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Louse flies (Diptera, Hippoboscidae) were collected off 38 species of birds from 16 genera of 14 families and 5 orders. Birds were captured in large funnel traps and mist nets on the Courish Spit (Kaliningrad Province, Russia). Five species of ornithophilic louse flies were found: Ornithomya avicularia (Linnaeus, 1758), O. chloropus (Bergroth, 1901), O. fringillina (Curtis, 1856), O. comosa (Austen, 1930), and Crataerina hirundinis (Linnaeus, 1758); besides, the mammalophilic species Lipoptena fortisetosa (Maa, 1965) was recorded on a non-specific bird host. The association of louse flies with their hosts, infestation parameters, collection data, and general geographic distribution of the recorded louse fly species are considered. The overall infestation of birds with louse flies was low. The louse fly fauna of Kaliningrad Province is compared with the faunas of the adjacent territories.

Introduction Osimertinib is the current recommended treatment for EGFR T790M–positive NSCLC after EGFR tyrosine kinase inhibitor therapy. However, resistance to osimertinib therapy is inevitably acquired after a period of effective treatment. We had a patient with EGFR L858R/T790M–positive NSCLC who initially responded to osimertinib therapy but eventually experienced development of resistance. Plasma cell–free DNA analysis revealed the occurrence of exon 16–skipping HER2, which may have resulted in the erb-b2 receptor tyrosine kinase 2 gene (HER2) splice variant HER2D16. HER2D16 has never been reported in lung cancer, and HER2D16-driven signaling is known to be regulated by Src kinase in breast cancer. We investigated the role of HER2D16 as an osimertinib-resistant mechanism. Methods We constructed and established H1975 cells stably expressing HER2D16. The dimeric formation of HER2D16 was tested by using nonreducing polyacrylamide gel electrophoresis. The effects of the study drugs on signaling transduction were examined by using Western blot. Synergistic effect was assessed by using the Chou-Talalay method. Results We found that HER2D16 can form a homodimer in NSCLC cells. HER2D16-expressing H1975 cells were resistant to osimertinib treatment. We also found that mutant EGFR and HER2D16 cooperated to activate downstream signaling for osimertinib resistance. In addition, cotreatment with osimertinib and an Src kinase inhibitor failed to reverse resistance, indicating that HER2D16-driven signaling in NSCLC did not occur through a canonical pathway. Finally, we revealed that the combination of osimertinib with the pan-HER small-molecule inhibitor afatinib could synergistically repress cell growth and signaling in H1975-HER2D16 cells. Conclusion HER2D16 can contribute to osimertinib resistance through an Src-independent pathway. HER2D16 should be included in the molecular diagnosis panel for lung cancer.

BACKGROUND This study assesses different technologies for detecting epidermal growth factor receptor (EGFR) mutations from circulating tumor DNA in patients with EGFR T790M-positive advanced non-small cell lung cancer (NSCLC) from the AURA3 study (NCT02151981), and it evaluates clinical responses to osimertinib and platinum-pemetrexed according to the plasma T790M status. METHODS Tumor tissue biopsy samples were tested for T790M during screening with the cobas EGFR Mutation Test (cobas tissue). Plasma samples were collected at screening and at the baseline and were retrospectively analyzed for EGFR mutations with the cobas EGFR Mutation Test v2 (cobas plasma), droplet digital polymerase chain reaction (ddPCR; Biodesix), and next-generation sequencing (NGS; Guardant360, Guardant Health). RESULTS With cobas tissue test results as a reference, the plasma T790M positive percent agreement (PPA) was 51% (110 of 215 samples) by cobas plasma, 58% (110 of 189) by ddPCR, and 66% (136 of 207) by NGS. Plasma T790M detection was associated with a larger median baseline tumor size (56 mm for T790M-positive vs 39 mm for T790M-negative; P

INTRODUCTION: EGFR mutated (EGFRm) NSCLC tumors occasionally express programmed cell death ligand 1 (PD-L1), although frequency and clinical relevance are not fully characterized. We report PD-L1 expression in patients with EGFRm advanced NSCLC and association with clinical outcomes following treatment with osimertinib or comparator EGFR tyrosine kinase inhibitors in the FLAURA trial (phase III, NCT02296125). METHODS: Of 231 tissue blocks available from the screened population (including EGFRm-positive and -negative samples), 197 had sufficient tissue for PD-L1 testing using the SP263 (Ventana, Tucson, Arizona) immunohistochemical assay. Tumor cell (TC) staining thresholds of PD-L1 TC greater than or equal to 1%, TC greater than or equal to 25%, and TC greater than or equal to 50% were applied. Progression-free survival (PFS) was investigator-assessed, per Response Evaluation Criteria in Solid Tumor, version 1.1, according to PD-L1 expressors (TC ≥ 1%) or negatives (TC < 1%) in randomized patients. RESULTS: PD-L1 staining was successful in 193 of 197 patient formalin-fixed paraffin-embedded blocks; of these, 128 of 193 were EGFRm-positive and 106 of 128 patients were randomized to treatment (osimertinib: 54; comparator: 52). At the PD-L1 TC greater than or equal to 25% threshold, 8% (10 of 128) of EGFRm-positive tumors expressed PD-L1 versus 35% (23 of 65) of EGFRm-negative tumors. With the TC greater than or equal to 1% threshold, 51% (65 of 128) versus 68% (44 of 65) were mutation-positive and -negative, respectively, and with the TC greater than or equal to 50% threshold, 5% (7 of 128) versus 28% (18 of 65), were mutation-positive and -negative, respectively. For PD-L1 expressors (TC ≥ 1%), median PFS was 18.4 months with osimertinib and 6.9 months with comparator (hazard ratio = 0.30; 95% confidence interval: 0.15-0.60). For PD-L1-negative patients (TC < 1%), median PFS was 18.9 months with osimertinib and 10.9 months with comparator (hazard ratio = 0.37; 95% confidence interval: 0.17-0.74). CONCLUSIONS: Clinical benefit with osimertinib was unaffected by PD-L1 expression status. ispartof: JOURNAL OF THORACIC ONCOLOGY vol:15 issue:1 pages:138-143 ispartof: location:United States status: published

