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Sample records for egfr fish assay

  1. Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation

    PubMed Central

    Hoshi, Hirotaka; Hiyama, Gen; Ishikawa, Kosuke; Inageda, Kiyoshi; Fujimoto, Jiro; Wakamatsu, Ai; Togashi, Takushi; Kawamura, Yoshifumi; Takahashi, Nobuhiko; Higa, Arisa; Goshima, Naoki; Semba, Kentaro; Watanabe, Shinya; Takagi, Motoki

    2016-01-01

    Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel cell

  2. Construction of a novel cell-based assay for the evaluation of anti-EGFR drug efficacy against EGFR mutation.

    PubMed

    Hoshi, Hirotaka; Hiyama, Gen; Ishikawa, Kosuke; Inageda, Kiyoshi; Fujimoto, Jiro; Wakamatsu, Ai; Togashi, Takushi; Kawamura, Yoshifumi; Takahashi, Nobuhiko; Higa, Arisa; Goshima, Naoki; Semba, Kentaro; Watanabe, Shinya; Takagi, Motoki

    2017-01-01

    Epidermal growth factor receptor (EGFR) overexpression and EGFR-mediated signaling pathway dysregulation have been observed in tumors from patients with various cancers, especially non-small cell lung cancer. Thus, several anti-EGFR drugs have been developed for cancer therapy. For patients with known EGFR activating mutations (EGFR exon 19 in-frame deletions and exon 21 L858R substitution), treatment with an EGFR tyrosine kinase inhibitor (EGFR TKI; gefitinib, erlotinib or afatinib) represents standard first-line therapy. However, the clinical efficacy of these TKIs is ultimately limited by the development of acquired drug resistance such as by mutation of the gatekeeper T790 residue (T790M). To overcome this acquired drug resistance and develop novel anti-EGFR drugs, a cell-based assay system for EGFR TKI resistance mutant-selective inhibitors is required. We constructed a novel cell-based assay for the evaluation of EGFR TKI efficacy against EGFR mutation. To this end, we established non-tumorigenic immortalized breast epithelial cells that proliferate dependent on EGF (MCF 10A cells), which stably overexpress mutant EGFR. We found that the cells expressing EGFR containing the T790M mutation showed higher resistance against gefitinib, erlotinib and afatinib compared with cells expressing wild-type EGFR. In contrast, L858R mutant-expressing cells exhibited higher TKI sensitivity. The effect of T790M-selective inhibitors (osimertinib and rociletinib) on T790M mutant-expressing cells was significantly higher than gefitinib and erlotinib. Finally, when compared with commercially available isogenic MCF 10A cell lines carrying introduced mutations in EGFR, our EGFR mutant-overexpressing cells exhibited obviously higher responsiveness to EGFR TKIs depending on the underlying mutations because of the higher levels of EGFR phosphorylation in the EGFR mutant-overexpressing cells than in the isogenic cell lines. In conclusion, we successfully developed a novel

  3. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  4. EGFR status in oral squamous cell carcinoma: comparing immunohistochemistry, FISH and CISH detection in a case series study

    PubMed Central

    Bernardes, Vanessa Fátima; Gleber-Netto, Frederico Omar; de Sousa, Sílvia Ferreira; Rocha, Rafael Malagoli; de Aguiar, Maria Cássia Ferreira

    2013-01-01

    Objectives To compare the immunohistochemistry (IHC) expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinomas (OSCC) with the gene amplification evaluated by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) and their association with clinicopathological parameters. Additionally, we tested the sensibility and specificity of CISH in comparison with FISH. Design Case series study Setting Oral surgery and pathology department in a school of dentistry. Participants 52 patients with histopathological diagnosis of OSCC. Methods Tumour tissue samples from 52 patients with OSCC were evaluated by IHC, FISH and CISH using tissue microarray technology. Clinicopathological data from all patients were collected. Results EGFR+ rates were 53.8% (28/52) by IHC, 5.8% (3/52) by CISH and 15.4% (8/52) by FISH. Amplification detected by CISH and FISH with IHC negative occurred in 3.8% (2/52), and one case (1.9%) showed amplification detected by CISH and FISH and protein overexpression concomitantly. There were 9.6% FISH+ cases with IHC and CISH negative rates and 6/8 (75%) FISH+ and also EGFR+ cases; however, an association between protein expression and gene amplification was not found for both techniques. IHC and FISH rates were not associated with clinicopathological features. CISH+ rates were associated with T3–T4 status. Compared with FISH assay, CISH reached a sensitivity of 37.5% and specificity of 100%. Conclusions There is no association between EGFR expression and gene amplification in OSCC when the IHC is driven to external epitopes of the protein. Although CISH demonstrates specificity, technical problems may influence sensibility when compared with FISH. PMID:23358562

  5. Standardisation of EGFR FISH in colorectal cancer: results of an international interlaboratory reproducibility ring study

    PubMed Central

    Sartore-Bianchi, Andrea; Fieuws, Steffen; Veronese, Silvio; Moroni, Mauro; Personeni, Nicola; Frattini, Milo; Torri, Valter; Cappuzzo, Federico; Borght, Sara Vander; Martin, Vittoria; Skokan, Margaret; Santoro, Armando; Gambacorta, Marcello; Tejpar, Sabine; Varella-Garcia, Marileila; Siena, Salvatore

    2015-01-01

    Aims Epidermal growth factor receptor (EGFR) gene copy number evaluated by fluorescence in situ hybridisation (FISH) can discriminate among KRAS wild-type patients those with better outcome to EGFR-targeted therapy in metastatic colorectal cancer, further enhancing selection of patients. Nevertheless, enumeration of gene copies is challenging and the lack of analytical standardisation has limited incorporation of the test into the clinical practice. We therefore assessed EGFR FISH interlaboratory consensus among five molecular diagnostic reference centres. Methods A set of 12 colorectal cancer samples circulated among laboratories, and samples were scored according to commonly agreed guidelines. Reproducibility was quantified using the standard error of measurement (SEM). Results A SEM of 0.865 and a within-subject coefficient of variation (WSCV) of 26.8% for mean EGFR gene/nuclei and a SEM of 0.235 and a WSCV of 19.4% for the mean EGFR gene/CEP7 ratio were observed. Measurement of the fraction of cells displaying chromosome 7 polysomy showed WSCV of 46.6%, 34.0% and 51.0% for percentage of cells displaying ≤2, ≥3 and ≥4 EGFR signals, respectively. Among different slides of the same specimen, the WSCV was 6.1% for mean EGFR gene/nuclei and 3.9% for mean of EGFR gene/CEP7 ratios. Conclusions Molecular diagnosis of EGFR gene copy number by FISH varied largely among pathology centres, with fluctuations covering the whole range of proposed cut-offs of predictive usefulness from literature. Definition of a detailed scoring system and implementation of comprehensive training programmes for laboratories are therefore necessary before including the test into clinical practice. PMID:22130903

  6. Cross-reactivity of EGFR mutation-specific immunohistochemistry assay in HER2-positive tumors.

    PubMed

    Verdu, Montse; Trias, Isabel; Roman, Ruth; Rodon, Natalia; Pubill, Carme; Arraiza, Nuria; Martinez, Begonya; Garcia-Pelaez, Beatriz; Serrano, Teresa; Puig, Xavier

    2015-09-01

    The coexpression of HER2 and EGFR L858R in a solitary nodule removed from the lung, whose mutation was not confirmed by molecular techniques, made us think about the possible existence of a cross-reaction between HER2 and the EGFR L858R-specific antibody. Our study was designed to further analyze the existence of this cross-reaction and stress the need to exclude a metastatic breast cancer when dealing with EGFR L858R-positive cases. The series consists of 42 primary breast carcinomas, 22 HER2 positive for overexpression and amplification, and 20 negative for both. EGFR mutations were studied by immunohistochemistry and confirmed using real-time PCR when positive. Immunohistochemistry assay with EGFR L858R was positive in 19 (86%) of the HER2-positive breast carcinomas and negative in all HER2-negative carcinomas. The EGFR L858R antibody gives false-positive results in most of the breast carcinomas with HER2 overexpression/amplification. As a consequence, it is essential to confirm any EGFR L858R-positive cases by molecular methods or at least discard the presence of HER2 overexpression/amplification before rendering a diagnosis. It is also important to consider that HER2 has been described in other carcinomas such as urothelial, gastric or ovarian, as well as lung, although infrequently.

  7. Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non–Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial

    PubMed Central

    Benlloch, Susana; Botero, Maria Luisa; Beltran-Alamillo, Jordi; Mayo, Clara; Gimenez-Capitán, Ana; de Aguirre, Itziar; Queralt, Cristina; Ramirez, Jose Luis; Cajal, Santiago Ramón y.; Klughammer, Barbara; Schlegel, Mariette; Bordogna, Walter; Chen, David; Zhang, Guili; Kovach, Barbara; Shieh, Felice; Palma, John F.; Wu, Lin; Lawrence, H. Jeffrey; Taron, Miquel

    2014-01-01

    The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials, anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs), and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing, using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples, the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing. PMID:24586842

  8. Detection of Epidermal Growth Factor Receptor Mutations in Lung Adenocarcinoma: Comparing Cobas 4800 EGFR Assay With Sanger Bidirectional Sequencing.

    PubMed

    Ardakani, Nima Mesbah; Giardina, Tindaro; Grieu-Iacopetta, Fabienne; Tesfai, Yordanos; Carrello, Amerigo; Taylor, Jeremy; Robinson, Cleo; Spagnolo, Dominic; Amanuel, Benhur

    2016-09-01

    Accurate detection of epidermal growth factor receptor (EGFR) mutations has a crucial role in the current treatment of patients with lung adenocarcinoma, and identification of clinically relevant mutations would qualify patients for treatment with tyrosine kinase inhibitors. Historically, Sanger sequencing has been used as the reference standard assay for EGFR mutational analysis; however, Cobas 4800 is a relatively new method. In the present study, we compared the performance of the Cobas assay against that of Sanger sequencing. A total of 493 consecutive formalin-fixed paraffin-embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations using both methods. After exclusion of the invalid results (n = 19), 474 samples from 455 patients were analyzed. The Cobas assay showed a mutation detection rate comparable to that of Sanger sequencing (18.1% vs. 17.9%, respectively; P < .05). Excellent agreement of 98.9% (κ, 0.964) was observed between the 2 methods. The Cobas assay is a fast and diagnostically robust platform with high analytical sensitivity; however, it is limited by its detection range and low tolerance to low DNA quality. Sanger sequencing is mostly affected by its lower analytic sensitivity. Ultimately, a dual testing strategy will be justified to increase the detection of novel mutations and reduce the false-negative results within an acceptable turnaround time. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  9. Pyrosequencing for EGFR mutation detection: diagnostic accuracy and clinical implications.

    PubMed

    Sahnane, Nora; Gueli, Rossana; Tibiletti, Maria G; Bernasconi, Barbara; Stefanoli, Michele; Franzi, Francesca; Pinotti, Graziella; Capella, Carlo; Furlan, Daniela

    2013-12-01

    EGFR-activating mutations predict responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) patients. Mutation screening is crucial to support therapeutic decisions and is commonly conducted using dideoxy sequencing, although its sensitivity is suboptimal in clinical settings. To evaluate the diagnostic performance of pyrosequencing and dideoxy sequencing, we examined EGFR mutation status in a retrospective cohort of 53 patients with NSCLCs clinically selected for TKI therapy and whose clinical outcome was available. Moreover, pyrosequencing quantitative results were compared with EGFR amplification data. EGFR mutations were investigated by pyrosequencing and by dideoxy sequencing. Detection rates of both methods were determined by titration assays using NCI-H1975 and HCC-827 cell lines. Increased EGFR copy number was assessed by fluorescence in situ hybridization (FISH). Pyrosequencing showed a higher detection rate than dideoxy sequencing. Tumor control rate of cases with mutant and wild-type EGFR was 86% and 29%, respectively. EGFR amplification was significantly associated with EGFR mutation and a positive correlation between high percentages of mutant alleles and clinical response to TKI was observed. We concluded that pyrosequencing is more sensitive than dideoxy sequencing in mutation screening for EGFR mutations. Detection rate of dideoxy sequencing was suboptimal when low frequencies of mutant alleles or low tumor cell contents were observed. Pyrosequencing enables quantification of mutant alleles that correlates well with increased EGFR copy number assessed by FISH. Pyrosequencing should be used in molecular diagnostic of NSCLC to appropriately select patients who are likely to benefit from TKI therapy.

  10. Validation of a Fish Short-term Reproduction Assay

    EPA Science Inventory

    The Fish Short-term Reproduction Assay is an in vivo assay conducted with fathead minnows and is designed to detect changes in spawning, gross morphology, histopathology, and specific biochemical endpoints that reflect disturbances in the hypothalamic-pituitary-gonadal (HPG) axis...

  11. Validation of a Fish Short-term Reproduction Assay

    EPA Science Inventory

    The Fish Short-term Reproduction Assay is an in vivo assay conducted with fathead minnows and is designed to detect changes in spawning, gross morphology, histopathology, and specific biochemical endpoints that reflect disturbances in the hypothalamic-pituitary-gonadal (HPG) axis...

  12. 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.

  13. Behavioral assay for assessing effects of pollutants on fish chemoreception

    SciTech Connect

    Lemly, A.D.; Smith, R.J.

    1986-04-01

    Behavioral assays are sensitive to sublethal levels of pollution but they usually require highly trained personnel and long observation periods. We describe a system that combines the sensitivity of a behavioral assay with commercially available automated monitoring equipment. The observation system consists of a special aquarium coupled to a recirculating water system, and an Opto-Varimex-Aqua activity tracking meter (Columbus Instruments, Columbus, Ohio) interfaced to a microcomputer. The tracking meter forms an intersecting, planar grid of light beams which, when interrupted by fish movements, is translated into a digitized signal and fed to the computer. The assay is based on the response of fish to natural chemical stimuli such as food odors or pheromones. When these stimulus solutions are injected into the water circulation the response of the fish is monitored by the computer system, which is capable of discriminating and quantifying changes in eight parameters. Normal responses to stimuli are compared with the response of fish that have been exposed to pollutants. We have successfully used this technique to examine effects of reduced pH on the response of fathead minnows, Pimephales promelas, to chemical feeding stimuli. The system should be easily adapted to any laboratory concerned with testing for effects of toxic substances, and will identify effects of pollution that have thus far been difficult or impossible to assess.

  14. Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.

    PubMed

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Iannaccone, Alessia; Chicchinelli, Nicoletta; Franco, Renato; Mancini, Rita; Pisconti, Salvatore; De Luca, Antonella; Botti, Gerardo; Morabito, Alessandro; Normanno, Nicola

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples.

  15. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    PubMed

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  16. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

    PubMed Central

    Hardison, D. Ransom; Holland, William C.; McCall, Jennifer R.; Bourdelais, Andrea J.; Baden, Daniel G.; Darius, H. Taiana; Chinain, Mireille; Tester, Patricia A.; Shea, Damian; Flores Quintana, Harold A.; Morris, James A.; Litaker, R. Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®- PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  17. MET FISH-positive status predicts short progression-free survival and overall survival after gefitinib treatment in lung adenocarcinoma with EGFR mutation.

    PubMed

    Noro, Rintaro; Seike, Masahiro; Zou, Fenfei; Soeno, Chie; Matsuda, Kuniko; Sugano, Teppei; Nishijima, Nobuhiko; Matsumoto, Masaru; Kitamura, Kazuhiro; Kosaihira, Seiji; Minegishi, Yuji; Yoshimura, Akinobu; Kubota, Kaoru; Gemma, Akihiko

    2015-02-06

    Lung adenocarcinoma patients with EGFR gene mutations have shown a dramatic response to gefitinib. However, drug resistance eventually emerges which limits the mean duration of response. With that in view, we examined the correlations between MET gene status as assessed by fluorescence in situ hybridization (FISH) with overall survival (OS) and progression-free survival (PFS) in adenocarcinoma patients with EGFR gene mutations who had received gefitinib therapy. We evaluated 35 lung cancer samples with EGFR mutation from adenocarcinoma patients who had received gefitinib. Gene copy numbers (GCNs) and amplification of MET gene before gefitinib therapy was examined by FISH. MET protein expression was also evaluated by immunohistochemistry (IHC). FISH assessment showed that of the 35 adenocarcinoma samples, 10 patients (29%) exhibited high polysomy (5 copies≦mean MET per cell) and 1 patient (3%) exhibited amplification (2≦MET gene (red)/CEP7q (green) per cell). IHC evaluation of MET protein expression could not confirm MET high polysomy status. The Eleven patients with MET FISH positivity had significantly shorter progression-free survival (PFS) and overall survival (OS) than the 24 patients who were MET FISH-negative (PFS: p = 0.001 and OS: p = 0.03). Median PFS and OS with MET FISH-positivity were 7.6 months and 16.8 months, respectively, whereas PFS and OS with MET FISH-negativity were 15.9 months and 33.0 months, respectively. Univariate analysis revealed that MET FISH-positivity was the most significant independent factor associated with a high risk of progression and death (hazard ratio, 3.83 (p = 0.0008) and 2.25 (p = 0.03), respectively). Using FISH analysis to detect high polysomy and amplification of MET gene may be useful in predicting shortened PFS and OS after Gefitinib treatment in lung adenocarcinoma. The correlation between MET gene status and clinical outcomes for EGFR-TKI should be further evaluated using large scale samples.

  18. Predicting fish acute toxicity using a fish gill cell line-based toxicity assay.

    PubMed

    Tanneberger, Katrin; Knöbel, Melanie; Busser, Frans J M; Sinnige, Theo L; Hermens, Joop L M; Schirmer, Kristin

    2013-01-15

    The OECD test guideline 203 for determination of fish acute toxicity requires substantial numbers of fish and uses death as an apical end point. One potential alternative are fish cell lines; however, several studies indicated that these appear up to several orders of magnitude less sensitive than fish. We developed a fish gill cell line-based (RTgill-W1) assay, using several measures to improve sensitivity. The optimized assay was applied to determine the toxicity of 35 organic chemicals, having a wide range of toxicity to fish, mode of action and physicochemical properties. We found a very good agreement between in vivo and in vitro effective concentrations. For up to 73% of the tested compounds, the difference between the two approaches was less than 5-fold, covering baseline toxicants but as well compounds with presumed specific modes of action, including reactivity, inhibition of acetylcholine esterase or uncoupling of oxidative phosphorylation. Accounting for measured chemical concentrations eliminated two outliers, the hydrophobic 4-decylaniline and the volatile 2,3-dimethyl-1,3-butadiene, with an outlier being operationally defined as a substance showing a more than 10-fold difference between in vivo/in vitro effect concentrations. Few outliers remained. The most striking were allyl alcohol (2700-fold), which likely needs to be metabolically activated, and permethrin (190-fold) and lindane (63-fold), compounds acting, respectively, on sodium and chloride channels in the brain of fish. We discuss further developments of this assay and suggest its use beyond predicting acute toxicity to fish, for example, as part of adverse outcome pathways to replace, reduce, or refine chronic fish tests.

  19. A universal assay for vitellogenin in fish mucus and plasma.

    PubMed

    Van Veld, Peter A; Rutan, Barbara J; Sullivan, Constance A; Johnston, L Danielle; Rice, Charles D; Fisher, Daniel F; Yonkos, Lance T

    2005-12-01

    Expression of vitellogenin (VTG) in male fish has become a widely used biomarker of exposure to environmental estrogens. Vitellogenin is usually measured in blood by immunoassays that require species-specific antibodies. In this paper, we describe a universal assay that is based on the high-molecular weight and extensive phosphoserine content of all VTGs. Plasma and mucosal proteins from Pimephales promelas and Fundulus heteroclitus and mucosal proteins from Gambusia holbrooki were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stained with a commercially available fluropore dye (Pro-Q Diamond), and visualized by ultraviolet transillumination. The method allowed sensitive detection of VTG in females and estradiol-treated males in all species tested. Quantitative analysis indicated that the phosphoprotein assay is at least as sensitive as antibody-based methods but is universal, offering the advantage of VTG measurement in multiple species.

  20. Exploring the impact of EGFR T790M neighboring SNPs on ARMS-based T790M mutation assay

    PubMed Central

    Xu, Sanpeng; Duan, Yaqi; Lou, Liping; Tang, Fengjuan; Shou, Juan; Wang, Guoping

    2016-01-01

    The present study aimed to explore the influence of T790M neighboring single nucleotide polymorphism (SNP) on the sensitivity of amplification refractory mutation system (ARMS)-based T790M mutation assay. Three ARMS-quantitative polymerase chain reaction (qPCR) systems (system 1 had a forward ARMS primer without rs1050171, system 2 included a forward ARMS primer with rs1050171 and system 3 contained the above two forward ARMS primers) were used to detect the T790M mutation in two series plasmid samples and genomic DNA (gDNA) of the cell line H1975. A total of 670 formalin-fixed paraffin-embedded (FFPE) tumor samples from non-small cell lung cancer patients were used to detect the epidermal growth factor receptor (EGFR) gene T790M mutation by direct sequencing and ARMS-qPCR. The ARMS-qPCR system 1 effectively detected samples with as low as 1% T790M mutant plasmid 1 (without rs1050171) and with 50% T790M mutant plasmid 2 (with rs1050171), while the ARMS-qPCR system 2 detected samples with 20 and 50% T790M mutant plasmid 1, in addition to samples with 1% T790M mutant plasmid 2. For the ARMS-qPCR system 3, samples with as low as 1% T790M mutant plasmids 1 or 2 were effectively detected. For gDNA analysis of the cell line H1975, the T790M mutation was effectively detected by the ARMS-qPCR systems 2 and 3 (~50% mutation rate), but was detected with a low mutation abundance by the ARMS-qPCR system 1 (~1% mutation rate). Of the 670 FFPE samples, 5 cases were identified to have the T790M mutation by sequencing and by the ARMS-qPCR system 1. One sample (named N067), which was considered as T790M-negative by sequencing, was demonstrated to have the T790M mutation using the ARMS-qPCR system 1. Sample N094, which was variant homozygous for rs1050171 and was indicated to be T790M-negative by sequencing and by the ARMS-qPCR system 1, was identified to have the T790M mutation with the ARMS-qPCR system 3. The A-variant allele frequency of rs1050171 was observed to be 28.2% in the

  1. Impact of concurrent PIK3CA mutations on response to EGFR tyrosine kinase inhibition in EGFR-mutant lung cancers and on prognosis in oncogene-driven lung adenocarcinomas

    PubMed Central

    Eng, Juliana; Woo, Kaitlin M.; Sima, Camelia S.; Plodkowski, Andrew; Hellmann, Matthew D.; Chaft, Jamie; Kris, Mark G.; Arcila, Maria E.; Ladanyi, Marc; Drilon, Alexander

    2016-01-01

    INTRODUCTION In patients with EGFR or KRAS-mutant lung adenocarcinomas, the prognostic impact of a concurrent PIK3CA mutation remains unclear. Although preclinical data suggest that sensitivity to EGFR tyrosine kinase inhibition (TKI) is decreased in EGFR-mutant lung cancers also harboring a PIK3CA mutation, this interaction has not been explored clinically. METHODS Patients with lung adenocarcinomas harboring a PIK3CA mutation concurrent with a separate driver mutation were identified via mutational hotspot testing, multiplex sizing assays, and FISH. Overall survival (OS) and outcomes with EGFR TKI monotherapy (EGFR-mutant) were estimated using Kaplan-Meier methods and compared between double mutant (EGFR or KRAS-mutant, concurrent PIK3C-mutant) and single mutant patients (EGFR or KRAS-mutant, PI3KCA wild-type) using log-rank tests. RESULTS In EGFR and KRAS-mutant lung cancers, a concurrent PIK3CA mutation was associated with a decrease in median OS: 18 vs 33 months (EGFR double n=10 vs single n=43 mutant, p=0.006), and 9 vs 16 months (KRAS double n=16 vs single n=47 mutant, p=0.020). In EGFR-mutant lung cancers, a concurrent PIK3CA mutation did not impact benefit from EGFR TKI monotherapy. Single vs double mutant: objective response rate 83% (n=29) vs 62% (n=6, p=0.80), median time to progression 11 (n=29) vs 8 months (n=6, p=0.84), and median duration of TKI therapy 15 (n=32) vs 15 months (n=10, p=0.65). CONCLUSION A concurrent PIK3CA mutation is a poor prognostic factor in patients with advanced EGFR- or KRAS-mutant lung adenocarcinomas. There was no evidence that clinical benefit from EGFR TKI monotherapy is affected by a concurrent PIK3CA mutation in EGFR-mutant lung cancers. PMID:26334752

  2. Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions.

    PubMed

    Vandersea, Mark W; Litaker, R Wayne; Yonnish, Bryan; Sosa, Emilio; Landsberg, Jan H; Pullinger, Chris; Moon-Butzin, Paula; Green, Jason; Morris, James A; Kator, Howard; Noga, Edward J; Tester, Patricia A

    2006-02-01

    The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.

  3. Molecular Assays for Detecting Aphanomyces invadans in Ulcerative Mycotic Fish Lesions

    PubMed Central

    Vandersea, Mark W.; Litaker, R. Wayne; Yonnish, Bryan; Sosa, Emilio; Landsberg, Jan H.; Pullinger, Chris; Moon-Butzin, Paula; Green, Jason; Morris, James A.; Kator, Howard; Noga, Edward J.; Tester, Patricia A.

    2006-01-01

    The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans. PMID:16461710

  4. Fluorescence In Situ Hybridization (FISH) Assays for Diagnosing Malaria in Endemic Areas

    PubMed Central

    Shah, Jyotsna; Mark, Olivia; Weltman, Helena; Barcelo, Nicolas; Lo, Wai; Wronska, Danuta; Kakkilaya, Srinivas; Rao, Aravinda; Bhat, Shalia T.; Sinha, Ruchi; Omar, Sabah; Moro, Manuel; Gilman, Robert H.; Harris, Nick

    2015-01-01

    Malaria is a responsible for approximately 600 thousand deaths worldwide every year. Appropriate and timely treatment of malaria can prevent deaths but is dependent on accurate and rapid diagnosis of the infection. Currently, microscopic examination of the Giemsa stained blood smears is the method of choice for diagnosing malaria. Although it has limited sensitivity and specificity in field conditions, it still remains the gold standard for the diagnosis of malaria. Here, we report the development of a fluorescence in situ hybridization (FISH) based method for detecting malaria infection in blood smears and describe the use of an LED light source that makes the method suitable for use in resource-limited malaria endemic countries. The Plasmodium Genus (P-Genus) FISH assay has a Plasmodium genus specific probe that detects all five species of Plasmodium known to cause the disease in humans. The P. falciparum (PF) FISH assay and P. vivax (PV) FISH assay detect and differentiate between P. falciparum and P. vivax respectively from other Plasmodium species. The FISH assays are more sensitive than Giemsa. The sensitivities of P-Genus, PF and PV FISH assays were found to be 98.2%, 94.5% and 98.3%, respectively compared to 89.9%, 83.3% and 87.9% for the detection of Plasmodium, P. falciparum and P. vivax by Giemsa staining respectively. PMID:26333092

  5. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater.

    PubMed

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen's' reports and fish community monitorings.

  6. A mercury saturation assay for measuring metallothionein in fish

    SciTech Connect

    Dutton, M.D. . Dept. of Zoology); Stephenson, M. . Environmental Science Branch); Klaverkamp, J.F. )

    1993-07-01

    An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution of enzymatic and nonenzymatic thiols, including rabbit liver MT-2, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of [sup 203]Hg in 10% trichloroacetic acid. Excess Hg was bound to an removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with [sup 203]Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.

  7. Survey of estrogenic activity in fish feed by yeast estrogen-screen assay.

    PubMed

    Matsumoto, Takeru; Kobayashi, Makito; Moriwaki, Toshihisa; Kawai, Shin'ichiro; Watabe, Shugo

    2004-10-01

    Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.

  8. Molecular assays in detecting EGFR gene aberrations: an updated HER2-dependent algorithm for interpreting gene signals; a short technical report.

    PubMed

    Tsiambas, Evangelos; Ragos, Vasileios; Lefas, Alicia Y; Georgiannos, Stavros N; Rigopoulos, Dimitrios N; Georgakopoulos, Georgios; Stamatelopoulos, Athanasios; Grapsa, Dimitra; Syrigos, Konstantinos

    2016-01-01

    Purpose: Among oncogenes that have already been identified and cloned, Epidermal Growth Factor Receptor (EGFR) remains one of the most significant. Understanding its deregulation mechanisms improves critically patients' selection for personalized therapies based on modern molecular biology and oncology guidelines. Anti-EGFR targeted therapeutic strategies have been developed based on specific genetic profiles and applied in subgroups of patients suffering by solid cancers of different histogenetic origin. Detection of specific EGFR somatic mutations leads to tyrosine kinase inhibitors (TKIs) application in subsets of them. Concerning EGFR gene numerical imbalances, identification of pure gene amplification is critical for targeting the molecule via monoclonal antibodies (mAbs). In the current technical paper we demonstrate the main molecular methods applied in EGFR analyses focused also on new data in interpreting numerical imbalances based on ASCO/ACAP guidelines for HER2 in situ hybridization (ISH) clarifications.

  9. An eDNA Assay to Monitor a Globally Invasive Fish Species from Flowing Freshwater

    PubMed Central

    Adrian-Kalchhauser, Irene; Burkhardt-Holm, Patricia

    2016-01-01

    Ponto-Caspian gobies are a flock of five invasive fish species that have colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby Neogobius melanostomus, figures among the 100 worst invaders in Europe. Current methods to detect the presence of Ponto-Caspian gobies involve catching or sighting the fish. These approaches are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile. To improve monitoring, we developed an assay based on the detection of DNA traces (environmental DNA, or eDNA) of Ponto-Caspian gobies in river water. The assay specifically detects invasive goby DNA and does not react to any native fish species. We apply the assay to environmental samples and demonstrate that parameters such as sampling depth, sampling location, extraction protocol, PCR protocol and PCR inhibition greatly impact detection. We further successfully outline the invasion front of Ponto-Caspian gobies in a large river, the High Rhine in Switzerland, and thus demonstrate the applicability of the assay to lotic environments. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than the conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by untrained individuals, and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s’ reports and fish community monitorings. PMID:26814998

  10. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    PubMed

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  11. New Applications of the Comet Assay: Comet-FISH and Transcription-Coupled DNA Repair

    PubMed Central

    Cox, Rachel A.; Hanawalt, Philip C.

    2009-01-01

    Transcription-coupled repair (TCR) is a pathway dedicated to the removal of damage from the template strands of actively transcribed genes. Although the detailed mechanism of TCR is not yet understood, it is believed to be triggered when a translocating RNA polymerase is arrested at a lesion or unusual structure in the DNA. Conventional assays for TCR require high doses of DNA damage for the statistical analysis of repair in the individual strands of DNA sequences ranging in size from a few hundred bases to 30 kb. The single cell gel electrophoresis (Comet) assay allows detection of single-or double-strand breaks at a 10 to 100-fold higher level of resolution. Fluorescence in situ hybridization (FISH) combined with the Comet assay (Comet-FISH) affords a heightened level of sensitivity for the assessment of repair in defined DNA sequences of cells treated with physiologically relevant doses of genotoxins. This approach also reveals localized susceptibility to chromosomal breakage in cells from individuals with hypersensitivity to radiation or chemotherapy. Several groups have reported preferential repair in transcriptionally active genes or chromosomal domains using Comet-FISH. The prevailing interpretation of the behavior of DNA in the Comet assay assumes that the DNA is arranged in loops and matrix-attachment sites; that supercoiled, undamaged loops are contained within the nuclear matrix and appear in Comet “heads”, and that Comet “tails” consist of relaxed DNA loops containing one or more breaks. According to this model, localization of FISH probes in Comet heads signifies that loops containing the targeted sequences are free of damage. This implies that preferential repair as detected by Comet-FISH might encompass large chromosomal domains containing both transcribed and non-transcribed sequences. We review the existing evidence and discuss the implications in relation to current models for the molecular mechanism of TCR. PMID:18291710

  12. The Comet-FISH assay for the analysis of DNA damage and repair.

    PubMed

    Spivak, Graciela

    2010-01-01

    In this chapter, I describe the alkaline single-cell gel electrophoresis (Comet assay) combined with fluorescence in situ hybridization (FISH) technology, used in our laboratory, to study the incidence and repair of lesions induced in human cells by ultraviolet light. The Comet-FISH method permits the simultaneous and comparative analysis of DNA damage and its repair throughout the genome and in defined chromosomal regions. This very sensitive approach can be applied to any lesion, such as those induced by chemical carcinogens and products of cellular metabolism that can be converted to DNA single- or double-strand breaks. The unique advantages and limitations of the method for particular applications are discussed.

  13. Important Considerations for Methemoglobin Measurement in Fish Blood: Assay Choice and Storage Conditions

    SciTech Connect

    Kuntz, Mel Anton; Rodnick, K. J.; J. A. Lacey

    2002-05-01

    Spectrophotometric assays of methaemoglobin (metHb) in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, tilapias Tilapia niloticus and Tilapia zillii and white sturgeon Acipenser transmontanus, under baseline conditions, were low (<4%) for each species, and yet higher than human values (<1%). MetHb results for a given fish species varied significantly between assays and two assays were deemed unacceptable for particular animals. For rainbow trout, white sturgeon, and the two species of tilapia, the Dubowski method gave uncharacteristically high estimates of metHb. MetHb could not measured in tilapia blood using the Evelyn & Malloy method due to spectral interference. Only the Horecker & Brackett assay worked well for all species. Storage conditions were extremely important in the quantification of metHb in rainbow trout blood. For consistent values, samples can be stored up to 4 h on ice (0 degrees C) or at least 20 days under liquid nitrogen (-196 degrees C). Auto-oxidation, however, elevates rainbow trout metHb at -20 and -80 degrees C. It should not be assumed that the blood of fishes and humans perform similarly during assays of metHb.

  14. Determination of PCBs in fish using enzyme-linked immunosorbent assay (ELISA)

    USGS Publications Warehouse

    Lasrado, J.A.; Santerre, C.R.; Zajicek, J.L.; Stahl, J.R.; Tillitt, D.E.; Deardorff, D.

    2003-01-01

    Polychlorinated biphenyls (PCBs) were determined in fish tissue using an enzyme-linked immunosorbent assay (ELISA). Standard curves for Aroclor 1248, 1254, and 1260 in catfish tissue were developed with ranges from 0.05 to 0.5 ppm and 0.5 to 5.0 ppm. Wild fish were initially analyzed using gas chromatography/electron-capture detection (GC/ECD) and those having residues within the standard curve ranges were analyzed with ELISA. Results obtained using ELISA and GC/ECD were not significantly different (p < 0.05) from 0.05 to 0.5 ppm. From 0.5 to 5.0 ppm, the standard curve for Aroclor 1254 was the best predictor of total PCB in wild fish samples.

  15. EGFR Promoter Methylation, EGFR Mutation, and HPV Infection in Chinese Cervical Squamous Cell Carcinoma.

    PubMed

    Zhang, Wei; Jiang, Yinghao; Yu, Qingmiao; Qiang, Shaoying; Liang, Ping; Gao, Yane; Zhao, Xingye; Liu, Wenchao; Zhang, Ju

    2015-10-01

    Therapy strategy toward epidermal growth factor receptor (EGFR) inhibition in cervical cancer has been ongoing. EGFR promoter methylation status and EGFR tyrosine kinase inhibitor-sensitive mutations in cervical cancer may be significant for clinical outcome prediction using anti-EGFR treatment. In this study, EGFR tyrosine kinase inhibitor-sensitive mutations, EGFR exons 18, 19, and 21 mutations, were detected by sequencing in a total of 293 Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation status was detected by an EGFR asymmetric PCR and hybridization-fluorescence polarization assay and sequencing in 293 Chinese cervical squamous cell carcinoma tissue samples. High-risk human papillomavirus (HPV) genotypes in 293 Chinese cervical squamous cell carcinoma tissue samples were detected by an asymmetric GP5+/6+ PCR and hybridization-fluorescence polarization assay. No EGFR exons 18, 19, and 21 mutations were detected, EGFR promoter methylation status was identified in 98 samples, and HPV 16 infection was the first frequent HPV genotype. The methylated EGFR promoter was identified most frequently in cervical squamous cell carcinoma samples with HPV 16 infection (53.4%). Statistical significant difference of EGFR promoter methylation prevalence was found between HPV 16 and other HPV genotypes (P<0.01). This study suggested that there was no EGFR tyrosine kinase inhibitor-sensitive mutation in EGFR exons 18, 19, and 21 in Chinese cervical squamous cell carcinoma tissue samples. EGFR promoter methylation was common and it might be associated with HPV 16 infection in Chinese cervical squamous cell carcinoma. The results provided a novel understanding and an applicable pharmacogenomic tool for individualized management of cervical cancer patients.

  16. Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: comparison of assay methods and strains.

    PubMed

    Burkholder, Joann M; Gordon, Andrew S; Moeller, Peter D; Law, J Mac; Coyne, Kathryn J; Lewitus, Alan J; Ramsdell, John S; Marshall, Harold G; Deamer, Nora J; Cary, S Craig; Kempton, Jason W; Morton, Steven L; Rublee, Parke A

    2005-03-01

    Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays.

  17. Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains

    PubMed Central

    Burkholder, JoAnn M.; Gordon, Andrew S.; Moeller, Peter D.; Law, J. Mac; Coyne, Kathryn J.; Lewitus, Alan J.; Ramsdell, John S.; Marshall, Harold G.; Deamer, Nora J.; Cary, S. Craig; Kempton, Jason W.; Morton, Steven L.; Rublee, Parke A.

    2005-01-01

    Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays. PMID:15728353

  18. Assessing the applicability of FISH-based prematurely condensed dicentric chromosome assay in triage biodosimetry.

    PubMed

    Suto, Yumiko; Gotoh, Takaya; Noda, Takashi; Akiyama, Miho; Owaki, Makiko; Darroudi, Firouz; Hirai, Momoki

    2015-03-01

    The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

  19. In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture

    USGS Publications Warehouse

    Troyer, R.M.; Garver, K.A.; Ranson, J.C.; Wargo, A.R.; Kurath, G.

    2008-01-01

    A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72 h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested. ?? 2008.

  20. In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture.

    PubMed

    Troyer, Ryan M; Garver, Kyle A; Ranson, Judith C; Wargo, Andrew R; Kurath, Gael

    2008-11-01

    A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested.

  1. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-09-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.

  2. Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

    PubMed Central

    Fairey, E R; Edmunds, J S; Deamer-Melia, N J; Glasgow, H; Johnson, F M; Moeller, P R; Burkholder, J M; Ramsdell, J S

    1999-01-01

    Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:10464070

  3. Application of a fish DNA damage assay as a biological toxicity screening tool for metal plating wastewater

    SciTech Connect

    Choi, K.; Zong, M.; Meier, P.G.

    2000-01-01

    The utility of a fish DNA damage assay as a rapid monitoring tool was investigated. Metal plating wastewater was chosen as a sample because it contains various genotoxic metal species. Fish DNA damage assay results were compared to data generated from the conventional whole effluent toxicity (WET) test procedure. The Microtox{reg_sign} assay (Azur Environmental, Carlsbad, CA, USA) using Vibrio fischeri was also employed. Eleven samples from two metal plating companies were collected for this evaluation. For the fish DNA damage assay, 7-d-old fathead minnow larvae, Pimephales promelas, were utilized. They were exposed to a series of dilutions at 20 C for 2 h. Whole effluent toxicity tests conducted in this study included two acute toxicity tests with Daphnia magna and fathead minnows and two chronic toxicity tests with Ceriodaphnia dubia and fathead minnows. The fish DNA damage assay showed good correlations with both the acute and chronic WET test results, especially with those obtained with fathead minnows. The kappa values, an index of agreement, between the fish DNA damage assay and WET tests were shown to be acceptable. These findings imply that this novel fish DNA damage assay has use as an expedient toxicity screening procedure since it produces comparable results to those of the acute and chronic fathead minnow toxicity tests.

  4. Microsomal EROD data of fish liver sample assay from species collected in the Salt and Gila Rivers, Arizona

    USGS Publications Warehouse

    Nicks, Diane

    2017-01-01

    This dataset includes microsomal ERDO data from an assay done with liver samples from several fish species that are found in Arizona at sites that are being assessed for PBDE contamination. The data was created in September and October 2016.

  5. An innovative and integrative assay for toxicity testing using individual fish embryos. Application to oxazepam.

    PubMed

    Granger Joly de Boissel, Philippine; Gonzalez, Patrice; Buleté, Audrey; Daffe, Guillemine; Clérandeau, Christelle; Vulliet, Emmanuelle; Cachot, Jérôme

    2017-08-01

    This paper describes the development of an integrative embryo-toxicity assay in Japanese medaka allowing analysis of several toxicological endpoints together in a same individual. In this assay, embryos are topically exposed, and survival, hatching success, malformations, biometry, behaviour, and target gene expression are subsequently analysed in each individual. This assay was applied to oxazepam, an anxiolytic pharmaceutical compound currently found in wastewater treatment plant effluent. Even if oxazepam accumulation in embryos was very low, it caused spinal and cardiac malformations, delayed growth, erratic swimming and deregulation of genes involved in apoptosis, DNA repair and mitochondrial metabolism. Relationship between gene deregulation, abnormal behaviour, and developmental anomalies was demonstrated. This assay is sensitive enough to detect adverse effects at low chemical concentrations and at multiple endpoints in a unique fish embryo. This integrative embryo-toxicity assay is a powerful tool to characterize the spectrum of effects of new chemicals and also to link effects induced at different molecular, tissue and physiological levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Mini-column assay for rapid detection of malachite green in fish.

    PubMed

    Shalaby, Ali R; Emam, Wafaa H; Anwar, Mervat M

    2017-07-01

    A simple, rapid and economical mini-column method for detecting malachite green (MG) residue in fish was developed. The method used a column with 2mm ID that was tightly packed with silica gel followed by alumina. Detection of MG was performed by viewing the developed mini-column at visible light by naked eye; where MG was seen as compact green band at the confluence of the silica gel layer with alumina layer. The limit of detection of the assay was 2ng which conform the minimum required performance limit (MRPL). Evaluation utility of the method indicated that all blank and spiked samples at levels below MRPL were assessed as accepted. The intensity of the green band increased whenever MG level in the extract increased; indicated that suggested mini-column technique could be used for semi-quantitative determination of MG in fish samples. The method can be used to select the questionable samples.

  7. Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines.

    PubMed

    Kono, Tomoya; Takayama, Hiroaki; Nagamine, Ryusuke; Korenaga, Hiroki; Sakai, Masahiro

    2013-01-15

    To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.

  8. Identification of DNA damage in marine fish Therapon jarbua by comet assay technique.

    PubMed

    Nagarani, N; Devi, V Janaki; Kumaraguru, A K

    2012-07-01

    The marine fish Therapon jarbua was exposed to acute concentration of mercuric chloride (HgCl2). In static acute toxicity bioassays at 24, 48, 72 and 96 hr LC50 values were estimated for each concentrations such as control, 2, 1, 0.5, 0.25 and 0.125 ppm, respectively. DNAdamage (single-strand break) was also studied in gill, kidney and blood tissues at single-cell levels in the specimens exposed to different acute doses of HgCl2, by applying single-cell electrophoresis (comet assay). Dose-dependent responses were observed in DNA damage in all tissues. A comparison of DNA damage in all tissue at two concentration namely, 0.125 and 0.25 ppm indicated that the gill cells (maximum damage as 249.3 and 289.7 AU) were more sensitive to the heavy metal exposure than kidney (maximum 225.17 AU) and blood cells (maximum 200.3 AU). This study explored the utility of the comet assay for in vivo laboratory studies using fish for screening the genotoxic potential for various agents.

  9. Developing immune function assays to monitor fish health in field studies

    SciTech Connect

    Rice, C.D.; Kergosien, D.H.; Adams, S.M. |

    1995-12-31

    The East Fork Poplar Creek system, a 24km long stream in TN that receives point source discharges of contaminants near its headwaters, was chosen to evaluate a field approach to fish immunotoxicology. Previous studies in this stream have shown that cytochrome P4501A activity, liver somatic indices, macrophage aggregates, and parasitic liver lesions are significantly elevated in sunfish with the degree of impact decreasing with distance from the contaminant source. Red-breasted sunfish were collected between May 23 and June 3 of 1994. Captured fish were anesthetized in MS-222 and processed by two different methods. One group was sacrificed at each sampling station, weights and lengths recorded, and the spleen and anterior kidney tissues removed and placed in buffer on ice. The other group was kept in MS-222 for 2 hr and transported to the laboratory. The spleen and anterior kidney from each fish were then prepared as a single cell suspension and shipped overnight to Mississippi State University. Cells were then washed by centrifugation and resuspended in appropriate media to evaluate PMA-stimulated phagocyte oxidative burst and non-specific cytotoxic cell (NCC) activity against K562 tumor targets. Oxidative burst responses were dramatically suppressed in both groups at stations near the headwaters but returned to reference levels further downstream. There were no differences between treatment groups at each station. NCC activities did not follow gradient-response patterns observed with phagocyte oxidative burst data and there were inconsistent differences between treatment groups at each station. These data show that simple immune function assays, such as phagocyte oxidative burst responses, can be used as an ancillary biomarker in fish health monitoring.

  10. Giemsa versus acridine orange staining in the fish micronucleus assay and validation for use in water quality monitoring.

    PubMed

    Polard, T; Jean, S; Merlina, G; Laplanche, C; Pinelli, E; Gauthier, L

    2011-01-01

    This study concerns a comparative analysis of the acridine orange and Giemsa staining procedures for the fish erythrocyte micronucleus assay. The goal was to optimize the assay in the context of field water monitoring. Fish (Carassius carassius) were exposed to a reference genotoxic agent, cyclophosphamide monohydrate 5 mg l(-1) for 2, 4, and 6 days before testing. Slides from each individual were scored using the two procedures. The results show that the assay was more sensitive when acridine orange was used. When slides were Giemsa stained, the presence of ambiguous artefacts, leading to false positives and increasing random variance, reduced the contrast between exposed and control samples. Acridine Orange staining was then applied in the context of water quality monitoring. Fish were exposed for 4 days to water sampled in two hydrological contexts: basal flow and spring flood. The results show that exposure to spring flood water in an agricultural stream can induce mutagenicity.

  11. EGFR expression and copy number changes in low T-stage oral squamous cell carcinomas.

    PubMed

    Rössle, Matthias; Weber, Claudia S; Züllig, Lena; Graf, Nicole; Jochum, Wolfram; Stöckli, Sandro J; Moch, Holger; Huber, Gerhard F

    2013-08-01

    EGFR-directed therapies are used to treat patients with advanced head and neck squamous cell carcinoma (SCC). As it is still unclear whether or not EGFR amplification represents an early or late event in head and neck SCC progression, we aimed to determine the frequency of abnormalities of EGFR protein and gene copy numbers in early oral SCC. A tissue microarray of cancer tissue from 120 patients with pT1/2 oral SCC was constructed. We investigated EGFR protein expression by immunohistochemistry. EGFR gene copy enumeration was performed using fluorescence in-situ hybridization (FISH) and the novel automated silver in-situ hybridization (SISH) technology. Of early oral SCC, 19.3% showed high, 57.1% moderate and 23.6% low EGFR expression. EGFR amplification/polysomy was identified in 8% and 9% of cases by FISH and SISH, respectively. EGFR-SISH had a high concordance with EGFR-FISH (kappa value = 1.0), and both methods showed high conformity with EGFR immunohistochemistry (P = 0.001 and P = 0.006, respectively). No correlation was found of EGFR protein expression or gene amplification status with pT or pN stage. Only a small subgroup of early oral SCC is characterized by EGFR amplification, which can be identified reliably using EGFR-SISH technology. This finding suggests that EGFR gene amplification mostly occurs in advanced stages of oral SCC. © 2013 John Wiley & Sons Ltd.

  12. An improved AhR reporter gene assay for analyzing dioxins in soil, sediment and fish.

    PubMed

    Chao, How-Ran; Wang, Ya-Fan; Wang, Yao-Nan; Lin, Ding-Yan; Gou, Yan-You; Chen, Chien-Yu; Chen, Kuan-Chung; Wu, Wen-Kai; Chiang, Bao-An; Huang, Yu-Ting; Hsieh, Lien-Te; Yeh, Kuei-Jyum C; Tsou, Tsui-Chun

    2012-10-01

    Our goal was to develop a fast-screening bioassay to determine dioxin levels in the environmental and biological samples from dioxin-contaminated areas. Our original dioxin-responsive-element (DRE)-driven luciferase bioassay (using Huh7-DRE-Luc cells) was modified by reducing the incubation temperature of the cell culture from 37 to 35°C and by adding phorbol-12-myristate-13-acetate, and the modified bioassay was used to examine samples from soil, sediment, and fish. The results of this bioassay were shown to be significantly related to those of the high-resolution gas chromatography/high-resolution mass spectrometry assay of dioxins. The correlative equation was: log (PCDD/Fs I-TEQs) = 1.19 × log (BEQs) - 1.15 with R(2) = 0.95 (p < 0.001).

  13. Micronuclei assay and FISH analysis in human lymphocytes treated with six metal salts.

    PubMed

    Migliore, L; Cocchi, L; Nesti, C; Sabbioni, E

    1999-01-01

    The capability of some metal compounds for inducing micronuclei (MN) in human lymphocytes was studied. In this investigation, Al (III), Cd (II), Hg (II), Sb (V), Te (VI), and Tl (I) salts were considered. The FISH (fluorescence in situ hybridization) technique with a centromeric probe was coupled with the MN assay in binucleated cells in order to detect both centromere-positive MN (C+ MN) due to malsegregation phenomena and centromere-negative MN (C- MN) due to chromosome breakage. The blood of two young nonsmoking male donors was employed for all experiments. In both donors, all the tested metal compounds, with the exception of Tl(2)SO(4), showed a statistically significant increase of MN compared to controls, at least at one dose. FISH analysis revealed an increase in the fraction of C+ MN for Al, Cd, and Hg compounds, and of C- MN for the Sb salt; however, this was not a statistically significant increase. A different efficiency was observed for the different metal compounds, in particular, KSbO(3) and CH(3)HgCl, which were highly genotoxic, whereas the others showed minimal effects. Copyright 1999 Wiley-Liss, Inc.

  14. Construction of a taste-blind medaka fish and quantitative assay of its preference-aversion behavior.

    PubMed

    Aihara, Y; Yasuoka, A; Iwamoto, S; Yoshida, Y; Misaka, T; Abe, K

    2008-11-01

    In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference-aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference-aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

  15. An ELISA assay for cytochrome P4501A in fish liver cells

    SciTech Connect

    Brueschweiler, B.J.; Fent, K. |; Wuergler, F.E. |

    1996-04-01

    An enzyme-linked immunosorbent assay (ELISA) for measuring cytochrome P4501A (CYP1A) expression in vitro in fish hepatoma cells is described. Cells were cultured as monolayers in 96-microwell cell culture plates and exposed to polychlorinated biphenyl (PCB) congeners 77, 105, 153, and 169; 3-methylcholanthrene (3-MC), and {beta}-naphthoflavone (BNF) for 3 d. Relative CYP1A protein content, CYP1A enzymatic activity, and total protein content were determined directly within the wells. At low concentrations of PCB 77, PCB 169, and 3-MC, the ethoxyresorufin-O-deethylase (EROD) activity was induced, but it was inhibited at high concentrations of these compounds. However, CYP1A protein content measured in an ELISA performed with intact cells increased monotonically in response to the concentration. No CYP1A induction was observed for PCB 105 and PCB 153. Because comparison between EROD activity and CYP1A amount gives information about the catalytic efficiency of CYP1A in the cells, this noncompetitive, solid-phase ELISA is recommended as a complementary method to the EROD assay. This novel ELISA method may be an accurate in vitro technique for a rapid and sensitive screening of CYP1A-inducible compounds.

  16. Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors.

