Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis  Tumour angiogenesis is regulated by many angiogenic molecules including VEGF, which are produced by tumour or stromal cells within the tumour microenvironment . Notch s pathway , 22. Mol pathway . The 2.3 pathway .Interestingly, in a retina model the expression of DLL4 was shown to fluctuate dynamically in individual ECs within sprouting vessels  while JAin vitro angiogenesis, on xenograft tumour growth and vasculature, and on tumour response to anti-VEGF therapy.Many studies have focused on the effects of endothelial DLL4 or JAG1 on tumour growth and vascularisation. However, whether there is any function interaction between DLL4 and JAG and how such an interaction would affect tumour angiogenesis and tumour growth and progression are unknown. Prospectively, it would be important for targeted therapy if they were antagonistic in tumour. Therefore, we investigated different effects of DLL4 and JAG1 on Both rhDLL4 and rrJAG1, when coated on plates , were capable of upregulating the expression of DLL4, JAG1, HEY1, and HEY2 in HMEC-1 at mRNA levels with an equal potency Figure . Howeverversus 2.8-fold)  Figure .FACS analysis for measuring the expression of hDLL4 or hJAG1 at protein levels in parental HMEC-1 cells that were sorted from co-culture of ligand-overexpressing HMEC-1 cells by mixing an equal amount of parental cells showed that mDLL4 stimulated the expression of endogenous hDLL4 significant stronger than mJAG1 did, whereas both mDLL4 and mJAG1 equally induced the expression of endogenous hJAG1 compared to the EV control Figure , consistThe role of these ligands in endothelial sprouting was investigated using hanging drop assays. mDLL4 decreased sprouting while mJAG1 promoted sprouting of HMEC-1 Figure . Blockinin vitro . mDLL4 expressed in U87 cells conferred tumours more resistance to anti-VEGF therapy as shown by the shorter delay for 5 days (mDLL4:control versus mDLL4:mAb) compared to the 14-day delay in EV-tumours (EV:control versus EV:mAb). Similarly, mJAG1 expressed in U87 also instigated tumour resistance to anti-VEGF therapy  compared to the 14-day delay in EV-tumours (EV:control versus EV:mAb). However, such resistance contributed by mJAG1  but dramatically postponed xenograft growth of either mDLL4-tumour  or bevacizumab therapy (mDLL4:DBZ+mAb versus mDLL4:mAb) . Combined DBZ with bevacizumab treatment (mJAG1:DBZ+mAb) showed a synergistic inhibition effect on the growth of mJAG1-tumours compared to DBZ (mJAG1:DBZ) or bevacizumab (mJAG1:mAb) alone  Figure  but suche Figure .versus I)  Figure .versus I-III) but did not affect pericyte coverage in mDLL4- and mJAG1-tumours (VIII-IX versus II-III).Bevacizumab reduced vessel density in EV-, mDLL4- and mJAG1-tumours (VII-IX versus I-II) but significantly decreased vessel size in EV, mDLL4 and mJAG1 groups (IV-VI versus I-III). Pericyte coverage was also significantly increased in DBZ-treated mDLL4 and mJAG1 groups (V-VI versus II-III).DBZ treatment increased vessel numbers in EV and mDLL4 groups (IV-V versus V-VI).The percentage of pericyte coverage in over-expressing tumours treated with both DBZ and bevacizumab was similar to that in tumours treated with DBZ alone  as DLL4 is induced by hypoxia through HIF-1α [versus VIII) as well as treated with the combination of DBZ and bevacizumab (XII versus XI) but more DLL4 expression occurred in the vasculature of mJAG1-tumour than in EV-tumours when treated with the combination (XII versus X). DLL4 stained positively on tumour and/or stromal cells, and increased especially in cells surrounding tumour vessels when EV-tumours and mJAG1-tumours were treated with bevacizumab (VII and IX) as well as treated with the combination (X and XII).Bevacizumab profoundly pruned the vessels leaving behind surviving vessels that expressed high levels of DLL4 in all three types of tumours (VII-IX). Increased DLL4 expression was observed in EV-tumours treated with bevacizumab compared to control , and detected endogenous expression of hDLL4 or hJAG1 in parental (neighbouring) cells by FACS staining with anti-DLL4 or JAG1 mAbs respectively . mJAG1 expressed in U87 cells also significantly stimulated endogenous hDLL4 expression in neighbouring U87 cells compared to both controls  but substantially weaker than mDLL4 did (mJAG1 versus mDLL4). Co-culture of mDLL4-expesssing U87 with HMEC-1 cells . Similarly, mJAG1 expressed in U87 cells greatly induced hDLL4 expression in HMEC-1 cells compared to EV control (mJAG1 versus EV) but significantly weaker than mDLL4 did (mJAG1 versus mDLL4). Mixture of mDLL4-expressing with mJAG1-expressing U87 cells induced hDLL4 expression in HMEC-1 as did by mDLL4-expressing U87 alone (mDLL4+mJAG1 versus mDLL4).To validate the ectively . mDLL4 eectively  greatly s Figure  demonstrIn vivo, both mDLL4 and mJAG1 increased the expression of DLL4 in tumour tissues but there was more induction by mDLL4 than by mJAG1 . JAG1 was abundantly expressed in mJAG1-expressing tumours ratifying the transduction efficacy . Inhibition with DBZ resulted in abated JAG1 expression in mDLL4-tumours compared to that of mDLL4-tumour control (V versus II) while bevacizumab had no significant effects on JAG1 expression in all three types of tumours compared to that of all control tumours (VII-IX versus I-III). Combined treatment with DBZ and bevacizumab dramatically reduced expression of endogenous JAG1 in both EV- and mDLL4-tumours (X-XI versus I-II).Endogenous JAG1 was weakly expressed in the vessels Figure . NotablyIn vitro co-culture experiments showed that either mDLL4 or mJAG1 expressed in U87 cells dramatically induced endogenous hJAG1 expression in neighbouring U87 cells . Co-culture of over-expressing U87 cells with HMEC-1 cells revealed that either mDLL4 or mJAG1 expressed in U87 cells significantly stimulated endogenous hJAG1 expression in HMEC-1 cells compared to the EV control . Both groups had an increase in hypoxia with either DBZ (IV-V) or bevacizumab treatment (VII-VIII) with an additive effect with combination treatment seen in mDLL4-tumours (XI). In contrast, expression of mJAG1 did not significantly reduce CAIX expression compared to EV-tumours (III versus I). Treatment of mJAG1-tumours with DBZ (VI) or bevacizumab (IX) did not significantly affect the level of CAIX expression compared to control-treated mJAG1 tumours (III) but the combination treatment significantly increased hypoxia (XII versus III).Consistent with our previous work with hDLL4 , mDLL4 sversus I) while treatment with DBZ obviated the effect and increased necrosis (IV-VI versus I-III). Bevacizumab treatment increased necrosis in EV-tumours (VII versus I) but not in mDLL4- and mJAG1-expressing tumours (VIII-IX versus II-III). Combined treatment with DBZ and bevacizumab enhanced necrosis in all groups (X-XII versus I-III).Both mDLL4 and mJAG1 significantly reduced tumour necrosis in either mDLL4- or mJAG1-tumours Figure  and 6D cTo further investigate the interplay between JAG1 and DLL4 in promoting tumour growth, we used specific anti-DLL4 mAb (MedImm) that recognises both human DLL4 and mouse DLL4 to block DLL4-induced Notch signalling in mJAG1-expressing tumours. Blocking DLL4-Notch signalling with anti-DLL4 mAb significantly delayed tumour growth Figure  and incrin vitro.mDLL4-induced Notch signalling appeared to be greater than mJAG1-induced signalling. mDLL4 increased endogenous DLL4 expression in a feed-forward manner  while mJin vitro but increased tumour growth in vivo as described for hDLL4 previously [via the ECs themselves or via tumour-EC interactions. Recently it was reported that tumour cells can differentiate into ECs [mDLL4 and mJAG1 reduced cell proliferation eviously , suggestinto ECs , 33 but into ECs  and JAG1into ECs  have recinto ECs , 36, 37;into ECs .via increased perfusion and decreased necrosis. Both ligands upregulated mHEY1 more than hHEY1, implying that paracrine signalling from tumour cells to stromal cells is stronger than autocrine signalling within tumour cells, supporting a vasculature-dependent mechanism for the enhanced tumour growth.Tumour immunostaining revealed different vessel phenotypes. mDLL4 decreased vessel number but harboured bigger vessels as described previously with hDLL4 , 7 whileIn vitro co-culture assays supported the findings in vivo. Both mDLL4 and mJAG1 expressed on different types of tumour cells strongly stimulated the expression of endogenous hDLL4 and hJAG1 in neighbouring cells including tumour cells themselves and HMEC-1 cells. Such autocrine and paracrine feed-forward signalling were much higher for induction of endogenous hDLL4 by mDLL4 than by mJAG1 in all types of tumour cells we have examined and in HMEC-1 ECs as well. Clearly, no antagonising effects between DLL4- and JAG1-Notch signalling were found.The effects of DLL4-Notch signalling on stromal cells in tumour microenvironment may be greater than that of mJAG1-Notch signalling. Indeed, endogenous DLL4 expression was higher in tumour vessels, particularly in those larger vessels which are known to have greater perfusion  and moreBoth mDLL4 and JAG1 xenografts had an increased sensitivity