR) model. In the 2D-QSAR model, the support vector machine (SVM) classifier combined with the feature selection method was applied to predict whether a compound was an EGFR inhibitor. As a result, the prediction accuracy of the 2D-QSAR model was 98.99% by using tenfold cross-validation test and 97.67% by using independent set test. Then, in the 3D-QSAR model, the model with q2 = 0.565 (cross-validated correlation coefficient) and r2 = 0.888 (non-cross-validated correlation coefficient) was built to predict the activity of EGFR inhibitors. The mean absolute error (MAE) of the training set and test set was 0.308 log units and 0.526 log units, respectively. In addition, molecular docking was also employed to investigate the interaction between EGFR inhibitors and EGFR. PMID:28630865

MT4-MMP and EGFR expression levels are key biomarkers for breast cancer patient response to chemotherapy and erlotinib

PubMed Central

Yip, Cassandre; Foidart, Pierre; Somja, Joan; Truong, Alice; Lienard, Mehdi; Feyereisen, Emilie; Schroeder, Hélène; Gofflot, Stéphanie; Donneau, Anne-Françoise; Collignon, Joëlle; Delvenne, Philippe; Sounni, Nor Eddine; Jerusalem, Guy; Noël, Agnès

2017-01-01

Background: Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC. Methods: We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays. Results: Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours. Conclusions: We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs. PMID:28196064

Erlotinib for Patients with EGFR Wild-Type Metastatic NSCLC: a Retrospective Biomarkers Analysis.

PubMed

Inno, Alessandro; Di Noia, Vincenzo; Martini, Maurizio; D'Argento, Ettore; Di Salvatore, Mariantonietta; Arena, Vincenzo; Schinzari, Giovanni; Orlandi, Armando; Larocca, Luigi Maria; Cassano, Alessandra; Barone, Carlo

2018-03-20

Erlotinib is approved for the treatment of patients with EGFR mutation positive, metastatic NSCLC. It is also approved as second/third line therapy for EGFR mutation negative patients, but in this setting the benefit of erlotinib is modest and there is no validated biomarker for selecting EGFR wild-type patients who may benefit the most from the treatment. We retrospectively assessed EGFR and K-RAS mutational status, and EGFR, c-MET and IGF1-R expression in tumor samples of 72 patients with metastatic NSCLC treated with erlotinib after at least one prior line of chemotherapy, from 2008 to 2012. We analyzed the association between biomarkers and outcome (RR, PFS, and OS). EGFR mutated patients achieved a better RR (56% vs 8%, p = .002), PFS (10 vs 3 months, HR 0.53, p = 0.48) and OS (20 vs 6 months, HR 0.55, p = .07), compared to EGFR wild-type patients. Among 63 EGFR wild-type patients, those with EGFR high-expression had a better outcome in terms of RR (40% vs 2%, p = .002), PFS (7.5 vs 2 months, HR 0.45, p = .007) and OS (30 vs 5 months, HR 0.34, p < .001) compared to patients with EGFR intermediate or low/negative-expression. IGF1-R expression, c-MET expression and K-RAS mutational status did not significantly affect the outcome; however, no patients with K-RAS mutation or c-MET high-expression achieved an objective response. In patients with metastatic, chemo-refractory EGFR wild-type NSCLC, EGFR high-expression may represent a positive predictor of activity for erlotinib, whereas K-RAS mutation and c-MET high-expression may predict lack of activity. These findings deserve further prospective evaluation.

Nuclear Factor-Kappa B Activity in the Host-Tumor Microenvironment of Ovarian Cancer

DTIC Science & Technology

2012-08-01

analysis was performed in the Vanderbilt University Small Animal Imaging Core using the Xenogen IVIS 200 bioluminescent image system with Living...progression through systemic NF-B inhibition is that anti-tumor cytotoxic macrophages 9 may require NF-B signaling for normal function, and NF...Shin, L Klampfer, LH Augenlicht, R Perez- Soler , JM Mariadason. PIK3CA/PTEN expression status predicts response of colon cancer cells to the EGFR

Xenogeneic Decellularized Scaffold: A Novel Platform for Ovary Regeneration

PubMed Central

Liu, Wen-Yue; Lin, Shi-Gang; Zhuo, Ru-Yi; Xie, Yuan-Yuan; Pan, Wei

2017-01-01

Women younger than 40 years may face early menopause because of premature ovarian failure (POF). The cause of POF can be idiopathic or iatrogenic, especially the cancer-induced oophorectomy and chemo- or radiation therapy. The current treatments, including hormone replacement therapy (HRT) and cryopreservation techniques, have increased risk of ovarian cancer and may reintroduce malignant cells after autografting. Decellularization technique has been regarded as a novel regenerative medicine strategy for organ replacement, wherein the living cells of an organ are removed, leaving the extracellular matrix (ECM) for cellular seeding. This study aimed to produce a xenogeneic decellularized ovary (D-ovary) scaffold as a platform for ovary regeneration and transplantation. We have developed a novel decellularization protocol for porcine ovary by treatment with physical, chemical, and enzymatic methods. Using hematoxylin and eosin (H&E) staining, DAPI staining, scanning electron microscopy (SEM), and quantitative analysis, this approach proved effective in removing cellular components and preserving ECM. Furthermore, the results of biological safety evaluation demonstrated that the D-ovary tissues were noncytotoxic for rat ovarian cells in vitro and caused only a minimal immunogenic response in vivo. In addition, the D-ovary tissues successfully supported rat granulosa cell penetration ex vivo and showed an improvement in estradiol (E2) hormone secretion. PMID:27981878

Xenogeneic Decellularized Scaffold: A Novel Platform for Ovary Regeneration.

PubMed

Liu, Wen-Yue; Lin, Shi-Gang; Zhuo, Ru-Yi; Xie, Yuan-Yuan; Pan, Wei; Lin, Xian-Feng; Shen, Fei-Xia

2017-02-01

Women younger than 40 years may face early menopause because of premature ovarian failure (POF). The cause of POF can be idiopathic or iatrogenic, especially the cancer-induced oophorectomy and chemo- or radiation therapy. The current treatments, including hormone replacement therapy (HRT) and cryopreservation techniques, have increased risk of ovarian cancer and may reintroduce malignant cells after autografting. Decellularization technique has been regarded as a novel regenerative medicine strategy for organ replacement, wherein the living cells of an organ are removed, leaving the extracellular matrix (ECM) for cellular seeding. This study aimed to produce a xenogeneic decellularized ovary (D-ovary) scaffold as a platform for ovary regeneration and transplantation. We have developed a novel decellularization protocol for porcine ovary by treatment with physical, chemical, and enzymatic methods. Using hematoxylin and eosin (H&E) staining, DAPI staining, scanning electron microscopy (SEM), and quantitative analysis, this approach proved effective in removing cellular components and preserving ECM. Furthermore, the results of biological safety evaluation demonstrated that the D-ovary tissues were noncytotoxic for rat ovarian cells in vitro and caused only a minimal immunogenic response in vivo. In addition, the D-ovary tissues successfully supported rat granulosa cell penetration ex vivo and showed an improvement in estradiol (E2) hormone secretion.

Continued EGFR-TKI with concurrent radiotherapy to improve time to progression (TTP) in patients with locally progressive non-small cell lung cancer (NSCLC) after front-line EGFR-TKI treatment.

PubMed

Wang, Y; Li, Y; Xia, L; Niu, K; Chen, X; Lu, D; Kong, R; Chen, Z; Sun, J

2018-03-01

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is the optimal treatment for EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, most patients developed systemic or local progression due to acquired EGFR-TKI resistance. This retrospective study aimed to evaluate the feasibility of continued EGFR-TKI with concurrent radiotherapy (CTCRT) in patients with local progression after front-line EGFR-TKI treatment. Advanced NSCLC patients with active EGFR mutation who received EGFR-TKI were treated with CTCRT after local progression. Medical data were analyzed for time to progression (TTP), progression-free survival (PFS), tumor response rate, overall survival (OS) and adverse events. A total of 50 irradiated lesions from 44 patients were included. Median TTP and PFS of measurable lesions (n = 31) were both significantly prolonged after local radiotherapy (TTP1 + TTP2 vs. TTP1: 21.7 vs. 16.0 months, P = 0.010; PFS1 + PFS2 vs. PFS1: 21.3 vs. 16.0 months, P = 0.027). For all lesions (n = 50), objective response rate (ORR) and local tumor control rate (LCR) were 54.0 and 84.0%, respectively. Median OS was 26.6 months. There were no serious adverse events before or after radiotherapy. The treatment modality of CTCRT is considerable and effective for EGFR-mutant NSCLC patients even with local failure from front-line EGFR-TKI treatment.

EGFR T790M mutation testing within the osimertinib AURA Phase I study.

PubMed

Dearden, Simon; Brown, Helen; Jenkins, Suzanne; Thress, Kenneth S; Cantarini, Mireille; Cole, Rebecca; Ranson, Malcolm; Jänne, Pasi A

2017-07-01

Reliable epidermal growth factor receptor (EGFR) mutation testing techniques are required to identify eligible patients with EGFR mutation/T790M positive advanced non-small cell lung cancer (NSCLC), for treatment with osimertinib (AZD9291), an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR-TKI-sensitizing and T790M resistance mutations over wild-type EGFR. There is no current consensus regarding the best method to detect EGFR T790M mutations. The aim of this study was to describe the concordance between local testing, which used a variety of methods, and central testing, using the cobas ® EGFR Mutation Test, for EGFR-sensitizing mutations and the T790M resistance mutation. Tumor samples were obtained from all patients screened for inclusion onto the osimertinib Phase I expansion component of the AURA Phase I/II study (NCT01802632). Samples underwent central laboratory testing for EGFR-sensitizing mutations and T790M resistance mutation using the cobas ® EGFR Mutation Test. Results were compared with local laboratory test results, based on other testing methodologies including Sanger sequencing, therascreen ® , PNAClamp™, and Sequenom MassARRAY ® . Central laboratory testing was successful in 99% of samples passing histopathology review and testing success rates were comparable across the three central laboratories. Concordance between central and local testing for common sensitizing mutations was high (>98%) and concordance for the T790M mutation was also high (>90%). Tumor heterogeneity, along with other technical factors may have influenced this result. Within the osimertinib AURA Phase I study, EGFR mutation testing across three centralized laboratories using the cobas ® EGFR Mutation Test was feasible and successful, with strong concordance between local and central laboratory results, including for T790M. The cobas ® EGFR Mutation Test has subsequently been approved as the companion diagnostic test for osimertinib in

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

PubMed

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Predictive biomarkers for response to EGFR-directed monoclonal antibodies for advanced squamous cell lung cancer.

PubMed

Bonomi, P D; Gandara, D; Hirsch, F R; Kerr, K M; Obasaju, C; Paz-Ares, L; Bellomo, C; Bradley, J D; Bunn, P A; Culligan, M; Jett, J R; Kim, E S; Langer, C J; Natale, R B; Novello, S; Pérol, M; Ramalingam, S S; Reck, M; Reynolds, C H; Smit, E F; Socinski, M A; Spigel, D R; Vansteenkiste, J F; Wakelee, H; Thatcher, N

2018-06-14

Upregulated expression and aberrant activation of the epidermal growth-factor receptor (EGFR) are found in lung cancer, making EGFR a relevant target for non-small-cell lung cancer (NSCLC). Treatment with anti-EGFR monoclonal antibodies (mAbs) is associated with modest improvement in overall survival in patients with squamous cell lung cancer (SqCLC) who have a significant unmet need for effective treatment options. While there is evidence that using EGFR gene copy number, EGFR mutation, and EGFR protein expression as biomarkers can help select patients who respond to treatment, it is important to consider biomarkers for response in patients treated with combination therapies that include EGFR mAbs. Randomized trials of EGFR-directed mAbs cetuximab and necitumumab in combination with chemotherapy, immunotherapy, or anti-angiogenic therapy in patients with advanced NSCLC, including SqCLC, were searched in the literature. Results of associations of potential biomarkers and outcomes were summarized. Results. Data from phase III clinical trials indicate that patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein (H-score of ≥ 200) and/or gene copy numbers of EGFR (e.g., ≥40% cells with ≥4 EGFR copies as detected by fluorescence in situ hybridization; gene amplification in ≥ 10% of analyzed cells) derive greater therapeutic benefits from EGFR-directed mAbs. Biomarker data are limited for EGFR mAbs used in combination with immunotherapy and are absent when used in combination with anti-angiogenic agents. Therapy with EGFR-directed mAbs in combination with chemotherapy is associated with greater clinical benefits in patients with NSCLC, including SqCLC, whose tumors express high levels of EGFR protein and/or have increased EGFR gene copy number. These data support validating the role of these as biomarkers to identify those patients who derive the greatest clinical benefit from EGFR mAb therapy. However, data on biomarkers

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers

PubMed Central

Essenburg, Curt J.; Turner, Lisa; Madaj, Zachary; Winn, Mary E.; Melnik, Marianne K.; Korkaya, Hasan; Maroun, Christiane R.; Christensen, James G.; Steensma, Matthew R.; Boerner, Julie L.; Graveel, Carrie R.

2016-01-01

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients. PMID:27655711

Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinoma.

