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PURPOSE Some gastric cancers harbor MET gene amplifications that can be targeted by selective MET inhibitors to achieve tumor responses, but resistance eventually develops. Savolitinib, a selective MET inhibitor, is beneficial for treating patients with MET-driven gastric cancer. Understanding the resistance mechanisms is important for optimizing postfailure treatment options. PATIENTS AND METHODS Here, we identified the mechanisms of acquired resistance to savolitinib in 3 patients with gastric cancer and MET-amplified tumors who showed a clinical response and then cancer progression. Longitudinal circulating tumor DNA (ctDNA) is useful for monitoring resistance during treatment and progression when rebiopsy cannot be performed. RESULTS Using a next-generation sequencing 100-gene panel, we identified the target mechanisms of resistance MET D1228V/N/H and Y1230C mutations or high copy number MET gene amplifications that emerge when resistance to savolitinib develops in patients with MET-amplified gastric cancer. CONCLUSION We demonstrated the utility of ctDNA in gastric cancer and confirmed this approach using baseline tumor tissue or rebiopsy.

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

Despite the fact that aortic valve replacement surgery is well developed and accompanied by high efficiency, it is still far from ideal and associated with a large number of complications. In this relation, the methods of surgical treatment based on valve-preserving philosophy surpass traditional prosthetics, since they may decrease risks at early and long-term periods after the operation, which is especially important in elderly and senile patients. The manuscript presents the results of a study on application of the ultrasonic decalcification in 70 patients with calcified aortic valve stenosis. After surgery, up to 13 months, a significant decrease in the systolic pressure gradient on the valve and an increase in the area of ​​the effective valve opening were observed. This shows the effectiveness of the ultrasonic decalcification in the elderly patient population.

Advanced non-small-cell lung cancer (NSCLC) patients with EGFR T790M-positive tumours benefit from osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). Here we show that the size of the EGFR T790M-positive clone impacts response to osimertinib. T790M subclonality, as assessed by a retrospective NGS analysis of 289 baseline plasma ctDNA samples from T790M‐positive advanced NSCLC patients from the AURA3 phase III trial, is associated with shorter progression-free survival (PFS), both in the osimertinib and the chemotherapy-treated patients. Both baseline and longitudinal ctDNA profiling indicate that the T790M subclonal tumours are enriched for PIK3CA alterations, which we demonstrate to confer resistance to osimertinib in vitro that can be partially reversed by PI3K pathway inhibitors. Overall, our results elucidate the impact of tumour heterogeneity on response to osimertinib in advanced stage NSCLC patients and could help define appropriate combination therapies in these patients., A recent clinical trial shows that a proportion of lung cancer patients carrying the EGFR T790M mutation develop resistance to osimertinib. In this study, the authors show that T790M subclonality is associated with worse clinical outcome likely through co-occurring PIK3CA alterations, which can be targeted by PI3K pathway inhibitors.

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

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes., BRAF or MEK1/2 inhibitors are cytostatic in melanoma and the surviving cells develop drug resistance. This study shows that the pro-survival pool is biased towards MCL1 in melanoma so that BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, improving tumour growth inhibition.

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

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.