to DBZ compared to EV-tumours, suggesting that Notch signalling is responsible for the enhanced tumour growth . We haveThe variation in vasculature phenotypes between mDLL4 and mJAG1 tumours may be explained by differences in downstream signalling activated by these ligands. DLL4 stimulation but not JAG1 was found to markedly induce EphrinB2 expression in ECs . EphrinBin vitro and in vivo, especially in tumour stromal cells. In addition both mDLL4- and mJAG1-tumours are resistant to bevacizumab treatment. If mJAG1 were reversing the level of VEGFR2 in tumour vessels a greater response to bevacizumab would be expected. However bevacizumab only partially reduced tumour growth and was capable of decreasing vessel density in both tumours.Expression of mJAG1 increased vessel numbers in xenograft tumours. Indeed, an increase in sprouting angiogenesis has been demonstrated in the retina of mice with JAG1 gain-of-function  and tumoTo investigate this further, we treated the mJAG1-tumours with anti-DLL4 blocking antibody that recognises both human DLL4 and mouse DLL4. If mJAG1 were antagonising DLL4 signalling, anti-DLL4 treatment should have a little effect. However, we found that anti-DLL4 antibody was still capable of inhibiting tumour growth of both EV-tumour and mJAG1-tumour significantly. Blocking DLL4 signalling using anti-DLL4 antibody effectively delayed tumour growth possibly by disrupting functional angiogenesis , 20, 47,versus angiogenesis, and different microenvironments between different tumour types.In all xenograft models we tested, the same effects of mDLL4 and mJAG1 on the vascular phenotype were found, that is, mDLL4 reduced vessel numbers but increased vessel sizes while mJAG1 only increased vessel numbers. However, only in U87 xenograft model both mDLL4 and mJAG1 increased the tumour growth. In fact, such variability in tumour response to Notch-regulated angiogenesis has been already noted  and poteAltogether, we propose a model Figure  to depicμg/ml streptomycin, 10mM L-glutamine, 10ng/ml epidermal growth factor (Sigma), and 1μg/ml hydrocortisone . Recombinant human DLL4  and rat JAG1  were purchased from R&D. Dibenzazepine (DBZ), a γ-secretase inhibitor, was purchased from Syncom (The Netherlands) at a purity of 99.8% at 232nm. Anti-DLL4 blocking mAb, a human IgG1 isotype that cross-reacts with both human DLL4 and mouse DLL4 (MedImm) was supplied as previously [Immortalised human dermal microvascular endothelial cells (HMEC-1) were obtained from from ATCC through the CDC . Human ueviously . Anti-JA5 cells per well (depending on cell lines) and incubated in the CO2 incubator at 37°C. Cells were counted everyday over 5-6 days using the Z2 Coulter Counter (Beckman Coulter).Cells were seeded onto 6-well plates at 0.5-2 × 10RNA extraction from tumour tissues and QPCR were performed as described previously . PrimersThe small interfering RNAs (siRNAs) were designed using the siDESIGN Center website  and synthesised by Eurogentec (Southampton). siRNA sequences are: scrambled control sense 3’-UCUGAAAAGCACGCUUGAC-5’ and antisense 3’-GUCAAGCGUGCUUUUCAGA-5’; siDLL4 Duplex1 sense 3’-CAACUAUGCUUGUGAAUGU-5’ and antisense 3’-ACAUUCACAAGCAUAGUUG-5’; siDLL4 Duplex2 sense 3’-ACACAAACCAGAAGAAGGA-5’ and antisense 3’-UCCUUCUUCUGGUUUGUGU-5’; siJAG1 Duplex1 sense 3’-AUCUUAUGAGGGAUUUACG-5’ and antisense 3’-CGUAAAUCCCUCAUAAGAU-5’; siJAG1 Duplex2 sense 3’-AACAGGACAAACAAACAGG-5’ and antisense 3’-CCUGUUUGUUUGUCCUGUU-5’. HMEC-1 cells were transfected with siRNA (20nM for siDLL4 and 5nM for siJAG1) using HiPerfect (QIAGEN) according to manufacturer's instructions.HMEC-1 cells were suspended in medium containing 0.2% (w/v) carboxymethylcellulose and seeded in non-adherent 60-microwell minitrays (Sigma). Spheroids with defined size and cell number (500 cells/spheroid) were generated overnight. They were then embedded into Matrigel in a 24-well plate and allowed to polymerize before adding medium. Pictures were taken at 10× magnifications using an Axiovert 100M microscope . Capillary sprouting was assessed by measuring the average length of three longest sprouts per spheroid [2 incubator at 37°C for 2-3 days until the confluence of cell growth. The cells were then harvested for FACS staining by incubating with a primary antibody at 4°C for 30 minutes. Primary antibodies used include MedImm human anti-DLL4 blocking mAb (which cross-reacts with both human DLL4 and mouse DLL4) and in-house made mouse anti-JAG1 mAbs (clone 65D reacts with human JAG1 only with a high affinity while clone 142B cross-reacts with human JAG1 and mouse JAG1 with a low affinity compared to 65D). After washed with PBS for 3 times each 3-5 minutes, the cells were incubated with PE- or APC-conjugated secondary antibodies at 4°C for 30 minutes. After washed with PBS, the cells were sorted for GFP-negative and GFP-positive portions on the BD Fortessa X-20 FACS machine and analysed for the expression of DLL4 and JAG1 with the FlowJo software.Cell co-culture of retrovirus transduced tumour cells or EC cells, which are expressing GFP protein encoded by the retrovirus vector, with an equal amount of each parental tumour or EC line  (50:50 ratio) were performed in 6-well plates or 10-cm cell culture dishes in the CO3 in size [3 in size. Mice were sacrificed when reach to the criteria defined in the animal project license.Animal experiments were performed as previously described  under Ho in size . MedImm The mouse CD31 and NG2 staining protocol on frozen sections and quantification were performed as described previously . For theP < 0.05, two asterisks where P < 0.01 and three asterisks where P < 0.001.Prism software was used to analyse the data. The nalysis of variance (ANOVA) test was used to compare mean values among three or more data sets, and the Bonferroni's post-test to compare any two data sets among the three or more sets. Non-parametric Kruskal-Wallis test with Dunn's post-test comparison was also employed. A generalized linear model with random effects for tumour growth was used to analysis growth curves. ANOVA F-test was used to assess significance between curve fits. Kaplan-Meier survival curves were used to analyse the percentage survival of mice. All data were presented as Mean±SEM except when indicated specifically in figure legends. Statistical significance was indicated in figures with an asterisk where


The complex interplay of the tumour microenvironment (TME) and its role in disease progression and response to therapy is poorly understood. The majority of studies to date focus on individual components or molecules within the TME and so lack the power correlative analysis. Here we have performed a multi-parameter analysis of the TME in 62 resectable non-small cell lung cancer (NSCLC) specimens detailing number and location of immune infiltrate, assessing markers of cancer-associated fibroblasts, caveolin-1 and tenascin-C, and correlating with clinicopathological details, as well as markers of disease progression such as epithelial-to-mesenchymal transition (EMT). The influence of individual parameters on overall survival was determined in univariate and multivariate analysis and the combination of risk factors and interplay between components analysed. Low numbers of CD8 T cells, low stromal levels of caveolin-1 or high levels of tenascin-C were significant prognostic markers of decreased overall survival in both univariate and multivariate analysis. Patients with two or more risk factors had dramatically reduced overall survival and those with all three a median survival of just 7.5 months. In addition, low levels of tumour E-cadherin correlated with reduced immune infiltrate into the tumour nests, possibly linking EMT to the avoidance of CD8 T cell control. The multicomponent approach has allowed identification of the dominant influences on overall survival, and exploration of the interplay between different components of the TME in NSCLC. The influence of the tumour microenvironment (TME) upon non-small cell lung cancer (NSCLC) remains poorly defined with conflicting reports in the literature surrounding the influence of immune components as well as cancer-associated fibroblasts (CAFs) on disease progression and outcome. The radical success of the early trials of PD1/PD-L1 checkpoint inhibitor immunotherapies appears to demonstrate that the TME has a guiding influence on NSCLC disease outcome and points the way for further exploration of TME-targeted therapies [The complexity of the TME, comprising fibroblasts, endothelial cells, pericytes, immune infiltrate and other cells along with their secretions and extracellular matrix, make it a challenging system to study and many reports focus on one part in isolation, potentially overlooking important interactions. The major non-tumour cell type found in the NSCLC microenvironment are fibroblasts, which surround and penetrate through the tumour in a web-like pattern. This produces an architecture common to many solid tumours where groups of tumour cells are clustered in so called nests (or islets) and separated from adjacent nests by a swathe of fibroblasts, often including the vasculature. The origin and even definition