PubMed

Han, Ji-Youn; Kim, Sun Hye; Lee, Yeon-Su; Lee, Seung-Youn; Hwang, Jung-Ah; Kim, Jin Young; Yoon, Sung Jin; Lee, Geon Kook

2014-08-01

To investigate the clinical utility of targeted next-generation sequencing (NGS) for predicting the responsiveness to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy, we compared the efficacy with conventional sequencing in never-smokers with lung adenocarcinoma (NSLAs). We obtained DNA from 48 NSLAs who received gefitinib or erlotinib for their recurrent disease after surgery. Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF, and PIK3CA mutations. We analyzed ALK, RET, and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-PCR and quantitative real-time PCR. After molecular screening, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL), an L858R mutation, or none of the above mutations. The 31 samples were divided into four groups: (1) responders to EGFR-TKIs with only 19DEL or L858R (n=15); (2) primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); (3) primary resistance to EGFR-TKI without any mutations (n=8); (4) responders to EGFR-TKI without any mutations (n=4). With NGS, all conventionally detected mutations were confirmed except for one L858R in group 2. Additional uncovered predictive mutations with NGS included one PIK3CA E542K in group 2, two KRAS (G12V and G12D), one PIK3CA E542K, one concomitant PIK3CA and EGFR L858R in group 3, and one EGFR 19DEL in group 4. Targeted NGS provided a more accurate and clinically useful molecular classification of NSLAs. It may improve the efficacy of EGFR-TKI therapy in lung cancer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor-{alpha} enhances corneal epithelial cell migration by promoting EGFR recycling.

PubMed

McClintock, Jennifer L; Ceresa, Brian P

2010-07-01

PURPOSE. The goal of this study was to determine the molecular mechanism by which transforming growth factor-alpha (TGF-alpha) is a more potent activator of epidermal growth factor receptor (EGFR)-mediated corneal wound healing than epidermal growth factor (EGF). METHODS. Telomerase immortalized human corneal epithelial (hTCEpi) cells and primary human corneal epithelial cells were tested for their ability to migrate in response to EGF and TGF-alpha. In parallel, the endocytic trafficking of the EGFR in response to these same ligands was examined using indirect immunofluorescence, immunoblots, and radioligand binding. RESULTS. TGF-alpha, compared with EGF, is a more potent activator of corneal epithelial cell migration. Although both TGF-alpha and EGF were able to induce EGFR internalization and phosphorylation, only those receptors that were stimulated with EGF progressed to lysosomal degradation. EGFRs stimulated with TGF-alpha recycled back to the plasma membrane, where they could be reactivated with ligand. CONCLUSIONS. This study reveals that EGFR-mediated cell migration is limited by ligand-stimulated downregulation of the EGFR. This limitation can be overcome by treating cells with TGF-alpha because TGF-alpha stimulates EGFR endocytosis, but not degradation. After internalization of the TGF-alpha/EGFR complex, EGFR recycles back to the plasma membrane, where it can be restimulated. This sequence of events provides the receptor multiple opportunities for stimulation. Thus, stimulation with TGF-alpha prolongs EGFR signaling compared with EGF.

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

DOE PAGES

Su, Xinhui; Cheng, Kai; Jeon, Jongho; ...

2014-06-27

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-Z EGFR:1907 and 18F-FBEM-Z EGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-Z EGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al 18F-NOTA-Zmore » EGFR:1907 and 18F-CBT-Z EGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z EGFR:1907 with NOTA and radiolabeling with Al 18F to produce Al 18F-NOTA-Z EGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole ( 18F-CBT) was conjugated to Cys-Z EGFR:1907 to produce 18F-CBT-Z EGFR:1907. Binding affinity and specificity of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al 18F-NOTA-Z EGFR:1907 or 18F-CBT-Z EGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-Z EGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al 18F-NOTA-Z EGFR:1907 demonstrated high in vitro and in

Comparison of Two Site-Specifically 18F-Labeled Affibodies for PET Imaging of EGFR Positive Tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Xinhui; Cheng, Kai; Jeon, Jongho

The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules 64Cu-DOTA-Z EGFR:1907 and 18F-FBEM-Z EGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with 64Cu, 18F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of 18F-FBEM-Z EGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of 18F labeled affibody probes (Al 18F-NOTA-Zmore » EGFR:1907 and 18F-CBT-Z EGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating Z EGFR:1907 with NOTA and radiolabeling with Al 18F to produce Al 18F-NOTA-Z EGFR:1907. In a second approach the prosthetic group 18F-labeled-2-cyanobenzothiazole ( 18F-CBT) was conjugated to Cys-Z EGFR:1907 to produce 18F-CBT-Z EGFR:1907. Binding affinity and specificity of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al 18F-NOTA-Z EGFR:1907 or 18F-CBT-Z EGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al 18F-NOTA-Z EGFR:1907 and 18F-CBT-Z EGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although 18F-CBT-Z EGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al 18F-NOTA-Z EGFR:1907 demonstrated high in vitro and in

EGFR alterations and EML4-ALK rearrangement in primary adenocarcinoma of the urinary bladder.

PubMed

Alexander, Riley E; Montironi, Rodolfo; Lopez-Beltran, Antonio; Williamson, Sean R; Wang, Mingsheng; Post, Kristin M; Sen, Joyashree D; Arnold, Ashley K; Zhang, Shaobo; Wang, Xiaoyan; Koch, Michael O; Hahn, Noah M; Masterson, Timothy A; MacLennan, Gregory T; Davidson, Darrell D; Compérat, Eva; Cheng, Liang

2014-01-01

The identification of mutations in epidermal growth factor receptor (EGFR) and translocations involving anaplastic lymphoma kinase (ALK) in lung adenocarcinoma has drastically changed understanding of the disease and led to the development of targeted therapies. Adenocarcinoma of the urinary bladder is rare and poorly understood at the molecular level. We undertook this study to determine whether EGFR mutations, increases in EGFR copy number, or ALK translocations are present in these tumors. Twenty-eight cases of primary bladder adenocarcinoma were analyzed. For EGFR mutational analysis, PCR-amplified products were analyzed on the Q24 Pyrosequencer with Qiagen EGFR Pyro Kits. All cases were analyzed via fluorescence in situ hybridization (FISH) using Vysis ALK Break Apart FISH Probes for detection of ALK chromosomal translocation and Vysis Dual Color Probes to assess for increased gene copy number of EGFR. None of the 28 cases examined showed mutational events in EGFR or ALK rearrangements. EGFR polysomy was seen in 10 out of 28 (36%) cases. No correlation with EGFR polysomy was seen in the tumors with respect to age, histologic subtypes, pathologic stage, or lymph node metastasis. In summary, EGFR mutations and ALK rearrangements do not appear to be involved in the development of primary adenocarcinoma of the urinary bladder. A subgroup of cases (36%), however, demonstrated increased gene copy number of EGFR by FISH.

Known and putative mechanisms of resistance to EGFR targeted therapies in NSCLC patients with EGFR mutations—a review

PubMed Central

Stewart, Erin L.; Tan, Samuel Zhixing; Liu, Geoffrey

2015-01-01

Lung cancer is the leading cause of cancer related deaths in Canada with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Tumor characterization can identify cancer-driving mutations as treatment targets. One of the most successful examples of cancer targeted therapy is inhibition of mutated epidermal growth factor receptor (EGFR), which occurs in ~10-30% of NSCLC patients. While this treatment has benefited many patients with activating EGFR mutations, almost all who initially benefited will eventually acquire resistance. Approximately 50% of cases of acquired resistance (AR) are due to a secondary T790M mutation in exon 20 of the EGFR gene; however, many of the remaining mechanisms of resistance are still unknown. Much work has been done to elucidate the remaining mechanisms of resistance. This review aims to highlight both the mechanisms of resistance that have already been identified in patients and potential novel mechanisms identified in preclinical models which have yet to be validated in the patient settings. PMID:25806347

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment.

PubMed

Lauriola, Mattia; Enuka, Yehoshua; Zeisel, Amit; D'Uva, Gabriele; Roth, Lee; Sharon-Sevilla, Michal; Lindzen, Moshit; Sharma, Kirti; Nevo, Nava; Feldman, Morris; Carvalho, Silvia; Cohen-Dvashi, Hadas; Kedmi, Merav; Ben-Chetrit, Nir; Chen, Alon; Solmi, Rossella; Wiemann, Stefan; Schmitt, Fernando; Domany, Eytan; Yarden, Yosef

2014-10-03

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

EGFR and HER2 signaling in breast cancer brain metastasis

PubMed Central

Sirkisoon, Sherona R.; Carpenter, Richard L.; Rimkus, Tadas; Miller, Lance; Metheny-Barlow, Linda; Lo, Hui-Wen

2016-01-01

Breast cancer occurs in approximately 1 in 8 women and 1 in 37 women with breast cancer succumbed to the disease. Over the past decades, new diagnostic tools and treatments have substantially improved the prognosis of women with local diseases. However, women with metastatic disease still have a dismal prognosis without effective treatments. Among different molecular subtypes of breast cancer, the HER2-enriched and basal-like subtypes typically have higher rates of metastasis to the brain. Basal-like metastatic breast tumors frequently express EGFR. Consequently, HER2- and EGFR-targeted therapies are being used in the clinic and/or evaluated in clinical trials for treating breast cancer patients with brain metastases. In this review, we will first provide an overview of the HER2 and EGFR signaling pathways. The roles that EGFR and HER2 play in breast cancer metastasis to the brain will then be discussed. Finally, we will summarize the preclinical and clinical effects of EGFR- and HER2-targeted therapies on breast cancer metastasis. PMID:26709660

Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

PubMed

Wee, Ping; Wang, Zhixiang

2017-01-01

Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

Benzo[g]quinazolin-based scaffold derivatives as dual EGFR/HER2 inhibitors.

PubMed

Ghorab, Mostafa M; Alsaid, Mansour S; Soliman, Aiten M; Al-Mishari, Abdullah A

2018-12-01

Targeting EGFR has proven to be beneficial in the treatment of several types of solid tumours. So, a series of novel 2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio)-N-substituted acetamide 5-19 were synthesised from the starting material 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4, to be evaluated as dual EGFR/HER2 inhibitors. The target compounds 5-19, were screened for their cytotoxic activity against A549 lung cancer cell line. The percentage inhibition of EGFR enzyme was measured and compared with erlotinib as the reference drug. Compounds 6, 8, 10, and 16 showed excellent EGFR inhibitory activity and were further selected for screening as dual EGFR/HER2 inhibitors. The four selected compounds showed IC 50 ranging from 0.009 to 0.026 µM for EGFR and 0.021 to 0.069 µM for the HER2 enzyme. Compound 8 was found to be the most potent in this study with IC 50 0.009 and 0.021 µM for EGFR and HER2, respectively.

Anti-EGFR monoclonal antibody in cancer treatment: in vitro and in vivo evidence

PubMed

Quatrale, Anna Elisa; Petriella, Daniela; Porcelli, Letizia; Tommasi, Stefania; Silvestris, Nicola; Colucci, Giuseppe; Angelo, Angelo; Azzariti, Amalia

2011-01-01

The complexity of EGFR signaling network suggests that the receptor could be promising targets for new personalised therapy. In clinical practice two strategies targeting the receptor are available; they utilise monoclonal antibodies, directed towards the extracellular domain of EGFR, and small molecule tyrosine kinase inhibitors, which bind the catalytic kinase domain of the receptor. In this review, we summarise currently known pre-clinical data on the antitumor effects of monoclonal antibodies, which bind to EGFR in its inactive configuration, competing for ligand binding and thereby blocking ligand-induced EGFR tyrosine kinase activation. As a consequence of treatment, key EGFR-dependent intracellular signals in cancer cells are affected. Data explaining the mechanisms of action of anti-EGFR monoclonal antibodies, currently used in clinical setting and under development for the treatment of solid tumors, are revised with the aim to provide an overview of the most important preclinical studies showing the impact of this class of EGFR targeted agents on tumor biology.

EGFR mutant allelic-specific imbalance assessment in routine samples of non-small cell lung cancer.

PubMed

Malapelle, Umberto; Vatrano, Simona; Russo, Stefania; Bellevicine, Claudio; de Luca, Caterina; Sgariglia, Roberta; Rocco, Danilo; de Pietro, Livia; Riccardi, Fernando; Gobbini, Elisa; Righi, Luisella; Troncone, Giancarlo

2015-09-01

In non-small cell lung cancer (NSCLC), the epidermal growth factor receptor (EGFR) gene may undergo both mutations and copy number gains. EGFR mutant allele-specific imbalance (MASI) occurs when the ratio of mutant-to-wild-type alleles increases significantly. In this study, by using a previously validated microfluidic-chip-based technology, EGFR-MASI occurred in 25/67 mutant cases (37%), being more frequently associated with EGFR exon 19 deletions (p=0.033). In a subset of 49 treated patients, we assessed whether MASI is a modifier of anti-EGFR treatment benefit. The difference in progression-free survival and overall survival between EGFR-MASI-positive and EGFR-MASI-negative groups of patients did not show a statistical significance. In conclusion, EGFR-MASI is a significant event in NSCLC, specifically associated with EGFR exon 19 deletions. However, EGFR-MASI does not seem to play a role in predicting the response to first-generation EGFR small molecules inhibitors. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Biomarkers that currently affect clinical practice: EGFR, ALK, MET, KRAS

PubMed Central

Vincent, M.D.; Kuruvilla, M.S.; Leighl, N.B.; Kamel–Reid, S.

