Research Grants Council Research Articles

Long-Run Movement and Predictability of Bond Spread for BRICS and PIIGS: The Role of Economic, Financial and Political Risks

Asia University, China Medical University Hospital, Hang Seng University of Hong Kong, Research Grants Council (RGC) of Hong Kong and Ministry of Science and Technology, Taiwan.

Randomized Trial of Silver Nitrate with Sodium Fluoride for Caries Arrest.

The aim of this noninferiority double-blind randomized clinical trial was to compare the effectiveness of the topical semiannual application of a 25% silver nitrate (AgNO3) solution followed by a 5% sodium fluoride (NaF) varnish with that of a 38% silver diamine fluoride (SDF) solution in arresting caries among preschool children. Healthy 3-y-old children with active dentine carious lesions were randomly allocated to 2 groups via computer-generated random numbers. Lesions in group A received applications of a 25% AgNO3 solution followed by a 5% NaF varnish semiannually (every 6 mo). Lesions in group B received semiannual applications of a 38% SDF solution followed by a placebo varnish. A trained examiner recorded the status of caries and oral hygiene at baseline and during follow-up examinations. The examiner, children, and their caretakers were blinded to the intervention allocation. This study adopted an intention-to-treat analysis. A noninferiority test was conducted for the data analysis. Group A's noninferiority was accepted if the lower limit of the 95% CI for the difference in the mean number of arrested surfaces was >-0.5. A total of 1,070 children were recruited at baseline, with 535 children in each group. After 18 mo, the mean ± SD number of arrested surfaces was 3.3 ± 3.4 in group A (n = 484) and 3.2 ± 3.5 in group B (n = 476; P = 0.664). The difference in the mean number of arrested surfaces between the groups was 0.092 (95% CI, -0.322 to 0.505). Apart from black staining on the arrested lesions, no other significant side effect was observed. A semiannual application of 25% AgNO3 followed by 5% NaF is no worse than a 38% SDF in arresting dentine caries among preschool children over 18 mo. The Hong Kong Research Grants Council (GRF 17107315) funded this trial, which was registered at ClinicalTrials.gov (NCT02019160). This randomized clinical trial found that silver nitrate solution followed by sodium fluoride varnish is effective in arresting dentine caries among preschool children. As silver nitrate followed by sodium fluoride is a noninvasive and simple protocol, it can be an alternative strategy to manage dental caries among young children, especially in countries where silver diamine fluoride is not available.

Topographic Modulation of Acetylcholine Receptors Diffusion Dynamics on Live Cell Membrane

In a recent single-molecule tracking experiment [1], the acetylcholine receptors (AChRs) on muscle cell membranes were found to display non-Gaussian statistics due to dynamic heterogeneity resulting from the complex cellular environment of the AChRs. In this study, we vary the cellular environment of AChRs by using the microgroove patterning techniques to control the spatial distribution of adhesion complexes on the basal membrane of live cells and study its effect on the diffusion dynamics of AChRs. Cultured Xenopus muscle cells are found to align along the microgroove patterns with their adhesion complexes, such as β1-integrins, predominantly anchored on the ridge region of the microgrooves, forming a stripe-like pattern. Interestingly, we find the movement of AChRs is largely restricted within the microgroove-aligned integrin stripes. Detailed statistical analysis of the AChR trajectories reveals that the AChRs are slowed down by the patterned integrins, and immobilization and clustering of the AChRs are correlated with the spatial distribution of integrins. The experiment demonstrates that the spatial distribution of integrins plays a crucial role in modulating the diffusion dynamics of AChRs. ∗The work was supported by the Research Grants Council of Hong Kong SAR. [1] W. He, H. Song, Y. Su, L. Geng, B. J. Ackerson, H. B. Peng, and P. Tong, Nature Communications, 7:11701 (2016).

Residential greenness and mortality in oldest-old women and men in China: a longitudinal cohort study