Background/Purpose: MET-amplification is a resistance mechanism seen in ≤22% of patients (pts) with epidermal growth factor receptor mutation positive (EGFRm) non-small cell lung cancer (NSCLC) following progression on osimertinib, a third generation (3G), irreversible, oral EGFR tyrosine kinase inhibitor (TKI). In the Ph1b TATTON (NCT02143466) expansion cohorts (Parts B and D), pts with MET-amplified EGFRm advanced NSCLC and progression on a prior EGFR TKI, received osimertinib 80 mg + savolitinib 600 or 300 mg once daily; savolitinib is an oral, potent and highly selective MET TKI. Part B was split into three cohorts by prior therapy and tumor T790M status. Part D pts had received no prior 3G EGFR TKI and were T790M negative. For pts who eventually develop resistance to the combination, it is unclear which driver mutation(s) mediates this resistance. Methods: In this analysis, paired plasma samples collected at baseline and following disease progression and/or treatment discontinuation up to data cut-off date 4 March 2020, were assessed. Next generation sequencing (Guardant Health, Guardant360 73 gene panel or Omni 500 gene panel) was used to analyze the plasma ctDNA samples. All 73 genes on the Guardant360 panel were included in the Omni 500 gene panel. Analyses from each pt were reported only for genes included across the panels used. Genomic alterations were identified using Guardant Health's pipeline, which included mutations and amplifications of EGFR and MET. Disease progression was assessed by the investigator, according to the RECIST version 1.1. Assessments were completed for pts with progression-free survival (PFS) of over 2 months. Results: Of 180 pts who received study treatment, 70 provided baseline and progression/discontinuation ctDNA plasma samples. Of these, 45 pts were used for the analysis: 18/70 were not evaluable for ctDNA detection and 7/70 had a PFS of 2 months or shorter. Of the evaluable samples, the following acquired mutations were recorded (exclusivity between genes in most pts): MET D1228X, Y1230X, L1212X (20%, 9/45), EGFR C797X (16%, 7/45), KRAS G12X, G13X (11%, 5/45) and PIK3CA E545K (4%, 2/45). Most pts who developed MET-based resistance (7/9) developed more than one MET mutation, suggestive of polyclonal resistance. Across both Parts B and D, the resistance profiles appeared similar by prior EGFR TKI status and by savolitinib dose. Conclusions: In this analysis, approximately half of all evaluable pts had an identifiable acquired resistance mechanism; resistance appeared to be predominantly mediated by either MET, EGFR or KRAS. Co-occurring mutations across multiple genes were rarely detected. However, multiple acquired mutations were often detected in a specific gene, particularly MET, suggesting individual tumors showed inherent resistance dependencies. Citation Format: Aleksandra Markovets, Ji-Youn Han, Byoung Chul Cho, Mireille Cantarini, Pasi A. Janne, Ryan Hartmaier. Acquired resistance in patients with EGFRm NSCLC following treatment with osimertinib plus savolitinib in the Ph1b TATTON study Parts B and D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT024.

Nartshuk, E.P., Matyukhin, A.V., Shokhrin, V.P., Markovets, M.Yu. (2019): New records of ornithophilous louse-flies (Diptera: Hippoboscidae: Ornithomyinae) from the Russian Far East. Far Eastern Entomologist 384: 15-20, DOI: 10.25221/fee.384.4, URL: http://dx.doi.org/10.25221/fee.384.4

