Christopher W. Dawson

Epstein-Barr virus (EBV), an agent with growth transforming potential for human B cells, is associated with certain B cell lymphomas in man and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma (NPC). Since B cell growth transformation is associated with the constitutive expression of a small number of EBV-coded latent proteins, the nuclear antigens EBNA 1, EBNA 2, EBNA 3 and EBNA-LP and the latent membrane protein (LMP), the present work sought to determine whether this same pattern of virus gene expression occurred in NPC. Tumour biopsies were taken from NPC patients from three areas of differing tumour incidence (Kenya, Algeria, Britain) and immediately snap-frozen, as were biopsies of non-EBV-related carcinomas for controls. Immunoblotting of PAGE-separated proteins with selected human sera identified 24 NPC biopsies clearly expressing EBNA 1. When the analysis was extended using selected human sera with antibodies against the other EBNAs, there was no detectable expression of EBNA 2, EBNA 3 or EBNA-LP in any of these 24 biopsies; their EBNA 2-negative status was confirmed using a monoclonal antibody (MAb) PE2 which was reactive in immunoblotting and in immunoprecipitation with EBNA 2A and EBNA 2B proteins. Similar experiments with two different LMP-specific MAbs, CS1 to 4 and S12, revealed heterogeneity between NPC biopsies; 9/24 biopsies were demonstrably LMP-positive, the degree of expression varying considerably between individual tumours in a manner which was not related to the level of EBNA 1 expression. None of the 24 NPC biopsies expressed detectable amounts of EBV lytic cycle antigens. A nude mouse-passaged NPC cell line, C15, likewise expressed EBNA 1 and LMP but none of the other EBV latent proteins nor lytic cycle antigens. This work identifies a novel type of EBV-cell interaction in NPC cells which is distinct from that seen in in vitro transformed B cell lines and from that seen to date in EBV-positive B cell lymphomas.

Although frequently expressed in EBV-positive malignancies, the contribution of the oncogenic latent membrane proteins, LMP1 and LMP2, to the pathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. As a key effector in EBV-driven B cell transformation and an established "transforming" gene, LMP1 displays oncogenic properties in rodent fibroblasts and induces profound morphological and phenotypic effects in epithelial cells. LMP1 functions as a viral mimic of the TNFR family member, CD40, engaging a number of signalling pathways that induce morphological and phenotypic alterations in epithelial cells. Although LMP2A plays an essential role in maintaining viral latency in EBV infected B cells, its role in epithelial cells is less clear. Unlike LMP1, LMP2A does not display "classical" transforming functions in rodent fibroblasts but its ability to engage a number of potentially oncogenic cell signalling pathways suggests that LMP2A can also participate in EBV-induced epithelial cell growth transformation. Here we review the effects of LMP1 and LMP2 on various aspects of epithelial cell behaviour highlighting key aspects that may contribute to the pathogenesis of NPC.

Since its discovery 50 years ago, Epstein-Barr virus (EBV) has been linked to the development of cancers originating from both lymphoid and epithelial cells. Approximately 95% of the world's population sustains an asymptomatic, life-long infection with EBV. The virus persists in the memory B-cell pool of normal healthy individuals, and any disruption of this interaction results in virus-associated B-cell tumors. The association of EBV with epithelial cell tumors, specifically nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma (EBV-GC), is less clear and is currently thought to be caused by the aberrant establishment of virus latency in epithelial cells that display premalignant genetic changes. Although the precise role of EBV in the carcinogenic process is currently poorly understood, the presence of the virus in all tumor cells provides opportunities for developing novel therapeutic and diagnostic approaches. The study of EBV and its role in carcinomas continues to provide insight into the carcinogenic process that is relevant to a broader understanding of tumor pathogenesis and to the development of targeted cancer therapies.

Abstract In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8+ T cells led to enhanced infiltration and granzyme B–mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFβ expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFβ may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFβ signals in the epithelium. Cancer Res; 76(9); 2525–39. ©2016 AACR.

The Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is a pleiotropic protein the activities of which include effects on gene expression and cell transformation, growth, and death.LMP1 has been shown to induce nuclear factor (NF)-B and c-Jun NH 2 -terminal kinase/AP-1 activities in target cells, and in this study we demonstrate that LMP1 also engages the p38 mitogen-activated protein kinase cascade, leading to activation of the transcription factor ATF2. Mutational analysis of the LMP1 cytoplasmic COOH terminus revealed that p38 activation occurs from both the tumor necrosis factor receptor-associated factor (TRAF)-interacting, membrane-proximal COOH-terminal activating region (CTAR)1 domain (amino acids 186 -231) and the extreme tumor necrosis factor receptor-associated death domain (TRADD) binding CTAR2 region (amino acids 351-386).Because LMP1 also engages signaling on the NF-B axis through CTAR1 and CTAR2, we have examined whether these two pathways are overlapping or independent.We have found that inhibition of p38 by the highly specific inhibitor SB203580 did not affect NF-B binding activity.Conversely, although the metabolic inhibitor D609 blocked NF-B activation, it did not impair the ability of LMP1 to signal on the p38 axis, suggesting that these two LMP1-mediated pathways are primarily independent.Divergence of signals must, however, occur downstream of TRAF2 as a dominant negative TRAF2 mutant that blocks LMP1-induced NF-B activation also inhibited p38 signaling.In addition, we have found that p38 inhibition significantly impaired LMP1-mediated interleukin-6 and -8 expression.Thus, p38 may play a significant cooperative role in regulating at least some of the pleiotropic activities of LMP1.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an integral membrane protein that functions as a constitutively activated member of the tumor necrosis factor receptor family. Whereas LMP1 has been shown to activate the NF-κB and mitogen-activated protein kinase pathways, these effects alone are unable to account for the profound oncogenic properties of LMP1. Here we show that LMP1 can activate phosphatidylinositol 3-kinase (PI3K), a lipid kinase responsible for activating a diverse range of cellular processes in response to extracellular stimuli. LMP1 was found to stimulate PI3K activity inducing phosphorylation and subsequent activation of Akt, a downstream target of PI3K responsible for promoting cell survival. Treatment of LMP1-expressing cells with the PI3K inhibitor LY294002 resulted in decreased cell survival. The tumor necrosis factor receptor-associated factor-binding domain of LMP1 was found to be responsible for PI3K activation. The ability of LMP1 to induce actin stress-fiber formation, a Rho GTPase-mediated phenomenon, was also dependent on PI3K activation. These data implicate PI3K activation in many of the LMP1-induced phenotypic effects associated with transformation and suggests that this pathway contributes both to the oncogenicity of this molecule and its role in the establishment of persistent EBV infection.

Expression of the Epstein – Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-κB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187 – 231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-κB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-κB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-κB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-κB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-κB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-κB activity, also blocked NF-κB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a key effector of EBV-mediated B cell transformation. LMP1 displays potent oncogenic properties in rodent fibroblasts, and induces a wide range of effects in B cells and epithelial cells. LMP1 functions as a constitutively active tumor necrosis factor receptor (TNFR) engaging a multitude of signaling pathways that include NF-kappaB, the mitogen-activated protein kinases (MAPKs), JNK, p38, the JAK/STAT pathway and, more recently, the small Rho GTPases. The constitutive activation of these signaling cascades explains LMP1's ability to induce such a diverse array of morphological and phenotypic effects in cells and provides an insight into how LMP1 may induce cell transformation. The frequent expression of LMP1 in undifferentiated nasopharyngeal carcinoma (NPC) points to a role for this viral oncoprotein as a key effector molecule in NPC pathogenesis.

The molecular mechanisms underlying the pathogenesis of the malignant Hodgkin's/Reed–Sternberg (HRS) cells of Hodgkin's lymphoma (HL) are largely unknown. This study investigates the contribution of phosphatidyl-inositide 3 kinase (PI3-kinase) and demonstrates that Akt, a substrate of PI3-kinase, is constitutively activated in HL-derived cell lines. Several downstream effectors of Akt signalling, including glycogen synthase kinase 3 (GSK-3) α and β and mTOR substrates 4E-BP1 and p70 S6 kinase, were also phosphorylated in HL cells. The mTOR inhibitor, rapamycin, inhibited phosphorylation of these proteins. Furthermore, LY294002 inhibited phosphorylation of p70 S6 kinase and 4E-BP1, suggesting that the phosphorylation of p70 S6 kinase and 4E-BP1 in HL cells is PI3-kinase dependent. Importantly, HRS cells of primary tumour samples not only expressed high levels of activated Akt but also displayed phosphorylation of downstream targets of Akt activation including GSK-3, 4E-BP1, and p70 S6 Kinase. Inhibition of PI3-kinase and mTOR showed only modest effects on cell survival at the lower serum concentrations. However, rapamycin and doxorubicin acted synergistically to reduce HL cell survival. A combination of rapamycin and chemotherapy should be investigated in the treatment of HL. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These 'Las-exposed' psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1–42 days post-first access to diseased plants (padp); all tested nymphs became adults 7–14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49–59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8–29% of the psyllids were infected following 1–14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14–28 days padp for nymphs and 21–35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease.

Non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c-Myc, and HIF-1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up-regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1-mediated aerobic glycolysis, cellular transformation (proliferation and anchorage-independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1-mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage-independent growth of NPC cells. Our current findings demonstrate that LMP1-mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV-driven pathogenesis of NPC.

Long-term survival (LTS) is uncommon for patients with pancreatic ductal adenocarcinoma (PDAC). We sought to identify factors that predict 10-year, LTS after resection of PDAC.We identified all patients with PDAC who underwent resection at UCLA after 1990 and included all patients eligible for observed LTS (1/1/1990-12/31/2004). An independent pathologist reconfirmed the diagnosis of PDAC in patients with LTS. Logistic regression was used to predict LTS on the basis of patient and tumor characteristics.Of 173 included patients, 53% were male, median age at diagnosis was 66 years, and median survival was 23 months. The rate of observed LTS was 12.1% (n = 21). Age, sex, number of lymph nodes evaluated, margin status, lymphovascular invasion, and adjuvant chemotherapy and radiation were not associated with LTS. The following were associated with LTS on bivariate analysis: low AJCC stage (Ia, Ib, IIa) (P = .034), negative lymph node status (P = .034), low grade (well-, moderately-differentiated) (P = .001), and absence of perineural invasion (P = .019). Only low grade (odds ratio 7.17, P = .012) and absent perineural invasion (odds ratio 3.28, P = .036) were independently associated with increased odds of LTS. Our multivariate model demonstrated good discriminatory power for LTS, as indicated by a c-statistic of 0.7856.Absence of perineural invasion and low tumor grade were associated with greater likelihood of LTS. Understanding the tumor biology of LTS may provide critical insight into a disease that is typically marked by aggressive behavior and limited survival.

Abstract Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. It is also characterized by heavy infiltration with non-malignant leucocytes. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways which collectively promote cell proliferation and survival, angiogenesis, invasiveness, and aerobic glycolysis. LMP1 also affects cell-cell interactions, antigen presentation, and cytokine and chemokine production. Here, we discuss how LMP1 modulates local immune responses that contribute to the establishment of the NPC tumor microenvironment. We also discuss strategies for targeting the LMP1 protein as a novel therapy for EBV-driven malignancies.

Abstract We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.

The Epstein–Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumours, including nasopharyngeal carcinoma (NPC), where it plays an essential role in EBV genome maintenance, replication and transcription. Previous studies suggest that EBNA1 may have additional effects relevant to oncogenesis, including enhancement of cell survival, raising the possibility that EBNA1 may influence cellular gene expression. We have recently demonstrated by gene expression microarray profiling in an NPC cell model that EBNA1 influences the expression of a range of cellular genes, including those involved in transcription, translation and cell signalling. Here, we report for the first time that EBNA1 enhances activity of the AP-1 transcription factor in NPC cells and demonstrate that this is achieved by EBNA1 binding to the promoters of c-Jun and ATF2, enhancing their expression. In addition, we demonstrate elevated expression of the AP-1 targets interleukin 8, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 α in response to EBNA1 expression, which enhances microtubule formation in an in vitro angiogenesis assay. Furthermore, we confirm elevation of VEGF and the phosphorylated isoforms of c-Jun and ATF2 in NPC biopsies. These findings implicate EBNA1 in the angiogenic process and suggest that this viral protein might directly contribute to the development and aggressively metastatic nature of NPC.