of CAFs remains controversial  and indeBiomarkers of CAFs are beginning to show promise in the prognosis of a number of solid tumours but have been most extensively studied in breast cancer. Caveolin-1, when expressed by fibroblasts, appears to negatively regulate tumour cell growth and the loss of expression is strongly correlated with reduced overall survival, tamoxifen resistance and metastasis in breast cancer , 5 and hImmune infiltrate is commonly observed in NSCLC and is chiefly composed of T cells and macrophages. There have been conflicting reports on the importance of the composition and influence of location on disease progression. Al-Shibli et al.  found a In order to better understand the influence of the TME we sought to profile the major cellular TME components namely, fibroblasts, T cells  and macrophages and relate these to patient survival in univariate and multivariate analysis in the context of the known clinicopathological data.A cohort of 62 tumour specimens from patients with operable NSCLC stage 1-3) were analysed for their immune infiltrate by immunohistochemical analysis. The number of each cell-type and location in tumour nest or stroma was determined for total T cell (CD3), CD8 T cells (CD8), T-reg (FoxP3) and macrophages (CD68) and related to survival by Kaplan-Meier analysis  was performed including covariates age, gender, stage of disease and disease sub-type. Results of multivariate analysis for our investigational markers are shown in Table We determined the total number of the three independent risk factors  that each patient had and plotted this, sum of total risk factor against overall survival Figure . Total rUsing a multi-parameter approach to profile components of the TME we have been able to identify low CD8 number and absence of infiltration into the tumour nests as well as the over-production of Tn-C or reduced production of Cav-1 by CAFs as significant predictors of shortened overall survival in NSCLC. Uniquely by measuring all the parameters together we were able to determine that low levels of tumour E-cadherin, which appeared predictive of worse overall survival in univariate but not multivariate analysis, was a confounder of immune infiltration: presence of E-cadherin at moderate or high level was critical for the presence of CD8+ T cell, FoxP3+ T-regulatory cells and CD68+ macrophages in the tumour nests. While we only analysed two indicative markers here (E-cadherin and vimentin) any shift to a mesenchymal phenotype caused by EMT would cause loss of E-Cadherin and may directly inhibit the CD8+ cytotoxic T cell control of cancer. The value of the multi-parameter approach was demonstrated by the strong predictive value of reduced overall survival for patients that had more than one of the three risk factors , with those with all three risk factors present having a median survival of just 7.5 months.nab-paclitaxel and carboplatin in advance squamous NSCLC high stromal Cav-1 correlated with improved response rate and increased survival [Whilst highly predictive of survival in breast cancer, low stromal Cav-1 has only recently been shown to be of prognostic significance in NSCLC. In a phase II trial of survival . The anasurvival , 24. Oursurvival . This meTenascin-C, a component of the extracellular matrix, is rarely seen in adult tissue other than in the bone marrow or lymphoid tissue or in cases of wound healing and solid tumours. While there is evidence in a number of tumour indications that larger isoforms are more predominantly expressed in tumour stroma there are few definitive reports correlating stromal Tn-C with disease progression or survival . Tn-C exIn this study we focussed on two proteins differentially expressed in cancer associated fibroblasts of other tumour indications but which have only very recently begun to be investigated in NSCLC. The results presented here indicate that a systematic investigation into role of cancer associated fibroblasts in the disease progression of NSCLC is warranted and may yield useful targets for future therapy.in situ by engaging tumour cells in the context of TGF-β1 rich microenvironment [RM) also includes up-regulation of checkpoint proteins including PD-1, rationalising the efficacy of PD-1 inhibitor trials and supported by in vitro tumour cell killing by CD8+ CD103+ cells when under PD-1 blockade [Cancer is often referred to as the wound that never heals because of the ever-present inflammation and similarities to the wound healing process . The proironment . The acqblockade . The maiBy simultaneously profiling the intertwined components of the TME, we have identified individual factors that predict overall survival, a connection between immune retention and E-cadherin and ultimately combinatorial risk factors which identify those with dramatically reduced overall survival.Fresh surgical material from tumour resections at Nottingham University Hospitals NHS Trust, were collected with informed patient consent and National Research Ethics Service (NRES) approval (NRES REC 10/H0405/6) between March 2010 and April 2012. Samples were used in accordance with NRES approval (NRES REC 08/H0403/37). Patients were followed up to April 2015. Histological subtype and tumour stage were determined by pathologists at Nottingham University Hospitals NHS Trust according to Word Health Organisation criteria .20) blocked with streptavidin and then biotin (Vector Laboratories). Sections were subsequently washed in buffer, blocked in 5% rabbit serum and then incubated with the primary antibody or isotype and concentration matched negative control. After washing, sections were incubated with a secondary biotinylated rabbit anti-mouse antibody (DAKO), washed and incubated in avidin and then biotin complex (ABC) solutions according to manufacturer's instructions (Vector Laboratories). Sections were washed incubated with 3,3’-diaminobenzidine (DAB) (DAKO), washed and counterstained with haematoxylin. Subsequently the sections were washed, dehydrated and cleared before mounting a coverslip with Distyrene Plasticizer Xylene (DPX).Serial 5μm thick sections of formalin-fixed and paraffin-embedded (FFPE) tissue were cut for immunohistochemical analysis by microtome and mounted on polylysine-coated slides. Following deparaffinisation and re-hydration antigen retrieval was performed by heating to 98°C in a temperature controlled microwave in retrieval buffer according to manufacturer's instruction. Sections were then quenched, blocked in a 3% hydrogen peroxide solution for 10 mins, washed  7 μg/ml, mouse anti-human CD8  3 μg/ml, mouse anti-human FOXP3  2.5 μg/ml, mouse anti-human CD68  0.02 μg/ml, mouse anti-human vimentin  4 μg/ml, mouse anti-human E-cadherin  3 μg/ml, mouse anti-human caveolin-1  0.5 μg/ml and mouse anti-human tenascin-C  5 μg/ml.2 field of view) using a Leica DFC480 colour-inverted microscope; with three tumour nest (TN) hotspot and three stromal (STR) hotspot images taken. The hotspots were defined as a field of view with the most apparent accumulation of stained cells in either areas of tumour epithelia or stromal cells (as determined by tissue architecture). The stained cells were subsequently counted using ImageJ version 1.46r, “cell counter” plug-in and a TN and STR total was obtained for each image. The sum of the TN and STR cell numbers from all 6 images was determined to allow the total ratio (TN/STR) to be calculated for a given tissue section.A total of six images for each tissue section were obtained at 20x magnification (18.2 mmOn six fields of view E-cadherin was quantified using ImageScope software (Version 11.1.2.760) with ‘Positive Pixel Count v9.1’ algorithm and thresholds set at high positive- 200, positive- 150, weak positive- 100. The algorithm generated an H-score using the formula: H-Score = (3 x percentage of high positive pixels) + (2 x percentage of medium positive pixels) + (1 x percentage of low positive pixels).Slides were visually scanned by two blinded operators for any vimentin staining in any tumour cells and presence of any vimentin in tumour was scored as positive.Sections were scored by three blinded assessors for the degree of stromal staining 0= 0-1%, 1= 2-10%, 2= 11-50%, 3=>50%. Where scores from each assessor varied the majority (2 of 3 scorers) was recorded.Kaplan-Meier survival analysis was performed in GraphPad Prism software (v6.04) and Log-rank (Mantel-Cox) test P value reported. Where appropriate the Gehan-Breslow-Wilcoxon test P value was reported which is weighted for early death. Where there were more than two groups log-ranks test for trend was also reported.For multivariate Cox-regression analysis data was analysed in SPSS Statistics  with gender, age, histological type and stage as covariates alongside data from immunohistochemical staining.Analysis of correlation between E-cadherin H-score and CD8 TN/STR ratio was performed in GraphPad Prism software using Spearman's non-parametric correlation. Difference in TIL TN/STR ratio or number of those patients with low E-cadherin H-Score and those with moderate or high E-cadherin was tested by Mann-Whitney unpaired non-parametric T-test (GraphPad Prism).