Background/Purpose Following disease progression on osimertinib, a third generation (3G), irreversible, oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), ≤22% of patients (pts) with EGFR mutation positive (EGFRm) non-small cell lung cancer (NSCLC) develop MET-amplification as a resistance mechanism to treatment. In the Ph1b TATTON (NCT02143466) Part B expansion cohort, pts with MET-amplified EGFRm advanced NSCLC and progression on a prior EGFR-TKI received osimertinib + savolitinib, an oral, potent and highly selective MET TKI. Previous analyses have compared MET patient selection assays and found that different methods identify different but partially overlapping proportions of pts with MET amplification or overexpression. Here, we report the response rates observed in the subgroups defined by the different central MET assays in TATTON Part B. Methods MET testing (central, or local with central confirmation) was performed on tumor tissue collected after the most recent therapy. MET assays included FISH (MET gene copy number ≥5 or MET-CEP7 ratio ≥2) and IHC (MET +3 expression in ≥50% of tumor cells). TATTON Part B was split into 3 pt cohorts: pts previously treated with a 3G EGFR TKI and not previously treated with a 3G EGFR TKI (T790M positive/negative). This analysis uses the final study data (cutoff 4 March 2020) to compare the objective response rate (ORR) of subgroups identified by each assay. Results For pts who had received prior treatment with a 3G EGFR TKI, ORR by MET assay was as follows: FISH-positive overall (n=53) 30%; FISH polysomy (n=18) 28%; FISH amplification (n=35) 31%; IHC (n=13) 46%. For FISH-positive tumors, ORR trended higher for tumors with ≥10 MET gene copies (n=29, 35%) vs tumors with 5-9 copies (n=24, 25%). For pts with no prior treatment with a 3G EGFR TKI (T790M negative), ORR was as follows: FISH-positive overall (n=34) 65%; FISH polysomy (n=14) 57%; FISH amplification (n=20) 70%; IHC (n=4) 75%. For FISH-positive tumors, ORR trended higher for tumors with ≥10 MET gene copies (n=14, 79%) vs tumors with 5-9 copies (n=20, 55%). In tumors with both central FISH and IHC data available (n=34), IHC positivity (n=20) and FISH positivity (n=27) were correlated, specifically IHC and FISH amplification: 13/16 cases of FISH amplification were also IHC positive. In FISH-positive tumors IHC expression tended to increase with increasing gene copy number (≥10), but outcomes were largely comparable. Conclusions Overall, the MET assays were comparable with regards to ORR, thus supporting continued use of both FISH and IHC for detection of MET in pts following disease progression on any 3G EGFR TKI, though IHC data were limited. Further study is warranted in this area: the SAVANNAH (NCT03778229; FISH and IHC) and ORCHARD (NCT03944772; NGS) studies will provide opportunity for further analyses. Citation Format: Ryan Hartmaier, Ji-Youn Han, Byoung Chul Cho, Aleksandra Markovets, Nisha Kurian, Mireille Cantarini, Pasi A. Janne. Tumor response and MET-detection methods exploratory biomarker analysis of Part B of the Ph 1b TATTON study [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 CT127.

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.

Background In the phase III FLAURA study (NCT02296125), the 3rd-generation EGFR-TKI osimertinib showed superior efficacy to comparator EGFR-TKIs in previously untreated EGFR-mutated (EGFRm) advanced non-small cell lung cancer (NSCLC). Here we report results from an exploratory analysis of ctDNA for the early detection of disease progression (PD) in FLAURA. Methods Treatment-naive patients (pts) with EGFRm (ex19del/L858R) locally advanced/metastatic NSCLC (n = 556) were randomised 1:1 (osimertinib 80 mg qd: comparator [gefitinib 250 mg qd/erlotinib 150 mg qd]). Plasma samples were collected on Days 1, 8 and 15, then every 21 days for weeks (W) 3–18, then every 6W thereafter. In pts who had a plasma sample on PD and/or discontinuation, ctDNA droplet digital PCR (ddPCR; Biodesix) for EGFRm (ex19del/L858R/T790M) was performed at all available time points and C797S for post-W6 time points. C797S and T790M were the only resistance mutations assayed. ctDNA progression was defined with respect to the nadir ctDNA result and its proximity to the ddPCR detection and quantification limits. Results The ctDNA progression analysis included 122/556 (22%) pts with valid longitudinal monitoring ddPCR data and RECIST PD by DCO1 (12 June 2017). Across both arms, ctDNA progression preceded or co-occurred with PD in 80/122 (66%) pts with 2.7 months (mo) median lead time; 9.5 mo median progression-free survival (mPFS; n = 80). Acquired C797S or T790M was detected in 57/122 (47%) pts with ctDNA progression (osimertinib 4/50 [8%] C797S, comparator 53/72 [74%] T790M); median time to detection was 16.7 and 8.4 mo for the osimertinib and comparator arms, respectively, mirroring overall mPFS. In pts with ctDNA progression and PD (n = 106), acquired T790M and C797S were detected either at the same time as, or earlier than PD in 41/106 (38%) pts (osimertinib 2/39 [5%], comparator 39/67 [58%]); median lead time was 1.4 mo. Conclusions ctDNA monitoring may allow for earlier identification of pts who progress on first-line EGFR-TKI therapy and the detection of EGFR-mediated resistance mechanisms in advance of PD in EGFRm NSCLC. Future work aims to explore early detection of non-EGFR-mediated resistance. Clinical trial identification NCT02296125. Editorial acknowledgement Donna Tillotson, PhD, of iMed Comms, Macclesfield, UK, an Ashfield Company, part of UDG Healthcare plc, funded by AstraZeneca in accordance with Good Publications Practice (GPP3) guidelines. Legal entity responsible for the study AstraZeneca. Funding AstraZeneca. Disclosure J.E. Gray: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Array; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Genentech; Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Advisory / Consultancy: Celgene; Honoraria (self), Advisory / Consultancy: Takeda. A. Markovets: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. N. Nogami: Honoraria (self): Pfizer Inc., Chugai Pharmaceutical Co. Ltd, Eli Lilly, Taiho Pharmaceutical Co. Ltd., AstraZeneca, Kyowa Hakko Kirin, Ono Pharmaceutical Co. Ltd., Bristol-Myers Squibb, MSD. B.C. Cho: Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Novartis; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Bayer, AstraZeneca, MOGAM Institute, Dong-A ST, Janssen, Yuhan, Ono Pharmaceutical, Dizal Pharma, MSD; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution), Licensing / Royalties: Champions Oncology; Honoraria (institution), Advisory / Consultancy: Boehringer Ingelheim, Roche, Bristol-Myers Squibb, Pfizer, Eli Lilly, Takeda; Shareholder / Stockholder / Stock options: TheraCanVac Inc. B. Chewaskulyong: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): AstraZeneca. M. Majem: Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony, Research grant / Funding (self), Travel / Accommodation / Expenses: Bristol-Myers Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy: Tesaro; Speaker Bureau / Expert testimony: Hellsin; Travel / Accommodation / Expenses: Lilly; Advisory / Consultancy: Takeda; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre; Speaker Bureau / Expert testimony: Amgen. N. Peled: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, MSD, Novartis, Pfizer, Roche, Takeda; Advisory / Consultancy: Eli Lilly; Honoraria (self): Foundation Medicine; Shareholder / Stockholder / Stock options: Novellus Dx; Honoraria (self): Guardant360. K. Vishwanathan: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. A. Todd: Full / Part-time employment: AstraZeneca. Y. Rukazenkov: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. M. Johnson: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. C. Barrett: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. J. Chmielecki: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. R. Hartmaier: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca; Shareholder / Stockholder / Stock options, Licensing / Royalties, Nfe2l2 exon 2 ano/or exon 3 loss from work conducted at Foundation Medicine; provisional patent filed: Foundation Medicine. S.S. Ramalingam: Advisory / Consultancy, Research grant / Funding (self): Amgen, AstraZeneca, Bristol-Myers Squibb, Merck; Advisory / Consultancy: AbbVie, Celgene, Genentech, Lilly, Loxo, Takeda; Research grant / Funding (self): Tesaro.