2012-01-01

New drugs such as pemetrexed, the epidermal growth factor receptor (egfr) tyrosine kinase inhibitors, and the Alk inhibitor crizotinib have recently enabled progress in the management of advanced non-small-cell lung cancer (nsclc). More drugs, especially Met inhibitors, will follow. However, the benefits of these agents are not uniform across the spectrum of nsclc, and optimizing their utility requires some degree of subgrouping of nsclc by the presence or absence of certain biomarkers. The biomarkers of current or imminent value are EGFR and KRAS mutational status, ALK rearrangements, and MET immunohistochemistry. As a predictor of benefit for anti-egfr monoclonal antibodies, EGFR immunohistochemistry is also of potential interest. Some of the foregoing biomarkers (EGFR, ALK, MET) are direct drivers of the malignant phenotype. As such, they are, quite rationally, the direct targets of inhibitory drugs. However, KRAS, while definitely a driver, has resisted attempts at direct pharmacologic manipulation, and its main value might lie in its role as part of an efficient testing algorithm, because KRAS mutations appear to exclude EGFR and ALK mutations. The indirect value of KRAS in determining sensitivity to other targeted agents or to pemetrexed remains controversial. The other biomarkers (EGFR, ALK, MET) may also have indirect value as predictors of sensitivity to chemotherapy in general, to pemetrexed specifically, and to radiotherapy and molecularly targeted agents. These biomarkers have all enabled the co-development of new drugs with companion diagnostics, and they illustrate the paradigm that will govern progress in oncology in the immediate future. However, in nsclc, the acquisition of sufficient biopsy material remains a stubborn obstacle to the evolution of novel targeted therapies. PMID:22787409

The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

PubMed Central

Boncompain, Gaelle; Laketa, Vibor; Poser, Ina; Beck, Martin; Bork, Peer

2016-01-01

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane. PMID:27872256

EGFR G796D mutation mediates resistance to osimertinib.

PubMed

Zheng, Di; Hu, Min; Bai, Yu; Zhu, Xuehua; Lu, Xuesong; Wu, Chunyan; Wang, Jiying; Liu, Li; Wang, Zheng; Ni, Jian; Yang, Zhenfan; Xu, Jianfang

2017-07-25

Osimertinib is an effective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved in multiple countries and regions for patients with EGFR T790M mutation-positive non-small cell lung cancer (NSCLC). Despite impressive initial tumor responses, development of drug resistance ultimately limits the benefit of this compound. Mechanisms of resistance to osimertinib are just beginning to emerge, such as EGFR C797S and L718Q mutations, BRAF V600E and PIK3CA E545K mutations, as well as ERBB2 and MET amplification. However, a comprehensive view is still missing. In this study, we presented the first case of Chinese NSCLC patient who developed resistance to osimertinib, and discovered de novo EGFR G796D mutation as a potential mechanism. Our findings provided insights into mechanisms of resistance to osimertinib and highlighted tumor heterogeneity and clonal evolution during the development of drug resistance.

EGFR G796D mutation mediates resistance to osimertinib

PubMed Central

Bai, Yu; Zhu, Xuehua; Lu, Xuesong; Wu, Chunyan; Wang, Jiying; Liu, Li; Wang, Zheng; Ni, Jian; Yang, Zhenfan; Xu, Jianfang

2017-01-01

Osimertinib is an effective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved in multiple countries and regions for patients with EGFR T790M mutation-positive non-small cell lung cancer (NSCLC). Despite impressive initial tumor responses, development of drug resistance ultimately limits the benefit of this compound. Mechanisms of resistance to osimertinib are just beginning to emerge, such as EGFR C797S and L718Q mutations, BRAF V600E and PIK3CA E545K mutations, as well as ERBB2 and MET amplification. However, a comprehensive view is still missing. In this study, we presented the first case of Chinese NSCLC patient who developed resistance to osimertinib, and discovered de novo EGFR G796D mutation as a potential mechanism. Our findings provided insights into mechanisms of resistance to osimertinib and highlighted tumor heterogeneity and clonal evolution during the development of drug resistance. PMID:28572531

Clinical efficacy of icotinib in lung cancer patients with different EGFR mutation status: a meta-analysis

PubMed Central

Xu, Ping; Xiang, Da-Xiong; Yang, Rui; Wei, Wei; Qu, Qiang

2017-01-01

Icotinib is a novel and the third listed epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), which exerts a good anti-tumor efficacy on non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs has been shown to be associated with the EGFR mutation status, especially exon 19 deletion (19Del) and exon 21 L858R mutation. Therefore, a meta-analysis was performed to assess the efficacy of icotinib in NSCLC patients harboring EGFR mutations (19Del or L858R) and wild type (19Del and L858R loci wild type). A total of 24 studies were included for comparing the objective response rate (ORR) in the EGFR wild type and mutant patients treated with icotinib. The ORRs of EGFR mutant patients (19Del or L858R) are better than those of EGFR wild type patients (OR = 7.03(5.09–9.71), P < 0.00001). The pooling ORs from 21 studies on the disease control rate (DCR) in EGFR mutant patients are better than those of EGFR wild type patients (OR = 10.54(5.72–19.43), P < 0.00001). Moreover, the ORRs of EGFR 19Del patients are better than those of EGFR L858R patients after pooling ORs of 12 studies (OR = 2.04(1.12–3.73), P = 0.019). However, there was no significant difference on DCRs of EGFR 19Del patients and those of EGFR L858R patients (OR = 2.01(0.94–4.32), P = 0.072). Our findings indicated that compared with EGFR wild type patients, EGFR mutant patients have better ORRs and DCRs after icotinib treatment; EGFR 19Del patients treated with icotinib have better ORRs than EGFR L858R patients. EGFR mutation status is a useful biomarker for the evaluation of icotinib efficacy in NSCLC patients. PMID:28430623

Clinical efficacy of icotinib in lung cancer patients with different EGFR mutation status: a meta-analysis.

PubMed

Qu, Jian; Wang, Ya-Nan; Xu, Ping; Xiang, Da-Xiong; Yang, Rui; Wei, Wei; Qu, Qiang

2017-05-16

Icotinib is a novel and the third listed epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), which exerts a good anti-tumor efficacy on non-small cell lung cancer (NSCLC). The efficacy of EGFR-TKIs has been shown to be associated with the EGFR mutation status, especially exon 19 deletion (19Del) and exon 21 L858R mutation. Therefore, a meta-analysis was performed to assess the efficacy of icotinib in NSCLC patients harboring EGFR mutations (19Del or L858R) and wild type (19Del and L858R loci wild type). A total of 24 studies were included for comparing the objective response rate (ORR) in the EGFR wild type and mutant patients treated with icotinib. The ORRs of EGFR mutant patients (19Del or L858R) are better than those of EGFR wild type patients (OR = 7.03(5.09-9.71), P < 0.00001). The pooling ORs from 21 studies on the disease control rate (DCR) in EGFR mutant patients are better than those of EGFR wild type patients (OR = 10.54(5.72-19.43), P < 0.00001). Moreover, the ORRs of EGFR 19Del patients are better than those of EGFR L858R patients after pooling ORs of 12 studies (OR = 2.04(1.12-3.73), P = 0.019). However, there was no significant difference on DCRs of EGFR 19Del patients and those of EGFR L858R patients (OR = 2.01(0.94-4.32), P = 0.072). Our findings indicated that compared with EGFR wild type patients, EGFR mutant patients have better ORRs and DCRs after icotinib treatment; EGFR 19Del patients treated with icotinib have better ORRs than EGFR L858R patients. EGFR mutation status is a useful biomarker for the evaluation of icotinib efficacy in NSCLC patients.

Osimertinib for EGFR T790M mutation-positive non-small cell lung cancer.

PubMed

Soejima, Kenzo; Yasuda, Hiroyuki; Hirano, Toshiyuki

2017-01-01

Significant advances have been made since the development of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) targeting EGFR mutations in non-small-cell lung cancer (NSCLC), however, lung cancer cells eventually acquire resistance to those agents. Osimertinib (AZD9291) has been developed as 3 rd generation EGFR-TKI with activities against sensitizing mutations and T790 M resistance mutation, which account for about 50% of the mechanisms of acquired resistance to 1 st or 2 nd generation EGFR-TKIs. A recent phase I/II clinical trial with osimertinib for advanced NSCLC patients with known sensitizing EGFR mutations and documented disease progression on prior EGFR-TKIs revealed promising effect with acceptable toxicities. Areas covered: This article summarizes current understanding and available preclinical and clinical data on osimertinib and also discusses future directions. The literature search included PubMed and the latest articles from international conferences. Expert commentary: The development of osimertinib has provided new therapeutic options for NSCLC patients harboring T790 M. Compared with other EGFR-TKIs including rociletinib, osimertinib seems to possess an advantage with respect to the effect and safety profile among existing EGFR-TKIs. However, tumor progression still occurs even when treating with osimertinib. A further understanding of the mechanisms of resistance is eagerly anticipated in order to develop next generation EGFR-TKIs.

Personalized treatment of EGFR mutant and ALK-positive patients in NSCLC.

PubMed

Somasundaram, Aswin; Socinski, Mark A; Burns, Timothy F

2014-12-01

The epidermal growth factor receptor (EGFR) is mutated in 15% of adenocarcinomas of the lung. In addition, the anaplastic lymphoma kinase (ALK) is altered in 8% of adenocarcinomas of the lung. Treatment of EGFR mutant and ALK translocation-positive tumors in NSCLC with tyrosine kinase inhibitors (TKI) results in a dramatic therapeutic response and has revolutionized therapy. Unfortunately, resistance to TKIs invariably develops. Many promising new therapies are under investigation to overcome the resistance. We analyzed the current primary literature and recent national meetings to evaluate the clinical characteristics and therapeutic implications of relevant treatments for EGFR mutant and ALK-positive NSCLC in the first-line, acquired resistance, and adjuvant settings. Treatment with EGFR TKIs in the first-line setting of EGFR mutant NSCLC results in a significant clinical benefit. Several promising third generation EGFR TKIs are being evaluated in Phase II and III trials in the acquired resistance setting. Crizotinib is superior to chemotherapy in the first-line setting for ALK-positive NSCLC. Ceritinib is effective and approved for ALK-positive NSCLC in the acquired resistance setting. Continued investigation is needed to develop novel therapies to overcome acquired resistance to TKIs.

RAB-7 Antagonizes LET-23 EGFR Signaling during Vulva Development in Caenorhabditis elegans

PubMed Central

Skorobogata, Olga; Rocheleau, Christian E.

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(−) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans. PMID:22558469

RAB-7 antagonizes LET-23 EGFR signaling during vulva development in Caenorhabditis elegans.

PubMed

Skorobogata, Olga; Rocheleau, Christian E

2012-01-01

The Rab7 GTPase regulates late endosome trafficking of the Epidermal Growth Factor Receptor (EGFR) to the lysosome for degradation. However, less is known about how Rab7 activity, functioning late in the endocytic pathway, affects EGFR signaling. Here we used Caenorhabditis elegans vulva cell fate induction, a paradigm for genetic analysis of EGFR/Receptor Tyrosine Kinase (RTK) signaling, to assess the genetic requirements for rab-7. Using a rab-7 deletion mutant, we demonstrate that rab-7 antagonizes LET-23 EGFR signaling to a similar extent, but in a distinct manner, as previously described negative regulators such as sli-1 c-Cbl. Epistasis analysis places rab-7 upstream of or in parallel to lin-3 EGF and let-23 EGFR. However, expression of gfp::rab-7 in the Vulva Presursor Cells (VPCs) is sufficient to rescue the rab-7(-) VPC induction phenotypes indicating that RAB-7 functions in the signal receiving cell. We show that components of the Endosomal Sorting Complex Required for Transport (ESCRT)-0, and -I, complexes, hgrs-1 Hrs, and vps-28, also antagonize signaling, suggesting that LET-23 EGFR likely transits through Multivesicular Bodies (MVBs) en route to the lysosome. Consistent with RAB-7 regulating LET-23 EGFR trafficking, rab-7 mutants have increased number of LET-23::GFP-positive endosomes. Our data imply that Rab7, by mediating EGFR trafficking and degradation, plays an important role in downregulation of EGFR signaling. Failure to downregulate EGFR signaling contributes to oncogenesis, and thus Rab7 could possess tumor suppressor activity in humans.

Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos

PubMed Central

Dackor, J.; Strunk, K. E.; Wehmeyer, M. M.; Threadgill, D. W.

2007-01-01

Homozygosity for the Egfrtm1Mag null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta was quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfrtm1Mag homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation. PMID:17822758

[Efficacy of chemotherapy after EGFR-TKIs resistance in 191 patients with Unknow EGFR gene mutation in advanced lung adenocarcinoma].