Re-exposure to chickenpox may boost varicella-zoster virus (VZV) immunity in the elderly. This secondary immune response is hypothesized to confer protection against herpes zoster. We longitudinally sampled 36 adults over the course of one year after re-exposure to chickenpox. The resulting 183 samples and those of 14 controls were assessed for VZV-specific T-cell immunity and antibody titres. The percentages of VZV-specific CD4+ IL-2-producing T-cells were increased in re-exposed grandparents compared to control participants up to 9 months after re-exposure. Using a longitudinal mixture modelling approach, we found that 25% and 17% of re-exposed grandparents showed a boosting of VZV-specific CD4+ IL-2-producing T-cells and VZV-specific antibodies, respectively. The antibody boosting occurred exclusively in cytomegalovirus (CMV) IgG-positive participants. CMV IgG-positive participants also had higher VZV IE62-specific CD4+ IFN-γ-producing T-cell percentages and VZV-specific antibody titres. The protective effect of re-exposure to chickenpox is likely limited, as boosting only occurred in 17–25% of the VZV re-exposed grandparents and for less than one year. As such, the analysis of the dynamics of the VZV-specific immune response after re-exposure to chickenpox can offer important insights into the fundamentals of the secondary immune response after real life re-exposure.Many studies have focused on the characterization of the immune response in an experimental design . However, the study of secondary immune responses in humans after a natural re-exposure has received far less attention. A notable exception is a series of studies that focused on immune responses following the re-exposure to varicella-zoster virus (VZV)5. This hypothesis has had an important influence on policy making concerning universal childhood VZV vaccination. Simulation models exploring this hypothesis concluded that diminished circulating chickenpox, after the introduction of universal chickenpox vaccination, would cause a temporary increase in herpes zoster incidence9. These simulation results are largely driven by the more intensive intergenerational contacts between children and their parents and grandparents and an assumed direct inverse proportionality between the number of contacts with chickenpox cases and the probability of developing herpes zoster. Since the disease burden - expressed as Quality Adjusted Life Years (QALYs) losses - is typically weighted 10 to 20 times more for an average herpes zoster case than for an average chickenpox case, the overall public health impact of universal childhood VZV vaccination produced by such simulations tends to be negative10. Observational data in countries with universal childhood VZV vaccination so far cannot convincingly reject the occurrence of such an undesirable population impact, leading to continuing hesitance towards introducing universal VZV vaccination in many countries worldwide.Furthermore, given that re-exposure to VZV is assumed to boost VZV cellular immunity (referred to as “exogenous boosting”) and that herpes zoster, the (symptomatic) reactivation of VZV that had previously remained latent in neural ganglia after chickenpox, is likely to be caused by a reduced level of VZV-specific cellular immunity, re-exposure to VZV was hypothesized to reduce the risk of herpes zoster4. The bulk of the burden of herpes zoster is, however, carried by older adults11. The boosting studies in young adults showed a boosting of the cellular immune response in 60–70% of participants, although the quantification was limited. In the current study, we set out to analyse the characteristics of the secondary immune response in grandparents, starting shortly after they contacted their grandchild experiencing chickenpox.Therefore, both for biomedical insights and public health, it is important to adequately assess the VZV-specific immune response following a re-exposure to chickenpox. Until now, only a few studies have investigated the VZV-specific immune response following re-exposure to varicella, and these have focused almost exclusively on re-exposed parentsThirty-six grandparents  were recruited after being re-exposed to chickenpox for a minimum of four hours and within five days of varicella exanthema eruption in their grandchildren. These chickenpox patients were either determined to be VZV PCR-positive by skin or saliva swabs (21/25 children) and/or clinically diagnosed by a medical doctor. The re-exposed grandparents were longitudinally sampled beginning “as soon as possible after re-exposure” and at 3 weeks, 6 weeks, 15 weeks, 30 weeks and up to 52 weeks after re-exposure. Fourteen individuals  without known re-exposures to chickenpox during the last year were selected as controls. Nine of the 14 controls were longitudinally sampled up to 52 weeks after the first sample was taken. Five controls contributed a single blood sample.This study was approved by the ethics board of the Antwerp University Hospital. All methods were performed in accordance with the relevant guidelines and regulations when applicable. Written informed consent was obtained from all study participants.Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus gradient separation  from freshly obtained heparinized blood. The PBMCs were frozen in 90% foetal bovine serum  supplemented with 10% dimethyl sulphoxide . Filled cryovials were placed in Nalgene cryoboxes before freezing at −80 °C, and after 24 hours, the cells were stored in liquid nitrogen. The thawing of the cells for batch analysis was performed in a water bath at 37 °C. After washing with pre-heated (37 °C) medium supplemented with 10% FBS, a flow cytometric analysis was performed. An intermittent batch-wise analysis was performed in order to minimize inter-assay variability.PBMCs were either unstimulated or stimulated overnight with phytohaemagglutinin  + ionomycin 1 µg/mL), VZV IE62 PepMix, VZV IE63 PepMix or VZV gE PepMix . The overnight stimulation was performed in the presence of GolgiPlug (BD). Afterwards, the cells were washed and stained using the antibody panel described in Table  µg/mL, VFlow cytometric analyses were performed on a BDFACS Aria II flow cytometer . FlowJo software  was used for data analysis.2 and 5% CO2. For the 1-week-culture protocol, 30 ng/mL interleukin (IL)-21 (at day 0) and 12.5 U/mL IL-2, 5 ng/mL IL-7 and 5 ng/mL IL-15 (at day +2)  were added to maintain the culture. At day +6, the culture was restimulated by adding 20 µg/mL VZV lysate. For both protocols, CD8+ T cells were isolated from the (re)stimulated PBMCs by positive selection using magnetic CD8 microbeads  according to the manufacturer’s instructions. In a second step, CD8+ CD137+ T cells were further isolated using a flow cytometric sorting process. Briefly, the isolated CD8+ T cells were stained with anti-CD3, anti-CD8, and anti-CD137 antibodies and a Live/Dead stain, followed by a sorting of the living CD3+CD8+CD137+ T cells with a FACSAria II instrument . Total DNA from the isolated cells was obtained using a QIAamp DNA Micro Kit  according to the manufacturer’s instructions. The eluates of the 1-day and 1-week cultures were pooled and frozen at −20 °C until further processing. The T-cell receptor beta (TCRβ) sequencing on the DNA was performed by Adaptive Biotechnologies .PBMCs from three re-exposed grandparents, collected approximately 15 weeks after re-exposure, were thawed and recovered overnight in Iscove’s Modified Dulbecco’s Medium . One half of the PBMCs was used in a 1-day-culture protocol, while the other half was used in a 1-week-culture protocol. In both protocols, the PBMCs were stimulated with 20 µg/mL Varicella Zoster grade 2 Antigen (VZV lysate)  at day 0 and incubated for 24 hours at 37 °C, 3.5% ODNA from the three grandparents was extracted from 200,000 CD8+ T-cells per subject at three different time points, without prior selection after stimulation, as detailed above for the VZV-specific TCR sequencing. The TCRβ sequencing was performed by Adaptive Biotechnologies .3. The presence of IgGs directed against CMV pp150, pp28, p38, and p52 in thawed serum was determined using a Roche Elecsys assay .Serum was stored at −80 °C until further processing. VZV-specific IgG antibody titres (mIU/ml) in thawed serum were determined using VZV-infected cell lysate on a Liaison XL instrument with a DiaSorin kit The data management and analyses were performed using SPSS v20 and Monolix v4.3.3. The results were termed “significant” if the p-value was < 0.05.3). Due to the scarcity of IL-2-producing CD8+ T-cells, these data were not shown in the paper. In addition, we used a data processing algorithm per setting  that excluded cytokine-producing T-cell percentages if (1) the stimulus did not evoke a significant cytokine response AND the count of parent cells was lower than 10000 or if (2) the stimulus did not evoke a significant cytokine response AND the background cytokine response (the cytokine response in a sample without stimulus) was higher than 0.15%. We used basic nonparametric statistics  to compare the immune responses between the different time points for the re-exposed grandparents and the median immune response for each control. Based on a qualitative assessment of previously published results13, the different time points were categorized as ≤1 week, >1 week and ≤4 weeks, >4 weeks and ≤7 weeks, >7 weeks and ≤20 weeks, >20 weeks and ≤39 weeks, and >39 weeks. Each time category thus contained the results from samples obtained within the defined time period corresponding to the category. Not all individuals could be sampled in each time category, so the number of included data points varies between time points. To formally identify different subsets of individuals, we also applied a mixture modelling approach that was based on the longitudinal dynamics of the VZV-specific immune response. This could be modelled using a system of differential equations (in the case of antibodies) or damped exponential equations (in the case of T-cells). To achieve this, we developed a longitudinal mixed (regression) model. These are mathematical models that include parameters with fixed and random components. Time is a continuous variable in these models. The fixed component of a parameter can be interpreted as a conditional mean value for the parameter. The random component, however, represents an individual-specific deviation from the conditional mean value. This means that heterogeneity can be modelled using random effects. In our models, the random parameters were assumed to follow a lognormal distribution. In addition to the longitudinal mixed modelling, we also used a between-subjects mixture modelling approach, as available in Monolix 4.3.3 14. Here, we assume that the immune response  of each individual can be described by different equations with (fixed + random) parameters. In the mixture modelling approach, the statistical algorithm will estimate for each individual which equation is most adequate in describing the individual-specific immune response at all time points. The longitudinal data analyses were performed using Monolix 4.3.3 software. The parameter estimates were obtained using the Maximum Likelihood Estimation by the Stochastic Approximation Expectation Maximization (SAEM) algorithm. Stochastic approximation was used to calculate the Fisher Information Matrix and subsequently the standard errors per parameter. Importance sampling was used to determine the log-likelihood per model. The models were compared using Akaike’s Information Criterion 15. In the first step, the application of random effects was assessed in a forward regression algorithm, where we started from no random effects (thus only fixed effects) and subsequently computed the AIC