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an oncogenic protein which has previously been shown to engage the NF-κB, stress-activated MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and extracellular-regulated kinase (ERK)-MAPK pathways. In this study, we demonstrate that LMP1 activates ERK-MAPK in epithelial cells via the canonical Raf-MEK-ERK-MAPK pathway but in a Ras-independent manner. In agreement with the results of a previous study (B. A. Mainou, D. N. Everly, Jr., and N. Raab-Traub, J. Virol. 81:9680-9692, 2007), we show that the ability of LMP1 to activate ERK-MAPK mapped to its CTAR1 domain, the TRAF binding domain previously implicated in PI 3-kinase activation. A role for ERK-MAPK in LMP1-induced epithelial cell motility was identified, as LMP1-expressing cells displayed increased rates of haptotactic migration compared to those of LMP1-negative cells. These data implicate the ERK-MAPK pathway in LMP1-induced effects associated with transformation, suggesting that this pathway may contribute to the oncogenicity of LMP1 through its ability to promote cell motility and to enhance the invasive properties of epithelial cells.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.In this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.Inhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

Although frequently expressed in Epstein–Barr virus (EBV)-positive malignancies, the contribution of the oncogenic latent membrane protein-1 (LMP1) to the pathogenesis of nasopharyngeal carcinoma remains to be fully defined. As a key effector in EBV-driven B-cell transformation in vitro, LMP1 also displays oncogenic properties in rodent fibroblasts, and exhibits similar effects in epithelial cells. LMP1 functions as a viral mimic of the TNFR family member, CD40, engaging a plethora of signaling pathways including: NF-κB, JNK/p38 (SAPK), PI3-kinase and ERK–MPK. The constitutive activation of these pathways appears central in the ability of LMP1 to induce multiple morphological and phenotypic alterations. Here we review the effects of LMP1 on epithelial cell growth transformation, and its putative role in the pathogenesis of nasopharyngeal carcinoma, focusing on key areas of proliferation, survival, cell motility and invasion.

A micro-array analysis using biopsies from patients with EBV-positive undifferentiated nasopharyngeal carcinoma (NPC) and from cancer-free controls revealed down-regulation of tumour suppressor genes (TSG) not previously associated with this disease; one such gene was the ataxia telangiectasia mutated (ATM) gene. Q-PCR confirmed down-regulation of ATM mRNA and ATM protein expression in tumour cells was weak or absent in almost all cases. In NPC cell lines, however, ATM was down-regulated only in the EBV-positive line, C666.1, and in none of five EBV-negative lines. In vitro infection of EBV-negative NPC cell lines with a recombinant EBV was followed by the down-regulation of ATM mRNA and protein, and only EBV-positive cells showed a defective DNA damage response following gamma-irradiation. Our data suggest that loss of ATM function could be an important step in the pathogenesis of NPC, and may have implications for the treatment of this disease.

Abstract Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer prevalent in south‐east Asia and southern China, where it constitutes a significant health burden. Although the close association of NPC with Epstein–Barr virus (EBV) infection has been known for more than four decades, the exact role that EBV plays in the pathogenesis of this malignancy is still unclear. While NPC tumours are known to express a number of EBV‐encoded proteins, they also express a large number of virus‐encoded microRNAs (miRNAs), the most abundant of which are those encoded from the BamHI‐A region of the viral genome: the so‐called BART miRNAs. miRNAs are small non‐coding mRNAs that negatively regulate the expression of various genes at the post‐transcriptional level. Accumulating evidence suggests that miRNAs play important roles in tumourigenesis. Here, we review the role of EBV‐encoded BART miRNAs in modulating apoptosis and host innate defence mechanisms and their contribution to NPC pathogenesis. The rationale and strategies for therapeutic targeting of BART miRNAs in EBV‐infected NPC are also discussed. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.

Nasopharyngeal carcinoma (NPC) is a cancer common in southern China and South East Asia that is causally linked to Epstein-Barr virus (EBV) infection. Here, we demonstrate that NPC displays frequent dysregulation of the Hedgehog (HH) pathway, a pathway implicated in the maintenance of stem cells, but whose aberrant activation in adult tissues can lead to cancer. Using authentic EBV-positive carcinoma-derived cell lines and nasopharyngeal epithelial cell lines latently infected with EBV as models for NPC in vitro, we show that EBV activates the HH signalling pathway through autocrine induction of SHH ligand. Moreover, we find that constitutive engagement of the HH pathway induces the expression of a number of stemness-associated genes and imposes stem-like characteristics on EBV-infected epithelial cells in vitro. Using epithelial cells expressing individual EBV latent genes detected in NPC, we show that EBNA1, LMP1, and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. Our findings not only identify a role for dysregulated HH signalling in NPC oncogenesis, but also provide a novel rationale for therapeutic intervention.

Older adults display alterations in neural reflections of conflict-related processing. We examined response times (RTs), error rates, and event-related potential (ERP; N2 and P3 components) indices of conflict adaptation (i.e., congruency sequence effects) a cognitive control process wherein previous-trial congruency influences current-trial performance, along with post-error slowing, correct-related negativity (CRN), error-related negativity (ERN) and error positivity (Pe) amplitudes in 65 healthy older adults and 94 healthy younger adults. Older adults showed generalized slowing, had decreased post-error slowing, and committed more errors than younger adults. Both older and younger adults showed conflict adaptation effects; magnitude of conflict adaptation did not differ by age. N2 amplitudes were similar between groups; younger, but not older, adults showed conflict adaptation effects for P3 component amplitudes. CRN and Pe, but not ERN, amplitudes differed between groups. Data support generalized declines in cognitive control processes in older adults without specific deficits in conflict adaptation.

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a characteristically dense stroma comprised predominantly of cancer-associated fibroblasts (CAF). CAFs promote tumor growth, metastasis, and treatment resistance. This study aimed to investigate the molecular changes and functional consequences associated with chemotherapy treatment of PDAC CAFs. Chemoresistant immortalized CAFs (R-CAF) were generated by continuous incubation in gemcitabine. Gene expression differences between treatment-naïve CAFs (N-CAF) and R-CAFs were compared by array analysis. Functionally, tumor cells (TC) were exposed to N-CAF– or R-CAF–conditioned media and assayed for migration, invasion, and viability in vitro. Furthermore, a coinjection (TC and CAF) model was used to compare tumor growth in vivo. R-CAFs increased TC viability, migration, and invasion compared with N-CAFs. In vivo, TCs coinjected with R-CAFs grew larger than those accompanied by N-CAFs. Genomic analysis demonstrated that R-CAFs had increased expression of various inflammatory mediators, similar to the previously described senescence-associated secretory phenotype (SASP). In addition, SASP mediators were found to be upregulated in response to short duration treatment with gemcitabine in both immortalized and primary CAFs. Inhibition of stress-associated MAPK signaling (P38 MAPK or JNK) attenuated SASP induction as well as the tumor-supportive functions of chemotherapy-treated CAFs in vitro and in vivo. These results identify a negative consequence of chemotherapy on the PDAC microenvironment that could be targeted to improve the efficacy of current therapeutic regimens. Implications: Chemotherapy treatment of pancreatic cancer–associated fibroblasts results in a proinflammatory response driven by stress-associated MAPK signaling that enhances tumor cell growth and invasiveness. Mol Cancer Res; 14(5); 437–47. ©2016 AACR.

Abstract Nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element‐binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV‐infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1‐mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1‐mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo . Our findings demonstrate that LMP1 activation of SREBP1‐mediated lipogenesis promotes tumor cell growth and is involved in EBV‐driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

The association of Epstein-Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1-AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1-AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK-MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1-AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1-induced cellular transformation (proliferation and anchorage-independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage-independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5-fluorouracil. The current findings demonstrate that LMP1-mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1-AMPK pathway in the EBV-driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC.

The repurposing of drugs is becoming increasingly attractive as it avoids the lengthy process and cost implications associated with bringing a novel drug to market. Itraconazole is a broad-spectrum anti-fungal agent. An emerging body of in vivo, in vitro and clinical evidence have confirmed that it also possesses antineoplastic activities and has a synergistic action when combined with other chemotherapeutic agents. It acts via several mechanisms to prevent tumour growth, including inhibition of the Hedgehog pathway, prevention of angiogenesis, decreased endothelial cell proliferation, cell cycle arrest and induction of auto-phagocytosis. These allow itraconazole, either alone or in combination with other cytotoxic agents, to increase drug efficacy and overcome drug resistance. This study reviews the reported literature on the use of itraconazole in a variety of malignancies and highlights the recent insights into the critical pathways acted upon to prevent tumour growth.

Objective In this study, we investigated if the presence of histologically abnormal epithelium adjacent to the primary tumour influenced the frequency, timing, and topography of local vulvar recurrences (LVR) following treatment for squamous cell carcinoma of the vulva (VSCC). Methods The study population comprised a cohort of 201 consecutive cases with incident VSCC. LVR were categorised as local relapses (LR) if they occurred <2 cm from the tumour margins, and as second field tumours (SFT) when ≥2 cm from these margins. Univariable and multivariable competing risk modelling was performed to identify the prognostic factors associated with local disease recurrence. Results The characterization of the epithelium adjacent to the invasive component was possible for 199 (99.0%) patients. Of these, 171 (85.9%) were found to have intraepithelial abnormalities found adjacent to the surgical specimen. Multivariable analyses revealed that, following adjustment, Lichen Sclerosis (LS) was associated with an increase in the incidence of LVR, LR and SFT (SHRs: 3.4, 2.7 and 4.4, respectively). Although the incidence of LR and SFT in women with LS associated VSCC was similar, the peak incidence of SFT occurred more than two years before that of LR. Conclusions Women with VSCC arising in a field of LS may continue to have an increased risk of developing LR and SFT for many years after resection of their primary tumour. Our study suggests that these women should be followed up more regularly so that LVR can be detected earlier; unless a more robust surveillance programme or chemopreventative treatments become available.

Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein-Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection.We studied the ability of LTP to reactivate latently infected EBV-positive cell lines in vitro and in vivo, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination.Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene BZLF1, the early gene BMRF1, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide > lenalidomide > thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells in vitro and in vivo EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3Kδ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation.We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. Clin Cancer Res; 22(19); 4901-12. ©2016 AACR.

Virus infection and replication are often associated with apoptosis and this effect is likely to be responsible for much of the pathology associated with infectious disease. Many viruses encode proteins which can inhibit apoptosis thereby either prolonging the survival of infected cells such that the production of progeny virus is maximised or facilitating the establishment of virus persistence. These viral proteins target the cellular pathways responsible for regulating apoptosis and have been instrumental in furthering our understanding of the apoptotic process. Many of the viruses associated with oncogenic transformation have adopted strategies for blocking apoptosis highlighting the centrality of this effect in carcinogensis. Understading the mechanisms by which viruses regulate apoptosis may lead to the development of novel therapies for both infectious disease and cancer.

The contribution of Epstein-Barr virus (EBV) strain variation to the pathogenesis of virus-associated tumours remains unknown. Given the central role of LMP1 in EBV-induced transformation, much interest has focused on the influence of LMP1 sequence variation on the signaling pathways and multiple downstream phenotypic consequences of LMP1 expression. The identification of LMP1 variants with a common 10-amino-acid deletion and additional point mutations (typified by the CAO-LMP1 isolate) in EBV strains associated with nasopharyngeal carcinoma prompted us to examine the effect of stable prototype B95.8-LMP1 and CAO-LMP1 expression on the phenotype and differentiation of SCC12F human epithelial cells. Both forms of LMP1 were able to induce expression of the antiapoptotic A20 protein and provide protection from tumour necrosis factor-alpha-induced cytotoxicity. Although B95.8-LMP1 induced growth inhibition, expression of certain cell surface molecules (CD40, CD44, and CD54), and secretion of interleukin-6 and -8 in SCC12F cells, stable CAO-LMP1 expression failed to elicit these effects. Furthermore, B95. 8-LMP1, but not CAO-LMP1, induced alterations in cell morphology and blocked epithelial cell differentiation. Both B95.8-LMP1 and CAO-LMP1 induced similar levels of nuclear factor-kappaB activation, but the ability of CAO-LMP1 to activate the AP-1 pathway was relatively impaired. These data highlight significant functional differences between the prototype B95.8-LMP1 and the CAO-LMP1 variant when stably expressed in human epithelial cells and suggest that continued analysis of LMP1 variants will help to further dissect the signaling pathways activated by LMP1 as well as provide insights into the contribution of LMP1 sequence variation to the pathogenesis of EBV-associated tumours.