    PubMed

    Oka, Tomohiro; Mitsui-Watanabe, Naoko; Tatarazako, Norihisa; Onishi, Yuta; Katsu, Yoshinao; Miyagawa, Shinichi; Ogino, Yukiko; Yatsu, Ryohei; Kohno, Satomi; Takase, Minoru; Kawashima, Yukio; Ohta, Yasuhiko; Aoki, Yasunobu; Guillette, Louis J; Iguchi, Taisen

    2013-09-01

    Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRβ) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Are Fish and Standardized FETAX Assays Protective Enough for Amphibians? A Case Study on Xenopus laevis Larvae Assay with Biologically Active Substances Present in Livestock Wastes

    PubMed Central

    Martini, Federica; Tarazona, José V.; Pablos, M. Victoria

    2012-01-01

    Biologically active substances could reach the aquatic compartment when livestock wastes are considered for recycling. Recently, the standardized FETAX assay has been questioned, and some researchers have considered that the risk assessment performed on fish could not be protective enough to cover amphibians. In the present study a Xenopus laevis acute assay was developed in order to compare the sensitivity of larvae relative to fish or FETAX assays; veterinary medicines (ivermectin, oxytetracycline, tetracycline, sulfamethoxazole, and trimethoprim) and essential metals (zinc, copper, manganese, and selenium) that may be found in livestock wastes were used for the larvae exposure. Lethal (LC50) and sublethal effects were estimated. Available data in both, fish and FETAX studies, were in general more protective than values found out in the current study, but not in all cases. Moreover, the presence of nonlethal effects, caused by ivermectin, zinc, and copper, suggested that several physiological mechanisms could be affected. Thus, this kind of effects should be deeply investigated. The results obtained in the present study could expand the information about micropollutants from livestock wastes on amphibians. PMID:22629159

  18. Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples

    PubMed Central

    Kojima, Takahiro; Nishimura, Kouichi; Kandori, Shuya; Kawahara, Takashi; Yoshino, Takayuki; Ueno, Satoshi; Iizumi, Yuichi; Mitsuzuka, Koji; Arai, Yoichi; Tsuruta, Hiroshi; Habuchi, Tomonori; Kobayashi, Takashi; Matsui, Yoshiyuki; Ogawa, Osamu; Sugimoto, Mikio; Kakehi, Yoshiyuki; Nagumo, Yoshiyuki; Tsutsumi, Masakazu; Oikawa, Takehiro; Kikuchi, Koji; Nishiyama, Hiroyuki

    2016-01-01

    Introduction and Objectives Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples. Materials and Methods The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections. Results FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3%) of non-muscle-invasive BC (NMIBC) and 2/44 (5%) muscle-invasive BC (MIBC) patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45%) NMIBC and 8/44 (18%) MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive. Conclusions We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors. PMID:27930669

  19. Ascaridoidea: a simple DNA assay for identification of 11 species infecting marine and freshwater fish, mammals, and fish-eating birds.

    PubMed

    Kijewska, Agnieszka; Rokicki, Jerzy; Sitko, Jiri; Wegrzyn, Grzegorz

    2002-05-01

    Eleven species belonging to superfamily Ascaridoidea, which infect marine and freshwater fish, mammals, and fish-eating birds, were analyzed using a PCR-RFLP method. The following species were investigated: Anisakis pegreffi, A. physeteris, and A. simplex (parasites of fish and mammals), Contracaecum osculatum, C. radiatum, and C. rudolphi (parasites of mammals and fish-eating birds), Hysterothylacium aduncum (a parasite of fish), Porrocaecum angusticolle, P. crassum, P. depressum, and P. ensicaudatum (parasites of fish-eating birds). PCR-amplified rDNA regions encompassing ITS1, 5.8S rDNA, and ITS2 produced on templates of genomic DNA isolated from all investigated species were digested with TaqI, AluI, BsuRI, and RsaI endonucleases. Restriction patterns showed that endonuclease TaqI is the most useful enzyme for identification of all investigated species. No variations in restriction patterns within each species were detected. Therefore, we propose that the PCR-RFLP assay described in this report may be used for identification of marine and freshwater parasites from superfamily Ascaridoidea.

  20. 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

  1. An automated assay for the assessment of cardiac arrest in fish embryo.

    PubMed

    Puybareau, Elodie; Genest, Diane; Barbeau, Emilie; Léonard, Marc; Talbot, Hugues

    2017-02-01

    Studies on fish embryo models are widely developed in research. They are used in several research fields including drug discovery or environmental toxicology. In this article, we propose an entirely automated assay to detect cardiac arrest in Medaka (Oryzias latipes) based on image analysis. We propose a multi-scale pipeline based on mathematical morphology. Starting from video sequences of entire wells in 24-well plates, we focus on the embryo, detect its heart, and ascertain whether or not the heart is beating based on intensity variation analysis. Our image analysis pipeline only uses commonly available operators. It has a low computational cost, allowing analysis at the same rate as acquisition. From an initial dataset of 3192 videos, 660 were discarded as unusable (20.7%), 655 of them correctly so (99.25%) and only 5 incorrectly so (0.75%). The 2532 remaining videos were used for our test. On these, 45 errors were made, leading to a success rate of 98.23%.

  2. Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

    PubMed

    Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

    2016-09-15

    A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.

  3. An evaluation of 2,4-dichlorophenoxyacetic acid in the Amphibian Metamorphosis Assay and the Fish Short-Term Reproduction Assay.

    PubMed

    Coady, Katherine; Marino, Troy; Thomas, Johnson; Sosinski, Lindsay; Neal, Barbara; Hammond, Larry

    2013-04-01

    2,4-Dichlorophenoxyacetic acid (2,4-D) was evaluated in both the Amphibian Metamorphosis Assay (AMA) and the Fish Short Term Reproduction Assay (FSTRA). In the AMA, tadpoles were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.273, 3.24, 38.0 and 113 mg acid equivalents (ae)/L for either seven or 21 days. In the FSTRA, fathead minnows were exposed to mean measured 2,4-D concentrations of 0 (water control), 0.245, 3.14, 34.0, and 96.5 mg ae/L for 21 days. The respective concentrations of 2,4-D were not overtly toxic to either Xenopus laevis tadpoles or fathead minnows (Pimephales promelas). In the AMA, there were no signs of either advanced or delayed development, asynchronous development, or significant histopathological effects of the thyroid gland among 2,4-D exposed tadpoles evaluated on either day seven or day 21 of the exposure. Therefore, following the AMA decision logic, 2,4-D is considered "likely thyroid inactive" in the AMA with a No Observable Effect Concentration (NOEC) of 113 mg ae 2,4-D/L. In the FSTRA, there were no significant differences between control and 2,4-D exposed fish in regard to fertility, wet weight, length, gonado-somatic indices, tubercle scores, or blood plasma concentrations of vitellogenin. Furthermore, there were no treatment-related histopathologic changes in the testes or ovaries in any 2,4-D exposed group. The only significant effect was a decrease in fecundity among fish exposed to 96.5 mg ae 2,4-D/L. The cause of the reduced fecundity at the highest concentration of 2,4-D tested in the assay was most likely due to a generalized stress response in the fish, and not due to a specific endocrine mode of action of 2,4-D. Based on fish reproduction, the NOEC in the FSTRA was 34.0 mg ae 2,4-D/L. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Genotoxicity of chlorpyrifos in freshwater fish Labeo rohita using Alkaline Single-cell Gel Electrophoresis (Comet) assay.

    PubMed

    Ismail, Muhammad; Khan, Qaiser Mahmood; Ali, Rahat; Ali, Tayyaba; Mobeen, Ameena

    2014-10-01

    Chlorpyrifos is a widely used insecticide of organophosphate group, which causes severe toxicological effects in non target aquatic organisms especially in fish. In the present study the genotoxic effects of sublethal concentrations of chlorpyrifos were observed in the erythrocytes and gill cells of Labeo rohita (commonly known as rohu) using the Alkaline Single-Cell Gel Electrophoresis (Comet) assay. Effects of chlorpyrifos on the behavior of the fish were also investigated. The 96 h LC50 value of chlorpyrifos, estimated by Trimmed Spearman-Karber (TSK) in static bioassay, was found to be 442.8 µg/L. On the basis of LC50 value, the fish were exposed to three sublethal concentrations of chlorpyrifos (SL-I ∼221.4 µg/L, SL- II ∼110.7 µg/L and SL-III ∼73.8 µg/L) for 96 h. Blood and gill samples were collected at every 24 h and were subjected to the Comet assay. The observed DNA damage was concentration dependent and time dependent and those levels of DNA damage in between the tested concentrations and times were significantly different (p < 0.01). It was also found that the gill cells are more sensitive to chlorpyrifos, though; it revealed more DNA damage as compared to the erythrocytes of fish. Fish exposed to different concentrations of chlorpyrifos showed different neurotoxic behavioral responses. It was concluded that chlorpyrifos is a genotoxic and neurotoxic insecticide causing DNA damage and neurotoxic effects in Labeo rohita.

  5. Tyrosine kinase inhibitors - small molecular weight compounds inhibiting EGFR.

    PubMed

    Hegymegi-Barakonyi, Bálint; Eros, Dániel; Szántai-Kis, Csaba; Breza, Nóra; Bánhegyi, Péter; Szabó, Gábor Viktor; Várkondi, Edit; Peták, István; Orfi, László; Kéri, György

    2009-06-01

    Abnormally elevated EGFR kinase activity can lead to various pathological states, including proliferative diseases such as cancer. The development of selective protein kinase inhibitors has become an important area of drug discovery for the potential treatment of a variety of solid tumors such as breast, ovarian and colorectal cancers, NSCLC, and carcinoma of the head and neck. There are three small molecule EGFR kinase inhibitor drugs in clinical use (gefitinib, erlotinib and lapatinib), and several others are currently undergoing clinical development. This review summarizes the development of EGFR kinase inhibitors, and includes descriptions of the binding modes, the importance of a multiple-targets strategy, the effects of sensitizing and resistance mutations in the EGFR, and molecular diagnostic approaches. In addition, the use of target fishing for selectivity profiling, off-target identification and quantitative structure-activity relationship modeling for the prediction of EGFR inhibition is discussed.

  6. Characterization of ISR region and development of a PCR assay for rapid detection of the fish pathogen Tenacibaculum soleae.

    PubMed

    López, Jose R; Hamman-Khalifa, Abdel M; Navas, José I; de la Herran, Roberto

    2011-11-01

    The aims of this work were to characterize the 16S-23S internal spacer region of the fish pathogen Tenacibaculum soleae and to develop a PCR assay for its identification and detection. All T. soleae strains tested displayed a single internal spacer region class, containing tRNA(I) (le) and tRNA(A) (la) genes; nevertheless, a considerable intraspecific heterogeneity was observed. However, this region proved to be useful for differentiation of T. soleae from related and non-related species. Species-specific primers were designed targeting the 16S rRNA gene and the internal spacer region region, yielding a 1555-bp fragment. Detection limit was of 1 pg DNA per reaction (< 30 bacterial cells) when using pure cultures. The detection level in the presence of DNA from fish or other bacteria was lower; however, 10 pg were detected at a target/background ratio of 1 : 10(5) . The PCR assay proved to be more sensitive than agar cultivation for the detection of T. soleae from naturally diseased fish, offering a useful tool for diagnosis and for understanding the epidemiology of this pathogen.

  7. Development of an optical surface plasmon resonance biosensor assay for (fluoro)quinolones in egg, fish, and poultry meat.

    PubMed

    Huet, A-C; Charlier, C; Singh, G; Godefroy, S Benrejeb; Leivo, J; Vehniäinen, M; Nielen, M W F; Weigel, S; Delahaut, Ph

    2008-08-15

    The aim of this study was to develop an optical biosensor inhibition immunoassay, based on the surface plasmon resonance (SPR) principle, for use as a screening test for 13 (fluoro)quinolones, including flumequine, used as veterinary drugs in food-producing animals. For this, we immobilised various quinolone derivatives on the sensor chip and tested binding of a range of different antibodies (polyclonal and one engineered antibody) in the presence and absence of free (fluoro)quinolones. The main challenge was to detect flumequine in an assay giving good results for the other compounds. One antigen-antibody combination proved satisfactory: polyclonal antibodies raised against a dual immunogen and, on the sensor chip, a fluoroquinolone derivative. It was the first time that this concept of the bi-active antibody was described in the literature. The assay, optimised for detection in three matrices (poultry muscle, fish, and egg), was tested on incurred samples prepared by liquid extraction followed by two washing steps. This rapid, simple method proved adequate for detecting at least 13 (fluoro)quinolones at concentrations below established maximum residue levels (MRLs). The reference molecule norfloxacin could be detected in the range of 0.1-10 microg kg(-1) in extracts of egg and poultry meat and in the range of 0.1-100 microg kg(-1) in extracts of fish. The determined midpoints of these calibration curves were about 1, 1.5 and 3 microg kg(-1) in poultry meat, egg and fish, respectively.

  8. Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry.

    PubMed

    Ramsdorf, Wanessa A; Guimarães, Fernando de S F; Ferraro, Marcos V M; Gabardo, Juarez; Trindade, Edvaldo da Silva; Cestari, Marta Margarete

    2009-02-19

    When environmental analysis is performed, the high number of samples required and handling conditions during the transport of these samples to the laboratory are common problems. The comet assay is a useful, highly sensitive tool in biomonitoring. Some studies in the literature aim to preserve slides in lysis solution for use in the comet assay. Until now, however, no efficient methodology for preserving blood samples for this assay has been described. Because of this, the present report aimed to establish the proper conditions for samples maintenance prior to comet assay analysis. Samples were conserved in three different solutions: a high protein concentration solution (fetal bovine serum-FBS), an anticoagulant agent (a calcium chelator - ethylenediaminetetracetic acid - EDTA), and a salt buffered solution (phosphate buffered saline-PBS). Therefore, peripheral blood samples of Rhamdia quelen specimens were collected and maintained in these solutions until testing at 72h. Analyses of DNA fragmentation via the comet assay and cell viability via flow cytometry were performed at intervals of 24h. The results showed that samples maintained in FBS were preserved better; this was followed by those preserved in PBS and then last by those preserved in EDTA. In conclusion, blood samples from freshwater fish can be preserved up to 48h in fetal bovine serum at 4 degrees C in the absence of light. In this period, no DNA fragmentation occurs. We thus describe an excellent method of sample conservation for subsequent analysis in the laboratory.

  9. Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.

    PubMed

    Urdaci, M C; Chakroun, C; Faure, D; Bernardet, J F

    1998-01-01

    A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum.

  10. Analytic validation of a clinical-grade PTEN immunohistochemistry assay in prostate cancer by comparison with PTEN FISH.

    PubMed

    Lotan, Tamara L; Wei, Wei; Ludkovski, Olga; Morais, Carlos L; Guedes, Liana B; Jamaspishvili, Tamara; Lopez, Karen; Hawley, Sarah T; Feng, Ziding; Fazli, Ladan; Hurtado-Coll, Antonio; McKenney, Jesse K; Simko, Jeffrey; Carroll, Peter R; Gleave, Martin; Lin, Daniel W; Nelson, Peter S; Thompson, Ian M; True, Lawrence D; Brooks, James D; Lance, Raymond; Troyer, Dean; Squire, Jeremy A

    2016-08-01

    PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated, and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to four-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for the absence of PTEN gene deletion (549/602 tumors with two copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for the presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for the presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed two intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had two copies of the PTEN gene detected by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions, or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number.

  11. Analytic Validation of a Clinical-Grade PTEN Immunohistochemistry Assay in Prostate Cancer by Comparison to PTEN FISH

    PubMed Central

    Lotan, Tamara L.; Wei, Wei; Ludkovski, Olga; Morais, Carlos L.; Guedes, Liana B.; Jamaspishvili, Tamara; Lopez, Karen; Hawley, Sarah T.; Feng, Ziding; Fazli, Ladan; Hurtado-Coll, Antonio; McKenney, Jesse K.; Simko, Jeffrey; Carroll, Peter R.; Gleave, Martin; Lin, Daniel W.; Nelson, Peter S.; Thompson, Ian M.; True, Lawrence D.; Brooks, James D.; Lance, Raymond; Troyer, Dean; Squire, Jeremy A.

    2016-01-01

    PTEN loss is a promising prognostic and predictive biomarker in prostate cancer. Because it occurs most commonly via PTEN gene deletion, we developed a clinical-grade, automated and inexpensive immunohistochemical assay to detect PTEN loss. We studied the sensitivity and specificity of PTEN immunohistochemistry relative to 4-color fluorescence in situ hybridization (FISH) for detection of PTEN gene deletion in a multi-institutional cohort of 731 primary prostate tumors. Intact PTEN immunostaining was 91% specific for absence of PTEN gene deletion, (549/602 tumors with 2 copies of the PTEN gene by FISH showed intact expression of PTEN by immunohistochemistry) and 97% sensitive for presence of homozygous PTEN gene deletion (absent PTEN protein expression by immunohistochemistry in 65/67 tumors with homozygous deletion). PTEN immunohistochemistry was 65% sensitive for presence of hemizygous PTEN gene deletion, with protein loss in 40/62 hemizygous tumors. We reviewed the 53 cases where immunohistochemistry showed PTEN protein loss and FISH showed 2 intact copies of the PTEN gene. On re-review, there was ambiguous immunohistochemistry loss in 6% (3/53) and failure to analyze the same tumor area by both methods in 34% (18/53). Of the remaining discordant cases, 41% (13/32) revealed hemizygous (n=8) or homozygous (n=5) PTEN gene deletion that was focal in most cases (11/13). The remaining 19 cases had 2 copies of the PTEN gene by FISH, representing truly discordant cases. Our automated PTEN immunohistochemistry assay is a sensitive method for detection of homozygous PTEN gene deletions. Immunohistochemistry screening is particularly useful to identify cases with heterogeneous PTEN gene deletion in a subset of tumor glands. Mutations, small insertions or deletions and/or epigenetic or microRNA-mediated mechanisms may lead to PTEN protein loss in tumors with normal or hemizygous PTEN gene copy number. PMID:27174589

  12. Screening for potential endocrine disruptors in fish: evidence from structural alerts and in vitro and in vivo toxicological assays.

    PubMed

    Nendza, Monika; Wenzel, Andrea; Müller, Martin; Lewin, Geertje; Simetska, Nelly; Stock, Frauke; Arning, Jürgen

    2016-01-01

    The European chemicals' legislation REACH aims to protect man and the environment from substances of very high concern (SVHC). Chemicals like endocrine disruptors (EDs) may be subject to authorization. Identification of (potential) EDs with regard to the environment is limited because specific experimental assessments are not standard requirements under REACH. Evidence is based on a combination of in vitro and in vivo experiments (if available), expert judgement, and structural analogy with known EDs. The objectives of this study are to review and refine structural alerts for the indication of potential estrogenic and androgenic endocrine activities based on in vitro studies; to analyze in vivo mammalian long-term reproduction studies with regard to estrogen- and androgen-sensitive endpoints in order to identify potential indicators for endocrine activity with regard to the environment; to assess the consistency of potential estrogenic and androgenic endocrine activities based on in vitro assays and in vivo mammalian long-term reproduction studies and fish life-cycle tests; and to evaluate structural alerts, in vitro assays, and in vivo mammalian long-term reproduction studies for the indication of potential estrogenic and androgenic endocrine disruptors in fish. Screening for potential endocrine activities in fish via estrogenic and androgenic modes of action based on structural alerts provides similar information as in vitro receptor-mediated assays. Additional evidence can be obtained from in vivo mammalian long-term reproduction studies. Conclusive confirmation is possible with fish life-cycle tests. Application of structural alerts to the more than 33,000 discrete organic compounds of the EINECS inventory indicated 3585 chemicals (approx. 11%) as potential candidates for estrogenic and androgenic effects that should be further investigated. Endocrine activities of the remaining substances cannot be excluded; however, because the structural alerts perform much

  13. Prediction oriented QSAR modelling of EGFR inhibition.

    PubMed

    Szántai-Kis, C; Kövesdi, I; Eros, D; Bánhegyi, P; Ullrich, A; Kéri, G; Orfi, L

    2006-01-01

    Epidermal Growth Factor Receptor (EGFR) is a high priority target in anticancer drug research. Thousands of very effective EGFR inhibitors have been developed in the last decade. The known inhibitors are originated from a very diverse chemical space but--without exception--all of them act at the Adenosine TriPhosphate (ATP) binding site of the enzyme. We have collected all of the diverse inhibitor structures and the relevant biological data obtained from comparable assays and built prediction oriented Quantitative Structure-Activity Relationship (QSAR) which models the ATP binding pocket's interactive surface from the ligand side. We describe a QSAR method with automatic Variable Subset Selection (VSS) by Genetic Algorithm (GA) and goodness-of-prediction driven QSAR model building, resulting an externally validated EGFR inhibitory model built from pIC50 values of a diverse structural set of 623 EGFR inhibitors. Repeated Trainings/Evaluations (RTE) were used to obtain model fitness values and the effectiveness of VSS is amplified by using predictive ability scores of descriptors. Numerous models were generated by different methods and viable models were collected. Then, intensive RTE were applied to identify ultimate models for external validations. Finally, suitable models were validated by statistical tests. Since we use calculated molecular descriptors in the modeling, these models are suitable for virtual screening for obtaining novel potential EGFR inhibitors.

  14. [EGFR and gefitinib (Iressa)].

    PubMed

    Noro, Rintaro; Gemma, Akihiko

    2008-07-01

    We summarized the relationship between EGFR gene polymorphisms/mutations and gefitinib(Iressa). The SNPs of CA-simple sequence repeat of EGFR intron 1 and promoter sequence were reported to be related to the sensitivity and/ or toxicity of gefitinib(Iressa). Many reports have shown that there are differences between gefitinib responders and non-responders in the frequency of activating mutations and/or amplification in the EGFR gene, which suggests that such mutations might be predictive markers for sensitivity to gefitinib. However, there are known to be gefitinib-sensitive and intermediate-sensitive tumors that have no activating mutations in the EGFR gene, including gene amplification. SNPs in EGFR gene may be one of candidates for predictors of sensitivity. On the other hand, the incidence of gefitinib-induced lung injury was 3.98% in Japan(case-control study, AstraZeneca), which is higher than the correspondence rate, 0.3%, in the USA(FDA Approval Letter). Further studies about the prediction of drug-induced interstitial lung disease including SNPs analyses of the EGFR gene and other related molecules.

  15. Development of critical life stage assays: Teratogenic effects of SRS effluent components on freshwater fish, gambusia

    SciTech Connect

    Guram, M.S.

    1990-11-01

    The final report of the research carried out at Voorhees College contains a composite compilation of the last two years work. The data note variation in the number of young fish delivered per female vary markedly between several ponds on the SRS and of SRS ponds. The reasons for this are unknown at present. Initial research was carried out on the effects on the developing fish fetus of various substances that may have produced these variations. Further study is necessary to identify the factors that produce the observed alterations. 7 figs., 5 tabs.

  16. Pitfalls in immunohistochemical assessment of EGFR expression in soft tissue sarcomas

    PubMed Central

    Kersting, C; Packeisen, J; Leidinger, B; Brandt, B; von Wasielewski, R; Winkelmann, W; van Diest, P J; Gosheger, G; Buerger, H

    2006-01-01

    Background New targeted cancer treatments acting against growth factor receptors such as the epidermal growth factor receptor (EGFR) necessitate selecting patients for treatment with these drugs. Besides carcinomas, soft tissue sarcomas (STS) express EGFR and might thereby be a promising target for this new therapeutic strategy. Objective To test and compare different EGFR antibodies to determine the frequency of EGFR expression in STS. Methods 302 consecutive specimens of STS were examined using the tissue microarray technique. EGFR expression levels were assessed by immunohistochemistry using five different commercially available antibodies. Gene amplification status was measured by fluorescence in situ hybridisation (FISH). Immunoreactivity and amplification status were correlated with clinicopathological features and follow up data available in 163 cases. Results EGFR expression frequency ranged between 0.3% and 52.9%, depending on the antibody and scoring method used. In all, 3.5% of the tumours showed egfr gene amplification by FISH, which correlated with EGFR expression for three antibodies. Only one antibody had independent prognostic value in multivariate analysis and correlated with an unfavourable outcome; egfr gene amplification status showed no correlation with clinical features. Conclusions Frequency of EGFR immunopositivity in STS strongly depends on the antibody used, and only one of five antibodies tested predicted an unfavourable clinical outcome. This indicates that choice of primary antibody and scoring system have a substantial impact on the determination of EGFR immunoreactivity. PMID:16461571

  17. Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.

    PubMed

    Selvaraju, Suresh B; Kapoor, Renuka; Yadav, Jagjit S

    2008-01-01

    Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.

  18. Prognostic and predictive value of EGFR in head and neck squamous cell carcinoma

    PubMed Central

    Bossi, Paolo; Resteghini, Carlo; Paielli, Nicholas; Licitra, Lisa; Pilotti, Silvana; Perrone, Federica

    2016-01-01

    EGFR is an extensively studied biomarker in head and neck squamous cell carcinoma (HNSCC). In this review, we discuss the prognostic and predictive role of EGFR in HNSCC, focusing on the different molecular alterations in specific treatment modalities such as radiotherapy alone (RT), combination of surgery, RT and chemotherapy (CT), EGFR inhibitors. We considered EGFR at different molecular levels: protein expression, protein activation, gene copy number, polymorphisms, mutation, EGFRvIII expression and EGFR ligand expression. Considering RT alone, evidence supports the predictive and prognostic role of high EGFR expression only when evaluated by quantitative assays: this may help select the patients who can mostly benefit from accelerated treatment. Conversely, no predictive biomarkers are available when treatment is a combination of surgery, CT and RT. For this combined treatment, several studies indicate that EGFR expression represents a good prognostic parameter only when measured by a “quantitative” or at least semi-quantitative method. With respect to EGFR inhibitors, neither EGFR expression nor increased gene copy number represent prognostic/predictive factors. If validated, nuclear EGFR, TGFα levels, EGFR phopshorylation and polymorphisms could represent additional prognostic factors in relation to combination of surgery, CT and RT, while EGFR polymorphisms and high amphiregulin levels could have prognostic value in patients treated with EGFR inhibitors. PMID:27556186

  19. Protein digestibility evaluations of meat and fish substrates using laboratory, avian and ileally cannulated dog assays

    USDA-ARS?s Scientific Manuscript database

    Fish and meat protein serves as important protein sources in the human and companion animal diets: however, limited information is available on differences in protein quality. Pollock fillet, and salmon fillet, beef loin, pork loin and chicken breast, were evaluated for protein quality and amino aci...

  20. A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.

    PubMed

    Sauer, Stephan; Burkett, Sandra S; Lewandoski, Mark; Klar, Amar J S

    2013-05-01

    Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

  1. A modified parallel artificial membrane permeability assay for evaluating the bioconcentration of highly hydrophobic chemicals in fish.

    PubMed

    Kwon, Jung-Hwan; Escher, Beate I

    2008-03-01

    Low cost in vitro tools are needed at the screening stage of assessment of bioaccumulation potential of new and existing chemicals because the number of chemical substances that needs to be tested highly exceeds the capacity of in vivo bioconcentration tests. Thus, the parallel artificial membrane permeability assay (PAMPA) system was modified to predict passive uptake/ elimination rate in fish. To overcome the difficulties associated with low aqueous solubility and high membrane affinity of highly hydrophobic chemicals, we measured the rate of permeation from the donor poly(dimethylsiloxane)(PDMS) disk to the acceptor PDMS disk through aqueous and PDMS membrane boundary layers and term the modified PAMPA system "PDMS-PAMPA". Twenty chemicals were selected for validation of PDMS-PAMPA. The measured permeability is proportional to the passive elimination rate constant in fish and was used to predict the "minimum" in vivo elimination rate constant. The in vivo data were very close to predicted values except for a few polar chemicals and metabolically active chemicals, such as pyrene and benzo[a]pyrene. Thus, PDMS-PAMPA can be an appropriate in vitro system for nonmetabolizable chemicals. Combination with metabolic clearance rates using a battery of metabolic degradation assays would enhance the applicability for metabolizable chemicals.

  2. Molecular genetic alterations in egfr CA-SSR-1 microsatellite and egfr copy number changes are associated with aggressiveness in thymoma.

    PubMed

    Conti, Salvatore; Gallo, Enzo; Sioletic, Stefano; Facciolo, Francesco; Palmieri, Giovannella; Lauriola, Libero; Evoli, Amelia; Martucci, Robert; Di Benedetto, Anna; Novelli, Flavia; Giannarelli, Diana; Deriu, Gloria; Granone, Pierluigi; Ottaviano, Margaret; Muti, Paola; Pescarmona, Edoardo; Marino, Mirella

    2016-03-01

    The key role of egfr in thymoma pathogenesis has been questioned following the failure in identifying recurrent genetic alterations of egfr coding sequences and relevant egfr amplification rate. We investigated the role of the non-coding egfr CA simple sequence repeat 1 (CA-SSR-1) in a thymoma case series. We used sequencing and egfr-fluorescence in situ hybridization (FISH) to genotype 43 thymomas; (I) for polymorphisms and somatic loss of heterozygosity of the non-coding egfr CA-SSR-1 microsatellite and (II) for egfr gene copy number changes. We found two prevalent CA-SSR-1 genotypes: a homozygous 16 CA repeat and a heterozygous genotype, bearing alleles with 16 and 20 CA repeats. The average combined allele length was correlated with tumor subtype: shorter sequences were significantly associated with the more aggressive WHO thymoma subtype group including B2/B3, B3 and B3/C histotypes. Four out of 29 informative cases analysed for somatic CA-SSR-1 loss of heterozygosity showed allelic imbalance (AI), 3/4 with loss of the longer allele. By egfr-FISH analysis, 9 out of 33 cases were FISH positive. Moreover, the two integrated techniques demonstrated that 3 out of 4 CA-SSR-1-AI positive cases with short allele relative prevalence showed significantly low or high chromosome 7 "polysomy"/increased gene copy number by egfr-FISH. Our molecular and genetic and follow up data indicated that CA-SSR-1-allelic imbalance with short allele relative prevalence significantly correlated with EGFR 3+ immunohistochemical score, increased egfr Gene Copy Number, advanced stage and with relapsing/metastatic behaviour in thymomas.

  3. EGFR-targeting peptide-coupled platinum(IV) complexes.

    PubMed

    Mayr, Josef; Hager, Sonja; Koblmüller, Bettina; Klose, Matthias H M; Holste, Katharina; Fischer, Britta; Pelivan, Karla; Berger, Walter; Heffeter, Petra; Kowol, Christian R; Keppler, Bernhard K

    2017-06-01

    The high mortality rate of lung cancer patients and the frequent occurrence of side effects during cancer therapy demonstrate the need for more selective and targeted drugs. An important and well-established target for lung cancer treatment is the occasionally mutated epidermal growth factor receptor (EGFR). As platinum(II) drugs are still the most important therapeutics against lung cancer, we synthesized in this study the first platinum(IV) complexes coupled to the EGFR-targeting peptide LARLLT (and the shuffled RTALLL as reference). Notably, HPLC-MS measurements revealed two different peaks with the same molecular mass, which turned out to be a transcyclization reaction in the linker between maleimide and the coupled cysteine moiety. With regard to the EGFR specificity, subsequent biological investigations (3-day viability, 14-day clonogenic assays and platinum uptake) on four different cell lines with different verified EGFR expression levels were performed. Unexpectedly, the results showed neither an enhanced activity nor an EGFR expression-dependent uptake of our new compounds. Consequently, fluorophore-coupled peptides were synthesized to re-evaluate the targeting ability of LARLLT itself. However, also with these molecules, flow cytometry measurements showed no correlation of drug uptake with the EGFR expression levels. Taken together, we successfully synthesized the first platinum(IV) complexes coupled to an EGFR-targeting peptide; however, the biological investigations revealed that LARLLT is not an appropriate peptide for enhancing the specific uptake of small-molecule drugs into EGFR-overexpressing cancer cells.

  4. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  5. COMPARISON OF AN IN VIVO FISH VTG ASSAY WITH YES AND E-SCREEN

    EPA Science Inventory

    This study compares the efficacy of two in vitro, estrogen-sensitive bioassays to rank the "relative estrogenicity" of five natural, pharmaceutical and xenoestrogens with a newly developed in vivo bioassay. The E-SCREEN (MCF-7 tumor cells) and YES (Yeast Estrogen Screen) assays w...

  6. Bridging the Gap From Screening Assays to Estrogenic Effects in Fish: Potential Roles of Multiple Estrogen Receptor Subtypes

    PubMed Central

    2015-01-01

    This study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human (h) estrogen receptor (ER)-based in vitro screening assays to predict estrogenic effects in aquatic species. Adult male Japanese medaka (Oryzias latipes) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon. All exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen (YES), which reports activation of hERα. These exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka. Effects of the same YES-calibrated solutions in the T47D-KBluc assay, which reports activation of hERα and hERβ, generally recapitulated observations in medaka. Using competitive ligand binding assays, it was found that the magnitude of vitellogenin/choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka ERβ subtypes, which are highly expressed in male medaka liver prior to estrogen exposure. Results support emerging evidence that ERβ subtypes are critically involved in the teleost estrogenic response, with the ERα:ERβ ratio being of particular importance. Accordingly, incorporation of multiple ER subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems, including complex estrogenic mixtures. PMID:24422420

  7. 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

  8. Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

    PubMed

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

    2012-10-04

    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 casitas B-lineage lymphoma-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.

  9. Epidermal Growth Factor Receptor (EGFR) Targeted Photoimmunotherapy (PIT) for the Treatment of EGFR expressing Bladder Cancer.

    PubMed

    Railkar, Reema; Krane, L Spencer; Li, Q Quentin; Sanford, Thomas; Siddiqui, Mohammad Rashid; Haines, Diana; Vourganti, Srinivas; Brancato, Sam J; Choyke, Peter L; Kobayashi, Hisataka; Agarwal, Piyush K

    2017-06-15

    The use of light as a means of therapy for bladder cancer (BC) has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of photo-therapy called photoimmunotherapy (PIT) which targets EGFR-expressing BC. Anti-EGFR antibody panitumumab (pan) was labeled with the photo-absorber (PA), IRDye 700Dx (IR700), to create a pan IR700 antibody-PA conjugate which is activated by near-infrared radiation (NIR). BC tissue microarray (TMA) and BC cell lines were analyzed for expression of EGFR. Mechanism of PIT induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most BCs have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Pan IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Pan alone, pan IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed towards cell death. NIR-PIT with pan IR700 reduced growth compared to only pan IR700 treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Pan IR700-induced PIT selectively kills EGFR-expressing BC cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of BC and ultimately in patients. Copyright ©2017, American Association for Cancer Research.

  10. Fish short-term reproduction assay (FSTRA) with atrazine and the Japanese medaka (Oryzias latipes).

    PubMed

    Hosmer, Alan J; Schneider, Suzanne Z; Anderson, Julie C; Knopper, Loren D; Brain, Richard A

    2017-02-15

    Breeding groups of Japanese medaka (Oryzias latipes) were exposed to atrazine at measured concentrations of 0.6, 5.5, and 53 µg/L for 35 d. Evaluated endpoints included survival, fecundity, fertility, growth (weight and length), behavior, secondary sex characteristics (anal fin papillae), gonad histopathology, and hepatic vitellogenin. No statistically significant effects of atrazine exposure on survival and growth of medaka were noted during the test, and mean survival was ≥97.5% in all treatment groups on Day 35. No significant effects of atrazine exposure on reproduction were observed. The number of mean cumulative eggs produced in the negative control, 0.6, 5.5, and 53 µg/L treatment groups was 7158, 6691, 6883, and 6856 eggs, respectively. The mean number of eggs per female reproductive day was 40.9, 38.2, 40.2, and 39.2 eggs per day, respectively. There were also no dose-dependent effects on mean anal fin papillae counts among male fish or expression of vtg-II in males or females. In addition, atrazine exposure was not related to the development stage of test fish, with testes stages ranging from 2 to 3 in all groups and ovaries ranging from stage 2 to 2.5. Overall, exposure to atrazine up to 53 µg/L for 35 d did not result in significant, treatment-related effects on measured endpoints related to survival, growth, or reproduction in Japanese medaka. This article is protected by copyright. All rights reserved.

  11. ADAM17 Transactivates EGFR Signaling during Embryonic Eyelid Closure

    PubMed Central

    Hassemer, Eryn L.; Endres, Bradley; Toonen, Joseph A.; Ronchetti, Adam; Dubielzig, Richard; Sidjanin, Duska J.

    2013-01-01

    Purpose. During mammalian embryonic eyelid closure ADAM17 has been proposed to play a role as a transactivator of epidermal growth factor receptor (EGFR) signaling by shedding membrane bound EGFR ligands. However, ADAM17 also sheds numerous other ligands, thus implicating ADAM17 in additional molecular pathways. The goal of this study was to experimentally establish the role of ADAM17 and determine ADAM17-mediated pathways essential for the embryonic eyelid closure. Methods. Wild-type (WT) and woe mice, carrying a hypomorphic mutation in Adam17, were evaluated using H&E and scanning electron microscopy. Expressions of ADAM17, EGFR, and the phosphorylated form EGFR-P were evaluated using immunohistochemistry. BrdU and TUNEL assays were used to evaluate cell proliferation and apoptosis, respectively. In vitro scratch assays of primary cultures were used to evaluate cell migration. Clinical and histologic analyses established if the hypermorphic EgfrDsk5 allele can rescue the woe embryonic eyelid closure. Results. woe mice exhibited a failure to develop the leading edge of the eyelid and consequently failure of the embryonic eyelid closure. Expression of ADAM17 was identified in the eyelid epithelium in the cells of the leading edge. ADAM17 is essential for epithelial cell migration, but does not play a role in proliferation and apoptosis. EGFR was expressed in both WT and woe eyelid epithelium, but the phosphorylated EGFR-P form was detected only in WT. The EgfrDsk5 allele rescued woe eyelid closure defects, but also rescued woe anterior segment defects and the absence of meibomian glands. Conclusions. We provide in vivo genetic evidence that the role of ADAM17 during embryonic eyelid closure is to transactivate EGFR signaling. PMID:23211830

  12. eGFR

    MedlinePlus

    ... Global Sites Search Help? Advertisement Proceeds from website advertising help sustain Lab Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising & Sponsorship: Policy | Opportunities Estimated Glomerular Filtration Rate (eGFR) ...

  13. Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay.

    PubMed

    Šrut, Maja; Traven, Luka; Štambuk, Anamaria; Kralj, Sonja; Žaja, Roko; Mićović, Vladimir; Klobučar, Göran I V

    2011-02-01

    The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.

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

    SciTech Connect

    Yu, Zhiwei; Cui, Binbin; Jin, Yinghu; Chen, Haipeng; Wang, Xishan

    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, 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.

  15. Affinity and Matrix Effects in Measuring Fish Plasma Vitellogenin Using Immunosorbent Assays: Considerations for Aquatic Toxicologists

    PubMed Central

    Bartell, Stephen E.; Schoenfuss, Heiko L.

    2012-01-01

    Enzyme-linked immunosorbent assays (ELISAs) are important tools in aquatic toxicology and have become crucial in assessing exposure concentrations in the aquatic environment and acute physiological responses in exposed organisms. These assays utilize the inherent properties of antibodies to recognize and selectively bind a target molecule, while largely ignoring other molecules to provide semiquantitative values. A variety of methodologies to measure plasma vitellogenin using ELISAs have generated widely divergent data. Limitations of the ELISA method are known in the wider immunology field, though aquatic toxicologists may be less familiar with these limitations. We evaluated several mechanisms contributing to the divergent vitellogenin data in the literature. Antibody affinities and the matrix in which standard curves are constructed are possible error generators. These errors can be amplified by large sample dilutions necessary to fall within the standard curve. It is important for the aquatic toxicology research community to realize the limitations and understand the pitfalls of absolute plasma vitellogenin data in their studies. PMID:23762638

  16. Overcoming EGFR(T790M) and EGFR(C797S) resistance with mutant-selective allosteric inhibitors.

    PubMed

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A; Eck, Michael J

    2016-06-02

    The epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harbouring activating mutations in the EGFR kinase, but resistance arises rapidly, most frequently owing to the secondary T790M mutation within the ATP site of the receptor. Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternative mechanisms of action. Here we describe the rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild-type receptor. The crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays. However, as a single agent it is not effective in blocking EGFR-driven proliferation in cells owing to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state. We observe marked synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR(L858R/T790M) and by EGFR(L858R/T790M/C797S), a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors.

  17. Gain of the EGFR gene located on 7p12 is a frequent and early event in squamous cell carcinoma of the lung.

    PubMed

    Kang, Ji Un; Koo, Sun Hoe; Kwon, Kye Chul; Park, Jong Woo; Jung, Sung Su

    2008-07-01

    Identification of molecular alterations in biological fluids has been proposed as a powerful tool for cancer diagnosis. The purpose of this study was to identify cells that carry chromosomal alterations indicative of malignancy-specifically, gains in the loci 5p15.2 (D5S23, D5S721), 6p11 approximately q11, 7p12 (EGFR), and 8q24.12 approximately q24.13 (MYC)-for the detection of lung cancer using induced sputum. The overall sensitivity of the multicolor fluorescence in situ hybridization (FISH) assay from 52 lung cancer patients was 71% and the specificity was 100% (15 of 15). The most frequently detected gains were at 7p12 (EGFR) in 17 of 24 completely resectable early-stage (II+IIIA) non-small cell lung cancers (NSCLC) (71%). There was a statistically significant increase in the proportion of cases with gains of EGFR in squamous cell carcinomas (SCC), compared with adenocarcinomas (AC) (82 vs. 43%, respectively; P = 0.017), and a higher average EGFR gene copy number in the SCCs than in the ACs (5.04 vs. 3.78, respectively; P = 0.013) in 41 NSCLCs. Conversely, a gain at the 6p11 approximately q11 and 8q24.12 approximately q24.13 (MYC) regions appears to have a higher frequency of gain in the ACs (71 and 86%, respectively) than in the SCCs (48 and 56%, respectively). The results of this study showed the potential utility of the LAVysion FISH assay for the detection of lung cancer by a noninvasive technique based on the analysis of genetic alterations of induced sputum. Defining abnormalities in sputum specimens as FISH aneusomy may be a possible diagnostic method for the early detection of lung cancer in screening of high-risk populations and monitoring for recurrence.

  18. EGFR signaling in renal fibrosis

    PubMed Central

    Zhuang, Shougang; Liu, Na

    2014-01-01

    Signaling through the epidermal growth factor receptor (EGFR) is involved in regulation of multiple biological processes, including proliferation, metabolism, differentiation, and survival. Owing to its aberrant expression in a variety of malignant tumors, EGFR has been recognized as a target in anticancer therapy. Increasingly, evidence from animal studies indicates that EGFR signaling is also implicated in the development and progression of renal fibrosis. The therapeutic value of EGFR inhibition has not yet been evaluated in human kidney disease. In this article, we summarize recent research into the role of EGFR signaling in renal fibrogenesis, discuss the mechanism by which EGFR regulates this process, and consider the potential of EGFR as an antifibrotic target. PMID:26312153

  19. TELEOST FISH (Solea solea): A NOVEL MODEL FOR ECOTOXICOLOGICAL ASSAY OF CONTAMINATED SEDIMENTS

    PubMed Central

    Ribecco, C; Hardiman, G; Sásik, R; Vittori, S; Carnevali, O

    2012-01-01

    Chemical analysis of sediment is not indicative of the downstream biological effects on aquatic organisms. In this study, the biological effects of sediment were examined using: Teleost fish, (Solea solea), Artemia and rotifers. Although chemicals levels were below the limits permissible by Italian law, S. solea juveniles exposed to sediment (0.3% w/v) for 96 hours, revealed significant induction in the expression levels of HSP70, ERα, TRα, RXRα, PPARα, β, CYP4501A1 and CYP3A mRNAs, suggesting the utility of this species as a novel biosensor. The bio-toxicity of the sediment was further validated by exposing Artemia and rotifers to concentrations of elutriate (derived from the sediment) from 10 to 100 % v/v (with a 50% mortality rate). These results suggest that sediment defined as moderately contaminated, solely on the basis of the chemical profile, may in fact cause harmful effects to aquatic organisms. This study highlights the need for biological approaches in the establishment of sediment toxicity levels. PMID:22217502

  20. A chick assay for determination of available iron from biological material and its application to fish protein concentrates.

    PubMed

    Soevik, T; Opstvedt, J; Braekkan, O R

    1979-04-01

    A dose response assay for the assessment of available iron in biological materials using chicks as experimental animals is described. Day-old chicks were fed on an iron-deficient diet for 2 to 3 weeks at which time the hematocrit had decreased to about 20%. After this depletion period, the standard groups were fed graded levels of iron sulphate up to a maximum level of 30 ppm iron. The test groups were fed two levels of biological material giving a total dietary iron concentration within the standard range. The response was measured by hemoglobin concentration (g/100 ml), and the dose by the consumption of iron per gram of body weight gain. Contents of available iron (relative to iron sulphate-iron) in the test substances were calculated from the dose response regression equations obtained on the standard groups, and hemoglobin (g/100 ml), feed consumption and body weight gain in the test groups. Statistical evaluation of the data revealed that the assay complied with the requirement for statistical and fundamental validity. Results from application of the method on six different samples of fish protein concentrate (FPC) are reported.

  1. Quantitative PCR assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and application to environmental samples and fishes from the Chesapeake Bay.

    PubMed

    Gauthier, D T; Reece, K S; Xiao, J; Rhodes, M W; Kator, H I; Latour, R J; Bonzek, C F; Hoenig, J M; Vogelbein, W K

    2010-09-01

    Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

  2. Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay▿

    PubMed Central

    Gauthier, D. T.; Reece, K. S.; Xiao, J.; Rhodes, M. W.; Kator, H. I.; Latour, R. J.; Bonzek, C. F.; Hoenig, J. M.; Vogelbein, W. K.

    2010-01-01

    Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass. PMID:20656856

  3. Diagnosis of adults Xp11.2 translocation renal cell carcinoma by immunohistochemistry and FISH assays: clinicopathological data from ethnic Chinese population

    PubMed Central

    Qu, Yuanyuan; Gu, Chengyuan; Wang, Hongkai; Chang, Kun; Yang, Xiaoqun; Zhou, Xiaoyan; Dai, Bo; Zhu, Yao; Shi, Guohai; Zhang, Hailiang; Ye, Dingwei

    2016-01-01

    This study aimed to assess the utility of transcription factor E3 (TFE3) break-apart fluorescence in situ hybridization (FISH) assay in diagnosis of Xp11.2 translocation renal cell carcinoma (Xp11.2 RCC) and to compare the clinicopathological features between adult Xp11.2 RCC and non-Xp11.2 RCC. 76 pathologically suspected Xp11.2 RCCs were recruited from our institution. Both TFE3 immunohistochemistry (IHC) and TFE3 FISH assay were performed for the entire cohort. The progression-free survival (PFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method. FISH analysis confirmed 30 Xp11.2 RCCs, including 28 cases with positive TFE3 immunostaining and 2 cases with negative immunostaining. The false-positive and false-negative rates were 6.7% (2/30) and 4.3% (2/46), respectively, for TFE3 IHC compared with FISH assay. Xp11.2 RCC was significantly associated with higher pathological stage and Fuhrman nuclear grade compared with non-Xp11.2 RCC (P < 0.05). The median PFS and OS for TFE3 FISH-positive group were 13.0 months (95% CI, 8.4–17.6 months) and 50.0 months (95% CI, 27.6–72.4 months), respectively, while the median PFS and OS had not been reached for TFE3 FISH-negative group. In conclusion, TFE3 break-apart FISH assay is a highly useful and standard diagnostic method for Xp11.2 RCC. Adult Xp11.2 RCC is clinically aggressive and often presents at advanced stage with poor prognosis. PMID:26880493

  4. Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

    PubMed

    Shah, Jyotsna; Weltman, Helena; Narciso, Patricia; Murphy, Christina; Poruri, Akhila; Baliga, Shrikala; Sharon, Leesha; York, Mary; Cunningham, Gail; Miller, Steve; Caviedes, Luz; Gilman, Robert; Desmond, Edward; Ramasamy, Ranjan

    2017-01-01

    Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or

  5. Cross-species conservation of endocrine pathways: a critical analysis of tier 1 fish and rat screening assays with 12 model chemicals.

    PubMed

    Ankley, Gerald T; Gray, L Earl

    2013-04-01

    Many structural and functional aspects of the vertebrate hypothalamic-pituitary-gonadal (HPG) axis are known to be highly conserved, but the significance of this from a toxicological perspective has received comparatively little attention. High-quality data generated through development and validation of Tier 1 tests for the U.S. Environmenal Protection Agency Endocrine Disruptor Screening Program (EDSP) offer a unique opportunity to compare responses of mammals versus fish to chemicals that may affect shared pathways within the HPG axis. The present study focuses on data generated with model chemicals that act (primarily) as estrogen receptor agonists (17α-ethynylestradiol, methoxychlor, bisphenol A), androgen receptor agonists (methyltestosterone, 17β-trenbolone), androgen receptor antagonists (flutamide, vincolozolin, p,p'-DDE), or inhibitors of different steroidogenic enzymes (ketoconazole, fadrozole, fenarimol, prochloraz). All 12 chemicals had been tested in the EDSP fish short-term (21 d) reproduction assay and in one or more of the four in vivo Tier 1 screens with rats (uterotrophic, Hershberger, male and female pubertal assays). There was a high concordance between the fish and rat assays with respect to identifying chemicals that impacted specific endocrine pathways of concern. Although most chemicals were detected as positive in both rat and fish assays, eliminating data from one class of vertebrate or the other would weaken the battery. For example, the effects of competitive inhibitors of steroid hormone synthesis were far more obvious in the fish assay, whereas the activity of androgen receptor antagonists was clearer in mammalian assays. The observations are significant both to the cross-species extrapolation of toxicity of HPG-active substances and the optimization of screening and testing frameworks for endocrine-disrupting chemicals. Copyright © 2013 SETAC.

  6. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely t...

  7. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely t...

  8. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrined-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  9. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals (poster)

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  10. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrined-disrupting chemicals

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  11. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals (poster)

    EPA Science Inventory

    The fish short term reproduction assay (FSTRA) is a key component of the USEPA endocrine disruptor screening program (EDSP). The FSTRA considers several mechanistic and apical responses in fathead minnows (Pimephales promelas) to determine whether an unknown chemical is likely to...

  12. Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

    PubMed

    Jedlinski, Adam; Garvin, Stina; Johansson, Ann-Charlotte; Edqvist, Per-Henrik; Ponten, Fredrik; Roberg, Karin

    2017-10-01

    The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation.

    PubMed

    Takezawa, Ken; Pirazzoli, Valentina; Arcila, Maria E; Nebhan, Caroline A; Song, Xiaoling; de Stanchina, Elisa; Ohashi, Kadoaki; Janjigian, Yelena Y; Spitzler, Paula J; Melnick, Mary Ann; Riely, Greg J; Kris, Mark G; Miller, Vincent A; Ladanyi, Marc; Politi, Katerina; Pao, William

    2012-10-01

    EGF receptor (EGFR)-mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKI). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Because afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. FISH analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR(T790M) were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR-TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR-mutant tumors with acquired resistance to EGFR-TKIs.

  14. Identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets by polyclonal antibody-based enzyme-linked immunosorbent assay.

    PubMed

    Asensio, Luis; González, Isabel; Rodríguez, Miguel A; Mayoral, Belén; López-Calleja, Inés; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2003-02-26

    An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the species identification of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. The assay was performed in two different formats, microtiter plates and immunostick tubes, and uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of grouper (anti-GSP), wreck fish (anti-WSP), and Nile perch (anti-PSP). The antibodies were made species-specific by blocking them with the heterologous soluble muscle proteins. Immunorecognition of polyclonal antibodies adsorbed to their specific fish samples was made with swine antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of the species studied.

  15. pEGFR-Tyr 845 expression as prognostic factors in oral squamous cell carcinoma

    PubMed Central

    Aquino, Gabriella; Pannone, Giuseppe; Santoro, Angela; Liguori, Giuseppina; Franco, Renato; Serpico, Rosario; Florio, Gianluca; De Rosa, Alfredo; Mattoni, Marilena; Cozza, Valentina; Botti, Gerardo; Losito, Simona; Longo, Francesco; Staibano, Stefania; Cuda, Giovanni; Lo Muzio, Lorenzo; Sbordone, Carolina; Bufo, Pantaleo; Grimaldi, Anna; Caraglia, Michele; Di Domenico, Marina

    2012-01-01

    The EGFR (epidermal growth factor receptor) a member of the family of transmembrane protein kinase receptors known as the erbB family shows a significant correlation with the presence of metastases and poorly differentiated oral cancer. Aim of the present work is to define the key-role of EGFR in oral cancer prognosis. We have analyzed the EGFR expression on 149 cases of oral squamous cell cancers (OSCC) and we have found that it was poorly expressed in normal oral epithelium, but its expression was significantly increased in OSCCs. Moreover, we have recorded that both pEGFR-Tyr 845 and pEGFR-Tyr 1068 were mainly distributed in high histological grading and in advanced stages. Western blotting has confirmed the total absence of EGFR phosphorylation in normal oral epithelium and the higher level of protein phosphorylation in representative cases of OSCCs. The EGF-R amplification was found by fluorescence in situ hybridization (FISH) in 14% of OSCC; interestingly, EGF-R amplification was mainly observed in OSCC with higher histological grading (G2 and G3) and advanced stage (pT4) sub-groups. Kaplan-Meyer survival analysis suggested that patients with positive pEGFR-Tyr 845 tumors had a worse prognosis and were bad responders to chemotherapy. These results confirm the central role of EGF-R activation status as a prognostic biomarker in OSCC. PMID:22825335

  16. 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

  17. 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

  18. Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands.