Circulating tumor DNA is released into the bloodstream from tumors and can reflect both intra-tumor heterogeneity and clonal evolution.1 As ctDNA levels are thought to reflect tumor burden, a decrease in ctDNA while on therapy may suggest treatment efficacy.2 We assessed whether longitudinal changes in ctDNA could supplement or improve RECIST-based measures for decision making during drug development. Plasma samples collected at regular intervals were analyzed to predict the risk of disease progression in patients with EGFRm (Ex19del or L858R) advanced NSCLC following initiation of first-line EGFR tyrosine kinase inhibitor (TKI) treatment in the FLAURA study (NCT02296125). Of 556 patients who received treatment (osimertinib 80 mg daily [QD] or comparator EGFR-TKI [gefitinib 250 mg QD or erlotinib 150 mg QD]), 353 (63%) had an available sample with detectable levels of ctDNA at baseline. Patients with both imaging and ctDNA samples at baseline and 3 additional timepoints (n=320, the analysis set) were divided (as implemented in caret R package)3 into training (n=259) and validation (n=61) cohorts. Clinical data from these patients (data cut-off: June 12 2017) were used to fit joint models of either ctDNA dynamics or sum of the longest tumor diameters (SLD) and progression-free survival (PFS). Longitudinal ctDNA levels were measured using droplet digital PCR (ddPCR; Biodesix®) for EGFR-TKI sensitizing mutations (Ex19del or L858R) in all available samples. Model covariates were selected based on their statistical significance for the longitudinal and hazard sub-models and were analyzed using JMbayes package as implemented in R software (3.5.1). Our analysis indicates that the joint model can independently characterize changes in ctDNA and SLD and can predict PFS in the validation cohort, using only ctDNA changes occurring within 6 weeks from the initiation of therapy. Similarly, in the training cohort, joint modelling indicates that the relationship between SLD and PFS using RECIST is consistent with the longitudinal ctDNA analysis. In the analysis set, our model estimated a PFS of 16.6 months (95% confidence interval [CI] 12.1, NC) for osimertinib (n=153) and 9.4 months (95% CI 8.0, 13.5) for comparator EGFR-TKI (n=167). Model-predicted PFS probability using either ctDNA or SLD data was similar, suggesting ctDNA dynamics may provide similar information to RECIST for clinical decision making. Based on this analysis, ctDNA dynamics could be utilized to predict RECIST-defined progression for decision making during drug development of treatments for patients with NSCLC.References: 1. Stewart CM, et al. J Pathol 2018;244(5):616-27 2. Herbreteau G, et al. J Thorac Dis 2019;11(Suppl 1):S113-26 3. R Core Team (2019). https://www.R-project.org/. Accessed Jan 27, 2020 Citation Format: Karthick Vishwanathan, James Dunyak, Philip Overend, Ryan Hartmaier, Aleksandra Markovets, Juliann Chmielecki, Ganesh Mugundu, Carl Barrett, Helen Tomkinson, Martin Johnson. Utility of longitudinal circulating tumor DNA (ctDNA) modeling to predict RECIST-defined progression in first-line patients with epidermal growth factor receptor mutation-positive (EGFRm) advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT024.

Osimertinib is a potent EGFR inhibitor (EGFRi) that is approved to treat patients with metastatic non-small cell lung cancer (NSCLC) with EGFR gene mutations. Despite the benefit observed for osimertinib many patients still develop resistance and relapse on treatment. One mechanism thought to cause resistance is de novo acquisition of resistant molecular changes in a drug tolerant persister (DTP) cell population. Here we characterized the molecular phenotypes of osimertinib DTPs to identify novel vulnerabilities to eliminate DTPs and prevent the emergence of drug resistance. We used a multiomics approach by combining transcriptomics (RNA-seq), proteomics (reverse phase protein array), and epigenomics assays (ATAC-seq) to profile osimertinib DTPs in four EGFRm NSCLC cell-lines (PC9, H1975, HCC2935, and HCC827). We compared DTPs generated by three weeks exposure to osimertinib to DMSO control, 24 hrs. acute treatment, and DTPs removed from drug. Due to the nature of the time-course we could disentangle effects of acute treatment from long-term DTPs; for example, we observed downregulation of cell-cycle and cell-cycle regulated processes in both acute treatment and DTPs. Phenotypically DTPs were in a non-proliferative state, morphologically larger, and resistant to apoptosis. Unsupervised clustering of multiomics data revealed that DTPs are distinct from acute treated and DMSO control cells at the epigenome, RNA, and protein-level. Interestingly, DTPs removed from drug for up to seven days do not fully return to control state but instead maintain some features in common with DTPs despite regaining proliferative ability. Motif analysis of regions that increased chromatin accessibility in Osimertinib DTPs identified an enrichment for TEAD transcription factor binding sites. TEAD transcription factors are regulated by the transcription co-activators YAP1/TAZ. When the Hippo pathway is on YAP1/TAZ is phosphorylated and localized to the cytoplasm, whereas when the pathway turns off YAP1/TAZ loses phosphorylation, translocate to the nucleus, and acts as a co-factor for TEAD transcription factors. We validated by Western blot that in osimertinib DTPs YAP1 loses phosphorylation indicating that the Hippo pathway switches from on to off. YAP1/TEAD downstream target genes also increase RNA expression in osimertinib DTPs. In addition to the Hippo pathway switch, and potentially related, we observe epithelial to mesenchymal (EMT) transition as a prominent molecular phenotype of DTPs across all four models. The EMT transition was evident at the transcriptome, proteome, and epigenetic level and is a prominent feature of surviving cells but not the 24 hrs. acute treatment. The chromatin accessibility changes at EMT regulated genes suggest the possibility that the EMT transition may be regulated by an epigenetic switch. Consistent with this idea we demonstrate that multiple drugs targeting the epigenome including HDAC inhibitors can effectively block regrowth of DTPs after osimertinib removal. Together our data suggests blocking protective changes to the chromatin landscape may represent a therapeutic strategy to target osimertinib DTPs. Citation Format: Steven W Criscione, Matthew Martin, Yi Yao, Aleksandra Markovets, Jingwen Zhang, Sladjana Gagrica, Oona Delpuech, Zhongwu Lai, Paul Smith, Jonathan Dry. The epigenome and gene expression landscape of osimeritib drug tolerant persister cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A117. doi:10.1158/1535-7163.TARG-19-A117