ABSTRACT The frequent expression of latent membrane proteins LMP2A and LMP2B in Epstein Barr virus (EBV)-associated tumors suggests that these proteins play a role in EBV-induced epithelial cell growth transformation. Expression of LMP2A and LMP2B had no effect on the morphology of squamous epithelial cells in monolayer culture, but their expression was associated with an increased capacity to spread and migrate on extracellular matrix. Although the mechanisms by which LMP2A and LMP2B promote cell spreading and motility are unclear, the use of selective pharmacological inhibitors has established a role for tyrosine kinases in this phenotype but ruled out contributions of phosphatidylinositol 3-kinase, extracellular signal-regulated kinase/mitogen-activated protein kinase, and protein kinase C. The ability of LMP2B to induce a phenotype that is virtually indistinguishable from that of LMP2A suggests that regions of the LMP2 protein in addition to the cytosolic amino terminus are capable of inducing phenotypic effects in epithelial cells. Thus, rather than serving to modulate the activity of LMP2A, LMP2B may directly engage signaling pathways to influence epithelial cell behavior such as cell adhesion and motility.

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.

The Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) oncogene can induce profound effects on epithelial growth and differentiation including many of the features of the epithelial-to-mesenchymal transition (EMT). To better characterise these effects, we used the well-defined Madin Darby Canine Kidney (MDCK) epithelial cell model and found that LMP1 expression in these cells induces EMT as defined by characteristic morphological changes accompanied by loss of E-cadherin, desmosomal cadherin and tight junction protein expression. The induction of the EMT phenotype required a functional CTAR1 domain of LMP1 and studies using pharmacological inhibitors revealed contributions from signalling pathways commonly induced by integrin–ligand interactions: extracellular signal-regulated kinases/mitogen-activated protein kinases (ERK-MAPK), PI3-Kinase and tyrosine kinases, but not transforming growth factor beta (TGFβ). More detailed analysis implicated the CTAR1-mediated induction of Slug and Twist in LMP1-induced EMT. A key role for β1 integrin signalling in LMP1-mediated ERK-MAPK and focal adhesion kianse (FAK) phosphorylation was observed, and β1 integrin activation was found to enhance LMP1-induced cell viability and survival. These findings support an important role for LMP1 in disease pathogenesis through transcriptional reprogramming that enhances tumour cell survival and leads to a more invasive, metastatic phenotype.

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

Approximately 20% of global cancer incidence is causally linked to an infectious agent. Epstein-Barr virus (EBV) accounts for around 1% of all virus-associated cancers and is associated with nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP1), the major oncoprotein encoded by EBV, behaves as a constitutively active tumour necrosis factor (TNF) receptor activating a variety of signalling pathways, including the three classic MAPKs (ERK-MAPK, p38 MAPK and JNK/SAPK). The present study identifies novel signalling properties for this integral membrane protein via the induction and secretion of activin A and TGFβ1, which are both required for LMP1's ability to induce the expression of the extracellular matrix protein, fibronectin. However, it is evident that LMP1 is unable to activate the classic Smad-dependent TGFβ signalling pathway, but rather elicits its effects through the non-Smad arm of TGFβ signalling. In addition, there is a requirement for JNK/SAPK signalling in LMP1-mediated fibronectin induction. LMP1 also induces the expression and activation of the major fibronectin receptor, α5β1 integrin, an effect that is accompanied by increased focal adhesion formation and turnover. Taken together, these findings support the putative role for LMP1 in the pathogenesis of NPC by contributing to the metastatic potential of epithelial cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The <i>EBER</i> genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These <i>EBER</i> transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of <i>EBER</i> RNA. These include phosphorylation of ATF2, which interacts with <i>EBER</i> promoters. A second is induction of TFIIIC, a pol III-specific factor that activates <i>EBER</i> genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate <i>EBER</i> expression <i>in vivo</i>, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential.

Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFβ-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised. In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFβ-SMAD-mediated transcription and renders cells refractory to TGFβ-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFβ-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFβ stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFβ. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFβ-induced Id1 upregulation. The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFβ-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation

Fish migrations through riverine systems can be energetically demanding, and the presence of fishways to facilitate upstream passage can add an additional energetic cost that may directly affect fitness. Successful fishway passage is a function of the ability of fish to select appropriate paths and swimming strategies that do not exceed their swimming capacity. Triaxial accelerometers were used to estimate the energetic expenditure of adult lake sturgeon (Acipenser fulvescens) swimming through a vertical slot fishway, to determine whether individual behaviour or path selection, resulting in differences in cumulative energy use, explain fishway passage success. Most individuals attempted to pass the fishway (n=30/44; 68%), although successful passage only occurred for a subset of those attempting (n=7/30; 23%). High-speed swimming was rarely observed during upstream passage through fishway basins, and was of short duration. Two turning basins delayed passage, subsequently resulting in a higher energetic cost. The rate at which energy was expended did not differ among successful and unsuccessful individuals, although successful sturgeon exhibited higher costs of transport (42.75 versus 25.85 J kg(-1) m(-1)). Energy expenditure metrics were not predictive of successful fishway passage, leading us to conclude that other endogenous or exogenous factors influence passage success. In a practical application of field measurements of energy expenditure, we demonstrate that fishway passage through a structure designed to facilitate migration does result in an energetic loss for lake sturgeon (3249-16,331 J kg(-1)), equivalent to individuals travelling 5.8-28.2 km in a lentic system.

BHRF1, a component of the restricted early antigen (EA) complex of the Epstein-Barr virus (EBV) lytic cycle, encodes a 17 kDa putative transmembrane protein with both sequence and functional homology to the Bcl-2 proto-oncogene. To determine whether there was any sequence variation over the BHRF1 open reading frame (ORF), 15 EBV isolates from different geographical regions and from both healthy donors and patients with EBV-associated diseases were sequenced. A small number of base changes which resulted in amino acid substitutions in the BHRF1 protein were found relative to the prototype B95.8 EBV sequence and these were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues. In transient transfection assays none of the mutations in the BHRF1 ORF from eight different EBV isolates had a significant effect on BHRF1 protein localization compared to the B95.8 BHRF1 protein. However, transient expression of the adenovirus 12 19K protein or Bcl-2 resulted in localization patterns distinct from that observed with BHRF1 protein. Whilst all eight EBV isolates and E1B-19K gave comparable levels of protection to the DNA-damaging agent c/s-platin, Bcl-2 did not afford significant protection. Thus, despite several amino acid changes in the BHRF1 ORF of some of the EBV isolates studied, the ability of the protein to protect against cis-platin induced apoptosis is conserved. The highly conserved nature of BHRF1 amongst different EBV isolates at both the sequence and functional level supports the proposed important role of BHRF1 in delaying cell death, thereby maximizing the production of progeny virus and facilitating the establishment of virus persistence.

The transforming growth factor-β (TGF-β) signalling pathway plays a critical role in carcinogenesis. It has a biphasic action by initially suppressing tumorigenesis but promoting tumour progression in the later stages of disease. Consequently, the functional outcome of TGF-β signalling is strongly context-dependent and is influenced by various factors including cell, tissue and cancer type. Disruption of this pathway can be caused by various means, including genetic and environmental factors. A number of human viruses have been shown to modulate TGF-β signalling during tumorigenesis. In this review, we describe how this pathway is perturbed in Epstein-Barr virus (EBV)-associated cancers and how EBV interferes with TGF-β signal transduction. The role of TGF-β in regulating the EBV life cycle in tumour cells is also discussed.

Significance Epstein−Barr virus (EBV) infection is implicated in the development of certain cancers; however, the mechanisms by which EBV contributes to the pathogenesis of epithelial cell malignancies remains unclear. This study highlights the association of EBV infection with aberrant modifications in histone bivalent marks, H3K4me3 and H3K27me3, in nasopharyngeal epithelial cells. Down-regulation of the DNA damage repair pathway genes, including MLH1 , associated with aberrant histone bivalent marks in a promoter hypermethylation-independent manner, was observed after EBV infection. This finding provides strong evidence linking EBV infection to epigenetic modifications in epithelial cells. This study also suggests that the level of MLH1 may be useful as a potential biomarker to evaluate the responsiveness of nasopharyngeal carcinoma patients to cisplatin-based therapies.

Epstein-Barr virus (EBV) is closely linked to the development of a number of human cancers. EBV-associated malignancies are characterized by a restricted pattern of viral latent protein expression which is sufficient for the virus to both initiate and sustain cell growth and to protect virus-infected cells from immune attack. Expression of these EBV proteins in malignant cells provides an attractive target for therapeutic intervention. Among the viral proteins expressed in the EBV-associated epithelial malignancies, the protein encoded by the BamHI-A rightward frame 1 (BARF1) is of particular interest. BARF1 is a viral oncoprotein selectively expressed in latently infected epithelial cancers, nasopharyngeal carcinoma (NPC) and EBV-positive gastric cancer (EBV-GC). Here, we review the roles of BARF1 in oncogenesis and immunomodulation. We also discuss potential strategies for targeting the BARF1 protein as a novel therapy for EBV-driven epithelial cancers.

Sequence variants of the Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) have been reported in association with EBV-linked malignancies but little is known about their effects on signalling pathways and phenotype. We have examined the ability of the nasopharyngeal carcinoma (NPC)-derived variant, CAO-LMP1 to activate the transcription factors NF-kappaB and AP-1 in epithelial cells. In this study, transient expression of CAO-LMP1 was found to activate higher levels of NF-kappaB and AP-1 than the prototype B95.8-LMP1 in human embryonic kidney (HEK) 293 cells and SV40-transformed keratinocytes (SVK). In addition, pulse-chase analysis revealed that CAO-LMP1 has a longer half-life than B95.8-LMP1. Chimera studies localised these phenomena to the transmembrane domains of CAO-LMP1, suggesting that this enhanced signalling capacity may be a consequence of its prolonged half-life. The ability of CAO-LMP1 to activate higher levels of NF-kappaB and AP-1 may contribute to its potent transforming properties.

The frequent coexpression of the EBV-encoded latent membrane proteins LMP1 and LMP2A/B in virus-associated tumors suggests that these two proteins may cooperate in the transformation process. While LMP2A is unable to directly activate the NF-kappaB and AP-1 pathways, we found that coexpression of LMP2A with LMP1 resulted in a significant enhancement of LMP1-mediated activation of these pathways. This enhancement was found to be critically dependent on the tyrosine residues present within the ITAM motif (Y74/Y85) and, to a lesser extent, the tyrosine at position 112 (Y112). Subsequent analysis revealed that LMP2A is able to stabilize and modulate the turnover of LMP1 by extending its half-life. This ability does not require a direct physical interaction between the two proteins but rather, results from an indirect effect of LMP2A on the turnover of the LMP1 protein. This study highlights an important role for LMP2A as a modulator of LMP1 activity in epithelial cells.

SCC12F cells are a line of keratinocytes that retain the capacity for terminal differentiation in vitro. We showed previously that the Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein 1 (LMP1) altered SCC12F morphology in vitro, downregulated cell-cell-adhesion molecule expression and promoted cell motility. In organotypic raft culture, LMP1-expressing cells failed to stratify and formed poorly organized structures which displayed impaired terminal differentiation. To understand better the mechanism(s) by which LMP1 induces these effects, we generated SCC12F cells in which LMP1 expression is inducible. Following induction, these cells exhibited phenotypic changes similar to those observed previously and allowed us to investigate the effects of LMP1 expression on cellular pathways associated with growth, differentiation and morphology. Using microarrays and a number of confirmatory techniques, we identified sets of differentially expressed genes that are characteristically expressed in inflammatory and hyperproliferative epidermis, including chemokines, cytokines and their receptors, growth factors involved in promoting epithelial cell motility and proliferation and signalling molecules that regulate actin filament reorganization and cell movement. Among the genes whose expression was differentially induced significantly by LMP1, the induction of IL-1beta and IL-1alpha was of particular interest, as many of the LMP1-regulated genes identified are established targets of these cytokines. Our findings suggest that alterations in the IL-1 signalling network may be responsible for many of the changes in host-cell gene expression induced in response to LMP1. Identification of these LMP1-regulated genes helps to define the mechanism(s) by which this oncoprotein influences cellular pathways that regulate terminal differentiation, cell motility and inflammation.