    PubMed

    Caillaud, A; Eixarch, H; de la Iglesia, P; Rodriguez, M; Dominguez, L; Andree, K B; Diogène, J

    2012-01-01

    The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20 mg tissue equivalent (TE) ml(-1) according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC(20)). The LOQ was estimated as 0.0096 ng CTX1B eq.g TE(-1) with 23-31% variability between experiments. These values were deemed sufficient even though quantification given at the IC(50) (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17-19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B g(-1) of TE ranging from 0.058 (± 0.012) to 6.23 (± 0.713) ng CTX1B eq.g TE(-1). The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.

  19. A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum.

    PubMed

    Zhang, Yang; Xiao, Jingfan; Wang, Qiyao; Zhang, Yuanxing

    2016-08-28

    Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method.

  20. [A monoclonal antibody-based indirect competitive inhibition enzyme-linked immunosorbent assay for detecting tetrodotoxin in puffer fish].

    PubMed

    Wang, J; Luo, X; Ji, R; Zhang, D

    1997-03-01

    The homogenized tissue samples of puffer fish were extracted with boiling 0.1% acetic acid solution for 2 times, then the combined filtrate was defatted with ether ethyl for 2 times and the water phase were diluted with 0.05 mol/L PBS for ELISA determination (adjusted pH to 6.5-7.0). The results showed that the minimum detecable concentration of tetrodotoxin was 1.0 x 10(-3) mg/L (0.1 ng/assay), and the standard curve was linear between 1.0 x 10(-2) and 1.0 mg/L. The recovery rates from tetrodotoxin spiked samples of this method were 73.8%-117.4%. The accuracy of ELISA was compared with the traditional mouse bioassay system. The results showed no significant differences between the two methods (for paired-t test, P > 0.2), and significant correlations in tetrodotoxin concentration were obtained (r = 0.916).

  1. Evaluation of heavy-metal ion toxicity in fish cells using a combined stress protein and cytotoxicity assay

    SciTech Connect

    Ryan, J.A.; Hightower, L.E. )

    1994-08-01

    All organisms, from bacteria and yeast to humans, respond to physical and chemical stressors by increasing the synthesis of a small group of cellular stress proteins.'' The authors have developed a simple in vitro system for quickly screening environmentally relevant stressors to detect stress-induced proteins that are good candidates for biomarkers. Polyacrylamide gel electrophoresis was used to detect stressor-induced, concentration-dependent changes in cellular stress protein levels in two fish cell culture systems, whereas simultaneous in vitro neutral red uptake cytotoxicity assays measured the stressors effect on cellular physiology. There was a direct concentration-dependent relationship between sublethal cytotoxic effects and the increases in stress protein levels. Increases of 50 to 200% were detected in stress proteins from desert topminnow, Poeciliopsis lucida, hepatoma-derived cell cultures exposed to cadmium or copper. Three proteins showed similar increases in winter flounder, Pleuronectes americanus, kidney cell cultures exposed to the same stressors. Increases in the evolutionarily conserved heat-shock protein hsp70 were detected in each experiment; its level increased with increasing stressor concentrations.

  2. The suitability of FISH chromosome painting and ESR-spectroscopy of tooth enamel assays for retrospective dose reconstruction.

    PubMed

    Sevan'kaev, Alexander; Khvostunov, Igor; Lloyd, David; Voisin, Philippe; Golub, Elena; Nadejina, Natalie; Nugis, Vladimir; Sidorov, Oleg; Skvortsov, Valeriy

    2006-02-01

    A comparative analysis of two groups of highly irradiated victims was carried out in order to evaluate the suitability of two assays for retrospective dose assessment: late translocations and electron spin resonance (ESR) dosimetry. The first group comprised 24 subjects who exhibited acute radiation syndrome (ARS) due to overexposure as a result of nuclear submarine accidents during the period 1961-1985. Their grades of ARS and individual doses were ascertained by Navy physicians who carried out primary examinations and treatment of the exposed seamen. Cytogenetic analyses were made 16-40 y after their accidents. During medical treatment seven tooth samples were collected for ESR analysis from this group. The second group consisted of ten highly irradiated men from the Chernobyl accident. Comparison was made between estimates of their average whole-body penetrating radiation doses derived from several biological parameters. In three cases ESR measurements on tooth enamel from this group were also made. Retrospective dosimetry using FISH translocations was attempted 10-13 y later. Yields of late translocations were in good agreement with initially estimated doses and with doses obtained by ESR spectroscopy analysis of tooth enamel long after exposure. It was concluded that both persisting stable translocations and ESR spectroscopy signals are suitable with similar efficiencies for retrospective biodosimetry after acute whole-body exposure.

  3. Development of a SYBR green I real-time PCR assay for specific identification of the fish pathogen Aeromonas salmonicida subspecies salmonicida.

    PubMed

    Fernández-Álvarez, Clara; González, Santiago F; Santos, Ysabel

    2016-12-01

    A SYBR Green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 ± 0.35 °C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The SYBR Green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.

  4. The First-in-class Anti-EGFR Antibody Mixture Sym004 Overcomes Cetuximab Resistance Mediated by EGFR Extracellular Domain Mutations in Colorectal Cancer.

    PubMed

    Sánchez-Martín, Francisco J; Bellosillo, Beatriz; Gelabert-Baldrich, Mariona; Dalmases, Alba; Cañadas, Israel; Vidal, Joana; Martinez, Alejandro; Argilés, Guillem; Siravegna, Giulia; Arena, Sabrina; Koefoed, Klaus; Visa, Laura; Arpí, Oriol; Horak, Ivan David; Iglesias, Mar; Stroh, Christopher; Kragh, Michael; Rovira, Ana; Albanell, Joan; Tabernero, Josep; Bardelli, Alberto; Montagut, Clara

    2016-07-01

    Approved anti-EGFR antibodies cetuximab and panitumumab provide significant clinical benefit in patients with metastatic colorectal cancer (MCRC). However, patients ultimately develop disease progression, often driven by acquisition of mutations in the extracellular domain (ECD) of EGFR. Sym004 is a novel 1:1 mixture of two nonoverlapping anti-EGFR mAbs that recently showed promising clinical activity in a phase I trial in MCRC. Our aim was to determine the efficacy of Sym004 to circumvent cetuximab resistance driven by EGFR ECD mutations. Functional studies were performed to assess drug-receptor binding as well as ligand-dependent activation of individual EGFR mutants in the presence of cetuximab, panitumumab, and Sym004. Cell viability and molecular effects of the drugs were assayed in cetuximab-resistant cell lines and in tumor xenograft models. Efficacy of Sym004 was evaluated in patients progressing to cetuximab that harbored EGFR mutation in the post-cetuximab tumor sample. Contrary to cetuximab and panitumumab, Sym004 effectively bound and abrogated ligand-induced phosphorylation of all individual EGFR mutants. Cells resistant to cetuximab harboring mutations in EGFR maintained sensitivity to Sym004, which was consistent with an effective suppression of EGFR downstream signaling, translating into profound and sustained tumor regression in the xenograft model. As proof-of-principle, a patient with a tumor harboring an EGFR mutation (G465R) following cetuximab therapy benefited from Sym004 therapy. Sym004 is an active drug in MCRC resistant to cetuximab/panitumumab mediated by EGFR mutations. EGFR mutations are potential biomarkers of response to Sym004 to be evaluated in ongoing large clinical trials. Clin Cancer Res; 22(13); 3260-7. ©2016 AACR. ©2016 American Association for Cancer Research.

  5. Highly Sensitive and Reliable Detection of EGFR Exon 19 Deletions by Droplet Digital Polymerase Chain Reaction.

    PubMed

    Oskina, Natalya; Oscorbin, Igor; Khrapov, Evgeniy; Boyarskikh, Ulyana; Subbotin, Dmitriy; Demidova, Irina; Imyanitov, Evgeny; Filipenko, Maxim

    2017-06-06

    Analysis of EGFR mutations is becoming a routine clinical practice but the optimal EGFR mutation testing method is still to be determined. We determined the nucleotide sequence of deletions located in exon 19 of the EGFR gene in lung tumor samples of patients residing in different regions of Russia (153 tumor DNA specimens), using Sanger sequencing. We developed a droplet digital polymerase chain reaction assay capable of detecting all common EGFR deletions in exon 19. We also compared the therascreen amplification refractory mutation system assay with a droplet digital polymerase chain reaction assay for the detection of all the deletions in our study. The droplet digital polymerase chain reaction assay demonstrated 100% sensitivity against polymerase chain reaction fragment length analysis and detected all possible types of deletions revealed in our study (22 types). At the same time, the therascreen EGFR RGQ PCR Kit was not able to detect deletions c.2252-2276>A and c.2253-2276 and showed low performance for another long deletion. Thus, we can conclude that the extraordinary length of deletions and their atypical locations (shift at the 3'-region compared to known deletions) could be problematic for the therascreen EGFR RGQ PCR Kit and should be taken into account during targeted mutation test development. However, droplet digital polymerase chain reaction is a promising and reliable assay that can be used as a diagnostic tool to genotype formalin-fixed paraffin-embedded cancer samples for EGFR or another clinically relevant somatic mutation.

  6. The Hippo coactivator YAP1 mediates EGFR overexpression and confers chemo-resistance in esophageal cancer

    PubMed Central

    Song, Shumei; Honjo, Soichiro; Jin, Jiankang; Chang, Shih-Shin; Scott, Ailing W; Chen, Qiongrong; Kalhor, Neda; Correa, Arlene M.; Hofstetter, Wayne L.; Albarracin, Constance T.; Wu, Tsung-Teh; Johnson, Randy L.; Hung, Mien-Chie; Ajani, Jaffer A.

    2015-01-01

    Purpose Esophageal cancer (EC) is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of EC patients. However, clinical trials using EGFR inhibitors have not provided benefit for EC patients. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. Experimental Design In this study, expression of YAP1 and EGFR were examined in EAC resistant tumor tissues vs sensitive tissues by immunohistochemistry. Western blot, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis and in vitro and in vivo functional assays were performed to elucidate the YAP1 mediate EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. Results We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant EC tissues compared to sensitive EC tissues. Further, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-FU and docetaxcel, while knockdown of YAP sensitizes EC cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. Conclusions Our data provide evidence that YAP1 up-regulation of EGFR plays an important role in conferring therapy resistance in EC cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in EC. PMID:25739674

  7. Monitoring of gefitinib sensitivity with radioiodinated PHY based on EGFR expression.

    PubMed

    Yoshimoto, Mitsuyoshi; Hirata, Masahiko; Kanai, Yasukazu; Naka, Sadahiro; Nishii, Ryuichi; Kagawa, Shinya; Kawai, Keiichi; Ohmomo, Yoshiro

    2014-01-01

    Epidermal growth factor receptor (EGFR) is attractive target for tumor diagnosis and therapy, as it is specifically and abundantly expressed in tumor cells. EGFR-tyrosine kinase (TK) inhibitors such as gefitinib and erlotinib are widely used in the treatment of non-small cell lung cancer (NSCLC). In this study, we investigated whether radioiodinated 4-(3-iodo-phenoxy)-6,7-diethoxy-quinazoline (PHY), which is a candidate EGFR-TK imaging agent for single photon emission computed tomography (SPECT) is able to predict gefitinib sensitivity. We used four NSCLC cell lines-A549 (wild-type EGFR), H1650 (mutant EGFR; del E746_A750), H1975 (mutant EGFR; L858R, T790M) and H3255 (mutant EGFR; L858R)-and one epidermoid carcinoma cell line, A431 (wild-type EGFR). Cell proliferation assay and Western blotting revealed that A431 and H3255 with high EGFR expression showed high sensitivity to gefitinib. On the other hand, A549, H1650 and H1975 showed much lower sensitivity to gefitinib. The blocking study revealed that gefitinib decreased tumor uptake in (125)I-PHY in A431-bearing mice. Moreover, in vivo tumor uptake of (125)I-PHY was correlated with the IC50 of gefitinib for cell proliferation. In the present study, tumor uptake of (125)I-PHY was correlated with the gefitinib sensitivity and this uptake was based on expression levels of EGFR, but not on mutation status. Although the mutation status is the most important factor for predicting gefitinib sensitivity, the abundant expression of EGFR is essential for therapy with EGFR-TK inhibitors. Therefore, radioiodinated PHY is a potential imaging agent to predict gefitinib sensitivity based on EGFR expression levels though further modifications of the imaging agent is needed to accurately estimate the mutation status.

  8. Strand-specific, real-time RT-PCR assays for quantification of genomic and positive-sense RNAs of the fish rhabdovirus, Infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Purcell, Maureen K.; Hart, S. Alexandra; Kurath, Gael; Winton, James R.

    2006-01-01

    The fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), is an important pathogen of salmonids. Cell culture assays have traditionally been used to quantify levels of IHNV in samples; however, real-time or quantitative RT-PCR assays have been proposed as a rapid alternative. For viruses having a single-stranded, negative-sense RNA genome, standard qRT-PCR assays do not distinguish between the negative-sense genome and positive-sense RNA species including mRNA and anti-genome. Thus, these methods do not determine viral genome copy number. This study reports development of strand-specific, qRT-PCR assays that use tagged primers for enhancing strand specificity during cDNA synthesis and quantitative PCR. Protocols were developed for positive-strand specific (pss-qRT-PCR) and negative-strand specific (nss-qRT-PCR) assays for IHNV glycoprotein (G) gene sequences. Validation with synthetic RNA transcripts demonstrated the assays could discriminate the correct strand with greater than 1000-fold fidelity. The number of genome copies in livers of IHNV-infected fish determined by nss-qRT-PCR was, on average, 8000-fold greater than the number of infectious units as determined by plaque assay. We also compared the number of genome copies with the quantity of positive-sense RNA and determined that the ratio of positive-sense molecules to negative-sense genome copies was, on average, 2.7:1. Potential future applications of these IHNV strand-specific qRT-PCR assays are discussed.

  9. Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

    PubMed Central

    Brinkmann, Markus; Blenkle, Henning; Salowsky, Helena; Bluhm, Kerstin; Schiwy, Sabrina; Tiehm, Andreas; Hollert, Henner

    2014-01-01

    Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We

  10. Evaluation of genotoxicity using the micronucleus assay and nuclear abnormalities in the tropical sea fish Bathygobius soporator (Valenciennes, 1837) (Teleostei, Gobiidae)

    PubMed Central

    2009-01-01

    The micronucleus and nuclear abnormalities assays have been used increasingly to evaluate genotoxicity of many compounds in polluted aquatic ecossystems. The aim of this study is to verify the efficiency of the micronucleus assay and nuclear abnormality assay in field and laboratory work, when using erythrocytes of the tropical marine fish Bathygobius soporator as genotoxicity biomarkers. Gill peripheral blood samples were obtained from specimens of Bathygobius soporator. In order to investigate the frequencies of micronuclei and to assess the sensitivity of species, the results were compared with samples taken at the reference site and maintained in the laboratory, and fish treated with cyclophosphamide. The micronucleus assay was efficient in demonstrating field pollution and reproducing results in the labotatory. There were significant higher frequencies of micronuclei in two sites subject to discharge of urban and industrial effluents. The nuclear abnormality assay did not appear to be an efficient tool for genotoxicity evaluation when compared with field samples taken at a reference site in laboratory, with a positive control. PMID:21637697

  11. Radionuclide therapy using ¹³¹I-labeled anti-epidermal growth factor receptor-targeted nanoparticles suppresses cancer cell growth caused by EGFR overexpression.

    PubMed

    Li, Wei; Liu, Zhongyun; Li, Chengxia; Li, Ning; Fang, Lei; Chang, Jin; Tan, Jian

    2016-03-01

    Anti-epidermal growth factor receptor (EGFR)-targeted nanoparticles can be used to deliver a therapeutic and imaging agent to EGFR-overexpressing tumor cells. (131)I-labeled anti-EGFR nanoparticles derived from cetuximab were used as a tumor-targeting vehicle in radionuclide therapy. This paper describes the construction of the anti-EGFR nanoparticle EGFR-BSA-PCL. This nanoparticle was characterized for EGFR-targeted binding and cellular uptake in EGFR-overexpressing cancer cells by using flow cytometry and confocal microscopy. Anti-EGFR and non-targeted nanoparticles were labeled with (131)I using the chloramine-T method. Analyses of cytotoxicity and targeted cell killing with (131)I were performed using the MTT assay. The time-dependent cellular uptake of (131)I-labeled anti-EGFR nanoparticles proved the slow-release effects of nanoparticles. A radioiodine therapy study was also performed in mice. The EGFR-targeted nanoparticle EGFR-BSA-PCL and the non-targeted nanoparticle BSA-PCL were constructed; the effective diameters were approximately 100 nm. The results from flow cytometry and confocal microscopy revealed significant uptake of EGFR-BSA-PCL in EGFR-overexpressing tumor cells. Compared with EGFR-BSA-PCL, BSA-PCL could also bind to cells, but tumor cell retention was minimal and weak. In MTT assays, the EGFR-targeted radioactive nanoparticle (131)I-EGFR-BSA-PCL showed greater cytotoxicity and targeted cell killing than the non-targeted nanoparticle (131)I-BSA-PCL. The radioiodine uptake of both (131)I-labeled nanoparticles, (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL, was rapid and reached maximal levels 4 h after incubation, but the (131)I uptake of (131)I-EGFR-BSA-PCL was higher than that of (131)I-BSA-PCL. On day 15, the average tumor volumes of the (131)I-EGFR-BSA-PCL and (131)I-BSA-PCL groups showed a slow growth relationship compared with that of the control group. The EGFR-targeted nanoparticle EGFR-BSA-PCL demonstrated superior cellular binding and uptake

  12. Accurate detection and quantification of the fish viral hemorrhagic Septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay.

    PubMed

    Pierce, Lindsey R; Willey, James C; Palsule, Vrushalee V; Yeo, Jiyoun; Shepherd, Brian S; Crawford, Erin L; Stepien, Carol A

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R(2) = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/10(6) actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics.

  13. Accurate Detection and Quantification of the Fish Viral Hemorrhagic Septicemia virus (VHSv) with a Two-Color Fluorometric Real-Time PCR Assay

    PubMed Central

    Palsule, Vrushalee V.; Yeo, Jiyoun; Shepherd, Brian S.; Crawford, Erin L.; Stepien, Carol A.

    2013-01-01

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting >80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain – IVb – appeared in the Great Lakes in 2003, has killed many game fish species in a series of outbreaks in subsequent years, and shut down interstate transport of baitfish. Cell culture is the diagnostic method approved by the USDA-APHIS, which takes a month or longer, lacks sensitivity, and does not quantify the amount of virus. We thus present a novel, easy, rapid, and highly sensitive real-time quantitative reverse transcription PCR (qRT-PCR) assay that incorporates synthetic competitive template internal standards for quality control to circumvent false negative results. Results demonstrate high signal-to-analyte response (slope = 1.00±0.02) and a linear dynamic range that spans seven orders of magnitude (R2 = 0.99), ranging from 6 to 6,000,000 molecules. Infected fishes are found to harbor levels of virus that range to 1,200,000 VHSv molecules/106 actb1 molecules with 1,000 being a rough cut-off for clinical signs of disease. This new assay is rapid, inexpensive, and has significantly greater accuracy than other published qRT-PCR tests and traditional cell culture diagnostics. PMID:23977162

  14. Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells

    PubMed Central

    Vouri, M; Croucher, D R; Kennedy, S P; An, Q; Pilkington, G J; Hafizi, S

    2016-01-01

    Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl–Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl–Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling. PMID:27775700

  15. Rapid detection of Flavobacterium columnare in fish using TaqMan real-time polymerase chain reaction assay

    USDA-ARS?s Scientific Manuscript database

    Columnaris disease caused by Flavobacterium columnare, a ubiquitous Gram-negative organism prevalent among both warm and cold water reared fish species is important from an economical perspective to the aquaculture industry, worldwide. Because, in commercial aquaculture large numbers of fish are rea...

  16. Biomonitoring of lead-contaminated Missouri streams with an assay for erythrocyte δ-aminolevulinic acid dehydratase activity in fish blood

    USGS Publications Warehouse

    Schmitt, C. J.; Wildhaber, M.L.; Hunn, J.B.; Nash, T.; Tieger, M. N.; Steadman, B. L.

    1993-01-01

    The activity of the enzyme δ-aminolevulinic acid dehydratase (ALA-D) in erythrocytes has long been used as a biomarker of lead exposure in humans and waterfowl and, more recently, in fishes. The assay was tested for ALA-D activity in fishes from streams affected by lead in combination with other metals from lead-zinc mining and related activities. Fishes (mostly catostomids) were collected from sites affected by historic and current mining activities, and from sites considered to be unaffected by mining (reference sites). A group of potentially toxic elements was measured in blood and carcass samples of individual fish, as were ALA-D activity, total protein (TP), and hemoglobin (Hb) in blood. Concentrations of mining-related metals (lead, zinc, and cadmium) were significantly greater (P<0.05) in fish blood and carcass at sites affected by historic mining activities than at reference and active mining sites. When analyzed by multiple regression, ALA-D activity, Hb, and TP accounted for 66% of blood-lead and 69% of carcass-lead variability. Differences among species were small. ALA-D activity as a biomarker adequately distinguished sites affected by bioavailable environmental lead. Zinc was the only other metal that affected ALA-D activity; it appeared to ameliorate the inactivation of ALA-D by lead.

  17. Differences in detection of Aeromonas salmonicida in covertly infected salmonid fishes by the stress-inducible furunculosis test and culture-based assays

    USGS Publications Warehouse

    Cipriano, R.C.; Ford, L.A.; Smith, D.R.; Schachte, J.H.; Petrie, C.J.

    1997-01-01

    Accurate detection of Aeromonas salmonicida subsp. salmonicida (the cause of furunculosis disease) in covertly infected salmonids is difficult and is a cause of concern for those involved in fish health inspection and resource management programs. In this study, we examined populations of brook trout Salvelinus fontinalis, Atlantic salmon Salmo salar, and lake trout Salvelinus namaycush that previously sustained natural episodes of furunculosis. Consequently, the sampled fish were presumed to harbor latent infections. Mucus, gill, liver, kidney, heart, spleen, and intestine samples (N = 100 fish per group sampled) were processed and examined by (1) direct dilution counts and (2) quadrant streaking after a 48-h pre-enrichment in trypticase soy broth (TSB). Another subsample of fish from each group was then subjected to stress-inducible furunculosis tests. Stress tests detected A. salmonicida in three of four groups of fish that were examined whereas the pathogen was detected in only two of the groups analyzed with culture-based assays. Although pre-enrichment in TSB enhanced detection within internal sampling sites including the liver, heart, spleen, and kidney, enrichment did not enhance detection from mucus, gill, or intestinal samples.

  18. 2,3,7,8 tetrachlorodibenzodioxin in fish from the Pigeon river of Eastern Tennessee: Its toxicity and mutagenicity as revealed by the Ames Salmonella Assay

    SciTech Connect

    Blevins, R.D. )

    1990-04-01

    Levels of 2,3,7,8 tetrachlorodibenzodioxin (TCDD)were determined in both striated muscle (fillets) and whole body extracts of fish specimens harvested during a two year period (1987-1989) from the Pigeon River (between Hartford and Newport) of Eastern Tennessee. Whole body (wet weight) fish extract levels as high as 117 {mu}g/kg body weight and composite fish fillet (wet weight) extract levels as high 87 {mu}g/kg fillet weight were observed. Pure TCDD was found to be highly toxic to the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA1535 at TCDD dosages which exceeded 825 ng/ml in the top agar of the Ames Salmonella assay. An 825 ng/ml TCDD dosage was not mutagenic to any of the tested Salmonella strains, (both with and without metabolic activation (S9) mix). However, when both acidic and alcohol fish extracts from the Pigeon River were tested for mutagenicity, several of the fish extracts were found to be mutagenic to Salmonella strains TA97, TA98, and TA100 (having mutagenic ratios which greatly exceeded the 2.5 {times} spontaneous ratio). These mutagenic extracts also demonstrated mutagenic dose-response curves. Other chemicals within the extracts as well as synergistic effects may account for the mutagenicity.

  19. Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish.

    PubMed

    Oplatowska, Michalina; Connolly, Lisa; Stevenson, Paul; Stead, Sara; Elliott, Christopher T

    2011-07-18

    Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable

  20. DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.

    PubMed

    Loh, W K W; Bond, P; Ashton, K J; Roberts, D T; Tibbetts, I R

    2014-08-01

    The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples.

  1. Use of the Comet-FISH Assay to Compare DNA Damage and Repair in p53 and hTERT Genes following Ionizing Radiation

    PubMed Central

    McKenna, Declan J.; Doherty, Bernadette A.; Downes, C. Stephen; McKeown, Stephanie R.; McKelvey-Martin, Valerie J.

    2012-01-01

    The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting. PMID:23145163

  2. Development and validation of a high performance liquid chromatography assay for 17alpha-methyltestosterone in fish feed.

    PubMed

    Marwah, Ashok; Marwah, Padma; Lardy, Henry

    2005-09-25

    17alpha-Methyltestosterone (MT) is used to manipulate the gender of a variety of fish species. A high performance liquid chromatography (HPLC) internal standard method for the determination of 17alpha-methyltestosterone in fish feed using 3beta-methoxy-17beta-hydroxyandrost-5-en-7-one as internal standard (IS) has been developed. The method has been validated for the quantitation of MT in fish feed using 245 nm UV absorbance as the parent wavelength and 255 nm as a qualifier wavelength. The method was validated in the concentration range of 15.0-120 mg/kg of 17alpha-methyltestosterone in fish feed. Method was also found to be suitable for other feeds.

  3. EGFR, KRAS, BRAF, and PIK3CA characterization in squamous cell anal cancer.

    PubMed

    Martin, Vittoria; Zanellato, Elena; Franzetti-Pellanda, Alessandra; Molinari, Francesca; Movilia, Alessandra; Paganotti, Alessia; Deantonio, Letizia; De Dosso, Sara; Assi, Agnese; Crippa, Stefano; Boldorini, Renzo; Mazzucchelli, Luca; Saletti, Piercarlo; Frattini, Milo

    2014-04-01

    Combined chemoradiation therapy is the gold standard in the treatment of squamous cell anal cancer (SCAC). However, even if the response rate is very high, many patients eventually relapse or experience a reccurrence, thus requiring an invasive surgical procedure that has severe side effects. Most SCAC tumors overexpress epidermal growth factor receptor (EGFR); therefore, it is reasonable to consider anti-EGFR drugs as a new treatment option, as demonstrated by anecdotal reports. Promising results obtained in other solid tumors, both squamous and non-squamous, have revealed that an increase in the EGFR gene copy number may predict the efficacy of anti-EGFR therapies, while the presence of mutations in downstream members of the EGFR pathway may confer resistance. These markers have been only sporadically considered in SCAC. We investigated the status of the EGFR gene using FISH and examined KRAS, BRAF, and PIK3CA hot-spots mutations using sequencing analysis in a cohort of 84 patients affected by SCAC. Twenty-eight patients (34%) showed an increase in EGFR gene copy number due to amplification (4%) or to polysomy (30%). KRAS and PIK3CA gene mutations were found in 4 (5%) and 13 patients (16%), respectively. No mutations were found in the BRAF gene. The characterization of the EGFR pathway may help in identifying different subgroups of SCAC that have specific molecular features, which may have implications in what targeted therapies are used to treat each patient.

  4. Concomitant high gene copy number and protein overexpression of IGF1R and EGFR negatively affect disease-free survival of surgically resected non-small-cell-lung cancer patients

    PubMed Central

    Flacco, A.; Bianconi, F.; Ragusa, M.; Vannucci, J.; Bellezza, G.; Chiari, R.; Minotti, V.; Pistola, L.; Tofanetti, F. R.; Siggillino, A.; Baldelli, E.; Sidoni, A.; Daddi, N.; Puma, F.; Varella-Garcia, M.; Crinò, L.

    2014-01-01

    Background Insulin-like growth factor 1 receptor (IGF1R) represents a novel molecular target in non-small-cell-lung cancer (NSCLC). IGF1R and epidermal growth factor receptor (EGFR) activation are essential to mediate tumor cell survival, proliferation, and invasion. This study investigates the prognostic role of IGF1R and EGFR in surgically resected NSCLC. Materials and methods IGF1R and EGFR copy number gain (CNG) were tested by fluorescence in situ hybridization (FISH) and protein expression by immunohistochemistry (IHC) in 125 stage I–II–IIIA NSCLC patients. Results Fourty-six tumors (40.3 %) were IGF1R FISH-positive (FISH+), and 76 (67.2 %) were EGFR FISH+. Tumors with concomitant IGF1R/EGFR FISH+ were observed in 34 cases (30.1 %). IGF1R and EGFR FISH+ were associated with SCC histology (p = 0.01 and p = 0.04, respectively). IGF1R and EGFR protein over-expression (IHC+) were detected in 45 (36.0 %) and 69 (55.2 %) cases, respectively. Tumors with concomitant IGF1R/EGFR IHC+ were detected in 31 (24.8 %) patients. IGF1R/EGFR FISH+ and IGF1R/EGFR IHC+ were significantly associated (χ2 = 4.02, p = 0.04). Patients with IGF1R/EGFR FISH+ and IGF1R/EGFR IHC+ were associated with shorter disease-free survival (DFS) (p = 0.05 and p = 0.05, respectively). Patients with concomitant IGF1R/EGFR FISH+/IHC+ had a worse DFS and overall survival (p = 0.005 and p = 0.01, respectively). The multivariate model confirmed that IGF1R/EGFR FISH+/IHC+ (hazard ratio (HR), 4.08; p = 0.01) and tumor stage (II–III vs I) (HR, 4.77; p = 0.003) were significantly associated with worse DFS. Conclusions IGF1R/EGFR FISH+ correlates with IGF1R/EGFR IHC+. IGF1R/EGFR FISH+/IHC+ is an independent negative prognostic factor for DFS in early NSCLC. These features may have important implications for future anti-IGF1R therapeutic approaches. PMID:23314677

  5. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array-based multiplex assay.

    PubMed

    Lievens, B; Frans, I; Heusdens, C; Justé, A; Jonstrup, S P; Lieffrig, F; Willems, K A

    2011-11-01

    Fish diseases can be caused by a variety of diverse organisms, including bacteria, fungi, viruses and protozoa, and pose a universal threat to the ornamental fish industry and aquaculture. The lack of rapid, accurate and reliable means by which fish pathogens can be detected and identified has been one of the main limitations in fish pathogen diagnosis and fish disease management and has consequently stimulated the search for alternative diagnostic techniques. Here, we describe a method based on multiplex and broad-range PCR amplification combined with DNA array hybridization for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV-1, CyHV-2 and CyHV-3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were targeted. For bacterial identification, the ribosomal RNA gene was used. The developed methodology permitted 100% specificity for the identification of the target species. Detection sensitivity was equivalent to 10 viral genomes or less than a picogram of bacterial DNA. The utility and power of the array for sensitive pathogen detection and identification in complex samples such as infected tissue is demonstrated in this study. © 2011 Blackwell Publishing Ltd.

  6. 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.

  7. A non-destructive BFCOD assay for in vivo measurement of cytochrome P450 3A (CYP3A) enzyme activity in fish embryos and larvae.

    PubMed

    Oziolor, Elias M; Carey, Alexis N; Matson, Cole W

    2017-08-01

    There is increasing interest in quantifying the exposure and effects of anthropogenic contaminants in fish. Determination of exposures in wild fish is routinely performed, but methods to investigate potential effects are less established. One of the most relevant approaches would be the use of in vivo assays, but existing assays are often limited to in vitro determination of enzyme activity. Many pharmaceuticals and some persistent pollutants activate, and are metabolized by cytochrome P4503A (CYP3A), which make it a relevant and desirable target for biomarker research. We altered the established 7-benzyloxy-4-trifluoromethylcoumarin-O-debenzylation (BFCOD) in vitro protocol for CYP3A activity determination, developing a rapid and inexpensive method to measure in vivo (and in ovo) CYP3A activity in two fish systems: Gulf killifish (Fundulus grandis) and zebrafish (Danio rerio) early life stages. Even with very low concentrations of 7-benzyloxy-4-trifluoromethyl coumarin (BFC, 0.06 µM or 20 µg/L), we were able to detect significant induction in CYP3A activity in embryos of F. grandis, as well as in larvae of D. rerio in response to benzo[a]pyrene (BaP) and fluoranthene (FL) exposures. Because of concerns regarding the possible contribution of CYP1A to BFCOD activity from previous research, we have used a CYP1A post-translational inhibitor (FL) in order to calculate the contribution of CYP1A to the BFCOD assay. We also dosed with benzo[k]fluoranthene (BkF) and showed significant induction of CYP1A activity, with no concurrent increase in CYP3A activity. In this paper, we have taken an established in vitro CYP3A activity assay, and utilized the reaction in a novel way to allow for the non-destructive determination of CYP3A. In summary, we describe a sensitive, cheap, fast and easy modified BFCOD assay for in ovo and in vivo determination of CYP3A activity for use in moderate throughput early-life-stage fish experiments.

  8. Study of 30 patients with unexplained developmental delay and dysmorphic features or congenital abnormalities using conventional cytogenetics and multiplex FISH telomere (M-TEL) integrity assay.

    PubMed

    Popp, Susanne; Schulze, Birgit; Granzow, Martin; Keller, Monika; Holtgreve-Grez, Heidi; Schoell, Brigitte; Brough, Michaela; Hager, Hans-Dieter; Tariverdian, Gholamali; Brown, Jill; Kearney, Lyndal; Jauch, Anna

    2002-07-01

    Cryptic subtelomeric chromosome rearrangements are a major cause of mild to severe mental retardation pointing out the necessity of sensitive screening techniques to detect such aberrations among affected patients. In this prospective study a group of 30 patients with unexplained developmental retardation and dysmorphic features or congenital abnormalities were analysed using the recently published multiplex FISH telomere (M-TEL) integrity assay in combination with conventional G-banding analysis. The patients were selected by one or more of the following criteria defined by de Vries et al.: (a) family history with two or more affected individuals, (b) prenatal onset growth retardation, (c) postnatal growth abnormalities, (d) facial dysmorphic features, (e) non-facial dysmorphism and congenital abnormalities. In addition, we included two patients who met these criteria and revealed questionable chromosome regions requiring further clarification. In four patients (13.3%) cryptic chromosome aberrations were successfully determined by the M-TEL integrity assay and in two patients with abnormal chromosome regions intrachromosomal aberrations were characterized by targetted FISH experiments. Our results accentuate the requirement of strict selection criteria prior to patient testing with the M-TEL integrity assay. Another essential precondition is high-quality banding analysis to identify structural abnormal chromosomes. The detection of familial balanced translocation carriers in 50% of the cases emphasizes the significance of such an integrated approach for genetic counselling and prenatal diagnosis.

  9. EGFR inhibitors and autophagy in cancer treatment.

    PubMed

    Cui, Jie; Hu, Yun-Feng; Feng, Xie-Min; Tian, Tao; Guo, Ya-Huan; Ma, Jun-Wei; Nan, Ke-Jun; Zhang, Hong-Yi

    2014-12-01

    Epidermal growth factor receptor (EGFR) inhibitor treatment is a strategy for cancer therapy. However, innate and acquired resistance is a major obstacle of the efficacy. Autophagy is a self-digesting process in cells, which is considered to be associated with anti-cancer drug resistance. The activation of EGFR can regulate autophagy through multiple signal pathways. EGFR inhibitors can induce autophagy, but the specific function of the induction of autophagy by EGFR inhibitors remains biphasic. On the one hand, autophagy induced by EGFR inhibitors acts as a cytoprotective response in cancer cells, and autophagy inhibitors can enhance the cytotoxic effects of EGFR inhibitors. On the other hand, a high level of autophagy after treatment of EGFR inhibitors can also result in autophagic cell death lacking features of apoptosis, and the combination of EGFR inhibitors with an autophagy inducer might be beneficial. Thus, autophagy regulation represents a promising approach for improving the efficacy of EGFR inhibitors in the treatment of cancer patients.

  10. The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

    PubMed

    Lewandowska, Marzena Anna; Czubak, Karol; Klonowska, Katarzyna; Jozwicki, Wojciech; Kowalewski, Janusz; Kozlowski, Piotr

    2015-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

  11. Lung adenocarcinoma: Sustained subtyping with immunohistochemistry and EGFR, HER2 and KRAS mutational status.

    PubMed

    Sousa, Vitor; Rodrigues, Carolina; Silva, Maria; Alarcão, Ana Maria; Carvalho, Lina

    2015-01-01

    Pulmonary adenocarcinomas are still in the process of achieving morphological, immunohistochemical and genetic standardization. The ATS/ERS/IASLC proposed classification for lung adenocarcinomas supports the value of the identification of histological patterns, specifically in biopsies. Thirty pulmonary adenocarcinomas were subjected to immunohistochemical study (CK7, CK5, 6, 18, CK20, TTF1, CD56, HER2, EGFR and Ki-67), FISH and PCR followed by sequencing and fragment analysis for EGFR, HER2 and KRAS. Solid pattern showed lower TTF1 and higher Ki-67 expression. TTF1 expression was higher in non-mucinous lepidic and micropapillary patterns when compared to acinar and solid and acinar, solid and mucinous respectively. Higher Ki67 expression was present in lepidic and solid patterns compared to mucinous. EGFR membranous staining had increasing expression from non-mucinous lepidic/BA pattern to solid pattern and micropapillary until acinar pattern. EGFR mutations, mainly in exon 19, were more frequent in females, together with non-smoking status, while KRAS exon 2 mutations were statistically more frequent in males, especially in solid pattern. FISH EGFR copy was correlated gross, with mutations. HER2 copy number was raised in female tumours without mutations, in all cases. Although EGFR and KRAS mutations are generally considered mutually exclusive, in rare cases they can coexist as it happened in one of this series, and was represented in acinar pattern with rates of 42.9% and 17.9%, respectively. EGFR mutations were more frequent in lepidic/BA and acinar patterns. Some cases showed different EGFR mutations. The differences identified between the adenocarcinoma patterns reinforce the need to carefully identify the patterns present, with implications in diagnosis and in pathogenic understanding. EGFR and KRAS mutational status can be determined in biopsies representing bronchial pulmonary carcinomas because when a mutation is present it is generally present in all the

  12. Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue

    PubMed Central

    2012-01-01

    Background Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent

  13. Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue.

    PubMed

    Hembrough, Todd; Thyparambil, Sheeno; Liao, Wei-Li; Darfler, Marlene M; Abdo, Joseph; Bengali, Kathleen M; Taylor, Paul; Tong, Jiefei; Lara-Guerra, Humberto; Waddell, Thomas K; Moran, Michael F; Tsao, Ming-Sound; Krizman, David B; Burrows, Jon

    2012-05-03

    Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in

  14. Sublethal toxicity of esbiothrin relationship with total antioxidant status and in vivo genotoxicity assessment in fish (Cyprinus carpio L., 1758) using the micronucleus test and comet assay.

    PubMed

    Selvi, Mahmut; Cavaş, Tolga; Cağlan Karasu Benli, A; Koçak Memmi, Burcu; Cinkılıç, Nilüfer; Dinçel, Aylin Sepici; Vatan, Ozgür; Yılmaz, Dilek; Sarıkaya, Rabia; Zorlu, Tolga; Erkoç, Figen

    2013-11-01

    Esbiothrin, synthetic pyrethroid with quick activity against insects, is widely used against household pests and in public health. Despite widespread use, data on ecotoxicity and genotoxic effects are extremely scarce. The aim of the present study is to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronucleus test and comet assay in peripheral blood erythrocytes. Effects of two sublethal exposure concentrations on plasma total antioxidant status (TAS mmol/L), and Hct values were examined. On the basis of the 96 h LC50 data from U.S. EPA ecotox database (32 μg/L) two sublethal exposure concentrations (5 and 10 μg/L) were used together with ethyl methanesulfonate (EMS) (5 mg/L) as positive control. Five fish were used for each dose/duration group (24, 48, and 72 h) under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. Plasma TAS (mmol/L) levels decreased in 24 h; an increase was observed slightly for 48 and obviously for 72 h in both exposure doses. Similarly, hematocrit (Hct) values differed between exposure duration but no significant differences in mean values were found between groups of the same exposure time. The general trend was a rise after 48 h, which decreased afterwards. Our results revealed significant increases in the frequencies of micronuclei and levels of DNA strand breaks and thus demonstrated the genotoxic potential of this pesticide on fish, a nontarget organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish.

  15. The association between EGFR variant III, HPV, p16, c-MET, EGFR gene copy number and response to EGFR inhibitors in patients with recurrent or metastatic squamous cell carcinoma of the head and neck

    PubMed Central

    2011-01-01

    Background We examine the potential prognostic and predictive roles of EGFR variant III mutation, EGFR gene copy number (GCN), human papillomavirus (HPV) infection, c-MET and p16INK4A protein expression in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN). Methods We analyzed the archival tumor specimens of 53 patients who were treated in 4 phase II trials for R/M SCCHN. Two trials involved the EGFR inhibitor erlotinib, and 2 trials involved non-EGFR targeted agents. EGFRvIII mutation was determined by quantitative RT-PCR, HPV DNA by Linear Array Genotyping, p16 and c-MET protein expression by immunohistochemistry, and EGFR GCN by FISH. Results EGFRvIII mutation, detected in 22 patients (42%), was associated with better disease control, but no difference was seen between erlotinib-treated versus non-erlotinib treated patients. EGFRvIII was not associated with TTP or OS. The presence of HPV DNA (38%), p16 immunostaining (32%), c-MET high expression (58%) and EGFR amplification (27%), were not associated with response, TTP or OS. Conclusion EGFRvIII mutation, present in about 40% of SCCHN, appears to be an unexpected prognostic biomarker associated with better disease control in R/M SCCHN regardless of treatment with erlotinib. Larger prospective studies are required to validate its significance. PMID:21352589

  16. Copy number gains in EGFR and copy number losses in PTEN are common events in osteosarcoma tumors.

    PubMed

    Freeman, Serena S; Allen, Steven W; Ganti, Ramapriya; Wu, Jianrong; Ma, Jing; Su, Xiaoping; Neale, Geoff; Dome, Jeffrey S; Daw, Najat C; Khoury, Joseph D

    2008-09-15

    Osteosarcoma cell lines and tumors have been shown to express epidermal growth factor receptor (EGFR) and harbor amplifications at the EGFR locus. In this study, the authors further analyzed the genomic features of EGFR in osteosarcoma tumors and investigated whether they correlate with phosphatase and tensin homolog (PTEN) expression and copy number status. EGFR and PTEN expression was assessed by immunohistochemistry (n = 28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix (Santa Clara, Calif) 50K single nucleotide polymorphism (SNP) arrays (n = 31) and fluorescence in situ hybridization (FISH) (n = 27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n = 34). In addition, EGFRvIII expression was assessed using reverse transcriptase polymerase chain reaction (n = 24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5-23 years) and median follow-up of 4.4 years. EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23 of 28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16 of 27 cases; 59%) showed gains at the EGFR locus using FISH (amplification in 4 of 27 [15%] and balanced chromosome 7 polysomy in 12 of 27 [44%]), and 12 cases showed deletions at the PTEN locus (biallelic deletions in 4 of 27 [15%] and monoallelic deletion in 9 of 27 [33%]). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected. EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus. (c) 2008 American Cancer Society.

  17. EGFR mutation testing on cytological and histological samples in non-small cell lung cancer: a Polish, single institution study and systematic review of European incidence.

    PubMed

    Szumera-Ciećkiewicz, Anna; Olszewski, Włodzimierz T; Tysarowski, Andrzej; Kowalski, Dariusz M; Głogowski, Maciej; Krzakowski, Maciej; Siedlecki, Janusz A; Wągrodzki, Michał; Prochorec-Sobieszek, Monika

    2013-01-01

    The targeted treatment of advanced non-small-cell lung cancer (NSCLC) depends on confirmation of activating somatic EGFR mutation. The aim of the study was to evaluate the incidence of EGFR mutations in NSCLC detected in cytological and histological material and present literature review on European EGFR mutation incidence. 273 patients with confirmed NSCLC were entered into the study: 189 histological, paraffin-embedded materials, 12 fresh and 72 fixed cytological specimens. DNA was extracted from both types of material and the EGFR mutation in exons 18-21 was analyzed by direct sequencing. In addition the EGFR gene copy number in cases with sufficient histological material (110 patients) was evaluated by fluorescent in situ hybridization (FISH) technique. The percentage of EGFR somatic mutations was 10.62%. FISH positive results (amplification or high polysomy of EGFR gene) were identified in 33 patients (30.0%). The strongest clinicopathological correlation with the EGFR mutation was found for histological type (adenocarcinoma; p < 0.01), gender (females; p < 0.01) and FISH positive result (p < 0.05). This is the first, single institution study that estimates the EGFR mutation incidence in the Polish population. Cytological material recovered from fixed preparations and stained with hematoxylin and eosin showed DNA quality comparable to fresh tumor cells and histological samples.

  18. Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer.

    PubMed

    Lococo, Filippo; Paci, Massimiliano; Rapicetta, Cristian; Rossi, Teresa; Sancisi, Valentina; Braglia, Luca; Cavuto, Silvio; Bisagni, Alessandra; Bongarzone, Italia; Noonan, Douglas M; Albini, Adriana; Maramotti, Sally

    2015-08-19

    Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.

  19. Overcoming EGFR T790M and C797S resistance with mutant-selective allosteric inhibitors

    PubMed Central

    Jia, Yong; Yun, Cai-Hong; Park, Eunyoung; Ercan, Dalia; Manuia, Mari; Juarez, Jose; Xu, Chunxiao; Rhee, Kevin; Chen, Ting; Zhang, Haikuo; Palakurthi, Sangeetha; Jang, Jaebong; Lelais, Gerald; DiDonato, Michael; Bursulaya, Badry; Michellys, Pierre-Yves; Epple, Robert; Marsilje, Thomas H.; McNeill, Matthew; Lu, Wenshuo; Harris, Jennifer; Bender, Steven; Wong, Kwok-Kin; Jänne, Pasi A.; Eck, Michael J.

    2016-01-01

    EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib and afatinib are approved treatments for non-small cell lung cancers harboring activating mutations in the EGFR kinase1,2, but resistance arises rapidly, most frequently due to the secondary T790M mutation within the ATP-site of the receptor.3,4 Recently developed mutant-selective irreversible inhibitors are highly active against the T790M mutant5,6, but their efficacy can be compromised by acquired mutation of C797, the cysteine residue with which they form a key covalent bond7. All current EGFR TKIs target the ATP-site of the kinase, highlighting the need for therapeutic agents with alternate mechanisms of action. Here we describe rational discovery of EAI045, an allosteric inhibitor that targets selected drug-resistant EGFR mutants but spares the wild type receptor. A crystal structure shows that the compound binds an allosteric site created by the displacement of the regulatory C-helix in an inactive conformation of the kinase. The compound inhibits L858R/T790M-mutant EGFR with low-nanomolar potency in biochemical assays, but as a single agent is not effective in blocking EGFR-driven proliferation in cells due to differential potency on the two subunits of the dimeric receptor, which interact in an asymmetric manner in the active state8. We observe dramatic synergy of EAI045 with cetuximab, an antibody therapeutic that blocks EGFR dimerization9,10, rendering the kinase uniformly susceptible to the allosteric agent. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by L858R/T790M EGFR and by L858R/T790M/C797S EGFR, a mutant that is resistant to all currently available EGFR TKIs. More generally, our findings illustrate the utility of purposefully targeting allosteric sites to obtain mutant-selective inhibitors. PMID:27251290

  20. Relative sensitivity of fish and mammalian cells to the antibiotic, trimethoprim: cytotoxic and genotoxic responses as determined by neutral red retention, Comet and micronucleus assays.

    PubMed

    Papis, Elena; Davies, Simon J; Jha, Awadhesh N

    2011-01-01

    Relative cytotoxicity and genotoxicity of a widely used antibiotic, trimethoprim (TRIMP) was evaluated under in vitro conditions using rainbow trout gonad-2 (RTG-2) and Chinese hamster ovary-K1 (CHO-K1) cells. Whilst cytotoxicity was determined using neutral red retention (NRR) assay, the genotoxicity was determined using single cell gel electrophoresis or the Comet assay and cytokinesis-block micronucleus (CBMN) assay. For NRR assay, concentration-dependent cytotoxic effect was observed for both the cell lines (estimated EC(50) values: 671.82 ± 21.78 and 611.6 ± 20.4 μg ml(-1) for RTG-2 and CHO-K1 cells, respectively). There was no statistically significant difference between the two cell lines for this assay. For the Comet assay, standard 6 h exposure to TRIMP did not show any positive response for any of the cell types used. However, 48 h exposure to RTG-2 cells showed a concentration-dependent induction of DNA damage (r = 0.86). The highest concentration of TRIMP used (i.e. 100 μg ml(-1)) showed relatively higher DNA damage, compared to ethyl methane sulfonate (EMS; 1 μg ml(-1) or 8 mM), a reference genotoxic agent, used concurrently. In contrast, 24 h exposure time for CHO-K1 cells did not show any concentration-dependent increase for this assay. For MN assay, a significant correlation was found between the MN induction and TRIMP concentration for both the cell lines (RTG-2: r = 0.68; CHO-K1: r = 0.79), although only the highest concentration used showed a significant increase for binucleated (BN) cell with micronuclei (BNMN). The study suggests that whilst the cells of different origin could exhibit similar cytotoxicity, they could display differential genotoxic effects. Furthermore, genotoxic effects of TRIMP are primarily exposure period dependent phenomena and, in addition to inhibiting the action of dihydrofolate reductase, oxidative stress could also contribute for the observed toxic effects, fish cells in general being more sensitive for genotoxic

  1. Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay.

    PubMed

    Sánchez-Fortún, S; Llorente, M T; Castaño, A

    2005-06-01

    A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluorescence reader at room temperature for less than 1h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC(50(48)) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC(50(48)) value, and the results obtained indicate a strain scission factor (SSF) of 0.126+/-0.014, 0.181+/-0.014, 0.217+/-0.013, and 0.245+/-0.013 in cell suspensions, and 0.077+/-0.019, 0.107+/-0.014, 0.151+/-0.014, and 0.202+/-0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023+/-0.009, 0.033+/-0.017, 0.068+/-0.012, and 0.088+/-0.015 in cell suspensions, and 0.033+/-0.010, 0.044+/-0.011, 0.080+/-0.009, and 0.093+/-0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2(1/100 IC(50(48))) and 300(1/10 IC(50(48))) Hg/L of tetrakis

  2. Increased epidermal growth factor receptor (EGFR) expression in malignant mammary phyllodes tumors.

    PubMed

    Tse, Gary M K; Lui, Philip C W; Vong, Joaquim S L; Lau, Kin-Mang; Putti, Thomas C; Karim, Rooshdiya; Scolyer, Richard A; Lee, C-Soon; Yu, Alex M C; Ng, David C H; Tse, Agnes K Y; Tan, Puay-Hoon

    2009-04-01

    Mammary phyllodes tumors are uncommon stromal-epithelial neoplasms, and are divided into benign, borderline malignant and frankly malignant groups on the basis of their histological features. Accumulating evidence shows that epidermal growth factor receptor (EGFR) is involved in the pathogenesis and progression of many malignancies. This study investigated 453 phyllodes tumors (296 benign, 98 borderline, 59 malignant) for EGFR expression using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for gene amplification. The staining was correlated to tumor margin status, degree of malignancy, stromal cellularity, mitotic activity, nuclear pleomorphism and stromal overgrowth. Cases with strong positive IHC staining were selected for FISH. The overall positive rate for EGFR was 16.2% (48/296), 30.6% (30/98) and 56% (33/59) for benign, borderline malignant and frankly malignant phyllodes tumors, respectively. FISH demonstrated egfr gene amplification in 8% of immunohistochemically positive cases. The results of this study provide strong evidence that EGFR overexpression is involved in the pathogenesis of phyllodes tumors, although gene amplification may not be the major underlying mechanism for overexpression.

  3. [Lung adenocarcinoma with concomitant EGFR mutation and ALK rearrangement].