value after adding a random effect to each of the parameters separately. In a second step, we started from the model with the best AIC for the fixed + random effect for a certain parameter and computed the AIC values again when adding random effects to the other parameters that yielded convergence in the first step (and so on). For several models, likelihood computation was not possible (no MCMC convergence or ill-defined model).Per individual, sample and stimulation condition, the net percentages of IFN-γ or IL-2-producing CD3+CD4+ T-cells and CD3+CD8+ T-cells were calculated using Fisher’s Exact test , central memory (CCR7+CD45RA−), late effector (CCR7−CD45RA+) and naïve (CCR7+CD45RA+) T-cells. Next, we assessed  for each subtype and for each VZV peptide  whether a change was noted for the T-cells from individuals who showed cytokine production against the peptide  were cultured using VZV-lysate. After one week, the CD137+CD8+ T-cells were sorted for DNA extraction. Next, TCRet al.17):1 and AB2 representing the post-re-exposure VZV-specific antibody titres for two different trajectories, pS the antibody production rate for short-living antibody secreting cells (SASC), S0 the initial SASC concentration, uS the SASC decay rate, pL the constant antibody production rate by long-living antibody-secreting cells (LASC), uAB the antibody decay rate, and iAB the initial antibody concentration . The main goal of the mathematical models in this paper was not to arrive at an exact representation of the underlying immunological processes, but rather to produce adequate model fits to our immunological measurements. Six out of the 36 (17%) re-exposed grandparents were found to follow the boosting dynamics represented by the differential equation for AB1, whereas 30/36 grandparents were found to have a constant value represented by the equation for AB2  for two different types of trajectories, a, b, c and d parameters for the damped exponential function, and e a constant number of cytokine-producing cells for individuals following equation T2.Next, we focused on longitudinal mixture modelling of VZV-specific cellular immunity. Here, due to higher experimental variability, we chose to work with more flexible curve fitting by means of a damped exponential equation model:We chose to only model CD4+ IL-2+, CD4+ IFN-γ+ and CD8+ IFN-γ+ T-cells against VZV IE62 because of the higher prevalence of zero value results for the other conditions. Moreover, no MCMC convergence was obtained for the CD4+ IFN-γ-producing T-cell dataset. Although the modelling analysis for the CD8+ IFN-γ-producing T-cell data showed MCMC convergence, the goodness of fit was much better for the CD4+ IL-2-producing T-cell data against VZV IE62  had an effect on exogenous boosting. Using a contingency table approach (Fisher’s Exact test) on the VZV antibody and CD4+ IL-2 VZV IE62 T-cell data, we found that gender had no significant effect on boosting nor did CMV seropositivity on the CD4+ IL-2+ boosting for VZV IE62-specific T-cells. However, all six individuals who were determined to have been exogenously boosted using the longitudinal mixture modelling approach were CMV seropositive versus 16/30 that were not boosted .Next, we assessed whether CMV seropositivity had an effect on the VZV-specific immune response 21. However, re-exposure to chickenpox has been shown to induce the boosting of VZV-specific T-cells in 60–70% of re-exposed parents4. The ad hoc approach towards defining “boosting” in those previous parent studies contrasts with our formal statistical approach to detect boosting events in the current study, and may have led to the finding of a relatively high proportion of boosted parents. In addition, it has been shown that the T-cell response after VZV vaccination decreases with age13. If this finding can be extrapolated to exogenous re-exposure, this could mean that grandparents are less susceptible to boosting than parents. The latter hypothesis is further supported by the recent suggestions that immunosenescence is not only caused by CMV-dependent mechanisms but CMV-independent mechanisms as well23.The lack of a pronounced and systematic boosting of VZV-specific immunity after exogenous re-exposure to VZV is noteworthy, as many vaccine-based studies have shown robust boosting of both VZV-specific antibodies and T-cells after vaccination, even in older individuals23. It has been argued that chronic CMV infection leads to an overall increased activation of the immune system23. Our study also showed overall higher  VZV-specific immune responses in CMV-seropositive grandparents. Together, our observations can be explained by a more activated and responsive immune system in CMV-seropositive adults. However, this does not align with our recent observation that CMV seropositivity is associated with an increased risk of HZ in adults younger than 50 years24. One alternative hypothesis could be that a chronic CMV infection, shown to have led to more differentiated and possibly less equipped T-cell immunity against various pathogens including VZV25, has weakened the immune barriers against VZV and thereby has led to a more pronounced in vivo spreading of VZV (and subsequent boosting). This hypothesis could explain why only CMV-seropositive grandparents showed a boosting of antibodies and why CMV-seropositive grandparents had higher cellular and humoral immune responses against VZV. Unfortunately, our study did not include a measurement of VZV viraemia. Interestingly, a recent serological study on VZV-specific antibody titres as a function of age also implied that chronic CMV infection could lead to more frequent VZV reactivation and subsequent boosting (termed endogenous boosting22). Both hypotheses (CMV as an activator of immunity versus CMV as a depressor of immunity) could explain the observations made in this study. More research is clearly needed to disentangle the effects of CMV and ageing on VZV dynamics.Interestingly, our study showed that CMV-seropositive grandparents were more likely to show boosting of VZV-specific antibodies after re-exposure to chickenpox. Some  studies have shown an increased immune response after vaccination against influenza in CMV-seropositive individuals6. Many cost-effectiveness studies have concluded that the decrease of contacts with chickenpox patients after the implementation of universal chickenpox vaccination would lead to a significant increase of herpes zoster incidence, which could overshadow the positive effects on the reduction of chickenpox26. These simulation models were based on the deterministic modelling estimation that re-exposure to chickenpox would lead to a duration of protection against herpes zoster for 20 years27. However, a recent individual-based model for VZV estimated the duration of exogenous boosting to last for 1–2 years9. Our experimental study supports the latter modelling estimation through our findings that (average) boosting of cellular immunity was not noticeable by one year after re-exposure (vs. control participants). This means that our data suggest that the cost-effectiveness analyses for chickenpox vaccination should be redone using the new experimental results, potentially leading to a policy in favour of universal chickenpox vaccination.The results from this study are of importance beyond the realm of fundamental immunology, as the exogenous boosting hypothesis is considered to be an obstacle for the implementation of universal chickenpox vaccination28. However, the estimated number of vesicles (more or less than 50) was in our study not associated with the boosting of antibodies or CD4+ IL-2-producing T-cells (data not shown). In addition, the lack of “true” baseline samples could have led to an underestimation of boosting. Furthermore, more frequent sampling during the first weeks after re-exposure could also give more insights into boosting dynamics, as recent studies on T-cell dynamics and TCR dynamics following VZV vaccination highlighted a peak in VZV-specific T-cells about one week after vaccination20. Future larger studies should also encompass both parents and grandparents so that the effect of immunosenescence could receive more attention. These larger studies will be more suitable for the mathematical classification of boosted and non-boosted individuals. Indeed, we noted that the formal classification of boosting did not correlate with our visual classification in all instances. Moreover, it would be interesting if other cytokines besides IFN-γ and IL-2 could also be included in the assessment.Our pilot study encountered several limitations, including the modest sample size, the exclusive focus on grandparents and the exclusive use of peptide mixes for the stimulation of PBMCs. We note that differences in exposure characteristics might also have had an effect on boosting potential as more infectious children (more than 50 vesicles) have been estimated to have mildly increased infectivityIn conclusion, we have found empirical evidence for short-lasting boosting of the cellular immune response against VZV in a minority of grandparents after re-exposure to chickenpox. CMV-seropositive individuals were shown to have higher cellular and humoral immune responses against VZV than CMV-seronegative individuals. In addition, the VZV-specific humoral immune response was boosted only in CMV-seropositive grandparents.Supplementary FiguresSupplementary Table 1Supplementary Table 2


Ailian Liu's degree should have appeared as PhD.In the article, “Malignant transformation of a mature teratoma of the adrenal gland: A rare case report and literature review”,


They are in close contact with SMCs, which possess multiple caveolae at the contact sites. Immunohistologically we identified such cells with antibodies against vimentin and PDGFRα, but not CD34 and cKIT. With Next Generation Sequencing we searched for highly expressed genes in the media of lymphatic collectors, and found therapeutic targets, suitable for acceleration of lymphatic contractility, such as neuropeptide Y receptors 1, and 5; tachykinin receptors 1, and 2; purinergic receptors P2RX1, and 6, P2RY12, 13, and 14; 5-hydroxytryptamine receptors HTR2B, and 3C; and adrenoceptors α2A,B,C. Our studies represent the first comprehensive characterization of human epifascial lymphatic collectors, as a prerequisite for diagnosis and therapy.Millions of patients suffer from lymphedema worldwide. Supporting the contractility of lymphatic collectors is an attractive target for pharmacological therapy of lymphedema. However, lymphatics have mostly been studied in animals, while the cellular and molecular characteristics of human lymphatic collectors are largely unknown. We studied epifascial lymphatic collectors of the thigh, which were isolated for autologous transplantations. Our immunohistological studies identify additional markers for LECs . We show and confirm differences between initial and collecting lymphatics concerning the markers ESAM1, D2-40 and LYVE-1. Our transmission electron microscopic studies reveal two types of smooth muscle cells (SMCs) in the media of the collectors with dark and light cytoplasm. We observed  The lymphatic vascular system is composed of initial lymphatics  pre-collectors, collectors, lymph nodes, and trunks . InitialDespite their great importance for the active transport of lymph, human lymphatic collectors have not been characterized well, at both morphological and molecular levels. The majority of studies were performed on animals such as dog, guinea pig, sheep, cow, rabbit, and rat –16. A feWhereas initial lymphatics do not possess any mural cells, not even pericytes, lymphatic collectors have a tunica media (media) and a tunica externa (adventitia). The phasic, peristaltic contractions of lymphatic collectors are based on the structure and function of smooth muscle cells (SMCs) in the media . The expvasa vasorum and ICLCs extending into the media. We confirm heterogeneity between LECs of initial lymphatics and collectors, identify new markers for LECs, show expression of vimentin and PDGFRα in ICLCs, and indicate molecular targets in the media of lymphatic collectors for the control of contractility.