British Journal of UrologyVolume 74, Issue 3 p. 302-307 Choosing the correct pain relief for extracorporeal lithotripsy C. DAWSON, C. DAWSON Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorJ.A. VALE, J.A. VALE Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorD.A. CORRY, D.A. CORRY Radiology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorN.P. COHEN, N.P. COHEN Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorJ. GALLAGHER, J. GALLAGHER Anaesthetics, St Bartholomew's Hospital, London, UKSearch for more papers by this authorI.B. NOCKLER, I.B. NOCKLER Radiology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorH.N. WHITFIELD, H.N. WHITFIELD Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this author C. DAWSON, C. DAWSON Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorJ.A. VALE, J.A. VALE Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorD.A. CORRY, D.A. CORRY Radiology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorN.P. COHEN, N.P. COHEN Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorJ. GALLAGHER, J. GALLAGHER Anaesthetics, St Bartholomew's Hospital, London, UKSearch for more papers by this authorI.B. NOCKLER, I.B. NOCKLER Radiology, St Bartholomew's Hospital, London, UKSearch for more papers by this authorH.N. WHITFIELD, H.N. WHITFIELD Departments of Urology, St Bartholomew's Hospital, London, UKSearch for more papers by this author First published: September 1994 https://doi.org/10.1111/j.1464-410X.1994.tb16615.xCitations: 29AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume74, Issue3September 1994Pages 302-307 RelatedInformation

Abstract Background Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. Results Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. Conclusions Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.

Objective The aim of the study was to improve the engagement of professional interpreters for women during labour. Methods The quality improvement initiative was co-designed by a multidisciplinary group at one Melbourne hospital and implemented in the birth suite using the plan-do-study-act framework. The initiative of offering women an interpreter early in labour was modified over cycles of implementation and scaled up based on feedback from midwives and language services data. Results The engagement of interpreters for women identified as requiring one increased from 28% (21/74) at baseline to 62% (45/72) at the 9th month of implementation. Conclusion Improving interpreter use in high-intensity hospital birth suites is possible with supportive leadership, multidisciplinary co-design and within a framework of quality improvement cycles of change. What is known about the topic? Despite Australian healthcare standards and policies stipulating the use of accredited interpreters where needed, studies indicate that services fall well short of meeting these during critical stages of childbirth. What does the paper add? Collaborative approaches to quality improvement in hospitals can significantly improve the engagement of interpreters to facilitate communication between health professionals and women with low English proficiency. What are the implications for practice? This language services initiative has potential for replication in services committed to improving effective communication between health professionals and patients.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) up-regulates the human leukocyte antigen (HLA) class I antigen presentation machinery (APM). This appears counterintuitive with immune evasion in EBV-associated tumours like nasopharyngeal carcinoma (NPC).Latent membrane protein 1-transfected epithelial cell lines were used as a model system to study the impact of LMP1 and c-Myc on HLA class I components. The expression of components of the HLA class I APM, c-Myc and Ki-67 was analysed in LMP1+ and LMP1- NPC by immunohistochemistry.In epithelial cells, LMP1 up-regulated HLA class I APM. This effect could be counteracted by c-Myc, which itself was up-regulated by LMP1 apparently through IL6 induction and Jak3/STAT3 activation. Studies of NPC biopsies revealed down-regulation of HLA class I APM expression. No difference was observed between LMP1+ and LMP1- NPC. However, expression of Ki-67 and c-Myc were up-regulated in LMP1+ tumours.These findings raise the possibility that c-Myc activation in NPC might antagonise the effect of LMP1 on HLA class I expression thus contributing to immune escape of tumour cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is a highly metastatic disease that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the contribution of lysophosphatidic acid (LPA) signalling to the pathogenesis of NPC. Here we demonstrate two distinct functional roles for LPA in NPC. First, we show that LPA enhances the migration of NPC cells and second, that it can inhibit the activity of EBV-specific cytotoxic T cells. Focusing on the first of these phenotypes, we show that one of the LPA receptors, LPA receptor 5 (LPAR5), is down-regulated in primary NPC tissues and that this down-regulation promotes the LPA-induced migration of NPC cell lines. Furthermore, we found that EBV infection or ectopic expression of the EBV-encoded LMP2A was sufficient to down-regulate LPAR5 in NPC cell lines. Our data point to a central role for EBV in mediating the oncogenic effects of LPA in NPC and identify LPA signalling as a potential therapeutic target in this disease.

The molecular events that drive the progression of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) are still to be elucidated. Here, we report for the first time the pathogenic significance of an NPC-associated gene, wingless-type MMTV integration site family, member 5A (WNT5A) and the contribution of EBV to its expression. WNT5A is a representative Wnt protein that activates non-canonical Wnt signalling. With regard to its role in carcinogenesis, there is conflicting evidence as to whether WNT5A has a tumour-promoting or tumour-suppressive role. We show that WNT5A is upregulated in primary NPC tissue samples. We also demonstrate that WNT5A expression was dramatically increased in NPC cell lines expressing the EBV-encoded LMP2A gene, suggesting that this EBV-encoded latent gene is responsible for upregulating WNT5A in NPC. In addition, in vitro WNT5A overexpression promotes the proliferation, migration and invasion of NPC cells. Our results not only reveal pro-tumorigenic effects of WNT5A in NPC but also suggest that WNT5A could be an important therapeutic target in patients with EBV-associated disease.

The γ‐herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ‐herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ‐herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ‐herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ‐herpesviruses alter metabolic phenotypes as a means to support viral infection and long‐term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ‐herpesviruses provide new opportunities for therapeutic intervention.

Abstract Background Helicobacter pylori ( H. pylori ) infection is associated with cognitive deficits in humans, an association potentially mediated or moderated by folate concentration or inflammation. Materials and Methods We used the National Health and Nutrition Examination Survey ( NHANES ) datasets to examine whether folate concentration or inflammation mediates or moderates the relationship between H. pylori and cognitive function. Models were performed using linear, Poisson, and zero‐inflated Poisson regression, and we performed separate analyses for groups aged 20–59 and 60–90 years with sample sizes ranging from 700 to 1700. Results We did not find evidence of mediation in either age group. In the 20‐ to 59‐year group, interactions between H. pylori and ferritin ( p values ranging from .004 to .039) were associated with worse processing speed, better working memory, and worse reaction time. Interactions between H. pylori and fibrinogen ( p values ranging from .023 to .045), C‐reactive protein ( CRP ) ( p = .023), and the inflammatory index ( p = .045) were associated with worse processing speed. In 60‐ to 90‐year‐olds, H. pylori interacted with ferritin and the inflammatory index to predict fewer mathematical errors ( p values of .036 and .023). Interactions with folate ( p values of .016 and .006) and C‐reactive protein ( p values ranging from &lt;.001 to .048) were inconsistent in directionality. Conclusions In this dataset, representative of the US population, inflammation and folate concentrations moderated but did not mediate the association between H. pylori seropositivity and cognition.

Approximately 15-20% of global cancer incidence is causally linked to viral infection, yet the low incidence of cancers in healthy infected individuals suggests that malignant conversion of virus-infected cells occurs after a long period as a result of additional genetic modifications. There are four families of viruses that are now documented to be involved in the development of human cancers which include members of the polyomavirus, hepadnavirus, papillomavirus and herpesvirus families. Although a number of these viruses are implicated in the aetiology of lymphomas or leukaemias, the vast majority are associated with malignancies of epithelial cells. In epithelial tissues, several classes of proteins are involved in maintaining tissue architecture, including those that promote cell-cell adhesion, and others, which mediate cell-matrix interactions. Proteins representative of all classes are frequently altered in malignant tumour cells that possess invasive and metastatic properties. Malignant tumour cells acquire mechanisms to degrade basement membranes and invade the underlying tissue. Many viruses encode proteins which engage signalling pathways that affect one or more of these mechanisms. It is believed that activation of these processes by chronic viral infection can, under certain circumstances, promote tumour cell invasion and metastasis. This review will take a brief look at the current knowledge of viral-induced alterations in cell motility and invasiveness in the context of tumour invasion and metastasis.

Pancreatic ductal adenocarcinoma (PDA) is a devastating malignancy with limited and modest clinical treatments. High-throughput technologies and accurate disease models now provide a comprehensive picture of the diverse molecular signaling pathways and cellular processes governing PDA tumorigenesis. Central among these is oncogenic KRAS, a mediator of cellular plasticity, metabolic reprogramming, and inflammatory and paracrine signaling required for tumor development and maintenance. Biological aggressiveness is further conferred by a highly fibrotic and immunosuppressive PDA microenvironment that also acts as a barrier to effective drug delivery. The regulation of these mechanisms and their implications for early cancer detection, chemoprevention and therapy are discussed.

Latent membrane protein 1 (LMP1), the major oncoprotein encoded by Epstein–Barr virus (EBV), is expressed at widely variable levels in undifferentiated nasopharyngeal carcinoma (NPC) biopsies, fueling intense debate in the field as to the importance of this oncogenic protein in disease pathogenesis. LMP1-positive NPCs are reportedly more aggressive, and in a similar vein, the presence of cancer-associated fibroblasts (CAFs) surrounding “nests” of tumour cells in NPC serve as indicators of poor prognosis. However, there is currently no evidence linking LMP1 expression and the presence of CAFs in NPC. In this study, we demonstrate the ability of LMP1 to recruit fibroblasts in vitro in an ERK-MAPK-dependent mechanism, along with enhanced viability, invasiveness and transformation to a myofibroblast-like phenotype. Taken together, these findings support a putative role for LMP1 in recruiting CAFs to the tumour microenvironment in NPC, ultimately contributing to metastatic disease.

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

Ratings of speech samples of children with cleft palate were obtained from speech clinicians in a Cleft Palate Clinic, speech clinicians in the public schools, parents of children with clefts, parents of children without clefts, children with clefts and children without clefts. Analyses of the obtained ratings suggest that nasality and articulation ratings obtained from adult groups do not differ appreciably. Correlations between ratings of nasality and articulation are interpreted as suggesting that speech clinicians are more likely to differentiate between these two variables than other listener groups.

Epstein–Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein–Barr virus (EBV) episome and by virtue of a glycine–alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.

We describe two new monoclonal antibodies specific for the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) that are suitable for the immunohistochemical analysis of routinely processed paraffin sections. These antibodies were applied to the immunohistochemical detection of LMP2A in Hodgkin's disease (HD). LMP2A-specific membrane staining was seen in the Hodgkin and Reed-Sternberg (HRS) cells of 22 of 42 (52%) EBV-positive HD cases, but not in 39 EBV-negative HD cases. In lymphoid tissues from patients with acute infectious mononucleosis (IM), interfollicular immunoblasts were shown to express LMP2A. This is the first demonstration of LMP2A protein expression at the single-cell level in EBV-associated lymphoproliferations in vivo. The detection of LMP2A protein expression in HD and IM is of importance in view of the proposed role of this protein for maintaining latent EBV infection and its possible contribution for EBV-associated transformation. Because LMP2A provides target epitopes for EBV-specific cytotoxic T cells, the expression of this protein in HRS cells has implications for the immunotherapeutic approaches to the treatment of HD.

Abstract Two human gamma‐herpesviruses, Epstein–Barr virus and Kaposi's sarcoma associated herpesvirus/human herpesvirus 8 display oncogenic potential, causing benign and malignant lymphoproliferative disorders in genetically susceptible or immunosuppressed individuals. As a family of viruses that establish persistent life‐long infections, herpesviruses have evolved strategies to limit innate antiviral responses and evade host immune surveillance. Herpesviruses have developed mechanisms to disrupt antigen presentation, pirate the production of immune regulating cytokines, and inhibit pro‐apoptotic signaling pathways. Although these strategies are designed to facilitate the long‐term persistence of herpesviruses, in certain circumstances they can contribute to viral‐driven carcinogenesis. J. Med. Virol. 84:272–281, 2012. © 2011 Wiley Periodicals, Inc.

Objective To compare the safety and efficacy of 10% sinecatechins (Veregen ® ) ointment against placebo in the treatment of usual type vulvar intraepithelial neoplasia (uVIN). Design A Phase II double‐blind randomised control trial. Setting A tertiary gynaecological oncology referral centre. Population All women diagnosed with primary and recurrent uVIN. Methods Eligible patients were randomised 1:1 to receive either sinecatechins or placebo ointment (applied three times daily for 16 weeks) and were followed up at 2, 4, 8, 16, 32 and 52 weeks. Main outcome measures The primary outcome measure, recorded at 16 and 32 weeks, was histological response (HR). Secondary outcome measures included clinical (CR) response, toxicity, quality of life and pain scores. Results There was no observed difference in HR between the two arms. However, of the 26 patients who were randomised, all 13 patients who received sinecatechins showed either complete (n = 5) or partial (n = 8) CR, when best CR was evaluated. In placebo group, three patients had complete CR, two had partial CR, six had stable disease and two were lost to follow up. Patients in the sinecatechins group showed a statistically significant improvement in best observed CR as compared with the placebo group ( P = 0.002). There was no difference in toxicity reported in either group. Conclusion Although we did not observe a difference in HR between the two treatment arms, we found that 10% sinecatechins application is safe and shows promise in inducing clinical resolution of uVIN lesions and symptom improvement, thus warranting further investigation in a larger multicentre study. Tweetable abstract A randomised control study indicating that sinecatechins ointment may be a novel treatment for uVIN.