    PubMed

    Caliez, J; Monnet, I; Pujals, A; Rousseau-Bussac, G; Jabot, L; Boudjemaa, A; Leroy, K; Chouaid, C

    2017-05-01

    Among patients with non-small-cell lung cancer, coexistence of EGFR mutation and ALK rearrangement is rare. We describe the clinical features of two patients with this double anomaly. A 62-year-old Caucasian non-smoking woman was diagnosed with cT4N0M0 lung adenocarcinoma. Initial biopsy showed EGFR mutation and ALK rearrangement. She received cisplatin-gemcitabine, followed by 17 months of gemcitabine. Owing to progression, she received erlotinib for 14 months, then paclitaxel for 6 months and finally crizotinib. A partial response was achieved and maintained for 24 months. A 45-year-old Caucasian woman, light smoker, was diagnosed with cT2N3M0 lung adenocarcinoma. Only EGFR mutation was found on initial analysis. She underwent treatment with cisplatin-gemcitabine and thoracic radiotherapy. Progression occurred after 8 months and afatinbib was started. Eight months later, progression was observed with a neoplasic pleural effusion in which tumor cells expressing ALK rearrangement were found. A new FISH analysis was performed on the initial tumor but did not find this rearrangement. Despite a third line of crizotinib, the patient died one month later. The literature shows 45 other cases of these two abnormalities, observed either from the start or during follow-up. EGFR's TKI were almost always given before ALK's TKI. Therapeutic strategy needs to be clarified in cases of double alteration. With regard to the second patient, appearance of ALK rearrangement may constitute a resistance mechanism to EGFR's TKI. Copyright © 2016 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  4. Polymorphisms in genes involved in EGFR-turnover are predictive for cetuximab efficacy in colorectal cancer

    PubMed Central

    Stintzing, Sebastian; Zhang, Wu; Heinemann, Volker; Neureiter, Daniel; Kemmerling, Ralf; Kirchner, Thomas; Jung, Andreas; Folwaczny, Matthias; Yang, Dongyun; Ning, Yan; Sebio, Ana; Stremitzer, Stefan; Sunakawa, Yu; Matsusaka, Satoshi; Yamauchi, Shinichi; Loupakis, Fotios; Cremolini, Chiara; Falcone, Alfredo; Lenz, Heinz-Josef

    2015-01-01

    Transmembrane receptors such as the epidermal growth factor receptor (EGFR) are regulated by their turnover, which is dependent on the ubiquitin-proteasome-system (UPS). We tested in two independent study cohorts whether single nucleotide polymorphisms (SNPs) in genes involved in EGFR turnover predict clinical outcome in cetuximab treated metastatic colorectal cancer patients. The following SNPs involved in EGFR degradation were analyzed in a screening cohort of 108 patients treated with cetuximab in the chemorefractory setting: c-CBL (rs7105971; rs4938637; rs4938638; rs251837), EPS15 (rs17567; rs7308; rs1065754), NAE1 (rs363169; rs363170; rs363172); SH3KBP1 (rs7051590; rs5955820; rs1017874; rs11795873); SGIP1 (rs604737; rs6570808; rs7526812); UBE2M (rs895364; rs895374); UBE2L3 (rs5754216). SNPs showing an association with response or survival were analyzed in BRAF and RAS wild-type samples from the FIRE-3 study. 153 FOLFIRI plus cetuximab treated patients served as validation set, 168 patients of the FOLFIRI plus bevacizumab arm served as controls. EGFR FISH was done in 138 samples to test whether significant SNPs were associated with EGFR expression. UBE2M rs895374 was significantly associated with PFS (logrank-p = 0.005; HR 0.60) within cetuximab treated patients. No association with bevacizumab treated patients (n=168) could be established (p= 0.56, HR: 0.90). rs895374 genotype did not affect EGFR FISH measurements. EGFR recycling is an interesting mechanism of secondary resistance to cetuximab in mCRC. This is the first report suggesting that germline polymorphisms in the degradation process predict efficacy of cetuximab in patients with mCRC. Genes involved in EGFR turnover may be new targets in the treatment of mCRC. PMID:26206335

  5. Development of a monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay for furaltadone metabolite AMOZ in fish and shrimp samples.

    PubMed

    Shen, Yu-Dong; Xu, Zhen-Lin; Zhang, Shi-Wei; Wang, Hong; Yang, Jin-Yi; Lei, Hong-Tao; Xiao, Zhi-Li; Sun, Yuan-Ming

    2012-11-07

    A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC(50) of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography-tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

  6. Simultaneous real-time assay of copper and cadmium ions by infrared photo diode electrode implanted in the muscle of live fish.

    PubMed

    Young Ly, Suw; Keun Kim, Jong

    2009-01-01

    The electrical circuit of an infrared photodiode electrode (IPE) was used in the simultaneous assay of copper and cadmium ions. The electrode's cyclic voltammetry (CV), chronoamperometry and square-wave (SW) stripping voltammetric optimum conditions were examined. Results for 0-160 mg L(-1) and 50-400 microg L(-1) SW Cu(II) Cd(II), the relative standard deviation of 0.158 Cu(II), 0.077 Cd(II) (n = 15) using 20.0 mg L(-1) have been obtained at optimum conditions. The low detection limit (S/N) was attained to be at 14.71 microg L(-1)(2.31 x 10(-7) mol L(-1)) Cu(II) and 18.42 microg L(-1)(1.63 x 10(-7) mol L(-1)) Cd(II). The handmade electrode was implanted deep in the muscle of live fish and interfaced with an electrochemical workstation. Real-time analytical application was performed on the online assay of living tissue as the specimen was moving. The methods are deemed useful in interfaced assay for physiological control, nanodiode fabrication, and in the production of laboratory on a biochip.

  7. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    PubMed

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

    2015-01-01

    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  8. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

    USGS Publications Warehouse

    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

    2016-01-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

  9. Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2.

    PubMed

    Wang, Ying-Nai; Lee, Heng-Huan; Lee, Hong-Jen; Du, Yi; Yamaguchi, Hirohito; Hung, Mien-Chie

    2012-05-11

    Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.

  10. 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

  11. Acquired RAS or EGFR mutations and duration of response to EGFR blockade in colorectal cancer

    PubMed Central

    Van Emburgh, Beth O.; Arena, Sabrina; Siravegna, Giulia; Lazzari, Luca; Crisafulli, Giovanni; Corti, Giorgio; Mussolin, Benedetta; Baldi, Federica; Buscarino, Michela; Bartolini, Alice; Valtorta, Emanuele; Vidal, Joana; Bellosillo, Beatriz; Germano, Giovanni; Pietrantonio, Filippo; Ponzetti, Agostino; Albanell, Joan; Siena, Salvatore; Sartore-Bianchi, Andrea; Di Nicolantonio, Federica; Montagut, Clara; Bardelli, Alberto

    2016-01-01

    Blockade of the epidermal growth factor receptor (EGFR) with the monoclonal antibodies cetuximab or panitumumab is effective in a subset of colorectal cancers (CRCs), but the emergence of resistance limits the efficacy of these therapeutic agents. At relapse, the majority of patients develop RAS mutations, while a subset acquires EGFR extracellular domain (ECD) mutations. Here we find that patients who experience greater and longer responses to EGFR blockade preferentially develop EGFR ECD mutations, while RAS mutations emerge more frequently in patients with smaller tumour shrinkage and shorter progression-free survival. In circulating cell-free tumour DNA of patients treated with anti-EGFR antibodies, RAS mutations emerge earlier than EGFR ECD variants. Subclonal RAS but not EGFR ECD mutations are present in CRC samples obtained before exposure to EGFR blockade. These data indicate that clonal evolution of drug-resistant cells is associated with the clinical outcome of CRC patients treated with anti-EGFR antibodies. PMID:27929064

  12. Radiation effects on cellularity, proliferation and EGFR expression in mouse bladder urothelium.

    PubMed

    Jaal, Jana; Dörr, Wolfgang

    2010-04-01

    This study was designed to determine changes in cell numbers, proliferation (using Ki-67) and EGFR expression in mouse bladder urothelium during the early and late radiation response. Groups of mice were irradiated with a single dose of 20 Gy and assayed 0-360 days later. Urothelial cells were counted. After immunohistochemistry, the absolute and relative numbers of Ki-67(+) and EGFR(+) cells were analyzed. Radiation exposure resulted in a decrease in total urothelial cell numbers to 49% by day 31, with restoration of cellularity by day 180. In contrast, at day 360, an increase in total cell number (143%) was seen. Slightly increased Ki-67 expression was found at days 120 and 180 after treatment, followed by a pronounced elevation at days 240 and 360. Compared to controls, higher EGFR expression was detected up to day 360 after irradiation. A positive correlation was found between total urothelial cells numbers and Ki-67 as well as EGFR expression. Radiation exposure results in an increased urothelial expression of EGFR that precedes urothelial restoration, indicating a contribution of the EGF/EGFR system to urothelial proliferation and differentiation. Further studies are needed to evaluate the impact of EGFR inhibition on radiation effects in the urinary bladder.

  13. Combination of BIBW2992 and ARQ 197 is effective against erlotinib-resistant human lung cancer cells with the EGFR T790M mutation.

    PubMed

    Qu, Geping; Liu, Changting; Sun, Baojun; Zhou, Changxi; Zhang, Zhijian; Wang, Peng

    2014-07-01

    Although the EGFR tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown marked effects against EGFR-mutated lung cancer, patients acquire resistance by various mechanisms, including the EGFR T790M mutation and Met induction, consequently suffering relapse. Thus, novel agents to overcome EGFR-TKI resistance are urgently needed. We aimed to investigate the inhibitory effects of a combination of BIBW2992 (irreversible EGFR inhibitor)/ARQ 197 (MET inhibitor) on the human lung adenocarcinoma cell line H1975. H1975 cells (harboring a T790M mutation in EGFR) were treated with erlotinib, BIBW2992 or ARQ 197 separately or with combinations of erlotinib/ARQ 197 or BIBW2992/ARQ 197. Cell growth, apoptosis and cell cycle distribution were evaluated by MTT assay, Annexin V/propidium iodide (PI) double staining and flow cytometry, respectively. EGFR, MET, AKT, ERK and the respective phosphorylated counterparts were detected by western blot analysis. Pathway-specific knockdown of MET and/or EGFR kinase signaling was achieved by siRNA interference. H1975 cells displayed EGFR and MET activation, and were resistant to erlotinib. The BIBW2992/ARQ 197 combination significantly inhibited growth, induced cell cycle arrest and apoptosis, and altered the phosphorylation of EGFR, MET, AKT and ERK1/2 in the H1975 cells. Phosphorylation of AKT and ERK1/2, downstream effectors of the EGFR and MET pathways, was not affected by the other tested treatments. Finally, knockdown of MET and/or EGFR in the H1975 cells confirmed the enhanced downstream inhibition of both MET and EGFR pathways. Combination of an irreversible EGFR inhibitor and MET inhibitor is effective in controlling H1975 cells with acquired resistance to erlotinib, by a mechanism involving the downregulation of PI3K/AKT and MEK/ERK signaling pathways.

  14. 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.

  15. Screening complex effluents for estrogenic activity with the T47D-KBluc cell bioassay: assay optimization and comparison with in vivo responses in fish.

    PubMed

    Wehmas, Leah C; Cavallin, Jenna E; Durhan, Elizabeth J; Kahl, Michael D; Martinovic, Dalma; Mayasich, Joe; Tuominen, Tim; Villeneuve, Daniel L; Ankley, Gerald T

    2011-02-01

    Wastewater treatment plant (WWTP) effluents can contain estrogenic chemicals, which potentially disrupt fish reproduction and development. The current study focused on the use of an estrogen-responsive in vitro cell bioassay (T47D-KBluc), to quantify total estrogenicity of WWTP effluents. We tested a novel sample preparation method for the T47D-KBluc assay, using powdered media prepared with direct effluent. Results of the T47D-KBluc assay were compared with the induction of estrogen receptor-regulated gene transcription in male fathead minnows (Pimephales promelas) exposed to the same effluents. Effluent samples for the paired studies were collected over the course of three months. According to the T47D-KBluc assay, the effluent estrogenicity ranged from 1.13 to 2.00 ng 17β-estradiol (E2) equivalents/L. Corresponding in vivo studies exposing male fathead minnows to 0, 10, 50, and 100% effluent dilutions demonstrated that exposure to 100% effluent significantly increased hepatic vitellogenin (VTG) and estrogen receptor α subunit transcripts relative to controls. The induction was also significant in males exposed to 250 ng E2/L or 100 ng E2/L. The in vitro and in vivo results support the conclusion that the effluent contains significant estrogenic activity, but there was a discrepancy between in vitro- and in vivo-based E2 equivalent estimates. Our results suggest that the direct effluent preparation method for the T47D-KBluc assay is a reasonable approach to estimate the estrogenicity of wastewater effluent.

  16. Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468

    PubMed Central

    2010-01-01

    Background Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. Methods The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. Results Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies. Conclusion

  17. Role of IKKalpha in the EGFR Signaling Regulation

    DTIC Science & Technology

    2011-09-01

    interaction with EGFR in Golgi apparatus and catalyzes EGFR S1026 phosphorylation. We found that EGFR S1026A possess a stronger tumorgenesis phenotype compare...reveals that both IKK! and EGFR colocaliBed in the Golgi apparatus under EGF stimulation (Fig. 3E). Since EGFR undergo ligand dependent...internaliBation, we hypothesis the physical contact of EGFR and IKK! appears at Golgi apparatus . Given the physical association between IKK! and EGFR, we

  18. Fish multigeneration test with preliminary short-term reproduction assay for estrone using Japanese medaka (Oryzias latipes).

    PubMed

    Nakamura, Ataru; Tamura, Ikumi; Takanobu, Hitomi; Yamamuro, Masumi; Iguchi, Taisen; Tatarazako, Norihisa

    2015-01-01

    The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste.Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l(–1) of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long-term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l(–1). No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l(–1) estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l(–1), suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis–ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis-ovais regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption.

  19. Genotoxicity evaluation of the herbicide Garlon(®) and its active ingredient (triclopyr) in fish (Anguilla anguilla L.) using the comet assay.

    PubMed

    Guilherme, Sofia; Santos, Maria A; Gaivão, Isabel; Pacheco, Mário

    2015-09-01

    Triclopyr-based herbicides are broadly used worldwide for site preparation and forest vegetation management. Thus, following application, these agrochemicals can inadvertently reach the aquatic ecosystems. Garlon(®) is one of the most popular commercial denominations of this group of herbicides, considered as highly toxic to fish, even by its manufacturer. Although DNA is frequently regarded as a target of pesticide toxicity, the genotoxic potential of Garlon(®) to fish remains completely unknown. Hence, the main goal of this study was to evaluate the genotoxicity of Garlon(®) and its active ingredient (triclopyr), clarifying the underlying mechanisms. Therefore, the comet assay, implemented as the standard procedure, with an extra step involving DNA lesion-specific repair enzymes (formamidopyrimidine DNA glycosylase and endonuclease III), was used to identify DNA damage in blood cells of Anguilla anguilla L. Short-term exposures (1 and 3 days) to Garlon(®) and triclopyr were carried out, adopting environmentally realistic concentrations (67.6 and 270.5 µg L(-1) Garlon(®) and 30 and 120 µg L(-1) triclopyr). The results concerning the nonspecific DNA damage proved the risk of the herbicide Garlon(®) and its active ingredient triclopyr in both tested concentrations and exposure lengths. In addition, the higher genotoxic potential of the formulation, in comparison with the active ingredient, was demonstrated. When the additional breaks corresponding to net enzyme-sensitive sites were considered, none of the conditions revealed significant levels of oxidative damage. This identification of the genotoxic properties of triclopyr-based herbicides to fish highlights the need to develop less hazardous formulations, as well as the adoption of mitigation measures related to the application of these agrochemicals in the framework of forestry and agriculture sustainable management.

  20. Short-term fish reproduction assays with methyl tertiary butyl ether with zebrafish and fathead minnow: Implications for evaluation of potential for endocrine activity.

    PubMed

    Mihaich, Ellen; Erler, Steffen; Le Blanc, Gerald; Gallagher, Sean

    2015-09-01

    The authors report on short-term fish reproduction assays in zebrafish and fathead minnow conducted to examine the potential for methyl tertiary butyl ether (MTBE) to cause effects on the endocrine system. Both studies were performed under good laboratory practice and in accordance with Organisation for Economic Co-operation and Development and US Environmental Protection Agency test guidelines. The results of the first study demonstrated that exposure to a high test concentration (147 mg/L) of MTBE impaired reproductive output of female zebrafish, evident by a reduction in fecundity. Based on the endpoints evaluated in the present study however, there was no supporting evidence to indicate that this effect was caused by disruption of or interaction with the endocrine system. In the second study, fathead minnows exposed to a wider but lower range of test concentrations showed no effects on any reproductive parameter of male or female fish, at the maximum recommended testing concentration of 100 mg/L (62 mg/L measured). The results of these 2 guideline studies indicate that MTBE does not interact with the hypothalamic-pituitary-gonadal axis of zebrafish or fathead minnow. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of SETAC.

  1. Development of a Sandwich Hybridization Assay to Rapidly Detect and Quantify Harmful Algal Bloom (hab) Species Linked with Coastal Fish Kills and Public Health Concerns

    NASA Astrophysics Data System (ADS)

    Greenfield, D.; Jones, W. J.; Mortensen, R.; Doll, C.; Reed, M.

    2016-02-01

    Molecular probe technologies have enabled more rapid and specific phytoplankton identification and enumeration compared to traditional technologies, such as microscopy. The method considered for this study is sandwich hybridization assay (SHA), a fast, efficient, and economic approach to detecting and quantifying plankton species. SHA employs two DNA probes, capture and signal, to detect ribosomal RNA (rRNA) sequences. The reaction entails an anti-digoxygenin horse-radish peroxidase (HRP) conjugate to which a HRP substrate is applied. The result is a colorimetric reaction, and the resultant absorbance can be used to estimate organism abundances. This project focuses on two raphidophyte HAB species that have been responsible for declining water quality and fish kills globally, but are particularly problematic in states along the southeastern (US) coast, Fibrocapsa japonica and Chattonella subsalsa. These species produce recurrent, dense blooms annually, and are directly linked with fish kills. Work presented here also includes the domoic acid (DA) producing diatom Pseudo-nitzschia pseudodelicatissima because DA has been associated with pigmy and dwarf sperm whale strandings in the southeastern US, and blooms of this genus are frequently reported in regional waters. Here we present findings from field samplings and multiple blooms (2014-2015) of all three species that occurred in South Carolina (US) and regional waters. We also provide updates on the development, validation, and quantification of SHA applications for each of these HAB species.

  2. Antioxidant activity of Cod (Gadus morhua) protein hydrolysates: in vitro assays and evaluation in 5% fish oil-in-water emulsion.

    PubMed

    Farvin, K H Sabeena; Lystbæk Andersen, Lisa; Hauch Nielsen, Henrik; Jacobsen, Charlotte; Jakobsen, Greta; Johansson, Inez; Jessen, Flemming

    2014-04-15

    Cod protein hydrolysates were fractionated according to the molecular mass into three fractions of >5 kDa, 3-5 kDa and <3 kDa using an ultrafiltration membrane system. The antioxidative activity of the crude hydrolysates and the fractions were investigated, both in in vitro assays (DPPH radical scavenging activity, reducing power, Fe²⁺ chelating activity and inhibition of lipid oxidation in liposome model system), and in 5% fish oil-in-water emulsions. The <3 kDa fractions had very good radical scavenging activity, Fe²⁺ chelating activity and reducing power while the fraction 3-5 kDa resulted in higher protection against oxidation in the liposome model system. When tested in 5% oil-in-water emulsions, all the fractions, including the crude protein hydrolysate, were able to protect fish oil against oxidation in an iron induced oxidation system. However, none of the peptide fractions were effective in preventing tocopherol loss and showed no tocopherol sparing property. Volatile oxidation products showed an interaction between the aldehydes and the protein/peptides added in the emulsions, and this needs further investigation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors.

    PubMed

    Choi, Yun Jung; Rho, Jin Kyung; Jeon, Byung-suk; Choi, Su Jin; Park, Su Cheol; Lee, Seung Sook; Kim, Hye-Ryoun; Kim, Cheol Hyeon; Lee, Jae Cheol

    2010-07-01

    H1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer. Three human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots. H1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors alpha-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR. Combined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.

  4. 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.

  5. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

    SciTech Connect

    Zhou, Wenjun; Ercan, Dalia; Chen, Liang; Yun, Cai-Hong; Li, Danan; Capelletti, Marzia; Cortot, Alexis B.; Chirieac, Lucian; Iacob, Roxana E.; Padera, Robert; Engen, John R.; Wong, Kwok-Kin; Eck, Michael J.; Gray, Nathanael S.; Jänne, Pasi A.

    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 potent 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.

  6. Specific PCR assays to determine bovine, porcine, fish and plant origin of gelatin capsules of dietary supplements.

    PubMed

    Lee, Jae-Hwang; Kim, Mi-Ra; Jo, Cheon-Ho; Jung, Yoo-Kyung; Kwon, Kisung; Kang, Tae Sun

    2016-11-15

    Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle, chicken muscle, egg, fish, milk and kidney.

    PubMed

    Wang, S; Xu, B; Zhang, Y; He, J X

    2009-05-01

    A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10μg/kg in kidney and 20μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015μg/kg, and the detection limits were 1.5μg/kg in pig muscle and 6μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r(2)) which was greater than 0.9.

  8. A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

    PubMed

    Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

    2015-04-07

    This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

  9. A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals.

    PubMed

    Ankley, Gerald T; Jensen, Kathleen M

    2014-11-01

    The fish short-term reproduction assay (FSTRA) is a key component of the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), which uses a weight-of-evidence analysis based on data from several assays to identify the potential for chemicals to act as agonists or antagonists of the estrogen or androgen receptors (ER and AR), or inhibitors of steroidogenic enzymes. The FSTRA considers a variety of mechanistic and apical responses in 21-d exposures with the fathead minnow (Pimephales promelas), including plasma steroid and vitellogenin (VTG; egg yolk protein) concentrations, secondary sex characteristics, gonad size and histopathology, and egg production. Although the FSTRA initially was described several years ago, recent data generation associated with implementation of the EDSP highlighted the need for more formal guidance regarding evaluation of information from the assay. The authors describe a framework for interpretation of FSTRA data relative to perturbation of endocrine pathways of concern to the EDSP. The framework considers end points individually and as suites of physiologically related responses relative to pathway identification. Sometimes changes in single end points can be highly diagnostic (e.g., induction of VTG in males by ER agonists, production of male secondary sex characteristics in females by AR agonists); in other instances, however, multiple, related end points are needed to reliably assess pathway perturbation (e.g., AR antagonism, steroid synthesis inhibition). In addition to describing an interpretive framework, the authors demonstrate its practical utility using publicly available FSTRA data for a wide range of known and hypothesized endocrine-disrupting chemicals. Environ Toxicol Chem 2014;33:2529-2540. Published 2014 Wiley Periodicals Inc., on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America. Published 2014 Wiley Periodicals Inc., on

  10. Dacomitinib Beats Gefitinib for EGFR(+) NSCLC.

    PubMed

    2017-08-01

    The irreversible EGFR inhibitor dacomitinib reduced the chance of lung cancer progression compared with an older, first-generation EGFR inhibitor, gefitinib, in a phase III trial. As reported at the 2017 American Society of Clinical Oncology Annual Meeting, the experimental drug also increased toxicity, which could limit its use, especially with a safer, more effective third-generation EGFR inhibitor not far behind in the development pipeline. ©2017 American Association for Cancer Research.

  11. [Therapeutic biomarkers of EGFR-TKI].

    PubMed

    Seike, Masahiro; Gemma, Akihiko

    2012-11-01

    Non-small cell lung cancer(NSCLC)patients with activating mutations of the epidermal growth factor receptor(EGFR)gene have shown a dramatic response to EGFR tyrosine kinase inhibitors(EGFR-TKI)such as gefitinib and erlotinib. EGFR activating mutations including exon 19 deletion and exon 21 L858R are recognized as markers ofthe sensitivity to EGFR-TKI therapy in NSCLC. However, the emergence of acquired resistance is virtually inevitable, thus limiting improvement in patient outcomes. Several acquired-resistance mechanisms and candidates, including exon 20 T790M secondary mutation, MET amplification, a high-level of HGF expression, PTEN downregulation, FAS-NF-κB pathway activation, epithelial-mesenchymal transition, and conversion to small cell lung cancer, have been identified. Understanding the mechanisms of acquired resistance to EGFR-TKI, followed by the development of molecular targeted drugs that can overcome the resistance, could serve as an important advance for targeting EGFR, which is activated in NSCLC. Further studies should be performed to clarify other mechanisms associated with the acquired resistance to EGFR-TKI. In this review, we summarize recent advances in the therapeutic biomarkers to EGFR-TKI.

  12. Comprehensive Genomic Profiling Identifies Frequent Drug-Sensitive EGFR Exon 19 Deletions in NSCLC not Identified by Prior Molecular Testing.

    PubMed

    Schrock, Alexa B; Frampton, Garrett M; Herndon, Dana; Greenbowe, Joel R; Wang, Kai; Lipson, Doron; Yelensky, Roman; Chalmers, Zachary R; Chmielecki, Juliann; Elvin, Julia A; Wollner, Mira; Dvir, Addie; -Gutman, Lior Soussan; Bordoni, Rodolfo; Peled, Nir; Braiteh, Fadi; Raez, Luis; Erlich, Rachel; Ou, Sai-Hong Ignatius; Mohamed, Mohamed; Ross, Jeffrey S; Stephens, Philip J; Ali, Siraj M; Miller, Vincent A

    2016-07-01

    Reliable detection of drug-sensitive activating EGFR mutations is critical in the care of advanced non-small cell lung cancer (NSCLC), but such testing is commonly performed using a wide variety of platforms, many of which lack rigorous analytic validation. A large pool of NSCLC cases was assayed with well-validated, hybrid capture-based comprehensive genomic profiling (CGP) at the request of the individual treating physicians in the course of clinical care for the purpose of making therapy decisions. From these, 400 cases harboring EGFR exon 19 deletions (Δex19) were identified, and available clinical history was reviewed. Pathology reports were available for 250 consecutive cases with classical EGFR Δex19 (amino acids 743-754) and were reviewed to assess previous non-hybrid capture-based EGFR testing. Twelve of 71 (17%) cases with EGFR testing results available were negative by previous testing, including 8 of 46 (17%) cases for which the same biopsy was analyzed. Independently, five of six (83%) cases harboring C-helical EGFR Δex19 were previously negative. In a subset of these patients with available clinical outcome information, robust benefit from treatment with EGFR inhibitors was observed. CGP identifies drug-sensitive EGFR Δex19 in NSCLC cases that have undergone prior EGFR testing and returned negative results. Given the proven benefit in progression-free survival conferred by EGFR tyrosine kinase inhibitors in patients with these alterations, CGP should be considered in the initial presentation of advanced NSCLC and when previous testing for EGFR mutations or other driver alterations is negative. Clin Cancer Res; 22(13); 3281-5. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Multifunctionalization of cetuximab with bioorthogonal chemistries and parallel EGFR profiling of cell-lines using imaging, FACS and immunoprecipitation approaches.

    PubMed

    Reschke, Melanie L; Uprety, Rajendra; Bodhinayake, Imithri; Banu, Matei; Boockvar, John A; Sauve, Anthony A

    2014-12-01

    The ability to derivatize antibodies is currently limited by the chemical structure of antibodies as polypeptides. Modern methods of bioorthogonal and biocompatible chemical modifications could make antibody functionalization more predictable and easier, without compromising the functions of the antibody. To explore this concept, we modified the well-known anti-epidermal growth factor receptor (EGFR) drug, cetuximab (Erbitux®), with 5-azido-2-nitro-benzoyl (ANB) modifications by optimization of an acylation protocol. We then show that the resulting ANB-cetuximab can be reliably modified with dyes (TAMRA and carboxyrhodamine) or a novel synthesized cyclooctyne modified biotin. The resulting dye- and biotin-modified cetuximabs were then tested across several assay platforms with several cell lines including U87, LN229, F98EGFR, F98WT and HEK293 cells. The assay platforms included fluorescence microscopy, FACS and biotin-avidin based immunoprecipitation methods. The modified antibody performs consistently in all of these assay platforms, reliably determining relative abundances of EGFR expression on EGFR expressing cells (LN229 and F98EGFR) and failing to cross react with weak to negative EGFR expressing cells (U87, F98WT and HEK293). The ease of achieving diverse and assay relevant functionalizations as well as the consequent rapid construction of highly correlated antigen expression data sets highlights the power of bioorthogonal and biocompatible methods to conjugate macromolecules. These data provide a proof of concept for a multifunctionalization strategy that leverages the biochemical versatility and antigen specificity of antibodies.

  14. Met Receptor Acts Uniquely for Survival and Morphogenesis of EGFR-Dependent Normal Mammary Epithelial and Cancer Cells

    PubMed Central

    Bersani, Francesca; Quaglino, Elena; Martignani, Eugenio; Baratta, Mario

    2012-01-01

    Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs. PMID:23028720

  15. Kinetic determination of vitellogenin induction in the epidermis of cyprinid and perciform fishes: Evaluation of sensitive enzyme-linked immunosorbent assays.

    PubMed

    Allner, Bernhard; Hennies, Mark; Lerche, Cristiano F; Schmidt, Thomas; Schneider, Klaus; Willner, Marco; Stahlschmidt-Allner, Petra

    2016-12-01

    Induction of vitellogenin (VTG) in male and immature fish is a standardized endpoint in endocrine-disruption testing. To establish a nondestructive swab sampling method, VTG induction in the epidermis of Cypriniformes and Perciformes species was investigated. Both VTG and estrogen receptor genes are expressed in epidermal cells. Immunoaffinity and mass fingerprint analyses show induction of identical VTG peptides in liver and epidermis. Induction of VTG by estradiol (E2) and bisphenol A (BPA) in the epidermis was quantified with homolog enzyme-linked immunosorbent assays. Initial values in juveniles and males were below 1 ng VTG/mL extraction buffer. Exposure to E2 led to values between 200 ng/mL and 4600 ng/mL in cyprinids and between 10 ng/mL and 81 ng/mL in perciforms. Exposure to BPA increased VTG amounts to 250 ng/mL in fathead minnows, 1360 ng/mL in goldfish, 100 ng/mL in zebrafish, and 12 ng/mL in bluegills. Serum VTG contents demonstrated a similar dose-response pattern in the epidermis and the blood. These results show that VTG induction may be reliably assessed in the skin mucus of fishes, demonstrating the suitability of this biological sample for investigating estrogenic activity in compliance with Organisation for Economic Co-operation and Development standard protocols. This broadens the perspectives in toxicological screening and environmental monitoring, reducing the number of tested animals and minimizing harmful effects for animals, allowing for follow-up of individual induction profiles. Environ Toxicol Chem 2016;35:2916-2930. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

  16. Increased incidence of micronuclei assessed with the micronucleus assay and the fluorescence in situ hybridization (FISH) technique in peripheral blood lymphocytes of nurses exposed to nitrous oxide.

    PubMed

    Lewińska, D; Stepnik, M; Krajewski, W; Arkusz, J; Stańczyk, M; Wrońska-Nofer, T

    2005-03-07

    It has been postulated that exposure to nitrous oxide and halogenated anaesthetics is associated with various adverse health effects such as neurological and reproductive abnormalities or impairment of hepatic functions. In spite of the quite well known genotoxic effects of exposure to nitrous oxide in vivo, the mechanisms of these effects are still not clear. The aim of this study was to assess the frequency of micronuclei and to identify the type of chromosomal damage (clastogenic or aneugenic) in peripheral blood lymphocytes of operating-room nurses exposed to nitrous oxide. The study group comprised 46 women working at departments where the concentration of nitrous oxide ranged from 14 to 2308 mg/m3. The control population was composed of 28 women employed in the same hospitals but in non-surgical departments. The clastogenic/aneugenic effect of nitrous oxide was evaluated in lymphocytes using the standard micronucleus (MN) assay in combination with the fluorescence in situ hybridization (FISH) technique with pancentromeric probes. The results show a significant increase of the MN frequency in lymphocytes of exposed nurses compared with the control group (4.36+/-2.23 versus 9.02+/-4.67). The multiple regression analysis revealed a statistically significant relationship (p=0.0009) between MN frequency and exposure status, indicating that the level of exposure was the main factor affecting chromosomal damage. As assessed by FISH analysis, the overall frequencies of centromere-positive MN in the control and exposed groups were 43 and 49%, respectively. The increase observed in the exposed group may suggest a slight, statistically insignificant pro-aneugenic effect of exposure to nitrous oxide.

  17. Application of enzyme-linked immunosorbent assay for measurement of polychlorinated biphenyls from hydrophobic solutions: Extracts of fish and dialysates of semipermeable membrane devices: Chapter 26

    USGS Publications Warehouse

    Zajicek, James L.; Tillitt, Donald E.; Huckins, James N.; Petty, Jimmie D.; Potts, Michael E.; Nardone, David A.

    1996-01-01

    Determination of PCBs in biological tissue extracts by enzyme-linked immunosorbent assays (ELISAs) can be problematic, since the hydrophobic solvents used for their extraction and isolation from interfering biochemicals have limited compatibility with the polar solvents (e.g. methanol/water) and the immunochemical reagents used in ELISA. Our studies of these solvent effects indicate that significant errors can occur when microliter volumes of PCB containing extracts, in hydrophobic solvents, are diluted directly into methanol/water diluents. Errors include low recovery and excess variability among sub-samples taken from the same sample dilution. These errors are associated with inhomogeneity of the dilution, which is readily visualized by the use of a hydrophobic dye, Solvent Blue 35. Solvent Blue 35 is also used to visualize the evaporative removal of hydrophobic solvent and the dissolution of the resulting PCB/dye residue by pure methanol and 50% (v/v) methanol/water, typical ELISA diluents. Evaporative removal of isooctane by an ambient temperature nitrogen purge with subsequent dissolution in 100% methanol gives near quantitative recovery of model PCB congeners. We also compare concentrations of total PCBs from ELISA (ePCB) to their corresponding concentrations determined from capillary gas chromatography (GC) in selected fish sample extracts and dialysates of semipermeable membrane device (SPMD) passive samplers using an optimized solvent exchange procedure. Based on Aroclor 1254 calibrations, ePCBs (ng/mL) determined in fish extracts are positively correlated with total PCB concentrations (ng/mL) determined by GC: ePCB = 1.16 * total-cPCB - 5.92. Measured ePCBs (ng/3 SPMDs) were also positively correlated (r2 = 0.999) with PCB totals (ng/3 SPMDs) measured by GC for dialysates of SPMDs: ePCB = 1.52 * total PCB - 212. Therefore, this ELISA system for PCBs can be a rapid alternative to traditional GC analyses for determination of PCBs in extracts of biota or in

  18. Low concentrations of caffeine induce asymmetric cell division as observed in vitro by means of the CBMN-assay and iFISH.

    PubMed

    Hatzi, Vasiliki I; Karakosta, Maria; Barszczewska, Katarzyna; Karachristou, Ioanna; Pantelias, Gabriel; Terzoudi, Georgia I

    2015-11-01

    The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism

  19. 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.

  20. EGFR — EDRN Public Portal

    Cancer.gov

    From NCBI Gene: The protein encoded by this gene is a transmembrane glycoprotein that is a member of the protein kinase superfamily. This protein is a receptor for members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor. Binding of the protein to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. Mutations in this gene are associated with lung cancer. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found for this gene. [provided by RefSeq, Jul 2010

  1. Pan-HER-An antibody mixture targeting EGFR, HER2 and HER3 abrogates preformed and ligand-induced EGFR homo- and heterodimers.

    PubMed

    Ellebaek, Sofie; Brix, Susanne; Grandal, Michael; Lantto, Johan; Horak, Ivan D; Kragh, Michael; Poulsen, Thomas Tuxen

    2016-11-01

    The human epidermal growth factor receptor (HER)-family is involved in development of many epithelial cancers. Therefore, HER-family members constitute important targets for anti-cancer therapeutics such as monoclonal antibodies (mAbs). A limitation to the success of single HER-targeting mAbs is development of acquired resistance through mechanisms such as alterted receptor dimerization patterns and dependencies. Pan-HER is a mixture of six mAbs simultaneously targeting epidermal growth factor receptor (EGFR), HER2 and HER3 with two mAbs against each receptor. Pan-HER has previously demonstrated broader efficacy than targeting single or dual receptor combinations also in resistant settings. In light of this broad efficacy, we decided to investigate the effect of Pan-HER compared with single HER-targeting with single and dual mAbs on HER-family cross-talk and dimerization focusing on EGFR. The effect of Pan-HER on cell proliferation and HER-family receptor degradation was superior to treatment with single mAbs targeting either single receptor, and similar to targeting a single receptor with two non-overlapping antibodies. Furthermore, changes in EGFR-dimerization patterns after treatment with Pan-HER were investigated by in situ proximity ligation assay and co-immunoprecipitation, demonstrating that Pan-HER and the EGFR-targeting mAb mixture efficiently down-regulate basal EGFR homo- and heterodimerization in two tested cell lines, whereas single mAbs had limited effects. Pan-HER and the EGFR-targeting mAb mixture also blocked EGF-binding and thereby ligand-induced changes in EGFR-dimerization levels. These results suggest that Pan-HER reduces the cellular capability to switch HER-dependency and dimerization pattern in response to treatment and thus hold promise for future clinical development of Pan-HER in resistant settings.

  2. Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs

    PubMed Central

    Zhang, Chengjuan; Wei, Bing; Li, Peng; Yang, Ke; Wang, Zhizhong; Ma, Jie; Guo, Yongjun

    2017-01-01

    Objective Epidermal growth factor receptor (EGFR) specific mutations have been known to improve survival of patients with non-small-cell lung carcinoma (NSCLC). However, whether there are any changes of EGFR mutations after targeted therapy and its clinical significance is unclear. This study was to identify the status of EGFR mutations after targeted therapy and predict the prognostic significance for NSCLC patients. Methods A total of forty-five (45) NSCLC patients who received EGFR-TKI therapy were enrolled. We identified the changes of EGFR mutations in plasma ctDNA by Amplification Refractory Mutation System (ARMS) PCR technology. Results In the 45 cases of NSCLC with EGFR mutations, the EGFR mutation status changed in 26 cases, in which, 12 cases (26.7%) from positive to negative, and 14 cases (31.1%) from T790M mutation negative to positive after TKI targeted therapy. The T790M occurance group had a shorter Progression -Free-Survival (PFS) than the groups of EGFR mutation undetected and EGFR mutation turned out to have no change after EGFR-TKI therapy (p < 0.05). Conclusions According to this study, it’s necessary to closely monitor EGFR mutations during follow-up to predict the prognosis of NSCLC patients who are to receive the TKI targeted therapy. PMID:28333951

  3. Genetic variability in EGFR, Src and HER2 and risk of colorectal adenoma and cancer

    PubMed Central

    Poole, Elizabeth M; Curtin, Karen; Hsu, Li; Kulmacz, Richard J; Duggan, David J; Makar, Karen W; Xiao, Liren; Carlson, Christopher S; Slattery, Martha L; Caan, Bette J; Potter, John D; Ulrich, Cornelia M

    2011-01-01

    The EGFR signaling pathway is involved in carcinogenesis at multiple sites, particularly colorectal cancer, and is a target of colorectal cancer chemotherapy. EGFR signaling is linked to pro-carcinogenic mechanisms, including cell proliferation, survival, angiogenesis, and more recently prostaglandin synthesis. Genetic variability in this pathway has not yet been studied in relation to colorectal carcinogenesis. In three case-control studies of colorectal adenoma (n=485 cases/578 controls), colon cancer (n=1424 cases/1780 controls) and rectal cancer (n=583 cases/775 controls), we investigated associations between candidate SNPs, tagSNPs and haplotypes in EGFR signaling (EGFR, Src, and HER2) and risk. We also examined associations with tumor subtypes: TP53 and KRAS2 mutations, CpG island methylator phenotype, and microsatellite instability. All three studies were genotyped using an identical Illumina GoldenGate assay, allowing thorough investigation of genetic variability across stages and locations of colorectal neoplasia. The EGFR tagSNP 142572T>C (rs3752651) CC genotype was associated with a suggested increased risk for both colon (OR: 1.40; 95% CI: 1.00-1.96; p-trend=0.04) and rectal cancer (OR: 1.39; 95% CI: 0.81-2.41; p-trend=0.65). In tumor subtype analyses, the association was limited to TP53-mutated colon tumors. Using the Chatterjee 1 df Tukey test to assess gene-gene interactions, we observed a statistically significant (p<0.01) interaction between SNPs in EGFR and Src for colorectal adenoma risk. The association with EGFR 142572 should be investigated in additional studies and the significant gene-gene interaction between EGFR and Src in relation to adenoma risk suggests that these two genes are jointly affecting early stages in colorectal carcinogenesis and requires further follow-up. PMID:22199994

  4. Prospective molecular marker analyses of EGFR and KRAS from a randomized, placebo-controlled study of erlotinib maintenance therapy in advanced non-small-cell lung cancer.

    PubMed

    Brugger, Wolfram; Triller, Nadja; Blasinska-Morawiec, Maria; Curescu, Stefan; Sakalauskas, Raimundas; Manikhas, Georgy Moiseevich; Mazieres, Julien; Whittom, Renaud; Ward, Carol; Mayne, Karen; Trunzer, Kerstin; Cappuzzo, Federico

    2011-11-01

    The phase III, randomized, placebo-controlled Sequential Tarceva in Unresectable NSCLC (SATURN; BO18192) study found that erlotinib maintenance therapy extended progression-free survival (PFS) and overall survival in patients with advanced non-small-cell lung cancer (NSCLC) who had nonprogressive disease following first-line platinum-doublet chemotherapy. This study included prospective analysis of the prognostic and predictive value of several biomarkers. Mandatory diagnostic tumor specimens were collected before initiating first-line chemotherapy and were tested for epidermal growth factor receptor (EGFR) protein expression by using immunohistochemistry (IHC), EGFR gene copy number by using fluorescent in situ hybridization (FISH), and EGFR and KRAS mutations by using DNA sequencing. An EGFR CA simple sequence repeat in intron 1 (CA-SSR1) polymorphism was evaluated in blood. All 889 randomly assigned patients provided tumor samples. EGFR IHC, EGFR FISH, KRAS mutation, and EGFR CA-SSR1 repeat length status were not predictive for erlotinib efficacy. A profound predictive effect on PFS of erlotinib relative to placebo was observed in the EGFR mutation-positive subgroup (hazard ratio [HR], 0.10; P < .001). Significant PFS benefits were also observed with erlotinib in the wild-type EGFR subgroup (HR, 0.78; P = .0185). KRAS mutation status was a significant negative prognostic factor for PFS. This large prospective biomarker study found that patients with activating EGFR mutations derive the greatest PFS benefit from erlotinib maintenance therapy. No other biomarkers were predictive for outcomes with erlotinib, although the study was not powered for clinical outcomes in biomarker subgroups other than EGFR IHC-positive [corrected]. KRAS mutations were prognostic for reduced PFS. The study demonstrated the feasibility of prospective tissue collection for biomarker analyses in NSCLC.

  5. Brief report: Clinical implications of variant ALK FISH rearrangement patterns

    PubMed Central

    Gao, Xin; Sholl, Lynette M.; Nishino, Mizuki; Heng, Jennifer; Jänne, Pasi A.; Oxnard, Geoffrey R.

    2015-01-01

    Introduction Break-apart fluorescence in situ hybridization (FISH) is the FDA-approved assay for detecting anaplastic lymphoma kinase (ALK) rearrangements in non-small cell lung cancer (NSCLC), identifying patients who can gain dramatic benefit from ALK kinase inhibitors. Assay interpretation can be technically challenging, and either splitting of the 5′ and 3′ probes or loss of the 5′ probe constitute rearrangement. We hypothesized that there may be clinical differences depending upon rearrangement pattern on FISH. Methods An IRB-approved database of NSCLC patients at Dana-Farber Cancer Institute was queried for ALK rearrangement. Clinical characteristics and response to crizotinib were reviewed. Immunohistochemistry (IHC) and targeted next-generation sequencing (NGS) were obtained when available. Results Of 1,614 NSCLC patients with ALK testing, 82 (5.1%) patients had ALK rearrangement by FISH: 30 with split signals, 25 with 5′ deletion, and 27 with details unavailable. Patients with 5′ deletion were older (p=0.01) and tended to have more extensive smoking histories (p=0.08). IHC was positive for ALK rearrangement in all 27 patients with FISH split signals, while 3 of 21 patients with FISH 5′ deletion had negative IHC (p=0.05). Targeted NGS on 2 of 3 cases with discordant FISH and IHC results did not identify ALK rearrangement, instead finding driver mutations in EGFR and KRAS. Patients with 5′ deletion treated with crizotinib had a smaller magnitude of tumor response (p=0.03). Conclusions Patients with 5′ deletion on ALK FISH harbor features less typical of ALK-rearranged tumors, potentially indicating that some cases with this variant are false-positives. Corroborative testing with IHC or NGS may be beneficial. PMID:26536196

  6. [Effect of EGFR-TKI on lymphangiogenesis of lung cancer with EGFR mutation].

    PubMed

    Cai, Minghui; Yang, Xinying; Jiang, Baohong; Teng, Fukang; Pan, Yan; Mao, Feng; Shen-Tu, Yang

    2014-12-01

    This study aims to explore the effect of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKIs) on the lymphangiogenesis of lung cancer with EGFR mutation, as well as to determine the function of EGFR targeted therapy in relation to the inhibition of lymphangiogenesis during lung cancer treatment. The EGFR double mutant lung cancer cell line NCI-H1975 is used to construct lung cancer xenograft models. The models are divided into two groups: the solvent control group and the EGFR-TKI treatment group. Each group includes five mice. The inhibitory effect of EGFR-TKI on the growth of transplanted tumors was observed. Immunohistochemical method and lymphatic endothelium specific antibody D2-40 were used in the experiment to observe the influence of EGFR-TKI on lymphangiogenesis in lung cancer. The weight and relative volume of tumors in the EGFR-TKI treated group were less than those in the solvent control group. The average lymphatic vessel density of EGFR-TKI-treated mice was 6.44 per case. This value was 10.70 per case in the solvent control group. Lower density of lymphangiogenesis was found in the EGFR-TKI treated group (P=0.023). The area and longest diameter of neonatal lymphatic vessel of the EGFR-TKI treated group were less than those of the solvent control group. Moreover, EGFR-TKI exhibited no significant effect on the invasion of tumor cells into the lymphatic vessel (P=0.519). EGFR-TKI can inhibit lymphangiogenesis in EGFR mutant lung cancer while suppressing vessel diameter and expansion area.

  7. Pharmacology of novel small-molecule tubulin inhibitors in glioblastoma cells with enhanced EGFR signalling.

    PubMed

    Phoa, Athena F; Browne, Stephen; Gurgis, Fadi M S; Åkerfeldt, Mia C; Döbber, Alexander; Renn, Christian; Peifer, Christian; Stringer, Brett W; Day, Bryan W; Wong, Chin; Chircop, Megan; Johns, Terrance G; Kassiou, Michael; Munoz, Lenka

    2015-12-15

    We recently reported that CMPD1, originally developed as an inhibitor of MK2 activation, primarily inhibits tubulin polymerisation and induces apoptosis in glioblastoma cells. In the present study we provide detailed pharmacological investigation of CMPD1 analogues with improved molecular properties. We determined their anti-cancer efficacy in glioblastoma cells with enhanced EGFR signalling, as deregulated EGFR often leads to chemoresistance. Eight analogues of CMPD1 with varying lipophilicity and basicity were synthesised and tested for efficacy in the cell viability assay using established glioblastoma cell lines and patient-derived primary glioblastoma cells. The mechanism of action for the most potent analogue 15 was determined using MK2 activation and tubulin polymerisation assays, together with the immunofluorescence analysis of the mitotic spindle formation. Apoptosis was analysed by Annexin V staining, immunoblotting analysis of bcl-2 proteins and PARP cleavage. The apoptotic activity of CMPD1 and analogue 15 was comparable across glioblastoma cell lines regardless of the EGFR status. Primary glioblastoma cells of the classical subtype that are characterized by enhanced EGFR activity were most sensitive to the treatment with CMPD1 and 15. In summary, we present mechanism of action for a novel small molecule tubulin inhibitor, compound 15 that inhibits tubulin polymerisation and mitotic spindle formation, induces degradation of anti-apoptotic bcl-2 proteins and leads to apoptosis of glioblastoma cells. We also demonstrate that the enhanced EGFR activity does not decrease the efficacy of tubulin inhibitors developed in this study.

  8. Accurate detection and quantification of the fish viral hemorrhagic septicemia virus (VHSv) with a two-color fluorometric real-time PCR assay

    USDA-ARS?s Scientific Manuscript database

    Viral Hemorrhagic Septicemia virus (VHSv) is one of the world's most serious fish pathogens, infecting > 80 marine, freshwater, and estuarine fish species from Eurasia and North America. A novel and especially virulent strain - IVb - appeared in the Great Lakes in 2003, killed many game fish species...

  9. ReadMax—a novel reading and scoring approach for EGFR gene copy number to predict therapeutic benefit of erlotinib treatment in EGFR wild‐type non‐small‐cell lung cancer

    PubMed Central

    Soulières, Denis; Klughammer, Barbara

    2015-01-01

    Abstract EGFR mutation testing is now well established as a means of selecting the optimal first‐line therapy for patients with advanced non‐small‐cell lung cancer (NSCLC). However, deciding on the correct treatment for EGFR wild‐type NSCLC remains a challenge. EGFR fluorescence in‐situ hybridization (FISH) testing of gene copy number has been a promising marker, but has provided mixed results despite attempts to standardize the reading and scoring process. The novel ReadMax reading and scoring system focuses on the most aberrant cells, to identify oncogene addiction, rather than taking a representative reading as in the Colorado method. The methodology was developed using historical samples from the TRUST and MERIT studies, followed by re‐reading of the samples from the SATURN trial. Analysis of samples using the ReadMax methodology revealed that progression‐free survival (PFS) and overall survival (OS) were improved in patients with ReadMax FISH‐positive (RM FISH+) tumours, compared with those whose tumours were not RM FISH+: PFS hazard ratios (HRs) were 0.52 for RM FISH+ versus 0.93 for not RM FISH+; OS HRs were 0.69 and 0.92, respectively. For PFS, HR for RM FISH+ versus not RM FISH+ in the SATURN erlotinib group was 0.53 (p = 0.003). The PFS and OS results were also similar in the EGFR wild‐type population (PFS HRs were 0.63 and 0.96; OS HRs were 0.61 and 0.84, respectively), although amplification of the EGFR gene in patients with EGFR wild‐type disease was not found to be predictive of treatment outcomes, which was unexpected but not unprecedented. KRAS status was not found to affect outcomes. Further experience is required to refine the ReadMax methodology and fully establish its validity and robustness. In conclusion, the ReadMax scoring system to identify patients with EGFR FISH‐positive NSCLC is a promising technique, which could improve treatment options and outcomes for patients with advanced NSCLC, in particular for EGFR

  10. The Cobas® EGFR Mutation Test v2 assay.

    PubMed

    Brown, Paul

    2016-02-01

    Paul Brown speaks to Gemma Westcott, Commissioning Editor: Paul Brown has served as the Head of Roche Molecular Diagnostics at Roche Diagnostics Corporation since February 2010 having previously held a variety of positions within Roche Pharma. After completing his doctorate in organic chemistry he was awarded a post-doctoral fellowship at the California Institute of Technology in Pasadena, CA, USA, but soon returned to his native UK to join Roche Pharma Research. Paul's first post within Roche was as group leader, doing drug discovery and making new small molecule drugs, but later moved into the business part of the company. Since then, he has enjoyed roles such as lifecycle leader for brands such as Tamiflu(®) and Xenical, vice president of sales and marketing of the pharmaceutical division and most recently general manager of Roche Pharma, Sweden.

