Developed from meta-analysis in 1992, the Prognosis After Resuscitation (PAR) score consists of seven, relatively straight-forward to calculate, variables with scores greater than 5 predicting nonsurvival [Previous local service reviews have collected data on hospital survival and PAR scoring between January 2002 and May 2008 . In addiSince 2002 a total of 225 post-cardiac arrest patients have been admitted to the ITU. Forty per cent survived until hospital discharge. The PAR score ranged between -2 and 18, with 0 being the most common score. Four patients from the 37 (13.5%), admitted to the ITU, with a PAR score of greater than 5 survived until hospital discharge. Forty-six per cent of patients with a PAR score of 5 or less survived to hospital discharge. See Figure Over the 8 years of review of our data we have only identified four patients where ongoing care was both appropriate and successful despite a PAR score greater than 5. We believe that these patients should have been admitted regardless of the PAR score due to the underlying pathology. The PAR score is an invaluable screening tool in justifying the decision not to admit a patient in whom it is felt critical care is not justified. However, caution must be used as the PAR score should be an aid to clinicians rather than the sole factor deciding appropriateness of critical care admission.


Metabolic transformations have been reported as involved in neoplasms survival. This suggests a role of metabolic pathways as potential cancer pharmacological targets. Modulating tumor's energy production pathways may become a substantial research area for cancer treatment. The significant role of metabolic deregulation as inducing transcriptional instabilities and consequently whole-system failure, is thus of foremost importance. By using a data integration approach that combines experimental evidence for high-throughput genome wide gene expression, a non-equilibrium thermodynamics analysis, nonlinear correlation networks as well as database mining, we were able to outline the role that transcription factors MEF2C and MNDA may have as main master regulators in primary breast cancer phenomenology, as well as the possible interrelationship between malignancy and metabolic dysfunction. The present findings are supported by the analysis of 1191 whole genome gene expression experiments, as well as probabilistic inference of gene regulatory networks, and non-equilibrium thermodynamics of such data. Other evidence sources include pathway enrichment and gene set enrichment analyses, as well as motif comparison with a comprehensive gene regulatory network (of homologue genes) in Arabidopsis thaliana. Our key finding is that the non-equilibrium free energies provide a realistic description of transcription factor activation that when supplemented with gene regulatory networks made us able to find deregulated pathways. These analyses also suggest a novel potential role of transcription factor energetics at the onset of primary tumor development.  Some of these glycolytic enzymes, as well as other modulators of tumor behavior, have recently been analyzed in search for a clue that inhibition of such enzymes or appropriate tuning of such modulators should deprive tumors of energy, while leaving non-transformed cells unaffected. Recent findings seem to point out to several so-called metabolic transformations that permit neoplasms survival, thus suggesting a role of metabolic pathways as potential pharmacological targets It is known that tumors could depend on energy production pathways that are different from those of normal cells. These unique pathways require in some cases the expression and function of so-called Thermodynamic studies at the transcriptional cancer is a transcriptional disorder disease energetic deregulationtranscriptional disorder leading both to cascade triggering and metabolic disorders related to neoplasm formation and development. For these reasons this paper will attempt to model the role of TFs at both the energetics (thermodynamic) level and the network approach.The role of gene interaction networks have also been extensively mentioned in relation to cancer phenomenology, it has been claimed that these network effects are, in fact much more important than individual gene contributions make sense out of the extremely large corpus of experimental evidence given, for instance by genome-wide expression analysis. With the continuous advent of novel techniques in high throughput molecular biology and the ‘omics maybe just one thing has been established: Complex biological systems need to be studied from several standpoints to unveil the actual mechanisms behind them. In the present case, our aim is to sketch some hints for a proposal of functional mechanisms behind gene expression