‘Lymphedema is a significant global problem, and its incidence will increase with a population that is living longer. In addition, because of the interrelationship between the lymphatic system and adipose tissue, the obesity epidemic has led to a rapid rise in this complication’ . InducinAutologous lymphatic collector transplantation (n = 22) was performed on breast cancer patients suffering from secondary lymphedema of the arm after standard treatment. A normal lymphatic collector was taken from the hypodermis of the thigh, transplanted into the axillary region and connected proximally and distally to lymphatic collectors as described previously . The colFor immunofluorescence studies, specimens were fixed for 20–25 min in 4% paraformaldehyde (PFA), rinsed in PBS, transferred into 10% and 30% sucrose in PBS, and embedded in tissue freeze medium . Sections of 12–14μm were incubated with the following primary antibodies: Rabbit-anti-human CCBE1 , rabbit-anti-human ESAM-1 , goat-anti-human smooth muscle α-actin (SMA) , mouse-anti-human CD31 , mouse-anti-human CD117 , mouse-anti-human D2-40 , rabbit-anti-human Lyve-1 , rabbit-anti-human Prox1 , mouse-anti-human vimentin , mouse-anti-human PDGFRα , mouse-anti-human CD34 . Secondary antibodies were: goat-anti-mouse Alexa 488/594, goat-anti-rabbit Alexa 594, donkey-anti-goat Alexa 488 . Sections were counter-stained with Dapi and mounted under cover slips with Fluoromount-G . Photos were taken with AxioImagerZ1 .Specimens were fixed with original Karnovsky’s fixative over-night , washed Lymphatic endothelial cells (LECs) were isolated from lymphatic malformations of two young children as described previously. The cells possess molecular characteristics of initial lymphatics . Purity Total RNA was isolated from LECs and BECs using peqGOLD Tri-Fast  according to the manufacturer’s instructions. Lymphatic collector samples of six female donors (age between 44 and 61 years) were isolated from surrounding connective tissue and snap-frozen in liquid nitrogen. The tissue was finely ground in liquid nitrogen using pestle and mortar, and immediately taken up in Tri-Fast reagent (PeqLab). Subsequently total RNA was prepared according to the manufacturer’s instructions.RNA quality was assessed by measuring the RIN (RNA Integrity Number) using an Agilent 2100 Bioanalyzer . Library preparation for RNA-Seq was performed using the TruSeq™ RNA Sample Prep Kit v2  starting from 1000 ng of total RNA. Accurate quantification of cDNA libraries was performed with the QuantiFluor™ dsDNA System (Promega). The size range of final cDNA libraries was determined applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent (average 350 bp). cDNA libraries were amplified and sequenced by use of cBot and HiSeq2000 from Illumina . Sequence images were transformed with Illumina software BaseCaller to bcl files, which were de-multiplexed to fastq files with CASAVA v1.8.2.Quality check was done with fastqc . Read alignment was performed using STAR v2.3.0 to the hg19 reference genome. Data were converted and sorted by samtools 0.1.19 and reads per gene were counted with htseq version 0.5.4.p3. Data analysis was performed using R/Bioconductor (3.0.2/2.12) loading DESeq, gplots and goseq packages. Candidate genes were filtered to a minimum of 1x fold change and FDR-corrected p-value <0.05. For functional analysis gene ontology enrichment was tested accounting for gene length via R-package goseq. Sequence data were deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE87360. A subset of data showing lymphatic collector-expressed genes are presented as For the treatment of secondary arm lymphedema in breast cancer patients, autologous transplantation of functional lymphatic collectors was performed. The transplants were taken from the thigh. In the hypodermis of the thigh, there are numerous lymphatic collectors so that a single collector can be removed without the risk of provoking leg lymphedema. The collectors of the thigh were identified surgically after intradermal injection of Patent Blue into the first interdigital spaces . The epiVasa vasorum of lymphatic collectors have previously been found in the adventitia [Vasa vasorum in the media were made up of capillaries with pericytes invested by a common basal lamina  LECs derived from initial lymphatics, and ii) BECs derived from dermal microvessels. The adventitia of the collectors was removed. Since the majority of cells in the collectors are SMCs, it could be expected that the collector-expressed genes found simultaneously in both of the lists were SMC genes. We set the threshold for the log2 fold change (Log2FC) at 2, and found 2518 candidate genes in collectors as compared to LECs , and 215The assumption that the majority the collector-expressed genes, which appeared in both of the lists, were SMC genes, was supported by the very high expression of: actin, both the smooth muscle-type (ACTA2) and the enteric-type (ACTG2), as well as desmin (DES), the myokine decorin (DCN) , and its2A,B,C , 5-hydroxytryptamine (serotonin) receptors HTR2B, and 3C; purinergic receptors , neuropeptide Y receptors 1 and 5 , tachykinin receptors 1 and 2 , and vasoactive intestinal peptide receptor 2 (VIPR2) as well as pituitary adenylate cyclase-activating polypeptide type I receptor 1 (ADCYAP1R1).Improving the contractility of lymphatic collectors is a promising therapeutic option for the treatment of lymphedema, which may be achieved by e.g. activation of the sympathetic, serotonergic, purinergic and neuropeptidergic systems. In superficial human thigh lymphatic collectors, we detected high expression of adrenoceptors αvasa vasorum, which we observed for the first time in the media of the largest collectors.The lymphatic vascular system consists of initial lymphatics, pre-collectors, collectors, lymph nodes, and trunks. These sections of the lymphovascular system differ with respect to their morphology, function and molecular setting. Initial lymphatics take up interstitial fluid, collectors and trunks possess transport functions, and lymph nodes facilitate the intimate contact between lymph and immune cells. While initial lymphatics are characterized by inter-endothelial valves, which allow the influx of interstitial fluid , lymphatCCBE1 gene cause a highly complex syndrome, the Hennekam syndrome. The affected children show multiple defects including mental retardation, facial dysplasia, hypoproteinemia, as well as generalized lymphedema [Additionally, we identified new markers for LECs of both initial and collecting vessels. These are CCBE1 and vimentin. CCBE1 is a large ECM protein with multiple binding capacities for e.g. calcium, collagen and VEGF-C . Mutatiomphedema . The conmphedema . The termphedema . Here weIn LECs of lymphatic collectors, we also identified expression of the WNT-signaling mediator β-catenin. WNT-signaling has rarely been studied in lymphatics (for review see: ). We havAutonomous, temperature-dependent contractions of lymphatic collectors have been observed quite some time ago, first in animals  and lateIn appr. 5% of cells derived from sheep lymphatic collectors, McCloskey and coworkers (1999) detected a hyperpolarization-activated inward current , indicative of pace-maker cells (ICLCs) . As notePDGFRα gene cause gastro-intestinal stroma tumor (GIST), a tumor originating from interstitial cells of Cajal in the gastro-intestinal tract [Interestingly, the expression of PDGFRα in human ICLCs is in line with the observation that mutations in the al tract . Howeveral tract . Using tal tract .Activation of SMCs in lymphatic collectors represents a promising target for pharmacological treatment of lymphedema. The SMCs of lymphatic collectors represent a functional syncytium . InteresA global characterization of human lymphatic collectors has not been performed yet. This knowledge, however, is the prerequisite for targeted therapeutic interventions to improve e.g. inflammatory diseases (lymphangitis) or contractile functions of collectors. The latter may be applicable to millions of patients worldwide suffering from secondary lymphedema. We therefore applied NGS and compared isolated thigh collectors with cultures of isolated LECs and BECs. We found 2518 collector-expressed genes in comparison to LECs, and 2151 collector-expressed genes in comparison to BECs. The overlapping genes in both lists mainly seem to represent SMC-expressed genes, as confirmed by the high expression of typical SMC genes highlighted in the However, although there is evidence that numerous of the genes listed in the 2 adrenoceptors ADRA2A, ADRA2B, and ADRA2C, which are coupled to G-proteins. Besides effects in the central nervous system, they control SMC functions such as contraction of spincters in the gastrointestinal (GI) tract, but also decrease of motility of SMCs in the GI tract, as well as constriction of certain arteries and veins [For the treatment of lymphedema adrenergic and peptidergic receptors appear to be of greatest interest. In animals, noradrenergic, purinergic, cholinergic and peptidergic nerves have been found along lymphatic collectors , and thend veins –69.The 5-hydroxytryptamine (serotonin) receptors HTR2B and HTR3C, which we found in the collectors, appear to be of special interest. Serotonin is a potent inducer of contraction of blood vessels, and also causes psychological effects in the central nervous system. Most of the body’s serotonin is located in the enterochromaffin cells of the GI tract, where it promotes motility . A similPurinergic receptors exist with numerous subtypes , and are widely distributed in the organism. In lymphatic collectors, we detected the purinergic receptors P2RX1, P2RX6, P2RY12, P2RY13, and P2RY14. P2Y receptors are G-protein-coupled receptors (GPCRs) . P2RY12 Neuropeptide Y (NPY) is a 36-amino acid peptide, which exerts a number of divers effects such as control of psychomotor activity, food intake and cardiovascular activity , 75. NPYVasoactive intestinal peptide (VIP) is a 28-amino acid peptide hormone, which stimulates contractility of the heart and relaxes SMCs of the aorta, trachea, gall bladder and gut . We deteex vivo in rat mesenteric lymphatics [Furthermore, in the collectors, we identified tachykinin receptors 1 and 2 , which are GPCRs that bind substance P (SP). SP, an 11-amino acid peptide, was originally identified by its ability to cause intestinal contractions . SP indumphatics , and, inmphatics . And amomphatics , 84.The huge number of specific agonists and antagonists that exist for the adrenergic, serotonergic, purinergic and peptidergic receptors in combination with the possibility to visualize pumping of lymphatic collectors live in the dermis of patients with the ICG method opens up a new field of therapeutic approaches for lymphedema.S1 TableGenes are listed alphabetically by HGNC Symbol. Log2FC: Log2 fold change; FDR: false discovery rate. Highly expressed SMC genes, which appear in both of the lists and are discussed in the manuscript, are marked in green.(PDF)Click here for additional data file.S2 TableGenes are listed alphabetically by HGNC Symbol. Log2FC: Log2 fold change; FDR: false discovery rate. Highly expressed SMC genes, which appear in both of the lists and are discussed in the manuscript, are marked in green.(PDF)Click here for additional data file.