SummaryBackgroundExposure to natural vegetation, or greenness, might affect health through several pathways, including increased physical activity and social engagement, improved mental health, and reductions in exposure to air pollution, extreme temperatures, and noise. Few studies of the effects of greenness have focused on Asia, and, to the best of our knowledge, no study has assessed the effect on vulnerable oldest-old populations. We assessed the association between residential greenness and mortality in an older cohort in China.MethodsWe used five waves (February, 2000–October, 2014) of the China Longitudinal Healthy Longevity Survey (CLHLS), a prospective cohort representative of the general older population in China. We assessed exposure to greenness through satellite-derived Normalised Difference Vegetation Index (NDVI) values in the 250 m and 1250 m radius around the residential address for each individual included in the study. We calculated contemporaneous NDVI values, cumulative NDVI values, and changes in NDVI from the start of the study over time. The health outcome of the study was all-cause mortality, excluding accidental deaths. Mortality rate ratios were estimated with Cox proportional hazards models, adjusted for age, sex, ethnicity, marital status, geographical region, childhood and adult socioeconomic status, social and leisure activity, smoking status, alcohol consumption, and physical activity.FindingsAmong 23 754 individuals (mean age at baseline 93 years [SD 7·5]) totaling 80 001 person-years, we observed 18 948 deaths during 14 years of follow-up, between June, 2000, and December, 2014. Individuals in the highest quartile of contemporaneous NDVI values had 27% lower mortality than those in the lowest quartile for the 250 m radius (hazard ratio [HR] 0·73, 95% CI 0·70–0·76), and 30% lower mortality for the 1250 m radius (0·70, 0·67–0·74). No clear association was observed for cumulative NDVI measurements and mortality. We did not detect an association between area-level changes in NDVI and mortality.InterpretationOur research suggests that proximity to more green space is associated with increased longevity, which has policy implications for the national blueprint of ecological civilisation and preparation for an ageing society in China.FundingBill & Melinda Gates Foundation, US National Institute on Aging, US National Institute of Health, Natural Science Foundation of China, UN Population Fund, China Social Sciences Foundation, and Hong Kong Research Grants Council.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 μg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. N/A. Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

Effectiveness of influenza vaccination on influenza-associated hospitalisations over time among children in Hong Kong: a test-negative case-control study

The protection conferred by influenza vaccination is generally thought to last less than a year, necessitating annual revaccination. However, the speed with which influenza vaccine effectiveness might decline during a year is unknown, which is of particular importance for locations with year-round influenza activity. We aimed to assess how influenza vaccine effectiveness changes by time intervals between vaccination and admission to hospital, taking advantage of almost year-round circulation of influenza in Hong Kong. In this test-negative case-control study, we analysed vaccine effectiveness in children (aged 6 months to 17 years) who were admitted to hospital in Hong Kong over 5 consecutive years (2012-17). We included those who were admitted to general wards in four public hospitals in Hong Kong with a fever (≥38°C) and any respiratory symptom, such as runny nose, cough, or sore throat. We used direct immunofluorescence assay and reverse transcription PCR to detect influenza virus infection, and recorded children's influenza immunisation history. We compared characteristics of positive cases and negative controls and examined how vaccine effectiveness changed by time between vaccination and admission to hospital with regression analyses. Between Sept 1, 2012, and Aug 31, 2017, we enrolled 15 695 children hospitalised for respiratory infections, including 2500 (15·9%) who tested positive for influenza A or B and 13 195 (84·1%) who tested negative. 159 (6·4%) influenza-positive cases and 1445 (11·0%) influenza-negative cases had been vaccinated. Most vaccinations were done by December of each influenza vaccination season. Influenza-related admissions to hospital occurred year-round, with peaks in January through March in most years and a large summer peak in 2016; pooled vaccine effectiveness for children of all ages was 79% (95% CI 42-92) for September to December, 67% (57-74) for January to April, and 43% (25-57) for May to August. Vaccine effectiveness against influenza A or B was estimated as 79% (95% CI 64-88) within 0·5-2 months of vaccination, 60% (46-71) within >2-4 months, 57% (39-70) within >4-6 months, and 45% (22-61) within >6-9 months. In separate analyses by type and subtype, we estimated that vaccine effectiveness declined by 2-5 percentage points per month. Influenza vaccine effectiveness decreased during the 9 months after vaccination in children in Hong Kong. Our findings confirm the importance of annual vaccination in children. Influenza vaccines that provide broader and longer-lasting protection are needed to provide year-round protection in regions with irregular influenza seasonality or lengthy periods of influenza activity. Health and Medical Research Fund, Hong Kong and the Research Grants Council, Hong Kong.

Cellular automaton modeling of pedestrian movement behavior on an escalator**Project supported by the National Natural Science Foundation of China (Grant Nos. 71473207 and 71871189), the Research Grant Council of the Hong Kong Administrative Region, China (Grant No. CityU118909), and the Open Fund of Beijing Engineering and Technology Research Center of Rail Transit Line Safety and Disaster Prevention (Grant No. RRC201701).

As a convenient passenger transit facility between floors with different heights, escalators have been extensively used in shopping malls, metro stations, airport terminals, etc. Compared with other vertical transit facilities including stairs and elevators, escalators usually have large transit capacity. It is expected to reduce pedestrian traveling time and thus improve the quality of pedestrian’s experiences especially in jamming conditions. However, it is noticed that pedestrians may present different movement patterns, e.g., queuing on each step of the escalator, walking on the left-side and meanwhile standing on the right-side of the escalator. These different patterns affect the actual escalator traffic volume and finally the passenger spatiotemporal distribution in different built environments. Thus, in the present study, a microscopic cellular automaton (CA) simulation model considering pedestrian movement behavior on escalators is built. Simulations are performed considering different pedestrian movement speeds, queuing modes, and segregation on escalators with different escalator speeds. The actual escalator capacities under different pedestrian movement patterns are investigated. It is found that walking on escalators will not always benefit escalator transit volume improvement, especially in jamming conditions.

