Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain

<p>Abstract</p> <p>Background</p> <p>The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated.</p> <p>Results</p> <p>We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms.</p> <p>Conclusion</p> <p>The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.</p

Inherited germline T790M mutation and somatic epidermal growth factor receptor mutations in non-small cell lung cancer patients.

Five cases of non-small cell lung cancer, associated with germline transmission of epidermal growth factor receptor (EGFR)-T790M mutation, have been reported1; these patients had family histories of lung cancer. The activity of gefitinib was tested in only two patients, who were both refractory to this drug.2 Herein, we describe a family of European descent in which two family members had non-small cell lung cancer associated with germline transmission of T790M mutation and who were treated with gefitinib

An Influenza A/H1N1/2009 Hemagglutinin Vaccine Produced in Escherichia coli

The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy.We expressed the globular HA receptor binding domain, referred to here as HA(63-286)-RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA(63-286)-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model.Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients

The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan–Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort, with P=0.00015 as the P-value for a test of the additional impact of markers on prognosis, after taking stage into consideration. Confirmation of the impact of these markers in independent studies will be necessary

<i>Gaia</i> Data Release 1. Summary of the astrometric, photometric, and survey properties

Context. At about 1000 days after the launch of Gaia we present the first Gaia data release, Gaia DR1, consisting of astrometry and photometry for over 1 billion sources brighter than magnitude 20.7. Aims. A summary of Gaia DR1 is presented along with illustrations of the scientific quality of the data, followed by a discussion of the limitations due to the preliminary nature of this release. Methods. The raw data collected by Gaia during the first 14 months of the mission have been processed by the Gaia Data Processing and Analysis Consortium (DPAC) and turned into an astrometric and photometric catalogue. Results. Gaia DR1 consists of three components: a primary astrometric data set which contains the positions, parallaxes, and mean proper motions for about 2 million of the brightest stars in common with the HIPPARCOS and Tycho-2 catalogues – a realisation of the Tycho-Gaia Astrometric Solution (TGAS) – and a secondary astrometric data set containing the positions for an additional 1.1 billion sources. The second component is the photometric data set, consisting of mean G-band magnitudes for all sources. The G-band light curves and the characteristics of ∼3000 Cepheid and RR-Lyrae stars, observed at high cadence around the south ecliptic pole, form the third component. For the primary astrometric data set the typical uncertainty is about 0.3 mas for the positions and parallaxes, and about 1 mas yr−1 for the proper motions. A systematic component of ∼0.3 mas should be added to the parallax uncertainties. For the subset of ∼94 000 HIPPARCOS stars in the primary data set, the proper motions are much more precise at about 0.06 mas yr−1. For the secondary astrometric data set, the typical uncertainty of the positions is ∼10 mas. The median uncertainties on the mean G-band magnitudes range from the mmag level to ∼0.03 mag over the magnitude range 5 to 20.7. Conclusions. Gaia DR1 is an important milestone ahead of the next Gaia data release, which will feature five-parameter astrometry for all sources. Extensive validation shows that Gaia DR1 represents a major advance in the mapping of the heavens and the availability of basic stellar data that underpin observational astrophysics. Nevertheless, the very preliminary nature of this first Gaia data release does lead to a number of important limitations to the data quality which should be carefully considered before drawing conclusions from the data

HAWC Study of Very-High-Energy γ\gamma-ray Spectrum of HAWC J1844-034

Recently, the region surrounding eHWC J1842-035 has been studied extensively by gamma-ray observatories due to its extended emission reaching up to a few hundred TeV and potential as a hadronic accelerator. In this work, we use 1,910 days of cumulative data from the High Altitude Water Cherenkov (HAWC) observatory to carry out a dedicated systematic source search of the eHWC J1842-035 region. During the search we have found three sources in the region, namely, HAWC J1844-034, HAWC J1843-032, and HAWC J1846-025. We have identified HAWC J1844-034 as the extended source that emits photons with energies up to 175 TeV. We compute the spectrum for HAWC J1844-034 and by comparing with the observational results from other experiments, we have identified HESS J1843-033, LHAASO J1843-0338, and TASG J1844-038 as very-high-energy gamma-ray sources with a matching origin. Also, we present and use the multi-wavelength data to fit the hadronic and leptonic particle spectra. We have identified four pulsar candidates in the nearby region from which PSR J1844-0346 is found to be the most likely candidate due to its proximity to HAWC J1844-034 and the computed energy budget. We have also found SNR G28.6-0.1 as a potential counterpart source of HAWC J1844-034 for which both leptonic and hadronic scenarios are feasible.Comment: 13 pages, 9 figures, published in Ap