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 (

Osimertinib is current recommended treatment for EGFR T790M-positive NSCLC following progression on prior EGFR tyrosine kinase inhibitor therapy. However, acquired resistance to osimertinib therapy inevitably developed after a period of effective treatment. We had a patient of EGFR L858R/T790M-positive NSCLC who initially responded to osimertinib therapy but eventually developed resistance. Plasma cell-free DNA analysis disclosed exon-16-skipping HER2 (HER2Δ16) in one osimertinib treated patient. HER2Δ16 was regarded as an oncogenic driver in breast cancer and has never been reported in lung cancer before. Therefore, it’s intriguing to investigate the role of HER2Δ16 in osimertinib resistance in NSCLC. The purpose of this study is to explore the mechanism of HER2Δ16-mediated resistance to osimertinib and to explore strategies to overcome the resistance. We constructed and established H1975 cells stably expressing either vehicle, WT-HER2 or HER2Δ16. We observed that without osimertinib treatment, there was no difference in cell growth and the response to EGF-stimulation. However, when treated with osimertinib, H1975-HER2Δ16 cells were more resistant under normal and EGF-stimulating conditions compared to vehicle or WT-HER2 expressing cells. Moreover, osimertinib treatment in H1975-HER2Δ16 cells had less inhibitory effect on phosphor-ERK and phosphor-AKT compared to vehicle or H1975-WT-HER2 cells. Interestingly, co-treatment of osimertinib with Src-kinase inhibitor, dasatinib, failed to reverse the resistance, indicating that HER2Δ16-driven osimertinib-resistance was independent of Src-kinase. Finally, we revealed that combination of osimertinib with the HER2 small molecular inhibitor, afatinib, can suppress cell growth in H1975-WT-HER2 and H1975-HER2Δ16 cells. In summary, we have shown that HER2Δ16, may be a resistance mechanism to osimertinib in EGFR mutated NSCLC and that HER2Δ16-driven resistance was Src-kinase independent. Moreover, we found the HER2Δ16-driven resistance could be rescued by combination of osimertinib with afatinib. Citation Format: Chia-Chi Hsu, Bin-Chi Liao, Wei-Yu Liao, Aleksandra Markovets, Daniel Stetson, Kenneth Thress, Chih-Hsin Yang. Exon-16-skipping HER2 contributes to osimertinib-resistance through Src-independent manner in EGFRL858R/T790M-positive non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4752.

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.