Sinonasal glomangiopericytoma is a benign rare tumour of pericytes that accounts for less than 0.5% of all sinonasal tumours. It is an indolent tumour with a macroscopic appearance of common inflammatory polyps.We report the case of a 55-year-old male who presented with right nasal obstruction. CT and MRI examinations demonstrated a soft-tissue mass that obstructed mainly the right nasal cavity. Biopsy revealed glomangiopericytoma. The tumour was treated with preoperative embolisation followed by complete endoscopic resection. Very few cases have been reported to be treated in this way. Sinonasal glomangiopericytoma (GPC) is a rare mesenchymal tumour of the sinonasal tract characterised by a prominent perivascular growth pattern with a low malignant potential –4. This We report the case of a 55-year-old male who was treated with preoperative embolisation with microparticles and complete endoscopic excision. We describe the clinicopathologic, radiologic, and surgical characteristics of the tumour and review the literature.A 55-year-old male presented to the ENT outpatient clinic complaining of right nasal blockage during the last six months. Endoscopy revealed a polypoid mass that filled the right nasal cavity and caused deviation of the lateral nasal wall. The patient’s medical history and laboratory studies were unremarkable.A computed tomography (CT) scan showed a well-defined, sharply enhancing mass lesion in the right nasal cavity extending into the posterior ethmoid air cells with minimum projection to the nasopharynx. The medial wall of the orbit was preserved, and there was no intracranial extension. The mass measured 4 x 3 x 1.5 cm. Retained secretions and inflammatory changes to the sphenoid and maxillary sinuses were also seen. No lymphadenopathy was identified .Magnetic Resonance Imaging (MRI) revealed an ovoid encapsulated mass occupying the right nasal cavity extending into the ethmoid air cells and nasopharynx. Enhancement of the mass was heterogeneous on T1-weighted images following intravenous administration of gadolinium. The signal was heterogeneous on T2-weighted images .A biopsy was performed after preparation for any possible bleeding including nasal vasoconstrictors and general hypotensive anaesthesia, and this showed glomangiopericytoma. Histological examination of the biopsy specimen showed sheets of benign spindled cells arranged in a syncytium separated by prominent vessels. Pleomorphism, necrosis, and increased mitoses were not present. Immunohistochemistry showed positivity to vimentin, SMA, with few cells positive with CD34 and negative reactions with S-100 protein, desmin, and myogenin .Because of the bleeding propensity of the tumour, preoperative embolisation was planned. Under general anesthesia and systemic heparinisation, the right common femoral artery was punctured and a 5F diagnostic catheter was used to selectively catheterise both external carotid arteries. The angiographic control demonstrated arterial supply to the mass by both internal maxillary arteries . Althougμm in size . The final angiographic control showed complete devascularisation of the tumour . The vessels were embolised with microparticles measuring 350–500 e tumour . Immediae tumour . CompletSinonasal glomangiopericytoma (GPC), also known as haemangiopericytoma (HPC), was first reported by Stout and Murray in 1942 [GPC is a rare, mesenchymal tumour of the sinonasal tract characterised by a prominent perivascular growth pattern with a low malignant potential, accounting for less than 0.5 % of sinonasal tumours. The proposed cell of origin is a modified perivascular glomus-like myoid cell , 3, 5.The World Health Organisation in 2005 identified it as a unique sinonasal low malignancy lesion and proposed the term ‘glomangiopericytoma’ and ‘myopericytoma’ because of its similarity with glomus tumours.This tumour has been known as HPC-like tumour or sinonasal HPC , 6.CTNNB1 gene [GPC belongs to the category of borderline and low-malignant-potential soft tissue tumours of the nose and paranasal sinuses. It differs from conventional soft tissue HPC in location, histologic findings. and biologic behaviour with lower rates of metastasis and mortality as compared to soft-tissue HPC –7. The pNB1 gene .Patients across a broad age range may be affected, predominantly in the sixth and seventh decade of life in both genders, with a small predilection in females. Clinically, it may present with nasal obstruction or repeated episodes of epistaxis. It only rarely may provoke pain because of local infiltration, headache, vision impairment, and local swelling , 7. SeveUsually because of the symptoms, patients are first evaluated endoscopically. Exact tumour extension is better appreciated with CT and MRI. Computed tomography invariably shows a soft-tissue mass with strong enhancement after contrast administration that may be mistaken for an inflammatory polyp. On T1-weighted MR images, the mass appears solid isointense with strong contrast enhancement, while on T2-weighted images the signal varies from iso- to hypo-intense, differentiating it from high-intensity inflammatory fluid. The vessel supply to the GPC is best displayed to digital subtraction angiography and is used to plan a preoperative embolisation. Transarterial embolisation is indicated in order to prevent massive surgical haemorrhage .et al [Sinonasal GPC is an indolent tumour with excellent prognosis after surgical resection. Most specimens are fragmented during endoscopic removal so the gross appearance of these lesions is generally not helpful. Watanabe et al  recognisDifferential diagnosis includes vascular and other tumours such as solitary fibrous tumour, lobular capillary haemangioma (pyogenic granuloma), angiofibroma, and glomus tumour. Solitary fibrous tumours (SFTs) consist of a cellular spindle cell proliferation effacing submucosal structures with staghorn-type vessels. They are rare in the sinonasal tract. Angiofibromas have abundant stromal collagen and a prominent vascular stroma. Lobular capillary haemangiomas (LCHs) in contrast to the GPC have a lobular architecture with staghorn-type vessels in the interlobular stroma . Glomus The therapy of choice is definitely wide field complete excision. Endoscopic surgery is preferred, because it offers more advantages compared with the external surgery. The overview is better and can establish more precisely the tumour insertion and its extension. Furthermore, loss of blood is decreased, the risk of damaging the lacrimal structures is reduced, and the natural physiology of the nose can be maintained. Factors not favourable for endoscopic surgery are septal deviation, tumours which are large or highly vascular and have orbital extension. Nevertheless, intracranial extension and pterygopalatine fossa involvement are not limits for endoscopic approaches , 15.When the tumour exhibits high vascularity, endoscopic resection is not excluded provided preoperative embolisation is performed. The benefits of this method are intraoperative bleeding reduction with better inspection of the tumour bed.We believe that preoperative embolisation prior to endoscopic excision to be the treatment of choice of large or highly vascularised tumour in order to lower the risk of the procedure. In the literature, preoperative embolisation before resection of GPC has been reported in less than 23 patients , 16.Local recurrence has been reported in 7–20% of cases, mainly because of incomplete surgical resection. Metastatic rates (5–10%) of these tumours are generally lower than those (12–60%) of HPC at other sites. Radiotherapy and chemotherapy can be used as both adjuvant therapy and palliation therapy for metastatic disease , 16.Sinonasal GPC has a relatively indolent behaviour with a potential for local recurrence but not metastatic disease. The diagnostic algorithm involves endoscopy, CT and MRI for lesion characterisation and definition of lesion extent. Angiography with superselective embolisation is indicated especially in the case of large or highly vascular tumours. It helps to reduce preoperative blood loss and operation time and adds to the safety of the procedure. Complete surgical resection is the mainstay of treatment and may be performed endoscopically, by external or combined approaches. Optimal clinical management requires a long-term follow-up of all cases.The authors declare that they have no competing interests.


The focus of this research was to present a data article for analyzing the cost of displacing a drilling fluid during the drilling operation. The cost of conventional Spud, KCl and Pseudo Oil base (POBM) muds used in drilling oil and gas wells are compared with that of a Reversible Invert Emulsion Mud. The cost analysis is limited to three sections for optimum and effective Comparison. To optimize drilling operations, it is important that we specify the yardstick by which drilling performance is measured. The most relevant yardstick is the cost per foot drilled. The data have shown that the prices for drilling mud systems are a function of the mud system formulation cost for that particular mud weight and maintenance per day. These costs for different mud systems and depend on the base fluid. The Reversible invert emulsion drilling fluid, eliminates the cost acquired in displacing Pseudo Oil Based mud (POBM) from the well, possible formation damage (permeability impairment) resulting from the use of viscous pill in displacing the POBM from the wellbore, and also eliminates the risk of taking a kick during mud change-over. With this reversible mud system, the costs of special fluids that are rarely applied for the well-completion purpose (cleaning of thick mud filter cake) may be reduced to the barest minimum. Specifications TableValue of the data•These data describe the volume and material estimate needed for each hole section and the type of mud necessary to achieve smooth drilling operation in each hole section.•The data showed the cost, the quantity of materials and sequence at which these materials are applied to achieve optimum displacement.•These data can be used to study the economic analysis of new mud systems proposed by researchers and also help to compare if these mud systems are economically viable.•These data can also be used to analyze and predict prices and/or build cost for drilling mud systems for a certain mud weight and daily maintenance expense.•The data reveals that cost varies according to the different mud types and are dependent on the base fluid phase.1The type of drilling fluid systems and the volume of drilling fluid needed for each hole section is summarized in The volume of drilling fluids needed for three sections was estimated and presented in The cost for drilling a typical well may be constant when drilled without any instability case. Instability during a drilling operation in wells can quickly escalate cost dramatically The build cost for a drilling fluid system is the price for the individual components and mixing requirements. The total build cost includes purchasing the initial drilling systems materials and the expenses involved with conditioning the drilling mud system in the well as it is drilled.2The water-based mud in the wellbore from the previous hole section is displaced and replaced with POBM drilling fluid. The first step is to lower the viscosity and gel strength of the water-based mud. The suggested method is to dilute the fluid with water to obtain a low rheology After drilling and prior to running completion hardware, the fluid in the borehole is often displaced to a water-based completion fluid, usually a solution of various salts. During this displacement, chemical washes and viscous spacers are placed in the solution to make surfaces water- wet, while helping to remove oil mud and residual oil-wet material from the borehole The viscosity and gel strengths of the POBM are low prior to displacement. The suggested method was to dilute the fluid with premix, base fluid or a thinner to obtain the low rheology if this is necessary. The optimal thinning of the POBM fluid will dictate how easy the mud will be displaced out of the hole.ID = Hole Internal Diameter, inchD = Hole Depth, ftThe volume of drilling fluid needed for each section was obtained using the equation below:Eqs. sxs = Sackslb/bbl = Pound per barrelbbl = BarrelEq.