Validation of standardized data formats and tools for ground-level particle-based gamma-ray observatories

Ground-based gamma-ray astronomy is still a rather young field of research,with strong historical connections to particle physics. This is why mostobservations are conducted by experiments with proprietary data and analysissoftware, as it is usual in the particle physics field. However in recentyears, this paradigm has been slowly shifting towards the development and useof open-source data formats and tools, driven by upcoming observatories such asthe Cherenkov Telescope Array (CTA). In this context, a community-driven,shared data format (the gamma-astro-data-format or GADF) and analysis toolssuch as Gammapy and ctools have been developed. So far these efforts have beenled by the IACT community, leaving out other types of ground-based gamma-rayinstruments.We aim to show that the data from ground particle arrays, such asthe High-Altitude Water Cherenkov (HAWC) observatory, is also compatible withthe GADF and can thus be fully analysed using the related tools, in this caseGammapy. We reproduce several published HAWC results using Gammapy and dataproducts compliant with GADF standard. We also illustrate the capabilities ofthe shared format and tools by producing a joint fit of the Crab spectrumincluding data from six different gamma-ray experiments. We find excellentagreement with the reference results, a powerful check of both the publishedresults and the tools involved. The data from particle detector arrays such asthe HAWC observatory can be adapted to the GADF and thus analysed with Gammapy.A common data format and shared analysis tools allow multi-instrument jointanalysis and effective data sharing. Given the complementary nature of pointingand wide-field instruments, this synergy will be distinctly beneficial for thejoint scientific exploitation of future observatories such as the SouthernWide-field Gamma-ray Observatory and CTA.<br

Gamma-ray Emission from Classical Nova V392 Per: Measurements from Fermi and HAWC

This paper reports on the $\gamma$-ray properties of the 2018 Galactic novaV392 Per, spanning photon energies $\sim$0.1 GeV to 100 TeV by combiningobservations from the Fermi Gamma-ray Space Telescope and the HAWC Observatory.In one of the most rapidly evolving $\gamma$-ray signals yet observed for anova, GeV $\gamma$ rays with a power law spectrum with index $\Gamma = 2.0 \pm0.1$ were detected over eight days following V392 Per's optical maximum. HAWCobservations constrain the TeV $\gamma$-ray signal during this time and alsobefore and after. We observe no statistically significant evidence of TeV$\gamma$-ray emission from V392 Per, but present flux limits. Tests of theextension of the Fermi/LAT spectrum to energies above 5 TeV are disfavored by 2standard deviations (95\%) or more. We fit V392 Per's GeV $\gamma$ rays withhadronic acceleration models, incorporating optical observations, and comparethe calculations with HAWC limits.<br

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

BACKGROUND: Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. METHODOLOGY AND PRINCIPAL FINDINGS: In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. CONCLUSIONS: We suggest that targeting HSP90 will have clinical impact for NSCLC patients

The High-Altitude Water Cherenkov (HAWC) Observatory in M\'exico: The Primary Detector

The High-Altitude Water Cherenkov (HAWC) observatory is a second-generation continuously operated, wide field-of-view, TeV gamma-ray observatory. The HAWC observatory and its analysis techniques build on experience of the Milagro experiment in using ground-based water Cherenkov detectors for gamma-ray astronomy. HAWC is located on the Sierra Negra volcano in M\'exico at an elevation of 4100 meters above sea level. The completed HAWC observatory principal detector (HAWC) consists of 300 closely spaced water Cherenkov detectors, each equipped with four photomultiplier tubes to provide timing and charge information to reconstruct the extensive air shower energy and arrival direction. The HAWC observatory has been optimized to observe transient and steady emission from sources of gamma rays within an energy range from several hundred GeV to several hundred TeV. However, most of the air showers detected are initiated by cosmic rays, allowing studies of cosmic rays also to be performed. This paper describes the characteristics of the HAWC main array and its hardware.Comment: Accepted for publications in Nuclear Inst. and Methods in Physics Research, A (2023) 168253 ( https://www.sciencedirect.com/science/article/abs/pii/S0168900223002437 ); 39 pages, 14 Figure