PubMed

He, Ping; Wang, Yan; Yang, Sheng; Yu, Shufei; Wang, Ziping; Li, Junling; Wang, Bin; Hao, Xuezhi; Wang, Hongyu; Hu, Xingsheng; Zhang, Xiangru; Shi, Yuankai

2013-10-20

Subsequent chemotherapy were needed in patients with advanced pulmonary adenocarcinoma experiencing disease progression after epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. The study is to explore factors potentially influencing efficacy of subsequent chemotherapy. One hundred and ninety-one patients with advanced lung adenocarcinoma, who were resistant from EGFR-TKIs and then received subsequent chemotherapy, were identified. Data of patient's characteristics, responses to chemotherapy and survival time were analyzed retrospectively. The overall response rate of the pemetrexed-based chemotherapy (9.3%) was higher than non-pemetrexed-based regimen (1.1%), P=0.011. Furthermore, the response in the second-line was more obvisous [objective response rate (ORR) 14.3% vs 3.7%, P=0.041]. The patients who achieved response of partial response (PR) showed longer progression-free survival (PFS) than those who achieved non-PR (PFS 10.1 months and 2.3 months, P=0.012). The patients treated with platinum-based chemotherapy had longer PFS and OS than those with non-platinum-based chemotherapy, therefore platinum-based regimen was independent prognosis factors for PFS and OS (PFS: RR=0.634, 95%CI: 0.466-0.832, P=0.004; OS: RR=0.666, 95%CI: 0.460-0.960, P=0.030), especially the pateients who were aquired EGFR-TKIs resistance and who got drmatic progression from EGFR-TKIs treatment might got more benefits from platinum-based chemotherapy. However there was no significant difference in ORR, PFS or OS between patients with TKIs primary resistance and acquired resistance, or between dramtic progression and gradual/local progression. The patients with advanced lung adenocarcinoma might get benefits from pemetrexed-based or platinum-based chemotherapy after they were EGFR-TKIs resistace.

Traumatic laryngotracheal stenosis treated by hyoid–sternohyoid osseomuscular flap combined with xenogenic acellular dermal matrix: A case report and literature review

PubMed Central

Yang, Hang; Chen, Zhe; Wang, Qin-Yin; Weng, Li-Xia; Wang, Fang; Wu, Ting-Ting; Zhou, Min-Li; Bao, Yang-Yang

2017-01-01

Objective The treatment of laryngotracheal stenosis is a major therapeutic challenge. Various treatments include observation, medical management, and surgical management. The most effective surgical management is resection and reconstruction. To the authors’ knowledge, no reports have described the use of xenogenic acellular dermal matrix (ADM) for laryngotracheal stenosis. Methods A 27-year-old man presented with hemoptysis of the neck due to a traffic accident. Emergency orotracheal intubation was performed. Tracheostomy was then performed under local anesthesia. Computed tomography revealed fractures of the right thyroid cartilage and posterior arc of the cricoid cartilage and stenosis of the subglottis and first and second tracheal rings. We used a composite hyoid–sternohyoid osseomuscular flap with xenogenic ADM and a straight silicone tube as a lumen stent to reconstruct the laryngotracheal stenosis. Results Surgical recovery was uneventful. The tracheotomy opening was changed to a metal tube 5 days postoperatively. Four months postoperatively, the silicone tube was endoscopically removed under local anesthesia. The patient was decannulated 20 days later. The patient satisfied with his voice, respiration, and deglutition at the 16-month postoperative follow-up. Conclusion The use of ADM for laryngotracheal stenosis may reduce the growth of granulation tissues and promote the repair process. PMID:28480810

Traumatic laryngotracheal stenosis treated by hyoid-sternohyoid osseomuscular flap combined with xenogenic acellular dermal matrix: A case report and literature review.

PubMed

Yang, Hang; Chen, Zhe; Zhou, Shui-Hong; Wang, Qin-Yin; Weng, Li-Xia; Wang, Fang; Wu, Ting-Ting; Zhou, Min-Li; Bao, Yang-Yang

2017-10-01

Objective The treatment of laryngotracheal stenosis is a major therapeutic challenge. Various treatments include observation, medical management, and surgical management. The most effective surgical management is resection and reconstruction. To the authors' knowledge, no reports have described the use of xenogenic acellular dermal matrix (ADM) for laryngotracheal stenosis. Methods A 27-year-old man presented with hemoptysis of the neck due to a traffic accident. Emergency orotracheal intubation was performed. Tracheostomy was then performed under local anesthesia. Computed tomography revealed fractures of the right thyroid cartilage and posterior arc of the cricoid cartilage and stenosis of the subglottis and first and second tracheal rings. We used a composite hyoid-sternohyoid osseomuscular flap with xenogenic ADM and a straight silicone tube as a lumen stent to reconstruct the laryngotracheal stenosis. Results Surgical recovery was uneventful. The tracheotomy opening was changed to a metal tube 5 days postoperatively. Four months postoperatively, the silicone tube was endoscopically removed under local anesthesia. The patient was decannulated 20 days later. The patient satisfied with his voice, respiration, and deglutition at the 16-month postoperative follow-up. Conclusion The use of ADM for laryngotracheal stenosis may reduce the growth of granulation tissues and promote the repair process.

Preclinical Comparison of Osimertinib with Other EGFR-TKIs in EGFR-Mutant NSCLC Brain Metastases Models, and Early Evidence of Clinical Brain Metastases Activity.

PubMed

Ballard, Peter; Yates, James W T; Yang, Zhenfan; Kim, Dong-Wan; Yang, James Chih-Hsin; Cantarini, Mireille; Pickup, Kathryn; Jordan, Angela; Hickey, Mike; Grist, Matthew; Box, Matthew; Johnström, Peter; Varnäs, Katarina; Malmquist, Jonas; Thress, Kenneth S; Jänne, Pasi A; Cross, Darren

2016-10-15

Approximately one-third of patients with non-small cell lung cancer (NSCLC) harboring tumors with EGFR-tyrosine kinase inhibitor (TKI)-sensitizing mutations (EGFRm) experience disease progression during treatment due to brain metastases. Despite anecdotal reports of EGFR-TKIs providing benefit in some patients with EGFRm NSCLC brain metastases, there is a clinical need for novel EGFR-TKIs with improved efficacy against brain lesions. We performed preclinical assessments of brain penetration and activity of osimertinib (AZD9291), an oral, potent, irreversible EGFR-TKI selective for EGFRm and T790M resistance mutations, and other EGFR-TKIs in various animal models of EGFR-mutant NSCLC brain metastases. We also present case reports of previously treated patients with EGFRm-advanced NSCLC and brain metastases who received osimertinib in the phase I/II AURA study (NCT01802632). Osimertinib demonstrated greater penetration of the mouse blood-brain barrier than gefitinib, rociletinib (CO-1686), or afatinib, and at clinically relevant doses induced sustained tumor regression in an EGFRm PC9 mouse brain metastases model; rociletinib did not achieve tumor regression. Under positron emission tomography micro-dosing conditions, [ 11 C]osimertinib showed markedly greater exposure in the cynomolgus monkey brain than [ 11 C]rociletinib and [ 11 C]gefitinib. Early clinical evidence of osimertinib activity in previously treated patients with EGFRm-advanced NSCLC and brain metastases is also reported. Osimertinib may represent a clinically significant treatment option for patients with EGFRm NSCLC and brain metastases. Further investigation of osimertinib in this patient population is ongoing. Clin Cancer Res; 22(20); 5130-40. ©2016 AACR. ©2016 American Association for Cancer Research.

EGFR amplification and expression in oral squamous cell carcinoma in young adults.

PubMed

Costa, V; Kowalski, L P; Coutinho-Camillo, C M; Begnami, M D; Calsavara, V F; Neves, J I; Kaminagakura, E

2018-07-01

The aim of this study was to investigate epidermal growth factor receptor (EGFR) gene alterations in two groups of patients with oral squamous cell carcinoma (OSCC) (a test group of subjects aged ≤40 years and a control group of subjects aged ≥50 years) and to associate the results with EGFR immunostaining, clinicopathological features, and the prognosis. Sixty cases of OSCC were selected (test group, n=21; control group, n=39). The tissue microarray technique was applied to ensure the uniformity of results. Gene amplification was analyzed by fluorescence in situ hybridization (FISH), and immunohistochemical staining for EGFR was analyzed using an automated imaging system. EGFR amplification was higher in the test group than in the control group (P=0.018) and was associated with advanced clinical stage (P=0.013), regardless of age. Patients with EGFR overexpression had worse survival rates, as did patients who had T3-T4 tumours and positive margins. EGFR overexpression has a negative impact on disease progression. Despite the higher amplification of EGFR in young adults, it does not significantly impact the survival rates of affected patients. Copyright © 2018 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Effect of Genetic African Ancestry on eGFR and Kidney Disease

PubMed Central

Nadkarni, Girish N.; Belbin, Gillian; Lotay, Vaneet; Wyatt, Christina; Gottesman, Omri; Bottinger, Erwin P.; Kenny, Eimear E.; Peter, Inga

2015-01-01

Self-reported ancestry, genetically determined ancestry, and APOL1 polymorphisms are associated with variation in kidney function and related disease risk, but the relative importance of these factors remains unclear. We estimated the global proportion of African ancestry for 9048 individuals at Mount Sinai Medical Center in Manhattan (3189 African Americans, 1721 European Americans, and 4138 Hispanic/Latino Americans by self-report) using genome-wide genotype data. CKD-EPI eGFR and genotypes of three APOL1 coding variants were available. In admixed African Americans and Hispanic/Latino Americans, serum creatinine values increased as African ancestry increased (per 10% increase in African ancestry, creatinine values increased 1% in African Americans and 0.9% in Hispanic/Latino Americans; P≤1x10−7). eGFR was likewise significantly associated with African genetic ancestry in both populations. In contrast, APOL1 risk haplotypes were significantly associated with CKD, eGFR<45 ml/min per 1.73 m2, and ESRD, with effects increasing with worsening disease states and the contribution of genetic African ancestry decreasing in parallel. Using genetic ancestry in the eGFR equation to reclassify patients as black on the basis of ≥50% African ancestry resulted in higher eGFR for 14.7% of Hispanic/Latino Americans and lower eGFR for 4.1% of African Americans, affecting CKD staging in 4.3% and 1% of participants, respectively. Reclassified individuals had electrolyte values consistent with their newly assigned CKD stage. In summary, proportion of African ancestry was significantly associated with normal-range creatinine and eGFR, whereas APOL1 risk haplotypes drove the associations with CKD. Recalculation of eGFR on the basis of genetic ancestry affected CKD staging and warrants additional investigation. PMID:25349204

Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLC.

PubMed

Gaber, Rania; Watermann, Iris; Kugler, Christian; Reinmuth, Nils; Huber, Rudolf M; Schnabel, Philipp A; Vollmer, Ekkehard; Reck, Martin; Goldmann, Torsten

2014-09-17

Epidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies. As a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes. EGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p=0.001). Overall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p=0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_165.

Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, Nicole M.; Ceresa, Brian P., E-mail: brian.ceresa@louisville.edu

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as amore » plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway.« less

Past Decline Versus Current eGFR and Subsequent Mortality Risk.

PubMed

Naimark, David M J; Grams, Morgan E; Matsushita, Kunihiro; Black, Corri; Drion, Iefke; Fox, Caroline S; Inker, Lesley A; Ishani, Areef; Jee, Sun Ha; Kitamura, Akihiko; Lea, Janice P; Nally, Joseph; Peralta, Carmen Alicia; Rothenbacher, Dietrich; Ryu, Seungho; Tonelli, Marcello; Yatsuya, Hiroshi; Coresh, Josef; Gansevoort, Ron T; Warnock, David G; Woodward, Mark; de Jong, Paul E

2016-08-01

A single determination of eGFR associates with subsequent mortality risk. Prior decline in eGFR indicates loss of kidney function, but the relationship to mortality risk is uncertain. We conducted an individual-level meta-analysis of the risk of mortality associated with antecedent eGFR slope, adjusting for established risk factors, including last eGFR, among 1.2 million subjects from 12 CKD and 22 other cohorts within the CKD Prognosis Consortium. Over a 3-year antecedent period, 12% of participants in the CKD cohorts and 11% in the other cohorts had an eGFR slope <-5 ml/min per 1.73 m(2) per year, whereas 7% and 4% had a slope >5 ml/min per 1.73 m(2) per year, respectively. Compared with a slope of 0 ml/min per 1.73 m(2) per year, a slope of -6 ml/min per 1.73 m(2) per year associated with adjusted hazard ratios for all-cause mortality of 1.25 (95% confidence interval [95% CI], 1.09 to 1.44) among CKD cohorts and 1.15 (95% CI, 1.01 to 1.31) among other cohorts during a follow-up of 3.2 years. A slope of +6 ml/min per 1.73 m(2) per year also associated with higher all-cause mortality risk, with adjusted hazard ratios of 1.58 (95% CI, 1.29 to 1.95) among CKD cohorts and 1.43 (95% CI, 1.11 to 1.84) among other cohorts. Results were similar for cardiovascular and noncardiovascular causes of death and stronger for longer antecedent periods (3 versus <3 years). We conclude that prior decline or rise in eGFR associates with an increased risk of mortality, independent of current eGFR. Copyright © 2016 by the American Society of Nephrology.

Is MPP a good prognostic factor in stage III lung adenocarcinoma with EGFR exon 19 mutation?