Experimental study on the regulation function of slit dam against debris flows

The authors received financial support from the National Natural Science Foundation of China (grant no. 11672318), the Youth Innovation Promotion Association, CAS, and the Chinese Academy of Sciences (CAS) Pioneer Hundred Talents Program, and from the research grant T22-603/15-N provided by the Research Grants Council of the Government of Hong Kong SAR, China.

Targeted Release of Endogenous Cations from Xenograft Bone Driven by Fluoride Incorporation Contributes to Enhanced Bone Regeneration

Xenograft, namely bone-devired biological apatite (BAp), is widely recognized as a favorable biomaterial in bone tissue engineering owing to biodegradability, biocompatibility, and osteoconductive properties. Substitutions of endogenous trace ions are thought to improve osteogenic capacity of xenograft when compared with synthetic hydroxyapatite (HAp). In order to modify the physicochemical and biological properties of apatite, different approaches to induce trace ion incorporation have been widely considered. In this study, we demonstrate that the incorporation of fluoride ion into porcine bone-derived biological apatite (pBAp) contributes to altered crystal morphology of the apatite, sustained release of fluoride, and targeted release of endogenous trace ions (e.g., magnesium and calcium) into the peripheral tissue microenvironment. This ionic balanced perimaterial microenvironment not only leads to superior proliferation and osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs), but also accelerates new bone formation of the calvarial defect on a rat model via the activation of Wnt/β-catenin signaling. These promising observations may attribute to the controlled release of endogenous trace ions from xenograft to the peripheral tissue microenvironment driven by fluoride ion incorporation. Lastly, this study may provide a new insight to strengthen the osteogenicity of xenografts for clinical applications in the future. Funding Statement: This work was supported by National Natural Science Foundation of China (81470783, 81400550, 31370957), the Natural Science Foundation of Guangdong Province (2015A030311051, 2016A030310173), Shenzhen Innovative Technology Commission Grant (Nos. JCYJ20140414090541811 and JCYJ20160429185449249), Hong Kong Research Grant Council General Research Funds (RGC GRF) (Nos. 718913E, 17214516 and 772113M) and Hong Kong Health and Medical Research Fund (No. 03142446). Declaration of Interests: The authors declare no conflict of interest. Ethical Approval Statement: All procedures involving laboratory animal usage were in accordance with the institutional guidelines and were approved by the Animal Care and Use Committee of the Sun Yat-sen University (IACUC-DB-16-0103).

Hepcidin Attenuates the Secondary Neuronal Injury after Intracerebral Hemorrhage in Rats

Iron plays a key role in secondary neuronal injury after intracerebral hemorrhage (ICH), and hepcidin is able to reduce brain iron in iron-overloaded rats by down-regulating iron transport proteins. These led us to hypothesize that hepcidin might reduce iron-mediated neurotoxicity by inhibiting iron accumulation in ICH brain. Here, we tested our hypothesis in 510 rats and demonstrated for the first time that hepcidin could significantly suppress the ICH-induced increase in iron and ferritin in brain tissues and cerebrospinal fluid by inhibiting expression of iron transport proteins, increase neuronal survival by attenuating ICH-induced apoptosis, neurodegeneration and brain edema, as well as effectively improve ICH-induced behavioral and cognitive deficit in rats. We concluded that hepcidin could effectively attenuate iron-mediated secondary neuronal injury after ICH in rats. This naturally existing protein which is biodegradable, nontoxic, and non-immunogenic can potentially be developed into a therapeutic drug for the treatment of ICH patients. Funding: The studies were supported by the National Natural Science Foundation of China (NSFC31330035-ZMQ, NSFC31571195-ZMQ), the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF14111815-KY, GRF 14167817-KY, GRF14107616-KY), Hong Kong Health & Medical Research Fund (HMRF: 03141436-KY), Hong Kong Innovation and Technology Fund (ITS/163/16-KY), and the National 973 Programs (2014CB541604-ZMQ). Conflict of interest: The authors declare that they have no conflict of interest. Ethical Approval Statement: The Health Department of Hong Kong and Shanghai Government and the Animal Research Ethics Committee of The Chinese University of Hong Kong and Fudan University approved the experimental procedures of this study and the care and handling of the animals were in accord with National Institutes of Health guidelines.