We investigated whether tumour markers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment (CYFRA 21-1) in pleural effusions and serum can be used to distinguish pleural effusion aetiology.During the first thoracentesis, we measured pleural fluid and serum tumour marker concentrations and calculated the pleural fluid/serum ratio for patients diagnosed with pleural effusion, using electrochemiluminescence immunoassays. Receiver operating characteristic (ROC) analysis was carried out and the Hanley and McNeil method was used to test the significance of the difference between the areas under ROC curves (AUCs). In order to detect which tumour marker best discriminates between malignant and non-malignant pleural effusions and to establish the predictive value of those markers, discriminant function analysis (DFA) and logistic regression analysis were utilized.Serum tumour markers CYFRA 21-1 and NSE as well as pleural NSE were good predictors of pleural effusion malignancy and their combined model was found statistically significant . Respective ROC analysis showed significant discrimination value of the combination of these three markers (AUC = 0.79).Serum markers showed superiority to pleural fluid markers in determining pleural fluid aetiology. Serum CYFRA 21-1 and NSE concentrations as well as pleural fluid NSE values had the highest clinical value in differentiating between malignant and non-malignant pleural effusions. The combination of these three markers produced a significant model to resolve pleural effusion aetiology. Pleural effusion is the accumulation of liquid in the pleural cavity and, in most cases, is considered a result of a systemic or intrathoracic process. This pathological entity is a frequent problem in pulmonology and, though its incidence varies with clinical background, 90% of all pleural effusions are attributed to congestive heart failure, malignant processes, and pneumonia . The differentiation of exudates and transudates requires the evaluation of various biochemical parameters and their comparison in pleural fluid and serum. When differentiating transudates from exudates using classical Light’s criteria, it is helpful to recognize the pathogenic mechanism resulting in the pleural effusion. Recognizing the correct pathogenic mechanism is also useful for the purpose of differential diagnosis , neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment (CYFRA 21-1).During the first thoracentesis, a total of 110 pleural fluid and serum samples were collected prospectively from 110 consecutive patients diagnosed with pleural effusion and admitted to the Department of Pulmonology at Clinical Hospital Centre in Rijeka, Croatia, between November 2013 and November 2014. Measurements were made at the time of admission to the Clinical Hospital Centre. After excluding 10 patients with unknown pleural effusion origin, 100 patients formed the study group. The 10 excluded patients were not followed up because 7 patients refused additional treatment and/or diagnostics and 3 suffered a fatal outcome .Informed consent was obtained from all patients included in the study. All procedures were performed in accordance to the ethical standards of the institutional and national research committee, and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study protocol was authorized by the ethics committee of the relevant Clinical Hospital Centre.Pleural effusion was diagnosed after thorough anamnesis, physical examination (lung percussion and auscultation), and chest x-ray. Pleural effusion was confirmed by thoracentesis.Blood was sampled by venipuncture of antecubital veins. Pleural fluid was collected by a needle inserted between the seventh and ninth rib spaces, between the posterior axillary line and midline (thoracentesis). Both blood and pleural fluid were collected in 7 mL biochemical tubes without anticoagulants . Blood and pleural specimens were centrifuged at a relative centrifugal force (RCF) of 1917xg for 10 minutes and the supernatant was analysed immediately. Blood specimens were tested at least 30 minutes after sampling, whereas pleural fluid samples were analysed immediately without previous storage. Tumour marker concentrations were determined by electrochemiluminescence immunoassays on the Cobas® e601 analyser . The readers of the tests and reference standards were professionals, blind to the results of the other test.Transudative and exudative pleural effusions were differentiated by Light’s criteria: effusion/serum protein ratio > 0.5, effusion/serum LD ratio > 0.6, effusion LD activity greater than two-thirds the upper limit of the laboratory’s reference range for serum LD  and Statistica version 13 . Variables were tested for normality using a Kolmogorov-Smirnov test. Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and those with non-normal distribution as median and interquartile range (IQR). To test the difference in tumour marker concentrations between malignant and benign pleural effusions, we used the non-parametric Mann-Whitney U-test because the variables were not normally distributed. To determine the clinical usefulness of tumour markers in the studied group and assess the performance of tumour markers in distinguishing malignant and non-malignant pleural effusions, the marker values were reviewed by a receiver operating characteristic (ROC) analysis. The area under the ROC curve (AUC) serves as an overall measure of a biomarker/diagnostic test’s accuracy , negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-)and the AUC. We used Hanley and McNeil’s method for pairwise comparison of ROC curves in order to test the difference between the AUCs. P < 0.05 was considered significant.In order to detect the tumour markers which best discriminate between malignant and non-malignant pleural effusions, discriminant function analysis (DFA) was employed. Logistic regression analysis was performed in order to establish the predictive value of those markers.We calculated the required sample size using MedCalc software assuming that the desired statistical power of the test is 90% with a confidence level of 95% and the ratio of sample sizes in the negative and positive groups is 1. We wanted to show that the expected AUC of 0.7 for a particular test is significant from the null hypothesis value of 0.5. The required sample size was calculated to be 82. The present study had a slightly larger sample size to ensure that we would have enough results if some of the patients were excluded later.The study group consisted of 73% males . The median age of the study group was 71 years (range 22 - 92 years). In 88 patients (88%), we found a unilateral pleural effusion, whereas 12 (12%) patients were diagnosed with a bilateral effusion. The time interval between defining the pleural effusion as malignant or non-malignant and the determination of tumour marker concentrations was approximately 7 days. No adverse events occurred as a result of venipuncture or thoracentesis.According to Light’s criteria, pleural exudates made up 71% (N = 71) of the study group; the other 29% (N = 29) were pleural transudates . The maiPrimary tumour sites in malignant pleural effusions were distributed as follows: lung (N = 17), pleura (N = 15), ovary (N = 4), colon (N = 4), stomach (N = 3), breast (N = 2), prostate (N = 2), malignant disease of unknown origin (N = 2), skin (N = 1), soft tissues (N = 1), pancreas (N = 1), oesophagus (N = 1), hypopharynx (N = 1), and lymph nodes (N = 1). The non-malignant or benign pleural effusion group (N = 45) consisted of all patients with pleural transudates or non-malignant pleural exudates, who were then compared to the patients diagnosed with malignant pleural effusions (N = 55). The median age of the patients with malignant pleural effusions was 70 (48 – 90) years; whereas the median age of the patients with non-malignant pleural effusions was 73 (22 – 92) years.The gender distribution was 39 males and 16 females in the malignant group and 34 males and 11 women in the non-malignant pleural effusion group. The concentrations of tumour markers CEA, NSE, CA-125, and CYFRA 21-1 in pleural fluid (P), serum (S) and their pleural fluid/serum ratios (R) in the malignant and non-malignant patient group are presented in The ROC analyses of the mentioned tumour markers  are shown in After pairwise comparison of all 12 ROC curves, significant differences were shown between AUCs of (S) CYFRA 21-1, (R) CEA, (P) CEA, (S) NSE, (P) NSE, (P) CYFRA 21-1, while no difference was found comparing AUCs of (P) CA-125, (R) CA-125, (S) CA-125 and (R) CYFRA 21-1 (data not shown). These results show that there are significant differences between diagnostic values of these tumour markers. However, there was no statistically significant difference between AUCs for tumour markers (S) CYFRA 21-1, (R) CEA, (P) CEA, (S) NSE, (P) NSE, (P) CYFRA 21-1 (data not shown) and therefore it cannot be concluded that any of them had greater diagnostic accuracy in distinguishing malignant from non-malignant pleural effusions. In order to better explain these results, DFA was performed.i.e. NSE) and two serum markers (i.e. NSE and CYFRA 21-1) discriminated well between groups with malignant and non-malignant pleural effusions. In order to establish the predictive value of those markers for classification with respect to malignancy of pleural effusion, logistic regression analysis was performed. The results presented in Sets of four tumour markers concentrations measured in pleural fluid and serum, as well as their ratios were analysed with respect to their discriminatory ability, and the results presented in Determining the diagnostic utility of tumour markers in pleural effusions, especially CEA, NSE, CA-125, and CYFRA 21-1, has many advantages with regard to both cost and the non-invasive approach, which is why we dedicated the entirety of this study to the analysis of these four markers. Targeted analysis of tumour markers in pleural effusions is acceptable, as the price of the test is not high. A cytomorphological finding of malignant cells in pleural effusions and immunocytochemical analysis can be used as alternative tests for greater diagnostic accuracy. Although these tumour markers exhibit great specificity, the low sensitivity of each individual marker limits their diagnostic value, and a few studies suggested they be used along with other pleural effusion tests for greater diagnostic accuracy , tumour markers with the highest specificity, PPV, and LR+ should be used. One marker can be great for confirming diagnosis but not so good or very bad at excluding the disease and Studies of the diagnostic value of tumour markers in pleural effusions are not rare, but they have not had definitive and clear results   , even though increased NSE concentrations have been reported in non-small cell lung cancer (NSCLC)  still recommends it to monitor the response to therapy in patients with epithelial ovarian cancer and to detect residual or recurrent disease in patients who have undergone first-line therapy and who would be considered for second-look procedures  or compare their diagnostic utility with more classical laboratory tests .The present study has some limitations. Although the patients were included as a consecutive series, the fact that the study was performed in the Department of Pulmonology has the consequence of exposure to more diseases related to lung pathology. In addition, the number of patients included in this study is representative of the size of the medical facility where the study was performed. Furthermore, the present study does not evaluate a larger number of different tumour markers (The advantage of this study is that it is a real-time study conducted in a Croatian population; differences exist in the expression of particular tumour markers in different races and populations due to genetic susceptibility.In conclusion, we have shown that certain tumour markers have a higher diagnostic value than others in Croatian patients. Serum markers were superior to pleural fluid markers in determining pleural fluid aetiology, according to this study. Serum CYFRA 21-1 concentrations, serum and pleural NSE values were found to be most significant predictors of pleural effusion malignancy and the combination of these three entities produced a significant model for distinguishing malignant from non-malignant pleural effusions. Further research in other populations and larger study groups is needed to determine whether measuring tumour markers in pleural effusions is appropriate in general.