There is no approved therapy specifically for the treatment of papillary renal cell carcinoma (PRCC). Advances in molecular profiling of PRCC have identified a segment of PRCC with 10% MET mutation rates (Linehan et al 2016). Chromosome 7 gain is a hallmark of PRCC and thought to occur at 50% frequency in PRCC (Jiang et al 1998). We undertook a retrospective analysis of archival tumors to evaluate MET pathway aberrations for correlation with efficacy in a phase II study of savolitinib (volitinib, AZD6094, HMPL-504) in patients with PRCC (NCT02127710). Archival diagnostic tumor samples were mandated for central confirmation of PRCC diagnosis, histological subtyping, and for exploratory biomarker analysis. Eighty-four archival tumors were obtained and profiled using four methods: H&E stain for PRCC histological subtype, immunohistochemistry (IHC) for c-Met protein expression (Ventana, CONFIRM), fluorescent in situ hybridization (FISH) for MET gene amplification (Abbott, VYSIS), and Next Generation Sequencing (NGS) as an orthogonal method for confirmation of MET amplifications, detection of HGF gene amplifications, MET mutations, chromosome 7 ploidy, and other exploratory genomic biomarkers (Foundation Medicine Inc., T7 panel). Eight patients with a confirmed partial response (PR) to savolitinib were used to define MET-driven PRCC. Retrospective analysis demonstrated that only NGS, not IHC, FISH or histological subtype, identified all PRs. NGS detected any one of the following biomarkers alone or in combination in PRs: chromosome 7 copy number gain, MET or HGF gene focal amplification, MET kinase domain mutations. The frequency of these biomarkers in the phase II population were 31.5%, 10%, 1%, and 7.5%, respectively and captured all responders (18% ORR); however, 42% of non-responders (PD and SD) were also classified as MET-driven by NGS. Focal MET amplifications were confirmed with MET FISH testing, but FISH was unable to identify gains in chromosome 7. IHC was not as sensitive as NGS (57% vs 100%) but had a higher specificity. Molecular characterization of MET status was more predictive of response to savolitinib than a classification based on pathology. Citation Format: Melanie M. Frigault, Sabina Signoretti, Aleksandra Markovets, Daniel Stetson, William Howat, Martine Roudier, Vincent Haddad, Brian Dougherty, Toni K. Choueiri, J. Carl Barrett. MET gene copy number gains evaluated by NGS is more predictive than other methods to enrich for papillary RCC patients sensitive to savolitinib, a selective MET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4541.

9027Background: In the Phase III AURA3 trial (NCT02151981), osimertinib, a third-generation EGFR-TKI, had significantly greater efficacy than platinum-pemetrexed in patients (pts) with advanced NSC...

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.

For the last decade, owing to methods of computerized neuroradiology, it has been established that the process of the human organism natural aging is accompanied by a gradual atrophic reduction of the brain tissue volume and a decrease of the cerebral blood flow level, while the intracranial cerebrospinal fluid volume increases. The goal of the present study was to elucidate functional significance of the above changes in terms of interaction of intracranial hemo- and liquorocirculation systems at various stages of ontogenesis of practically healthy people. The total of 122 people aged from 6 to 100 years were examined at rest and during performance of goal-oriented functional physiological tests with simultaneous continuous recording of parameters of cerebral blood flow and liquorodynamics by methods of transcranial dopplerography and rheoencephalography. The obtained data are processed by means of the pattern and phasic two-dimensional analysis with use of special computer programs. In the same age groups, the brain neurophysiological activity was evaluated by applying special psychological tests. It has been shown that with age, on the background of a decrease of hemodynamic parameters of cerebral blood supply, there is observed an enhancement of the liquorodynamic factor of cerebral blood circulation due to an increase of the liquor volume and facilitation of its translocation in the single craniospinal cavity. The enhancement of the liquor-dependent mechanism of compensation of intracranial pulse oscillations of the blood volume is particularly expressed both in children and in elderly people due to a relatively high liquor volume. Owing to the improvement of intracranial liquorodynamic processes, the change of the cerebral blood circulation is compensated, which is confirmed by results of performed psychophysiological studies.