PubMed

Zhang, Tian; Wang, Jing; Su, Yanjun; Chen, Xi; Yan, Qingna; Li, Qi; Sun, Leina; Wang, Yuwen; Er, Puchun; Pang, Qingsong; Wang, Ping

2017-06-20

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein encoded by a gene located in the short arm of chromosome 7. This study aimed to investigate the clinicopathologic characteristics of classic EGFR exon mutation in Chinese patients with TMN stage III lung adenocarcinoma who received radical surgery. A total of 1,801 lung adenocarcinomas were analyzed for mutations in EGFR; 35% exhibited mutation of classic EGFR exons. Clinical and pathologic characteristics of patients with EGFR exon 19 mutation were compared with those who harbored EGFR exon 21 mutation. Patients with EGFR exon 19 mutation had a higher overall survival (OS, p=0.023) than those harboring EGFR exon 21 mutation. Our results demonstrated that patients with a micropapillary pattern (MPP) pathologic type in EGFR exon 19 mutation had a higher OS (p=0.022), and patients with exon 19 mutation were more sensitive to EGFR-tyrosine kinase inhibitors (p=0.032). The results of the current study can be used in decision-making regarding the treatment of patients with classic EGFR exon mutations.

Concordant and Discordant EGFR Mutations in Patients with Multifocal Adenocarcinomas: Implications for EGFR Targeted Therapy

PubMed Central

Chuang, Jody C.; Shrager, Joseph B.; Wakelee, Heather A.; Neal, Joel W.

2016-01-01

Purpose Adenocarcinoma remains the most common subtype of lung cancer in the United States. Most patients present with tumors that are invasive and often metastatic, but some patients develop multiple precursor in-situ or minimally invasive adenocarcinoma tumors which can be synchronous and metachronous. These precursor lesions harbor the same spectrum of genetic mutations found in pure-invasive adenocarcinomas, such as EGFR, KRAS and p53 mutations. It is less clear, however, if separate lesions in patients who present with multifocal disease share common underlying genetic driver mutations. Methods Here we review the relevant literature on molecular driver alterations in adenocarcinoma precursor lesions. We then report four cases with multifocal EGFR mutant adenocarcinomas in whom we performed molecular testing on 2 separate lesions. Findings In two of these patients, the mutations are concordant, and in two cases the mutations are discordant. A review of the literature demonstrates increasing evidence that lesions with discordant mutations may confer a more favorable prognosis, since they are unlikely to represent metastases. Implications Our findings suggest that the emergence of the dominant EGFR driver alteration is often independent between lesions in patients with multifocal adenocarcinomas, and thus the same targeted therapy may not be effective for all lesions. However, genetic testing of multiple lesions can help distinguish separate primary tumors from metastatic disease. PMID:27368115

Acquired EGFR T790M Mutation After Relapse Following EGFR-TKI Therapy: A Population-based Multi-institutional Study.

PubMed

Kaburagi, Takayuki; Kiyoshima, Moriyuki; Nawa, Takeshi; Ichimura, Hideo; Saito, Takefumi; Hayashihara, Kenji; Yamada, Hideyasu; Satoh, Hiroaki; Endo, Takeo; Inage, Yoshihisa; Saito, Kazuhito; Inagaki, Masaharu; Hizawa, Nobuyuki; Sato, Yukio; Ishikawa, Hiroichi; Sakai, Mitsuaki; Kamiyama, Koichi; Kikuchi, Norihiro; Nakamura, Hiroyuki; Furukawa, Kinya; Kodama, Takahide; Yamashita, Takaaki; Nomura, Akihiro; Yoshida, Susumu

2018-05-01

To describe the prevalence and determinants of acquired epidermal growth factor receptor (EGFR) T790M gene mutation in a clinical practice setting. We performed a retrospective chart review study between January 2013 and November 2017 across multiple institutes, covering a population of 3 million people. We reviewed the charts of 233 patients non-small cell lung cancer with EGFR mutations. Of them, 99 (42.5%) patients had acquired T790M mutations in EGFR. Patients ≥75 years old and patients with an exon 19 deletion had higher rates of acquired T790M mutation than did younger patients and those with an exon 21 L858R mutation. In 75 patients treated with afatinib, 34 (45.3%) patients had acquired T790M mutation. The sensitivity of T790M mutation detection was lower in plasma specimens than in biopsy specimens. This population-based study confirms previous studies and highlights potential determinants of acquired T790M mutation to be considered in clinical practice. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

PubMed

Zhou, Xiangjun; Zhang, Wei; Yao, Qisheng; Zhang, Hao; Dong, Guie; Zhang, Ming; Liu, Yutao; Chen, Jian-Kang; Dong, Zheng

2017-06-01

Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing. Copyright © 2017 the American Physiological Society.

Impact of Weight Loss at Presentation on Survival in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR-TKI) Sensitive Mutant Advanced Non-small Cell Lung Cancer (NSCLC) Treated with First-line EGFR-TKI.

PubMed

Lin, Liping; Zhao, Juanjuan; Hu, Jiazhu; Huang, Fuxi; Han, Jianjun; He, Yan; Cao, Xiaolong

2018-01-01

Purpose The aim of this study is to evaluate the impact of weight loss at presentation on treatment outcomes of first-line EGFR-tyrosine kinase inhibitors (EGFR-TKI) in EGFR-TKI sensitive mutant NSCLC patients. Methods We retrospectively analyzed the clinical outcomes of 75 consecutive advanced NSCLC patients with EGFR-TKI sensitive mutations (exon 19 deletion or exon 21 L858R) received first-line gefitinib or erlotinib therapy according to weight loss status at presentation in our single center. Results Of 75 EGFR-TKI sensitive mutant NSCLC patients, 49 (65.3%) patients had no weight loss and 26 (34.7%) had weight loss at presentation, the objective response rate (ORR) to EGFR-TKI treatment were similar between the two groups (79.6% vs. 76.9%, p = 0.533). Patients without weight loss at presentation had significantly longer median progression free survival (PFS) (12.4 months vs. 7.6 months; hazard ratio [HR] 0.356, 95% confidence interval [CI] 0.212-0.596, p < 0.001) and overall survival (OS) (28.5 months vs. 20.7 months; HR 0.408, 95% CI 0.215-0.776, p = 0.006) than those with weight loss at presentation; moreover, the stratified analysis by EGFR-TKI sensitive mutation types also found similar trend between these two groups except for OS in EGFR exon 21 L858R mutation patients. Multivariate analysis identified weight loss at presentation and EGFR-TKI sensitive mutation types were independent predictive factors for PFS and OS. Conclusions Weight loss at presentation had a detrimental impact on PFS and OS in EGFR-TKI sensitive mutant advanced NSCLC patients treated with first-line EGFR-TKI. It should be considered as an important factor in the treatment decision or designing of EGFR-TKI clinical trials.

Should EGFR mutations be tested in advanced lung squamous cell carcinomas to guide frontline treatment?

PubMed

Chiu, Chao-Hua; Chou, Teh-Ying; Chiang, Chi-Lu; Tsai, Chun-Ming

2014-10-01

There is no argument over using epidermal growth factor receptor (EGFR) mutation status to guide the frontline treatment for advanced lung adenocarcinoma (LADC); however, the role of the testing in lung squamous cell carcinoma (LSQC) remains controversial. Currently, the guidelines/consensus statements regarding EGFR mutation testing in LSQC are not consistent among different oncology societies. American Society of Clinical Oncology recommends performing EGFR mutation testing in all patients; European Society for Medical Oncology, College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology, and National Comprehensive Cancer Network suggest for some selected group. EGFR mutation is rarely found in LSQC; however, more importantly, it is not a valid predictive biomarker for EGFR tyrosine kinase inhibitors (EGFR-TKI) in LSQC as it has been shown in LADC. Available data showed that the response rate and progression-free survival in EGFR mutant LSQC patients treated with EGFR-TKI are not better than that observed in patients treated with platinum-doublet chemotherapy in the first-line setting. Therefore, in contrast to advanced LADC, EGFR mutation testing may not be necessarily performed upfront in advanced LSQC because not only the mutation rate is low, but also the predictive value is insufficient. For LSQC patients with known sensitizing-EGFR mutations, both conventional chemotherapy and EGFR-TKI are acceptable frontline treatment options.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

PubMed

Pescador, David; Ibáñez-Fonseca, Arturo; Sánchez-Guijo, Fermín; Briñón, Jesús G; Arias, Francisco Javier; Muntión, Sandra; Hernández, Cristina; Girotti, Alessandra; Alonso, Matilde; Del Cañizo, María Consuelo; Rodríguez-Cabello, José Carlos; Blanco, Juan Francisco

2017-08-01

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas

PubMed Central

Fukuoka, Hidenori; Cooper, Odelia; Ben-Shlomo, Anat; Mamelak, Adam; Ren, Song-Guang; Bruyette, Dave; Melmed, Shlomo

2011-01-01

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease. PMID:22105169

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

PubMed

Garousi, Javad; Andersson, Ken G; Dam, Johan H; Olsen, Birgitte B; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Ståhl, Stefan; Löfblom, John; Thisgaard, Helge; Tolmachev, Vladimir

2017-07-20

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111 In-labelled DOTA-conjugated Z EGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z EGFR:2377 . DOTA-Z EGFR:2377 was labelled with 57 Co (T 1/2  = 271.8 d), 55 Co (T 1/2  = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68 Ga (T 1/2  = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57 Co was used in animal studies. Both 57 Co-DOTA-Z EGFR:2377 and 68 Ga-DOTA-Z EGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z EGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57 Co-DOTA-Z EGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68 Ga-DOTA-Z EGFR:2377 . The results of this study suggest that the positron-emitting cobalt isotope 55 Co would be an optimal label for DOTA-Z EGFR:2377 and further development should concentrate on this radionuclide as a label.

Loss of EGFR confers acquired resistance to AZD9291 in an EGFR-mutant non-small cell lung cancer cell line with an epithelial-mesenchymal transition phenotype.

PubMed

Xu, Jing; Zhao, Xiaoting; He, Dengfeng; Wang, Jinghui; Li, Weiying; Liu, Yinghui; Ma, Li; Jiang, Mei; Teng, Yu; Wang, Ziyu; Gu, Meng; Wu, Jianbin; Wang, Yue; Yue, Wentao; Zhang, Shucai

2018-05-24

AZD9291 is an irreversible, small-molecule inhibitor which has potency against mutant EGFR- and T790M-resistant mutation. Despite the encouraging efficacy in clinical, the acquired resistance will finally occur. Further study will need to be done to identify the acquired resistance mechanisms and determine the next treatment. We established an AZD9291-resistant cell line (HCC827/AZDR) from parental HCC827 cell line through stepwise pulsed selection of AZD9291. The expression of EGFR and its downstream pathways were determined by western blot analysis or immunofluorescence assay. The sensitivity to indicated agents were evaluated by MTS. Compared with parental HCC827 cells, the HCC827/AZDR cells showed high resistance to AZD9291 and other EGFR-TKIs, and exhibited a mesenchymal-like phenotype. Almost complete loss of EGFR expression was observed in HCC827/AZDR cells. But the activation of downstream pathway, MAPK signaling, was found in HCC827/AZDR cells even in the presence of AZD9291. Inhibition of MAPK signaling had no effect on cell viability of HCC827/AZDR and could not reverse AZD9291 resistance because of the subsequent activation of AKT signaling. When treated with the combination of AKT and MAPK inhibitor, HCC827/AZDR showed remarkable growth inhibition. Loss of EGFR could be proposed as a potential acquired resistance mechanism of AZD9291 in EGFR-mutant NSCLC cells with an EMT phenotype. Despite the loss of EGFR, the activation of MAPK pathway which had crosstalk with AKT pathway could maintain the proliferation and survival of resistant cells. Blocking MAPK and AKT signaling may be a potential therapeutic strategy following AZD9291 resistance.

COX-2/EGFR expression and survival among women with adenocarcinoma of the lung

PubMed Central

Van Dyke, Alison L.; Cote, Michele L.; Prysak, Geoffrey M.; Claeys, Gina B.; Wenzlaff, Angie S.; Murphy, Valerie C.; Lonardo, Fulvio; Schwartz, Ann G.

2008-01-01

Previous studies suggest that cyclooxygenase-2 (COX-2) expression may predict survival among patients with non-small cell lung cancer. COX-2 may interact with epidermal growth factor receptor (EGFR), suggesting that combined COX-2/EGFR expression may provide predictive value. The extent to which their independent or combined expression is associated with prognosis in women with adenocarcinoma of the lung is unknown. In the present study, we examined relationships between COX-2 expression (n = 238), EGFR expression (n = 158) and dual COX-2/EGFR expression (n = 157) and survival among women with adenocarcinoma of the lung. Overall survival was estimated by constructing Cox proportional hazards models adjusting for other significant variables and stratifying by stage at diagnosis and race. Clinical or demographic parameters were not associated with either COX-2 or EGFR expression. Patients with COX-2-positive tumors tended to have poorer prognosis than did patients with COX-2-negative tumors [hazard ratio (HR) 1.67, 95% confidence interval (CI) 1.01–2.78]. African-Americans with COX-2-positive tumors had a statistically non-significant higher risk of death than African-Americans with COX-2-negative tumors (HR 5.58, 95% CI 0.64–48.37). No association between COX-2 expression and survival was observed among Caucasians (HR 1.29, 95% CI 0.72–2.30). EGFR expression was associated with a 44% reduction in the risk of death (HR 0.56, 95% CI 0.32–0.98). COX-2−/EGFR+ tumor expression, but not COX-2+/EGFR+ tumor expression, was associated with survival when compared with other combined expression results. In conclusion, COX-2 and EGFR expression, but not combined COX-2+/EGFR+ expression, independently predict survival of women with adenocarcinoma of the lung. PMID:18453539

Mechanisms of skin aging induced by EGFR inhibitors.