Selective and Suppressive Effects of Antibiotics on Donor and Recipient Strains in Gut Microflora Determine Transmission Efficiency of blaNDM-1-Bearing Plasmids

Animal and human gastrointestinal tract is the key site from which resistant bacterial pathogens emerge. In this work, we tested whether antibiotics of different functional categories exhibited differential potential in promoting transmission of multidrug resistance-encoding plasmids among the gut microflora by measuring the population size of both donor and potential recipient strains before, during and after the treatment process. In this study, we showed that drugs that are commonly used to treat Gram negative bacterial infections such as ampicillin and amoxicillin could enrich both carbapenem resistant enterobacteriaceae (CRE) and antibiotic-susceptible E. coli in the gastrointestinal tract, thereby promoting transmission of the blaNDM-1-bearing plasmid in the gut microbiome. In contrast, meropenem was found to minimize the population of CRE in the gut microbiome, hence treatment with this drug exhibited drastically lower potential to promote transmission of the blaNDM-1-bearing plasmid to the recipient strains. We further showed that whether an antibiotic enriches or suppresses the population size of donor and recipient strains in the gastrointestinal tract is actually dependent on the relative inhibitory effects of the drug on the firmicutes or proteobacteria, and that an increased population size of proteobacteria due to a higher suppressive effect on firmicutes is a key factor in enhancing the efficiency of transmission of the blaNDM-1-bearing plasmid and hence dissemination of carbapenem resistant strains. In conclusion, this study identified and characterized the factors that affected the transmission of MDR plasmid in gut microbiome and its underlying mechanisms. Such findings can help establish new guidelines for prudent antibiotic usage to minimize the chance of dissemination of resistance mobile elements among the GI tract microflora. Funding Statement: The research was supported by the Collaborative Research Fund (C5026-16G) of the Hong Kong Research Grant Council and the Health and Medical Research Fund (HMRF) of the Food and Health Bureau, the Government of the Hong Kong SAR (14130402 to SC). Declaration of Interests: All authors declared no conflict of interests. Ethics Approval Statement: The experimental protocol was approved by the Research Animal Care and Use Committee of the Hong Kong Polytechnic University.

Tropism, replication competence, and innate immune responses of influenza virus: an analysis of human airway organoids and ex-vivo bronchus cultures

Human airway organoids are three-dimensional cultures derived from stem cells, which self-organise in ex-vivo conditions to form so-called mini-airways. The cellular morphology of these cultures is physiologically similar to the human airway, with cilia, goblet cells, and club cells facing the inner lumen and basal cells situated at the outer layer. The aim of this study was to compare replication competence, tissue tropism, and host responses elicited by human and avian strains of influenza A virus in ex-vivo human bronchus and human airway organoids. Between Sept 29, 2016, and Jan 4, 2017, we obtained ex-vivo cultures of the human bronchus and cultured human airway organoids from lung stem cells obtained from human lung tissues removed as part of the routine clinical care of patients undergoing surgical resection at the Department of Cardiothoracic Surgery, University of Hong Kong, Queen Mary Hospital, Hong Kong. We compared viral replication competence, tissue tropism, and cytokine and chemokine induction of avian influenza A viruses isolated from humans (Sh2/H7N9, H5N1/483, H5N6/39715), and human H1N1pdm/415742 in airway organoids and ex-vivo bronchus explant cultures. Virus tropism and replication kinetics of human and avian influenza A viruses in human airway organoids mimicked those found in ex-vivo cultures of human bronchus explants. In both airway organoids and bronchus explants, influenza A H1N1 subtype (H1N1) and avian influenza A H7N9 viruses replicated to significantly higher titres than did the highly pathogenic avian influenza (HPAI) H5N1, whereas HPAI H5N6 replication was moderate. H1N1, H7N9, and H5N6 viruses infected ciliated cells and goblet cells, but not basal cells in both airway organoids and bronchus explants. The expression of cytokines, interleukin 6, and interferon β, and the chemokine regulated-on-activation, normal T-cell expressed and secreted, was significantly higher in human airway organoids infected with HPAI H5N1 virus than H1N1pdm/415742, Sh2/H7N9, and H5N6/39715 viruses, and the expression of monocyte chemoattractant protein-1 was significantly higher in human organoids infected with HPAI H5N1 virus than H1N1pdm/415742 and Sh2/H7N9 viruses. Human airway organoid cultures provided results that were comparable to those observed in human ex-vivo bronchus cultures, and thus provide an alternative physiologically relevant experimental model for investigating virus tropism and replication competence that could be used to assess the pandemic threat of animal influenza viruses. US National Institute of Allergy and Infectious Diseases, Research Grants Council of the Hong Kong Special Administrative Region.