  11. EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules.

    PubMed

    Ellina, Maria-Ioanna; Bouris, Panagiotis; Aletras, Alexios J; Theocharis, Achilleas D; Kletsas, Dimitris; Karamanos, Nikos K

    2014-08-01

    ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression. Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules. EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF-EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF-EGFR network. The EGF-EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2. The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF-EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Newly Designed Break-Apart and ASPL-TFE3 Dual-Fusion FISH Assay Are Useful in Diagnosing Xp11.2 Translocation Renal Cell Carcinoma and ASPL-TFE3 Renal Cell Carcinoma

    PubMed Central

    Chen, Xiancheng; Yang, Yang; Gan, Weidong; Xu, Linfeng; Ye, Qing; Guo, Hongqian

    2015-01-01

    Abstract The diagnosis of Xp11.2 translocation renal cell carcinoma (tRCC), which relies on morphology and immunohistochemistry (IHC), is often either missed in the diagnosis or misdiagnosed. To improve the accuracy of diagnosis of Xp11.2 tRCC and ASPL-TFE3 renal cell carcinoma (RCC), we investigated newly designed fluorescence in situ hybridization (FISH) probes (diagnostic accuracy study). Based on the genetic characteristics of Xp11.2 tRCC and the ASPL-TFE3 RCC, a new break-apart TFE3 FISH probe and an ASPL-TFE3 dual-fusion FISH probe were designed and applied to 65 patients with RCC who were <45 years old or showed suspicious microscopic features of Xp11.2 tRCC in our hospital. To test the accuracy of the probes, we further performed reverse transcriptase–polymerase chain reaction (PCR) on 8 cases for which frozen tissues were available. Among the 65 cases diagnosed with RCC, TFE3 IHC was positive in 24 cases. Twenty-two cases were confirmed as Xp11.2 tRCC by break-apart TFE3 FISH, and 6 of these cases were further diagnosed as ASPL-TFE3 RCC by ASPL-TFE3 dual-fusion FISH detection. Importantly, reverse transcriptase–PCR showed concordant results with the results of FISH assay in the 8 available frozen cases. The break-apart and ASPL-TFE3 dual-fusion FISH assay can accurately detect the translocation of the TFE3 gene and ASPL-TFE3 fusion gene and can thus serve as a valid complementary method for diagnosing Xp11.2 tRCC and ASPL-TFE3 RCC. PMID:25984679

  13. Molecular confirmation of t(6;11)(p21;q12) renal cell carcinoma in archival paraffin-embedded material using a break-apart TFEB FISH assay expands its clinicopathologic spectrum.

    PubMed

    Argani, Pedram; Yonescu, Raluca; Morsberger, Laura; Morris, Kerry; Netto, George J; Smith, Nathan; Gonzalez, Nilda; Illei, Peter B; Ladanyi, Marc; Griffin, Constance A

    2012-10-01

    A subset of renal cell carcinomas (RCCs) is characterized by t(6;11)(p21;q12), which results in fusion of the untranslated Alpha (MALAT1) gene to the TFEB gene. Only 21 genetically confirmed cases of t(6;11) RCCs have been reported. This neoplasm typically demonstrates a distinctive biphasic morphology, comprising larger epithelioid cells and smaller cells clustered around basement membrane material; however, the full spectrum of its morphologic appearances is not known. The t(6;11) RCCs differ from most conventional RCCs in that they consistently express melanocytic immunohistochemical (IHC) markers such as HMB45 and Melan A and the cysteine protease cathepsin K but are often negative for epithelial markers such as cytokeratins. TFEB IHC has been proven to be useful to confirm the diagnosis of t(6;11) RCCs in archival material, because native TFEB is upregulated through promoter substitution by the gene fusion. However, IHC is highly fixation dependent and has been proven to be particularly difficult for TFEB. A validated fluorescence in situ hybridization (FISH) assay for molecular confirmation of the t(6;11) RCC in archival formalin-fixed, paraffin-embedded material has not been previously reported. We report herein the development of a break-apart TFEB FISH assay for the diagnosis of t(6;11)(p21;q12) RCCs. We validated the assay on 4 genetically confirmed cases and 76 relevant expected negative control cases and used the assay to report 8 new cases that expand the clinicopathologic spectrum of t(6;11) RCCs. An additional previously reported TFEB IHC-positive case was confirmed by TFEB FISH in 46-year-old archival material. In conclusion, TFEB FISH is a robust, clinically validated assay that can confirm the diagnosis of t(6;11) RCC in archival material and should allow a more comprehensive clinicopathologic delineation of this recently recognized neoplastic entity.

  14. Dominant Enhancers of Egfr in Drosophila Melanogaster: Genetic Links between the Notch and Egfr Signaling Pathways

    PubMed Central

    Price, J. V.; Savenye, E. D.; Lum, D.; Breitkreutz, A.

    1997-01-01

    The Drosophila epidermal growth factor receptor (EGFR) is a key component of a complex signaling pathway that participates in multiple developmental processes. We have performed an F(1) screen for mutations that cause dominant enhancement of wing vein phenotypes associated with mutations in Egfr. With this screen, we have recovered mutations in Hairless (H), vein, groucho (gro), and three apparently novel loci. All of the E(Egfr)s we have identified show dominant interactions in transheterozygous combinations with each other and with alleles of N or Su(H), suggesting that they are involved in cross-talk between the N and EGFR signaling pathways. Further examination of the phenotypic interactions between Egfr, H, and gro revealed that reductions in Egfr activity enhanced both the bristle loss associated with H mutations, and the bristle hyperplasia and ocellar hypertrophy associated with gro mutations. Double mutant combinations of Egfr and gro hypomorphic alleles led to the formation of ectopic compound eyes in a dosage sensitive manner. Our findings suggest that these E(Egfr)s represent links between the Egfr and Notch signaling pathways, and that Egfr activity can either promote or suppress Notch signaling, depending on its developmental context. PMID:9383058

  15. EGFR inhibitor and chemotherapy combinations for acquired TKI resistance in EGFR-mutant NSCLC models.

    PubMed

    Laurila, Niina; Koivunen, Jussi P

    2015-07-01

    Acquired resistance to EGFR TKIs is the most important limiting factor for treatment efficiency in EGFR-mutant NSCLC. Although the continuation of EGFR TKI beyond disease progression in combination with chemotherapy is often suggested as a strategy for treating acquired resistance, the optimal treatment sequence for EGFR TKI and chemotherapy is unknown. In the current work, NSCLC cell lines PC9ER, H1975 and HCC827GR, representing the acquired TKI resistance genotypes (T790M, cMET), were exposed to a chemotherapeutic agent, cisplatin or paclitaxel, in combination with EGFR TKIs (erlotinib, WZ4002) in vitro and analysed for cytotoxicity and apoptotic response. The result showed that all the combinations of EGFR TKIs with a chemotherapeutic agent tested had a synergistic effect on cytotoxicity and increased the apoptotic response. The sequences involving a chemotherapeutic agent concurrently with an EGFR TKI or preceding it were the most efficient strategies. Our in vitro models suggest that the combination of an EGFR TKI and chemotherapy is beneficial in cases of acquired EGFR TKI resistance. Furthermore, the sequence of chemotherapy followed by EGFR TKI is significantly more powerful than the reversed order, so that an intercalated approach is likely to be the most active strategy in clinical use and ought to be tested in a randomized clinical trial.

  16. Investigational EGFR-targeted therapies in HNSCC

    PubMed Central

    Cassell, Andre; Grandis, Jennifer R.

    2010-01-01

    Importance of the Field The epidermal growth factor receptor (EGFR) is an established therapeutic target in head and neck squamous cell carcinoma (HNSCC). The EGFR-targeting monoclonal antibody cetuximab (™Erbitux) was FDA-approved for use in HNSCC in 2006. The molecular basis for the efficacy of an antibody approach compared with inhibition of EGFR tyrosine kinase function using small molecule inhibitors, or downregulation of protein expression via antisense strategies remains incompletely understood. Areas covered in this review A literature search was performed to identify studies elucidating mechanisms of action of several approaches to targeting EGFR in HNSCC (monoclonal antibodies, tyrosine kinase inhibitors, antisense approaches, and ligand toxin conjugates). What the reader will gain Monoclonal antibodies decrease tumor growth via receptor endocytosis and recruitment of host immune defenses. Tyrosine kinase inhibitors bind to the ATP binding pocket of the tyrosine kinase domain, inhibiting signaling. Antisense approaches decrease EGFR expression with high specificity although drug delivery remains problematic. Ligand-toxin conjugates facilitate the entry of toxin and the ADP-ribosylation of the ribosome, thereby inhibiting translation. Take home message Elucidation mechanisms by which these different strategies inhibit EGFR function may enhance the development of more effective treatments for HNSCC and enable prospective identification of individuals who will benefit from EGFR inhibition. PMID:20415598

  17. Correlation of EGFR, pEGFR and p16INK4 expressions and high risk HPV infection in HIV/AIDS-related squamous cell carcinoma of conjunctiva

    PubMed Central

    2014-01-01

    Background Squamous cell carcinoma of conjunctiva has increased tenfold in the era of HIV/AIDS. The disease pattern has also changed in Africa, affecting young persons, with peak age-specific incidence of 30-39 years, similar to that of Kaposi sarcoma, a well known HIV/AIDS defining neoplasm. In addition, the disease has assumed more aggressive clinical course. The contributing role of exposure to high risk HPV in the development of SCCC is still emerging. Objective The present study aimed to investigate if immunohistochemical expressions of EGFR, pEGFR and p16, could predict infection with high risk HPV in HIV-related SCCC. Methods FFPE tissue blocks of fifty-eight cases diagnosed on hematoxylin and eosin with SCCC between 2005-2011, and subsequently confirmed from medical records to be HIV positive at the department of human pathology, UoN/KNH, were used for the study. Immunohistochemistry was performed to assess the expressions of p16INK4A, EGFR and pEGFR. This was followed with semi-nested PCR based detection and sequencing of HPV genotypes. The sequences were compared with the GenBank database, and data analyzed for significant statistical correlations using SPSS 16.0. Ethical approval to conduct the study was obtained from KNH-ERC. Results Out of the fifty-eight cases of SCCC analyzed, twenty-nine (50%) had well differentiated (grade 1), twenty one (36.2%) moderately differentiated (grade 2) while eight (13.8%) had poorly differentiated (grade 3) tumours. Immunohistochemistry assay was done in all the fifty eight studied cases, of which thirty nine cases (67.2%) were positive for p16INK4A staining, forty eight cases (82.8%) for EGFR and fifty one cases (87.9%) showed positivity for p-EGFR. HPV DNA was detected in 4 out of 40 SCCC cases (10%) in which PCR was performed, with HPV16 being the only HPV sub-type detected. Significant statistical association was found between HPV detection and p16INK4 (p=0.000, at 99% C.I) and EGFR (p=0.028, at 95% C.I) expressions

  18. Emergence of RET rearrangement co-existing with activated EGFR mutation in EGFR-mutated NSCLC patients who had progressed on first- or second-generation EGFR TKI.

    PubMed

    Klempner, Samuel J; Bazhenova, Lyudmila A; Braiteh, Fadi S; Nikolinakos, Petros G; Gowen, Kyle; Cervantes, Claudia M; Chmielecki, Juliann; Greenbowe, Joel R; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M; Ou, Sai-Hong Ignatius

    2015-09-01

    The gatekeeper mutation T790M mutation is the responsible for the majority of the resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with EGFR-mutated non-small cell lung cancer (NSCLC). Other previously described resistance mechanisms include HER2 amplification, MET amplification, PIK3CA mutation, epithelial-mesenchymal transition (EMT), small cell transformation have also been identified. However other resistance mechanisms remains to be discovered. Hybrid-capture based comprehensive genomic profiling (CGP) was performed on pre- and post-EGFR TKI progression EGFR-mutated NSCLC tumor samples during routine clinical care. We identify two paired pre- and post-EGFR TKI progression EGFR-mutated NSCLC patient tumor samples where both post EGFR TKI samples harbored in-frame CCDC6-RET rearrangements but not in the pre-EGFR TKI tumor samples. Furthermore analysis of the clinical database revealed one additional NCOA4-RET rearrangement co-existing with activated EGFR mutation in an EGFR-mutated NSCLC patient who had progressed on afatinib. None of the known resistance mechanisms to EGFR TKI including EGFR T790M, EGFR amplification, HER2 amplification, MET amplification, PIK3CA mutation, BRAF mutation, EMT or small cell transformation was identified in the three post progression samples that now harbored RET rearrangements. This is the first report of RET rearrangement co-existing with activated EGFR mutations in EGFR-mutated patients who had progressed on either first- or second generation EGFR TKI. As such, RET rearrangement may serve as a potential resistance mechanism to EGFR TKI in EGFR-mutated NSCLC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Whacking a mole-cule: clinical activity and mechanisms of resistance to third generation EGFR inhibitors in EGFR mutated lung cancers with EGFR-T790M.

    PubMed

    Costa, Daniel B; Kobayashi, Susumu S

    2015-12-01

    Epidermal growth factor receptor (EGFR) mutations, especially EGFR-exon 19 deletions and EGFR-L858R, are the most frequent actionable genomic events in lung adenocarcinomas. Tumors arise due to constitutively activated EGFR signaling and are susceptible to EGFR tyrosine kinase inhibitors (TKIs). First generation EGFR TKIs (gefitinib and erlotinib) and the second generation EGFR TKI afatinib are approved worldwide. Although targeted therapies against EGFR mutants induce dramatic initial responses, acquired resistance (through multiple biological mechanisms) to erlotinib, gefitinib and afatinib emerges within the first 1-2 years of continued monotherapy. EGFR-T790M accounts for more than half of acquired resistance to first or second generation EGFR TKIs by modifying ATP affinity and drug binding kinetics. Two new studies have shown that two covalent pyrimidine inhibitors-AZD9291 and rociletinib of EGFR-T790M (i.e., third generation EGFR TKIs) shown remarkable clinical activity in patients with acquired resistance to erlotinib, gefitinib and afatinib when the tumor carries EGFR-T790M in conjunction with an activating mutation. However, and regrettably, acquired resistance to these third generation EGFR TKIs has already been reported in preclinical models and clinical specimens; such as a tertiary mutation at EGFR-C797S that prevents covalent binding of EGFR TKIs. The experience with sequential EGFR TKI monotherapy highlights tumor heterogeneity and adaptability (i.e., relentless game of whack-a-mole played between TKIs and cancer), and will help shape future clinical development of novel combinatory approaches to manage EGFR mutated lung adenocarcinomas.

  20. Evaluation of EGFR as a prognostic and diagnostic marker for head and neck squamous cell carcinoma patients

    PubMed Central

    Polanska, Hana; Raudenska, Martina; Hudcová, Kristyna; Gumulec, Jaromir; Svobodova, Marketa; Heger, Zbynek; Fojtu, Michaela; Binkova, Hana; Horakova, Zuzana; Kostrica, Rom; Adam, Vojtech; Kizek, Rene; Masarik, Michal

    2016-01-01

    Approximately 90% of all head and neck tumors are squamous cell carcinomas. The overall survival of patients with head and neck squamous cell carcinoma (HNSCC) is low (≤50%). A non-invasive marker of disease progression is sorely required. The present study focused on the plasmatic levels of epidermal growth factor receptor (EGFR) in HNSCC patients (N=92) compared with healthy (N=29) and diabetic [type 2 diabetes mellitus (T2DM); N=26] controls. Enzyme-linked immunosorbent assay using antibodies against the extracellular region of EGFR (L25-S645) was performed. No significant changes were observed between diabetic and healthy controls. However, there were significantly higher EGFR plasma levels in HNSCC patients compared with both control groups (P=0.001 and 0.005, respectively). Receiver operating characteristic curve analysis identified a sensitivity of 76.09%, a specificity of 67.27% and an area under curve of 0.727 for this comparison. No significant association was observed between EGFR plasma levels and tumor stage, tumor grade, lymph node or distant metastasis occurrence, smoking habit or hypertension. However, the presence of human papillomavirus infection and T2DM in HNSCC patients had borderline effect on the plasma EGFR levels. Survival analysis revealed no significant influence of plasmatic EGFR levels on the overall and disease-specific survival of HNSCC patients. In conclusion, EGFR plasma levels appear to be a relatively promising diagnostic, but poor prognostic, HNSCC marker. PMID:27602151

  1. NF-κB acts downstream of EGFR in regulating low dose cadmium induced primary lung cell proliferation.

    PubMed

    Kundu, Subhadip; Sengupta, Suman; Bhattacharyya, Arindam

    2013-12-01

    Apart from cytotoxicity cadmium has no special attributes towards cell's physiological function. The role of cadmium with respect to cell growth is still under debate. Mitogen activated protein kinase and Ca(2+)/calmodulin dependent protein kinase dependent pathways are the two elaborately studied concerning cadmium induced cell proliferation. Low concentration of cadmium chloride (2.5 μM) was applied to mice primary lung epithelial cells and cell proliferation was measured both by cell cycle analysis and Brdu incorporation assay. Effects of differential dose of cadmium chloride on lung epithelial cells were evaluated morphologically by atomic force microscopy. RT-PCR and western blot altogether corroborated the specific signalling pathways concerning cadmium induced lung cell proliferation. Cadmium induced lung epithelial cells which over-expressed EGFR, were transfected with siEGFR, revealed downstream molecules and RNAi induced EGFR silencing. Use of siEGFR effectively prevents expression of proinflammatory and cell proliferative markers. Moreover N-acetyl cysteine and ascorbic acid mediated inhibition of EGFR and downstream signalling molecules indicate the involvement of reactive oxygen species. Exposure to low concentration of cadmium promotes the growth of primary mice lung epithelial cell by EGFR signalling. We have also transfected the primary lung epithelial cell with siRNA against the regulatory subunit of nuclear factor-κB (NF-κB) and the data shows that cadmium induced lung cell proliferation is the effect of EGFR mediated NF-κB activation.

  2. Novel Selective and Potent EGFR Inhibitor that Overcomes T790M-Mediated Resistance in Non-Small Cell Lung Cancer.

    PubMed

    Li, Yanxia; Song, Zhendong; Jin, Yue; Tang, Zeyao; Kang, Jian; Ma, Xiaodong

    2016-11-02

    Treating patients suffering from EGFR mutant non-small cell lung cancer (NSCLC) with first-generation EGFR tyrosine kinase inhibitors (EGFR TKI) provides excellent response rates. However, approximately 60% of all patients ultimately develop drug resistance due to a second T790M EGFR TKI mutation. In this study, we report the novel molecule N-(3-((5-chloro-2-(4-((1-morpholino)methyl)phenylamino)-4-pyrimidinyl)amino)phenyl)acrylamide (DY3002) to preferentially inhibit the EGFR T790M mutant (EGFR(T790M)) (IC50 = 0.71 nM) over wild-type EGFR (IC50 = 448.7 nM) in kinase assays. Compared to rociletinib (SI = 21.4) and osimertinib (SI = 40.9), it significantly increased selectivity (SI = 632.0) against EGFR(T790M) over wild-type EGFR. Furthermore, in cell-based tests, DY3002, with an IC50 value of 0.037 μM, exhibited enhanced inhibitory potency against H1975 cells. Moreover, AO/EB and DAPI staining assays as well as flow cytometer analyses indicated that DY3002 possesses superior biological properties compared to alternatives. In addition, a rat oral glucose tolerance test revealed that treatment with high drug doses (50 mg/kg) of DY3002 did not result in hyperglycemia, suggesting a reduction of side effects in NSCLC patients will be achievable relative to established EGFR inhibitors. In summary, our findings indicate DY3002 as a promising preclinical candidate for effective treatment of patients with EGFR(T790M)-mutated NSCLC.

  3. Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)

    PubMed Central

    Nitta, Hiroaki; Hauss-Wegrzyniak, Beatrice; Lehrkamp, Megan; Murillo, Adrian E; Gaire, Fabien; Farrell, Michael; Walk, Eric; Penault-Llorca, Frederique; Kurosumi, Masafumi; Dietel, Manfred; Wang, Lin; Loftus, Margaret; Pettay, James; Tubbs, Raymond R; Grogan, Thomas M

    2008-01-01

    Background Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. Methods The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Results Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO

  4. Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

    PubMed

    Sakuma, Yuji; Matsukuma, Shoichi; Nakamura, Yoshiyasu; Yoshihara, Mitsuyo; Koizume, Shiro; Sekiguchi, Hironobu; Saito, Haruhiro; Nakayama, Haruhiko; Kameda, Yoichi; Yokose, Tomoyuki; Oguni, Sachiko; Niki, Toshiro; Miyagi, Yohei

    2013-10-01

    Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.

  5. Role of IKK-alpha in EGFR Signaling Regulation

    DTIC Science & Technology

    2013-09-01

    IKKα forms a specific interaction with EGFR in Golgi apparatus and catalyzes EGFR S1026 phosphorylation. We found that EGFR S1026A possess a stronger...other mechanisms (6, 7) or biological functions of EGFR that have yet to be discovered may have important roles in breast cancer. Overexpression of...factor, plays an important role in many cancer types. The modification patterns of EGFR are critical for its function and the understanding of these

  6. 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.

  7. Co-inhibition of EGFR and IGF1R synergistically impacts therapeutically on adrenocortical carcinoma

    PubMed Central

    Lian, Jianpo; Wang, Xiaojing; Ning, Guang; Wang, Weiqing; Zhu, Yu

    2016-01-01

    Purpose Adrenocortical carcinoma (ACC) is a rare tumor with very poor prognosis and no effective treatment. The aim of this study was to explore a novel therapy co-targeting EGFR and IGF1R in vitro and vivo. Methods The expression of EGFR and IGF1R were evaluated in a series of adrenocortical tumors by immunohistochemistry. Cell viability of ACC cell lines H295R and SW13 were determined by MTT assay after treatment with the combination of EGFR inhibitor Erlotinib and IGF1R inhibitor NVP-AEW541. Apoptosis was assessed by flow cytometry. The mechanism within intracellular signaling pathways was analyzed by Western blot. Mice bearing human ACC xenografts were treated with Erlotinib and NVP-AEW541, and the effects on tumour growth were assessed. Results Our results show a significant over-expression of EGFR (66.67%) and IGF1R (80.0%) in ACC. Besides, the co-overexpression of EGFR and IGF1R was seen in 8/15 ACCs, as compared with ACAs (P<0.05). Erlotinib and NVP-AEW541 significantly inhibited cell viability and induced apoptosis by blocking phosphorylation of MEK/ERK and AKT, respectively. Meanwhile, we found that single inhibition of IGF1R induced compensatory activation of MEK/ERK, leading to sustained activation of mTOR, which represent as aggregation of EGFR and IGF1R downstream components. More importantly, the combination of Erlotinib and NVP-AEW541 enhances anti-tumour efficacy compared to treatment with either agent alone or to untreated control in vitro and vivo. Conclusions In conclusion, coinhibition therapy targeting EGFR and IGF1R may be considerable for treatment of ACC in the future. PMID:27105537

  8. MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab

    PubMed Central

    2014-01-01

    Background Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. Methods miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. Results In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. Conclusions miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy. PMID:24650032

  9. Androgen Receptor Promotes Tamoxifen Agonist Activity by Activation of EGFR in ERα-Positive Breast Cancer

    PubMed Central

    Ciupek, Andrew; Rechoum, Yassine; Gu, Guowei; Gelsomino, Luca; Beyer, Amanda R.; Brusco, Lauren; Covington, Kyle R.; Tsimelzon, Anna; Fuqua, Suzanne A. W.

    2015-01-01

    Purpose Tamoxifen (Tam) resistance represents a significant clinical problem in estrogen receptor (ER) -positive breast cancer. We previously showed that decreased expression of Rho guanine nucleotide dissociation inhibitor (Rho GDI), a negative regulator of the Rho GTPase pathway, is associated with Tam resistance. We now discover that androgen receptor (AR) is overexpressed in cells with decreased Rho GDI and seek to determine AR’s contribution to resistance. Methods We engineered ER -positive cell lines with stable knock-down (KD) of Rho GDI (KD cells). Resistance mechanisms were examined using microarray profiling, protein-interaction studies, growth and reporter gene assays, and Western blot analysis combined with a specific AR antagonist and other signaling inhibitors. Results Tam-resistant tumors and cell lines with low Rho GDI levels exhibited upregulated AR expression. Microarray of Rho GDI KD cells indicated that activation of EGFR and ER was associated with Tam treatment. When AR levels were elevated interaction between AR and EGFR was detected. Constitutive and Tam-induced phosphorylation of EGFR and ERK1/2 was blocked by the AR antagonist Enzalutamide, suggesting that AR-mediated EGFR activation was a mechanism of resistance in these cells. Constitutive ERα phosphorylation and transcriptional activity was inhibited by Enzalutamide and the EGFR inhibitor gefitinib, demonstrating that AR-mediated EGFR signaling activated ER. Tam exhibited agonist activity in AR over-expressing cells, stimulating ERα transcriptional activity and proliferation, which was blocked by Enzalutamide and gefitinib. Conclusions We describe a novel model of AR-mediated Tam resistance through activation of EGFR signaling leading to ER activation in ER -positive cells with low expression of Rho GDI. PMID:26487496

  10. Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2.

    PubMed

    Rexer, Brent N; Ghosh, Ritwik; Narasanna, Archana; Estrada, Mónica Valeria; Chakrabarty, Anindita; Song, Youngchul; Engelman, Jeffrey A; Arteaga, Carlos L

    2013-10-01

    Mutations in receptor tyrosine kinase (RTK) genes can confer resistance to receptor-targeted therapies. A T798M mutation in the HER2 oncogene has been shown to confer resistance to the tyrosine kinase inhibitor (TKI) lapatinib. We studied the mechanisms of HER2-T798M-induced resistance to identify potential strategies to overcome that resistance. HER2-T798M was stably expressed in BT474 and MCF10A cells. Mutant cells and xenografts were evaluated for effects of the mutation on proliferation, signaling, and tumor growth after treatment with combinations of inhibitors targeting the EGFR/HER2/HER3/PI3K axis. A low 3% allelic frequency of the T798M mutant shifted 10-fold the IC50 of lapatinib. In mutant-expressing cells, lapatinib did not block basal phosphorylation of HER2, HER3, AKT, and ERK1/2. In vitro kinase assays showed increased autocatalytic activity of HER2-T798M. HER3 association with PI3K p85 was increased in mutant-expressing cells. BT474-T798M cells were also resistant to the HER2 antibody trastuzumab. These cells were sensitive to the pan-PI3K inhibitors BKM120 and XL147 and the irreversible HER2/EGFR TKI afatinib but not the MEK1/2 inhibitor CI-1040, suggesting continued dependence of the mutant cells on ErbB receptors and downstream PI3K signaling. BT474-T798M cells showed increased expression of the EGFR ligands EGF, TGFα, amphiregulin, and HB-EGF. Addition of the EGFR neutralizing antibody cetuximab or lapatinib restored trastuzumab sensitivity of BT474-T798M cells and xenografts, suggesting that increased EGFR ligand production was causally associated with drug resistance. Simultaneous blockade of HER2 and EGFR should be an effective treatment strategy against HER2 gene-amplified breast cancer cells harboring T798M mutant alleles. ©2013 AACR.

  11. 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.

  12. 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

  13. 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.

  14. Screening for EGFR Mutations in Patients with Head and Neck Cancer Treated with Gefitinib on a Compassionate-Use Program: A Hellenic Cooperative Oncology Group Study

    PubMed Central

    Murray, Samuel; Bobos, Mattheos; Angouridakis, Nikolaos; Nikolaou, Angelos; Linardou, Helena; Razis, Evangelia; Fountzilas, George

    2010-01-01

    Background and Aim. EGFR is commonly expressed in cancers of the head and neck (H and N), and anti-EGFR agents have demonstrated improvements in outcomes (TTP and OS). The aim of this study was to determine EGFR gene status in H and N cancer patients treated with gefitinib and to correlate mutational status with clinico-pathological data and response. Patients and Methods. Patients with histologically confirmed H and N cancer having failed prior treatment for advanced disease entered this compassionate-use-program. Nineteen patients received gefitinib. EGFR expression was assessed by IHC, gene copy number by FISH, and mutation analysis was conducted for EGFR (18-21), KRAS, BRAF (V600E), and HER-2 exon 20. An additional TKI naive cohort of 73 patients was also screened. Results. Mutations were detected in 6/19 patients (3× EGFR, 1× KRAS, and 2× HER2-exon 20). There were no significant differences in TTP or OS for patients with somatic EGFR mutations. No BRAF mutations were detected. Conclusions. The incidence of EGFR mutations in H and N cancer in this study was 5.3%. No statistically relevant correlations between mutation or gene gain and response or survival were observed. Due to the limited number of patients and low incidence of genetic aberrations in the genes analyzed, additional studies are warranted. PMID:21274259

  15. Kaempferol inhibits cell proliferation and glycolysis in esophagus squamous cell carcinoma via targeting EGFR signaling pathway.

    PubMed

    Yao, Shihua; Wang, Xiaowei; Li, Chunguang; Zhao, Tiejun; Jin, Hai; Fang, Wentao

    2016-08-01

    Antitumor activity of kaempferol has been studied in various tumor types, but its potency in esophagus squamous cell carcinoma is rarely known. Here, we reported the activity of kaempferol against esophagus squamous cell carcinoma as well as its antitumor mechanisms. Results of cell proliferation and colony formation assay showed that kaempferol substantially inhibited tumor cell proliferation and clone formation in vitro. Flow cytometric analysis demonstrated that tumor cells were induced G0/G1 phase arrest after kaempferol treatment, and the expression of protein involved in cell cycle regulation was dramatically changed. Except the potency on cell proliferation, we also discovered that kaempferol had a significant inhibitory effect against tumor glycolysis. With the downregulation of hexokinase-2, glucose uptake and lactate production in tumor cells were dramatically declined. Mechanism studies revealed kaempferol had a direct effect on epidermal growth factor receptor (EGFR) activity, and along with the inhibition of EGFR, its downstream signaling pathways were also markedly suppressed. Further investigations found that exogenous overexpression of EGFR in tumor cells substantially attenuated glycolysis suppression induced by kaempferol, which implied that EGFR also played an important role in kaempferol-mediated glycolysis inhibition. Finally, the antitumor activity of kaempferol was validated in xenograft model and kaempferol prominently restrained tumor growth in vivo. Meanwhile, dramatic decrease of EGFR activity and hexokinase-2 expression were observed in kaempferol-treated tumor tissue, which confirmed these findings in vitro. Briefly, these studies suggested that kaempferol, or its analogues, may serve as effective candidates for esophagus squamous cell carcinoma management.

  16. Apigenin induces apoptosis and impairs head and neck carcinomas EGFR/ErbB2 signaling.

    PubMed

    Masuelli, Laura; Marzocchella, Laura; Quaranta, Alessandro; Palumbo, Camilla; Pompa, Giorgio; Izzi, Valerio; Canini, Antonella; Modesti, Andrea; Galvano, Fabio; Bei, Roberto

    2011-01-01

    The development of head and neck squamous cell carcinomas (HNSCCs) is a multistep process progressing from precancerous lesions to highly malignant tumors. A critical role in HNSCCs development and progression is played by EGFR family members including EGFR and ErbB2. The aim of this study was to investigate the effect of apigenin, a low molecular weight flavonoid contained in fruits and vegetables, on growth and survival and on EGFR/ErbB2 signaling in cell lines derived from HNSCCs of the tongue (CAL-27, SCC-15) or pharynx (FaDu). Using sulforhodamine B assay, FACS analysis and activated caspase-3 detection by immunofluorescence, we here demonstrate that apigenin dose-dependently inhibits survival and induces apoptosis of HNSCC cells. Further, by performing western blotting with antibodies specific for phosphorylated EGFR, ErbB2, Erk1/2 and Akt we demonstrate that apigenin reduces ligand-induced phosphorylation of EGFR and ErbB2 and impairs their downstream signaling. On the whole, our results suggest that apigenin properties might be exploited for chemoprevention and/or therapy of head and neck carcinomas.

  17. GALNT6 expression enhances aggressive phenotypes of ovarian cancer cells by regulating EGFR activity.

    PubMed

    Lin, Tzu-Chi; Chen, Syue-Ting; Huang, Min-Chuan; Huang, John; Hsu, Chia-Lang; Juan, Hsueh-Fen; Lin, Ho-Hsiung; Chen, Chi-Hau

    2017-03-28

    Ovarian cancer is the most lethal of the gynecologic malignancies. N-acetylgalactosaminyltransferase 6 (GALNT6), an enzyme that mediates the initial step of mucin type-O glycosylation, has been reported to regulate mammary carcinogenesis. However, the expression and role of GALNT6 in ovarian cancer are still unclear. Here we showed that high GALNT6 expression correlates with increased recurrence, lymph node metastasis, and chemoresistance in ovarian endometrioid and clear cell carcinomas; and higher GALNT6 levels are significantly associated with poorer patient survivals. GALNT6 knockdown with two independent siRNAs significantly suppressed viability, migration, and invasion of ovarian cancer cells. Using phospho-RTK array and Western blot analyses, we identified EGFR as a critical target of GALNT6. GALNT6 knockdown decreased phosphorylation of EGFR, whereas GALNT6 overexpression increased the phosphorylation. Lectin pull-down assays with Vicia villosa agglutinin (VVA) indicated that GALNT6 was able to modify O-glycans on EGFR. Moreover, the GALNT6-enhanced invasive behavior was significantly reversed by erlotinib, an EGFR inhibitor. Our results suggest that GALNT6 expression is associated with poor prognosis of ovarian cancer and enhances the aggressive behavior of ovarian cancer cells by regulating EGFR activity.

  18. High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsies

    PubMed Central

    Do, Hongdo; Krypuy, Michael; Mitchell, Paul L; Fox, Stephen B; Dobrovic, Alexander

    2008-01-01

    Background Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. Methods We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. Results All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. Conclusion This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings. PMID:18495026

  19. A peptide antigen derived from EGFR T790M is immunogenic in non-small cell lung cancer

    PubMed Central

    OFUJI, KAZUYA; TADA, YOSHITAKA; YOSHIKAWA, TOSHIAKI; SHIMOMURA, MANAMI; YOSHIMURA, MAYUKO; SAITO, KEIGO; NAKAMOTO, YASUNARI; NAKATSURA, TETSUYA

    2015-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, have demonstrated marked clinical activity against non-small cell lung cancer (NSCLC) harboring activating epidermal growth factor receptor (EGFR) mutations. However, in most cases, patients develop acquired resistance to EGFR-TKI therapy. The threonine to methionine change at codon 790 of EGFR (EGFR T790M) mutation is the most common acquired resistance mutation, and is present in ~50% cases of TKI resistance. New treatment strategies for NSCLC patients harboring the EGFR T790M mutation are required. We evaluated the immunogenicity of an antigen derived from EGFR with the T790M mutation. Using BIMAS we selected several EGFR T790M-derived peptides bound to human leukocyte antigen (HLA)-A*02:01. T790M-A peptide (789–797) (IMQLMPFGC)-specific cytotoxic T lymphocytes (CTLs) were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ healthy donors. An established T790M-A-specific CTL line showed reactivity against the NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2−, T790M+), and the corresponding wild-type peptide (ITQLMPFGC)-pulsed T2 cells using an interferon-γ (IFN-γ) enzyme-linked immuno spot (ELISPOT) assay. This CTL line also demonstrated peptide-specific cytotoxicity against H1975-A2 cells. This finding suggests that the EGFR T790M mutation-derived antigen could be a new target for cancer immunotherapy. PMID:25532027

  20. EGFR Mutation status in Japanese lung cancer patients: genotyping analysis using LightCycler.

    PubMed

    Sasaki, Hidefumi; Endo, Katsuhiko; Konishi, Akimitsu; Takada, Minoru; Kawahara, Masaaki; Iuchi, Keiji; Matsumura, Akihide; Okumura, Meinoshin; Tanaka, Hisaichi; Kawaguchi, Tomoya; Shimizu, Toshiki; Takeuchi, Hiroshi; Yano, Motoki; Fukai, Ichiro; Fujii, Yoshitaka

    2005-04-15

    Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in approximately 25% of Japanese lung cancer patients. These EGFR mutations are reported to be correlated with clinical response to gefitinib therapy. However, DNA sequencing using the PCR methods described to date is time-consuming and requires significant quantities of DNA; thus, this existing approach is not suitable for a routine pretherapeutic screening program. We have genotyped EGFR mutation status in Japanese lung cancer patients, including 102 surgically treated lung cancer cases from Nagoya City University Hospital and 16 gefitinib-treated lung cancer cases from Kinki-chuo Chest Medical Center. The presence or absence of three common EGFR mutations were analyzed by real-time quantitative PCR with mutation-specific sensor and anchor probes. In exon 21, EGFR mutations (CTG --> CGG; L858R) were found from 8 of 102 patients from Nagoya and 1 of 16 from Kinki. We also detected the deletion mutations in exon 19 from 7 of 102 patients from Nagoya (all were deletion type 1a) and 4 of 16 patients from Kinki (one was type 1a and three were type 1b). In exon 18, one example of G719S mutation was found from both Nagoya and Kinki. The L858R mutation was significantly correlated with gender (women versus men, P < 0.0001), Brinkman index (600 < or = versus 600, P = 0.001), pathologic subtypes (adenocarcinoma versus nonadenocarcinoma, P = 0.007), and differentiation status of the lung cancers (well versus moderately or poorly, P = 0.0439), whereas the deletion mutants were not. EGFR gene status, including the type of EGFR somatic mutation, was correlated with sensitivity to gefitinib therapy. For example, some of our gefitinib-responsive patients had L858R or deletion type 1a mutations. On the other hand, one of our gefitinib-resistant patients had a G719S mutation. Using the LightCycler PCR assay, the EGFR L858R mutation status might correlate with gender, pathologic subtypes, and

  1. Co-activation of STAT3 and YES-Associated Protein 1 (YAP1) Pathway in EGFR-Mutant NSCLC.

    PubMed

    Chaib, Imane; Karachaliou, Niki; Pilotto, Sara; Codony Servat, Jordi; Cai, Xueting; Li, Xuefei; Drozdowskyj, Ana; Servat, Carles Codony; Yang, Jie; Hu, Chunping; Cardona, Andres Felipe; Vivanco, Guillermo Lopez; Vergnenegre, Alain; Sanchez, Jose Miguel; Provencio, Mariano; de Marinis, Filipo; Passaro, Antonio; Carcereny, Enric; Reguart, Noemi; Campelo, Charo Garcia; Teixido, Christina; Sperduti, Isabella; Rodriguez, Sonia; Lazzari, Chiara; Verlicchi, Alberto; de Aguirre, Itziar; Queralt, Cristina; Wei, Jia; Estrada, Roger; Puig de la Bellacasa, Raimon; Ramirez, Jose Luis; Jacobson, Kirstine; Ditzel, Henrik J; Santarpia, Mariacarmela; Viteri, Santiago; Molina, Migual Angel; Zhou, Caicun; Cao, Peng; Ma, Patrick C; Bivona, Trever G; Rosell, Rafael

    2017-09-01

    The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is limited by adaptive activation of cell survival signals. We hypothesized that both signal transducer and activator of transcription 3 (STAT3) and Src-YES-associated protein 1 (YAP1) signaling are dually activated during EGFR TKI treatment to limit therapeutic response. We used MTT and clonogenic assays, immunoblotting, and quantitative polymerase chain reaction to evaluate the efficacy of EGFR TKI alone and in combination with STAT3 and Src inhibition in three EGFR-mutant NSCLC cell lines. The Chou-Talalay method was used for the quantitative determination of drug interaction. We examined tumor growth inhibition in one EGFR-mutant NSCLC xenograft model (n = 4 mice per group). STAT3 and YAP1 expression was evaluated in tumors from 119 EGFR-mutant NSCLC patients (64 in an initial cohort and 55 in a validation cohort) by quantitative polymerase chain reaction. Kaplan-Meier and Cox regression analyses were used to assess the correlation between survival and gene expression. All statistical tests were two-sided. We discovered that lung cancer cells survive initial EGFR inhibitor treatment through activation of not only STAT3 but also Src-YAP1 signaling. Cotargeting EGFR, STAT3, and Src was synergistic in two EGFR-mutant NSCLC cell lines with a combination index of 0.59 (95% confidence interval [CI] = 0.54 to 0.63) for the PC-9 and 0.59 (95% CI = 0.54 to 0.63) for the H1975 cell line. High expression of STAT3 or YAP1 predicted worse progression-free survival (hazard ratio [HR] = 3.02, 95% CI = 1.54 to 5.93, P = .001, and HR = 2.57, 95% CI = 1.30 to 5.09, P = .007, respectively) in an initial cohort of 64 EGFR-mutant NSCLC patients treated with firstline EGFR TKIs. Similar results were observed in a validation cohort. Our study uncovers a coordinated signaling network centered on both STAT3 and Src-YAP signaling

  2. Comparison of an enzyme-linked immunosorbent assay (ELISA) to gas chromatography (GC) - measurement of polychlorinated biphenyls (PCBs) in selected US fish extracts

    USGS Publications Warehouse

    Zajicek, J.L.; Tillitt, D.E.; Schwartz, T.R.; Schmitt, C.J.; Harrison, R.O.

    2000-01-01

    The analysis of PCBs in fish tissues by immunoassay methods was evaluated using fish collected from a US monitoring program, the National Contaminant Biomonitoring Program of the US Department of Interior, Fish and Wildlife Service. Selected composite whole fish samples, which represented widely varying concentrations and sources of PCBs, were extracted and subjected to congener PCB analysis by gas chromatography (GC) and total PCB analysis using an ELISA (ePCBs) calibrated against technical Aroclor 1248. PCB congener patterns in these fishes were different from the patterns found in commercial Aroclors or their combinations as demonstrated by principal component analysis of normalized GC congener data. The sum of the PCB congeners measured by GC (total-PCBs) ranged from 37 to 4600 ng/g (wet weight). Concentrations of PCBs as determined by the ELISA method were positively correlated with total-PCBs and the ePCBs/total-PCBs ratios for individual samples ranged from 1 to 6. Ratios of ePCBs/total-PCBs for dilutions of Aroclors 1242, 1254, and 1260 and for matrix spikes range from 0.6 for 1242 to 2.5 for 1254 and 1260. These results suggest that higher chlorinated PCB congeners have higher affinity for the anti-PCB antibodies. Partial least squares with latent variable analysis of GC and ELISA data of selected Aroclors and fish samples also support the conclusion that ELISA derived PCB concentrations are dependent on the degree on chlorination.

  3. Electrochemical aptamer/antibody based sandwich immunosensor for the detection of EGFR, a cancer biomarker, using gold nanoparticles as a signaling probe.

    PubMed

    Ilkhani, Hoda; Sarparast, Morteza; Noori, Abolhassan; Zahra Bathaie, S; Mousavi, Mir F

    2015-12-15

    Detection of epidermal growth factor receptor (EGFR) in biological fluids is of paramount importance, since it has significant application in cancer diagnosis, drug development, and therapy monitoring. EGFR is a cancer biomarker, and its overexpression is associated with the development of some types of cancer. Herein, we report on the development of a sensitive and selective electrochemical aptamer/antibody (Apt/Ab) sandwich immunosensor for detection of EGFR. In this study, a biotinylated anti-human EGFR Apt was immobilized on streptavidin-coated magnetic beads (MB) and served as a capture probe. A polyclonal anti-human EGFR Ab was conjugated to citrate-coated gold nanoparticles (AuNPs) and used as a signaling probe. In the presence of EGFR, an Apt-EGFR-Ab sandwich was formed on the MB surface. The extent of the complexation was evaluated by differential pulse voltammetry of AuNPs after their dissolution in HCl. Under optimal conditions, the dynamic concentration range of the immunosensor for EGFR spanned from 1 to 40 ng/mL, with a low detection limit of 50 pg/mL, and RSD percent of less than 4.2%. The proposed approach takes advantage of sandwich assay for high specificity, MBs for fast separation, and electrochemical method for cost-effective and sensitive detection. In this proof-of-principle study, we demonstrate the potential clinical efficacy of the immunosensor for monitoring of chemotherapy effectiveness in breast cancer samples.

  4. Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration.

    PubMed

    Wodziak, Dariusz; Dong, Aiwen; Basin, Michael F; Lowe, Anson W

    2016-01-01

    A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis.

  5. Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration

    PubMed Central

    Wodziak, Dariusz; Dong, Aiwen; Basin, Michael F.; Lowe, Anson W.

    2016-01-01

    A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis. PMID:27764193

  6. Low Incidence along with Low mRNA Levels of EGFR(vIII) in Prostate and Colorectal Cancers Compared to Glioblastoma.

    PubMed

    Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

    2017-01-01

    Background: The presence as well as the potential role of EGFR(vIII) in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFR(vIII) mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFR(vIII) and EGFR(WT) mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFR(vIII) expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFR(vIII) expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFR(vIII) varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFR(vIII) mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFR(vIII) plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFR(vIII) importance should not be underestimated even in tumors with relatively low expression of this oncogene.

  7. Cytotoxic Effects of PEGylated Anti-EGFR Immunoliposomes Combined with Doxorubicin and Rhenium-188 Against Cancer Cells.

    PubMed

    Hsu, Wei-Chuan; Cheng, Chu-Nian; Lee, Te-Wei; Hwang, Jeng-Jong

    2015-09-01

    We aimed to construct epidermal growth factor receptor (EGFR)-targeting cetuximab-immunoliposomes (IL-C225) for targeted delivery of doxorubicin and rhenium-188 (Re-188) to EGFR(+) cancer cells. Synthesized IL-C225 was analyzed by dynamic light scattering, transmission electron microscopy, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Cell binding and internalization were examined using doxorubicin-loaded IL-C225 (DXR-IL-C225) with confocal microscopy. IL-C225 combined with doxorubicin and Re-188 ((188)Re-DXR-IL-C225) was synthesized, and the cytotoxic effects of (188)Re-DXR-IL-C225 were analyzed in EGFR(+) cancer cells using cell viability assays. IL-C225 bound to EGFR on A431 cancer cells and was rapidly internalized. Furthermore, IL-C225 localized within the tumor cells efficiently. (188)Re-DXR-IL-C225 exhibited outstanding cytotoxic effects against EGFR(+) cancer cells in vitro and showed superior cytotoxic effects compared to DXR-IL-C225 or (188)Re-IL-C225 alone. The new formulation of (188)Re-DXR-IL-C225 may be a potential theranostic vehicle for delivery of drugs in the treatment of EGFR-overexpressing human cancer. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  8. 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.

  9. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations.

    PubMed

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-06-01

    This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer.This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated.Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23-52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2-26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7-14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856).Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation.

  10. The content of mutant EGFR DNA correlates with response to EGFR-TKIs in lung adenocarcinoma patients with common EGFR mutations

    PubMed Central

    Hung, Ming-Szu; Lung, Jr-Hau; Lin, Yu-Ching; Fang, Yu-Hung; Hsieh, Meng-Jer; Tsai, Ying-Huang

    2016-01-01

    Abstract This study aimed to elucidate the association of the content of mutant epidermal growth factor receptor (EGFR) deoxyribonucleic acid (DNA) with the treatment response to EGFR-tyrosine kinase inhibitor (TKI) and survival in patients with lung cancer. This retrospective cohort study included 77 lung adenocarcinoma patients with common EGFR mutations from December 2012 to February 2015. The content of mutant EGFR DNA in lung cancer tissues was determined using an Amplification Refractory Mutation System. The association of the amount of mutant EGFR DNA with treatment response, the clinical variables, and the progression-free survival (PFS) after EGFR-TKI therapy were evaluated. Using the amount of mutant EGR DNA above 4.77% as the cut-off value, the sensitivity to predict EGFR-TKI responder is 82.0% and the specificity is 75.0% (area under the curve [AUC]: 0.734, P = 0.003). The high content of mutant EGFR DNA is an independent factor associated with the response to EGFR-TKIs (odds ratio: 13.07, 95% confidence interval [CI]: 3.23–52.11, P = 0.0003). A significantly longer PFS was observed in the group with the high content of mutant EGFR DNA (26.3 months, 95% CI: 12.2–26.3) compared with the low content of mutant EGFR DNA groups (12.3 months, 95% CI: 5.7–14.8, P = 0.0155). A better predictive value of the content of mutant EGFR DNA was noted in patients with exon 19 deletions (AUC: 0.892, P < 0.0001) than exon 21 L858R mutations (AUC: 0.675, P = 0.0856). Our results show that the content of mutant EGFR DNA is associated with the clinical response to EGFR-TKIs, especially in patients with exon 19 deletions mutation. PMID:27368002

  11. Optimizing the sequence of anti-EGFR targeted therapy in EGFR-mutant lung cancer

    PubMed Central

    Meador, Catherine B.; Jin, Hailing; de Stanchina, Elisa; Nebhan, Caroline A.; Pirazzoli, Valentina; Wang, Lu; Lu, Pengcheng; Vuong, Huy; Hutchinson, Katherine E.; Jia, Peilin; Chen, Xi; Eisenberg, Rosana; Ladanyi, Marc; Politi, Katerina; Zhao, Zhongming; Lovly, Christine M.; Cross, Darren A. E.; Pao, William

    2015-01-01

    Metastatic EGFR-mutant lung cancers are sensitive to the first- and second- generation EGFR tyrosine kinase inhibitors (TKIs), gefitinib, erlotinib, and afatinib, but resistance develops. Acquired resistance (AR) to gefitinib or erlotinib occurs most commonly (>50%) via the emergence of a second-site EGFR mutation, T790M. Two strategies to overcome T790M-mediated resistance are dual inhibition of EGFR with afatinib plus the anti-EGFR antibody, cetuximab (A+C), or mutant-specific EGFR inhibition with AZD9291. A+C and AZD9291 are now also being tested as first-line therapies, but whether these therapies will extend progression-free survival or induce more aggressive forms of resistance in this setting remains unknown. We modeled resistance to multiple generations of anti-EGFR therapies preclinically in order to understand the effects of sequential treatment with anti-EGFR agents on drug resistance and determine the optimal order of treatment. Using a panel of erlotinib/afatinib-resistant cells including a novel patient-derived cell line (VP-2), we found that AZD9291 was more potent than A+C at inhibiting cell growth and EGFR signaling in this setting. 4 of 4 xenograft-derived A+C-resistant cell lines displayed in vitro and in vivo sensitivity to AZD9291, but 4 of 4 AZD9291-resistant cell lines demonstrated cross-resistance to A+C. Addition of cetuximab to AZD9291 did not confer additive benefit in any preclinical disease setting. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences. This paradigm is applicable to other tumor types in which multiple generations of inhibitors are now available. PMID:25477325

  12. 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

  13. EGFR genomic alterations in cancer: prognostic and predictive values.

    PubMed

    Bronte, Giuseppe; Terrasi, Marianna; Rizzo, Sergio; Sivestris, Nicola; Ficorella, Corrado; Cajozzo, Massimo; Di Gaudio, Francesca; Gulotta, Gaspare; Siragusa, Sergio; Gebbia, Nicola; Russo, Antonio

    2011-06-01

    The role of EGFR in cancer development and progression has been recognized for long time in a variety of human malignancies including lung, head and neck, colon, breast, ovary and glioma. Recently its role as a target of antineoplastic agents has also been identified and a variety of EGFR-targeted drugs is already being used in a clinical setting and others are at present under investigation. Many data involving EGFR protein expression are now available for the choice of anti-EGFR monoclonal antibodies in colorectal cancer and with regard to EGFR gene mutations for the choice of tyrosine kinase inhibitors in lung cancer. Other EGFR-related molecular factors, including the EGFR gene copy number, are currently under investigation. This review summarizes both preclinical and clinical available data regarding EGFR genomic alterations as prognostic and predictive factors.

  14. Small Molecule Inhibitors of EGFR Ectodomain for Breast Cancer Therapy

    DTIC Science & Technology

    2010-08-01

    3451 Walnut Street Franklin Bldg P-221 Philadelphia, PA 19104-6205 9. SPONSORING / MONITORING AGENCY... wild type EGFR. We used structure based approaches to design low molecular weight compounds targeted to the ectodomain of EGFR that act by inhibiting

  15. Aberrant large tumor suppressor 2 (LATS2) gene expression correlates with EGFR mutation and survival in lung adenocarcinomas

    PubMed Central

    Luo, Susan Y.; Sit, Ko-Yung; Sihoe, Alan D.L.; Suen, Wai-Sing; Au, Wing-Kuk; Tang, Ximing; Ma, Edmond S.K.; Chan, Wai-Kong; Wistuba, Ignacio I.; Minna, John D.; Tsao, George S.W.; Lam, David C.L.