See “Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells,” by Gürlevik E, Fleischmann-Mundt B, Brooks J, et al, on page 338. See “Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells,” by Gürlevik E, Fleischmann-Mundt B, Brooks J, et al, on page 338. With an overall 5-year survival rate of only 7%, pancreatic ductal adenocarcinoma (PDA) has now eclipsed breast cancer as the third leading cause of cancer death in the United States1American Cancer SocietyCancer facts & figures 2016. American Cancer Society, Atlanta2016: 1-66Google Scholar and it is predicted to be second by 2030.2Rahib L. Smith B.D. Aizenberg R. et al.Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States.Cancer Res. 2014; 74: 2913-2921Crossref PubMed Scopus (4229) Google Scholar Despite improvements in surgical and clinical management, the majority of patients with localized PDA who undergo successfully R0 surgical resection (ie, resection followed by absence of cancerous cells seen microscopically at the margins) succumb to recurrent local or distant metastatic disease. Clinical trials demonstrate single-agent 5-fluorouracil or gemcitabine adjuvant chemotherapy provides overall and disease-free survival benefit, and further adjuvant trials are addressing the efficacy of multi-agent chemotherapy regimens shown to be beneficial in advanced, inoperable PDA.3Paulson A.S. Tran Cao H.S. Tempero M.A. et al.Therapeutic advances in pancreatic cancer.Gastroenterology. 2013; 144: 1316-1326Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 4Garrido-Laguna I. Hidalgo M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies.Nat Rev Clin Oncol. 2015; 12: 319-334Crossref PubMed Scopus (438) Google Scholar The number and types of adjuvant trials has lagged far behind those for advanced stage PDA, in part owing to the limited number of patients who undergo surgery. Assessment of adjuvant systemic therapies are complicated by further variables introduced by the surgical procedure, inconsistencies in pathologic evaluation (ie, margin status), and extended length of study.4Garrido-Laguna I. Hidalgo M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies.Nat Rev Clin Oncol. 2015; 12: 319-334Crossref PubMed Scopus (438) Google Scholar Although it may be appropriate in some instances to extrapolate clinical trial findings for advanced PDA to the adjuvant setting, differences in tumor burden along with the physiologic differences after surgical resection of primary tumor are biological confounders and important caveats to such cross-comparisons. One approach for accelerating the development and validation of most promising forms of adjuvant therapy would be initial assessment in preclinical models. Genetically engineered mouse models of PDA have been key factors in advancing our understanding of PDA biology, providing unique insights into the role of the tumor microenvironment and inflammatory response, while also serving as important platforms for assessing therapeutic agents.5Perez-Mancera P.A. Guerra C. Barbacid M. et al.What we have learned about pancreatic cancer from mouse models.Gastroenterology. 2012; 142: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar However, most PDA genetically engineered mouse models best recapitulate locally advanced or metastatic disease. They are also further complicated by variable time to onset, tumor multifocality, and other histologic abnormalities such as acinar to ductal metaplasia and dysplasia that diffusely involves the pancreas. PDA mouse models amenable to surgical resection and adjuvant treatment while also replicating the tumor microenvironment and intact immune response in which PDA naturally arise have been lacking. Gürlevik et al6Gürlevik E. Fleischmann-Mundt B. Brooks J. et al.Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells.Gastroenterology. 2016; 151: 338-350PubMed Google Scholar now present a rapid and flexible system for reproducibly modeling a localized form PDA amenable to surgical resection and suitable for assessing the efficacy adjuvant systemic therapies in relation to tumor recurrence and overall survival. In this model, a plasmid carrying a Cre recombinase was electroporated using tweezers-type electrode in the pancreas of LSL-Kras-G12Dxp53fl/fl mice. These animals developed single tumor nodules at the site of electroporation. Histologically, the tumors showed moderately differentiated PDA at day 21 postelectroporation without any observable metastatic spread. To closely resemble the human PDA, the authors introduced to the model a transposon encoding constitutively active form of Akt2, a kinase known to promotes invasive growth and metastasis.7Ruggeri B.A. Huang L. Wood M. et al.Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas.Mol Carcinog. 1998; 21: 81-86Crossref PubMed Scopus (287) Google Scholar, 8Hezel A.F. Kimmelman A.C. Stanger B.Z. et al.Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (875) Google Scholar Tumors carrying activated Kras-G12D, Akt2 and p53 deletion showed accelerated primary tumor growth, local invasion and distant metastasis and significantly reducing survival, while preserving the overall histologic features of PDA, including its stromal microenvironment. In their model, mice receiving adjuvant gemcitabine after R0 surgical resection had an improvement in overall survival attributable to a reduction in local, but not metastatic, tumor recurrence—a finding that parallels that observed for patients who received adjuvant gemcitabine in the large phase III CONKO-001 clinical trial that established the efficacy of gemcitabine in the adjuvant setting.9Oettle H. Neuhaus P. Hochhaus A. et al.Adjuvant chemotherapy with gemcitabine and long-term outcomes among patients with resected pancreatic cancer: the CONKO-001 randomized trial.JAMA. 2013; 310: 1473-1481Crossref PubMed Scopus (1184) Google Scholar Beyond offering important proof-of-principle for the model itself, this provocative observation raises important questions about the therapeutic mechanism of action for gemcitabine, given its effects were limited to the control of residual/recurrent local disease without apparent influence on metastatic disease. Accumulating data in PDA and other cancer types indicate cytotoxic chemotherapy facilitates protumorigenic and antitumorigenic effects linked to the stromal microenvironment and immune response. The immunomodulatory effects of chemotherapy range from suppressive to stimulatory depending upon tumor type, specific cytotoxic agent, and its dosing regimen.10Zheng Y. Dou Y. Duan L. et al.Using chemo-drugs or irradiation to break immune tolerance and facilitate immunotherapy in solid cancer.Cell Immunol. 2015; 294: 54-59Crossref PubMed Scopus (58) Google Scholar, 11Kersten K. Salvagno C. de Visser K.E. Exploiting the Immunomodulatory Properties of Chemotherapeutic Drugs to Improve the Success of Cancer Immunotherapy.Front Immunol. 2015; 6: 516Crossref PubMed Scopus (58) Google Scholar For instance, gemcitabine given at concentrations similar to or below those used for patients can attenuate immune suppression through reductions in either total numbers or phenotypic subsets of myeloid-derived suppressor cells (MDSCs).10Zheng Y. Dou Y. Duan L. et al.Using chemo-drugs or irradiation to break immune tolerance and facilitate immunotherapy in solid cancer.Cell Immunol. 2015; 294: 54-59Crossref PubMed Scopus (58) Google Scholar Low-dose gemcitabine reduces regulatory T-cell (Treg) numbers to modestly improve survival in a murine orthotopic PDA model.12Shevchenko I. Karakhanova S. Soltek S. et al.Low-dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer.Int J Cancer. 2013; 133: 98-107Crossref PubMed Scopus (127) Google Scholar Low-dose (metronomic) cytotoxic chemotherapy can selectively ablate Tregs relative to other T-cell populations,11Kersten K. Salvagno C. de Visser K.E. Exploiting the Immunomodulatory Properties of Chemotherapeutic Drugs to Improve the Success of Cancer Immunotherapy.Front Immunol. 2015; 6: 516Crossref PubMed Scopus (58) Google Scholar offering rationale for the use of low-dose cyclophosphamide in combination with GVAX and CRS-207 vaccines in recent promising clinical trial for metastatic PDA.13Le D.T. Wang-Gillam A. Picozzi V. et al.Safety and survival with GVAX pancreas prime and Listeria Monocytogenes-expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer.J Clin Oncol. 2015; 33: 1325-1333Crossref PubMed Scopus (414) Google Scholar Furthermore, higher dose gemcitabine selectively eliminates splenic MDSCs and increase the anti-tumor activity of natural killer (NK) and CD8+ T cells in mice bearing large tumors,14Suzuki E. Kapoor V. Jassar A.S. et al.Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity.Clin Cancer Res. 2005; 11: 6713-6721Crossref PubMed Scopus (852) Google Scholar whereas data from PDA patients receiving gemcitabine points to reduction in the number of Tregs15Homma Y. Taniguchi K. Nakazawa M. et al.Changes in the immune cell population and cell proliferation in peripheral blood after gemcitabine-based chemotherapy for pancreatic cancer.Clin Transl Oncol. 2014; 16: 330-335Crossref PubMed Scopus (63) Google Scholar and increased CD14+ monocytes and myeloid dendritic cells in peripheral blood (PB).16Soeda A. Morita-Hoshi Y. Makiyama H. et al.Regular dose of gemcitabine induces an increase in CD14+ monocytes and CD11c+ dendritic cells in patients with advanced pancreatic cancer.Jpn J Clin Oncol. 2009; 39: 797-806Crossref PubMed Scopus (50) Google Scholar Gürlevik et al6Gürlevik E. Fleischmann-Mundt B. Brooks J. et al.Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells.Gastroenterology. 2016; 151: 338-350PubMed Google Scholar extend these observations to the adjuvant treatment setting to identify a novel immunomodulatory activity for gemcitabine localized to the remnant pancreas after R0 surgical resection. Adjuvant gemcitabine selectively reduced the Cd11b+Gr1lowF4/80+ subset of MDSCs and increased the number of NK cells without altering Tregs or macrophage polarization (Figure 1). These changes were specific to remnant pancreas and not observed in PB. Depletion of NK cells, but not CD8+ T cells, reversed survival advantage and inhibition of local tumor recurrence seen with gemcitabine, mechanistically linking its therapeutic action to an enhanced NK cell response after tumor resection.6Gürlevik E. Fleischmann-Mundt B. Brooks J. et al.Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells.Gastroenterology. 2016; 151: 338-350PubMed Google Scholar NK cells bridge innate and adaptive immunity and mediate antitumor activity through constitutive cytotoxic activity and secretion of immunomodulatory cytokines. NK cell number and functional activities are subject to modulation by cellular and soluble factors and their engagement of activating or inhibiting receptors on NK cells.17Krasnova Y. Putz E.M. Smyth M.J. et al.Bench to bedside: NK cells and control of metastasis.Clin Immunol. 2015; PubMed Google Scholar Although increased NK cells observed in their adjuvant model would seem to be tied to alterations in MDSCs and their immunosuppressive activity, it is possible that gemcitabine may have also acted directly on residual tumor cells to modulate their susceptibility to NK immune surveillance. For example, gemcitabine can up-regulate the expression of NK group 2 member D (NKG2D) ligand on lung cancer cells through a DNA stress/ATM-dependent mechanism, leading to augmentation in NK tumor cell recognition and cytolysis.18Okita R. Wolf D. Yasuda K. et al.Contrasting effects of the cytotoxic anticancer drug gemcitabine and the EGFR tyrosine kinase inhibitor gefitinib on NK cell-mediated cytotoxicity via regulation of NKG2D ligand in non-small-cell lung cancer cells.PLoS One. 2015; 10: e0139809Crossref Scopus (26) Google Scholar Gemcitabine prosurvival effects on local disease recurrence in this adjuvant model bring focus on the implication of primary tumor resection not only in terms of eradicating immunomodulatory activity mediated by the primary tumor, but also in relation to local and systemic inflammatory responses induced by the surgical procedure itself. A clinical study of NK cell function in PB after pancreaticoduodenectomy for PDA finds a transient reduction in NK cytotoxicity within the first week after surgery followed by a subsequent normalization or augmentation of NK activity at 30 days, the magnitude of which was correlated with improved survival.19Iannone F. Porzia A. Peruzzi G. et al.Effect of surgery on pancreatic tumor-dependent lymphocyte asset: modulation of natural killer cell frequency and cytotoxic function.Pancreas. 2015; 44: 386-393PubMed Google Scholar With patient tissue evaluation not possible in the postoperative setting, the adjuvant mouse model presented here by Gürlevik et al highlights the further likelihood of a clinically important, tissue-localized NK responses that may differ from that seen in PB and which could be augmented to further therapeutic advantage through the use of cytotoxic agents or other immune modulators. As mentioned, both the timing and dose of gemcitabine or other cytotoxic agent may be further critical variables in unmasking an innate and/or adaptive immune response able to inhibit local disease recurrence or subsequent metastasis. Of potential importance, the initiation of gemcitabine immediately after surgery in this adjuvant model contrasts the clinical scenario in which patients only start adjuvant chemotherapy after a period of recovery from surgery. It is unclear whether a unique window of opportunity exists in the period immediately before, during, or after surgery to most effectively intervene with a therapy that augments local immune surveillance by NK cells. Beyond this issue, there are known differences in functional NK cell subpopulations present in PB or secondary lymphoid tissues, as well as differences in NK cell functional activity observed between PB, the primary tumor, the premetastatic niche, and established metastatic tumor sites.17Krasnova Y. Putz E.M. Smyth M.J. et al.Bench to bedside: NK cells and control of metastasis.Clin Immunol. 2015; PubMed Google Scholar These differences are likely to have important implications for NK cell-targeted agents in patients with either early or later stage PDA and could be explored further in this adjuvant model by instead introducing genetic alterations that mediate slower disease kinetics to allow their evaluation. Indeed, the flexibility and apparent ease by which different combinations of molecular alterations can be introduced readily into this electroporation-based model are one of its greatest strengths, offering the opportunity to mechanistically assess the importance that individual or combination alterations play in regulating various aspects of PDA biology, including modulation of an immunostimulatory or immunohibitory tumor microenvironment. Such mechanistic insights could eventually be leveraged to identify prognostic or predictive biomarkers useful in individually tailoring PDA immunotherapy regimens based on a combination of tumor profiling and immune cell analysis of tumor and/or blood samples. Finally, although not addressed in their paper,6Gürlevik E. Fleischmann-Mundt B. Brooks J. et al.Administration of gemcitabine after pancreatic tumor resection in mice induces an antitumor immune response mediated by natural killer cells.Gastroenterology. 2016; 151: 338-350PubMed Google Scholar this model also offers a suitable platform for exploring and validating the use of neoadjuvant therapeutic approaches before surgery, an area of intense interest and debate in the field. Administration of Gemcitabine After Pancreatic Tumor Resection in Mice Induces an Antitumor Immune Response Mediated by Natural Killer CellsGastroenterologyVol. 151Issue 2PreviewEven after potentially curative R0 resection, patients with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis owing to high rates of local recurrence and metastasis to distant organs. However, we have no suitable transgenic animal models for surgical interventions. Full-Text PDF Covering the CoverGastroenterologyVol. 151Issue 2PreviewIn this retrospective cohort study, continuation of low-dose aspirin use was associated with a nearly 3-fold increased risk of recurrent lower GI bleeding, but reduced risk of serious cardiovascular events and death. Full-Text PDF