Abstract 5347: Somatic mutation and overexpression of anaplastic lymphoma kinase (ALK) are uncommon events in Asian head and neck cancers

Abstract Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is frequently translocated in anaplastic large cell lymphoma (ALCL) and non-small cell lung cancer (NSCLC), etc. Gain-of-function mutations, largely mutations associated with resistance to ALK inhibitors, have been identified in neuroblastoma, colon adenocarcinoma and prostate adenocarcinoma, etc. Pan-cancer analysis of whole-exome sequencing data of >25 cancer types showed a mutational rate ranging from 0-9.4% across pan-cancers (www.cbioportal.org). In cancers of the head and neck, the potential role of ALK on precision treatment has been demonstrated in an Asian head and neck sarcomatoid carcinoma patient bearing an ALK aberration (Kim et al., 2015). In this study, we sought to examine the mutational rates of ALK in the US-TCGA HNSCC Provisional cohort, in a small Asian HNSCC cohort, as well as in Nasopharyngeal carcinoma (NPC, an Asian-prevalent HNC). Analysis of the whole-exome sequencing (WES) data of the US-TCGA HNSCC Provisional cohort (N=527; largely Caucasian ethnicity, 85.6%; www.cbioportal.org) revealed a somatic mutation rate of 4.0% (21 out of 527 patients, with 18 non-synonymous and 3 synonymous mutations) and a gene amplification rate of 0.4% (2 out of 522 tumors with copy number change data available). In contrast, the Asian prevalent Epstein-Barr Virus-associated NPC (N=102 micro-dissected tumors with paired blood samples analyzed by WES), no somatic ALK mutation was identified. Using a small Asian HNSCC cohort (N=16), next-generation sequencing (NGS) analysis with an ALK exon coverage of ~90% revealed no somatic ALK mutation, but the presence of Asian-predilected SNPs with unknown clinical or biological significance. Lastly, ALK protein expression, analyzed by Western blotting, was found to be low or largely undetectable in 25 Asian HNSCC tumors analyzed thus far. Yet, we cannot exclude the possibility of focal ALK expression in HNSCC tumors, which can be largely obscured by Western blotting. In conclusion, somatic mutation of ALK appears to be rare events in Asian HNSCC and EBV-associated Nasopharyngeal carcinoma. Acknowledgements: VWYL receives funding supports from the Research Grant Council, Hong Kong (General Research Fund: #1711484, #17121616; Theme-based Research: T12-401/13-R) and the Hong Kong Cancer Fund, Hong Kong. KWL receives funding from the Research Grant Council (Theme-based Research: T12-401/13-R), Hong Kong. WP is supported by Faculty Postdoctoral Fellowship Scheme from the Faculty of Medicine, Chinese University of Hong Kong. JYKC receives funding support by the Dr Stanley Ho Medical Foundation. Reference Kim, S. M., Kim, M. J., Jung, H. A., Sun, J. M., Choi, Y. L., Ko, Y. H., . . . Ahn, M. J. (2015) Presence of anaplastic lymphoma kinase translocation in sarcomatoid carcinoma of head and neck and treatment effect of crizotinib: A case series. Head Neck, 37(5), E66-69 Citation Format: Wenying Piao, Peony Hiu Yan Poon, Lan Wang, Chin Wang Lau, Jason Ying Kuen Chan, Yuxiong Su, Amy Bik Wan Chan, Hoi Lam Ngan, Kwok Wai Lo, Vivian Wai Yan Lui. Somatic mutation and overexpression of anaplastic lymphoma kinase (ALK) are uncommon events in Asian head and neck cancers [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 5347.

Abstract 3079: Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell

Abstract How oncogenic virus rewires glucose flux to regulate cell motility is not well-understood. Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis. Here we demonstrate that EBV-encoded LMP1 enhances glycolysis thereby accelerating cell mobility of nasopharyngeal epithelial (NPE) cells. Activation of IGF1/mTORC2/AKT/PDHE1α signaling which mediates Histone 3 acetylation is involved in cell motility in EBV-infected NPE cells. Blocking the glucose uptake by pharmacological inhibitors effectively suppress glycolysis as well as the cell motility, linking the glucose metabolism to cell movement. We further show that mTORC2/AKT mediated phosphorylation of pyruvate decarboxylase kinase 1 (PDHK1) and its substrate, pyruvate decarboxylase E1α (PDHE1α) component plays a significant role in cell motility. PDHE1α knockdown effectively blocks LMP1-induced cell motility while overexpression of its phosphomimetic mutant S293A but not S293D inhibits LMP1-induced cell motility, supporting the involvement of PDHE1α in enhanced cell motility is depended on Ser293 phosphorylation. Furthermore, PDHE1α translocation into nucleus enhances Histone 3 acetylation and its binding to the promoter region of snail to enhance cell motility. Finally, we find that LMP1 increases the mRNA expression and the secretion of the ligand IGF1, which promotes phosphorylation of IGF1R as the key regulator for LMP1-induced mTORC2/AKT activation. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells to promote cell motility involving activation of mTORC2/AKT/PDHE1α. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong. Citation Format: Jun Zhang, Weitao Lin, Chi Man Tsang, Teng Fei Liu, Wing Chong Tang, Yim Ling Yip, Wen Deng, Lin Jia, Sai Wah Tsao. Epstein Barr virus-encoded LMP1 reprograms glucose metabolism to enhance cell motility in nasopharyngeal epithelial cell [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 3079.