Resistance to targeted EGFR inhibitors is likely to develop in EGFR-mutant lung cancers. Early identification of innate or acquired resistance mechanisms to these agents is essential to direct development of future therapies. We describe the detection of heterogeneous mechanisms of resistance within populations of EGFR-mutant cells (PC9 and/or NCI-H1975) with acquired resistance to current and newly developed EGFR tyrosine kinase inhibitors, including AZD9291. We report the detection of NRAS mutations, including a novel E63K mutation, and a gain of copy number of WT NRAS or WT KRAS in cell populations resistant to gefitinib, afatinib, WZ4002, or AZD9291. Compared with parental cells, a number of resistant cell populations were more sensitive to inhibition by the MEK inhibitor selumetinib (AZD6244; ARRY-142886) when treated in combination with the originating EGFR inhibitor. In vitro, a combination of AZD9291 with selumetinib prevented emergence of resistance in PC9 cells and delayed resistance in NCI-H1975 cells. In vivo, concomitant dosing of AZD9291 with selumetinib caused regression of AZD9291-resistant tumors in an EGFRm/T790M transgenic model. Our data support the use of a combination of AZD9291 with a MEK inhibitor to delay or prevent resistance to AZD9291 in EGFRm and/or EGFRm/T790M tumors. Furthermore, these findings suggest that NRAS modifications in tumor samples from patients who have progressed on current or EGFR inhibitors in development may support subsequent treatment with a combination of EGFR and MEK inhibition. Cancer Res; 75(12); 2489–500. ©2015 AACR.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by

Recently, the lineage hTURDUS2 of Haemoproteus minutus (Haemosporida, Haemoproteidae) was reported to cause mortality in captive parrots. This parasite lineage is widespread and prevalent in the blackbird Turdus merula throughout its entire distribution range. Species identity of other closely related lineages recently reported in dead parrots remains unclear, but will be important to determine for a better understanding of the epidemiology of haemoproteosis. Using polymerase chain reaction (PCR)-based and microscopic methods, we analyzed 265 blood samples collected from 52 species of wild birds in Eurasia (23 samples from Kamchatka Peninsula, 73 from Sakhalin Island, 150 from Ekaterinburg and 19 from Irkutsk regions of Russia). Single infections of the lineages hTURDUS2 (hosts are redwing Turdus iliacus and fieldfare Turdus pilaris), hTUPHI1 (song thrush Turdus philomelos) and hTUCHR01 (fieldfare, redwing, song thrush and brown-headed thrush Turdus chysolaus) were detected. We identified species of these haemoproteids based on morphology of their blood stages and conclude that these lineages belong to H. minutus, a widespread parasite of different species of thrushes (genus Turdus), which serve as reservoir hosts of this haemoproteid infection. Phylogenetic analysis shows that the lineages hTURDUS2, hTUCHR01 and hTUPHI1 of H. minutus are closely related to Haemoproteus pallidus (lineages hPFC1 and hCOLL2), Haemoproteus pallidulus (hSYAT03), and Haemoproteus sp. (hMEUND3); genetic distance among their mitochondrial cytochrome b (cyt b) lineages is small (

Accurate variant calling in next generation sequencing (NGS) is critical to understand cancer genomes better. Here we present VarDict, a novel and versatile variant caller for both DNA- and RNA-sequencing data. VarDict simultaneously calls SNV, MNV, InDels, complex and structural variants, expanding the detected genetic driver landscape of tumors. It performs local realignments on the fly for more accurate allele frequency estimation. VarDict performance scales linearly to sequencing depth, enabling ultra-deep sequencing used to explore tumor evolution or detect tumor DNA circulating in blood. In addition, VarDict performs amplicon aware variant calling for polymerase chain reaction (PCR)-based targeted sequencing often used in diagnostic settings, and is able to detect PCR artifacts. Finally, VarDict also detects differences in somatic and loss of heterozygosity variants between paired samples. VarDict reprocessing of The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma dataset called known driver mutations in KRAS, EGFR, BRAF, PIK3CA and MET in 16% more patients than previously published variant calls. We believe VarDict will greatly facilitate application of NGS in clinical cancer research.

// Ryan E. Henry 1,* , Evan R. Barry 1,* , Lillian Castriotta 1 , Brendon Ladd 1 , Aleksandra Markovets 1 , Garry Beran 2 , Yongxin Ren 3 , Feng Zhou 3 , Ammar Adam 1 , Michael Zinda 1 , Corinne Reimer 1 , Weiguo Qing 3 , Weiguo Su 3 , Edwin Clark 1 , Celina M. D’Cruz 1 and Alwin G. Schuller 1 1 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Waltham, MA, USA 2 AstraZeneca Pharmaceuticals PLC, Oncology Bioscience, Alderley Park, UK 3 Hutchison Medi Pharma Ltd, Zhangjiang Hi-Tech Park, Shanghai, China * These authors have contributed equally to this work Correspondence to: Alwin G. Schuller, email: // Celina M. D’Cruz, email: // Keywords : MET, MYC, NSCLC, acquired resistance, savolitinib Received : January 14, 2016 Accepted : July 13, 2016 Published : July 26, 2016 Abstract Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib (volitinib, AZD6094, HMPL-504), a potent and selective MET inhibitor, in NSCLC. In vitro , savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo , savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET -amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.

Late-hatched Great Tits Parus major have often a lower local recruitment rate than those hatched early. However, estimates of recruitment usually neglect differential disappearance due to dispersal. We report on the proportions of late-hatched breeders in two Great Tit breeding populations (near St. Petersburg, NW Russia, and on the Courish Spit, Eastern Baltic). Using a novel method, late-hatched birds (hatching in July) were distinguished in the next breeding season by the incomplete moult of their tail feathers. Based on this method, we were able to estimate the percentage of late-hatched individuals in all first-year birds, including immigrants. The proportion of late hatched breeders was considerable in some years, and appeared related to the proportion of late fledglings in the previous year. In the Courish Spit population we found no significant difference in the global recruitment rate (accounting for both local birds and immigrants) of early- and late-hatched Great Tits. In the St. Petersburg population, the annual percentage of late-fledged breeders was significantly lower than the percentage of late-hatched fledglings in the previous year. However, in the St. Petersburg population we cannot definitely conclude that the global recruitment rate of late fledglings was lower than that of early fledglings because of peculiarities of the study site. We also can suggest that it may be a result of competition between early and late fledglings, winter residents and newcomers in spring (especially in males), as well as a result of different survival of early- and late-born individuals during the first year.

