Current insights into the aetiology, pathobiology, and management of local disease recurrence in squamous cell carcinoma of the vulva.

Squamous cell carcinoma of the vulva is predominantly a disease of the elderly, where the mainstay of treatment is radical surgery. Local vulval recurrence (LVR) is a significant problem for these patients, and the rates of recurrence have not improved over the last three decades. Disappointingly, we still lack an understanding of how LVRs develop, and the best approach to prevent and manage the condition. This review discusses recent insights into the key prognostic factors that influence the risk of recurrence, focusing on the role of tumour-adjacent non-neoplastic epithelial disorders, which are thought to play a causative role. TWEETABLE ABSTRACT: A review that discusses the key prognostic factors that influence local recurrence in vulval cancer.

Adjacent Lichen Sclerosis predicts local recurrence and second field tumour in women with vulvar squamous cell carcinoma.

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 <2cm from the tumour margins, and as second field tumours (SFT) when ≥2cm 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.

Modular versus all-polyethylene tibial components: comparison of pre- and early post-operative patient scores in total knee replacement.

INTRODUCTION: All-polyethylene (AP) tibial components of total knee replacement (TKR) are substantially cheaper than their modular counterparts. It is well established that their survivorship and radiographic outcomes are comparable. In this study, patient-derived outcome measures were used to compare these two implant types. METHODS: A cohort of 456 primary TKRs (142 AP, 314 modular) were assessed with preoperative and 1-year post-operative Oxford Knee Score, Western Ontario and McMaster Universities Arthritis Index and Short Form - 12 scores. RESULTS: Both groups performed well with no significant difference in improvement and final scores at 1 year. Although there was a significant difference in mean age among the groups (P < 0.001) age-adjusted scores continued to show no significant difference between the two groups. DISCUSSION: Our results support the more frequent use of AP tibial components for uncomplicated TKR.

c-Myc and EBV-LMP1: two opposing regulators of the HLA class I antigen presentation machinery in epithelial cells.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The EBV-encoded latent membrane proteins, LMP2A and LMP2B, limit the actions of interferon by targeting interferon receptors for degradation.

Although frequently expressed in Epstein-Barr virus (EBV)-positive malignancies, the role that latent membrane protein 2A and 2B (LMP2A and LMP2B) have in the oncogenic process remains obscure. Here we show a novel function for these proteins in epithelial cells, namely, their ability to modulate signalling from type I/II interferon receptors (IFNRs). We show that LMP2A- and LMP2B-expressing epithelial cells show decreased responsiveness to interferon (IFN)alpha and IFNgamma, as assessed by STAT1 phosphorylation, ISGF3 and GAF-mediated binding to IFN-stimulated response element and IFNgamma-activated factor sequence elements and luciferase reporter activation. Transcriptional profiling highlighted the extent of this modulation, with both viral proteins impacting 'globally' on IFN-stimulated gene expression. Although not affecting the levels of cell-surface IFNRs, LMP2A and LMP2B accelerated the turnover of IFNRs through processes requiring endosome acidification. This function may form part of EBV's strategy to limit anti-viral responses and define a novel function for LMP2A and LMP2B in modulating signalling from receptors that participate in innate immune responses.

Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGFbeta signaling pathways.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

The Epstein-Barr virus encoded latent membrane protein 2A augments signaling from latent membrane protein 1.

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.

The transmembrane domains of the EBV-encoded latent membrane protein 1 (LMP1) variant CAO regulate enhanced signalling activity.

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 expression and function of Epstein-Barr virus encoded latent genes.

The association of Epstein-Barr virus (EBV) with various malignancies is well established but the pattern of EBV latent gene expression in these different tumours is variable, reflecting distinct aspects of the virus-cell interaction. These different forms of EBV latency are associated with phenotypic variation and highlight the influence of EBV latent proteins on cell growth and survival. The EBV latent proteins have distinct functions associated with the maintenance of EBV infection and the control of various signalling and transcriptional pathways that facilitate the proliferation and survival of infected cells. Understanding the function of these EBV latent proteins will not only provide insight into the mechanisms governing fundamental cell processes but will also identify targets for novel treatment.

Identification of functional differences between prototype Epstein-Barr virus-encoded LMP1 and a nasopharyngeal carcinoma-derived LMP1 in human epithelial cells.

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.

CTL control of EBV in nasopharyngeal carcinoma (NPC): EBV-specific CTL responses in the blood and tumors of NPC patients and the antigen-processing function of the tumor cells.