Abstract 4278: Somatic MAPK pathway mutations are associated with high mutational burden and good survival in head and neck squamous cell carcinoma (HNSCC)

Abstract Somatic mutations of the MAPK pathway occur in ~14% of head and neck squamous cell carcinoma (HNSCC; TCGA Provisional; www.cbioportal.org). Notably, mutations found in MAPK signaling components are apparent hotspot or activating mutations, potentially driving activation of the MAPK pathway. These mutations include HRAS p.G12V/D/A/C/S & p.G13V/R, MAPK1 p.E322K/*, BRAF p.D594N & p.V600E mutations. We found that this MAPK pathway mutated subset of HNSCC tumors have a higher mutational burden than MAPK-wildtype (WT) tumors. The average rate of non-synonymous mutations in MAPK pathway mutated tumors is 265.1±42.84 mutation/tumor vs 143.5±5.83 mutation/tumor in WT tumors in the TCGA cohort (P=0.0005). MAPK pathway mutations are not associated with HPV positivity (P=0.5815). Subsequent analysis shows that this subset of MAPK pathway-mutated tumors is unexpectedly associated with TP53 wildtype status (P=0.005584), as well as CASP8 mutations [29.3%; 27/92 cases vs. a 6.5% rate (27/418 cases) in the MAPK pathway WT group; P=9.91 x10-9]. Based on the current survival data of the TCGA Provisional cohort, it appears that patients whose tumors bearing HRAS, BRAF, MAPK1, MAP2K1, and RPS6KA1 mutations have a long median survival of 7.94 years (95.3 months; P=0.0165) vs. patients without these somatic mutations. Though TP53 WT status is a well-recognized biomarker for good prognosis in HNSCC, it only predicts with a median survival of ~45.8 months in this very same cohort. Thus, our findings suggest that MAPK pathway mutations may serve as potential genomic biomarkers for good prognosis in HNSCC. Recently, both HRAS and MAPK1 hotspot mutations have been shown to serve as potential neo-antigens in cervical cancer. Further investigation is needed to determine if these somatic MAPK pathway mutations are capable of mounting an anti-cancer immunity (via hotspot neo-antigens or neo-antigens due to higher mutational burden per se) in these patients, which may contribute to their improved survival. Acknowledgements: VWYL received funding supports from the Research Grant Council, Hong Kong (General Research Fund: #1711484, #17121616; Theme-based Research: T12-401/13-R) and the Hong Kong Cancer Fund, Hong Kong. Citation Format: Hoi-Lam Ngan, Andrew Y. Fong, Sharon Chan, Shing Chun Yu, Laura R. H. Ip, Jennifer R. Grandis, Vivian Lui. Somatic MAPK pathway mutations are associated with high mutational burden and good survival in head and neck squamous cell carcinoma (HNSCC) [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 4278.

Abstract 3084: Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell

Abstract Epstein-Barr virus (EBV) infection of preinvasive nasopharyngeal epithelium is believed to be an essential initiation event during nasopharyngeal carcinoma pathogenesis, but the underlying mechanisms are largely undefined. Here we demonstrate that EBV-encoded LMP1 reprograms the glucose metabolism from oxidative phosphorylation to glycolysis of nasopharyngeal epithelial (NPE) is a major process for NPC pathogenesis. RNA-seq analysis indicates that EBV infection reprograms the glucose metabolism from glycolysis and oxidative phosphorylation and enhances the metabolic associated pathways. Our study show EBV encoded LMP1 is important for this event, EBV infection and LMP1 expression enhance the extracellular acidification rate and decrease the oxygen consumption rate in NPE cells. We further show that activation of mTORC2/AKT signaling is involved in this metabolic reprogramming in EBV-infected NPE cells. Genetic or pharmacologic inhibition of the mTORC2/AKT signaling blocks the LMP1-induced glycolysis. In summary, this study provides evidences of involvement of EBV infection in metabolic reprogramming of NPE cells. Acknowledgment: This project was supported by funding from the Research Grants Council, Hong Kong: General Research Fund (17120814, 17161116, 17111516, 171110315), Area of Excellence (AoE/M-06/08), Theme-Based Research Scheme (T12-401/13-R), Collaborative Research Fund (Project reference: C7027-16G); Health and Medical Research Fund of Hong Kong (12110782, 13120872, 04151726); and CRCG, University of Hong Kong, Hong Kong. Citation Format: Jun Zhang, Weitao Lin, Chi-Man Tsang, Teng Fei Liu, W C. Tang, Tim Ling Yip, Wen Deng, Lin Jia, Sai-Wah Tsao. Epstein Barr virus-encoded LMP1 activates the mTORC2 signaling pathway to reprogram glucose metabolism in nasopharyngeal epithelial cell [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 3084.