PubMed

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner U; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E; Homey, Bernhard

2016-10-01

The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls. EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.

Mechanisms of skin aging induced by EGFR inhibitors

PubMed Central

Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Hevezi, Peter; Bölke, Edwin; Sohn, Dennis; Jänicke, Reiner; Belum, Viswanath Reddy; Robert, Caroline; Lacouture, Mario E.; Homey, Bernhard

2017-01-01

BACKGROUND The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. OBJECTIVE To evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. PATIENTS AND METHODS Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (< 25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. RESULTS Progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA β-Gal activity. There was significantly decreased baseline expression in EGFR-density in aged skin, when compared to young controls. CONCLUSIONS EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents. PMID:27165055

Antitumor efficacy of triple monoclonal antibody inhibition of epidermal growth factor receptor (EGFR) with MM151 in EGFR-dependent and in cetuximab-resistant human colorectal cancer cells

PubMed Central

Napolitano, Stefania; Martini, Giulia; Martinelli, Erika; Della Corte, Carminia Maria; Morgillo, Floriana; Belli, Valentina; Cardone, Claudia; Matrone, Nunzia; Ciardiello, Fortunato; Troiani, Teresa

2017-01-01

Purpose We investigated the effect of triple monoclonal antibody inhibition of EGFR to overcome acquired resistance to first generation of anti-EGFR inhibitors. Experimental design MM151 is a mixture of three different monoclonal IgG1 antibodies directed toward three different, non-overlapping, epitopes of the EGFR. We performed an in vivo study by using human CRC cell lines (SW48, LIM 1215 and CACO2) which are sensitive to EGFR inhibitors, in order to evaluate the activity of MM151 as compared to standard anti-EGFR mAbs, such as cetuximab, as single agent or in a sequential strategy of combination MM151 with irinotecan (induction therapy) followed by MM151 with a selective MEK1/2 inhibitor (MEKi) (maintenance therapy). Furthermore, the ability of MM151 to overcome acquired resistance to cetuximab has been also evaluated in cetuximab-refractory CRC models. Results MM151 shown stronger antitumor activity as compared to cetuximab. The maintenance treatment with MM151 plus MEKi resulted the most effective therapeutic modality. In fact, this combination caused an almost complete suppression of tumor growth in SW48, LIM 1215 and CACO2 xenografts model at 30 week. Moreover, in this treatment group, mice with no evidence of tumor were more than double as compared to single agent treated mice. Its superior activity has also been demonstrated, in cetuximab-refractory CRC models. Conclusions These results provide experimental evidence that more efficient and complete EGFR blockade may determine better antitumor activity and could contribute to prevent and/or overcome acquired resistance to EGFR inhibitors. PMID:29137301

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

PubMed

Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

2017-08-01

Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR

Design, Synthesis and Evaluation of Ribose-modified Anilinopyrimidine Derivatives as EGFR Tyrosine Kinase Inhibitors

NASA Astrophysics Data System (ADS)

Hu, Xiuqin; Wang, Disha; Tong, Yi; Tong, Linjiang; Wang, Xia; Zhu, Lili; Xie, Hua; Li, Shiliang; Yang, You; Xu, Yufang

2017-11-01

The synthesis of a series of ribose-modified anilinopyrimidine derivatives was efficiently achieved by utilizing DBU or tBuOLi-promoted coupling of ribosyl alcohols with 2,4,5-trichloropyrimidine as key step. Preliminary biological evaluation of this type of compounds as new EGFR tyrosine kinase inhibitors for combating EGFR L858R/T790M mutant associated with drug resistance in the treatment of non-small cell lung cancer revealed that 3-N-acryloyl-5-O-anilinopyrimidine ribose derivative 1a possessed potent and specific inhibitory activity against EGFR L858R/T790M over WT EGFR. Based upon molecular docking studies of the binding mode between compound 1a and EGFR, the distance between the Michael receptor and the pyrimidine scaffold is considered as an important factor for the inhibitory potency and future design of selective EGFR tyrosine kinase inhibitors against EGFR L858R/T790M mutants.

The influence of platelet-rich fibrin on angiogenesis in guided bone regeneration using xenogenic bone substitutes: a study of rabbit cranial defects.

PubMed

Yoon, Jong-Suk; Lee, Sang-Hwa; Yoon, Hyun-Joong

2014-10-01

The purpose of this study was to investigate the influence of platelet-rich fibrin (PRF) on angiogenesis and osteogenesis in guided bone regeneration (GBR) using xenogenic bone in rabbit cranial defects. In each rabbit, 2 circular bone defects, one on either side of the midline, were prepared using a reamer drill. Each of the experimental sites received bovine bone with PRF, and each of the control sites received bovine bone alone. The animals were sacrificed at 1 week (n = 4), 2 weeks (n = 3) and 4 weeks (n = 3). Biopsy samples were examined histomorphometrically by light microscopy, and expression of vascular endothelial growth factor (VEGF) was determined by immunohistochemical staining. At all experimental time points, immunostaining intensity for VEGF was consistently higher in the experimental group than in the control group. However, the differences between the control group and the experimental group were not statistically significant in the histomorphometrical and immunohistochemical examinations. The results of this study suggest that PRF may increase the number of marrow cells. However, PRF along with xenogenic bone substitutes does not show a significant effect on bony regeneration. Further large-scale studies are needed to confirm our results. Copyright © 2014 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

PubMed Central

Tomas, Alejandra; Vaughan, Simon O.; Burgoyne, Thomas; Sorkin, Alexander; Hartley, John A.; Hochhauser, Daniel; Futter, Clare E.

2015-01-01

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance. PMID:26066081

Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: An Evidence-Based Analysis.

PubMed

2010-01-01

sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%. In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting? A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying-Nai; Wang, Hongmei; Yamaguchi, Hirohito

2010-09-03

Research highlights: {yields} ARF1 activation is involved in the EGFR transport to the ER and the nucleus. {yields} Assembly of {gamma}-COP coatomer mediates EGFR transport to the ER and the nucleus. {yields} Golgi-to-ER retrograde trafficking regulates nuclear transport of EGFR. -- Abstract: Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored.more » Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH{sub 2}-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with {gamma}-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.« less

Involvement of lipid rafts in adhesion-induced activation of Met and EGFR.

PubMed

Lu, Ying-Che; Chen, Hong-Chen

2011-10-27

Cell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure. Human epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy. We demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts. Our results suggest for the first time that cell adhesion to a

Loss of EGFR-ASAP1 signaling in metastatic and unresectable hepatoblastoma.

PubMed

Ranganathan, Sarangarajan; Ningappa, Mylarappa; Ashokkumar, Chethan; Higgs, Brandon W; Min, Jun; Sun, Qing; Schmitt, Lori; Subramaniam, Shankar; Hakonarson, Hakon; Sindhi, Rakesh

2016-12-02

Hepatoblastoma (HBL), the most common childhood liver cancer is cured with surgical resection after chemotherapy or with liver transplantation if local invasion and multifocality preclude resection. However, variable survival rates of 60-80% and debilitating chemotherapy sequelae argue for more informed treatment selection, which is not possible by grading the Wnt-β-catenin over activity present in most HBL tumors. A hypothesis-generating whole transcriptome analysis shows that HBL tumors removed at transplantation are enriched most for cancer signaling pathways which depend predominantly on epidermal growth factor (EGF) signaling, and to a lesser extent, on aberrant Wnt-β-catenin signaling. We therefore evaluated whether EGFR, ASAP1, ERBB2 and ERBB4, which signal downstream after ligation of EGF, and which show aberrant expression in several other invasive cancers, would also predict HBL tumor invasiveness. Immunohistochemistry of HBL tumors (n = 60), which are histologically heterogeneous, shows that compared with well-differentiated fetal cells, less differentiated embryonal and undifferentiated small cells (SCU) progressively lose EGFR and ASAP1 expression. This trend is exaggerated in unresectable, locally invasive or metastatic tumors, in which embryonal tumor cells are EGFR-negative, while SCU cells are EGFR-negative and ASAP1-negative. Loss of EGFR-ASAP1 signaling characterizes undifferentiated and invasive HBL. EGFR-expressing HBL tumors present novel therapeutic targeting opportunities.

EGFR inhibition attenuates diabetic nephropathy through decreasing ROS and endoplasmic reticulum stress

PubMed Central

Zhong, Peng; Wang, Jingying; Weng, Qiaoyou; Qian, Yuanyuan; Han, Jibo; Zou, Chunpeng; Liang, Guang

2017-01-01

Diabetic nephropathy (DN) is a progressive kidney disease due to glomerular capillary damage in diabetic patients. Endoplasmic reticulum (ER) stress caused by reactive oxygen species (ROS) is associated with DN progression. Epidermal growth factor receptor (EGFR) mediates oxidative stress and damage of cardiomyocytes in diabetic mice. Here we demonstrated that AG1478, a specific inhibitor of EGFR, blocked EGFR and AKT phosphorylation in diabetic mice. Oxidative stress and ER stress markers were eliminated after AG1478 administration. AG1478 decreased pro-fibrotic genes TGF-β and collagen IV. Furthermore, we found that high glucose (HG) induced oxidative stress and ER stress, and subsequently increased ATF4 and CHOP. These changes were eliminated by either AG1478 or ROS scavenger N-acetyl-L-cysteine (NAC) administration. These results were confirmed by knock-down approaches in renal mesangial SV40 cells. However, AG1478, not NAC, reversed HG induced EGFR and AKT phosphorylation. These results suggest that EGFR/AKT/ROS/ER stress signaling plays an essential role in DN development and inhibiting EGFR may serve as a potential therapeutic strategy in diabetic kidney diseases. PMID:28427241

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR.

PubMed

Hwang, Ki-Eun; Kwon, Su-Jin; Kim, Young-Suk; Park, Do-Sim; Kim, Byoung-Ryun; Yoon, Kwon-Ha; Jeong, Eun-Taik; Kim, Hak-Ryul

2014-05-01

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib-simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular Basis for Necitumumab Inhibition of EGFR Variants Associated with Acquired Cetuximab Resistance.

PubMed

Bagchi, Atrish; Haidar, Jaafar N; Eastman, Scott W; Vieth, Michal; Topper, Michael; Iacolina, Michelle D; Walker, Jason M; Forest, Amelie; Shen, Yang; Novosiadly, Ruslan D; Ferguson, Kathryn M

2018-02-01

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR . ©2017 American Association for Cancer Research.

Large-scale manufacturing of GMP-compliant anti-EGFR targeted nanocarriers: production of doxorubicin-loaded anti-EGFR-immunoliposomes for a first-in-man clinical trial.

PubMed

Wicki, Andreas; Ritschard, Reto; Loesch, Uli; Deuster, Stefanie; Rochlitz, Christoph; Mamot, Christoph

2015-04-30

We describe the large-scale, GMP-compliant production process of doxorubicin-loaded and anti-EGFR-coated immunoliposomes (anti-EGFR-ILs-dox) used in a first-in-man, dose escalation clinical trial. 10 batches of this nanoparticle have been produced in clean room facilities. Stability data from the pre-GMP and the GMP batch indicate that the anti-EGFR-ILs-dox nanoparticle was stable for at least 18 months after release. Release criteria included visual inspection, sterility testing, as well as measurements of pH (pH 5.0-7.0), doxorubicin HCl concentration (0.45-0.55 mg/ml), endotoxin concentration (<1.21 IU/ml), leakage (<10%), particle size (Z-average of Caelyx ± 20 nm), and particle uptake (uptake absolute: >0.50 ng doxorubicin/μg protein; uptake relatively to PLD: >5 fold). All batches fulfilled the defined release criteria, indicating a high reproducibility as well as batch-to-batch uniformity of the main physico-chemical features of the nanoparticles in the setting of the large-scale GMP process. In the clinical trial, 29 patients were treated with this nanoparticle between 2007 and 2010. Pharmacokinetic data of anti-EGFR-ILs-dox collected during the clinical study revealed stability of the nanocarrier in vivo. Thus, reliable and GMP-compliant production of anti-EGFR-targeted nanoparticles for clinical application is feasible. Copyright © 2015 Elsevier B.V. All rights reserved.

Detecting and treating breast cancer resistance to EGFR inhibitors

DOEpatents

Moonlee, Sun-Young; Bissell, Mina J.; Furuta, Saori; Meier, Roland; Kenny, Paraic A.