The increased usage of circulating tumor DNA (ctDNA) sequencing for oncology clinical research demonstrates a critical need for sensitive and specific testing. While we have observed a high degree of concordance between single tumor mutations in tumor and plasma, several recent studies have highlighted a lack of concordance between plasma and tumor panel NGS gene panel testing due to biological and technical factors. To explore further these factors and benchmark ctDNA NGS testing services, a set of matched plasma, tumor, and normal samples from 24 subjects were acquired from three biobanking companies. Replicate 2 ml-plasma samples were tested by four ctDNA sequencing companies, and matching tumor/normal samples were tested by two tumor sequencing companies. Concordance was measured by comparing plasma mutations to tumor mutations as well as comparing mutations among the same plasma tested by the ctDNA companies. While our experience with NGS of matched samples from clinical trials typically identifies ~30% of patients with no detectable mutation and therefore likely not shedding tumor DNA, with the retrospectively collected commercial samples ~60% lacked detectable high confidence mutations, likely due to quality control issues with sample collection. We also found variation in the concordance of ctDNA mutation detection rates among the four vendors, due to significant differences in DNA yield and assay sensitivity. While factors such as tumor heterogeneity and timing of plasma-tumor collection can lower concordance rates, the majority of discordance in our study was due to technical rather than biological variation. Assay analytical variance and the impact of reporting false positive variants are key factors that need to be addressed as plasma-based NGS testing is more widely incorporated into translational and clinical research. Examples illustrating the complexity of the analyses and giving support for confidence in ctDNA testing results will be given. Citation Format: Daniel Stetson, Brian Dougherty, Ambar Ahmed, Tristan Lubinski, Aleksandra Markovets, Kenneth Thress, Robert McEwen, Gaia Schiavon, David Whitston, Barrett Nuttall, J. Carl Barrett. Examination of analytical factors impacting concordance of plasma-tumor testing by next-generation sequencing (NGS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-249. doi:10.1158/1538-7445.AM2017-LB-249

9018 Background: Osimertinib, an EGFR T790M-selective tyrosine kinase inhibitor (TKI), provides high and durable response rates in patients (pts) with T790M positive advanced NSCLC whose disease has progressed following EGFR-TKI therapy. We investigated whether changes in the levels of plasma EGFRmutations post-osimertinib treatment are associated with clinical outcome. Methods: We studied pts from the AURA Phase I study (NCT01802632) with acquired resistance to EGFR-TKIs, T790M positive baseline genotyping (in tumor or plasma), and plasma samples collected at baseline and 6 weeks (wks) post- osimertinib (20–240 mg daily) treatment. Plasma samples were analyzed for the presence of detectable EGFR mutations (Ex19del, L858R and T790M) using BEAMing digital PCR (Sysmex/Inostics). In pts with detectable plasma EGFR mutations at baseline, clinical outcomes (by investigator, per RECIST 1.1) were compared for pts with or without detectable plasma EGFRmutations at 6 wks. Results: Evaluable baseline and 6 wk plasma samples were collected from 160 pts with T790M positive genotyping, of whom 143 had detectable EGFR mutations at baseline (median allelic fraction for Ex19del: 7.09%; L858R: 3.81%; T790M: 2.12%). In this cohort, the overall median progression-free survival (mPFS) was 9.3 months (m; 95% CI 8.2, 9.7). Clearance of plasma EGFRmutations at 6 wks was seen in 92/143 (64%) pts. mPFS was longer in pts with plasma clearance (10.9 m; 95% CI 9.5, 15.2) compared with pts without (5.5 m; 95% CI 3.9, 6.7), as was objective response rate (ORR; 70%; 95% CI 59%, 79% vs. 35%; 95% CI 22%, 50%). Conclusions: Clearance of plasma EGFR mutations after 6 wks of osimertinib therapy appears to be associated with improved ORR and mPFS in pts with T790M positive NSCLC. Evidence or lack of such a “plasma response” measured at 6 wks could, potentially, be used to predict subsequent outcomes on therapy. Further research is needed to better understand whether continued detection of plasma EGFR mutations at 6 wks may indicate the presence of heterogeneous resistance mechanisms, which could, potentially, be targeted by combination therapies. Validation of these results in an independent cohort of pts is ongoing. Clinical trial information: NCT02228369.

Slow rhythmic oscillations in the human cranial cavity were studied using two noninvasive methods: the bioimpedance method (volume ratios between liquid media in the cranial cavity) and transcranial ultrasound Doppler echography (variation in the blood flow in the middle cerebral artery). The combination of these methods made it possible to estimate the intracranial hemodynamics. Simultaneous recording of these parameters and their spectral analysis were carried out in healthy subjects and patients with intracranial hypertension syndrome and disturbed cerebrospinal fluid (CSF) flow. The parameters were recorded at rest and immediately after manual (osteopathic) correction. The recording and analysis were performed using a Macintosh-IIsi PC and the Chart-3.52, Cricket Graph-3.32, and Canvas-3.5 software. It was found that slow oscillations of the bioimpedance (BIM) in the frequency range 0.08–0.2 Hz were of intracranial origin and were related to the mechanisms of regulation of the blood supply to and oxygen consumption by cerebral tissue, as well as with the dynamics of the CSF circulation.

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.

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.