in cancer and cell energetics. The analysis work-flow for the present study was as follows . DI is useful to see also :Statistical pre-processing of the microarray gene expression data.Determination of differentially expressed genes and statistical significance assessment.Data mining for functional features within the statistically significant differential expression gene set.Non-equilibrium thermodynamics calculations , 3, 4, 5Probabilistic inference of gene regulatory networks.Pathway statistical enrichment analysis.Search for common non-linear correlations found for human MEF2C in this work  that areGene Set Enrichment design matrix which gives a representation of the different RNA targets which have been hybridized to the arrays. The second one, or contrast matrix which allows the coefficients defined by the design matrix to be combined into contrasts of interest. Each contrast corresponds to a comparison of interest between the RNA targets After pre-processing  of the samples Once we had a set of differentially expressed genes, we proceeded to implement a data mining search over it. Search parameters include the following constraints:Genes that are well known transcription factors, reported not only by sequence homology but also by actual experimental evidence.Genes that have been associated in the literature with the presence of breast cancer (higher scores) or any other tumors -liquid neoplasms were excluded- (lower scores).Genes whose protein products are related to cellular level metabolic pathways.Genes whose transcripts possess a complete physicochemical characterization, e.g. Affymetrix® calibration probes have reported free energies of formation.Core set consisted in four genes, namely MNDA, POU2AF1, MEF2C and SMAD3. In what follows we will analyze in detail the non-equilibrium thermodynamics of transcription as well as the regulatory network structure of such genes within a sample set of 1191 microarrays.From the set of genes included in the GeneChip® under study (namely Affymetrix HGU133-A) which were statistically significant in their differential expression between tumors and controls, we built sets that satisfy the aforementioned constraints. Then we made the intersection set of all these. This set, that we will call hereon a On general grounds, the finding of relevant genes associated with a cancer phenotype is based on determining features such as differential expression patterns. However, two important issues that should be also taken into account besides the gene expression levels, are the energetics within the cell and the physicochemical properties of the biomolecules involved in transcriptional regulation (transcription factors in particular). Both features could be responsible for TF activity since they affect the mechanism behind the activation of target genes according to previous specific cellular energetic conditions.An interesting trend in the transcriptional energetics in some well-studied genes upstream in the regulatory cascade) in order to kick-start such processes and, by taking into account that transcriptional regulation has been characterized as an activated process In brief, since master TFs are needed in early stages of whole-genome transcription  A non-equilibrium thermodynamical theory of gene regulation has already been proposed The present theoretical framework mass flux of these species As is usual in non-equilibrium thermodynamics we will assume that a generalized entropy-like function The non-equilibrium Gibbs free energy for a mixture of If we recall from reference The constitutive equations are,chemical potentialsIrreversible coupling is given by give:(8)Substitutaffinity of transcription i.e. the thermodynamic conjugate variable to the probe intensity chemical affinity for the gene expression process. As gene expression is a process that follows thermal activation kinetics, If we change variables in spike-in experimental data provided by the gene-chip manufacturer). Since these experiments have been made without the use of any knock-out or knock-down techniques , the gene expression levels The related chemical potential of transcription Since we statistical significance in the inference and based on their IBS index value we selected a set of 712 genes. The optimal network (within the given approximations) was found by a Maximum Entropy Method as is shown elsewhere in silico and database-mining) methods To deconvolute a Gene Regulatory Network (GRN) related to primary breast cancer we applied a methodology based on a local pattern-sharing measure as surrogate to actual gene-gene interactions to our dataset(see Reactome BNIP3 (Affymetrix-probe ID 201848_s_at) expression level. BNIP3 is a well known marker of progression and malignancy in primary breast cancer that correlates