2016-04-05

The application describes therapeutic compositions and methods for treating cancer. For example, therapeutic compositions and methods related to inhibition of FAM83A (family with sequence similarity 83) are provided. The application also describes methods for diagnosing cancer resistance to EGFR inhibitors. For example, a method of diagnosing cancer resistance to EGFR inhibitors by detecting increased FAM83A levels is described.

EPHA2 Blockade Overcomes Acquired Resistance to EGFR Kinase Inhibitors in Lung Cancer.

PubMed

Amato, Katherine R; Wang, Shan; Tan, Li; Hastings, Andrew K; Song, Wenqiang; Lovly, Christine M; Meador, Catherine B; Ye, Fei; Lu, Pengcheng; Balko, Justin M; Colvin, Daniel C; Cates, Justin M; Pao, William; Gray, Nathanael S; Chen, Jin

2016-01-15

Despite the success of treating EGFR-mutant lung cancer patients with EGFR tyrosine kinase inhibitors (TKI), all patients eventually acquire resistance to these therapies. Although various resistance mechanisms have been described, there are currently no FDA-approved therapies that target alternative mechanisms to treat lung tumors with acquired resistance to first-line EGFR TKI agents. Here we found that EPHA2 is overexpressed in EGFR TKI-resistant tumor cells. Loss of EPHA2 reduced the viability of erlotinib-resistant tumor cells harboring EGFR(T790M) mutations in vitro and inhibited tumor growth and progression in an inducible EGFR(L858R+T790M)-mutant lung cancer model in vivo. Targeting EPHA2 in erlotinib-resistant cells decreased S6K1-mediated phosphorylation of cell death agonist BAD, resulting in reduced tumor cell proliferation and increased apoptosis. Furthermore, pharmacologic inhibition of EPHA2 by the small-molecule inhibitor ALW-II-41-27 decreased both survival and proliferation of erlotinib-resistant tumor cells and inhibited tumor growth in vivo. ALW-II-41-27 was also effective in decreasing viability of cells with acquired resistance to the third-generation EGFR TKI AZD9291. Collectively, these data define a role for EPHA2 in the maintenance of cell survival of TKI-resistant, EGFR-mutant lung cancer and indicate that EPHA2 may serve as a useful therapeutic target in TKI-resistant tumors. ©2016 American Association for Cancer Research.

Monitoring of Circulating Tumor Cells and Their Expression of EGFR/Phospho-EGFR During Combined Radiotherapy Regimens in Locally Advanced Squamous Cell Carcinoma of the Head and Neck

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tinhofer, Ingeborg, E-mail: ingeborg.tinhofer@charite.de; Hristozova, Tsvetana; Stromberger, Carmen

2012-08-01

Purpose: The numbers of circulating tumor cells (CTCs) and their expression/activation of epidermal growth factor receptor (EGFR) during the course of combined chemo- or bioradiotherapy regimens as potential biomarkers of treatment efficacy in squamous cell carcinoma of the head and neck (SCCHN) were determined. Methods and Materials: Peripheral blood samples from SCCHN patients with locally advanced stage IVA/B disease who were treated with concurrent radiochemotherapy or induction chemotherapy followed by bioradiation with cetuximab were included in this study. Using flow cytometry, the absolute number of CTCs per defined blood volume as well as their expression of EGFR and its phosphorylatedmore » form (pEGFR) during the course of treatment were assessed. Results: Before treatment, we detected {>=}1 CTC per 3.75 mL blood in 9 of 31 patients (29%). Basal expression of EGFR was detected in 100% and pEGFR in 55% of the CTC+ cases. The frequency of CTC detection was not influenced by induction chemotherapy. However, the number of CTC+ samples significantly increased after radiotherapy. This radiation-induced increase in CTC numbers was less pronounced when radiotherapy was combined with cetuximab compared to its combination with cisplatin/5-fluorouracil. The former treatment regimen was also more effective in reducing pEGFR expression in CTCs. Conclusions: Definitive radiotherapy regimens of locally advanced SCCHN can increase the number of CTCs and might thus contribute to a systemic spread of tumor cells. Further studies are needed to evaluate the predictive value of the radiation-induced increase in CTC numbers and the persistent activation of the EGFR signalling pathway in individual CTC+ cases.« less

Prognostic significance of epidermal growth factor receptor (EGFR) over expression in urothelial carcinoma of urinary bladder.

PubMed

Hashmi, Atif Ali; Hussain, Zubaida Fida; Irfan, Muhammad; Khan, Erum Yousuf; Faridi, Naveen; Naqvi, Hanna; Khan, Amir; Edhi, Muhammad Muzzammil

2018-06-07

Epidermal growth factor receptor (EGFR) has been shown to have abnormal expression in many human cancers and is considered as a marker of poor prognosis. Frequency of over expression in bladder cancer has not been studied in our population; therefore we aimed to evaluate the frequency and prognostic significance of EGFR immunohistochemical expression in locoregional population. We performed EGFR immunohistochemistry on 126 cases of bladder cancer and association of EGFR expression with tumor grade, lamina propria invasion, deep muscle invasion and recurrence of disease was evaluated. High EGFR expression was noted in 26.2% (33 cases), 15.1% (19 cases) and 58.7% (74 cases) revealed low and no EGFR expression respectively. Significant association of EGFR expression was noted with tumor grade, lamina propria invasion, deep muscle invasion and recurrence status while no significant association was seen with age, gender and overall survival. Kaplan- Meier curves revealed significant association of EGFR expression with recurrence while no significant association was seen with overall survival. Significant association of EGFR overexpression with tumor grade, muscularis propria invasion and recurrence signifies its prognostic value; therefore EGFR can be used as a prognostic biomarker in Urothelial bladder carcinoma.

Sex difference in EGFR pathways in mouse kidney-potential impact on the immune system.

PubMed

Liu, Fengxia; Jiao, Yan; Jiao, Yun; Garcia-Godoy, Franklin; Gu, Weikuan; Liu, Qingyi

2016-11-24

Epidermal growth factor receptor (Egfr) has been the target of several drugs for cancers. The potential gender differences in genes in the Egfr axis have been suggested in humans and in animal models. Female and male mice from the same recombinant inbred (RI) strain have the same genomic components except the sex difference. A population of different RI mouse strains allows to conduct precise analysis of molecular pathways and regulation of Egfr between female and male mice. The whole genome expression profiles of 70 genetically diverse RI strains of mice were used to compare three major molecular aspects of Egfr gene: the relative expression levels, gene network and expression quantitative trait loci (eQTL) that regulate the expression of Egfr between female and male mice. Our data showed that there is a significant sex difference in the expression levels in kidney. A considerable number of genes in the gene network of Egfr are sex differentially expressed. The expression levels of Egfr in mice are statistical significant different between C57BL/6 J (B6) and DBA/2 J (D2) genotypes in male while no difference in female mice. The eQTLs that regulate the expression levels of Egfr between female and male mice are also different. Furthermore, the differential expression levels of Egfr showed significantly different correlations with two known biological traits between male and female mice. Overall there is a substantial sex difference in the Egfr pathways in mice. These data may have significant impact on drug target design, development, formulation, and dosage determinant for women and men in clinical trials.

Detection of Rare Mutations in EGFR-ARMS-PCR-Negative Lung Adenocarcinoma by Sanger Sequencing.

PubMed

Liang, Chaoyue; Wu, Zhuolin; Gan, Xiaohong; Liu, Yuanbin; You, You; Liu, Chenxian; Zhou, Chengzhi; Liang, Ying; Mo, Haiyun; Chen, Allen M; Zhang, Jiexia

2018-01-01

This study aimed to identify potential epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer that went undetected by amplification refractory mutation system-Scorpion real-time PCR (ARMS-PCR). A total of 200 specimens were obtained from the First Affiliated Hospital of Guangzhou Medical University from August 2014 to August 2015. In total, 100 ARMS-negative and 100 ARMS-positive specimens were evaluated for EGFR gene mutations by Sanger sequencing. The methodology and sensitivity of each method and the outcomes of EGFR-tyrosine kinase inhibitor (TKI) therapy were analyzed. Among the 100 ARMS-PCR-positive samples, 90 were positive by Sanger sequencing, while 10 cases were considered negative, because the mutation abundance was less than 10%. Among the 100 negative cases, three were positive for a rare EGFR mutation by Sanger sequencing. In the curative effect analysis of EGFR-TKIs, the progression-free survival (PFS) analysis based on ARMS and Sanger sequencing results showed no difference. However, the PFS of patients with a high abundance of EGFR mutation was 12.4 months [95% confidence interval (CI), 11.6-12.4 months], which was significantly higher than that of patients with a low abundance of mutations detected by Sanger sequencing (95% CI, 10.7-11.3 months) (p<0.001). The ARMS method demonstrated higher sensitivity than Sanger sequencing, but was prone to missing mutations due to primer design. Sanger sequencing was able to detect rare EGFR mutations and deemed applicable for confirming EGFR status. A clinical trial evaluating the efficacy of EGFR-TKIs in patients with rare EGFR mutations is needed. © Copyright: Yonsei University College of Medicine 2018

Cetuximab Resistance in Head and Neck Cancer Is Mediated by EGFR-K521 Polymorphism.

PubMed

Braig, Friederike; Kriegs, Malte; Voigtlaender, Minna; Habel, Beate; Grob, Tobias; Biskup, Karina; Blanchard, Veronique; Sack, Markus; Thalhammer, Anja; Ben Batalla, Isabel; Braren, Ingke; Laban, Simon; Danielczyk, Antje; Goletz, Steffen; Jakubowicz, Elzbieta; Märkl, Bruno; Trepel, Martin; Knecht, Rainald; Riecken, Kristoffer; Fehse, Boris; Loges, Sonja; Bokemeyer, Carsten; Binder, Mascha

2017-03-01

Head and neck squamous cell carcinomas (HNSCC) exhibiting resistance to the EGFR-targeting drug cetuximab poses a challenge to their effective clinical management. Here, we report a specific mechanism of resistance in this setting based upon the presence of a single nucleotide polymorphism encoding EGFR-K 521 (K-allele), which is expressed in >40% of HNSCC cases. Patients expressing the K-allele showed significantly shorter progression-free survival upon palliative treatment with cetuximab plus chemotherapy or radiation. In several EGFR-mediated cancer models, cetuximab failed to inhibit downstream signaling or to kill cells harboring a high K-allele frequency. Cetuximab affinity for EGFR-K 521 was reduced slightly, but ligand-mediated EGFR activation was intact. We found a lack of glycan sialyation on EGFR-K 521 that associated with reduced protein stability, suggesting a structural basis for reduced cetuximab efficacy. CetuGEX, an antibody with optimized Fc glycosylation targeting the same epitope as cetuximab, restored HNSCC sensitivity in a manner associated with antibody-dependent cellular cytotoxicity rather than EGFR pathway inhibition. Overall, our results highlight EGFR-K 521 expression as a key mechanism of cetuximab resistance to evaluate prospectively as a predictive biomarker in HNSCC patients. Further, they offer a preclinical rationale for the use of ADCC-optimized antibodies to treat tumors harboring this EGFR isoform. Cancer Res; 77(5); 1188-99. ©2016 AACR . ©2016 American Association for Cancer Research.

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres as a therapeutic target against EGFR-positive non-small cell lung cancer.

PubMed

Cheng, Chun-Chia; Chou, Kuei-Fang; Wu, Cheng-Wen; Su, Nai-Wen; Peng, Cheng-Liang; Su, Ying-Wen; Chang, Jungshan; Ho, Ai-Sheng; Lin, Huan-Chau; Chen, Caleb Gon-Shen; Yang, Bi-Ling; Chang, Yu-Cheng; Chiang, Ya-Wen; Lim, Ken-Hong; Chang, Yi-Fang

2018-02-01

YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Radiological Features of Brain Metastases from Non-small Cell Lung Cancer Harboring EGFR Mutation.

PubMed

Takamori, Shinkichi; Toyokawa, Gouji; Shimokawa, Mototsugu; Kinoshita, Fumihiko; Kozuma, Yuka; Matsubara, Taichi; Haratake, Naoki; Akamine, Takaki; Mukae, Nobutaka; Hirai, Fumihiko; Tagawa, Tetsuzo; Oda, Yoshinao; Iwaki, Toru; Iihara, Koji; Honda, Hiroshi; Maehara, Yoshihiko

2018-06-01

To investigate the radiological features on computed tomography (CT) of brain metastasis (BM) from epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). Thirty-four patients with NSCLC with BMs who underwent surgical resection of the BMs at the Department of Neurosurgery, Kyushu University from 2005 to 2016 were enrolled in the study. The EGFR statuses of the 34 BMs were investigated. Radiological features, including the number, size, and location of the tumor, were delineated by CT. Patients with EGFR-mutated BMs had significantly higher frequencies of multiple metastases than those with the non-EGFR-mutated type (p=0.042). BMs harboring mutations in EGFR were more frequently observed in the central area of the brain compared to those without mutations in EGFR (p=0.037). Careful follow-up of patients with EGFR-mutated NSCLC may be necessary given the high frequencies of multiple BMs and their location in the central area of the brain. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

HER2 and EGFR overexpression support metastatic progression of prostate cancer to bone

PubMed Central

Day, Kathleen C.; Hiles, Guadalupe Lorenzatti; Kozminsky, Molly; Dawsey, Scott J.; Paul, Alyssa; Broses, Luke J.; Shah, Rajal; Kunja, Lakshmi P.; Hall, Christopher; Palanisamy, Nallasivam; Daignault-Newton, Stephanie; El-Sawy, Layla; Wilson, Steven James; Chou, Andrew; Ignatoski, Kathleen Woods; Keller, Evan; Thomas, Dafydd; Nagrath, Sunitha; Morgan, Todd; Day, Mark L.