    2015-01-01

    Background Large tumor suppressor 2 (LATS2) gene is a putative tumor suppressor gene with potential roles in regulation of cell proliferation and apoptosis in lung cancer. The aim of this study is to explore the association of aberrant LATS2 expression with EGFR mutation and survival in lung adenocarcinoma (AD), and the effects of LATS2 silencing in both lung AD cell lines. Methods LATS2 mRNA and protein expression in resected lung AD were correlated with demographic characteristics, EGFR mutation and survival. LATS2-specific siRNA was transfected into four EGFR wild-type (WT) and three EGFR mutant AD cell lines and the changes in LATS2 expression and relevant signaling molecules before and after LATS2 knockdown were assayed. Results Fifty resected lung AD were included (M:F = 23:27, smokers:non-smokers = 19:31, EGFR mutant:wild-type = 21:29) with LATS2 mRNA levels showed no significant difference between gender, age, smoking and pathological stages while LATS2 immunohistochemical staining on an independent set of 79 lung AD showed similar trend. LATS2 mRNA level was found to be a significant independent predictor for survival status (disease-free survival RR = 0.217; p = 0.003; Overall survival RR = 0.238; p = 0.036). siRNA-mediated suppression of LATS2 expression resulted in augmentation of ERK phosphorylation in EGFR wild-type AD cell lines with high basal LATS2 expression, discriminatory modulation of Akt signaling between EGFR wild-type and mutant cells, and induction of p53 accumulation in AD cell lines with low baseline p53 levels. Conclusions LATS2 expression level is predictive of survival in patients with resected lung AD. LATS2 may modulate and contribute to tumor growth via different signaling pathways in EGFR mutant and wild-type tumors. PMID:24976335

  16. Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

    PubMed Central

    2013-01-01

    Background Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting. Results A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants. Conclusions Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome. PMID:23806066

  17. Radiolabeling and biological evaluation of DOTA-Ph-Al derivative conjugated to anti-EGFR antibody ior egf/r3 for targeted tumor imaging and therapy.

    PubMed

    Pnwar, Puja; Iznaga-Escobar, Normando; Mishra, Pushpa; Srivastava, Vibha; Sharma, Rakesh Kumar; Chandra, Ramesh; Mishra, Anil K

    2005-08-01

    An appropriate bifunctional chelating agent namely DOTA-Ph-Al was developed for the conjugation with biological vectors (anti EGFr antibody). We hereby report the synthesis of p-bromoacetamidobenzyl derivative of DOTA and its conjugation to monoclonal antibody anti-EGFR ior egf/r3. Immunoconjugate was prepared by conjugation of p-bromoacetamidobenzyl derivative of DOTA with ior egf/r3. Modified antibody was purified by size exclusion chromatography. DOTA-Ph-Al-ior egf/r3 exhibited quantitative 99mTc-labeling (>96%) with specific activity 10-20 mCi/mg of protein and 90Y-labeling with specific activity 2-5 mCi/mg. Immunoreactivity was determined by flow cytometry. Receptor ligand assay on murine cell line EAT and human tumor cell line U-87MG showed Kd = 2.87 nM and 4.86 nM respectively. The stability in serum indicated that 99mTc remained bound to antibodies up to 24h and 98% 90Y was associated with the mAb for five days. Biodistribution characteristics of Ab-conjugate radiolabeled to 99mTc and 90Y radionuclide was examined in BALB/c mice grafted with EAT and athymic mice with U-87MG cell line demonstrated high tumor uptake with 5.5 +/- 1.3 and 7.85 +/- 1.2%ID/g at four and 24 h for 99mTc- DOTA-Ph-AI-ior egf/r3 in EAT tumors after post injection respectively. Maximal radiotracer uptake peaked 17.6 +/- 2.5%ID/g in EAT tumor and 12.89 +/- 0.66% ID/g in U-87MG tumor at 48h for 90Y. The drug excreted through renal routes as the activity in the kidneys was 13.42 +/- 0.33%ID/g at 1 h and 4.51 +/- 1.2%ID/g at 4 h for 99mTc- DOTA-Ph-Al-ior egf/r3.

  18. Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia.

    PubMed

    Lindhagen, Elin; Eriksson, Anna; Wickström, Malin; Danielsson, Katarina; Grundmark, Birgitta; Henriksson, Roger; Nygren, Peter; Aleskog, Anna; Larsson, Rolf; Höglund, Martin

    2008-11-01

    Gefitinib inhibits epidermal growth factor receptor (EGFR) signalling, but may also act by non-EGFR dependent mechanisms. We have investigated the activity of gefitinib in haematological tumour cells, in particular acute myeloblastic leukaemia (AML). Cytotoxic activity of gefitinib, alone or in combination with standard anti-leukaemic drugs, was assessed by the short-term fluorometric microculture cytotoxicity assay in tumour cells from 117 patients representing five haematological and five non-haematological malignancies. In AML, the EGFR status was analysed by immunochemistry. Gefitinib-induced apoptosis was investigated in a subset of AML samples, as well as in the leukaemia cell line MV-4-11, using a multiparametric high content screening assay. To confirm activation of caspase-3 in cells treated with gefitinib, a blocking test was carried out in which MV4-11 cells were pretreated with the specific caspase inhibitor DEVD-FMK. Gefitinib showed highest cytotoxic activity in AML (n = 19) with many samples being sensitive at concentrations achievable in clinical practice (<10 microM), and no difference between previously untreated and relapsed patients. No correlation between the activity of gefitinib and standard antileukaemic drugs (cytarabine, doxorubicin, etoposide) was observed. Combining gefitinib with these drugs resulted in mainly additive or synergistic (etoposide) effects, with no evidence of sequence dependency. The AML cells did not express the EGFR. Gefitinib induced apoptosis, which was at least partly mediated by activation of the caspase-3 pathway. In vitro, gefitinib has significant cytotoxic activity in AML by inducing apoptosis through non-EGFR dependent pathways.

  19. The EGFR Is Required for Proper Innervation to the Skin

    PubMed Central

    Maklad, Adel; Nicolai, Jodi R.; Bichsel, Kyle J.; Evenson, Jackie E.; Lee, Tang-Cheng; Threadgill, David W.; Hansen, Laura A.

    2008-01-01

    EGFR family members are essential for proper peripheral nervous system development. A role for EGFR itself in peripheral nervous system development in vivo, however, has not been reported. We investigated whether EGFR is required for cutaneous innervation using Egfr null and skin-targeted Egfr mutant mice. Neuronal markers; including PGP9.5, GAP-43, acetylated tubulin, and neurofilaments; revealed that Egfr null dorsal skin was hyperinnervated with a disorganized pattern of innervation. In addition, receptor subtypes such as lanceolate endings were disorganized and immature. To determine whether the hyperinnervation phenotype resulted from a target-derived effect of loss of EGFR, mice lacking EGFR expression in the cutaneous epithelium were examined. These mice retained other aspects of the cutaneous Egfr null phenotype but exhibited normal innervation. The sensory deficits in Egfr null dorsal skin were not associated with any abnormality in the morphology or density of dorsal root ganglion (DRG) neurons or Schwann cells. However, explant and dissociated cell cultures of DRG revealed more extensive branching in Egfr null cultures. These data demonstrate that EGFR is required for proper cutaneous innervation during development and suggest that it limits axonal outgrowth and branching in a DRG-autonomous manner. PMID:18830272

  20. Sensitive genotyping of somatic mutations in the EGFR, KRAS, PIK3CA, BRAF genes from NSCLC patients using hydrogel biochips.

    PubMed

    Emelyanova, Marina; Arkhipova, Ksenia; Mazurenko, Natalia; Chudinov, Alexander; Demidova, Irina; Zborovskaya, Irina; Lyubchenko, Lyudmila; Zasedatelev, Alexander; Nasedkina, Tatiana

    2015-04-01

    Targeted inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR gene and key effectors of the EGFR-signaling pathway (KRAS, BRAF, PIK3CA) are associated with sensitivity to these drugs. We developed a highly sensitive LUNG CANCER (LC)-biochip approach for the detection of the most common EGFR, KRAS, PIK3CA, and BRAF gene mutations. The locked nucleic acid clamp PCR technique was used to increase the sensitivity of the assay, then allele-specific hybridization of a fluorescently labeled target on a biochip was performed. To prove the feasibility of the approach, clinical samples from 112 patients with NSCLC were analyzed. A total of 14 EGFR (12.5%) mutations, 21 (18.8%) KRAS mutations, 12 (10.7%) PIK3CA mutations, and 1 BRAF mutation (0.9%) were found. We compared the results with those from direct sequencing. We detected 50 different mutations by the LC-biochip assay and only 33 of them were found by direct sequencing. To demonstrate that the LC-biochip assay did not give false-positive results, the 17 specimens with discordant results were subjected to locked nucleic acid clamp PCR followed by sequencing. The results of this analysis were identical to the results obtained by the LC-biochip assay indicating that the biochip-based assay was both accurate and reliable. This approach was able to detect approximately 0.5% of mutated alleles in wild-type DNA background. The biochip-based assay is a reliable and inexpensive method for the identification of NSCLC patients, who may respond to a specific targeted therapy.

  1. Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer

    PubMed Central

    Madden, Julie M; Mueller, Kelly L; Bollig-Fischer, Aliccia; Stemmer, Paul; Mattingly, Raymond R; Boerner, Julie L

    2014-01-01

    Purpose Triple negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Methods Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 μM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation while MTT assays determined the synergistic effect of inhibitor combination. A dual luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Results Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Conclusions Taken together these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play in important role in regulating EGFR and mTOR inhibitor synergy and warrants further investigation. PMID:25129346

  2. Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer.

    PubMed

    Madden, Julie M; Mueller, Kelly L; Bollig-Fischer, Aliccia; Stemmer, Paul; Mattingly, Raymond R; Boerner, Julie L

    2014-09-01

    Triple-negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study, we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 μM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation, while MTT assays determined the synergistic effect of inhibitor combination. A Dual-Luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Taken together, these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play an important role in regulating EGFR and mTOR inhibitor synergy and warrant further investigation.

  3. Discovery of (R)-1-(3-(4-Amino-3-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (CHMFL-EGFR-202) as a Novel Irreversible EGFR Mutant Kinase Inhibitor with a Distinct Binding Mode.

    PubMed

    Wang, Aoli; Li, Xixiang; Wu, Hong; Zou, Fengming; Yan, Xiao-E; Chen, Cheng; Hu, Chen; Yu, Kailin; Wang, Wenchao; Zhao, Peng; Wu, Jiaxin; Qi, Ziping; Wang, Wei; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shanchun; Yun, Cai-Hong; Liu, Jing; Liu, Qingsong

    2017-03-22

    On the basis of Ibrutinib's core pharmacophore, which was moderately active to EGFR T790M mutant, we discovered novel epidermal growth factor receptor (EGFR) inhibitor compound 19 (CHMFL-EGFR-202), which potently inhibited EGFR primary mutants (L858R, del19) and drug-resistant mutant L858R/T790M. Compound 19 displayed a good selectivity profile among 468 kinases/mutants tested in the KINOMEscan assay (S score (1) = 0.02). In particular, it did not exhibit apparent activities against INSR and IGF1R kinases. The X-ray crystal structure revealed that this class of inhibitors formed a covalent bond with Cys797 in a distinct "DFG-in-C-helix-out" inactive EGFR conformation. Compound 19 displayed strong antiproliferative effects against EGFR mutant-driven nonsmall cell lung cancer (NSCLC) cell lines such as H1975, PC9, HCC827, and H3255 but not the wild-type EGFR expressing cells. In the H1975 and PC9 cell-inoculated xenograft mouse models, compound 19 exhibited dose-dependent tumor growth suppression efficacy without obvious toxicity. Compound 19 might be a potential drug candidate for EGFR mutant-driven NSCLC.

  4. EGFR mutation testing in blood for guiding EGFR tyrosine kinase inhibitor treatment in patients with nonsmall cell lung cancer

    PubMed Central

    Yuan, Jin-Qiu; Zhang, Yue-Lun; Li, Hai-Tao; Mao, Chen

    2017-01-01

    Abstract Background: Epidermal growth factor receptor (EGFR) mutation testing in tumor tissue is now a common practice in selecting non-small cell lung cancer (NSCLC) patients for EGFR tyrosine kinase inhibitor (TKI) treatment. However, tumor tissues are often absent or insufficient for the testing. Blood is a potential substitute providing a noninvasive, easily accessible and repeatedly measureable source of genotypic information. However which is the best blood EGFR mutation testing method remains unclear. We undertake this study to investigate the best blood EGFR mutation testing method for selecting EGFR TKI treatment in patients with NSCLC. Methods: This study was registered in PROSPERO (CRD42017055263). PubMed, EMBASE, Cochrane library, and NIHR Health Technology Assessment program will be searched. Studies fulfill the following criteria will be eligible: (1) randomized controlled trials or cohort studies; (2) included patients with NSCLC; (3) reported response, progression-free survival, or overall survival for EGFR TKI by the EGFR mutation status in blood sample. Diagnostic accuracy of blood EGFR mutation tests for predicting response to TKI will be pooled. Tumor response, progression-free survival, and overall survival according to different blood EGFR mutation testing methods will be evaluated and compared. Results: Based published data and combined analysis, this study will quantitatively compare the blood EGFR mutation testing methods according to their accuracy for predicting treatment response and relationship with clinical outcome in NSCLC patients treated with EGFR TKIs. Conclusion: This protocol will determine the best blood EGFR mutation testing method. PMID:28207548

  5. Balancing potency, metabolic stability and permeability in pyrrolopyrimidine-based EGFR inhibitors.

    PubMed

    Han, Jin; Henriksen, Silje; Nørsett, Kristin G; Sundby, Eirik; Hoff, Bård Helge

    2016-11-29

    The present study describes our continuous effort to develop epidermal growth factor receptor (EGFR) inhibitors based on the 6-aryl-pyrrolo[2,3-d]pyrimidin-4-amine scaffold. The activity-ADME space has been evaluated by synthesizing 43 new structures, including four variations of the 4-amino group and 34 different substitution patterns in the 6-aryl moiety. Most of the new pyrrolopyrimidines were highly active, with twelve analogues possessing lower IC50 values than the commercial drug Erlotinib in enzymatic assays. Ten EGFR inhibitors were also profiled in cell studies using the Ba/F3-EGFR(L858R) reporter cells, and all revealed nanomolar activity. However, some of the privileged structures in terms of potency had ADME short-comings: compounds containing amides, sulfonamides, amine and hydroxymethyl substituents in the 6-aryl group had low permeability and high efflux, derivatives having (R)-3-amino-3-phenylpropan-1-ol at C-4 induced hERG inhibition properties, and metabolic lability was seen for compounds having (S)-2-methoxy-1-phenylethan-1-amine at C-4. Based on a trade-off between enzymatic activity, cellular potency and ADME properties, (S)-2-phenyl-2-((6-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)ethan-1-ol appeared as the most promising drug candidate. Cellular studies indicate this compound to have therapeutic use in EGFR driven diseases. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. miR-27a suppresses EV71 replication by directly targeting EGFR.

    PubMed

    Zhang, Lianglu; Chen, Xiong; Shi, Yingying; Zhou, Bingfei; Du, Chen; Liu, Yongjuan; Han, Song; Yin, Jun; Peng, Biwen; He, Xiaohua; Liu, Wanhong

    2014-12-01

    Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has broken out several times and was accompanied by neurological disease. microRNAs, a class of small non-coding RNAs that are approximately 20 nucleotides long, play important roles in the regulation of various biological processes, including antiviral defense. However, the roles of miRNAs in EV71 replication and pathogenesis are not well understood. In this study, we found that the expression of miR-27a was significantly decreased in EV71-infected cells. Interestingly, the over-expression of miR-27a could inhibit EV71 replication, as measured by virus titration, qPCR, and Western blotting. We identified EGFR mRNA is a bona fide target of miR-27a by computational analysis and luciferase reporter assays. Furthermore, miR-27a could decrease EGFR expression, as measured by qPCR and Western blotting. Moreover, the inhibition of EGFR expression by miR-27a decreased the phosphorylation of Akt and ERK, which facilitate EV71 replication. These results suggest that miR-27a may have antiviral activity against EV71 by inhibiting EGFR.

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

    PubMed

    Sullivan, Ivana; Planchard, David

    2016-01-01

    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

  8. 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

  9. Effects of an Alkaline Diet on EGFR-TKI Therapy in EGFR Mutation-positive NSCLC.

    PubMed

    Hamaguchi, Reo; Okamoto, Toshihiro; Sato, Masaaki; Hasegawa, Michiko; Wada, Hiromi

    2017-09-01

    The acidic tumor microenvironment is associated with progression of cancers. The purpose of this study was to investigate the association between an alkaline diet and the effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) patients. Eleven advanced or recurrent NSCLC patients with EGFR mutations treated with EGFR-TKI after being instructed to follow an alkaline diet were retrospectively evaluated. The median progression-free survival (PFS) and overall survival (OS) were 19.5 (range=3.1-33.8) and 28.5 (range=15.4-46.6) months. The average dosage of EGFR-TKI was 56±22% of the standard dosage. Urine pH was significantly increased after the alkaline diet (6.00±0.38 vs. 6.95±0.55; p<0.05). An alkaline diet may enhance the effect of EGFR-TKI treatment in NSCLC patients with EGFR mutations. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  10. Imaging of EGFR and EGFR Tyrosine Kinase Overexpression in Tumors by Nuclear Medicine Modalities

    PubMed Central

    Mishani, Eyal; Abourbeh, Galith; Eiblmaier, Martin; Anderson, Carolyn J

    2008-01-01

    Protein tyrosine kinases (PTKs) play a pivotal role in signal transduction pathways and in the development and maintenance of various cancers. They are involved in multiple processes such as transcription, cell cycle progression, proliferation, angiogenesis and inhibition of apoptosis. Among the PTKs, the EGFR is one of the most widely studied and has emerged as a promising key target for the treatment of cancer. Indeed, several drugs directed at this receptor are FDA-approved and many others are at various stages of development. However, thus far, the therapeutic outcome of EGFR-targeted therapy is suboptimal and needs to be refined. Quantitative PET molecular imaging coupled with selective labelled biomarkers may facilitate in vivo EGFR-targeted drug efficacy by noninvasively assessing the expression of EGFR in tumor, guiding dose and regime by measuring target drug binding and receptor occupancy as well as potentially detecting the existence of a primary or secondary mutation leading to either drug interaction or failure of EGFR recognition by the drug. This review describes the attempts to develop labelled EGFR molecular imaging agents that are based either on low molecular weight tyrosine kinase inhibitors or monoclonal antibodies directed to the extracellular binding domain of the receptor to be used in nuclear medicine modalities. PMID:18991714

  11. Honokiol inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors

    PubMed Central

    Leeman-Neill, Rebecca J.; Cai, Quan; Joyce, Sonali C.; Thomas, Sufi M.; Bhola, Neil E.; Neill, Daniel B.; Arbiser, Jack L.; Grandis, Jennifer R.

    2010-01-01

    Purpose To investigate the utility of honokiol, a naturally-occurring compound, in the treatment of head and neck squamous cell carcinoma (HNSCC) as well as its ability to target the epidermal growth factor receptor (EGFR), a critical therapeutic target in HNSCC, and to enhance the effects of other EGFR-targeting therapies. Experimental Design Human HNSCC cell lines and the xenograft animal model of HNSCC were employed to test the effects of honokiol treatment. Results Honokiol was found to inhibit growth in human HNSCC cell lines, with EC50s ranging from 3.3-7.4 μM, and to induce apoptosis, as shown through annexin V staining. These effects were associated with inhibition of EGFR signaling, including downstream inhibition of MAPK, Akt and STAT3 and expression of STAT3 target genes, Bcl-XL and cyclin D1. Furthermore, honokiol enhanced the growth inhibitory and anti-invasion activity of the EGFR-targeting agent, erlotinib. While HNSCC xenograft models did not demonstrate significant inhibition of in vivo tumor growth with honokiol treatment alone, the combination of honokiol plus cetuximab, an FDA-approved EGFR inhibitor for this malignancy, significantly enhanced growth inhibition. Finally, HNSCC cells rendered resistant to erlotinib retained sensitivity to the growth inhibitory effects of honokiol. Conclusions These results suggest that honokiol may be an effective therapeutic agent in HNSCC where it can augment the effects of EGFR inhibitors and overcome drug resistance. PMID:20388852

  12. Should EGFR tyrosine kinase inhibitors be used in non-small cell lung cancer in the absence of EGFR mutations? No, EGFR TKIs should be reserved for patients with EGFR mutations.

    PubMed

    Riely, Gregory J

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) that block epidermal growth factor receptor (EGFR) clearly work best in patients who have non-small cell lung cancer (NSCLC) with EGFR mutations, but are they worth using in patients without these mutations? In this month's Counterpoints, Dr Frances A. Shepherd says that there is a role for EGFR TKIs in patients with wild-type EGFR disease. Dr Gregory J. Riely, however, says that the level of toxicity associated with EGFR TKIs outweighs any slight chance of benefit for these patients, who have multiple other treatment options.

  13. Application of endocrine disruptor screening program fish short-term reproduction assay: Reproduction and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus) exposed to Bermuda pond sediment.

    PubMed

    Fort, Douglas J; Mathis, Michael; Fort, Chelsea E; Fort, Hayley M; Bacon, Jamie P

    2015-06-01

    A modified tier 1 Endocrine Disruptor Screening Program (EDSP) 21-d fish short-term reproduction assay (FSTRA) was used to evaluate the effects of sediment exposure from freshwater and brackish ponds in Bermuda on reproductive fecundity and endocrine function in fathead minnow (Pimephales promelas) and killifish (Fundulus heteroclitus). Reproductively active male and female fish were exposed to control sediment and sediment from 2 freshwater ponds (fathead minnow) and 2 marine ponds (killifish) contaminated with polyaromatic hydrocarbons and metals via flow-through exposure for 21 d. Reproductive fecundity was monitored daily. At termination, the status of the reproductive endocrine system was assessed by the gonadosomatic index, gonadal histology, plasma steroids (estrogen [E2], testosterone [T], and 11-ketotestosterone [11-KT]), steroidogenic enzymes (aromatase and combined 3β/17β -hydroxysteroid dehydrogenase [3β/17β-HSD]), and plasma vitellogenin (VTG). Decreased reproductive fecundity, lower male body weight, and altered endocrinological measures of reproductive status were observed in both species. Higher plasma T levels in female minnows and 11-KT levels in both male and female minnows and female killifish exposed to freshwater and brackish sediments, respectively. Decreased female E2 and VTG levels and gonadal cytochrome P19 (aromatase) activity were also found in sediment exposed females from both species. No effect on female 3β/17β-HSD activity was found in either species. The FSTRA provided a robust model capable of modification to evaluate reproductive effects of sediment exposure in fish. © 2015 SETAC.

  14. 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

  15. 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

  16. Epiregulin and EGFR interactions are involved in pain processing.

    PubMed

    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; Gkogkas, Christos; Sonenberg, Nahum; Greenspan, Joel D; Fillingim, Roger B; Ohrbach, Richard; 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

    2017-09-01

    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.

  17. Simple assay for analyzing five microcystins and nodularin in fish muscle tissue: hot water extraction followed by liquid chromatography-tandem mass spectrometry.

    PubMed

    Bogialli, Sara; Bruno, Milena; Curini, Roberta; Di Corcia, Antonio; Laganá, Aldo; Mari, Barbara

    2005-08-24

    A simple, specific, and sensitive procedure for determining six cyanotoxins, that is, microcystins RR, LR, YR, LA, and LW and nodularin, in fish muscle tissue is presented. This method is based on the matrix solid-phase dispersion technique with heated water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissue by 4 mL of water acidified to pH 2 and heated at 80 degrees C. After acidification and filtration, 0.2 mL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, with at least two precursor ion > product ion transitions selected for each target compound. Analyte recovery ranged between 61 and 82% and was not substantially affected by either the analyte concentrations or the type of fish. The nonexcellent recovery of some of the microcystins was traced to binding of these compounds to protein phosphatases in fish tissue occurring during sample treatment. The existence of covalently bound microcystins in fish has been evidenced by several studies. Compared to an older sample preparation procedure, this one extracted larger amounts of the analytes in a simpler and much more rapid way. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 1.6 and 4.0 ng/g. The effects of temperature and volume of the extractant on the analyte recovery were studied.

  18. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining (1)H NMR metabolomics and conventional biochemical assays.

    PubMed

    Cappello, Tiziana; Brandão, Fátima; Guilherme, Sofia; Santos, Maria Ana; Maisano, Maria; Mauceri, Angela; Canário, João; Pacheco, Mário; Pereira, Patrícia

    2016-04-01

    Oxidative stress has been described as a key pathway to initiate mercury (Hg) toxicity in fish. However, the mechanisms underlying Hg-induced oxidative stress in fish still need to be clarified. To this aim, environmental metabolomics in combination with a battery of conventional oxidative stress biomarkers were applied to the gills of golden grey mullet (Liza aurata) collected from Largo do Laranjo (LAR), a confined Hg contaminated area, and São Jacinto (SJ), selected as reference site (Aveiro Lagoon, Portugal). Higher accumulation of inorganic Hg and methylmercury was found in gills of fish from LAR relative to SJ. Nuclear magnetic resonance (NMR)-based metabolomics revealed changes in metabolites related to antioxidant protection, namely depletion of reduced glutathione (GSH) and its constituent amino acids, glutamate and glycine. The interference of Hg with the antioxidant protection of gills was corroborated through oxidative stress endpoints, namely the depletion of glutathione peroxidase and superoxide dismutase activities at LAR. The increase of total glutathione content (reduced glutathione+oxidized glutathione) at LAR, in parallel with GSH depletion aforementioned, indicates the occurrence of massive GSH oxidation under Hg stress, and an inability to carry out its regeneration (glutathione reductase activity was unaltered) or de novo synthesis. Nevertheless, the results suggest the occurrence of alternative mechanisms for preventing lipid peroxidative damage, which may be associated with the enhancement of membrane stabilization/repair processes resulting from depletion in the precursors of phosphatidylcholine (phosphocholine and glycerophosphocholine), as highlighted by NMR spectroscopy. However, the observed decrease in taurine may be attributable to alterations in the structure of cell membranes or interference in osmoregulatory processes. Overall, the novel concurrent use of metabolomics and conventional oxidative stress endpoints demonstrated to be

  19. EGFR-targeted delivery of DOX-loaded Fe3O4@ polydopamine multifunctional nanocomposites for MRI and antitumor chemo-photothermal therapy.

    PubMed

    Mu, Xupeng; Zhang, Fuqiang; Kong, Chenfei; Zhang, Hongmei; Zhang, Wenjing; Ge, Rui; Liu, Yi; Jiang, Jinlan

    2017-01-01

    Multifunctional nanocomposites that have multiple therapeutic functions together with real-time imaging capabilities have attracted intensive concerns in the diagnosis and treatment of cancer. This study developed epidermal growth factor receptor (EGFR) antibody-directed polydopamine-coated Fe3O4 nanoparticles (Fe3O4@PDA NPs) for magnetic resonance imaging and antitumor chemo-photothermal therapy. The synthesized Fe3O4@PDA-PEG-EGFR-DOX NPs revealed high storage capacity for doxorubicin (DOX) and high photothermal conversion efficiency. The cell viability assay of Fe3O4@PDA-PEG-EGFR NPs indicated that Fe3O4@ PDA-PEG-EGFR NPs had no cell cytotoxicity. However, Fe3O4@PDA-PEG-EGFR-DOX NPs could significantly decrease cell viability (~5% of remaining cell viability) because of both photothermal ablation and near-infrared light-triggered DOX release. Meanwhile, the EGFR-targeted Fe3O4@PDA-PEG-EGFR-DOX NPs significantly inhibited the growth of tumors, showing a prominent in vivo synergistic antitumor effect. This study demonstrated the potential of using Fe3O4@PDA NPs for combined cancer chemo-photothermal therapy with increased efficacy.

  20. EGFR-targeted delivery of DOX-loaded Fe3O4@ polydopamine multifunctional nanocomposites for MRI and antitumor chemo-photothermal therapy

    PubMed Central

    Mu, Xupeng; Zhang, Fuqiang; Kong, Chenfei; Zhang, Hongmei; Zhang, Wenjing; Ge, Rui; Liu, Yi; Jiang, Jinlan

    2017-01-01

    Multifunctional nanocomposites that have multiple therapeutic functions together with real-time imaging capabilities have attracted intensive concerns in the diagnosis and treatment of cancer. This study developed epidermal growth factor receptor (EGFR) antibody-directed polydopamine-coated Fe3O4 nanoparticles (Fe3O4@PDA NPs) for magnetic resonance imaging and antitumor chemo-photothermal therapy. The synthesized Fe3O4@PDA-PEG-EGFR-DOX NPs revealed high storage capacity for doxorubicin (DOX) and high photothermal conversion efficiency. The cell viability assay of Fe3O4@PDA-PEG-EGFR NPs indicated that Fe3O4@ PDA-PEG-EGFR NPs had no cell cytotoxicity. However, Fe3O4@PDA-PEG-EGFR-DOX NPs could significantly decrease cell viability (~5% of remaining cell viability) because of both photothermal ablation and near-infrared light-triggered DOX release. Meanwhile, the EGFR-targeted Fe3O4@PDA-PEG-EGFR-DOX NPs significantly inhibited the growth of tumors, showing a prominent in vivo synergistic antitumor effect. This study demonstrated the potential of using Fe3O4@PDA NPs for combined cancer chemo-photothermal therapy with increased efficacy. PMID:28435266

  1. Lupeol evokes anticancer effects in oral squamous cell carcinoma by inhibiting oncogenic EGFR pathway.

    PubMed

    Rauth, Sanchita; Ray, Sudipta; Bhattacharyya, Sayantan; Mehrotra, Debapriya Ghosh; Alam, Neyaz; Mondal, Goutam; Nath, Partha; Roy, Asoke; Biswas, Jaydip; Murmu, Nabendu

    2016-06-01

    Epidermal growth factor receptor (EGFR) pathway is overexpressed in head and neck cancer (HNC). Lupeol, a natural triterpene (phytosterol found in fruits, vegetables, etc.), has been reported to be effective against multiple cancer indications. Here we investigate the antitumor effects of Lupeol and underlying mechanism in oral cancer. Lupeol-induced antitumor response was evaluated in two oral squamous cell carcinoma (OSCC) cell lines (UPCI:SCC131 and UPCI:SCC084) by viability (MTT), proliferation, and colony formation assays. Lupeol-mediated induction of apoptosis was examined by caspase 3/7 assay and flow cytometry. Effect of Lupeol on EGFR in the presence or absence of EGF was delineated by Western blot. The mRNA stability assay was performed to check the role of Lupeol on COX-2 mRNA regulation. Lupeol inhibited proliferation of OSCC cells in vitro by inducing apoptosis 48 h post treatment. Ligand-induced phosphorylation of EGFR and subsequent activation of its downstream molecules such as protein kinase B (PKB or AKT), I kappa B (IκB), and nuclear factor kappa B (NF-κB) was also found to be, in part, suppressed. Interestingly, Lupeol suppressed expression of COX-2 at mRNA and protein level in a time-dependent manner. Primary explants from oral squamous cell carcinoma tissues further confirmed significant inhibition of proliferation (Ki67) in Lupeol-treated explants as compared to untreated control at 48 h. Together these data suggest that Lupeol may act as a potent inhibitor of the EGFR signaling in OSCC and therefore imply its role in triggering antitumor efficacy.

  2. Low EGFR/MET ratio is associated with resistance to EGFR inhibitors in non-small cell lung cancer.

    PubMed

    Park, Silvia; Langley, Emma; Sun, Jong-Mu; Lockton, Steve; Ahn, Jin Seok; Jain, Anjali; Park, Keunchil; Singh, Sharat; Kim, Phillip; Ahn, Myung-Ju

    2015-10-13

    Although activating mutations in the epidermal growth factor receptor (EGFR) gene are predictive markers for response to EGFR inhibitors, 30-40% of EGFR-mutant non-small cell lung cancer (NSCLC) patients are de novo non-responders. Hence, we sought to explore additional biomarkers of response. We conducted a prospective pilot study to characterize the expression and/or activation of key receptor tyrosine kinases (RTKs) in stage IIIB-IV NSCLC tumors. A total of 37 patients were enrolled and 34 underwent EGFR inhibitor treatment. As expected, patients bearing activating EGFR mutations showed increased progression free survival (PFS) compared to patients with wild-type EGFR status (9.3 vs 1.4 months, p = 0.0629). Analysis of baseline tumor RTK profiles revealed that, regardless of EGFR mutation status, higher levels of EGFR relative to MET correlated with longer PFS. At multiple EGFR/MET ratio cut-offs, including 1, 2 and 3, median PFS according to below vs. above cut-offs were 0.4 vs. 6.1 (p = 0.0001), 0.5 vs. 9.3 (p = 0.0006) and 1.0 vs. 11.2 months (p = 0.0008), respectively. The EGFR/MET ratio measured in tumors at baseline may help identify NSCLC patients most likely to benefit from prolonged PFS when treated with EGFR inhibitors.

  3. 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.

  4. Low EGFR/MET ratio is associated with resistance to EGFR inhibitors in non-small cell lung cancer

    PubMed Central

    Sun, Jong-Mu; Lockton, Steve; Ahn, Jin Seok; Jain, Anjali; Park, Keunchil; Singh, Sharat; Kim, Phillip; Ahn, Myung-Ju

    2015-01-01

    Purpose Although activating mutations in the epidermal growth factor receptor (EGFR) gene are predictive markers for response to EGFR inhibitors, 30–40% of EGFR-mutant non-small cell lung cancer (NSCLC) patients are de novo non-responders. Hence, we sought to explore additional biomarkers of response. Methods We conducted a prospective pilot study to characterize the expression and/or activation of key receptor tyrosine kinases (RTKs) in stage IIIB-IV NSCLC tumors. A total of 37 patients were enrolled and 34 underwent EGFR inhibitor treatment. Results As expected, patients bearing activating EGFR mutations showed increased progression free survival (PFS) compared to patients with wild-type EGFR status (9.3 vs 1.4 months, p = 0.0629). Analysis of baseline tumor RTK profiles revealed that, regardless of EGFR mutation status, higher levels of EGFR relative to MET correlated with longer PFS. At multiple EGFR/MET ratio cut-offs, including 1, 2 and 3, median PFS according to below vs. above cut-offs were 0.4 vs. 6.1 (p = 0.0001), 0.5 vs. 9.3 (p = 0.0006) and 1.0 vs. 11.2 months (p = 0.0008), respectively. Conclusion The EGFR/MET ratio measured in tumors at baseline may help identify NSCLC patients most likely to benefit from prolonged PFS when treated with EGFR inhibitors. PMID:26439803

  5. 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

  6. Chromosomal aneusomy in sputum, as detected by Fluorescence In Situ Hybridization (FISH) predicts lung cancer incidence

    PubMed Central

    Varella-Garcia, Marileila; Schulte, Aline P.; Wolf, Holly J.; Feser, William J; Zeng, Chan; Braudrick, Sarah; Yin, Xiang; Hirsch, Fred R.; Kennedy, Timothy C.; Keith, Robert L.; Barón, Anna E.; Belinsky, Steven A.; Miller, York E.; Byers, Tim; Franklin, Wilbur A.

    2009-01-01

    Lung cancer is usually disseminated at diagnosis making prognosis poor. Smokers are at high risk for lung cancer and are targets for prevention and early detection strategies. Sputum is a potential source for lung cancer biomarkers, but no test is currently available with sufficient sensitivity and specificity for clinical screening utility. Chromosomal aneusomy (CA) was measured in sputum samples collected prospectively from 100 incident lung cancer cases and 96 controls matched on age, gender, and date of collection. The CA-FISH assay was performed using a four-target DNA FISH probe including EGFR, MYC, 5p15 and CEP6. Sensitivity for a positive CA-FISH assay (abnormal for ≥ 2 of the 4 markers) was substantially higher for samples collected within 18 months (76%) than >18 months before lung cancer diagnosis (31%). Specificity for a positive FISH by this same definition was 85%. Among subjects providing sputum sample within 18 months before diagnosis, sensitivity was higher for squamous cell cancers (94%) than for other histologic types (69%). The adjusted odds ratios for specimens collected within 18 months of cancer diagnosis were higher using the CA-FISH assay (OR=27.2, 95% CI 7.8 to 94.1) than previous studies assessing cytologic atypia (OR=2.3, CI 0.8 to 6.4) or gene promoter methylation (OR=6.5; CI 1.2 to 35.5). In conclusion, chromosomal aneusomy in sputum is a promising biomarker for prediction of lung cancer risk. Evaluation of the 4-DNA targets was more effective than any single marker and had highest sensitivity for samples collected ≤ 18 months to lung cancer diagnosis and patients diagnosed with squamous cell carcinoma. PMID:20332298

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

    PubMed

    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.

  8. Insulin activates EGFR by stimulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells.

    PubMed

    Shin, Miyoung; Yang, Eun Gyeong; Song, Hyun Kyu; Jeon, Hyesung

    2015-06-01

    The expression of epidermal growth factor receptor (EGFR) is an important diagnostic marker for triple-negative breast cancer (TNBC) cells, which lack three hormonal receptors: estrogen and progesterone receptors as well as epidermal growth factor receptor 2. EGFR transactivation can cause drug resistance in many cancers including TNBC, but the mechanism underlying this phenomenon is poorly defined. Here, we demonstrate that insulin treatment induces EGFR activation by stimulating the interaction of EGFR with insulin-like growth factor receptor 1 (IGF-1R) in the MDA-MB-436 TNBC cell line. These cells express low levels of EGFR, while exhibiting high levels of IGF-1R expression and phosphorylation. Low-EGFRexpressing MDA-MB-436 cells show high sensitivity to insulinstimulated cell growth. Therefore, unexpectedly, insulin stimulation induced EGFR transactivation by regulating its interaction with IGF-1R in low-EGFR-expressing TNBC cells.

  9. 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

  10. Copper improves the anti-angiogenic activity of disulfiram through the EGFR/Src/VEGF pathway in gliomas.

    PubMed

    Li, Yi; Fu, Shi-Yuan; Wang, Li-Hui; Wang, Fang-Yang; Wang, Nan-Nan; Cao, Qi; Wang, Ya-Ting; Yang, Jing-Yu; Wu, Chun-Fu

    2015-12-01

    Disulfiram (DSF) possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. DSF also potently inhibits angiogenesis, but the effect of Cu on this anti-angiogenic activity is unknown. Here we show that DSF inhibits the proliferation, migration, invasion, adhesion and complex tube formation of human umbilical vascular endothelial cells (HUVECs). Aortic ring assays and Matrigel plug assays revealed that DSF significantly inhibited the formation of microvessels. Importantly, Cu improved the anti-angiogenic activity of DSF in all these assays, while copper alone had no effect. DSF/Cu treatment of U87 human glioblastoma cells resulted in suppression of VEGF secretion through the EGFR/c-Src/VEGF pathway. Reduction of EGFR phosphorylation disables recruitment of multiple Src homology 2 (SH2) domains, resulting in transcriptional down-regulation of VEGF. The role of EGFR/c-Src/VEGF pathway was further confirmed by using specific inhibitor, which significantly improved the anti-angiogenic activity of DSF/Cu. DSF/Cu also exerted increased anti-tumor effects on subcutaneous and intracerebral U87 xenograft models by reducing microvessel density (MVD) and VEGF expression. These results indicate that Cu improves the anti-angiogenic activity of DSF by targeting the EGFR/Src/VEGF signaling pathway, thus providing a rationale for the use of DSF/Cu rather than DSF alone as an angiogenesis inhibitor in clinical applications. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Utility of Gene Expression and Ex vivo Steroid Production in a 96 h Assay for Predicting Impacts of Endocrine Active Chemicals on Fish Reproduction.

    EPA Science Inventory

    Development of efficient test methods that can generate reliable data to inform risk assessment is an on-going challenge in the field of ecotoxicology. In the present study we evaluated whether a 96 h in vivo assay focused on a small number of quantitative real-time polymerase ch...

  12. Utility of Gene Expression and Ex vivo Steroid Production in a 96 h Assay for Predicting Impacts of Endocrine Active Chemicals on Fish Reproduction.

    EPA Science Inventory

    Development of efficient test methods that can generate reliable data to inform risk assessment is an on-going challenge in the field of ecotoxicology. In the present study we evaluated whether a 96 h in vivo assay focused on a small number of quantitative real-time polymerase ch...

  13. Cytomorphologic features of advanced lung adenocarcinomas tested for EGFR and KRAS mutations: a retrospective review of 50 cases.

    PubMed

    Marotti, Jonathan D; Schwab, Mary C; McNulty, Nancy J; Rigas, James R; DeLong, Peter A; Memoli, Vincent A; Tsongalis, Gregory J; Padmanabhan, Vijayalakshmi

    2013-01-01

    Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin-embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real-time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group.

  14. Bisphosphonates enhance antitumor effect of EGFR-TKIs in patients with advanced EGFR mutant NSCLC and bone metastases

    PubMed Central

    Zhang, Guowei; Cheng, Ruirui; Zhang, Zengli; Jiang, Tao; Ren, Shengxiang; Ma, Zhiyong; Zhao, Sha; Zhou, Caicun; Zhang, Jun

    2017-01-01

    Whether bisphosphonates could enhance the effect of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC) patients with EGFR mutation and bone metastases (BM) remains unknown. EGFR mutation status were collected from 1560 patients with NSCLC and BM. 356 NSCLC patients with EGFR mutation and BM were identified. Among them, 91 patients received EGFR-TKIs alone and 105 patients received EGFR-TKIs plus bisphosphonates as first-line therapy. Comparing to TKIs alone, EGFR-TKIs plus bisphosphonates had a statistically significant longer progression-free survival (PFS: 11.6 vs. 9.3 months; HR = 0.68, P = 0.009), while a similar overall survival (OS: 20.5 vs. 19.5 months; HR = 0.95, P = 0.743) in patients with EGFR-mutant NSCLC and BM. The incidence of skeletal-related events in combined group was numerically lower than that in EGFR-TKIs alone group (29.7% vs. 39.4%, P = 0.147). In multivariate analysis, EGFR mutation was found to be a significant independent prognostic factor for OS in NSCLC patients with BM (HR = 0.710, P = 0.021). In conclusion, EGFR mutation was the significant independent prognostic factor for OS and the addition of bisphosphonates to EGFR-TKIs could enhance the antitumor effect of EGFR-TKIs in patients with EGFR-mutant NSCLC and BM. PMID:28211502

  15. Anticancer drugs exert differential apoptotic and cytotoxic effects on glioblastoma primary cultures with various EGFR and bcl-2 profiles.

    PubMed

    Pédeboscq, Stéphane; L'Azou, Béatrice; Passagne, Isabelle; De Giorgi, Francesca; Ichas, François; Liguoro, Dominique; Pometan, Jean-Paul; Cambar, Jean

    2009-01-01

    The aim of this study was to determine the apoptotic and cytotoxic effects induced on glioblastoma cells by various anticancer agents that possess different mechanisms of action (alkylating drugs, anti-EGFR (Epidermal Growth Factor receptor), proteasome inhibitor). Primary cell cultures were obtained from patients who underwent surgery for their glioblastoma. The cytotoxic effects of drugs were determined by MTT (dimethylthiazolyl diphenyl tetrazolium bromide) assay and apoptosis was evaluated by measuring mitochondrial potential by flow cytometry. Biological markers (EGFR, bcl-2) were studied by a immunoblotting technique to find out predictive markers of response. We found a large interindividual sensitivity, thus confirming the interest of the primary cultures. New proteasome inhibitor bortezomib had considerable cytotoxic and apoptotic potential in glioblastoma, even at very low concentrations. Moreover, the characterization of patients' cells for EGFR and bcl-2 status could constitute an interest, with the evaluation of other markers, in the study of expected chemotherapy response.

  16. Antimicrobial assays of natural extracts and their inhibitory effect against Listeria innocua and fish spoilage bacteria, after incorporation into biopolymer edible films.

    PubMed

    Iturriaga, L; Olabarrieta, I; de Marañón, I Martínez

    2012-08-01

    The antimicrobial activity of twelve natural extracts was tested against two fish spoilage bacteria (Pseudomonas fluorescens and Aeromonas hydrophila/caviae) and Listeria innocua, in order to assess their potential utilization in the preservation and safety of minimally processed fish products. After a screening of the active extracts by agar diffusion and vapour diffusion methods, oregano and thyme essential oils and citrus extract were selected. The minimum inhibitory concentration (MIC) of the selected extracts was determined by disc diffusion method against target bacteria and at two temperatures: bacteria's optimal growth temperature (30 °C or 37 °C) and refrigeration temperature (4 °C). Due to its better solubility, lack of odour and greater inhibitory effect obtained against L. innocua at refrigerated temperature, citrus extract was selected and incorporated at 1% (v/v) into different biopolymer film forming solutions (gelatin, methyl cellulose and their blend 50:50 w/w). The antimicrobial activity of the developed films was then evaluated, just after preparation of the films and after one month of storage at 43±3% relative humidity and 24±3 °C. Regardless of the biopolymer matrix, all the developed films showed antimicrobial activity against the target bacteria. The most sensitive bacterium towards active films was L. innocua while P. fluorescens appeared as the most resistant one, in accordance with the previously performed antimicrobial tests for pure extracts. The differences in activity of the films between the tested two temperatures were not significant except for L. innocua, for which three times higher inhibition diameters were observed at refrigerated temperature. The inhibitory effectiveness of the films against the tested strains was maintained regardless of the biopolymer matrix for at least one month. Therefore, these edible films show potential for their future use in fresh fish fillets preservation.

  17. Association of EGFR Exon 19 Deletion and EGFR-TKI Treatment Duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients

    PubMed Central

    Matsuo, Norikazu; Azuma, Koichi; Sakai, Kazuko; Hattori, Satoshi; Kawahara, Akihiko; Ishii, Hidenobu; Tokito, Takaaki; Kinoshita, Takashi; Yamada, Kazuhiko; Nishio, Kazuto; Hoshino, Tomoaki

    2016-01-01

    The most common event responsible for resistance to first- and second-generation (1st and 2nd) epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) is acquisition of T790M mutation. We examined whether T790M is related to clinicopathologic or prognostic factors in patients with relapse of EGFR mutant non-small cell lung cancer (NSCLC) after treatment with 1st or 2nd EGFR-TKIs. We retrospectively reviewed the T790M status and clinical characteristics of 73 patients with advanced or recurrent NSCLC who had been treated with EGFR-TKIs and undergone rebiopsy at Kurume University Hospital between March 2005 and December 2015. T790M mutation was more frequent in patients with EGFR exon 19 deletion mutation (63%, 26/41) than in those with L858R mutation (38%, 12/32) (p = 0.035). The median total duration of 1st or 2nd EGFR-TKI treatment was significantly longer in patients with T790M mutation than in those without (15.3 months vs 8.1 months, p < 0.001). Multivariate analysis revealed that the type of EGFR mutation and the total duration of EGFR-TKI treatment were significantly associated with T790M prevalence. Patients with EGFR exon 19 deletion mutation who receive long-term EGFR-TKI therapy show a high prevalence of T790M mutation. The present data are potentially important for clinical decision-making in NSCLC patients with EGFR mutation. PMID:27811988

  18. The S492R EGFR ectodomain mutation is never detected in KRAS wild-type colorectal carcinoma before exposure to EGFR monoclonal antibodies.

    PubMed

    Esposito, Claudia; Rachiglio, Anna Maria; La Porta, Maria Libera; Sacco, Alessandra; Roma, Cristin; Iannaccone, Alessia; Tatangelo, Fabiana; Forgione, Laura; Pasquale, Raffaella; Barbaro, Americo; Botti, Gerardo; Ciardiello, Fortunato; Normanno, Nicola

    2013-12-01

    The activity of the epidermal growth factor receptor (EGFR) antibodies cetuximab and panitumumab in metastatic colorectal carcinoma (mCRC) is significantly limited by molecular mechanisms leading to intrinsic or acquired resistance. The S492R mutation of the EGFR, which is caused by either the 1476C>A or the 1474A>C substitution, interferes with binding to cetuximab but not to panitumumab, and has been detected in mCRC with acquired resistance to cetuximab. Since mechanisms of acquired and intrinsic resistance to EGFR monoclonal antibodies in CRC significantly overlap, we evaluated the frequency of the S492R mutation in a series of KRAS-exon 2 wild-type CRC patients. Genomic DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues that were obtained from 505 systemic therapy-naïve CRC patients. A PCR/sequencing method for the detection of the S492R mutation was developed, by using as positive control a plasmid in which the 1474A>C mutation was generated by site directed mutagenesis. The lowest level of detection of this assay was approximately 10% mutant DNA in a background of wild-type DNA. PCR sequencing analysis revealed no S492R mutations in any of the analyzed 505 CRC specimens. Our findings suggest that the S492R mutation is not involved in primary resistance to cetuximab in CRC. Therefore, patients with mCRC should not be routinely screened for this mutation prior therapy with cetuximab.

  19. EGFR regulates macrophage activation and function in bacterial infection.

    PubMed

    Hardbower, Dana M; Singh, Kshipra; Asim, Mohammad; Verriere, Thomas G; Olivares-Villagómez, Danyvid; Barry, Daniel P; Allaman, Margaret M; Washington, M Kay; Peek, Richard M; Piazuelo, M Blanca; Wilson, Keith T

    2016-09-01

    EGFR signaling regulates macrophage function, but its role in bacterial infection has not been investigated. Here, we assessed the role of macrophage EGFR signaling during infection with Helicobacter pylori, a bacterial pathogen that causes persistent inflammation and gastric cancer. EGFR was phosphorylated in murine and human macrophages during H. pylori infection. In human gastric tissues, elevated levels of phosphorylated EGFR were observed throughout the histologic cascade from gastritis to carcinoma. Deleting Egfr in myeloid cells attenuated gastritis and increased H. pylori burden in infected mice. EGFR deficiency also led to a global defect in macrophage activation that was associated with decreased cytokine, chemokine, and NO production. We observed similar alterations in macrophage activation and disease phenotype in the Citrobacter rodentium model of murine infectious colitis. Mechanistically, EGFR signaling activated NF-κB and MAPK1/3 pathways to induce cytokine production and macrophage activation. Although deletion of Egfr had no effect on DC function, EGFR-deficient macrophages displayed impaired Th1 and Th17 adaptive immune responses to H. pylori, which contributed to decreased chronic inflammation in infected mice. Together, these results indicate that EGFR signaling is central to macrophage function in response to enteric bacterial pathogens and is a potential therapeutic target for infection-induced inflammation and associated carcinogenesis.

  20. 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

  1. Jab1 is a target of EGFR signaling in ERα-negative breast cancer

    PubMed Central

    Wang, Jiaxu; Barnes, Rebecca O; West, Nathan R; Olson, Melanie; Chu, Jenny E; Watson, Peter H

    2008-01-01

    Introduction c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα- phenotype. Methods MDA-MB-231 and MDA-MB-468 ERα-/EGFR+ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry. Results EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004). Conclusion Jab1 is a target of EGFR signaling in ERα- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα- breast cancer. PMID:18534028

  2. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  3. Novel Quinazoline Derivatives Bearing Various 4-Aniline Moieties as Potent EGFR Inhibitors with Enhanced Activity Against NSCLC Cell Lines.

    PubMed

    Wang, Changyan; Sun, Yajun; Zhu, Xingqi; Wu, Bin; Wang, Qiao; Zhen, Yuhong; Shu, Xiaohong; Liu, Kexin; Zhou, Youwen; Ma, Xiaodong

    2016-04-01

    A class of novel quinazoline derivatives bearing various C-4 aniline moieties was synthesized and biologically evaluated as potent epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Most of these inhibitors are comparable to gefitinib in inhibiting these cancer cell lines, and several of them even displayed superior inhibitory activity. In particular, analogue 5b with an IC50 of 0.10 μm against the EGFR wild-type A431 cells and 5c with an IC50 of 0.001 μm against the gefitinib-sensitive HCC827 cells (EGFR del E746-A750) was identified as highly active EGFR inhibitors. It was also significant that the discovered analogue 2f, not only has high potency against the gefitinib-sensitive cells (IC50 = 0.031 μm), but also possesses remarkably improved activity against the gefitinib-resistant cells. In addition, the enzymatic assays and the Western blot analysis for evaluating the effects of the typical inhibitors indicated that these molecules strongly interfere with the EGFR target.

  4. Anti-EGFR-Conjugated Hollow Gold Nanospheres Enhance Radiocytotoxic Targeting of Cervical Cancer at Megavoltage Radiation Energies

    NASA Astrophysics Data System (ADS)

    Liu, Jiao; Liang, Ying; Liu, Ting; Li, Dengke; Yang, Xingsheng

    2015-05-01

    The study aimed to confirm that anti-epidermal growth factor receptor (EGFR) monoclonal antibody-conjugated hollow gold nanospheres (anti-EGFR/HGNs) can be selectively uptaken by cervical cancer cells and induce its apoptosis when combined with radiotherapy, as a result enhancing radiosensitivity of cervical cancer cells. HGNs with a mean diameter of 54.6 ± 7.11 nm and wall thickness of 5.01 ± 2.23 nm were viewed by transmission electron microscopy (TEM). Cell uptake was assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The cytotoxicity on HeLa cells, which were used in our experiment, was assessed by CCK-8 assay. Cell cycle and apoptosis were examined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry (FCM). The expression of several critical apoptosis-related proteins, including Bcl-2, Bax, Bad, and active caspase 3, was tested by western blot analysis. Cells treated by anti-EGFR/HGNs showed an obvious increase in nanoparticle uptake compared to naked HGNs. Anti-EGFR/HGNs combined with radiation resulted in a significant growth inhibition, compared with radiation combined with naked HGNs. Anti-EGFR/HGNs remarkably increased the ratio of HeLa cells in the G2/M phase and induced more apoptosis by an obvious deregulation of Bcl-2 and upregulation of Bax, Bad, and caspase 3 when combined with radiation. Therefore, anti-EGFR/HGNs can increase the targeted uptake of HGNs by HeLa cells and enhance radiocytotoxic targeting of cervical cancer at megavoltage radiation energies.