both with lab tests and clinical trials A curated set of 1191 whole genome gene expression profiles was generated Microarray pre-processing of the data was performed by using the affy library in BioConductor running under [R] on a 128 Gb RAM 8-Power5+ dual core-processor, symmetric multiprocessing (SMP) unit by IBM. Whereas all statistical tests were performed on a Dell Precision Series 8 Gb RAM QuadCore Workstation by using limma package in [R]/BioConductor. Information theoretical measures (Information Based Similarity) were calculated with the ibs program. Such information theoretical measures were used to infer regulatory interactions between TFs and target genes, i.e. to deconvolute the associated gene regulatory network MNDA, POU2AF1, MEF2C and SMAD3.From the set of differentially expressed genes, data mining techniques were implemented to determine a set of genes that at the same time were involved in metabolic activity at the cell level and/or in cancer; and possess experimental data to accurately determine the parameters involved in our non-equilibrium thermodynamic model. We have then come to investigate cell energetics in relation with mRNA expression of the following TFs: MNDA acts as a transcriptional activator/repressor in the myeloid lineage YY1POU2AF1 is a transcriptional coactivator Tumor suppressor region in 11q22-23 and it is suspected to modify non-coding effects on gene expression POU2AF1 amplification has been detected in multiple myeloma cells and this copy number variation also reflected in over-expression at both the mRNA and protein levels MEF2C is a transcription activator which binds specifically to the MEF2 element present in the regulatory regions of many muscle-specific genes. In fact, p38, ERK5, and MEF2C itself have been recently described as novel downstream Brk (PTK6) effector pathways c-JUN to the cyclin D1 promoter MEF2C is a transcriptional enhancer whose biological function in human breast cancer is still unknown. However, it has been shown that its chromosomal localization is assigned to the so-called mammary cancer susceptibility 1 locus (Mcs1) on chromosome 2q1 segregating with the sensitivity to mammary cancer development in a murine model SMAD3 is a transcriptional modulator activated by TGF-TGF-TGF-SMAD3 and SMAD2 knockdown, respectively, and this could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo In view of the importance of these genes in the onset and development of breast cancer, we have decided to investigate both the energetics and connectivity of their functions in both normal and neoplastic cells. We will discuss the role of TF activity, activation energies and chemical potentials of transcription as outlined and we will suggest some routes to follow to further understand the role of metabolic changes (both at energetics and pathways level) in breast malignancy.The role of integrative analysis in modern (high throughput) genomics is to present a basis for hypotheses generation that may be tested in more specific and detailed studies. In this sense our non-equilibrium thermodynamics calculations (plus some assumptions regarding energy release within the cell) supply a means to try to unveil causal structure of the regulatory interactions from correlation analysis (such as the network study presented here), thus providing a more appropriate frame for study.MNDA, POU2AF1, MEF2C and SMAD3 are shown in MEF2C are present in almost all stages of tumor progression. This gene also showed the lowest variance between sampling points as it could be seen in POU2AF1 expression levels were almost as low as those of MEF2C, its variance was much greater and in some instances mRNA concentrations doubling its baseline level are present. SMAD3 was in general found to present medium expression levels and medium variance. MNDA showed the highest concentrations (about 3.5 times that of MEF2C in some instances) and also showed the greater variability. With regards to transcriptional affinities (thermodynamic conjugate variables). We can however notice higher variability around the mean behavior which points out to the possible presence of activation processes taking place. If we consider that transcriptional affinity and transcription level are conjugate (i.e. its product is an energy term), the fact that affinities (for example in the case of MNDA) present a pattern of variance different from that of the expression levels, imply the existence of energy fluctuations that may be due to activation processes. This assertion becomes clearer when we consider the time evolution of the chemical potential  as well as the higher variance. SMAD3 also presented relatively low chemical potentials and medium values of variance. In accordance with mRNA concentration profiles, POU2AF1 showed medium chemical