2016-01-01

Activation of the epidermal growth factor receptors EGFR (ErbB1) and HER2 (ErbB2) drive the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. PMID:27793843

Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop.

PubMed

Pirker, Robert; Herth, Felix J F; Kerr, Keith M; Filipits, Martin; Taron, Miquel; Gandara, David; Hirsch, Fred R; Grunenwald, Dominique; Popper, Helmut; Smit, Egbert; Dietel, Manfred; Marchetti, Antonio; Manegold, Christian; Schirmacher, Peter; Thomas, Michael; Rosell, Rafael; Cappuzzo, Federico; Stahel, Rolf

2010-10-01

Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations. The "EGFR testing in NSCLC: from biology to clinical practice" International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group. Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests. The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.

Icotinib in Patients with Pretreated Advanced Esophageal Squamous Cell Carcinoma with EGFR Overexpression or EGFR Gene Amplification: A Single-Arm, Multicenter Phase 2 Study.

PubMed

Huang, Jing; Fan, Qingxia; Lu, Ping; Ying, Jianming; Ma, Changwu; Liu, Wei; Liu, Ying; Tan, Fenlai; Sun, Yan

2016-06-01

Epidermal growth factor receptor (EGFR) has been reported to be overexpressed and amplified in a high percentage of patients with esophageal squamous cell carcinoma (ESCC). The activity of icotinib, an EGFR tyrosine kinase inhibitor, was assessed in previously treated ESCC with EGFR overexpression or amplification. For this phase 2, single-arm, multicenter trial undertaken at six hospitals in China, we included Chinese patients with previously treated, histologically confirmed advanced ESCC and EGFR overexpression (immunohistochemical staining sore of 3+) or amplification (positive fluorescence in situ hybridization result). These patients received oral icotinib (250 mg, three times daily).The primary end point was the proportion of patients with objective responses as assessed by an independent radiology review committee. Between December 5, 2013, and May 28, 2015, a total of 281 patients were screened. Fifty-four eligible patients were enrolled. Nine responses were observed, including one complete response and eight partial responses, and 16 patients had stable disease, resulting in a 16.7% objective response rate (95% confidence interval [CI]: 6.7-26.6) and 46.3% disease control rate (95% CI: 33.0-59.6). The median progression-free survival and overall survival times were 52 (95% CI: 40-95) days and 153 (95% CI: 139-218) days, respectively. A total of 43 patients experienced at least one adverse event, but most were only grade 1 to 2 in severity. The most frequent was rash (48.1%), followed by diarrhea (22.2%). Icotinib showed favorable activity in patients with advanced, previously treated ESCC with EGFR overexpression or amplification. These findings suggest further research into EGFR overexpression or amplification for selecting responsive patients. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Targeting the Kinase-Independent Pro-survival Function of EGFR in Prostate Cancer

DTIC Science & Technology

2013-06-01

AD AWARD NUMBER: W81XWH-09-1-0343 TITLE: Targeting the Kinase-Independent Pro -survival Function of EGFR in Prostate Cancer...SUBTITLE Targeting the Kinase-Independent, Pro -survival Function of EGFR in Prostate Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-09-1...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The proposed study, targeting the kinase independent pro -survival function of EGFR in prostate

[Current Possibilities for Predicting Responses to EGFR Blockade in Metastatic Colorectal Cancer].

PubMed

Němeček, R; Svoboda, M; Slabý, O

2016-01-01

The combination of modern systemic chemotherapy and anti-EGFR monoclonal antibodies improves overall survival and quality of life for patients with metastatic colorecal cancer. By contrast, the addition of anti-EGFR therapy to the treatment regime of resistant patients may lead to worse progression-free survival and overall survival. Therefore, identifying sensitive and resistant patients prior to targeted therapy of metastatic colorecal cancer is a key point during the initial decision making process. Previous research shows that primary resistance to EGFR blockade is in most cases caused by constitutive activation of signaling pathways downstream of EGFR. Of all relevant factors (mutation of KRAS, NRAS, BRAF, and PIK3CA oncogenes, inactivation of tumor suppressors PTEN and TP53, amplification of EGFR and HER2, and expression of epiregulin and amphiregulin, mikroRNA miR-31-3p, and miR-31-5p), only evaluation of KRAS and NRAS mutations has entered routine clinical practice. The role of the other markers still needs to be validated. The ongoing benefit of anti-EGFR therapy could be indicated by specific clinical parameters measured after the initiation of targeted therapy, including early tumor shrinkage, the deepness of the response, or hypomagnesemia. The accuracy of predictive dia-gnostic tools could be also increased by examining a combination of predictive markers using next generation sequencing methods. However, unjustified investigation of many molecular markers should be resisted as this may complicate interpretation of the results, particularly in terms of their specific clinical relevance. The aim of this review is to describe current possibilities with respect to predicting responses to EGFR blockade in the context of the EGFR pathway, and the utilization of such results in routine clinical practice.

Evolution of the EGFR pathway in Metazoa and its diversification in the planarian Schmidtea mediterranea.

PubMed

Barberán, Sara; Martín-Durán, José M; Cebrià, Francesc

2016-06-21

The EGFR pathway is an essential signaling system in animals, whose core components are the epidermal growth factors (EGF ligands) and their trans-membrane tyrosine kinase receptors (EGFRs). Despite extensive knowledge in classical model organisms, little is known of the composition and function of the EGFR pathway in most animal lineages. Here, we have performed an extensive search for the presence of EGFRs and EGF ligands in representative species of most major animal clades, with special focus on the planarian Schmidtea mediterranea. With the exception of placozoans and cnidarians, we found that the EGFR pathway is potentially present in all other analyzed animal groups, and has experienced frequent independent expansions. We further characterized the expression domains of the EGFR/EGF identified in S. mediterranea, revealing a wide variety of patterns and localization in almost all planarian tissues. Finally, functional experiments suggest an interaction between one of the previously described receptors, Smed-egfr-5, and the newly found ligand Smed-egf-6. Our findings provide the most comprehensive overview to date of the EGFR pathway, and indicate that the last common metazoan ancestor had an initial complement of one EGFR and one putative EGF ligand, which was often expanded or lost during animal evolution.

Evolution of the EGFR pathway in Metazoa and its diversification in the planarian Schmidtea mediterranea

PubMed Central

Barberán, Sara; Martín-Durán, José M.; Cebrià, Francesc

2016-01-01

The EGFR pathway is an essential signaling system in animals, whose core components are the epidermal growth factors (EGF ligands) and their trans-membrane tyrosine kinase receptors (EGFRs). Despite extensive knowledge in classical model organisms, little is known of the composition and function of the EGFR pathway in most animal lineages. Here, we have performed an extensive search for the presence of EGFRs and EGF ligands in representative species of most major animal clades, with special focus on the planarian Schmidtea mediterranea. With the exception of placozoans and cnidarians, we found that the EGFR pathway is potentially present in all other analyzed animal groups, and has experienced frequent independent expansions. We further characterized the expression domains of the EGFR/EGF identified in S. mediterranea, revealing a wide variety of patterns and localization in almost all planarian tissues. Finally, functional experiments suggest an interaction between one of the previously described receptors, Smed-egfr-5, and the newly found ligand Smed-egf-6. Our findings provide the most comprehensive overview to date of the EGFR pathway, and indicate that the last common metazoan ancestor had an initial complement of one EGFR and one putative EGF ligand, which was often expanded or lost during animal evolution. PMID:27325311

EGFR Signaling in the Brain Is Necessary for Olfactory Learning in "Drosophila" Larvae

ERIC Educational Resources Information Center

Rahn, Tasja; Leippe, Matthias; Roeder, Thomas; Fedders, Henning

2013-01-01

Signaling via the epidermal growth factor receptor (EGFR) pathway has emerged as one of the key mechanisms in the development of the central nervous system in "Drosophila melanogaster." By contrast, little is known about the functions of EGFR signaling in the differentiated larval brain. Here, promoter-reporter lines of EGFR and its most prominent…

Discovery of a series of novel phenylpiperazine derivatives as EGFR TK inhibitors

NASA Astrophysics Data System (ADS)

Sun, Juan; Wang, Xin-Yi; Lv, Peng-Cheng; Zhu, Hai-Liang

2015-09-01

Human epidermal growth factor receptor (EGFR) is an important drug target that plays a fundamental role in signal transduction pathways in oncology. We report herein the discovery of a novel class of phenylpiperazine derivatives with improved potency toward EGFR. The biological activity of compound 3p as inhibitor of EGFR was further investigated both in vitro and in vivo. Notably, compound 3p exhibited an IC50 in the nanomolar range in A549 cell cultures and induced a cessation of tumor growth with no toxicity, as determined by loss of body weight and death of treated mice. Compoutational docking studies also showed that compound 3p has interaction with EGFR key residues in the active site.

The combi-targeting concept: in vitro and in vivo fragmentation of a stable combi-nitrosourea engineered to interact with the epidermal growth factor receptor while remaining DNA reactive.

PubMed

Qiu, Qiyu; Domarkas, Juozas; Banerjee, Ranjita; Merayo, Nuria; Brahimi, Fouad; McNamee, James P; Gibbs, Bernard F; Jean-Claude, Bertrand J

2007-01-01

JDA58 (NSC 741282), a "combi-molecule" optimized in the context of the "combi-targeting concept," is a nitrosourea moiety tethered to an anilinoquinazoline. Here, we sought to show its binary epidermal growth factor receptor (EGFR)/DNA targeting property and to study its fragmentation in vitro and in vivo. The fragmentation of JDA58 was detected in cells in vitro and in vivo by fluorescence microscopy and tandem mass spectrometry. EGFR phosphorylation and DNA damage were determined by Western blotting and comet assay, respectively. Tumor data were examined for statistical significance using the Student's t test. JDA58 inhibited EGFR tyrosine kinase (IC(50), 0.2 micromol/L) and blocked EGFR phosphorylation in human DU145 prostate cancer cells. It induced significant levels of DNA damage in DU145 cells in vitro or in vivo and showed potent antiproliferative activity both in vitro and in a DU145 xenograft model. In cell-free medium, JDA58 was hydrolyzed to JDA35, a fluorescent amine that could be observed in tumor cells both in vitro and in vivo. In tumor cells in vitro or in vivo, or in plasma collected from mice, the denitrosated species JDA41 was the predominant metabolite. However, mass spectrometric analysis revealed detectable levels of the hydrolytic product JDA35 in tumor cells both in vitro and in vivo. The results in toto suggest that growth inhibition in vitro and in vivo may be sustained by the intact combi-molecule plus JDA35 plus JDA41, three inhibitors of EGFR, and the concomitantly released DNA-damaging species. This leads to a model wherein a single molecule carries a complex multitargeted-multidrug combination.

Bone Tissue Engineering Under Xenogeneic-Free Conditions in a Large Animal Model as a Basis for Early Clinical Applicability.

PubMed

Weigand, Annika; Beier, Justus P; Schmid, Rafael; Knorr, Tobias; Kilian, David; Götzl, Rebekka; Gerber, Thomas; Horch, Raymund E; Boos, Anja M

2017-03-01

For decades, researchers have been developing a range of promising strategies in bone tissue engineering with the aim of producing a significant clinical benefit over existing therapies. However, a major problem concerns the traditional use of xenogeneic substances for the expansion of cells, which complicates direct clinical transfer. The study's aim was to establish a totally autologous sheep model as a basis for further preclinical studies and future clinical application. Ovine mesenchymal stromal cells (MSC) were cultivated in different concentrations (0%, 2%, 5%, 10%, and 25%) of either autologous serum (AS) or fetal calf serum (FCS). With an increase of serum concentration, enhanced metabolic activity and proliferation could be observed. There were minor differences between MSC cultivated in AS or FCS, comparing gene and protein expression of osteogenic and stem cell markers, morphology, and osteogenic differentiation. MSC implanted subcutaneously in the sheep model, together with a nanostructured bone substitute, either in stable block or moldable putty form, induced similar vascularization and remodeling of the bone substitute irrespective of cultivation of MSC in AS or FCS and osteogenic differentiation. The bone substitute in block form together with MSC proved particularly advantageous in the induction of ectopic bone formation compared to the cell-free control and putty form. It could be demonstrated that AS is suitable for replacement of FCS for cultivation of ovine MSC for bone tissue engineering purposes. Substantial progress has been made in the development of a strictly xenogeneic-free preclinical animal model to bring future clinical application of bone tissue engineering strategies within reach.

A novel anti-EGFR monoclonal antibody inhibiting tumor cell growth by recognizing different epitopes from cetuximab.

PubMed

Hong, Kwang-Won; Kim, Chang-Goo; Lee, Seung-Hyun; Chang, Ki-Hwan; Shin, Yong Won; Ryoo, Kyung-Hwan; Kim, Se-Ho; Kim, Yong-Sung

2010-01-01

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.