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.

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.

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.

BHRF1, a viral homologue of the Bcl-2 oncogene, disturbs epithelial cell differentiation.

Epstein-Barr virus (EBV) is associated with tumours of both lymphoid and epithelial origin. Whilst a role for EBV latent genes in the development of these malignancies is accepted, it is also possible that viral proteins involved in EBV replication may influence the oncogenic process. BHRF1 is an immediate early protein which has homology with the Bcl-2 oncogene and can protect B cells from apoptosis. In vivo this protein is most abundantly expressed in the upper layers of oral 'hairy' leukoplakia (HL), a benign hyperparakeratotic tongue lesion which represents a focus EBV replication. We have transfected BHRF1 into the human squamous cell carcinoma line SCC12F which retains several features of normal keratinocytes behaviour in vitro. BHRF1 expression in these epithelial cells is associated with a delay in the commitment of cells to terminal differentiation, increased resistance to the DNA damaging drug, cis-platin and enhanced survival under conditions of serum deprivation. As the differentiation of epithelial cells is an apoptotic process, this data strongly suggests that BHRF1 expression delays the terminal differentiation of epithelial cells through the prevention of apoptosis. This effect of BHRF1, which may normally function to promote productive EBV infection, could contribute to the development of EBV-associated tumours.

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.

CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator.

CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.

Altered interstitial fluid space dynamics and postresuscitation hypertension.

Hypertensin occurred 24 to 48 hours after resuscitation in 35 of 86 injured patients, who had combined systolic and diastolic hypertensin (150/100 mmHg) for six or more consecutive hours. Plasma volume (PV), RBC volume, extracellular fluid (ECF) volume by the inulin dilution technique, renal plasma flow, glomerular filtration rate, and peripheral renin levels were measured in hypertensive and nonhypertensive patients an average of 40 hours after injury. The hypertensive patients had an average mean arterial pressure (MAP) of 114 mmHg, compared with 95 mmHg in the nonhypertensive patients. The RBC volume and ECF were comparable for both groups, whereas PV was increased in the hypertensive patients (3.6 L vs 3.3 L). Calculated interstitial fluid space (IFS) volume was greater in the nonhypertensive patients, as was the ratio PV/IFS. The MAP in both groups correlated directly with PV/IFS and serum albumin concentrations, and inversely with peripheral renin concentrations. This suggests that postresuscitative hypertension is not due to fluid overload but rather to the fluid maldistribution related to altered IFS compliance as reflected by the increased PV/IFS.

Anergy and altered lymphocyte function in the injured patient.

The failure of the host-defense mechanism following trauma has been recognized, but the site of the deficit is unknown. The immunologic competence of 16 patients, including 15 who had minor injuries and required less than 4 transfusions, was prospectively studied with skin testing, leukocyte counts, and protein electrophoresis. The stimulation ratio (SR) and index of lymphocyte response to phytohemagglutanin (PHA) were measured as the ratio of thymidine uptake in stimulated cells to that of resting cells in both pooled normal serum as well as each patient's serum. Six patients, including the one patient with major injury, had no response to any of the four skin test antigens and were considered anergic (AN). Ten patients with minor trauma responded to at least one of the antigen skin tests and were considered nonanergic (NA). The anergic patients had significantly more shock and more blood transfusions and had significantly lower serum albumin levels. There was no statistical difference between the anergic and nonanergic groups in leukocyte count, absolute lymphocyte count, gamma-globulin fraction, or age. The average PHA Stimulation Ratio of patient lymphocytes in patient serum was significantly higher than the PHA Stimulation Ratio of patient lymphocytes in control serum. This suggests that the lymphocytes of injured patients respond in a greater magnitude when bathed in autologous-serum than when bathed in control pooled serum. Furthermore, the autologous serum did not inhibit the PHA response of control or normal lymphocytes. The presence of an enhancing factor in injured patients' sera or an absence of a supressor substance may be responsible for this phenomenon.

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.

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.

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 latent membrane protein inhibits human epithelial cell differentiation.

Epstein-Barr virus (EBV), a human herpesvirus, is strongly linked with two relatively rare forms of B-cell lymphoma and with a much more prevalent epithelial malignancy, undifferentiated nasopharyngeal carcinoma (NPC). The availability of suitable culture systems has allowed detailed analysis of EBV-induced growth transformation in B lymphocytes, but little is known about the virus--epithelial cell interaction or about the possible effector role of viral proteins in the pathogenesis of NPC. Here we describe an experimental system to monitor the effects of introduced viral or cellular genes upon human epithelial cell growth and differentiation. We transfected a human epithelial cell line, which retains several features of normal keratinocyte behaviour in vitro, with the EBV gene encoding latent membrane protein (LMP), one of only two viral proteins known to be expressed in NPC cells in vivo. LMP expression was accompanied by changes in the epithelial cell surface phenotype, mimicking surface changes observed in NPC cells, and by severe impairment of the cellular response to differentiation signals. The ability of LMP to inhibit terminal differentiation indicates a mechanism whereby EBV infection of squamous epithelium could contribute to the multi-step pathogenesis of NPC.

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.

Viruses and apoptosis.

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 carcinogenesis. Understanding the mechanisms by which viruses regulate apoptosis may lead to the development of novel therapies for both infectious disease and cancer.

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.

Identification of a human epithelial cell surface protein sharing an epitope with the C3d/Epstein-Barr virus receptor molecule of B lymphocytes.

This work examines the basis for our earlier observation that certain monoclonal antibodies (MAbs) specific for the B-cell-associated C3d/Epstein-Barr virus (EBV) receptor molecule CD21 also react with the surface of some epithelial cells. Of 9 proven anti-CD21 MAbs now examined on frozen sections of human nasopharynx, tonsil and ecto-cervix, only 3 (HB5, anti-B2, AB1) showed staining of stratified epithelium; 2 of these (HB5, anti-B2) also reacted with the surface of epithelial cells freshly dispersed from these sites. The proportion of HB5- and anti-B2-reactive cells in primary epithelial cultures fell to a low but stable level within days of explantation, while almost all permanently established epithelial cell lines, whether SV40 virus-transformed or of malignant origin, were not reactive with either MAb. This contrasts with the pattern of expression of another surface marker also found selectively on cells of the lymphoid and epithelioid lineages, the CDw40 antigen. Staining with CDw40 MAbs on epithelial sections was usually restricted to the basal (proliferating) layer, but the proportion of CDw40-positive cells increased to a relatively high level in normal epithelial cultures; furthermore, most epithelial cell lines expressed this antigen. Immunoprecipitation from the surface of metabolically labelled epithelial cells with the anti-CD21 MAb HB5 yielded a protein of approximate MW 200 kDa, clearly different in size from the 145 kDa CD21 molecule on B cells. This 200 kDa protein was identified on fresh ecto-cervical epithelium, on primary cultures of a laryngeal carcinoma and on one unusual SV40-transformed epithelial cell line. We conclude that stratified human epithelial cells express a 200 kDa surface molecule which is antigenically related to, but not identical with, the CD21 antigen on B cells. It remains to be seen whether this epithelial cell protein can function as an EBV receptor.

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.