potentials but large variance and MEF2C showed the largest mean values of chemical potentials but a lower variability.The parameters needed to calculate the intensity-dependent concentration, the expression affinity and the chemical potential of transcription for finities  similar otential . The checore regulation genes could be seen in The results of the inferred non-linear correlation network centered in the transcriptional regulation partners for the The structure of the GRN  resembleH. Sapiens and A. Thaliana. In particular, from our core genes, human MEF2C belongs to the MADS-box genes family The somewhat special role of MEF2C related interactions is worth-mentioning, in particular with regards to the regulatory effects of its MADS-box structure. It was previously shown that MADS-box gene transcription factor is a common regulator extremely conserved across plant and animal kingdoms. This is particularly the case in either Arabidopsis homolog genes with TAF12 and NRPB9A/B. The one-to-one gene interaction in human between MEF2C and POLR2I founded in the present work corresponds to 25 MEF2C-MADS-box conserved domain genes and NRPB9A interactions as results from a search in the Arabidopsis thaliana root transcriptional interactions databaseFrom the inferred gene regulatory network, MEF2C target genes: TAF12 and POLR2I correspond to be In order to sketch the role of specific biochemical sets of reactions related to energetic and transcriptional deregulation processes in the regulatory networks just referred, we performed pathway-related analyses in the list of 712 genes obtained from the IBS calculations. In particular, we made a pathway enrichment analysis for the statistical over-representation of pathways in this gene set.Activation of ATR in response to replication stress, Association of RAD51 with RAD52:DNA double-strand break ends, as well as metabolic activity related pathways like Cholesterol biosynthesis and Addition of galactose by beta 4-galactosyltransferases. With regards to the activation of ATR in response to replication stress rad3, a cell cycle checkpoint gene required for cell cycle arrest and DNA damage repair in response to DNA damage core set as it could be verified in a closer analysis of the network depicted in Significantly enriched pathways include cancer related pathways such as Transcriptional deregulation also occurred in genes participating in metabolic pathways, for instance, Further insight in the mechanisms that connect transcriptional deregulation in cancer and metabolic abnormalities could be found if we bring to attention a set of genes, consisting in PRKAA2, PRKAB2, CREB1, MAP3K1, DUSP4, TLR3, JUN, UBE2V1, TLR2 and MEF2C. All these genes turned out to be involved in the enrichment analyses both in the case of cancer-related pathways as well as metabolism-related pathways. PRKAA2 and PRKAB2 are AMPK subunits, CREB1 is a MAP kinase target and the rest are related with Toll-like receptor activity. Hence, kinase activity and signalling dysfunction seem likely to play a central role in cancer development, well beyond the usual RAS-ERK pathway. For more information, please refer to permutations - 1000, scoring scheme - weighted and metric - Signal2Noise.Previous analysis in this work were mostly derived from the list of differentially expressed genes i.e. single-gene analysis. In order to include the collective behavior of genes within pathways and functional modules, we performed Gene Set Enrichment Among the resulting statistically significant enriched gene sets  and 273  are shown in sion see . Module on biological samples could be done, due to lack of sample-availability. However, an indirect validation procedure is performed by means of data mining for experimental results in the literature supporting our findings. For instance, MEF2C was identified as a candidate gene/molecular marker for the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) and evaluated by RT-PCR. In reference MEF2C was shown to be up-regulated in IDC compared with DCIS  In a study of differential expression in human breast epithelial cell lines irradiated with low doses of high linear energy transfer radiation, and treated with estrogen assessed with cDNA expression arrays, activity of sion see  and it wMEF2C, SMAD3 and POU2AF1 can be found as deregulated within two different platforms (HGU133A & HGU133plus2) in the BarCode database MEF2C was differentially expressed in more than 60% of breast epithelium tumors samples (HGU133A) and more than 95% of breast stroma tumors samples (HGU133plus2). MNDA expression in breast carcinomas was examined on the EMBL-EBI database. MNDA was reported up-regulated in both studies.Expression of MNDA, POU2AF1, MEF2C and SMAD3) and high for genes with no-known TF-activity . One possible