  5. Radiosensitization of EGFR/HER2 positive pancreatic cancer is mediated by inhibition of Akt independent of Ras mutational status

    PubMed Central

    Kimple, Randall J.; Vaseva, Angelina V.; Cox, Adrienne D.; Baerman, Kathryn M.; Calvo, Benjamin F.; Tepper, Joel E.; Shields, Janiel M.; Sartor, Carolyn I.

    2009-01-01

    Purpose Epidermal growth factor receptor family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations. Experimental Design Expression of EGFR family members in pancreatic cancer lines was assessed by qRT-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity. Results Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and ERK was seen in lines expressing mutant K-ras. Overexpression of constitutively-active K-ras(G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MEK/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio 1.2–1.8) or nelfinavir (enhancement ratio 1.2–1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5). Conclusions Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other PI3K/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status. PMID:20103665

  6. Nimotuzumab Induces NK Cell Activation, Cytotoxicity, Dendritic Cell Maturation and Expansion of EGFR-Specific T Cells in Head and Neck Cancer Patients.

    PubMed

    Mazorra, Zaima; Lavastida, Anabel; Concha-Benavente, Fernando; Valdés, Anet; Srivastava, Raghvendra M; García-Bates, Tatiana M; Hechavarría, Esperanza; González, Zuyen; González, Amnely; Lugiollo, Martha; Cuevas, Iván; Frómeta, Carlos; Mestre, Braulio F; Barroso, Maria C; Crombet, Tania; Ferris, Robert L

    2017-01-01

    Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab's capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a (51)Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated.

  7. Nimotuzumab Induces NK Cell Activation, Cytotoxicity, Dendritic Cell Maturation and Expansion of EGFR-Specific T Cells in Head and Neck Cancer Patients

    PubMed Central

    Mazorra, Zaima; Lavastida, Anabel; Concha-Benavente, Fernando; Valdés, Anet; Srivastava, Raghvendra M.; García-Bates, Tatiana M.; Hechavarría, Esperanza; González, Zuyen; González, Amnely; Lugiollo, Martha; Cuevas, Iván; Frómeta, Carlos; Mestre, Braulio F.; Barroso, Maria C.; Crombet, Tania; Ferris, Robert L.

    2017-01-01

    Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab’s capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a 51Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated. PMID:28674498

  8. Computational analysis of EGFR inhibition by Argos.

    PubMed

    Reeves, Gregory T; Kalifa, Rachel; Klein, Daryl E; Lemmon, Mark A; Shvartsman, Stanislav Y

    2005-08-15

    Argos, a secreted inhibitor of the Drosophila epidermal growth factor receptor, and the only known secreted receptor tyrosine kinase inhibitor, acts by sequestering the EGFR ligand Spitz. We use computational modeling to show that this biochemically-determined mechanism of Argos action can explain available genetic data for EGFR/Spitz/Argos interactions in vivo. We find that efficient Spitz sequestration by Argos is key for explaining the existing data and for providing a robust feedback loop that modulates the Spitz gradient in embryonic ventral ectoderm patterning. Computational analysis of the EGFR/Spitz/Argos module in the ventral ectoderm shows that Argos need not be long-ranged to account for genetic data, and can actually have very short range. In our models, Argos with long or short length scale functions to limit the range and action of secreted Spitz. Thus, the spatial range of Argos does not have to be tightly regulated or may act at different ranges in distinct developmental contexts.

  9. NSCLC harboring EGFR exon-20 insertions after the regulatory C-helix of kinase domain responds poorly to known EGFR inhibitors.

    PubMed

    Yang, Mengmeng; Xu, Xiaoxi; Cai, Jie; Ning, Jinying; Wery, Jean Pierre; Li, Qi-Xiang

    2016-07-01

    Anecdote clinical observations hint that non-small cell lung cancer (NSCLC) with exon-20 insertions might respond poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), contrasting to those with classic mutations. Lack of patient-derived experimental models has been a major hurdle for the discovery of new treatment for the diseases. We established two NSCLC-PDXs harboring two different exon-20 insertions, LU0387-adenocarcinoma (ADC) with a nine-base insertion at 2319 (H773-V774insNPH) and LU3075-squamous cell carcinoma (SCC) with a nine-base insertion at 2316 (P772-H773insDNP). Both insertions immediately follow the regulatory C-helix of the kinase domain. Contrary to the generally good responses to EGFR inhibitors observed in PDXs with classic mutations, both exon-20 insertions are largely resistant to cetuximab and TKIs in vivo, suggesting fundamental difference from the classic EGFR mutations, consistent with the poor response rate to TKI seen in anecdotal clinic reports. It is worth noting that although responses are generally poor, they differ between the two exon-20 mutants depending on the type of TKI. In vitro drug sensitivity assays using established primary cell lines from our two PDXs largely confirmed the in vivo data. Our data from patient-derived experimental models confirmed that exon-20 insertions in domain immediately following the C-helix confer poor response to all known EGFR inhibitors, and suggested that these models can be utilized to facilitate the discovery of new therapies targeting NSCLC harboring exon-20 insertions.

  10. A systematic profile of clinical inhibitors responsive to EGFR somatic amino acid mutations in lung cancer: implication for the molecular mechanism of drug resistance and sensitivity.

    PubMed

    Ai, Xinghao; Sun, Yingjia; Wang, Haidong; Lu, Shun

    2014-07-01

    Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of patients with non-small cell lung cancer (NSCLC). However, a large number of somatic mutations in such protein have been observed to cause drug resistance or sensitivity during pathological progression, limiting the application of reversible EGFR tyrosine kinase inhibitor therapy in NSCLC. In the current work, we describe an integration of in silico analysis and in vitro assay to profile six representative EGFR inhibitors against a panel of 71 observed somatic mutations in EGFR tyrosine kinase domain. In the procedure, the changes in interaction free energy of inhibitors with EGFR upon various mutations were calculated one by one using a rigorous computational scheme, which was preoptimized based on a set of structure-solved, affinity-known samples to improve its performance in characterizing the EGFR-inhibitor system. This method was later demonstrated to be effective in inferring drug response to the classical L858R and G719S mutations that confer constitutive activation for the EGFR kinase. It is found that the Staurosporine, a natural product isolated from the bacterium Streptomyces staurosporeus, exhibits selective inhibitory activity on the T790M and T790M/L858R mutants. This finding was subsequently solidified by in vitro kinase assay experiment; the inhibitory IC50 values of Staurosporine against wild-type, T790M and T790M/L858R mutant EGFR were measured to be 937, 12 and 3 nM, respectively.

  11. Induced sensitivity to EGFR inhibitors is mediated by palmitoylated cysteine 1025 of EGFR and requires oncogenic Kras.

    PubMed

    Kharbanda, Akriti; Runkle, Kristin; Wang, Wei; Witze, Eric S

    2017-11-04

    Currently, there are no effective therapeutic strategies targeting Kras driven cancers, and therefore, identifying new targeted therapies and overcoming drug resistance have become paramount for effective long-term cancer therapy. We have found that reducing expression of the palmitoyl transferase DHHC20 increases cell death induced by the EGFR inhibitor gefitinib in Kras and EGFR mutant cell lines, but not MCF7 cells harboring wildtype Kras. We show that the increased gefitinib sensitivity in cancer cells induced by DHHC20 inhibition is mediated directly through loss of palmitoylation on a previously identified cysteine residue in the C-terminal tail of EGFR. We utilized an EGFR point mutant in which the palmitoylated cysteine 1025 is mutated to alanine (EGFR(C1025A)), that results in receptor activation. Expression of the EGFR mutant alone in NIH3T3 cells does not increase sensitivity to gefitinib-induced cell death. However, when EGFR(C1025A) is expressed in cells expressing activated Kras(G12V), EGFR inhibitor induced cell death is increased. Surprisingly, lung cancer cells harboring the EGFR inhibitor resistant mutation, T790M, become sensitive to EGFR inhibitor treatment when DHHC20 is inhibited. Finally, the small molecule, 2-bromopalmitate, which has been shown to inhibit palmitoyl transferases, acts synergistically with gefitinib to induce cell death in the gefitinib resistant cell line NCI-H1975. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Gefitinib Treatment in EGFR Mutated Caucasian NSCLC

    PubMed Central

    Ostoros, Gyula; Cobo, Manuel; Ciuleanu, Tudor; Cole, Rebecca; McWalter, Gael; Walker, Jill; Dearden, Simon; Webster, Alan; Milenkova, Tsveta; McCormack, Rose

    2014-01-01

    Introduction: In the phase IV, open-label, single-arm study NCT01203917, first-line gefitinib 250 mg/d was effective and well tolerated in Caucasian patients with epidermal growth factor receptor (EGFR) mutation-positive non–small-cell lung cancer (previously published). Here, we report EGFR mutation analyses of plasma-derived, circulating-free tumor DNA. Methods: Mandatory tumor and duplicate plasma (1 and 2) baseline samples were collected (all screened patients; n = 1060). Preplanned, exploratory analyses included EGFR mutation (and subtype) status of tumor versus plasma and between plasma samples. Post hoc, exploratory analyses included efficacy by tumor and plasma EGFR mutation (and subtype) status. Results: Available baseline tumor samples were 1033 of 1060 (118 positive of 859 mutation status known; mutation frequency, 13.7%). Available plasma 1 samples were 803 of 1060 (82 positive of 784 mutation status known; mutation frequency, 10.5%). Mutation status concordance between 652 matched tumor and plasma 1 samples was 94.3% (95% confidence interval [CI], 92.3–96.0) (comparable for mutation subtypes); test sensitivity was 65.7% (95% CI, 55.8–74.7); and test specificity was 99.8% (95% CI, 99.0–100.0). Twelve patients of unknown tumor mutation status were subsequently identified as plasma mutation-positive. Available plasma 2 samples were 803 of 1060 (65 positive of 224 mutation status-evaluable and -known). Mutation status concordance between 224 matched duplicate plasma 1 and 2 samples was 96.9% (95% CI, 93.7–98.7). Objective response rates are as follows: mutation-positive tumor, 70% (95% CI, 60.5–77.7); mutation-positive tumor and plasma 1, 76.9% (95% CI, 65.4–85.5); and mutation-positive tumor and mutation-negative plasma 1, 59.5% (95% CI, 43.5–73.7). Median progression-free survival (months) was 9.7 (95% CI, 8.5–11.0; 61 events) for mutation-positive tumor and 10.2 (95% CI, 8.5–12.5; 36 events) for mutation-positive tumor and plasma 1

  13. Mechanisms of resistance to EGFR tyrosine kinase inhibitors

    PubMed Central

    Huang, Lihua; Fu, Liwu

    2015-01-01

    Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies. PMID:26579470

  14. 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

  15. EGFR Signaling in Breast Cancer: Bad to the Bone

    PubMed Central

    Foley, John; Nickerson, Nicole; Nam, Seugyoon; Allen, Kah Tan; Gilmore, Jennifer L.; Nephew, Kenneth P.; Riese, David J.

    2010-01-01

    The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. This family includes EGFR/ErbB1/HER1, ErbB2/HER2/Neu ErbB3/HER3, and ErbB4/HER4. For many years it was believed that EGFR plays a minor role in the development and progression of breast malignancies. However, recent findings have led investigators to revisit these beliefs. Here we will review these findings and propose roles that EGFR may play in breast malignancies. In particular, we will discuss the potential roles that EGFR may play in triple-negative tumors, resistance to endocrine therapies, maintenance of stem-like tumor cells, and bone metastasis. Thus, we will propose the contexts in which EGFR may be a therapeutic target. PMID:20813200

  16. Determination of HER-2 status and chromosome 17 polysomy in breast carcinomas comparing HercepTest and PathVysion FISH assay.

    PubMed

    Varshney, Dolly; Zhou, Yvonne Y; Geller, Stephen A; Alsabeh, Randa

    2004-01-01

    We evaluated and compared 2 HER-2 tests (immunohistochemical analysis [HercepTest, DAKO, Carpinteria, CA] and fluorescence in situ hybridization [FISH]) and assessed chromosome 17 polysomy status in relation to these tests. HER-2 status was obtained in 690 cases. The rinse step in the HercepTest before and after addition of the visualization reagent was 2 minutes in 188 cases and was increased to 5 minutes in 600 cases. HercepTest with both rinse steps was performed on duplicate slides in 98 cases. Chromosome 17 ploidy status based on FISH results was determined in 687 cases. Weak overexpression (2+) of HER-2 protein was not due to gene amplification in a majority of cases (67/76 [88%]). A small subset of breast carcinomas (19/687 [2.8%]) strongly overexpressed (3+) HER-2 protein without gene amplification. The aneuploidy rate was similar in negative and 2+ cases (60/141 [42.5%] and 12/26 [46%]), compared with 86% (18/21) in 3+ cases. The incidence of polysomy 17 in 2+ nonamplified cases (3/67 [4%]) was similar to that seen in negative cases (5.5%), in contrast with 47% (9/19) of 3+ nonamplified cases. Adding a longer rinse step to the HercepTest converted a subset (3/10 [30%]) of weakly positive cases to negative cases. Weak overexpression of HER-2 protein in a majority of cases seems to represent an artifactual staining pattern. Chromosome 17 polysomy is a major factor in strong HER-2 protein overexpression in 3+ nonamplified cases.

  17. Comparison of the sensitivity of four native Canadian fish species to 17-α ethinylestradiol, using an in vitro liver explant assay.

    PubMed

    Beitel, Shawn C; Doering, Jon A; Eisner, Bryanna K; Hecker, Markus

    2015-12-01

    Exposure to environmental estrogens and other endocrine-active chemicals can impact reproduction of freshwater fishes. While extensive data exists regarding the effect of estrogens on standard laboratory species, little is known about the sensitivity of freshwater fishes native to North America to these compounds. Current testing strategies for the toxicological assessment of contaminants still rely heavily on studies with live animals, which poses increasing concerns from an economical and ethical perspective. Therefore, the aim of the present study was to investigate the sensitivity of four native species, namely, northern pike (Esox lucius), walleye (Sander vitreus), white sucker (Catostomus commersoni), and juvenile white sturgeon (Acipenser transmontanus), to an environmental estrogen, 17α-ethinylestradiol (EE2), using an in vitro tissue explant approach. Transcript abundances of vitellogenin (VTG) as well as the estrogen receptors (ER) α and β were used as the measuring endpoints as they represent well established biomarkers previously used to assess exposure to estrogens. Transcript abundance of VTG was upregulated in a concentration-dependent manner in each species. Liver explants of male walleye were found to have the greatest sensitivity to EE2, with a lowest observable effect concentration of 300 ng/L (1.0 nM) for VTG transcript abundance, with juvenile white sturgeon having the greatest magnitude of VTG transcript upregulation in exposed tissue (15-fold relative to control). Exposure of liver explants to EE2 resulted in no alteration in transcript abundance of ERβ, whereas upregulation of ERα was observed in northern pike only. Based on in vitro expression of VTG, the species tested were among the species with greatest sensitivity to environmental estrogens tested to date.

  18. 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

  19. 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

  20. Nuclear EGFR as a molecular target in cancer.

    PubMed

    Brand, Toni M; Iida, Mari; Luthar, Neha; Starr, Megan M; Huppert, Evan J; Wheeler, Deric L

    2013-09-01

    The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell's nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.

  1. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab.

    PubMed

    Brand, Toni M; Iida, Mari; Wheeler, Deric L

    2011-05-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance.

  2. Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II.

    PubMed

    Peng, Kesong; Tian, Xinqiao; Qian, Yuanyuan; Skibba, Melissa; Zou, Chunpeng; Liu, Zhiguo; Wang, Jingying; Xu, Zheng; Li, Xiaokun; Liang, Guang

    2016-03-01

    Cardiac hypertrophy is an important risk factor for heart failure. Epidermal growth factor receptor (EGFR) has been found to play a role in the pathogenesis of various cardiovascular diseases. The aim of this current study was to examine the role of EGFR in angiotensin II (Ang II)-induced cardiac hypertrophy and identify the underlying molecular mechanisms. In this study, we observed that both Ang II and EGF could increase the phospohorylation of EGFR and protein kinase B (AKT)/extracellular signal-regulated kinase (ERK), and then induce cell hypertrophy in H9c2 cells. Both pharmacological inhibitors and genetic silencing significantly reduced Ang II-induced EGFR signalling pathway activation, hypertrophic marker overexpression, and cell hypertrophy. In addition, our results showed that Ang II-induced EGFR activation is mediated by c-Src phosphorylation. In vivo, Ang II treatment significantly led to cardiac remodelling including cardiac hypertrophy, disorganization and fibrosis, accompanied by the activation of EGFR signalling pathway in the heart tissues, while all these molecular and pathological alterations were attenuated by the oral administration with EGFR inhibitors. In conclusion, the c-Src-dependent EGFR activation may play an important role in Ang II-induced cardiac hypertrophy, and inhibition of EGFR by specific molecules may be an effective strategy for the treatment of Ang II-associated cardiac diseases. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  3. 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

  4. Molecular mechanisms of resistance to the EGFR monoclonal antibody cetuximab

    PubMed Central

    Brand, Toni M; Iida, Mari

    2011-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase belonging to the HER family of receptor tyrosine kinases. Receptor activation upon ligand binding leads to down stream activation of the PI3K/AKT, RAS/RAF/MEK/ERK and PLCγ/PKC pathways that influence cell proliferation, survival and the metastatic potential of tumor cells. Increased activation by gene amplification, protein overexpression or mutations of the EGFR has been identified as an etiological factor in a number of human epithelial cancers (e.g., NSCLC, CRC, glioblastoma and breast cancer). Therefore, targeting the EGFR has been intensely pursued as a cancer treatment strategy over the last two decades. To date, five EGFR inhibitors, including three small molecule tyrosine kinase inhibitors (TKIs) and two monoclonal antibodies have gained FDA approval for use in oncology. Both approaches to targeting the EGFR have shown clinical promise and the anti-EGFR antibody cetuximab is used to treat HNSCC and CRC. Despite clinical gains arising from use of cetuximab, both intrinsic resistance and the development of acquired resistance are now well recognized. In this review we focus on the biology of the EGFR, the role of EGFR in human cancer, the development of antibody-based anti-EGFR therapies and a summary of their clinical successes. Further, we provide an in depth discussion of described molecular mechanisms of resistance to cetuximab and potential strategies to circumvent this resistance. PMID:21293176

  5. EGFR induces DNA decomposition via phosphodiester bond cleavage

    PubMed Central

    Tong, Yongpeng; Li, Shuiming; Huang, Chunliu

    2017-01-01

    EGFR may induce DNA degradation. This activity had not been previously described as an EGRF function. To confirm this unexpected activity, testing of EGFR in the presence of ATP and either 5A, 5C, 5G, 5T, or 5U oligonucleotides was performed. HPLC-MS analysis demonstrated that 5A and 5U levels significantly decreased in the presence of EGFR. Furthermore, fragments 4A and 4U were produced in 5A+EGFR+ATP and in 5U+EGFR+ATP reaction mixtures, respectively, but not in EGFR-negative controls. Degradation of Poly(A), Poly(C), Poly(G), Poly(I), Poly(T), and Poly(U) oligomers in the presence of EGFR and ATP correlated with the lower ability of reaction products to pair with complementary oligonucleotides. Gel electrophoresis showed that breakdown products migrated more quickly than controls, especially after addition of paired (complementary) oligomers, Poly(A) and Poly(U). Furthermore, λ DNA reaction products also migrated more quickly after incubation with EGFR. The results suggest that EGFR can induce breakage of certain types of nucleotide phosphodiester bonds, especially within the A residues of DNA or U residues of RNA, to induce DNA or RNA decomposition, respectively. This activity may be important in EGRF signaling, DNA degradation, or repair in normal or cancer cell activities. PMID:28272528

  6. Recent updates on third generation EGFR inhibitors and emergence of fourth generation EGFR inhibitors to combat C797S resistance.

    PubMed

    Patel, Harun; Pawara, Rahul; Ansari, Azim; Surana, Sanjay

    2017-05-11

    EGFR T790M mutation leads to resistance to most of clinically available EGFR TKIs. Third-generation EGFR TKIs against the T790M mutation have been in active clinical development, which includes osimertinib, rociletinib, HM61713, ASP8273, EGF816, and PF-06747775. On the other hand recently EGFR C797S mutation was reported to be a leading mechanism of resistance to the third-generation inhibitors. The C797S mutation appears to be an ideal target for overcoming the acquired resistance to the third generation inhibitors. This review summarizes the third generation inhibitors, synthesis, their mechanism of resistance and latest development on the discovery of a fourth-generation EGFR TKIs and U to Y allosteric strategies to combat the C797S EGFR resistance problem. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    SciTech Connect

    Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  8. 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

  9. Impacts of EGFR mutation and EGFR-TKIs on incidence of brain metastases in advanced non-squamous NSCLC.

    PubMed

    Wang, Bao-Xiao; Ou, Wei; Mao, Xiao-Yong; Liu, Zui; Wu, Hui-Qi; Wang, Si-Yu

    2017-09-01

    Brain metastases remain lethal in lung cancer patients. The impacts of epidermal growth factor receptor (EGFR) mutations and EGFR tyrosine kinase inhibitors (TKIs) on the incidence of brain metastases in patients with advanced non-squamous non-small cell lung cancer (NSCLC) are still uncertain. A total of 1672 patients with advanced non-squamous NSCLC with a definitive report on EGFR mutation status between January 2005 and June 2013 were retrospectively analyzed. The impacts of EGFR mutation status and EGFR TKIs use on the incidence of brain metastases and survival were investigated. Of the 1672 patients, 465 (27.8%) had an EGFR mutation, and 1207 (72.2%) did not. Four hundred and eighteen (25.0%) patients had baseline brain metastases. The cumulative incidence of brain metastases for patients in EGFR+ group was significantly higher than patients in EGFR- group (HR, 1.27; 95% CI 1.06-1.52; P=0.008). The cumulative incidence of brain metastases was also higher for patients who received an EGFR-TKI as their first-line treatment than those who received other first-line treatment (HR, 1.36; 95% CI 1.14-1.64; P=0.001). Patients harboring EGFR mutations had prolonged overall survival (OS) than patients with wild-type EGFR (HR, 0.47; 95% CI 0.41-0.54; P<0.001; median, 25.2 vs. 12.9 months). Both the EGFR mutation-positive status and the use of a TKI are associated with higher incidence of brain metastases for patients with advanced non-squamous NSCLC. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition

    PubMed Central

    Owen, William; Weitsman, Gregory; Fruhwirth, Gilbert; Dunn, Robert G.; Neat, Michael J.; McCaughan, Frank; Parker, Peter; Ng, Tony; Santis, George

    2017-01-01

    Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy. PMID:28141869

  11. MET-EGFR dimerization in lung adenocarcinoma is dependent on EGFR mtations and altered by MET kinase inhibition.

    PubMed

    Ortiz-Zapater, Elena; Lee, Richard W; Owen, William; Weitsman, Gregory; Fruhwirth, Gilbert; Dunn, Robert G; Neat, Michael J; McCaughan, Frank; Parker, Peter; Ng, Tony; Santis, George

    2017-01-01

    Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.

  12. Characterization of a complex chromosomal rearrangement using chromosome, FISH, and microarray assays in a girl with multiple congenital abnormalities and developmental delay.

    PubMed

    Hemmat, Morteza; Yang, Xiaojing; Chan, Patricia; McGough, Robert A; Ross, Leslie; Mahon, Loretta W; Anguiano, Arturo L; Boris, Wang T; Elnaggar, Mohamed M; Wang, Jia-Chi J; Strom, Charles M; Boyar, Fatih Z

    2014-01-01

    Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes (3, 7 and 12), seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient's developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient.

  13. Characterization of a complex chromosomal rearrangement using chromosome, FISH, and microarray assays in a girl with multiple congenital abnormalities and developmental delay

    PubMed Central

    2014-01-01

    Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes (3, 7 and 12), seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient’s developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient. PMID:25478007

  14. Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer

    PubMed Central

    Guerrab, Abderrahim El; Bamdad, Mahchid; Kwiatkowski, Fabrice; Aubel, Corinne

    2016-01-01

    Triple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy. PMID:27655662

  15. A semi-automated FISH-based micronucleus-centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation

    PubMed Central

    VRAL, ANNE; DECORTE, VEERLE; DEPUYDT, JULIE; WAMBERSIE, ANDRÉ; THIERENS, HUBERT

    2016-01-01

    The aim of the present study was to perform cytogenetic analysis by means of a semi-automated micro-nucleus-centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere-negative micronucleus (MNCM−) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low-dose' (0–100 mGy) MNCM− dose-response curve to evaluate the sensitivity and suitability of the micronucleus-centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi-automated scoring of MNCM− enables the detection of a dose of 50 mGy. The comparison of MNCM− yields in medical radiation workers and control individuals showed enhanced MNCM− scores in the medical radiation workers group (P=0.15). The highest MNCM− scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM− scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the

  16. ERβ localization influenced outcomes of EGFR-TKI treatment in NSCLC patients with EGFR mutations

    PubMed Central

    Wang, Zhijie; Li, Zhenxiang; Ding, Xiaosheng; Shen, Zhirong; Liu, Zhentao; An, Tongtong; Duan, Jianchun; Zhong, Jia; Wu, Meina; Zhao, Jun; Zhuo, Minglei; Wang, Yuyan; Wang, Shuhang; Sun, Yu; Bai, Hua; Wang, Jie

    2015-01-01

    Effects of estrogen receptorβ (ERβ) localization on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC) are unknown. First, we analyzed the relationship between ERβ localization determined by immunohistochemistry and EGFR-TKI outcomes in 184 patients with advanced NSCLC and found that ERβ expression localized in the cytoplasm and/or nucleus. The frequency of cytoplasmic ERβ (c-ERβ) and nuclear ERβ (n-ERβ) co-expression was 12% (22/184). C-ERβ and n-ERβ co-expression was correlated with poor median progression-free survival compared to patients without co-expression. In subsequent in vitro experiments, PC9 cells transfected with ERβ isoform1 (ERβ1, strong expression of both c-ERβ and n-ERβ) were more resistant to gefitinib than PC9 cells transfected with ERβ isoform2 or 5 (ERβ2 or ERβ5, strong expression of ERβ in cytoplasm but not nucleus). Resistance was identified due to interactions between ERβ1 and other isoforms, and mediated by activation of non-genomic pathways. Moreover, gefitinib resistance was reversed by a combination treatment with gefitinib and fulvestrant, both in cell lines and in one NSCLC patient. These results suggested that c-ERβ and n-ERβ co-expression was a potential molecular indicator of EGFR-TKI resistance, which might be overcome by combining EGFR-TKI and ER antagonist. PMID:26096604

  17. Immunohistochemical detection of EGFR in paraffin-embedded tumor tissues: variation in staining intensity due to choice of fixative and storage time of tissue sections.

    PubMed

    Atkins, Derek; Reiffen, Karl-August; Tegtmeier, Conny Lund; Winther, Henrik; Bonato, Marcellus S; Störkel, Stephan

    2004-07-01

    The epidermal growth factor receptor (EGFR) is highly expressed in a variety of solid malignant tumors and its expression has been correlated with disease progression and poor survival. With the advent of targeted therapies, especially IMC-C225 (Cetuximab), a monoclonal antibody (MAb) directed against the EGFR, there is an increasing interest in immunohistochemistry (IHC)-based EGFR screening methods using paraffin-embedded tumor specimens to select cancer patients eligible for treatment with Cetuximab. With the EGFRpharmDX kit, a complete assay for demonstration of EGFR is now available. Because no information about the preservation of the EGFR under various conditions of fixation is available, we performed a prospective study on a panel of commonly used fixatives to determine optimal tissue preservation protocols. The stability of the epitope on cut tissue sections stored for a period up to 24 month was also tested using material originating from patients with head and neck cancer, non-small-cell lung carcinomas, and colorectal adenocarcinomas. Depending on the fixative used and the time of storage of cut tissue sections, a variation in the determined level of EGFR expression was demonstrated compared with the most optimal fixation procedure.

  18. Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

    PubMed Central

    2011-01-01

    Background Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment. The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness. Results In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation. Conclusion Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC

  19. HER2 overexpression reverses the relative resistance of EGFR-mutant H1975 cell line to gefitinib.

    PubMed

    Xu, Jing; Shen, Li; Zhang, Bi-Cheng; Xu, Wen-Hong; Ruan, Shu-Qin; Pan, Chi; Wei, Qi-Chun

    2016-12-01

    Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) that has been demonstrated to be clinically useful for the treatment of patients with non-small cell lung cancer (NSCLC). However, ~50% of patients do not respond to EGFR TKI treatment through the emergence of mutations, such as T790M. Therefore, it is important to determine which patients are eligible for treatment with gefitinib. As a preferred dimerization partner for EGFR, the role of EGFR 2 (HER2) in mediating sensitivity to gefitinib is poorly understood. In the present study, full-length human HER2 cDNA was introduced to the NSCLC cell lines H1975 and H1299, which have a low endogenous expression level of HER2. In addition, it was observed in the present study that the H1975 cell line harbored the L858R and T790M mutations in the EGFR kinase domain. Western blot analysis and MTT assay were used to evaluate the TKI sensitivity of HER2 expression status, and the activation of HER3 and HER2 downstream effectors. The results indicated that the sensitivity of H1975 cells to gefitinib was restored by the overexpression of HER2, which stimulated HER2-driven signaling cascades accompanied by the activation of protein kinase B. By contrast, ectopic HER2 overexpression in H1299 cells did not significantly alter the sensitivity to gefitinib treatment. In conclusion, the current study results suggested that the relatively resistance of the H1975 cell line to gefitinib could be reversed by the overexpression of HER2. Therefore, the expression of HER2 could also be considered when evaluate the patients' potential response to gefitinib, particularly in the subgroup of lung cancer patients who harbor an EGFR mutation.

  20. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

    PubMed

    da Rosa, Marina Rolo Pinheiro; Falcão, Aline Semblano Carreira; Fuzii, Hellen Thais; da Silva Kataoka, Maria Sueli; Ribeiro, André L R; Boccardo, Enrique; de Siqueira, Adriane Sousa; Jaeger, Ruy G; de Jesus Viana Pinheiro, João; de Melo Alves Júnior, Sérgio

    2014-11-01

    Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion

  1. One Fish Two Fish.

    ERIC Educational Resources Information Center

    Hoffman, Michele

    1998-01-01

    This activity explains fisheries resource management to seven-year olds. First-grade students learn concepts such as offspring viability, life expectancy, and distribution of species, which help to determine when, where, and how people fish and the importance of fishing responsibly. Lists materials, procedures, and extensions. (SJR)

  2. One Fish Two Fish.

    ERIC Educational Resources Information Center

    Hoffman, Michele

    1998-01-01

    This activity explains fisheries resource management to seven-year olds. First-grade students learn concepts such as offspring viability, life expectancy, and distribution of species, which help to determine when, where, and how people fish and the importance of fishing responsibly. Lists materials, procedures, and extensions. (SJR)

  3. The effect of insecticides chlorpyrifos, α-cypermethrin and imidacloprid on primary DNA damage, TP 53 and c-Myc structural integrity by comet-FISH assay.

    PubMed

    Zeljezic, Davor; Vinkovic, Benjamin; Kasuba, Vilena; Kopjar, Nevenka; Milic, Mirta; Mladinic, Marin

    2017-09-01

    In parallel with the continuous use of conventional insecticides, introduction of more environmentally friendly substances continues to grow in modern agriculture. In the present study, we evaluated chlorpyrifos, and imidacloprid and α-cypermethrin as two representatives of green insecticides for their genotoxic activity. We conducted a 14-day treatment in extended human lymphocytes cultures using real life exposure relevant concentrations. An alkaline comet assay was used to detect primary DNA damage. Simultaneously, the effect on the specific action towards the TP 53 and c-Myc genes in terms of fragmentation and copy number were determined. Both genes are responsible for cell cycle regulation; thus playing an active role in carcinogenesis. Contrary to what was expected, imidacloprid showed the highest genotoxicity potential, irrespective of the fact that none of the insecticides induced a significant level of primary DNA damage at all tested concentrations. Similar, no significant effect towards the TP 53 and c-Myc gene was recorded. The present study indicates that low level use of chlorpyrifos as a conventional insecticide and imidacloprid and α-cypermethrin as green insecticides does not pose a risk to DNA in general, nor to the TP 53 and c-Myc gene structural integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. MicroRNA-137 inhibits growth of glioblastoma through EGFR suppression

    PubMed Central

    Zhang, Zhenxing; Song, Xiaofeng; Tian, He; Miao, Ye; Feng, Xu; Li, Yang; Wang, Honglei

    2017-01-01

    Aberrant expression of certain microRNAs (miRNAs) has been shown to contribute to the development of Glioblastoma multiforme (GBM). However, the involvement of miR-137 in the carcinogenesis of GBM has not been reported. Here, we showed that miR-137 levels in GBM tissues were significantly lower than the paired normal brain tissue in patients’ specimens. Moreover, low miR-137 levels in GBM tissue were associated with poor prognosis. In vitro, overexpression of miR-137 decreased GBM cell growth and increased cell apoptosis, while depletion of miR-137 enhanced cell growth and decreased cell apoptosis. Combined bioinformatics analysis and dual luciferase reporter assay showed that miR-137 may target the 3’-UTR of the epidermal growth factor receptor (EGFR) to reduce its protein translation, resulting in suppression of EGFR signaling in GBM cells. Together, our data suggest that reduction in miR-137 levels in GBM tissues may increase cell growth and decrease cell apoptosis, possibly through suppression of EGFR. PMID:28386374

  5. Detecting and treating breast cancer resistance to EGFR inhibitors

    SciTech Connect

    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.

  6. A Novel Technique to Detect EGFR Mutations in Lung Cancer.

    PubMed

    Liu, Yuanbin; Lei, Ting; Liu, Zhiyu; Kuang, Yanbin; Lyu, Jianxin; Wang, Qi

    2016-05-23

    Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples.

  7. Mechanisms of resistance to EGFR-targeted drugs: lung cancer.

    PubMed

    Morgillo, Floriana; Della Corte, Carminia Maria; Fasano, Morena; Ciardiello, Fortunato

    2016-01-01

    Despite the improvement in clinical outcomes derived by the introduction of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) in the treatment of patients with advanced non-small cell lung cancer (NSCLC) whose tumours harbour EGFR-activating mutations, prognosis remains unfavourable because of the occurrence of either intrinsic or acquired resistance. We reviewed the published literature and abstracts of oral and poster presentations from international conferences addressing EGFR-TKIs resistance mechanisms discovered in preclinical models and in patients with NSCLC. The molecular heterogeneity of lung cancer has several implications in terms of possible mechanisms of either intrinsic or acquired resistance to EGFR-targeted inhibitors. Several mechanisms of resistance have been described to EGFR-TKIs, such as the occurrence of secondary mutation (T790M, C797S), the activation of alternative signalling (Met, HGF, AXL, Hh, IGF-1R), the aberrance of the downstream pathways (AKT mutations, loss of PTEN), the impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism) and histological transformation. Although some of the mechanisms of resistance have been identified, much additional information is needed to understand and overcome resistance to EGFR-TKI agents. The majority of resistance mechanisms described are the result of a selection of pre-existing clones; thus, studies on the mechanisms by which subclonal alterations have an impact on tumour biology and influence cancer progression are extremely important in order to define the best treatment strategy.

  8. A functional role for EGFR signaling in myelination and remyelination.

    PubMed

    Aguirre, Adan; Dupree, Jeff L; Mangin, J M; Gallo, Vittorio

    2007-08-01

    Cellular strategies for oligodendrocyte regeneration and remyelination involve characterizing endogenous neural progenitors that are capable of generating oligodendrocytes during normal development and after demyelination, and identifying the molecular signals that enhance oligodendrogenesis from these progenitors. Using both gain- and loss-of-function approaches, we explored the role of epidermal growth factor receptor (EGFR) signaling in adult myelin repair and in oligodendrogenesis. We show that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter-driven overexpression of human EGFR (hEGFR) accelerated remyelination and functional recovery following focal demyelination of mouse corpus callosum. Lesion repopulation by Cspg4+ (also known as NG2) Ascl1+ (also known as Mash1) Olig2+ progenitors and functional remyelination were accelerated in CNP-hEGFR mice compared with wild-type mice. EGFR overexpression in subventricular zone (SVZ) and corpus callosum during early postnatal development also expanded this NG2+Mash1+Olig2+ progenitor population and promoted SVZ-to-lesion migration, enhancing oligodendrocyte generation and axonal myelination. Analysis of hypomorphic EGFR-mutant mice confirmed that EGFR signaling regulates oligodendrogenesis and remyelination by NG2+Mash1+Olig2+ progenitors. EGFR targeting holds promise for enhancing oligodendrocyte regeneration and myelin repair.

  9. Synergistic effects of erlotinib and everolimus on bronchial carcinoids and large-cell neuroendocrine carcinomas with activated EGFR/AKT/mTOR pathway.

    PubMed

    Bago-Horvath, Zsuzsanna; Sieghart, Wolfgang; Grusch, Michael; Lackner, Andreas; Hayden, Hubert; Pirker, Christine; Komina, Oxana; Węsierska-Gądek, Józefa; Haitel, Andrea; Filipits, Martin; Berger, Walter; Schmid, Katharina

    2012-01-01

    Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear. Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting. Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib. Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted. Copyright © 2012 S. Karger AG, Basel.

  10. A combination of gefitinib and FOLFOX-4 as first-line treatment in advanced colorectal cancer patients. A GISCAD multicentre phase II study including a biological analysis of EGFR overexpression, amplification and NF-kB activation

    PubMed Central

    Cascinu, S; Berardi, R; Salvagni, S; Beretta, G D; Catalano, V; Pucci, F; Sobrero, A; Tagliaferri, P; Labianca, R; Scartozzi, M; Crocicchio, F; Mari, E; Ardizzoni, A

    2007-01-01

    Interesting activity has been reported by combining chemotherapy with cetuximab. An alternative approach for blocking EGFR function has been the development of small-molecule inhibitors of tyrosine kinase domain such as gefitinib. We designed a multicentre phase II study in advanced colorectal cancer combining gefitinib+FOLFOX in order to determine the activity and to relate EGFR expression and gene amplification and NF-kB activation to therapeutic results. Patients received FOLFOX-4 regimen plus gefitinib as first-line treatment. Tumour samples were analysed for EGFR protein expression by immunohistochemical analysis and for EGFR gene amplification by fluorescence in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH) and NF-kB activation. Forty-three patients were enrolled into this study; 15 patients experienced a partial response (response rate=34.9%), whereas other 12 (27.9%) had a stable disease. Median progression-free survival (PFS) was 7.8 months and median overall survival (OS) was 13.9 months. We did not find any relationship with EGFR overexpression, gene amplification, while NF-kB activation was associated with a resistance to therapy. Gefitinib does not seem to increase the activity of FOLFOX in advanced colorectal cancer even in patients overexpressing EGFR or with EGFR amplification. Furthermore, while NF-kB activation seems to predict resistance to chemotherapy as demonstrated ‘in vitro' models, gefitinib does not overcome this mechanism of resistance, as reported for cetuximab. PMID:18059397

  11. Imaging of EGFR expression in murine xenografts using site-specifically labelled anti-EGFR 111In-DOTA-Z EGFR:2377 Affibody molecule: aspect of the injected tracer amount.

    PubMed

    Tolmachev, Vladimir; Rosik, Daniel; Wållberg, Helena; Sjöberg, Anna; Sandström, Mattias; Hansson, Monika; Wennborg, Anders; Orlova, Anna

    2010-03-01

    Overexpression of epidermal growth factor receptor (EGFR) is a prognostic and predictive biomarker in a number of malignant tumours. Radionuclide molecular imaging of EGFR expression in cancer could influence patient management. However, EGFR expression in normal tissues might complicate in vivo imaging. The aim of this study was to evaluate if optimization of the injected protein dose might improve imaging of EGFR expression in tumours using a novel EGFR-targeting protein, the DOTA-Z(EGFR:2377) Affibody molecule. An anti-EGFR Affibody molecule, Z(EGFR:2377), was labelled with (111)In via the DOTA chelator site-specifically conjugated to a C-terminal cysteine. The affinity of DOTA-Z(EGFR:2377) for murine and human EGFR was measured by surface plasmon resonance. The cellular processing of (111)In-DOTA-Z(EGFR:2377) was evaluated in vitro. The biodistribution of radiolabelled Affibody molecules injected in a broad range of injected Affibody protein doses was evaluated in mice bearing EGFR-expressing A431 xenografts. Site-specific coupling of DOTA provided a uniform conjugate possessing equal affinity for human and murine EGFR. The internalization of (111)In-DOTA-Z(EGFR:2377) by A431 cells was slow. In vivo, the conjugate accumulated specifically in xenografts and in EGFR-expressing tissues. The curve representing the dependence of tumour uptake on the injected Affibody protein dose was bell-shaped. The highest specific radioactivity (lowest injected protein dose) provided a suboptimal tumour-to-blood ratio. The results of the biodistribution study were confirmed by gamma-camera imaging. The (111)In-DOTA-Z(EGFR:2377) Affibody molecule is a promising tracer for radionuclide molecular imaging of EGFR expression in malignant tumours. Careful optimization of protein dose is required for high-contrast imaging of EGFR expression in vivo.

  12. Dual-targeting organometallic ruthenium(II) anticancer complexes bearing EGFR-inhibiting 4-anilinoquinazoline ligands.

    PubMed

    Zhang, Yang; Zheng, Wei; Luo, Qun; Zhao, Yao; Zhang, Erlong; Liu, Suyan; Wang, Fuyi

    2015-08-07

    We have recently demonstrated that complexation with (η(6)-arene)Ru(II) fragments confers 4-anilinoquinazoline pharmacophores a higher potential for inducing cellular apoptosis while preserving the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine (Chem. Commun., 2013, 49, 10224-10226). Reported herein are the synthesis, characterisation and evaluation of the biological activity of a new series of ruthenium(ii) complexes of the type [(η(6)-arene)Ru(N,N-L)Cl]PF6 (arene = p-cymene, benzene, 2-phenylethanol or indane, L = 4-anilinoquinazolines). These organometallic ruthenium complexes undergo fast hydrolysis in aqueous solution. Intriguingly, the ligation of (arene)Ru(II) fragments with 4-anilinoquinazolines not only makes the target complexes excellent EGFR inhibitors, but also confers the complexes high affinity to bind to DNA minor grooves while maintaining their reactivity towards DNA bases, characterising them with dual-targeting properties. Molecular modelling studies reveal that the hydrolysis of these complexes is a favourable process which increases the affinity of the target complexes to bind to EGFR and DNA. In vitro biological activity assays show that most of this group of ruthenium complexes are selectively active inhibiting the EGF-stimulated growth of the HeLa cervical cancer cell line, and the most active complex [(η(6)-arene)Ru(N,N-L13)Cl]PF6 (, IC50 = 1.36 μM, = 4-(3'-chloro-4'-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline) is 29-fold more active than its analogue, [(η(6)-arene)Ru(N,N-ethylenediamine)Cl]PF6, and 21-fold more active than gefitinib, a well-known EGFR inhibitor in use clinically. These results highlight the strong promise to develop highly active ruthenium anticancer complexes by ligation of cytotoxic ruthenium pharmacophores with bioactive organic molecules.

  13. CD44 correlates with clinicopathological characteristics and is upregulated by EGFR in breast cancer

    PubMed Central

    Xu, Hanxiao; Wu, Kongju; Tian, Yijun; Liu, Qian; Han, Na; Yuan, Xun; Zhang, Lu; Wu, Gen Sheng; Wu, Kongming

    2016-01-01

    Cluster of differentiation 44 (CD44), a well-known transmembrane glycoprotein, serves as a promoting factor in the carcinogenesis and progression of a variety of neoplasms. Previous studies have demonstrated that aberrant expression of CD44 was associated with the initiation, invasion, metastasis, and therapy-resistance of breast cancer, but whether there was any association between CD44 and pathological characteristics of breast cancer or epidermal growth factor receptor (EGFR) has not been clearly elucidated. In this study, we utilized public microarray data analysis and tissue microarray technologies to display that CD44 level was enhanced in breast cancer and was significantly correlated with histological grade and the status of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 (HER2) and EGFR. Furthermore, mRNA expression of CD44 in breast tumors was positively correlated with basal cytokeratin markers KRT5 and KRT17, but inversely associated with luminal marker FOXA1. Besides, Kaplan-Meier analysis showed that high CD44 mRNA level had adverse impact on the progression-free survival of patients with HER2-expressing or basal-like breast cancer. Functionally, inhibition of EGFR activity by erlotinib impaired the invasion and migration ability of breast cancer cell lines. Western blot assays demonstrated that erlotinib treatment decreased the expression of CD44, accompanied with the reduced protein levels of mesenchymal and cancer stem cell markers. Collectively, this study suggested that the expression of CD44 was upregulated by EGFR pathway and CD44 had a robust impact on the development of breast cancer. PMID:27499099

  14. Detection of T790M, the Acquired Resistance EGFR Mutation, by Tumor Biopsy versus Noninvasive Blood-Based Analyses.

    PubMed

    Sundaresan, Tilak K; Sequist, Lecia V; Heymach, John V; Riely, Gregory J; Jänne, Pasi A; Koch, Walter H; Sullivan, James P; Fox, Douglas B; Maher, Robert; Muzikansky, Alona; Webb, Andrew; Tran, Hai T; Giri, Uma; Fleisher, Martin; Yu, Helena A; Wei, Wen; Johnson, Bruce E; Barber, Thomas A; Walsh, John R; Engelman, Jeffrey A; Stott, Shannon L; Kapur, Ravi; Maheswaran, Shyamala; Toner, Mehmet; Haber, Daniel A

    2016-03-01

    The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non-small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management. As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy. We compared the T790M genotype from tumor biopsies with analysis of simultaneously collected circulating tumor cells (CTC) and circulating tumor DNA (ctDNA). T790M genotypes were successfully obtained in 30 (75%) tumor biopsies, 28 (70%) CTC samples, and 32 (80%) ctDNA samples. The resistance-associated mutation was detected in 47% to 50% of patients using each of the genotyping assays, with concordance among them ranging from 57% to 74%. Although CTC- and ctDNA-based genotyping were each unsuccessful in 20% to 30% of cases, the two assays together enabled genotyping in all patients with an available blood sample, and they identified the T790M mutation in 14 (35%) patients in whom the concurrent biopsy was negative or indeterminate. Discordant genotypes between tumor biopsy and blood-based analyses may result from technological differences, as well as sampling different tumor cell populations. The use of complementary approaches may provide the most complete assessment of each patient's cancer, which should be validated in predicting response to T790M-targeted inhibitors. ©2015 American Association for Cancer Research.

  15. mAb MDR1-modified chitosan nanoparticles overcome acquired EGFR-TKI resistance through two potential therapeutic targets modulation of MDR1 and autophagy.

    PubMed

    Zheng, Yan; Su, Chang; Zhao, Liang; Shi, Yijie

    2017-10-04

    Tyrosine kinase inhibitors (TKIs) that act against the epithelial growth factor receptor (EGFR) were once widely used in chemotherapy for many human cancers. However, acquired chemoresistance occurred in almost all patients, limiting the clinical application of EGFR-TKI. Thus far, no effective methods existing can resolve this problem. Designing a therapeutic treatment with a specific multi-target profile has been regarded as a possible strategy to overcome acquired EGFR-TKI resistance. MDR1 antibody-modified chitosan nanoparticles loading gefitinib and autophagy inhibitor chloroquine were prepared by ionic crosslinking and electrostatic attracting method. MTT assay, flow cytometry analysis and western blot assay were all performed to confirm the effect of different formulations of gefitinib on the proliferation of SMMC-7721/gefitinib cells. The preparations demonstrated their multi-target potential to achieve both tumor-targeting selectivity and the desired antitumor effects by blocking cell-surface MDR1 and inhibiting autophagy. mAb MDR1-modified CS NPs, when combined with the co-delivery of gefitinib and chloroquine, showed targeting and therapeutic potential on enhancing the delivery of anticancer drugs and inducing significant cell apoptosis against acquired EGFR-TKI resistance through the modulation of autophagy and while blocking the activity of the MDR1 receptor. A new approach to design an excellent nanoparticle drug-delivery system can overcome acquired EGFR-TKI resistance against various multiple antitumor targets.

  16. Hyperfiltration Affects Accuracy of Creatinine eGFR Measurement

    PubMed Central

    Huang, Shih-Han S.; Sharma, Ajay P.; Yasin, Abeer; Lindsay, Robert M.; Clark, William F.

    2011-01-01

    Summary Background and objectives Surrogate markers such as creatinine, cystatin C (CysC), and beta trace protein (BTP) have been used to estimate GFR (eGFR). The accuracy of eGFR may be altered with hyperfiltration and differences in filtration fraction (FF). It is hypothesized that the accuracy of creatinine for eGFR may be affected by hyperfiltration and different effective renal plasma flow (ERPF). Design, setting, participants, & measurements A total of 127 pediatric patients with various renal diseases underwent simultaneous measurements of GFR using 51Cr-EDTA renal scan and ERPF (131I-hippurate clearance) to calculate the FF (FF = GFR/ERPF). The eGFRs were calculated using the commonly used Schwartz (creatinine), Filler (CysC), and Benlamri (BTP) formulas. Agreement of the eGFRs with the measured isotope GFRs was assessed by Bland–Altman plots. Correlation analysis was performed using nonparametric tests to compare FF with eGFR − GFR. Results The 127 children at a median age (with 25th percentile, 75th percentile) of 11.9 (8.5, 14.9) years had a mean 51Cr EDTA-GFR of 100.6 ± 32.1 ml/min per 1.73 m2 and a median 131I-hippurate clearance (ERPF) of 588 (398,739) ml/min per 1.73 m2. Mean FF was 17.7 ± 4.5% with no correlation between the FF and the error (eGFR − GFR) for CysC and BTP eGFR, whereas there was a significant negative correlation between the error for Schwartz eGFR and FF. Conclusions There is a significant negative correlation between the error for the Schwartz eGFR and the FF. CysC and BTP are not affected by differences in FF. PMID:20966120

  17. EGFR Expression in Gallbladder Carcinoma in North America

    PubMed Central

    Kaufman, Matthew; Mehrotra, Bhoomi; Limaye, Sewanti; White, Sherrie; Fuchs, Alexander; Lebowicz, Yehuda; Nissel-Horowitz, Sandy; Thomas, Adrienne

    2008-01-01

    BACKGROUND: Increased epidermal growth factor receptor (EGF receptor) expression has been noted in various cancers and has become a useful target for therapeutic interventions. Small studies from Asia and Australia have demonstrated EGFR over-expression in gallbladder cancer. We sought to evaluate the expression of EGFR in a series of 16 gallbladder cancer patients from North America. METHODS: Using tumor registry data, we identified 16 patients diagnosed with gall bladder carcinoma at our medical center between the years of 1998 and 2005. We performed a retrospective review of these patients' charts, obtained cell blocks from pathology archives and stained for EGFR and Her2/neu. RESULTS: Fifteen of sixteen patients were noted to over-express EGFR. Three were determined 1+, nine were 2+ and three were 3+. Eight patients had poorly differentiated adenocarcinoma, six had moderately differentiated and two had well-differentiated tumors. In this small series, there was a trend toward shorter survival and more poorly differentiated tumors in patients with greater intensity of EGFR expression. One patient was EGFR negative but 3+ for erb-2/Her 2-neu expression. No patient co-expressed EGFR and Her-2-neu. Median survival of patients in this series was 17 months. CONCLUSION: In view of our observations confirming the over-expression of EGFR in our patient population in North America, and the recent success of EGFR targeted therapies in other solid tumors that over-express EGFR, it may now be appropriate to evaluate agents targeting this pathway either as single agents or in combination with standard chemotherapy. PMID:18825277

  18. Bisphosphonates inactivate human EGFRs to exert antitumor actions

    PubMed Central

    Yuen, Tony; Stachnik, Agnes; Iqbal, Jameel; Sgobba, Miriam; Gupta, Yogesh; Lu, Ping; Colaianni, Graziana; Ji, Yaoting; Zhu, Ling-Ling; Kim, Se-Min; Li, Jianhua; Liu, Peng; Izadmehr, Sudeh; Sangodkar, Jaya; Bailey, Jack; Latif, Yathin; Mujtaba, Shiraz; Epstein, Solomon; Davies, Terry F.; Bian, Zhuan; Zallone, Alberta; Aggarwal, Aneel K.; Haider, Shozeb; New, Maria I.; Sun, Li; Narla, Goutham; Zaidi, Mone

    2014-01-01

    Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers. PMID:25453081

  19. A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): Old successes and future perspectives

    PubMed Central

    Russo, Alessandro; Franchina, Tindara; Rita Ricciardi, Giuseppina Rosaria; Picone, Antonio; Ferraro, Giuseppa; Zanghì, Mariangela; Toscano, Giuseppe; Giordano, Antonio; Adamo, Vincenzo

    2015-01-01

    The discovery of Epidermal Growth Factor Receptor (EGFR) mutations in Non Small Cell Lung Cancer (NSCLC) launched the era of personalized medicine in advanced NSCLC, leading to a dramatic shift in the therapeutic landscape of this disease. After ten years from the individuation of activating mutations in the tyrosine kinase domain of the EGFR in NSCLC patients responding to the EGFR tyrosine kinase inhibitor (TKI) Gefitinib, several progresses have been done and first line treatment with EGFR TKIs is a firmly established option in advanced EGFR-mutated NSCLC patients. During the last decade, different EGFR TKIs have been developed and three inhibitors have been approved so far in these selected patients. However, despite great breakthroughs have been made, treatment of these molecularly selected patients poses novel therapeutic challenges, such as emerging of acquired resistance, brain metastases development or the need to translate these treatments in earlier clinical settings, such as adjuvant therapy. The aim of this paper is to provide a comprehensive review of the major progresses reported so far in the EGFR inhibition in this molecularly-selected subgroup of NSCLC patients, from the early successes with first generation EGFR TKIs, Erlotinib and Gefitinib, to the novel irreversible and mutant-selective inhibitors and ultimately the emerging challenges that we, in the next future, are called to deal with. PMID:26308162

  20. 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

  1. 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

    2017-04-08

    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.