British Journal of UrologyVolume 74, Issue 4 p. 397-404 The long-term results of treatment of urinary stones C. DAWSON, C. DAWSON St Bartholomew's Hospital, London, UK C. Dawson, FRCS, formerly Urology Research Fellow now Urology Registrar, Battle Hospital, Oxford Road, Reading RG3 1AG. UK.Search for more papers by this authorH.N. WHITFIELD, H.N. WHITFIELD St Bartholomew's Hospital, London, UK H.N. Whitfield, MA, MChir, FRCS, Consultant Urologist, now at Central Middlesex Hospital, Park Royal, London, UK.Search for more papers by this author C. DAWSON, C. DAWSON St Bartholomew's Hospital, London, UK C. Dawson, FRCS, formerly Urology Research Fellow now Urology Registrar, Battle Hospital, Oxford Road, Reading RG3 1AG. UK.Search for more papers by this authorH.N. WHITFIELD, H.N. WHITFIELD St Bartholomew's Hospital, London, UK H.N. Whitfield, MA, MChir, FRCS, Consultant Urologist, now at Central Middlesex Hospital, Park Royal, London, UK.Search for more papers by this author First published: October 1994 https://doi.org/10.1111/j.1464-410X.1994.tb00412.xCitations: 11 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat References 1 Delius M., Brendel W. Historical roots of lithotripsy. J. Lith Stone Dis 1990; 2: 161–3. 2 Hausler E. Kiefer W. Beruhrungsfreie Zerstorung fester Einschlusse in flussiger Umgebung. Verh Deutsch Physikal Gesellschaft 1973: 6: 692. 3 Rassweiler J., Aiken P. ESWL 1990 — state of the art. Urol Res 1990; 18 (suppll): S13–S23. 4 Guzina T., Babic M., Alagic MD et al Extracorporeal shock wave lithotripsy with Dornier MPL-9000 in 2005 patients. J. Endourol 1992; 6: 393–402. 5 Grabe M., Kinn A-C, Ahlgren G. et al Treatment of renal and ureteric stones with lithocut C-3000 lithotripter. J. Endourol 1992; 6: 403–6. 6 Netto NR, Gustavo CL, Claro JFA. Extracorporeal shock wave lithotripsy with Lithostar lithotriptor. Endourology 1992; 40: 430–4. 7 Dickinson AJ, Cranston D., Doble A. et al The mobile lithotriptor: an answer for the smaller centre. Br J Urol. 1993; 71: 396–400. 8 Mobley TB, Myers DA, Grine WB et al Low energy lithotripsy with the Lithostar: treatment results with 19 962 renal and ureteral calculi. J. Urol. 1993: 149: 1419–24. 9 Tolley DA, Wallace DMA Tiptaft RC. First UK Consensus Conference on Lithotriptor Terminology -1989. Br J Urol. 1991: 67: 9–12. 10 Dawson C., Whitfield HN. Morphological changes after lithotripsy. Eur Urol Update Series 1993; 2: 130–5. 11 de Almeida Claro J., Lima ML. Ferreira U. et al. Blood pressure changes after extracorporeal shock wave lithotripsy in normotensive patients. J. Urol. 1993; 150: 1765–7. 12 Kohrmann KU, Rassweiler J., Aiken P. The recurrence rate of stones following ESWL. World J Urol. 1993: 11: 26–30. 13 Wulfsohn MA. Pyelocaliceal diverticula. J. Urol. 1980; 123: 1–8. 14 Psihramis KE. Dretler SP. Extracorporeal shock wave lithotripsy of caliceal diverticula calculi. J. Urol. 1987; 138: 707–11. 15 Holmes SAV, Whitfield HN. Management of complex renal calculi. World J Urol. 1993; 11: 31–6. 16 Streem SB, Yost A. Treatment of caliceal diverticular calculi with extracorporeal shock wave lithotripsy: patient selection and extended followup. J. Urol. 1992: 148: 1043–6. 17 Fitzpatrick JM. The management of complex urinary calculi. Eur Urol Update Series 1993; 2: 50–5. 18 Esuvaranathan K., Tan EC, Tung KH et al Stones in horseshoe kidneys: results of treatment by extracorporeal shock wave lithotripsy and endourology. J. Urol. 1991; 146: 1213–15. 19 Locke DR. Newman RC, Steinbock GS et al Extracorporeal shock wave lithotripsy in horseshoe kidneys. Urology 1990; 35: 407–11. 20 Smith JE, Van Arsdalen KN, Hanno PM et al Extracorporeal Shock wave lithotripsy treatment of calculi in horseshoe kidneys. J. Urol. 1989; 142: 683–6. 21 Jones DJ, Wickham JEA, Kellett MJ. Percutaneous nephrolithotomy for calculi in horseshoe kidneys. J. Urol. 1991; 145: 481–3. 22 Vandeursen H., Baert L. Prophylactic role of extracorporeal shock wave lithotripsy in the management of nephro-calcinosis. Br J Urol. 1993; 71: 392–5. 23 Holmes SAV, Eardley I., Corry DA et al The use of extracorporeal shock wave lithotripsy for medullary sponge kidneys. Br J Urol. 1992; 70: 352–4. 24 Miller K., Bachor R., Sauter T. et al ESWL monotherapy for large stones and staghorn calculi. Urologia Internationalis 1990; 45: 95–8. 25 Vandeursen H., Baert L. Extracorporeal shock wave lithotripsy monotherapy for staghorn stones with the second generation lithotriptors. J. Urol. 1990; 143: 252–6. 26 Wirth MP, Theiss M., Frohmuller HG. Primary extracorporeal shock wave lithotripsy of staghorn renal calculi. Urol Int 1992: 48: 71–5. 27 Lam HS, Lingeman JE, Barron M. et al Staghorn calculi: analysis of treatment results between initial percutaneous nephrostolithotomy and extracorporeal shock wave lithotripsy monotherapy with reference to surface area. J Urol. 1992; 147: 1219–25. 28 Preminger GM. Management of ureteral calculi: the debate continues.. J Urol. 1992; 148: 1102–4. 29 Drach GW. Urinary lithiasis: etiology, diagnosis, and medical management. In PC Walsh, AB Retik, TA Stamey ED Vaughan eds. Campbell's Urology. 6th edn. Philadelphia: W.B. Saunders 1992: 2085–156. 30 Holm-Nielsen A., Jorgensen T., Mogensen P. et al The prognostic value of probe renography in ureteric stone obstruction. Br J Urol. 1981; 53: 504–7. 31 Eisenberger F. Schmidt AS. Extracorporeal shock wave lithotripsy. Curr Opin Urol. 1993; 3: 309–12. 32 Mueller SC, Wilbert D., Thueroff JW et al Extracorporeal shock wave lithotripsy of ureteral stones: clinical experience and experimental findings. J Urol. 1986; 135: 831–4. 33 Hofbauer J., Tuerk C., Hobarth K. et al ESWL in situ or ureteroscopy for ureteric stones. World J Urol. 1993; 11: 54–8. 34 Cass AS. Do upper ureteral stones need to be manipulated (push back) into the kidneys before extracorporeal shock wave lithotripsy? J. Urol. 1992; 147: 349–51. 35 Kirkali Z., Mungan U., Sade M. Extracorporeal electromagnetic shock wave lithotripsy of ureteric stones in situ. J Endourol 1992; 6: 411–16. 36 Mitre AI, Chambo JL, Nahas WC et al Ureteral calculi: extracorporeal shock wave lithotripsy performed in situ on an outpatient basis. World J Urol. 1992; 10: 213–15. 37 Rassweiler J., Henkel TO, Joyce AD et al Extracorporeal shock wave lithotripsy of ureteric stones with the Modulith SL20. Br J Urol. 1992; 70: 594–9. 38 Erturk E., Herrman E., Cockett ATK. Extracorporeal shock wave lithotripsy for distal ureteral stones. J. Urol. 1993; 149: 1425–6. 39 Anderson K. Keetch D., Albala D. et al Optimal therapy for the distal ureteric stone: lithotripsy vs ureteroscopy. J. Urol. 1993; 149: 220A. 40 Chaussy CG, Fuchs GJ. Ureteroscopy versus extracorporeal shock wave lithotripsy for the treatment of distal ureteric stones. J. Urol. 1993; 149: 220A. 41 Dretler SP. Stone fragility: a new therapeutic distinction. In JE Lingeman DE Newman eds, Shock Wave Lithotripsy: State of the Art. New York: Plenum 1988: 141–5. 42 Dretler SP. The clinical significance of variations in urinary stone fragility. J. Lith Stone Dis 1989; 1: 192–203. 43 Cohen NP. Parkhouse H., Scott ML et al Prediction of response to lithotripsy — the use of scanning electron microscopy and x-ray energy dispersive spectroscopy. Br J Urol. 1992; 70: 469–73. 44 Moon KL, Hricak H., Margolis AR et al Nuclear magnetic-resonance imaging characteristics of gallstones in vitro. Radiology 1983; 148: 753–6. 45 Baron RL. Shuman WP, Lee SP et al MR appearance of gallstones in vitro at 1.5 T: correlation with chemical composition. AJR 1989: 153: 497–502. 46 Stoller MI, Floth A., Hricak H. et al Magnetic Resonance imaging of Renal Calculi: an in vitro study. J. Lith Stone Dis 1991; 3: 162–4. 47 Speller RD, Horrocks JA. Photon scattering — a ‘new’ source of information in medicine and biology? Phys Med Biol 1991; 36: 1–6. Citing Literature Volume74, Issue4October 1994Pages 397-404 ReferencesRelatedInformation

The expression of p53 in a large panel of adenovirus (Ad) 2/5- and 12-transformed human, rat, and mouse cells has been examined. In all cases, in the absence of the larger Ad E1B protein, the level of p53 is very low. In human and rat cells when the Ad 12 E1B 54K polypeptide is expressed, p53 is much more abundant, although this is not the case in Ad 12 E1-transformed mouse cells. We conclude that expression of p53 is determined by virus serotype, host cell type, and viral proteins expressed. p53 in Ad 12 E1-transformed human cells is wild type but has an extended half-life. Stabilization is not through protein-protein interaction with the Ad E1B protein. The level of expression of c-Myc is also elevated in Ad-transformed human cells but this does not correlate with the presence of the E1B protein or with p53. However, Northern blot analysis indicates a direct correlation between mRNA and protein levels. We conclude that c-Myc is regulated at the transcriptional level, whereas p53 is regulated at the post-translational level in adenovirus transformants.