exception in the group of genes studied is GLDC which codes for a glycine-dehydrogenase enzyme that is anchored to the mitochondrion and has no reported evidence of transcription factor activity yet is grouped with the TF genes in the set of low chemical potential. This fact supports the TFlock-in-the-trigger for transcriptional cascading. It is important to stress that the chemical potentials spontaneous transcription do not show a big difference between TFs and Target Genes (TGs) at low gene expression intensity levels  and metabolic control pathways . Some affected genes are actually part of both kinds of pathways. These genes are correlated with MEF2C expression . As we massume that, in effect, there is a kind of control loop between MNDA and MEF2C somehow fine-tuned by the action of POU2AF1 and SMAD3 (and their interactors); then we may sketch a possible causal scenario if we consider that energy is been released gradually within the cells. MNDA is present at high concentrations (balance between the concentrations of MNDA and MEF2C. If energy release within the tumor cells occurs as a consequence of gradual activated processes, then MNDA should be transcribed first and MEF2C expression may be regulated by MNDA and its interactors. Once MEF2C is being produced, then activation of its many transcription targets should cause abnormal regulation in the already mentioned cancer and metabolic related pathways, thus causing diseased cellular states and, ultimately, carcinogenesis.Let us recall the results of the thermodynamic analysis and the correlation network analyses. In trations  almost atrations  and henctrations  because trations  so it isin vivo conditions is now becoming possible. In particular with regards to experimental verification of our findings, there have been several instances in which related work has been done. One approach to provide real-time semi-quantitative analysis of transcription is the imaging of reporter gene expression, for example, using firefly luciferase With regards to the design of possible experimental protocols to verify some of these findings, we must notice that experimental techniques in genomics are rapidly evolving, in such a way that probing the cell in real time under almost MNDA, POU2AF1, MEF2C and SMAD3 and how can this energetic constrains be related with the presence of primary breast carcinomas. In doing so we studied systematically mRNA levels for high throughput, whole-genome gene expression profiling. A set of 1191 publicly available microarrays was first studied by inferring gene regulatory networks based in a non-linear correlation measure between gene expression vectors arranged in a surrogate dynamic model of tumor progression. In the other hand, a non-equilibrium thermodynamic formalism was used to calculate the concentration-dependent gene expression intensity, the chemical potentials of transcription and their associated affinities in order to establish energetic constraints that help us to evaluate the biological hypotheses. Hence, a connection was established between mRNA concentration patterns -as given by experimental gene expression profiles- and local cell energetics - by means of these irreversible thermodynamical quantities. By analyzing the different patterns of gene expression for the selected genes, the corresponding non-equilibrium energetics, as well as their correlation structure as given by network analysis, an integrative model for the action of a core set of master regulator genes was developed. We analyzed in the surrogate dynamic model already mentioned to look up for transcriptional regulation paths. All these analyses suggest a novel potential role of transcription factor energetics in tumor development.In this paper, we have analyzed the role that thermodynamic fluctuations in energy at the cell-level play in the synthesis of transcription factors master regulators in primary breast cancer phenomenology, as well as the possible interrelationship between malignancy and metabolic dysfunction. Nevertheless, we can never emphasize enough that these findings should be regarded as hypothesis generators rather than as conclusive results. However, we believe that systematic studies relying in data integration guided by well-founded physical principles rather than intuition may become mandatory in time. This work intends to point-out in such direction. However, further, deeper investigations are needed in this direction in order that our understanding of these extremely complex phenomena will be substantially increased.In particular, by using a data integration approach that combines experimental evidence for high throughput genome wide gene expression, a non-equilibrium thermodynamics analysis, nonlinear correlation networks as well as database mining, we were able to hypothesize about the role that transcription factors MEF2C and MNDA may have as