Undifferentiated nasopharyngeal carcinoma (NPC) is latently infected with EBV and expresses a restricted number of viral proteins. Studies in healthy virus carriers have demonstrated that at least some of these proteins can act as targets for HLA class I-restricted CTLs. Therefore we have explored the possibility of a CTL-based therapy for NPC by characterizing EBV-specific CTL responses in 10 newly diagnosed NPC cases and 21 healthy virus carriers from Southeast Asia. Using the autologous EBV-transformed lymphoblastoid cell line, virus-specific CTL were reactivated in vitro from PBMC, cloned, and screened for cytotoxicity against target cells expressing individual EBV proteins from recombinant vaccinia vectors. EBV-specific CTLs were identified in 6 of 10 patients and 14 of 21 controls and mainly targeted the EBV nuclear Ag 3 (EBNA3) family of viral latent proteins. However, in 3 of 10 patients and 11 of 21 controls, CTLs specific for the NPC-associated protein LMP2 were also detected, albeit at low frequency. EBV-specific CTLs were detected in tumor biopsy material obtained from 3 of 6 of the patients, indicating that functional CTL are present at the tumor site, but none was specific for tumor-associated viral proteins. To assess the Ag-presenting function in NPC we studied two NPC-derived cell lines (C15 and c666.1) and demonstrated that both were capable of processing and presenting endogenously synthesized protein to HLA class I-restricted CTL clones. Overall, our data provide a sound theoretical basis for therapeutic strategies that aim to boost or elicit LMP2-specific CTL responses in NPC patients.

Inductive learning approaches to rainfall-runoff modelling.

Trying to model the rainfall-runoff process is a complex activity as it is influenced by a number of implicit and explicit factors--for example, precipitation distribution, evaporation, transpiration, abstraction, watershed topography, and soil types. However, this kind of forecasting is particularly important as it is used to predict serious flooding, estimate erosion and identify problems associated with low flow. Inductive learning approaches (e.g. decision trees and artificial neural networks) are particularly well suited to problems of this nature as they can often interpret underlying factors (such as seasonal variations) which cannot be modelled by other techniques. In addition, these approaches can easily be trained on the explicit factors (e.g. rainfall) and the inexplicit factors (e.g. abstraction) that affect river flow. Inductive learning approaches can also be extended to account for new factors that emerge over a period of time. This paper evaluates the application of decision trees and two artificial neural network models (the multilayer perceptron and the radial basis function network) to river flow forecasting in two flood prone UK catchments using real hydrometric data. Comparisons are made between the performance of these approaches and conventional flood forecasting systems.

Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production.

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)-kappaB and c-Jun NH2-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-kappaB 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-kappaB binding activity. Conversely, although the metabolic inhibitor D609 blocked NF-kappaB 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-kappaB 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.

CD40 and epithelial cells: across the great divide.

The widespread expression of CD40 in normal epithelial cells and carcinoma cells suggests that this receptor has important, additional influences beyond that of regulating immune responses. Here, Lawrence Young and colleagues discuss the effect of CD40 ligation on epithelial cells and consider the role of this pathway in the pathogenesis and treatment of carcinomas.

Functional differences between BHRF1, the Epstein-Barr virus-encoded Bcl-2 homologue, and Bcl-2 in human epithelial cells.

BHRF1, a component of the restricted early antigen complex of the Epstein-Barr virus lytic cycle, encodes a 17-kDa protein with both sequence and functional homology to the antiapoptotic Bcl-2 oncogene. Recent work has suggested that BHRF1 behaves like Bcl-2 in protecting cells from apoptosis induced by a range of stimuli. In this study, the effect of BHRF1 and Bcl-2 on the growth and differentiation of the SCC12F human epithelial cell line was examined. The levels of stable transfected BHRF1 expression achievable in SCC12F cells was consistently lower than that obtained with Bcl-2. While both BHRF1 and Bcl-2 inhibited epithelial differentiation, the effect of Bcl-2 was more pronounced, resulting in an almost complete blockade of differentiation in organotypic raft cultures. However, BHRF1-expressing SCC12F cells proliferated at a much higher rate than SCC12F cells expressing Bcl-2, and this effect was supported by cell cycle analysis which demonstrated that BHRF1, but not Bcl-2, promotes rapid transit through the cell cycle. These data highlight important differences between BHRF1 and Bcl-2 and suggest that BHRF1 may function to promote the survival and proliferation of lytically infected cells. The proliferative properties of BHRF1 described in this study, together with the demonstration that other oncogenic gamma herpesviruses encode Bcl-2 homologues, suggests that these proteins may serve to increase the susceptibility of virus-infected cells to oncogenic transformation, thereby contributing to the development of virus-associated tumors.

Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis.

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.

Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors.

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB 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-kappaB 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-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB 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-kappaB 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-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB 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.