  2. EGFR Soluble Isoforms and Their Transcripts Are Expressed in Meningiomas

    PubMed Central

    Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%.Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  3. [Epidermal growth factor receptor (EGFR) mutation status before and after acquired resistance to EGFR tyrosine kinase inhibitors in patients with lung adenocarcinoma].

    PubMed

    Ye, S B; Li, R; Shi, S S

    2017-02-08

    Objective: To investigate epidermal growth factor receptor (EGFR) mutation status in lung adenocarcinomas before and after acquiring resistance to EGFR tyrosine kinase inhibitors (TKIs) using ARMS method followed by further verification using droplet digital PCR technique. Methods: Twenty qualified patients were included, among them 13 were male and 7 were female patients. Before EGFR-TKIs treatment, 5 patients were EGFR wild-type by ARMS, and the other 15 patients had L858R or 19-del point mutations. The time to progression varied from 4 to 18 months. Mutation of exons 18, 19, 20 and 21 were detected by ARMS, and were verified by droplet digital PCR system method. Results: EGFR wild-type status was unchanged before and after acquired resistance to EGFR-TKIs in 5 lung adenocarcinoma patients. Alteration of EGFR mutation status occurred in 10 of the 15 patients with pre-treatment L858R or 19-del mutations. Among them, T790M mutation was found in 8 patients, L858R became G719X plus S768I mutation in one patient, and 19-del converted into wild-type in one other patient. Conclusions: T790M mutation is the primary type of EGFR mutation in lung adenocarcinomas with acquired resistance to EGFR-TKIs therapy. Acquired resistance to EGFR-TKIs dose not lead to the alteration of EGFR status in pre-treatment EGFR wild-type patients, but can alter EGFR mutation status in pre-treatment EGFR mutant patients.

  4. EGFR mediates hyperlipidemia-induced renal injury via regulating inflammation and oxidative stress: the detrimental role and mechanism of EGFR activation

    PubMed Central

    Fang, Qilu; Zou, Chunpeng; Zhong, Peng; Lin, Feng; Li, Weixin; Wang, Lintao; Zhang, Yali; Zheng, Chao; Wang, Yi; Li, Xiaokun; Liang, Guang

    2016-01-01

    Previous studies have implicated inflammation, oxidative stress, and fibrosis as key factors in the development of obesity-induced kidney diseases. Epidermal growth factor receptor (EGFR) plays an important role in cancer development. Recently, the EGFR pathway has been increasingly implicated in chronic cardiovascular diseases via regulating inflammation and oxidative stress. However, it is unclear if EGFR is involved in obesity-related kidney injury. Using ApoE−/− and C57BL/6 mice models and two specific EGFR inhibitors, we investigated the potential effects of EGFR inhibition in the treatment of obesity-related nephropathy and found that EGFR inhibition alleviates renal inflammation, oxidative stress and fibrosis. In NRK-52E cells, we also elucidated the mechanism behind hyperlipidemia-induced EGFR activation. We observed that c-Src and EGFR forms a complex, and following PA stimulation, it is the successive phosphorylation, not formation, of the c-Src/EGFR complex that results in the subsequent cascade activation. Second, we found that TLR4 regulates the activation EGFR pathway mainly through the phosphorylation of the c-Src/EGFR complex. These results demonstrate the detrimental role of EGFR in the pathogenesis of obesity-related nephropathy, provide a new understanding of the mechanism behind hyperlipidemia/FFA-induced EGFR activation, and support the use of EGFR inhibitors in the treatment of obesity-induced kidney diseases. PMID:27014908

  5. Combinatorial signaling by the Frizzled/PCP and Egfr-pathways during planar cell polarity establishment in the Drosophila eye

    PubMed Central

    Weber, Ursula; Pataki, Csilla; Mihaly, Jozsef; Mlodzik, Marek

    2008-01-01

    Summary Frizzled (Fz)/PCP signaling regulates planar, vectorial orientation of cells or groups of cells within whole tissues. Although Fz/PCP signaling has been analyzed in several contexts, little is known about nuclear events acting downstream of Fz/PCP signaling in the R3/R4 cell fate decision in the Drosophila eye or in other contexts. Here we demonstrate a specific requirement for Egfr-signaling and the transcription factors Fos (AP-1), Yan and Pnt in PCP dependent R3/R4 specification. Loss and gain-of-function assays suggest that the transcription factors integrate input from Fz/PCP and Egfr-signaling and that the ETS factors Pnt and Yan cooperate with Fos (and Jun) in the PCP-specific R3/R4 determination. Our data indicate that Fos (either downstream of Fz/PCP signaling or parallel to it) and Yan are required in R3 to specify its fate (Fos) or inhibit R4 fate (Yan), and that Egfr-signaling is required in R4 via Pnt for its fate specification. Taken together with previous work establishing a Notch-dependent Su(H) function in R4, we conclude that Fos, Yan, Pnt, and Su(H) integrate Egfr, Fz, and Notch-signaling input in R3 or R4 to establish cell fate and ommatidial polarity. PMID:18291359

  6. Mig-6 controls EGFR trafficking and suppresses gliomagenesis.

    PubMed

    Ying, Haoqiang; Zheng, Hongwu; Scott, Kenneth; Wiedemeyer, Ruprecht; Yan, Haiyan; Lim, Carol; Huang, Joseph; Dhakal, Sabin; Ivanova, Elena; Xiao, Yonghong; Zhang, Hailei; Hu, Jian; Stommel, Jayne M; Lee, Michelle A; Chen, An-Jou; Paik, Ji-Hye; Segatto, Oreste; Brennan, Cameron; Elferink, Lisa A; Wang, Y Alan; Chin, Lynda; DePinho, Ronald A

    2010-04-13

    Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

  7. Mig-6 controls EGFR trafficking and suppresses gliomagenesis

    PubMed Central

    Ying, Haoqiang; Zheng, Hongwu; Scott, Kenneth; Wiedemeyer, Ruprecht; Yan, Haiyan; Lim, Carol; Huang, Joseph; Dhakal, Sabin; Ivanova, Elena; Xiao, Yonghong; Zhang, Hailei; Hu, Jian; Stommel, Jayne M.; Lee, Michelle A.; Chen, An-Jou; Paik, Ji-Hye; Segatto, Oreste; Brennan, Cameron; Elferink, Lisa A.; Wang, Y. Alan; Chin, Lynda; DePinho, Ronald A.

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking. PMID:20351267

  8. Predictive radiogenomics modeling of EGFR mutation status in lung cancer

    PubMed Central

    Gevaert, Olivier; Echegaray, Sebastian; Khuong, Amanda; Hoang, Chuong D.; Shrager, Joseph B.; Jensen, Kirstin C.; Berry, Gerald J.; Guo, H. Henry; Lau, Charles; Plevritis, Sylvia K.; Rubin, Daniel L.; Napel, Sandy; Leung, Ann N.

    2017-01-01

    Molecular analysis of the mutation status for EGFR and KRAS are now routine in the management of non-small cell lung cancer. Radiogenomics, the linking of medical images with the genomic properties of human tumors, provides exciting opportunities for non-invasive diagnostics and prognostics. We investigated whether EGFR and KRAS mutation status can be predicted using imaging data. To accomplish this, we studied 186 cases of NSCLC with preoperative thin-slice CT scans. A thoracic radiologist annotated 89 semantic image features of each patient’s tumor. Next, we built a decision tree to predict the presence of EGFR and KRAS mutations. We found a statistically significant model for predicting EGFR but not for KRAS mutations. The test set area under the ROC curve for predicting EGFR mutation status was 0.89. The final decision tree used four variables: emphysema, airway abnormality, the percentage of ground glass component and the type of tumor margin. The presence of either of the first two features predicts a wild type status for EGFR while the presence of any ground glass component indicates EGFR mutations. These results show the potential of quantitative imaging to predict molecular properties in a non-invasive manner, as CT imaging is more readily available than biopsies. PMID:28139704

  9. EGFR Amplification and Glioblastoma Stem-Like Cells

    PubMed Central

    Liffers, Katrin; Lamszus, Katrin

    2015-01-01

    Glioblastoma (GBM), the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells). GS-cells can be maintained in vitro using serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR) gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retained EGFR amplification could overcome the limitations of current in vitro model systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with different EGFR status in order to maintain EGFR-dependent intratumoral heterogeneity in vitro. Further, it will summarize the current knowledge about the impact of EGFR amplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM. PMID:26136784

  10. EGFR mutation and lobar location of lung adenocarcinoma.

    PubMed

    Tseng, Chien-Hua; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Ho, Chao-Chi; Hsia, Te-Chun; Su, Kang-Yi; Wu, Ming-Fang; Chiu, Kuo-Liang; Liu, Chien-Ming; Wu, Tzu-Chin; Chen, Hung-Jen; Chen, Hsuan-Yu; Chang, Chi-Sheng; Hsu, Chung-Ping; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Lin, Chin-Hung; Chen, Jeremy J W; Chen, Kuan-Yu; Liao, Wei-Yu; Shih, Jin-Yuan; Yu, Sung-Liang; Yu, Chong-Jen; Yang, Pan-Chyr; Yang, Tsung-Ying; Chang, Gee-Chen

    2016-02-01

    The objective of this study was to investigate the associations among lung cancer location, and epidermal growth factor receptor (EGFR) mutation status. Treatment-naive, pathologically confirmed lung adenocarcinomas with tumor specimens available for genetic analysis were included from 2011 through 2014. Overall, 1771 patients with lung adenocarcinoma were included for analysis, after excluding those with carcinoma not otherwise specified, or synchronous multiple primary lung cancers. The median age was 64 years, and the female:male and never smoker:ever smoker ratios were 930:855 (52:48%) and 1167:604 (65:35%), respectively. The EGFR mutation rate was 56%. Among patients, 1093 (62%) had primary tumors in the upper lobes. Compared with the characteristics of the EGFR wild-type, tumors with EGFR activating mutations were more common in women (P < 0.001), never smokers (P < 0.001), and in the upper lobes (P = 0.004). Among EGFR activating mutations, compared with the EGFR exon 19 deletion, L858R mutation were more common in women (P = 0.002), never smokers (P = 0.038), and the upper lobes P < 0.0005). The present study is the first to address that different pulmonary lobar locations might harbor different EGFR mutation subtypes. We demonstrated that adenocarcinomas with L858R mutation, rather than exon 19 deletion or wild-type EGFR gene, prefer to locate over the upper lungs. This phenomenon was more significant in females and never-smokers, implying the result of complex interactions between genetic susceptibility and environmental factors. Therefore, EGFR L858R mutation and exon 19 deletion may not be identical disease entity from the point of carcinogenesis.

  11. 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.

  12. Fish Allergy

    MedlinePlus

    ... can react to touching fish or breathing in vapors from cooking fish. A fish allergy can cause ... hives red spots swelling a drop in blood pressure , causing lightheadedness or loss of consciousness Your child ...

  13. City Fishing.

    ERIC Educational Resources Information Center

    Lange, Robert E.

    1979-01-01

    A program of supplying opportunities for fishing at locations within and near urban areas was developed. This effort included stocking, management of bodies of water for fishing, and presentation of fishing clinics for urban fishermen. (RE)

  14. Fish Hearing.

    ERIC Educational Resources Information Center

    Blaxter, J. H. S.

    1980-01-01

    Provides related information about hearing in fish, including the sensory stimulus of sound in the underwater environment, mechanoreceptors in fish, pressure perception and the swimbladder, specializations in sound conduction peculiar to certain fish families. Includes numerous figures. (CS)

  15. Alpha-Particle Emitting 213Bi-Anti-EGFR Immunoconjugates Eradicate Tumor Cells Independent of Oxygenation

    PubMed Central

    Gaertner, Florian C.; Bruchertseifer, Frank; Morgenstern, Alfred; Essler, Markus; Senekowitsch-Schmidtke, Reingard

    2013-01-01

    Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that cell-bound α-particle emitting 213Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with 213Bi-anti-EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing 213Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml–1.48 MBq/ml) and irradiation with increasing doses of photons (0.5–12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by 213Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting 213Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, 213Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors. PMID:23724085

  16. GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness.

    PubMed

    Zajac, Mateusz; Law, Jeffrey; Cvetkovic, Dragana Donna; Pampillo, Macarena; McColl, Lindsay; Pape, Cynthia; Di Guglielmo, Gianni M; Postovit, Lynne M; Babwah, Andy V; Bhattacharya, Moshmi

    2011-01-01

    Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.

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

    PubMed

    Hirano, Toshiyuki; Yasuda, Hiroyuki; Tani, Tetsuo; 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

    2015-11-17

    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.

  18. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer.

    PubMed

    Park, Heae Surng; Jang, Min Hye; Kim, Eun Joo; Kim, Hyun Jeong; Lee, Hee Jin; Kim, Yu Jung; Kim, Jee Hyun; Kang, Eunyoung; Kim, Sung-Won; Kim, In Ah; Park, So Yeon

    2014-09-01

    Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

  19. miR-217 and CAGE form feedback loop and regulates the response to anti-cancer drugs through EGFR and HER2

    PubMed Central

    Han, Minho; Lee, Hansoo; Lee, Yun Sil; Choe, Jongseon; Kim, Young Myeong; Jeoung, Dooil

    2016-01-01

    MicroRNA array analysis revealed that miR-217 expression was decreased in anti-cancer drug-resistant Malme3MR cancer cells. CAGE, a cancer/testis antigen, was predicted as a target of miR-217. Luciferase activity and ChIP assays revealed a negative feedback relationship between CAGE and miR-217. miR-217 and CAGE oppositely regulated the response to anti-cancer drugs such as taxol, gefitinib and trastuzumab, an inhibitor of HER2. miR-217 negatively regulated the tumorigenic, metastatic, angiogenic, migration and invasion potential of cancer cells. The xenograft of Malme3MR cells showed an increased expression of pEGFRY845. CAGE and miR-217 inhibitor regulated the expression of pEGFRY845. CAGE showed interactions with EGFR and HER2 and regulated the in vivo sensitivity to trastuzumab. The down-regulation of EGFR or HER2 enhanced the sensitivity to anti-cancer drugs. CAGE showed direct regulation of HER2 and was necessary for the interaction between EGFR and HER2 in Malme3MR cells. miR-217 inhibitor induced interactions of CAGE with EGFR and HER2 in Malme3M cells. The inhibition of EGFR by CAGE-binding GTGKT peptide enhanced the sensitivity to gefitinib and trastuzumab and prevented interactions of EGFR with CAGE and HER2. Our results show that miR-217-CAGE feedback loop serves as a target for overcoming resistance to various anti-cancer drugs, including EGFR and HER2 inhibitors. PMID:26863629

  20. Nicotine induces resistance to erlotinib via cross-talk between α 1 nAChR and EGFR in the non-small cell lung cancer xenograft model.

    PubMed

    Li, Heyan; Wang, Shuo; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Iwama, Eiji; Fujii, Akiko; Ota, Keiichi; Hidaka, Noriko; Kawano, Yuko; Nakanishi, Yoichi

    2015-04-01

    Given our previously published study, α 1 nicotinic acetylcholine receptor (nAChR) plays an essential role in nicotine-induced cell signaling and nicotine-induced resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) in non-small cell lung cancer (NSCLC) PC9 cells. The aim of this study was to investigate the potential mechanism between nAChR and EGFR for nicotine-induced resistance to EGFR-TKI erlotinib in the NSCLC xenograft model. We identified the role of nicotine to EGFR/AKT/ERK pathways and to erlotinib-resistance in NSCLC PC9 and HCC827 cells by MTS assay and western blot. Then, we established the PC9 xenograft model with nicotine exposure and treated mice with erlotinib combined with vehicle or nicotine. We confirmed the effects of nicotine on EGFR/AKT/ERK pathways and determined nicotine's potential in preventing from the effect of erlotinib on NSCLC cells. Then, we showed that nicotine exposures can promote tumor growth and induce resistance to erlotinib in the PC9 xenograft model. Our results also indicated that chronic oral administration of nicotine can cause more significant erlotinib-resistance compared with acute i.v. injection of nicotine through activating α 1 nAChR and EGFR pathways. These results suggest that nicotine contributes to the progression and erlotinib-resistance of the NSCLC xenograft model via the cooperation between nAChR and EGFR. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients

    PubMed Central

    Srivastava, Raghvendra M.; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-bae, Jie; Wang, Lin; Seethala, Raja R.; Branstetter, Barton F.; Ferrone, Soldano; Ferris, Robert L.

    2015-01-01

    The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was utilized to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. PMID:25972070

  2. Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR*

    PubMed Central

    Jung, Sung Keun; Lee, Mee-Hyun; Lim, Do Young; Kim, Jong Eun; Singh, Puja; Lee, Sung-Young; Jeong, Chul-Ho; Lim, Tae-Gyu; Chen, Hanyong; Chi, Young-In; Kundu, Joydeb Kumar; Lee, Nam Hyouck; Lee, Charles C.; Cho, Yong-Yeon; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2014-01-01

    Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR. PMID:25368326

  3. Isoliquiritigenin induces apoptosis and inhibits xenograft tumor growth of human lung cancer cells by targeting both wild type and L858R/T790M mutant EGFR.

    PubMed

    Jung, Sung Keun; Lee, Mee-Hyun; Lim, Do Young; Kim, Jong Eun; Singh, Puja; Lee, Sung-Young; Jeong, Chul-Ho; Lim, Tae-Gyu; Chen, Hanyong; Chi, Young-In; Kundu, Joydeb Kumar; Lee, Nam Hyouck; Lee, Charles C; Cho, Yong-Yeon; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2014-12-26

    Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. STAT1-Induced HLA Class I Upregulation Enhances Immunogenicity and Clinical Response to Anti-EGFR mAb Cetuximab Therapy in HNC Patients.

    PubMed

    Srivastava, Raghvendra M; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-Bae, Jie; Wang, Lin; Seethala, Raja R; Branstetter, Barton F; Ferrone, Soldano; Ferris, Robert L

    2015-08-01

    The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies, and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was used to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1(-/-) cells. Cetuximab enhanced HNC cell recognition by EGFR853-861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271-279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. ©2015 American Association for Cancer Research.

  5. Imaging EGFR distribution using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Lucas, L.; Chen, X. K.; Smith, A.; Korbelik, M.; Zeng, H.; Lee, P. W. K.; Hewitt, K. C.

    2009-02-01

    The purpose of this study is to explore the feasibility of using Surface Enhanced Raman Spectroscopy (SERS) to image the distribution of Epidermal Growth Factor Receptor (EGFR) in cells. To accomplish this task, 30 nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per ml) are incubated with cells (106 per ml) of the A431 human epidermoid carcinoma cell line and normal human bronchial epithelial (NHBE) cells. Using the 632.8 nm excitation line of a He-Ne laser, Raman spectroscopy measurements are performed using a point mapping scheme. SERS signals are observed with an overall enhancement of 4-7 orders of magnitude. Raman intensity maps of the 1480 and 1583 cm-1 peaks correlate well with the expected distribution of AuNPs and EGFR. Normal cells show little to no enhancement. The results therefore present a simple yet effective means to image EGFR over-expression.

  6. EGFR targeted therapy in lung cancer; an evolving story.

    PubMed

    Bartholomew, C; Eastlake, L; Dunn, P; Yiannakis, D

    2017-01-01

    Specific oncogenes with driver mutations, such as the Epidermal Growth Factor Receptor (EGFR 1) gene can lead to non-small-cell lung cancer formation. Identification of these oncogenes, their driver mutations and downstream effects allow the targeting of these pathways by drugs. Such personalised therapy has become an important strategy in combating lung cancer and highlights the need to test for these mutations. Tyrosine Kinase Inhibitors (TKIs) against EGFR, such as Erlotinib, are able to halt these tumour promoting properties in non-small-cell lung cancers. Third generation EGFR TKIs, such as Osimertinib, are focussing on resulting acquired TKI resistance. Here we report the clinical course of a patient with metastatic non-small-cell lung cancer who has undergone EGFR targeted therapy and been further challenged by TKI acquired resistance. Her extended survival and maintained quality of life are a consequence of these modern, genotype-targeted, personalised metastatic non-small-cell lung cancer therapies.

  7. Regulatory mechanisms of EGFR signalling during Drosophila eye development.

    PubMed

    Malartre, Marianne

    2016-05-01

    EGFR signalling is a well-conserved signalling pathway playing major roles during development and cancers. This review explores what studying the EGFR pathway during Drosophila eye development has taught us in terms of the diversity of its regulatory mechanisms. This model system has allowed the identification of numerous positive and negative regulators acting at specific time and place, thus participating to the tight control of signalling. EGFR signalling regulation is achieved by a variety of mechanisms, including the control of ligand processing, the availability of the receptor itself and the transduction of the cascade in the cytoplasm. Ultimately, the transcriptional responses contribute to the establishment of positive and negative feedback loops. The combination of these multiple mechanisms employed to regulate the EGFR pathway leads to specific cellular outcomes involved in functions as diverse as the acquisition of cell fate, proliferation, survival, adherens junction remodelling and morphogenesis.

  8. Deubiquitination of EGFR by Cezanne-1 contributes to cancer progression

    PubMed Central

    Pareja, Fresia; Ferraro, Daniela Aleida; Rubin, Chanan; Cohen-Dvashi, Hadas; Zhang, Fan; Aulmann, Sebastian; Ben-Chetrit, Nir; Pines, Gur; Navon, Roy; Crosetto, Nicola; Köstler, Wolfgang; Carvalho, Silvia; Lavi, Sara; Schmitt, Fernando; Dikic, Ivan; Yakhini, Zohar; Sinn, Peter; Mills, Gordon B.; Yarden, Yosef

    2011-01-01

    Once stimulated, the epidermal growth factor receptor (EGFR) undergoes self-phosphorylation, which, on the one hand, instigates signaling cascades, and on the other hand, recruits CBL ubiquitin ligases, which mark EGFRs for degradation. Using RNA interference screens, we identified a deubiquitinating enzyme, Cezanne-1, that opposes receptor degradation and enhances EGFR signaling. These functions require the catalytic and ubiquitin-binding domains of Cezanne-1, and they involve physical interactions and trans-phosphorylaton of Cezanne-1 by EGFR. In line with the ability of Cezanne-1 to augment EGF-induced growth and migration signals, the enzyme is overexpressed in breast cancer. Congruently, the corresponding gene is amplified in approximately one third of mammary tumors, and high transcript levels predict an aggressive disease course. In conclusion, deubiquitination by Cezanne-1 curtails degradation of growth factor receptors, thereby promotes oncogenic growth signals. PMID:22179831

  9. Management of egfr tki–induced dermatologic adverse events

    PubMed Central

    Melosky, B.; Leighl, N.B.; Rothenstein, J.; Sangha, R.; Stewart, D.; Papp, K.

    2015-01-01

    Targeting the epidermal growth factor receptor (egfr) pathway has become standard practice for the treatment of advanced non-small-cell lung cancer. Compared with chemotherapy, egfr tyrosine kinase inhibitors (tkis) have been associated with improved efficacy in patients with an EGFR mutation. Together with the increase in efficacy comes an adverse event (ae) profile different from that of chemotherapy. That profile includes three of the most commonly occurring dermatologic aes: acneiform rash, stomatitis, and paronychia. Currently, no randomized clinical trials have evaluated the treatments for the dermatologic aes that patients experience when taking egfr tkis. Based on the expert opinion of the authors, some basic strategies have been developed to manage those key dermatologic aes. Those strategies have the potential to improve patient quality of life and compliance and to prevent inappropriate dose reductions. PMID:25908911

  10. 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

    deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib). Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined. EGFR MUTATION TESTING The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement

  11. Assessment of DDR2, BRAF, EGFR and KRAS mutations as therapeutic targets in non-adenocarcinoma lung cancer patients.

    PubMed

    Yashima, Hideaki; Shimizu, Kimihiro; Araki, Takuya; Aomori, Tohru; Ohtaki, Yoichi; Nagashima, Toshiteru; Enokida, Yasuaki; Atsumi, Jun; Nakamura, Tomonori; Takeyoshi, Izumi; Yamamoto, Koujirou

    2014-09-01

    Molecular-targeted therapy has not been established in non-adenocarcinoma lung cancer (non-AdLC), as no targets that affect the clinical efficacy of molecular-targeted drugs have yet been identified. In this study, we investigated the frequency of genetic variations in discoidin domain receptor 2 (DDR2), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) in non-AdLC patients, in order to evaluate the possibility of genetic mutations in these genes being used as therapeutic targets for the treatment of patients with non-AdLC. For this purpose, we enrolled 150 non-AdLC patients who had undergone surgery at the Gunma University Hospital between December, 2003 and December, 2012. Genetic mutations in the EGFR, KRAS, DDR2 and BRAF genes were detected by a sequencing method or probe assay using DNA derived from cancer tissues. No somatic mutations in DDR2 or BRAF were detected in non-AdLC patients. Conversely, genetic mutations in EGFR exon 19 were found in 3 squamous cell carcinoma (SCC) and 3 adenosquamous carcinoma patients, whereas KRAS codon 12 mutations were also found in 3 SCC patients and 1 large-cell neuroendocrine carcinoma patient. EGFR and KRAS mutations were mutually exclusive. This study indicated that, although DDR2 and BRAF mutations may only rarely be used as therapeutic targets, EGFR and KRAS mutations may represent candidate therapeutic targets, at least in the non-AdLC patients investigated.

  12. Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.

    PubMed

    Singer, Josef; Fazekas, Judit; Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

    2014-07-01

    Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a "caninized" version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer.

  13. Simultaneous Inhibition of EGFR/VEGFR and Cyclooxygenase-2 Targets Stemness-Related Pathways in Colorectal Cancer Cells

    PubMed Central

    Valverde, Araceli; Peñarando, Jon; Cañas, Amanda; López-Sánchez, Laura M.; Conde, Francisco; Hernández, Vanessa; Peralbo, Esther; López-Pedrera, Chary; de la Haba-Rodríguez, Juan; Aranda, Enrique; Rodríguez-Ariza, Antonio

    2015-01-01

    Despite the demonstrated benefits of anti-EGFR/VEGF targeted therapies in metastatic colorectal cancer (mCRC), many patients initially respond, but then show evidence of disease progression. New therapeutic strategies are needed to make the action of available drugs more efficient. Our study aimed to explore whether simultaneous targeting of EGFR/VEGF and cyclooxygenase-2 (COX-2) may aid the treatment and management of mCRC patients. The dual tyrosine kinase inhibitor AEE788 and celecoxib were used to inhibit EGFR/VEGFR and COX-2, respectively, in colorectal cancer cells. COX-2 inhibition with celecoxib augmented the antitumoral and antiangiogenic efficacy of AEE788, as indicated by the inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, down-regulation of VEGF production by cancer cells and reduction of cell migration. These effects were related with a blockade in the EGFR/VEGFR signaling axis. Notably, the combined AEE788/celecoxib treatment prevented β-catenin nuclear accumulation in tumor cells. This effect was associated with a significant downregulation of FOXM1 protein levels and an impairment in the interaction of this transcription factor with β-catenin, which is required for its nuclear localization. Furthermore, the combined treatment also reduced the expression of the stem cell markers Oct 3/4, Nanog, Sox-2 and Snail in cancer cells, and contributed to the diminution of the CSC subpopulation, as indicated by colonosphere formation assays. In conclusion, the combined treatment of AEE788 and celecoxib not only demonstrated enhanced anti-tumoral efficacy in colorectal cancer cells, but also reduced colon CSCs subpopulation by targeting stemness-related pathways. Therefore, the simultaneous targeting of EGFR/VEGF and COX-2 may aid in blocking mCRC progression and improve the efficacy of existing therapies in colorectal cancer. PMID:26107817

  14. Osimertinib (AZD9291) decreases programmed death ligand-1 in EGFR-mutated non-small cell lung cancer cells.

    PubMed

    Jiang, Xiao-Ming; Xu, Yu-Lian; Huang, Mu-Yang; Zhang, Le-Le; Su, Min-Xia; Chen, Xiuping; Lu, Jin-Jian

    2017-09-07

    Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In NSCLC patients, an EGFR mutation is likely to be correlated with high levels of expression of programmed death ligand-1 (PD-L1). Here, we showed that osimertinib decreased PD-L1 expression in human EGFR mutant NSCLC cells in vitro. Osimertinib (125 nmol/L) markedly suppressed PD-L1 mRNA expression in both NCI-H1975 and HCC827 cells. Pretreatment with the N-linked glycosylation inhibitor tunicamycin, osimertinib clearly decreased the production of new PD-L1 protein probably due to a reduction in mRNA. After blocking transcription and translation processes with actinomycin D and cycloheximide, respectively, osimertinib continued to reduce the expression of PD-L1, demonstrating that osimertinib might degrade PD-L1 at the post-translational level, which was confirmed by a cycloheximide chase assay, revealing that osimertinib (125 nmol/L) decreased the half-life of PD-L1 from approximately 17.8 h and 13.8 h to 8.6 h and 4.6 h, respectively, in NCI-H1975 and HCC827 cells. Pretreatment with the proteasome inhibitors (MG-132 or bortezomib) blocked the osimertinib-induced degradation of PD-L1, but an inhibitor of autophagy (chloroquine) did not. In addition, inhibition of GSK3β by LiCl prevented osimertinib-induced PD-L1 degradation. The results demonstrate that osimertinib reduces PD-L1 mRNA expression and induces its protein degradation, suggesting that osimertinib may reactivate the immune activity of T cells in the tumor microenvironment in EGFR-mutated NSCLC patients.

  15. Osimertinib benefit in EGFR-mutant NSCLC patients with T790M-mutation detected by circulating tumour DNA.

    PubMed

    Remon, J; Caramella, C; Jovelet, C; Lacroix, L; Lawson, A; Smalley, S; Howarth, K; Gale, D; Green, E; Plagnol, V; Rosenfeld, N; Planchard, D; Bluthgen, M V; Gazzah, A; Pannet, C; Nicotra, C; Auclin, E; Soria, J C; Besse, B

    2017-04-01

    Approximately 50% of epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) patients treated with EGFR tyrosine kinase inhibitors (TKIs) will acquire resistance by the T790M mutation. Osimertinib is the standard of care in this situation. The present study assesses the efficacy of osimertinib when T790M status is determined in circulating cell-free tumour DNA (ctDNA) from blood samples in progressing advanced EGFR-mutant NSCLC patients. ctDNA T790M mutational status was assessed by Inivata InVision™ (eTAm-Seq™) assay in 48 EGFR-mutant advanced NSCLC patients with acquired resistance to EGFR TKIs without a tissue biopsy between April 2015 and April 2016. Progressing T790M-positive NSCLC patients received osimertinib (80 mg daily). The objectives were to assess the response rate to osimertinib according to Response Evaluation Criteria in Solid Tumours (RECIST) 1.1, the progression-free survival (PFS) on osimertinib, and the percentage of T790M positive in ctDNA. The ctDNA T790M mutation was detected in 50% of NSCLC patients. Among assessable patients, osimertinib gave a partial response rate of 62.5% and a stable disease rate of 37.5%. All responses were confirmed responses. After median follow up of 8 months, median PFS by RECIST criteria was not achieved (95% CI: 4-NA), with 6- and 12-months PFS of 66.7% and 52%, respectively. ctDNA from liquid biopsy can be used as a surrogate marker for T790M in tumour tissue.

  16. Generation of a Canine Anti-EGFR (ErbB-1) Antibody for Passive Immunotherapy in Dog Cancer Patients

    PubMed Central

    Wang, Wei; Weichselbaumer, Marlene; Matz, Miroslawa; Mader, Alexander; Steinfellner, Willibald; Meitz, Sarah; Mechtcheriakova, Diana; Sobanov, Yuri; Willmann, Michael; Stockner, Thomas; Spillner, Edzard; Kunert, Renate; Jensen-Jarolim, Erika

    2014-01-01

    Passive immunotherapy with monoclonal antibodies represents a cornerstone of human anticancer therapies, but has not been established in veterinary medicine yet. As the tumor-associated antigen EGFR (ErbB-1) is highly conserved between humans and dogs, and considering the effectiveness of the anti-EGFR antibody cetuximab in human clinical oncology, we present here a “caninized” version of this antibody, can225IgG, for comparative oncology studies. Variable region genes of 225, the murine precursor of cetuximab, were fused with canine constant heavy gamma and kappa chain genes, respectively, and transfected into Chinese hamster ovary (CHO) DUKX-B11 cells. Of note, 480 clones were screened and the best clones were selected according to productivity and highest specificity in EGFR-coated ELISA. Upon purification with Protein G, the recombinant cetuximab-like canine IgG was tested for integrity, correct assembly, and functionality. Specific binding to the surface of EGFR-overexpressing cells was assessed by flow cytometry and immunofluorescence; moreover, binding to canine mammary tissue was demonstrated by immunohistochemistry. In cell viability and proliferation assays, incubation with can225IgG led to significant tumor cell growth inhibition. Moreover, this antibody mediated significant tumor cell killing via phagocytosis in vitro. We thus present here, for the first time, the generation of a canine IgG antibody and its hypothetical structure. On the basis of its cetuximab-like binding site, on the one hand, and the expression of a 91% homologous EGFR molecule in canine cancer, on the other hand, this antibody may be a promising research compound to establish passive immunotherapy in dog patients with cancer. PMID:24755200

  17. Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4-ALK-rearranged non-small-cell lung cancer harbored coexisting EGFR mutation.

    PubMed

    Miyanaga, Akihiko; Shimizu, Kumi; Noro, Rintaro; Seike, Masahiro; Kitamura, Kazuhiro; Kosaihira, Seiji; Minegishi, Yuji; Shukuya, Takehito; Yoshimura, Akinobu; Kawamoto, Masashi; Tsuchiya, Shinichi; Hagiwara, Koichi; Soda, Manabu; Takeuchi, Kengo; Yamamoto, Nobuyuki; Mano, Hiroyuki; Ishikawa, Yuichi; Gemma, Akihiko

    2013-05-29

    The EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non-small-cell lung cancers (NSCLCs). The EML4-ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS. We report that a case of EML4-ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor. We described the first clinical report of a patient with EML4-ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4-ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4-ALK-positive NSCLC.

  18. Anti-EGFR activation, anti-proliferative and pro-apoptotic effects of polyclonal antibodies induced by EGFR-based cancer vaccine.

    PubMed

    Ramírez, Belinda Sánchez; Alpízar, Yeranddy Aguiar; Fernández, Diana Rosa Hernández; Hidalgo, Greta Garrido; Capote, Ailem Rabasa; Rodríguez, Rolando Pérez; Fernández, Luis Enrique

    2008-09-08

    Up to now clinical experiences focusing EGF receptor, an attractive target for cancer therapy, have been limited to passive therapies, suggesting that therapeutic cancer vaccines inducing anti-epidermal growth factor receptor (EGFR) antibodies could also work. Here, the humoral immune response induced in mice with a vaccine formulation containing the human EGFR-extracellular domain and very small-sized proteoliposomes (VSSP), a novel nanoparticulated adjuvant was assessed. In vaccinated mice sera average of the specific polyclonal antibodies (PAb) titers was 10(-5). Anti-EGFR PAb were able to bind EGFR+ tumor cell lines, expressing different levels of the molecule. Noteworthy, the presence of Cetuximab only partially inhibited the vaccine-induced antibodies binding to H125 cells. Anti-EGFR PAb abrogated ligands-dependent EGFR phosphorylation, provoking tumor cells apoptosis. The described EGFR-based vaccine might be a superior therapeutic approach for patients with EGFR+ tumors.

  19. Expression and clinical value of EGFR in human meningiomas

    PubMed Central

    Backer-Grøndahl, Thomas; Ytterhus, Borgny; Granli, Unn S.; Lydersen, Stian; Gulati, Sasha; Torp, Sverre H.

    2017-01-01

    Background Meningiomas are common intracranial tumors in humans that frequently recur despite having a predominantly benign nature. Even though these tumors have been shown to commonly express EGFR/c-erbB1 (epidermal growth factor receptor), results from previous studies are uncertain regarding the expression of either intracellular or extracellular domains, cellular localization, activation state, relations to malignancy grade, and prognosis. Aims This study was designed to investigate the expression of the intracellular and extracellular domains of EGFR and of the activated receptor as well as its ligands EGF and TGFα in a large series of meningiomas with long follow-up data, and investigate if there exists an association between antibody expression and clinical and histological data. Methods A series of 186 meningiomas consecutively operated within a 10-year period was included. Tissue microarrays were constructed and immunohistochemically analyzed with antibodies targeting intracellular and extracellular domains of EGFR, phosphorylated receptor, and EGF and TGFα. Expression levels were recorded as a staining index (SI). Results Positive immunoreactivity was observed for all antibodies in most cases. There was in general high SIs for the intracellular domain of EGFR, phosphorylated EGFR, EGF, and TGFα but lower for the extracellular domain. Normal meninges were negative for all antibodies. Higher SIs for the phosphorylated EGFR were observed in grade II tumors compared with grade I (p = 0.018). Survival or recurrence was significantly decreased in the time to recurrence analysis (TTR) with high SI-scores of the extracellular domain in a univariable survival analysis (HR 1.152, CI (1.036–1.280, p = 0.009)). This was not significant in a multivariable analysis. Expression of the other antigens did not affect survival. Conclusion EGFR is overexpressed and in an activated state in human meningiomas. High levels of ligands also support this growth factor

  20. Role of IKKalpha in the EGFR Signaling Regulation

    DTIC Science & Technology

    2012-09-01

    strategies for targeting various EGFR-associated cancers and/or non-cancerous diseases . In current study, we identified that EGFR serine phosphorylation...Rigo C, Piccinin S, Mizzau M, Sonego M, Fabris M, et al. Twist is substrate for caspase cleavage and proteasome-mediated degradation. Cell Death Differ...2008;133:704–15. 31. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease . Cell 2009;139:871–90. 32

  1. Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions.

    PubMed

    Mayo-de-Las-Casas, C; Jordana-Ariza, N; Garzón-Ibañez, M; Balada-Bel, A; Bertrán-Alamillo, J; Viteri-Ramírez, S; Reguart, N; Muñoz-Quintana, M A; Lianes-Barragan, P; Camps, C; Jantús, E; Remon-Massip, J; Calabuig, S; Aguiar, D; Gil, M L; Viñolas, N; Santos-Rodríguez, A K; Majem, M; García-Peláez, B; Villatoro, S; Pérez-Rosado, A; Monasterio, J C; Ovalle, E; Catalán, M J; Campos, R; Morales-Espinosa, D; Martínez-Bueno, A; González-Cao, M; González, X; Moya-Horno, I; Sosa, A E; Karachaliou, N; Rosell, R; Molina-Vila, M A

    2017-09-01

    In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

  2. Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

    PubMed Central

    Ma, Jian Jun; Xu, Peng; Zhang, Wei; Li, Yu Mei; Fu, Qiang; Zhu, Guang Feng; Xue, Wei; Lei, Yong Hua; Gao, Jing Yu; Wang, Juan Ying; Shao, Chen; Yi, Cheng Gang; Wang, He

    2013-01-01

    Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment. PMID:23950876

  3. 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

  4. EGFR exon 20 insertion mutation in Japanese lung cancer.

    PubMed

    Sasaki, Hidefumi; Endo, Katsuhiko; Takada, Minoru; Kawahara, Masaaki; Kitahara, Naoto; Tanaka, Hisaichi; Okumura, Meinoshin; Matsumura, Akihide; Iuchi, Keiji; Kawaguchi, Tomoya; Kawano, Osamu; Yukiue, Haruhiro; Yokoyama, Tomoki; Yano, Motoki; Fujii, Yoshitaka

    2007-12-01

    Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small cell lung cancer (NSCLC), especially in female, never smoker patients with adenocarcinoma. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine to arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib. On the other hand, previous report has shown that the insertion mutation at exon 20 is related to gefitinib resistance. We investigated the exon 20 EGFR mutation statuses in 322 surgically treated non-small cell lung cancer cases. Two hundred and five adenocarcinoma cases were included. The presence or absence of EGFR mutations of kinase domains was analyzed by direct sequences. EGFR insertion mutations at exon 20 were found from 7 of 322 (2.17%) lung cancer patients. We also detected the 18 deletion type mutations in exon 19, and 25 L858R type mutations in exon 21. There was a tendency towards higher exon 20 insertion ratio in never smoker (never smoker 4.4% versus smoker 1.3%, p=0.0996) and female (female 4.5% versus male 1.3%, p=0.0917). Two exon 20 insertion cases were treated with gefitinib and failed to response. EGFR insertion mutation in exon 20 could not be ignored from Japanese lung cancers.

  5. Prevalence of EGFR Mutations in Lung Cancer in Uruguayan Population.

    PubMed

    Berois, Nora; Touya, Diego; Ubillos, Luis; Bertoni, Bernardo; Osinaga, Eduardo; Varangot, Mario

    2017-01-01

    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. We examined EGFR mutations in exons 18 through 21 in 289 NSCLC Uruguayan patients by PCR-direct sequencing. 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. 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.

  6. Mechanisms underlying skin disorders induced by EGFR inhibitors

    PubMed Central

    Holcmann, Martin; Sibilia, Maria

    2015-01-01

    The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is frequently mutated or overexpressed in a large number of tumors such as carcinomas or glioblastoma. Inhibitors of EGFR activation have been successfully established for the therapy of some cancers and are more and more frequently being used as first or later line therapies. Although the side effects induced by inhibitors of EGFR are less severe than those observed with classic cytotoxic chemotherapy and can usually be handled by out-patient care, they may still be a cause for dose reduction or discontinuation of treatment that can reduce the effectiveness of antitumor therapy. The mechanisms underlying these cutaneous side effects are only partly understood. Important questions, such as the reasons for the correlation between the intensity of the side effects and the efficiency of treatment with EGFR inhibitors, remain to be answered. Optimized adjuvant strategies to accompany anti-EGFR therapy need to be found for optimal therapeutic application and improved quality of life of patients. Here, we summarize current literature on the molecular and cellular mechanisms underlying the cutaneous side effects induced by EGFR inhibitors and provide evidence that keratinocytes are probably the optimal targets for adjuvant therapy aimed at alleviating skin toxicities. PMID:27308503

  7. The EGFR Family: Not So Prototypical Receptor Tyrosine Kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph; Ferguson, Kathryn M.

    2014-01-01

    The epidermal growth factor receptor (EGFR) was among the first receptor tyrosine kinases (RTKs) for which ligand binding was studied and for which the importance of ligand-induced dimerization was established. As a result, EGFR and its relatives have frequently been termed “prototypical” RTKs. Many years of mechanistic studies, however, have revealed that—far from being prototypical—the EGFR family is quite unique. As we discuss in this review, the EGFR family uses a distinctive “receptor-mediated” dimerization mechanism, with ligand binding inducing a dramatic conformational change that exposes a dimerization arm. Intracellular kinase domain regulation in this family is also unique, being driven by allosteric changes induced by asymmetric dimer formation rather than the more typical activation-loop phosphorylation. EGFR family members also distinguish themselves from other RTKs in having an intracellular juxtamembrane (JM) domain that activates (rather than autoinhibits) the receptor and a very large carboxy-terminal tail that contains autophosphorylation sites and serves an autoregulatory function. We discuss recent advances in mechanistic aspects of all of these components of EGFR family members, attempting to integrate them into a view of how RTKs in this important class are regulated at the cell surface. PMID:24691965

  8. Differential Efficacy of Combined Therapy With Radiation and AEE788 in High and Low EGFR-Expressing Androgen-Independent Prostate Tumor Models

    SciTech Connect

    Huamani, Jessica; Willey, Christopher; Thotala, Dinesh; Niermann, Kenneth J.; Reyzer, Michelle; Leavitt, Lauren; Jones, Cameron; Fleishcher, Arthur; Caprioli, Richard; Hallahan, Dennis E.; Kim, Dong Wook Nathan

    2008-05-01

    Purpose: To determine the efficacy of combining radiation (XRT) with a dual epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor inhibitor, AEE788, in prostate cancer models with different levels of EGFR expression. Methods and Materials: Immunoblotting was performed for EGFR, phosphorylated-EGFR, and phosphorylated-AKT in prostate cancer cells. Clonogenic assays were performed on DU145, PC-3, and human umbilical vein endothelial cells treated with XRT {+-} AEE788. Tumor xenografts were established for DU145 and PC-3 on hind limbs of athymic nude mice assigned to four treatment groups: (1) control, (2) AEE788, (3) XRT, and (4) AEE788 + XRT. Tumor blood flow and growth measurements were performed using immunohistochemistry and imaging. Results: AEE788 effectively decreased phosphorylated-EGFR and phosphorylated-AKT levels in DU145 and PC-3 cells. Clonogenic assays showed no radiosensitization for DU145 and PC-3 colonies treated with AEE788 + XRT. However, AEE788 caused decreased proliferation in DU145 cells. AEE788 showed a radiosensitization effect in human umbilical vein endothelial cells and increased apoptosis susceptibility. Concurrent AEE788 + XRT compared with either alone led to significant tumor growth delay in DU145 tumors. Conversely, PC-3 tumors derived no added benefit from combined-modality therapy. In DU145 tumors, a significant decrease in tumor blood flow with combination therapy was shown by using power Doppler sonography and tumor blood vessel destruction on immunohistochemistry. Maldi-spectrometry (MS) imaging showed that AEE788 is bioavailable and heterogeneously distributed in DU145 tumors undergoing therapy. Conclusions: AEE788 + XRT showed efficacy in vitro/in vivo with DU145-based cell models, whereas PC-3-based models were adequately treated with XRT alone without added benefit from combination therapy. These findings correlated with differences in EGFR expression and showed effects on both tumor cell

  9. Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6

    PubMed Central

    YI, YONG WEON; YOU, KYUSIC; BAE, EDWARD JEONG; KWAK, SAHNG-JUNE; SEONG, YEON-SUN; BAE, INSOO

    2015-01-01

    Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB-231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB-231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB-231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through down-regulation of RPS6. PMID:25955731

  10. Dual inhibition of EGFR and MET induces synthetic lethality in triple-negative breast cancer cells through downregulation of ribosomal protein S6.

    PubMed

    Yi, Yong Weon; You, Kyusic; Bae, Edward Jeong; Kwak, Sahng-June; Seong, Yeon-Sun; Bae, Insoo

    2015-07-01

    Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB‑231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB‑231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB‑231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through downregulation of RPS6.

  11. Tyrosine phosphoproteomics identified both co-drivers and co-targeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer

    PubMed Central

    Smith, Matthew A.; Lopez, Alex S.; Bai, Yun; Li, Jiannong; Fang, Bin; Koomen, John; Rawal, Bhupendra; Fisher, Kate J.; Chen, Y. Ann; Kitano, Michiko; Morita, Yume; Yamaguchi, Haruka; Shibata, Kiyoko; Okabe, Takafumi; Okamoto, Isamu; Nakagawa, Kazuhiko; Haura, Eric B.

    2014-01-01

    Purpose Irreversible EGFR-tyrosine kinase inhibitors (TKIs) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gate-keeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass-spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined. Results We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src-family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK-inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced anti-tumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions Our results identified both co-drivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in NSCLC patients with acquired T790M. PMID:24919575

  12. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  13. Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

    PubMed Central

    Schäfer, A.; Pahnke, A.; Schaffert, D.; van Weerden, W.M.; de Ridder, C.M.A.; Rödl, W.; Vetter, A.; Spitzweg, C.; Kraaij, R.; Wagner, E.

    2011-01-01

    Abstract The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes. PMID:21644815

  14. EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib

    PubMed Central

    Gallant, Jean-Nicolas; Sheehan, Jonathan H.; Shaver, Timothy M.; Bailey, Mark; Lipson, Doron; Chandramohan, Raghu; Brewer, Monica Red; York, Sally J.; Kris, Mark G.; Pietenpol, Jennifer A.; Ladanyi, Marc; Miller, Vincent A.; Ali, Siraj M.; Meiler, Jens; Lovly, Christine M.

    2015-01-01

    Oncogenic EGFR mutations are found in 10-35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). In analyzing the tumor from a 33-year-old male never smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18-25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD-transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI, afatinib. The patient eventually progressed, at which time, re-sequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target. PMID:26286086

  15. Pooled analysis of clinical outcome for EGFR TKI-treated patients with EGFR mutation-positive NSCLC.

    PubMed

    Paz-Ares, Luis; Soulières, Denis; Moecks, Joachim; Bara, Ilze; Mok, Tony; Klughammer, Barbara

    2014-08-01

    Patients with non-small-cell lung cancer (NSCLC) appear to gain particular benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitors (TKI) if their disease tests positive for EGFR activating mutations. Recently, several large, controlled, phase III studies have been published in NSCLC patients with EGFR mutation-positive tumours. Given the increased patient dataset now available, a comprehensive literature search for EGFR TKIs or chemotherapy in EGFR mutation-positive NSCLC was undertaken to update the results of a previously published pooled analysis. Pooling eligible progression-free survival (PFS) data from 27 erlotinib studies (n = 731), 54 gefitinib studies (n = 1802) and 20 chemotherapy studies (n = 984) provided median PFS values for each treatment. The pooled median PFS was: 12.4 months (95% accuracy intervals [AI] 11.6-13.4) for erlotinib-treated patients; 9.4 months (95% AI 9.0-9.8) for gefitinib-treated patients; and 5.6 months (95% AI 5.3-6.0) for chemotherapy. Both erlotinib and gefitinib resulted in significantly longer PFS than chemotherapy (permutation testing; P = 0.000 and P = 0.000, respectively). Data on more recent TKIs (afatinib, dacomitinib and icotinib) were insufficient at this time-point to carry out a pooled PFS analysis on these compounds. The results of this updated pooled analysis suggest a substantial clear PFS benefit of treating patients with EGFR mutation-positive NSCLC with erlotinib or gefitinib compared with chemotherapy.

  16. EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth.

    PubMed

    Chabu, Chiswili; Li, Da-Ming; Xu, Tian

    2017-03-10

    Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, we show that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog.

  17. EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth

    PubMed Central

    Chabu, Chiswili; Li, Da-Ming; Xu, Tian

    2017-01-01

    Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, we show that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog. PMID:28281543

  18. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer.

    PubMed

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel; Teixidó, Cristina; Karachaliou, Niki; Pedersen, Martin Haar; Castellví, Josep; Garzón, Mónica; Codony-Servat, Carles; Codony-Servat, Jordi; Giménez-Capitán, Ana; Drozdowskyj, Ana; Viteri, Santiago; Larsen, Martin R; Lassen, Ulrik; Felip, Enriqueta; Bivona, Trever G; Ditzel, Henrik J; Rosell, Rafael

    2017-09-04

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that