Background Haemoproteus parasites are widespread, and several species cause diseases both in birds and blood-sucking insects. These pathogens are transmitted by dipterans belonging to the Ceratopogonidae and Hippoboscidae, however certain vector species remain unknown for the majority of Haemoproteus spp. Owls are often infected by Haemoproteus parasites, but experimental studies on vectors of these infections are lacking. The aim of this study was to investigate sporogonic development of two widespread Haemoproteus parasites of owls, H. noctuae and H. syrnii in experimentally infected biting midges Culicoides impunctatus and Culicoides nubeculosus. We also followed in vitro sporogonic development of these infections and determined their phylogenetic relationships with Haemoproteus spp., for which vectors have been identified. Methods Wild-caught C. impunctatus and laboratory reared C. nubeculosus were infected experimentally by allowing them to take blood meals on one individual long-eared owl (Asio otus) and one tawny owl (Strix aluco) harbouring mature gametocytes of H. noctuae (lineage hCIRCUM01) and H. syrnii (hCULCIB01), respectively. The engorged insects were maintained in the laboratory at 16–18 °C, and dissected at intervals in order to follow the development of ookinetes, oocysts and sporozoites. We also observed in vitro development of sexual stages of both parasites by exposure of infected blood to air. The parasite lineages were determined by polymerase chain reaction-based methods. Bayesian phylogeny was constructed in order to determine the relationships of owl parasites with other avian Haemoproteus spp., for which vectors have been identified. Results Both H. noctuae and H. syrnii completed sporogony in C. nubeculosus, and H. noctuae completed sporogony in C. impunctatus. Ookinetes, oocysts and sporozoites of these parasites were reported and described. Gametes and ookinetes of both species readily developed in vitro. In accordance with sporogony data, the phylogenetic analysis placed both parasite lineages in a clade of Culicoides spp.-transmitted avian Haemoproteus (Parahaemoproteus) spp. Conclusions Culicoides nubeculosus and C. impunctatus are vectors of H. noctuae and H. syrnii. Phylogenies based on cytochrome b gene indicate parasite-vector relationships, and we recommend using them in predicting possible parasite-vector relationships and planning research on avian Haemoproteus spp. vectors in wildlife.

Here we studied cell-free plasma DNA (cfDNA) collected from subjects with advanced lung cancer whose tumors had developed resistance to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) AZD9291. We first performed next-generation sequencing of cfDNA from seven subjects and detected an acquired EGFR C797S mutation in one; expression of this mutant EGFR construct in a cell line rendered it resistant to AZD9291. We then performed droplet digital PCR on serial cfDNA specimens collected from 15 AZD9291-treated subjects. All were positive for the T790M mutation before treatment, but upon developing AZD9291 resistance three molecular subtypes emerged: six cases acquired the C797S mutation, five cases maintained the T790M mutation but did not acquire the C797S mutation and four cases lost the T790M mutation despite the presence of the underlying EGFR activating mutation. Our findings provide insight into the diversity of mechanisms through which tumors acquire resistance to AZD9291 and highlight the need for therapies that are able to overcome resistance mediated by the EGFR C797S mutation.

Species of Leucocytozoon (Haemosporida, Leucocytozoidae) traditionally have been described based on morphological characters of their blood stages and host cells, with limited information on their avian host specificity. Based on the current taxonomy, Leucocytozoon toddi is the sole valid species of leucocytozoids parasitizing falconiform birds. Using a nested polymerase chain reaction protocol, we determined the prevalence of Leucocytozoon infection in 5 species of diurnal raptors from California. Of 591 birds tested, 177 (29.9%) were infected with Leucocytozoon toddi. Subsequent phylogenetic analysis of the cytochrome b gene revealed that distinct haplotypes are present in hawks of these genera. Haplotypes present in Buteo spp. are not found in Accipiter spp., and there is a 10.9% sequence divergence between the 2 lineage clades. In addition, Leucocytozoon sp. from Accipiter spp. from Europe group more closely with parasites found in Accipiter spp. from California than the same California Accipiter species do with their sympatric Buteo spp. Similarly, a Leucocytozoon haplotype from a Common Buzzard (Buteo buteo) from Kazakhstan forms a monophyletic lineage with a parasite from B. jamaicensis from California. These results suggest that Leucocytozoon toddi is most likely a group of cryptic species, with 1 species infecting Buteo spp. and 1 or more species, or subspecies, infecting Accipiter spp.

The most critical, and dangerous, time of the avian life cycle probably is the migratory period when not only does a migrating bird have to decide on the routes to take to and from the wintering grounds but also when to rest, where and for how long. To understood the migrants decision we studied migratory European Bee-eaters (Merops apiaster) along the Arava Valley, Israel, during their spring migration stopover immediately after crossing the combined ecological barrier of the Sahel, Sahara, and Sinai deserts. We evaluated the effects of body mass and age on the decision to stopover as a function of the distance from the northern edge of the deserts northwards up to the Dead Sea basin. We propose a new “drop-out hypothesis” wherein the weaker individuals dropout earlier from a migratory flock that is moving northwards from an ecological barrier. Most of the birds that drop out at the stopover sites closest to the ecological barrier are juveniles, on their first migration from Africa and that lack experience of the northbound flight. The proportion of adults increases as the flock moves northwards. The evolutionary and conservation implications of this study stress the importance of conservation of not only a single stopover site along the migratory route but that of several points staggered out such that they will allow individuals with varying degrees of body condition to advance in their desired direction with minimum stress.