Abstract 2462: Epstein-Barr virus-encoded microRNAs regulate the expression of BRCA1 in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is an aggressive epithelial malignancy and is endemic in southern China and Southeast Asia. It is well-known to be associated with Epstein-Barr virus (EBV) infection. The miRNAs derived from EBV BamH1-A rightward transcripts (miR-BARTs) are abundantly expressed in NPC and have been suggested that can contribute to cancer development by regulating several gene expression. In this study, we established comprehensive expression profile of miRNAs in an EBV positive NPC cell line and 5 xenografts by small RNA sequencing. By using the prediction algorithm of miRanda and RNAhybrid, in silico analysis identified that several abundantly expressed miRNAs from viral and cellular genomes could negatively regulate BRCA1 expression via the 3' untranslated region (3'UTR). Notably, the regulatory role of miR-BART17-5p, miR-BART19-3p, miR182-5p on endogenous BRCA1 expression was demonstrated by transient transfection assays in HeLa cells and the direct recognitions were verified by luciferase reporter assays, while the downregulation of BRCA1 in primary NPCs was substantiated in the immunohistochemical (IHC) staining in a cohort of 61 histologically normal nasopharyngeal (NP) and 37 primary NPC cases (p<0.001). Taken togethher, we showed that the highly expressed miRNAs could work co-operatively to modulate BRCA1 activity. It has been reported that BRCA1 deficient tumor cells are defective in repairing the DNA double-strand breaks (DSBs) via error-free homologous recombination (HR). The impaired DSB repair by EBV latent infection may induce genome instability during NPC tumorigenesis and also contribute to sensitizing the tumor cells to ionizing radiation and DNA-damaging agent treatments. Acknowledgement: Theme-based Research Scheme (T12-401/13-R) and GRF (14104415, 14138016), Research Grant Council, Hong Kong Citation Format: Ka-Hei Lam, Raymond Wai-Ming Lung, Pok-Man Hau, Kevin Yuk-Lap Yip, George Sai-Wah Tsao, Ken Hung-On Yu, Joanna Hung-Man Tong, Kwok-Wai Lo, Ka-Fai To. Epstein-Barr virus-encoded microRNAs regulate the expression of BRCA1 in nasopharyngeal carcinoma [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 2462.

Abstract 482: Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalence in southern China and Southeast Asia. It is well-known to be associated with latent Epstein-Barr virus (EBV) infection. By using small RNA sequencing, we previously established a comprehensive miRNA profile in a panel of NPC samples and revealed that the EBV-encoded miRNAs derived from BamHI-A rightward transcripts (miR-BARTs) are abundantly expressed. The important roles of miR-BARTs in cancer development have been reported extensively. In the present study, we identified multiple putative binding sites of miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p on the 3'-UTR of a critical DNA double-strand break (DSB) repair gene, Ataxia-Telangiectasia-Mutant (ATM). Notably, the expression of these 4 miR-BARTs represented more than 10% of all EBV-encoded miRNAs in NPC cells while downregulation of ATM expression was observed in our local primary NPC samples in both qRT-PCR and immunohistochemical (IHC) staining analysis. In addition, the abilities of miR-BARTs to downregulate ATM expression were demonstrated in the transient transfection experiments. By manipulating the miR-BARTs expression in epithelial cell lines, we further revealed that these four viral miRNAs could inhibit both ionizing radiation (IR)-induced ATM kinase activity and BZLF1-induced viral lytic reactivation. In conclusion, our findings suggest that miR-BART5-5p, BART7-3p, BART9-3p and BART14-3p work co-operatively to modulate DNA damage response and to maintain viral latency, contributing to the NPC tumorigenesis. Acknowledgement: Theme-based Research Scheme (T12-401/13-R) and GRF (14104415 and 14138016), Research Grant Council, Hong Kong. Citation Format: Raymond W. Lung, Po-Man Hau, Joanna H. Tong, Ka-Hei Lam, Anthony W. Chan, Kevin Y. Yip, George S. Tsao, Kwok-Wai Lo, Ka-Fai To. Epstein-Barr virus-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma [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 482.

Abstract 3168: Serglycin promotes migration and invasion of esophageal cancer cells