The clinical use of Sodium nitroprusside (SNP) may be associated with an alteration of platelet function. The main focus of this study was the effect of SNP on platelet aggregation in the absence or presence of endothelial cells.Methods: Platelets were incubated with different concentrations of SNP with and without endothelial cells. Platelet aggregation was induced by ADP.Results: Platelet aggregation was significantly inhibited by all concentrations of SNP. Endothelial cells significantly increased this inhibitory effect of SNP. Time course studies showed an inverse correlation of incubation time to platelet aggregation inhibition in the absence of endothelial cells, and a direct correlation in the presence of endothelial cells. Blocking platelet and endothelial cell guanylate cyclase with 1 H--oxadiazolo quinoxalin-1-one (ODQ), or pretreatment of the endothelial cells with cyclooxygenase – inhibitors, had no influence on the increased inhibitory effect of the endothelial cells. Cyanide reversed the inhibitory effect of SNP completely.Conclusion: Endothelial cells play an important role in the SNP mediated inhibition of platelet aggregation. The effect is reversible only by cyanide, not by blocking classical NO signal transduction. The administration of Sodium nitroprusside (SNP) is a highly effective strategy in pulmonary and systemic hypertension crisis, both in adults and in neonates,2. The mAs well as this NO dependent effect other mechanisms seem to have an important impact on the inhibitory action of SNP in the presence of endothelial cells. Levin et al. already mentioned that the supernatant taken from SNP-incubated endothelial cells, had a surprising inhibitory effect on platelet aggregation not due to NO alone.In the present study we wanted to evaluate the role of endothelial cells on the antiaggregatory effect of SNP. An additional aim was to investigate wether the time span between the incubation of platelets with SNP and measurement of the antiaggregatory effect provides an explanation for the sometimes contradictory results of in vitro and in vivo studies. The third goal was to test the reversibility of the antiaggregatory effects of SNP by cyanide and classical NO pathway inhibition.Sodium nitroprusside (SNP), Cyanide and ADP were purchased from Sigma laboratories and 1 H-oxadia-zoloquinoxalin-1-one (ODQ) was purchased from Alexis Biochemicals. PBS was obtained from Boehringer, Germany and Protaminhydrochloride (Protamin ICN 1000) was from ICN, Frankfurt, Germany. Acetyl sali-cylic acid (ASS) was obtained from Bayer, Leverkusen, Germany and indomethacin (Liomethacin) was purchased from Chiesi Pharmaceutica, Parma, Italy.Blood samples Venous blood was drawn from 4 healthy volunteers and coagulation was prevented immediately using 3.8% sodium citrate 1:10. The volunteers took no medication two weeks prior to blood sampling.For the endothelial cell experiments we used a well established configuration using human umbilical endothelial cells (HUVEC). HUVEC were isolated from fresh human umbilical cord vein by enzymatic disaggregation with collagenase, and were used between passages 2 and 5. HUVEC were cultured as previously described, the plaThe effect of different concentrations of SNP and different incubation times was tested. After adding SNP to 5 mL of whole blood, samples were placed on culture plates. A new plate was used for each concentration and incubation time. Samples without SNP were used as controls. To study the effect of endothelial cells on aggregation, the incubation was performed on culture plates with, and without endothelial cells.SNP concentrations were 100 μM, 500 μM and 1000 μM. Time course studies were performed with 100 μM SNP, and incubation times of the whole blood samples were 0, 1, 2 and 4 hours.As total centrifugation time was 20 minutes (s. below) and SNP was added to the whole blood sample before centrifugation contact time between SNP and platelets was at least 20 minutes. Samples were kept at 37°C during the whole experiment.We wished to study the mechanism by which SNP inhibits platelet aggregation. NO signal transduction by cGMP was inhibited using the guanylate cyclase inhibitor 1 H- oxadiazoloquinoxalin-1-one (ODQ). ODQ (100 μM) was added to the sample 2 minutes before adding 100 μM SNP to allow the reaction of ODQ with the guanylate cylcase. Platelet rich plasma was then obtained from the blood sample. The platelet function was tested using the Born method. Aggregation was induced with ADP (20 μM) after the platelet count had been adjusted to 250,000/μL.To study whether SNP has an effect on the endothelial production of prostaglandins by activating cyclooxygenases (COX I and II), two cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin were used. Endothelial cells were incubated for 5 min with 3 ml of either 1 mM acetylsalicylic acid or 10 μM indomethacin. Sufficient suppression of the cyclooxygenases in HUVEC by ASA or indomethacin has been previously described. After tTo study the effect of cyanide on the effect of SNP, we added cyanide to whole blood in one set-up. In another experiment we added cyanide to platelet rich plasma (PRP) after the addition of ADP. A control group proved that cyanide itself has no effect on platelet aggregation.For preparation of platelet-rich plasma the whole blood samples were centrifuged for 10 minutes at 800 r.p.m. Platelet-poor plasma was obtained by further centrifugation of PRP at 3800 r.p.m for 10 minutes. Platelet count was then adjusted with PPP to 250,000/μL. Platelet response to ADP (20 μM) was recorded with a Lumiaggregometer .Data represent the mean +/− SD of at least 4 experiments.The levels of aggregation given in percent were detected every 20 seconds for 6 minutes. The following parameters were used for statistical analysis: For each experiment, all values of the aggregation curves were compared. Data were analyzed with Prism 3.0 using the Kruskal-Wallis-test and p ≤ 0.05 was accepted as being significant. In case of multiple comparisons p – values were corrected according to Dunn. Results were grouped according to incubation time: Group 1 = no incubation time, group 2 = incubation time of 1 hour, group 3 = incubation time of 2 hours, group 4 = incubation time of 4 hours.st and 3rd column). We could show that ADP induced thrombocyte aggregation was intact even 4 h after blood sampling, with and without endothelial cells.The difference in platelet aggregation of controls with, and without, endothelial cells was not significant different. .After 1 hour of incubation SNP inhibited platelet aggregation in a dose-dependent manner. 100 μM, 500 μM and 1000 μM of SNP were tested (data not shown). This was statistically significant for all concentrations of SNP. These experiments were not repeated with endothelial cells. Time course studies were performed with 100 μM of SNP, as the 100 μmol curve at one hour was at the middle of normal aggregation and total inhibition, so changes in both directions would be detectable. A time dependent reduction of platelet aggregation could be detected with an inverse correlation to incubation time. Endothelial cells significantly increased the SNP – mediated inhibition of platelet function after 2 and 4 hours of incubation Incubation with ODQ decreased the effect on platelet aggregation at 0 h of incubation time. ODQ did not inhibit the SNP mediated-effect on platelet aggregation later in the presence of endothelial cells. .Since endothelial cells play an important role in the SNP-induced inhibition of platelet aggregation, the effect of COX inhibitors and of ODQ was tested only after incubation with endothelial cells. The cox inhibitors acetyl salicylic acid and indomethacin  did not influence the effect of SNP on platelet aggregation. The results were therefore combined. The pretreatment of endothelial cells with COX inhibitors did not interfere with the effect of SNP incubation .Cyanide (2000 μM) alone did not influence platelet aggregation. Cyanide added to whole blood prior to addition of SNP repressed the effect of SNP in all cases. The addition of cyanide after the preparation of platelet-rich plasma, prior to the addition of ADP only inhibited the SNP effect of the endothelial cell experiments (Additional fileIn our study we could clearly demonstrate that endothelial cells play an important role in the antiaggregatory effect of SNP. Incubation with endothelial cells increased both the inhibitory effect on platelet aggregation and the duration of this effect. Levin et al. already mentioned that the supernates taken from SNP-incubated endothelial cells had an inhibiting effect on platelet aggregation. They spOur results without endothelial cells show an effect of SNP on platelets which is in accordance with other in vitro studies,26,30.Another interesting fact is that the addition of cyanide is able to reverse the effect, not only after immediate administration, as shown by Brune et al., but eveIn summary, our results show that endothelial cells play an important role in the SNP- mediated inhibition of platelet activation, which can be reversed only by cyanide. We could demonstrate that this effect is not mediated by activation of endothelial cyclooxygenases, as the inhibition of cyclooxygenases had no influence on the results. It is not dependent on cGMP, as it is not influenced by the guanylate cyclase inhibitor ODQ. Thus we suggest a cGMP independent, possibly not yet known pathway for platelet inhibition by SNP/endothelial