Between 1984 and 2002, a total of 7643 Bluethroats were ringed and 1054 of these were re-captured at the International Birding and Research Center (IBRCE) in Eilat, Israel (29°33′N, 34°57′E). The autumn migration of Bluethroat begins in September and ends in early December (median 01-Nov). The spring migration starts in early March and continues till May (median 21-Mar). Wintering Bluethroats arrive during October and November and stay in Eilat until March and April. The Bluethroats remain at the winter quarters for up to 4 months, though some individuals could linger for as long as more than 5 months. The majority of the wintering population was males (65.3%) and adult birds (66.1%), the sex ratio during winter (65:35) did not differ significantly from the sex ratio during autumn migration (60:40). The ratio between young and adult birds at Eilat during winter (66:34) differed significantly from the autumn migration (75:25). Wintering site fidelity for six seasons (consecutive autumn through spring) from 1996 to 2001 was 1.8%. Wintering site fidelity of Bluethroats when compared between birds that winter for the second and third season shows a recovery rate of 37.1%.

Targeted sequencing is used increasingly in clinics to guide therapeutic decisions by measuring mutations, small indels and/or rearrangements in cancer genes. An ability to use the same platform to detect additional oncogene activation (or tumor suppressor loss) through copy number changes could significantly expand the number of patients able to benefit from targeted therapies. Many currently available tools either requires a matched normal, works only for whole genome or exome sequencing, or don't work for PCR-based targeted sequencing. In addition, no tools available to detect breakpoints within a gene from targeted sequencing. A versatile copy number analysis tool from sequencing is needed to maximize the value of the sequencing routinely applied in clinics. Here we presented a novel computational tool, Seq2C (Sequencing To Copy Number), which is versatile to handle various situations and reports aberrations at gene level ready for interpretation. Seq2C works at cohort level and does not require a matched ‘normal’, though it can optionally use one or more normal samples for small ( Another distinct feature differentiating Seq2C from other currently available tools is that Seq2C identifies breakpoints and detects one or more exon deletion or duplication rearrangements within a gene, which is common in tumor suppressors as a mechanism to lose function. In addition, it can also detect potential fusions in genes such as ALK and ERG, where a copy number change is often accompanied with the fusion and a breakpoint can thus be called by Seq2C. Furthermore, it can predict gender from exome or whole genome sequencing. We applied Seq2C to exome sequencing of CCLE cell lines, and showed that it produced gene level copy number data highly correlated to those derived from microarrays, the current gold standard. Interestingly, Seq2C identified one or more exon deletions in several common tumor suppressors, such as TP53, PTEN, CDKN2A, NF1, STK11 and RB1, in cell lines with no known aberrations. They were supported by RNA-Seq data. It also correctly called known ERG fusion in prostate cell line NCI-H660 and identified a previously un-reported EML4-ALK fusion in a pancreatic cancer cell line SNU-324, which is confirmed by RNA-Seq data, suggesting EML4-ALK is not limited to lung cancer. In conclusion, Seq2C is a versatile copy number analysis tool for sequencing and will be useful for cancer research. Seq2C is freely available in GitHub. Citation Format: Zhongwu Lai, Aleksandra Markovets, Jonathan Dry. Seq2C: from sequence to copy number for cancer samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5268.

Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. Aberrant receptor tyrosine kinase (RTK) signaling is a well-documented driver of disease onset and progression in multiple cancer types, including non-small cell lung cancer (NSCLC), where the cMET RTK contributes to tumor progression, maintenance and resistance to targeted therapies. Here, we explore the therapeutic potential of the potent and selective cMET inhibitor savolitinib (volitinib, AZD6094, HMPL-504) in NSCLC and begin to elucidate mechanisms of acquired savolitinib resistance in preclinical models. Using in vitro proliferation assays and immunoblot analysis, we determine that savolitinib rapidly inhibits cMET auto-phosphorylation/activation and reduces the viability of NSCLC cell lines NCI-H1993 and EBC-1 with a GI50 of 4.20 nM and 2.14 nM, respectively. In vivo, once daily treatment of NCI-H1993 xenografts with 3.0 mg/kg savolitinib significantly slows tumor growth, whereas treatment of EBC-1 xenografts with 30.0 mg/kg results in tumor stasis. Importantly, we observe tumor regressions in a patient-derived xenograft model of a NSCLC lymph node metastasis, HLXF-036LN, dosed with savolitinib 50.0 mg/kg once daily. Pharmacodynamic analysis of in vitro and in vivo models shows that savolitinib sensitivity correlates with blockade of PI3K/AKT and MAPK signaling, and interestingly, with cMYC (MYC) protein down-regulation. To elucidate mechanisms of acquired resistance in NSCLC, we generated savolitinib resistance in vitro using the NCI-H1993 and EBC-1 cell lines and further sub-cloned resistant NCI-H1993 cells to study the heterogeneity of resistance mechanisms. Using small-molecule screening, phospho-protein arrays and interrogation of signaling pathway activity by immunoblot, we identify 1) deregulated mTORC1/2 signaling and 2) the uncoupling of MYC expression from cMET activation as commonly contributing to resistance in all clones tested. RNA interference (siRNA) and MYC over-expression experiments confirm the novel finding that sustained MYC expression can partially drive resistance to a tyrosine kinase inhibitor such as savolitinib. Additionally, we identify clone-specific resistance mechanisms arising via a previously-described switch to EGFR dependence or by our novel finding of a de novo requirement for PIM signaling. Taken together, this work demonstrates the preclinical efficacy of savolitinib in NSCLC and provides an initial characterization of potential resistance mechanisms, identifying core resistance targets and clone-specific vulnerabilities that could be exploited to counter acquired savolitinib resistance that may emerge in the clinic. Citation Format: Ryan E. Henry, Evan R. Barry, Brendon Ladd, Aleksandra Markovets, Garry J. Beran, Yongxin Ren, Feng Zhou, Lillian Castriotta, Ammar Adam, Weiguo Qing, Weiguo Su, Edwin Clark, Celina M. D'Cruz, Alwin Schuller. Acquired resistance to the cMET inhibitor savolitinib in lung cancer models through EGFR/mTOR/MYC deregulation and adoption of PIM signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C22.