Abstract Esophageal cancer is the eighth most common cancer in the world and it ranks sixth in mortality rate among cancer diseases. While patients with early stage esophageal cancer without metastasis have a better chance of being cured by surgery, most patients present with locally advanced or metastatic disease. Unfortunately, some common cancer biomarkers such as carcinoembryonic antigen (CEA) and CA19-9 have low sensitivity and specificity in esophageal cancer. A better understanding of the molecular mechanisms underlying esophageal cancer progression is needed to identify novel diagnostic markers and therapeutic targets for intervention. We previously identified serglycin (SRGN) as the top upregulated gene in a highly invasive esophageal squamous cell carcinoma (ESCC) cell subline. Serglycin is a proteoglycan with a core protein and glycosaminoglycan (GAG) chains. Its significance in tumorigenesis is not well understood. The GAG chains have the ability of binding specific growth factors and ligands, which may play an important role in facilitating cancer cell migration and invasion. Immunohistochemical analysis showed that serglycin expression was higher in ESCC compared with normal esophageal epithelium, and in lymph node metastases compared with primary ESCC. Ectopic overexpression of serglycin in ESCC cells enhanced their migration and invasion potential in vitro. Knockdown of serglycin reduced ESCC cell migration and invasion. In invasion chamber assays, conditioned medium (CM) from SRGN-overexpressing ESCC cells stimulated the migration and invasion ability of less invasive ESCC cells. Conditioned medium from ESCC cells expressing serglycin that lacks the GAG attachment site had no such effects. Chemokine antibody array analysis showed that midkine (MDK), a heparin-binding growth factor, was the top upregulated protein in the CM of SRGN-overexpressing cells. Analysis of TCGA and GTEx datasets showed that MDK expression was higher in many cancer types such as breast cancer, colon adenocarcinoma, glioblastoma and lung cancer, than in corresponding normal tissues. Western blot analysis showed that forced expression of serglycin, but not serglycin without the GAG binding domain, led to increase of MDK in the CM of multiple ESCC cell lines. Forced expression of serglycin also increased the expression level of phosphorylated extracellular signal-regulated kinase (p-ERK) in ESCC cells. Taken together, serglycin may play an important role in the progression ESCC by promoting migration and invasion of cancer cells. These effects may be mediated through activation of the ERK signaling pathway. Whether MDK is involved in this regulatory mechanism warrants further investigation. [This study was supported by Research Grants Council of the Hong Kong SAR, China, GRF Project No. 17111016] Citation Format: Yun ZHU, Bin Li, Wenwen Xu, Aky Lam, Simon Law, ALM Cheung. Serglycin promotes migration and invasion of esophageal cancer cells [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 3168.

Abstract 3419: Genomic characterization of mutation hotspots related to chemotherapy responses in esophageal squamous-cell carcinoma with archived formalin-fixed paraffin-embedded tissues

Abstract Introduction: Currently neoadjuvant chemoradiation therapy (CRT) prior to surgery is the most common treatment for Esophageal Squamous-cell Carcinoma (ESCC) patients. Given the fact that traditional TNM staging of the patients before treatment could not reflect their overall survival, a recent study conducted in Hong Kong demonstrated that the percentage of viable tumor cells remaining in the primary tumor after the CRT therapy was a prognostic marker for overall survival. Clinicians defined patients with no viable tumor cells (0%) as good responders, while the others having greater than 50% tumor cells remaining (50-100%) were considered poor responders. They observed that good responders show significantly better five-year survival rate. However, little is known about the mutation profiles between good and poor responders stratified in this way. Methods: Taking advantage of archived formalin-fixed paraffin-embedded (FFPE) tissues with comprehensive clinical outcomes, I apply a novel approach to systematically screen for possible somatic mutations that are associated with patients' responses to CRT in ESCC. I obtained 100 archived FFPE blocks from Queen Mary Hospital, Hong Kong, 50 each representing either good responders or poor responders. DNA libraries were constructed to target 131 cancer-related genes with TOMA Os-seq, which adopted single-stranded ligation to minimize the loss of input FFPE DNA. Then, libraries were sequenced on Illumina HiSeq X Ten. The final list of somatic mutations was filtered with in-house pipeline, excluding both germline variants from public databases and in-house Hong Kong database. Copy number variations (CNVs) were also identified with a pre-designed control sample in the same sequencing batch. Results: One test run on samples with good quality DNA showed high sensitivity and specificity up to 100% for pre-designed mutations. For CNV, the sensitivity was 87.5% and the specificity was 97.5%. Trials of sequencing with several ESCC FFPE samples revealed deleterious mutations from genes including TP53, BRCA2, and those mutations were successfully validated by Sanger sequencing. Groups of critical protein-altered mutations and CNVs that distinguish good and poor responders of ESCC will be presented in detail. Conclusion: In all, FFPE blocks are valuable sources to uncover the biomarkers for CRT response in ESCC. I expect those biomarkers can aid future evaluation of newly diagnostic patients to avoid unnecessary efforts in treating patients that are unsuitable for the traditional therapy and thus facilitate effective treatments. Acknowledgements: Theme-based Research Scheme grant provided by the Hong Kong Research Grants Council (T12-701/17-R to MLL). Citation Format: Chen Guo, Josephine Ko, Wei Dai, Lihua Tao, Simon Law, Anthony Wing Ip Lo, Maria Li Lung. Genomic characterization of mutation hotspots related to chemotherapy responses in esophageal squamous-cell carcinoma with archived formalin-fixed paraffin-embedded tissues [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 3419.