Evidence suggests that lichen sclerosus (LS) is the primary aetiological factor for local vulval recurrence (LVR) in vulval squamous cell carcinoma (VSCC). The long-term application of topical corticosteroids is believed to prevent LVR. Patients treated for LS-associated VSCC at a gynaecological cancer centre were invited to complete a questionnaire to evaluate whether they are receiving corticosteroids. 55 of the 95 eligible patients (58%) completed the questionnaire; LS was treated in 69%, with steroids given to 84.2%. Most received steroids >3 months, but discontinued treatment once asymptomatic. An online survey was distributed to 313 British Gynaecological Cancer Society members to determine whether gynaecological oncologists prescribe corticosteroids for LS following VSCC surgery. 41 consultants (13.1%) completed the survey; 70.7% prescribe topical corticosteroids (potent/very potent in 79.3%), and 58.6% treat >1 year. Our findings demonstrate that patients are more likely to be given topical corticosteroids if symptomatic of LS. Furthermore, although treatment regimens vary, the majority of respondents advocate the use of very potent steroids and would support a tertiary chemopreventative trial. Impact statement What is already known on this subject: Local vulval recurrence (LVR) affects approximately one in four women who have received surgery for vulval squamous cell carcinoma (VSCC). What the results of this study add: Lichen sclerosus (LS), an inflammatory dermatosis, is recognised as the likely primary aetiological factor for LVR. Although there is evidence to suggest that long-term topical corticosteroid use in patients with residual LS may prevent LVR, the extent to which women were given topical steroids following surgery remains unclear. Our patient questionnaire evaluates if these patients are already receiving topical steroids, along with the strength of such steroids and duration of treatment. The consultant survey determines whether clinicians currently prescribe topical steroids following VSCC surgery, as well as the strength and duration of steroid therapy. What the implications are of these findings for clinical practice and/or further research: We aim to establish whether the gynaecological oncology community believe that long-term steroids may prevent LVR in women with LS-associated VSCC and whether they would support and recruit to a multicentre tertiary chemopreventative trial. These findings could influence a future clinical trial and may alter the ongoing management of these women.

The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) protein is expressed in all virus-associated malignancies, where it performs an essential role in the maintenance, replication and transcription of the EBV genome. In recent years, it has become apparent that EBNA1 can also influence cellular gene transcription. Here, we demonstrate that EBNA1 is able to stimulate the expression of the Transforming growth factor-beta (TGFβ) superfamily member, bone morphogenic protein 2 (BMP2), with consequential activation of the BMP signalling pathway in carcinoma cell lines. We show that BMP pathway activation is associated with an increase in the migratory capacity of carcinoma cells, an effect that can be ablated by the BMP antagonist, Noggin. Gene expression profiling of authentic EBV-positive nasopharyngeal carcinoma (NPC) tumours revealed the consistent presence of BMP ligands, established BMP pathway effectors and putative target genes, constituting a prominent BMP "signature" in this virus-associated cancer. Our findings show that EBNA1 is the major viral-encoded protein responsible for activating the BMP signalling pathway in carcinoma cells and supports a role for this pathway in promoting cell migration and possibly, metastatic spread.

British Journal of UrologyVolume 73, Issue 2 p. 129-135 Does lithotripsy cause hearing loss? C. DAWSON, Corresponding Author C. DAWSON *Departments of Urology, St Bartholomew's Hospital, London, UK FRCS, Urology Research Fellow.St Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK.Search for more papers by this authorA. CHILCOTT-JONES, A. CHILCOTT-JONES †Departments of Audiology, St Bartholomew's Hospital, London, UK Chief Audiologist.Search for more papers by this authorD. A. CORRY, D. A. CORRY *Departments of Urology, St Bartholomew's Hospital, London, UK DCR(R) DMU, Superintendent III Radiographer.Search for more papers by this authorN. P. COHEN, N. P. COHEN *Departments of Urology, St Bartholomew's Hospital, London, UK FRCS, Urology Registrar, now at Aberdeen Royal Infirmary, Aberdeen, UK.Search for more papers by this authorH. O. L. WILLIAMS, H. O. L. WILLIAMS †Departments of Audiology, St Bartholomew's Hospital, London, UK FRCS, Senior Registrar.Search for more papers by this authorI. B. NOCKLER, I. B. NOCKLER ‡Departments of Radiology, St Bartholomew's Hospital, London, UK FRCR, Consultant Radiologist.Search for more papers by this authorH. N. WHITFIELD, H. N. WHITFIELD *Departments of Urology, St Bartholomew's Hospital, London, UK MA, MChir FRCS, Consultant Urologist, now at Central Middlesex Hospital, Park Royal, London, UK.Search for more papers by this author C. DAWSON, Corresponding Author C. DAWSON *Departments of Urology, St Bartholomew's Hospital, London, UK FRCS, Urology Research Fellow.St Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK.Search for more papers by this authorA. CHILCOTT-JONES, A. CHILCOTT-JONES †Departments of Audiology, St Bartholomew's Hospital, London, UK Chief Audiologist.Search for more papers by this authorD. A. CORRY, D. A. CORRY *Departments of Urology, St Bartholomew's Hospital, London, UK DCR(R) DMU, Superintendent III Radiographer.Search for more papers by this authorN. P. COHEN, N. P. COHEN *Departments of Urology, St Bartholomew's Hospital, London, UK FRCS, Urology Registrar, now at Aberdeen Royal Infirmary, Aberdeen, UK.Search for more papers by this authorH. O. L. WILLIAMS, H. O. L. WILLIAMS †Departments of Audiology, St Bartholomew's Hospital, London, UK FRCS, Senior Registrar.Search for more papers by this authorI. B. NOCKLER, I. B. NOCKLER ‡Departments of Radiology, St Bartholomew's Hospital, London, UK FRCR, Consultant Radiologist.Search for more papers by this authorH. N. WHITFIELD, H. N. WHITFIELD *Departments of Urology, St Bartholomew's Hospital, London, UK MA, MChir FRCS, Consultant Urologist, now at Central Middlesex Hospital, Park Royal, London, UK.Search for more papers by this author First published: February 1994 https://doi.org/10.1111/j.1464-410X.1994.tb07479.xCitations: 9 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume73, Issue2February 1994Pages 129-135 RelatedInformation

Objective Dysregulation of the Hedgehog (Hh) pathway has been described in a variety of cancers, including cervical cancer, a disease which shares a common aetiology with vulval squamous cell carcinoma (VSCC). Here, we investigate a large number of primary VSCC cases for evidence of Hedgehog pathway activation and examine the implications of pathway activity on clinical outcomes in a cohort of patients with primary VSCC. Methods Archival histology blocks containing VSCC and histologically normal adjacent epithelium were retrieved from a cohort of 91 patients who underwent treatment for primary VSCC. Immunohistochemistry staining was undertaken to assess for the expression of key Hh pathway components (SHH, PTCH1, GLI1). A competing risks statistical model was used to evaluate the implications of the levels of key Hh pathway components on clinical outcomes. Results We show that 92% of primary VSCC cases over-expressed one or more components of the Hh signalling pathway when compared to the adjacent normal epithelium. While expression of SHH and GLI1 did not correlate with any clinicopathological criteria, over- or under-expression of PTCH1 was associated with a reduced or increased risk of developing a local disease recurrence, respectively. In VSCC arising on a background of Lichen Sclerosus, the risk of local recurrence was potentiated in cases where PTCH1 was under-expressed. Conclusions Our findings reveal, for the first time, that the Hh pathway is activated in VSCC and that PTCH1 expression can be used as a biomarker to stratify patients and inform clinicians of the risk of their local recurrence, particularly in cases of VSCC associated with LS.

The symptoms and signs of prostate cancer are initially closely similar to those of benign prostatic hyperplasia—obstruction of the urethra by the malignant gland may lead to hesitancy, poor urinary flow, intermittent flow, post-micturition dribbling, and incomplete emptying. Secondary bladder instability may ensue and give rise to urinary frequency, nocturia, and urgency. #### Haemospermia The patient may also present with haematuria, typically reporting that the blood appears at the beginning of an otherwise clear urinary stream. Although this suggests a cause localised to the lower urinary tract, all such patients should be investigated and managed along the lines described for patients with haematuria (see next article in the series). #### Onset Investigations begin in the same way as for patients with benign prostatic hyperplasia—a full history of the main symptoms, followed by a thorough clinical examination and digital rectal examination. There are few clinical signs in early prostate cancer, but digital rectal examination will often detect an early lesion. In prostate cancer either the normal smooth surface of the prostate may be replaced by a hard nodule or the gland itself may be enlarged, hard, and “craggy” to the touch. The normal midline sulcus may be lost. The serum prostate specific antigen level should be established. Normal ranges depend on the assay used. Normal ranges for prostate specific antigen

To determine the number of patients with breast problems referred to general surgical clinics in a district general hospital and to assess the effect of changes implemented following the previous study on waiting time, investigations performed, and management of the patients.Two prospective outpatient audits with patient details recorded on questionnaires by the medical staff.The general surgical outpatient clinics of a single general surgical firm at Newbury District Hospital, Berkshire.Those patients attending the above clinics during two 3-month periods, 1 October to 31 December 1989 (Study 1), and 16 April to 19 July 1990 (Study 2).Of new referrals, 25% were for a breast problem. The waiting time fell from a median of 22 days in Study 1 to 10 days in Study 2. There was no significant difference between the studies for the proportion of each type of investigation performed. Between 80% and 85% of new patients did not need admission for an operation; however, of those operations performed, 65% were for carcinoma. The number of patients diagnosed as having carcinoma was the same in the two studies.That 25% of new, and up to 40% of follow-up patients seen in a general surgical clinic have breast problems. Many patients do not regard their symptoms as worrying and will not attend early clinic appointments even if these are offered. Writing to patients and general practitioners with the results of investigations ensures quicker receipt of the diagnosis and treatment plan, and reduces follow-up attendance. Only 15-20% of new patients need admission for an operation, and carcinoma is found in only 13-17%. Open access to the clinics does not result in general practitioners referring patients unnecessarily with breast problems.

As many as 15% of couples fail to conceive a child after a year of unprotected intercourse. It is important to investigate both partners fully to evaluate properly where the problem lies. This article considers only the male factors in subfertility. Contribution to subfertility View this table: Men in whom one or both testicles were undescended at birth have lower semen quality than normal, regardless of whether an early orchiopexy was performed. Spermatogenesis can also be impaired if a patient has had testicular pain in childhood or adolescence (signifying an episode of torsion); has had mumps orchitis after puberty; or has used certain prescribed drugs or has misused drugs. As spermatozoa take up to three months to mature fully, seminal analysis may also yield abnormal results for a while if a patient has had a fever. #### Important factors Several surgical operations may also impair fertility. Retrograde ejaculation may occur in about 40% of men after a bladder neck incision and is even more common after transurethral prostatectomy. The vas deferens and the testicular blood supply may both be damaged during repair of inguinal hernia. Dissection of retroperitoneal lymph nodes may affect the emission and ejaculation of semen by interrupting the sympathetic nervous system. #### Drugs that may inhibit spermatogenesis A general physical inspection will confirm that a patient has normal secondary sexual characteristics. Abnormalities such as hepatomegaly or gynaecomastia may suggest hypogonadism or hormonal abnormalities. The testes should be confirmed to lie vertically in the scrotum and be assessed for size and consistency. The vas deferens, reported to be absent in 2% of infertile men, should be carefully palpated. Finally, the patient should stand while the scrotum is examined for the presence of a varicocele. #### Frequency of sexual intercourse

Objective In view of changing landscape of surgical treatment for LUTS secondary to BPE, this audit was undertaken to assess key aspects of the processes and outcomes of the current interventional treatments for BPE, across different units in the UK. Materials and method A multi-institutional snapshot audit was conducted for patients undergoing interventions for LUTS/BPE over 8-week period. Using Delphi process two-part proforma was designed to capture data. Results 529 patients were included across 20 NHS trusts in England and Wales. Median age was 73 years. Indications for surgery were acute retention (47%) and LUTS (45%). 80% of patients had prior medical therapy. TURP formed the commonest procedure. 27% patients had &lt;23 hour hospital stay. Immediate (21%) and delayed (18%) complications were Clavien-Dindo &lt;2 category. High proportion of patients reported residual symptoms. Type and indication of surgery were significant predictor of complications, length of stay and failure of TWOC outcomes, on multivariate analyses. There were variations in departmental processes, 50% centres used PROMs. Conclusion Monopolar TURP still remains the commonest intervention for BPE. Most departments are adopting newer technologies. The audit identified opportunities for development of consistent, effective and patient centric practices as well as need for large-scale focused studies.