Background Recent advances in genomics and proteomics have allowed us to study the nuances of the Warburg effect – a long-standing puzzle in cancer energy metabolism – at an unprecedented level of detail. While modern next-generation sequencing technologies are extremely powerful, the lack of appropriate data analysis tools makes this study difficult. To meet this challenge, we developed a novel application for comparative analysis of gene expression and visualization of RNA-Seq data. Results We analyzed two biological samples (normal human brain tissue and human cancer cell lines) with high-energy, metabolic requirements. We calculated digital topology and the copy number of every expressed transcript. We observed subtle but remarkable qualitative and quantitative differences between the citric acid (TCA) cycle and glycolysis pathways. We found that in the first three steps of the TCA cycle, digital expression of aconitase 2 (ACO2) in the brain exceeded both citrate synthase (CS) and isocitrate dehydrogenase 2 (IDH2), while in cancer cells this trend was quite the opposite. In the glycolysis pathway, all genes showed higher expression levels in cancer cell lines; and most notably, digital gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase (ENO) were considerably increased when compared to the brain sample. Conclusions The variations we observed should affect the rates and quantities of ATP production. We expect that the developed tool will provide insights into the subtleties related to the causality between the Warburg effect and neoplastic transformation. Even though we focused on well-known and extensively studied metabolic pathways, the data analysis and visualization pipeline that we developed is particularly valuable as it is global and pathway-independent.

UnlabelledThe incidence of age-related macular degeneration (AMD), the main cause of blindness in older patients in the developed countries, is increasing with the ageing population. At present there is no effective treatment for the prevailing geographic atrophy, dry AMD, whereas antiangiogenic therapies successful used in managing the wet form of AMD. Recently we showed that mitochondria-targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium (SkQ1) is able to prevent the development and moreover caused regression of pre-existing signs of the retinopathy in OXYS rats, an animal model of AMD. Here we examine the effects of SkQ1 on expression of key regulators of angiogenesis vascular endothelial growth factor A (VEGF) and its antagonist pigment epithelium-derived factor (PEDF) genes in the retina of OXYS rats as evidenced by real-time PCR and an ELISA test for VEGF using Wistar rats as control. Ophthalmoscopic examinations confirmed that SkQ1 supplementation (from 1.5 to 3 months of age, 250 nmol/kg) prevented development while eye drops SkQ1 (250 nM, from 9 to 12 months) caused some reduction of retinopathy signs in OXYS rats and did not reveal any negative effects on the control Wistar rat's retina. Prevention of premature retinopathy by SkQ1 was connected with an increase of VEGF mRNA and protein in OXYS rat's retina up to the levels corresponding to the Wistar rats, and did not involve changes in PEDF expression. In contrast the treatment with SkQ1 drops caused a decrease of VEGF mRNA and protein levels and an increase in the PEDF mRNA level in the middle-aged OXYS rats, but in Wistar rats the changes of gene expression were the opposite.ConclusionsThe beneficial effects of SkQ1 on retinopathy connected with normalization of expression of VEGF and PEDF in the retina of OXYS rats and depended on age of the animals and the stage of retinopathy.

Pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the world, remains poorly understood. This makes it necessary to create animal models for studying AMD pathogenesis and to design new therapeutic approaches. Here we showed that retinopathy in OXYS rats is similar to human AMD according to clinical signs, morphology, and vascular endothelium growth factor (VEGF) and pigment epithelium-derived factor (PEDF) genes expression. Clinical signs of retinopathy OXYS rats manifest by the age 3 months against the background of significantly reduced expression level of VEGF and PEDF genes due to the decline of the amount of retinal pigment epithelium (RPE) cells and alteration of choroidal microcirculation. The disruption in OXYS rats' retina starts at the age of 20 days and appears as reduce the area of RPE cells but does not affect their ultrastructure. Ultrastructural pathological alterations of RPE as well as develop forms of retinopathy are observed in OXYS rats from age 12 months and manifested as excessive accumulation of lipofuscin in RPE regions adjacent to the rod cells, whirling extentions of the basement membrane into the cytoplasm. These data suggest that primary cellular degenerative alterations in the RPE cells secondarily lead to choriocapillaris atrophy and results in complete loss of photoreceptor cells in the OXYS rats' retina by the age of 24 months.

Background One of the major challenges in biology is the correct identification of promoter regions. Computational methods based on motif searching have been the traditional approach taken. Recent studies have shown that DNA structural properties, such as curvature, stacking energy, and stress-induced duplex destabilization (SIDD) are useful in promoter prediction, as well. In this paper, the currently used SIDD energy threshold method is compared to the proposed artificial neural network (ANN) approach for finding promoters based on SIDD profile data. Results When compared to the SIDD threshold prediction method, artificial neural networks showed noticeable improvements for precision, recall, and F-score over a range of values. The maximal F-score for the ANN classifier was 62.3 and 56.8 for the threshold-based classifier. Conclusions Artificial neural networks were used to predict promoters based on SIDD profile data. Results using this technique were an improvement over the previous SIDD threshold approach. Over a wide range of precision-recall values, artificial neural networks were more capable of identifying distinctive characteristics of promoter regions than threshold based methods.

Analysis of the mean date of first captures and median arrival dates of spring migration for 34 species of birds at Eilat, Israel, revealed that the earlier a species migrates through Eilat, the greater is the inter-annual variation in the total time of its passage. Birds arrive during spring migration in Eilat in four structured and independent waves. The annual fluctuation in the initial arrival dates (initial capture dates) and median dates (median date of all captures), not including recaptures, did not depend on the length of the migratory route. This implies that migrants crossing the Sahara desert depart from their winter quarters on different Julian days in different years. We suggest that negative correlations between the median date of the spring migration of early and late migrants depends upon the easterly (Hamsin) wind period. Moreover, we believe that the phenology of all birds during spring migration in Eilat is possibly also determined by external factors such as weather conditions on the African continent or global climatic processes in the Northern hemisphere. Orphean Warblers (Sylvia hortensis) show a strong positive correlation (rs=-0.502) of initial capture date with calendar years, whereas other species such as Barred Warbler (S. nisoria; rs = -0.391) and Spotted Flycatcher (Muscicapa striata; rs = -0.398) display an insignificant trend. The Dead Sea Sparrow (Passer moabiticus) and Red-Backed Shrike (Lanius collurio) are positively correlated regarding initial arrival date and medians of spring migration.

Cancer genomes are known to harbor a wide range of mutations, including complex variants with combination of insertion and deletion (InDels). Next generation sequencing (NGS) has revolutionized our understanding of mutations in cancer. Most of current variant callers from NGS focused on single nucleotide variants (SNV) or short InDels. However, detection of complex variants remains a challenge and off limits to most current variant callers. In addition, efficient analysis of ultra-deep targeted sequencing without downsampling for low frequency mutations in heterogeneous cancer samples, which is becoming more routine, is also a challenge and not handled well by most current variant callers. Here, we describe VarDict, a novel and versatile variant caller for ultra-deep targeted deep sequencing, exome, whole genome, and RNA-seq. VarDict is designed for heterogeneous cancer genomes and is able to simultaneously call SNVs (Single-Nucleotide Variants), MNVs (Multiple-Nucleotide Variants), InDels (user-defined sizes), and complex variants (combination of aforementioned events). VarDict handles ultra-deep sequencing of runs up to mean coverage of 1M without down-sampling or significant loss of performance. It performs local realignment around InDels on the fly and rescues soft-clipped reads for more accurate estimation of allele frequencies as well as allowing calls of InDels only supported by soft-clipped reads. In addition, it performs amplicon aware variant calling for PCR-based targeted sequencing by avoiding calling variants in PCR primers, discounting primer depths, and importantly, detecting variants with amplicon-bias, a common artifact for PCR based targeted sequencing. VarDict can also be run in paired mode to identify somatic or LOH variants, as well as variants whose allele frequencies have shifted significantly. It thus enables paired DNA-seq and RNA-seq variant calling that most variant callers do not handle well. To demonstrate the value of VarDict in practice, we applied VarDict on the WGS of NA12878, and compared the result to the calls made in Genome In A Bottle (GiaB). VarDict was able to call >96% of variants in GiaB. In addition, it found many more variants likely missed by GiaB, especially complex variants, many of which were never categorized before. We further applied VarDict in the ICGC-TCGA DREAM Mutation Calling challenge (syn312572). We found it to be as sensitive as the more commonly used somatic SNP callers like MuTect, Freebayes, and VarScan in calling SNPs but more sensitive in calling InDels than an array of other variant callers, including VarScan, FreeBayes, and Scalpel. VarDict is fully open source, implemented in Perl, and uses memory efficiently, regardless of the depth, making it a HPC cluster friendly tool. VarDict has further been integrated into bcbio-nextgen, an open source framework for scalable NGS analysis for ease of deployment. VarDict is freely available in GitHub (https://github.com/AstraZeneca-NGS/VarDict). Citation Format: Zhongwu Lai, Aleksandra Markovets, Miika Ahdesmaki, Justin Johnson. VarDict: A novel and versatile variant caller for next-generation sequencing in cancer research. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2015-4864

This paper examines the phenology and biometrics of Bluethroats staging in the Eilat region. This is of special interest because of the extreme conditions with which this temperate zone breeding species has to contend because Eilat is a desert habitat and is the last green area before the crossing of the deserts in autumn or after it in spring. Data were collected during 20 spring and 18 autumn migration seasons in the years 1984–2003, and a total of 7,464 Bluethroat were recorded. The number of trapped birds was much higher in autumn than in spring. The majority of Bluethroats caught in both the autumn and spring migrations were juveniles. We found differences in sex ratio in the individual age classes only in the autumn wherein among both adults and juveniles, males were in greater numbers. We also found significant differences in the dates of ringed birds from different sex–age classes in the spring and in autumn migrations. In spring, males from both age classes were caught earlier than females. In autumn, adult birds arrived earlier than juveniles. We think that it is important to identify and conserve the high quality stopover habitats such as Eilat wherein not only Bluethroats have been shown to stopover but also several hundred other species.