Bladder outflow obstruction due to benign prostatic hyperplasia is a common problem in elderly men, and by the age of 80 years most men will have developed symptoms attributable to this disorder. The symptoms may be either obstructive or irritative in type. Less common causes of bladder outflow obstruction include bladder neck obstruction and urethral stricture. #### Obstructive symptoms The aetiology of the irritative symptoms is poorly understood. Obstruction of the urethra by an enlarged prostate leads to a poor urinary flow. The bladder may take longer to generate a high enough pressure to start the flow of urine, and this pressure may not be sustained over the (prolonged) voiding cycle. This leads to hesitancy and intermittency. Other causes of irritative symptoms which merit attention are bladder cancer, urinary tract infection, urethral stricture, bladder diverticula, bladder calculus, and neuropathic bladder dysfunction. #### Irritative symptoms The assessment of a patient with outflow obstruction begins with a thorough history and examination. Symptom scores such as the American Urological Association's score are seldom used except in clinical trials. The clinical examination of a patient should focus on the urinary tract, especially the external genitalia, and should also include a digital rectal examination. A digital rectal examination is minimally invasive, allows a rough estimation of the size of the prostate, will detect locally advanced prostate cancer, and permits an assessment of rectal sphincter tone (which may be relevant to the presenting history). A urine culture should be performed to exclude urinary infection or haematuria, and the serum electrolytes should be checked for satisfactory renal function. Assessment procedure for patients with bladder outflow obstruction. Uroflometry is an important procedure that is often neglected but should be performed before surgery is considered. In some centres this …

Abstract Introduction Thalidomide (THAL), and the IMiDs® immunomodulatory agents lenalidomide (LEN), and pomalidomide (POM) are all approved for use in multiple myeloma (MM) either as single agents, or in combination with dexamethasone (DEX). Despite the enhanced efficacy of these novel agents, concern has arisen as to the increased incidence of secondary primary malignancies (SPM). For example, the IFM 2005-002 trial reported cases of lymphoblastic leukemia and Hodgkin’s disease (HD) following LEN use (Attal, Lauwers-Cances et al. 2012) in MM patients on maintenance therapy. Also, a recent case report described a MM patient who developed HD who had been treated with salvage therapy containing THAL(Chim, Choi et al. 2013), and two publications reported EBV reactivation in MM patients treated with LEN (Kneppers, van der Holt et al. 2011; Kroger, Zabelina et al. 2013). As HD is causally linked to EBV, this raises the question as to whether the IMiDs reactivate latent EBV infection in normal memory B-cells, and thereby increase the risk of EBV-related malignancies. To this end, we have investigated the ability of the IMiD’s to induce reactivation of latently infected B-cell lines. Methods A panel of latently infected EBV-positive B-cell lines including Burkitt’s lymphoma (BL) cells and lymphoblastoid cell lines (LCL) were treated with either LEN, THAL or POM, and the status of the EBV lytic cycle was evaluated using in vitro and in vivo models. Results Treatment of BL and LCL cell lines with physiological concentrations of IMiDs (1-5 μM) induced the immediate early gene BZLF1 and the early gene BMRF1. Interestingly, the ability to induce EBV reactivation was in their potency order (i.e. POM&gt;LEN&gt;THAL). The IMiD’s also induced lytic cell death, as an LCL carrying a BZLF1-deleted EBV, which is incapable of undergoing a lytic cycle, showed no change in cell viability, compared to wild-type cells which had increased cell death. The addition of the nucleoside analogue ganciclovir (GCV) enhanced the cytotoxic effect of LEN and POM alone in BL cells lines. An in vivo xenograft model of BL demonstrated that the combination of LEN and GCV was highly efficacious at suppressing tumor cell growth, thus confirming the ability of LEN to stimulate the EBV-lytic life cycle. The ability to induce EBV reactivation was directly related to the stimulation of phosphatidylinositol-3 kinase (PI3K) signaling, which was completely blockaded by the PI3K-δ inhibitor, CAL101. The combination of LEN with either, DEX or rituximab, induced increased BMRF1 compared to the LEN alone. Conclusions The IMiD class of drugs has a potent ability to reactivate the lytic cycle in B-cells latently infected with EBV. We hypothesize that the IMiD’s reactivate latently infected resting memory B cells through enhancing PI3K signaling. This reactivation may be further potentiated when the IMiDs are used in combination with rituximab or DEX, which may simultaneously enhance the EBV lytic cycle and suppress the host immune response. These findings suggest the possibility that immunocompromised patients who receive IMiDs should be monitored for evidence of EBV reactivation. Also, this may suggest a mechanism by which patients may develop EBV-associated SPM, an effect which is similar to the methotrexate induced EBV-positive lymphomas seen in rheumatoid arthritis patients (Feng, Cohen et al. 2004). References Attal, M., V. Lauwers-Cances, et al. (2012). “Lenalidomide maintenance after stem-cell transplantation for multiple myeloma.” The New England journal of medicine 366(19): 1782-1791. Chim, C. S., P. T. Choi, et al. (2013). “Hodgkin's lymphoma as a second cancer in multiple myeloma never exposed to lenalidomide.” Annals of hematology 92(6): 855-857. Feng, W. H., J. I. Cohen, et al. (2004). “Reactivation of latent Epstein-Barr virus by methotrexate: a potential contributor to methotrexate-associated lymphomas.” Journal of the National Cancer Institute 96(22): 1691-1702. Kneppers, E., B. van der Holt, et al. (2011). “Lenalidomide maintenance after nonmyeloablative allogeneic stem cell transplantation in multiple myeloma is not feasible: results of the HOVON 76 Trial.” Blood 118(9): 2413-2419. Kroger, N., T. Zabelina, et al. (2013). “Toxicity-reduced, myeloablative allograft followed by lenalidomide maintenance as salvage therapy for refractory/relapsed myeloma patients.” Bone marrow transplantation 48(3): 403-407. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

&lt;p&gt;Contains the text for the supplementary figures.&lt;/p&gt;

&lt;p&gt;EBV viral loads in patient serum samples on a LEN, THAL, DEX combination.&lt;/p&gt;

&lt;p&gt;1.Contains schematic of a LEN, THAL DEX dosing schedule. 2. EBV viral load in patients who received LEN, THAL, DEX with VTX or no VTX.&lt;/p&gt;

&lt;p&gt;Combination indices of LEN, THAL or POM in combination with GCV.&lt;/p&gt;

&lt;p&gt;Combination indices of LEN, THAL or POM in combination with GCV.&lt;/p&gt;

&lt;p&gt;EBV viral loads in patient serum samples on a LEN, THAL, DEX combination.&lt;/p&gt;

&lt;p&gt;1.Contains schematic of a LEN, THAL DEX dosing schedule. 2. EBV viral load in patients who received LEN, THAL, DEX with VTX or no VTX.&lt;/p&gt;

&lt;p&gt;Contains the text for the supplementary figures.&lt;/p&gt;

&lt;p&gt;P values of mouse tumor volumes over time&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein–Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We studied the ability of LTP to reactivate latently infected EBV-positive cell lines &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene &lt;i&gt;BZLF1&lt;/i&gt;, the early gene &lt;i&gt;BMRF1&lt;/i&gt;, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide &gt; lenalidomide &gt; thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3Kδ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. &lt;i&gt;Clin Cancer Res; 22(19); 4901–12. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;&lt;b&gt;Purpose:&lt;/b&gt; Lenalidomide, thalidomide, and pomalidomide (LTP) are immunomodulatory agents approved for use in multiple myeloma, but in some settings, especially with alkylating agents, an increase in Hodgkin lymphoma and other secondary primary malignancies (SPM) has been noted. Some of these malignancies have been linked to Epstein–Barr virus (EBV), raising the possibility that immunomodulatory drugs disrupt latent EBV infection.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Experimental Design:&lt;/b&gt; We studied the ability of LTP to reactivate latently infected EBV-positive cell lines &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;, and evaluated the EBV viral load in archived serum samples from patients who received a lenalidomide, thalidomide, and dexamethasone (LTD) combination.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; Treatment of EBV-infected B-cell lines with LTP at physiologically relevant concentrations induced the immediate early gene &lt;i&gt;BZLF1&lt;/i&gt;, the early gene &lt;i&gt;BMRF1&lt;/i&gt;, and the late proteins VCA and BCFR1. This occurred in the potency order pomalidomide &gt; lenalidomide &gt; thalidomide, and the nucleoside analogue ganciclovir enhanced the cytotoxic effects of lenalidomide and pomalidomide in Burkitt lymphoma cells &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. EBV reactivation was related to PI3K stimulation and Ikaros suppression, and blocked by the PI3Kδ inhibitor idelalisib. Combinations of lenalidomide with dexamethasone or rituximab increased EBV reactivation compared with lenalidomide alone and, importantly, lenalidomide with melphalan produced even greater reactivation.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Conclusions:&lt;/b&gt; We conclude LTP may reactivate EBV-positive resting memory B cells thereby enhancing EBV lytic cycle and host immune suppression. &lt;i&gt;Clin Cancer Res; 22(19); 4901–12. ©2016 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;P values of mouse tumor volumes over time&lt;/p&gt;

Most patients with urinary stones have microscopic haematuria detected by routine urine analysis. An absence of red cells in the urine suggests an alternative diagnosis. #### Presenting features of urinary stone disease An x ray film of the kidneys, ureters, and bladder shows the position and size of the kidneys as well as the presence of any calculi, which usually obstruct at the pelviureteric junction, the point where the ureter crosses the iliac arteries at the pelvic brim, or the vesicoureteric junction. All patients suspected of having a urinary stone should then have intravenous urography, although renal ultrasonography is acceptable in patients who are either allergic to contrast medium or pregnant. Obstruction may be detected by the delayed appearance or persistence of the nephrogram phase on the intravenous urogram or by the finding of caliceal dilatation in an ultrasound scan. #### Differential diagnosis of renal colic Renography after injection of technetium-99m mercapto acetyltriglycine is not routinely used and …

The inability to directly infect epithelial cells in vitro with Epstein-Barr virus (EBV) () has led to the development of epithelial cell-culture models to examine virus-induced growth transformation and productive infection.

BackgroundEpigallocathechin-3-gallate (EGCG), the major bioactive polyphenol in green tea, has anticarcinogenic properties in vitro and in vivo. Several recent reports have shown that EGCG affects the expression of human papillomavirus (HPV)-encoded E6 and E7, two oncoproteins required for HPV-driven oncogenesis. Here, we aimed to explore the effects of EGCG on keratinocyte proliferation and differentiation, in addition to HPV18 replication, in a three-dimensional organotypic raft culture system.MethodsOrganotypic raft cultures of HPV18-infected keratinocytes cultured at the air–liquid interface for 10 days were treated with EGCG for an additional 10 days before fixation and processing. Raft sections were stained with antibodies specific for various cell proliferation and keratinocyte differentiation markers in addition to tumour suppressor genes. Western blotting was performed on EGCG-treated cells to measure the level of HPV18 E6 and E7 protein expression.FindingsEGCG treatment blocked the ability of HPV18-positive keratinocytes to generate hyperplastic epithelium in raft culture. EGCG reduced cell proliferation as assessed by bromodeoxyuridine label incorporation and Ki67 staining; it upregulated expression of several tumour suppressor genes (p53 [TP53], p21, pRb), and impaired productive viral replication (as assessed by HPV18 E4 protein expression), but did not have an effect on keratinocyte differentiation. In culture, EGCG treatment promoted degradation of the E6 and E7 proteins and restored tumour suppressor gene expression.InterpretationThe results of our preclinical study suggest that EGCG inhibits the proliferation of HPV18-infected keratinocytes by enhancing the turnover and degradation of the E6 and E7 proteins, resulting in re-expression of several key tumour suppressor genes. These findings suggest that EGCG could potentially reverse the dysplastic changes induced by oncogenic HPV strains and could be used clinically to treat HPV-induced neoplasia.FundingCancer Research UK.