cells.The identification of this unknown antiaggregatory metabolite could be of great interest, since its effect on ADP-dependent platelet aggregation is long-lasting, but reversible by cyanide. We speculate that other less toxic ion metabolites could also reverse this effect, thus offering the opportunity for antidot treatment.All authors have read the manuscript and conflict of interest statement and have approved their submission for publication; all authors declare that they do not have competing interests.H-GT was performing the experiments and wrote the manuscript. MR and JK established the born method for measuring platelet aggregation and gave helpful input on pre-analytical pitfalls when working with platelets. WR and JD established the endothelial cultures in the lab and gave relevant input on the test up during incubation. All authors have read and approved the final manuscript.Comparison of aggregation at timepoints 0, 1, 2 and 4 hours.Click here for file


Staphylococcus aureus (MRSA) remain high in most countries. Screening for MRSA carriage followed by barrier precautions for documented carriers ) has been successful in some, but not all settings. Moreover, different strategies have been proposed, but comparative studies determining their relative effects and costs are not available. We, therefore, used a mathematical model to evaluate the effect and costs of different S&I strategies and to identify the critical parameters for this outcome. The dynamic stochastic simulation model consists of 3 hospitals with general wards and intensive care units (ICUs) and incorporates readmission of carriers of MRSA. Patient flow between ICUs and wards was based on real observations. Baseline prevalence of MRSA was set at 20% in ICUs and hospital-wide at 5%; ranges of costs and infection rates were based on published data. Four S&I strategies were compared to a do-nothing scenario: S&I of previously documented carriers (“flagged” patients); S&I of flagged patients and ICU admissions; S&I of flagged and group of “frequent” patients; S&I of all hospital admissions . Evaluated levels of efficacy of S&I were 10%, 25%, 50% and 100%. Our model predicts that S&I of flagged and S&I of flagged and ICU patients are the most cost-saving strategies with fastest return of investment. For low isolation efficacy universal screening and S&I of flagged and “frequent” patients may never become cost-saving. Universal screening is predicted to prevent hardly more infections than S&I of flagged and “frequent” patients, albeit at higher costs. Whether an intervention becomes cost-saving within 10 years critically depends on costs per infection in ICU, costs of screening and isolation efficacy.Nosocomial infection rates due to antibiotic-resistant bacteriae, e.g., methicillin-resistant  Staphylococcus aureus (MRSA) allows for strategies where carriers are treated with barrier precautions, e.g., isolation in single-bedrooms. At least theoretically, this should prevent spread of these bacteria. Several screen-and-isolate studies have been performed. However, the outcome was not unequivocal, possibly because clinical trials to determine the optimal screening strategy would necessitate long periods of follow-up due to stochasticity. In the absence of direct evidence we have used mathematical modelling to quantify the theoretical effectiveness and expenses of different screen-and-isolate strategies in hospitals with a high prevalence of antibiotic-resistant bacteria. We find that a strategy to screen patients who were previously known as carriers, possibly combined with screening of ICU-patients is the most cost-saving strategy for the best estimate of isolation efficacy of 25%. With a high efficacy of isolation all strategies are expected to become cost-saving compared to the do-nothing scenario.Within hospitals antibiotic-resistance of bacteria is common and it complicates treatment of bacterial infections. Screening of patients on admission for carriage of methicillin-resistant  Staphylococcus aureus (MRSA) may cause severe infections in hospitalized patients, such as bloodstream infections, surgical wound infections and pneumonia. These infections are associated with increased mortality rates, longer length of hospital stay and higher health care costs compared to methicillin-sensitive strains Methicillin-resistant It has become increasingly clear that rapid identification of carriage of MRSA, followed by implementation of barrier precautions for carriers, could be a powerful tool in controlling nosocomial spread Here we have performed multiple scenario analyses of a mathematical model to compare the effects and costs of different “screen and isolate” (S&I) strategies, with special emphasis of such a strategy in ICU populations.We have used an extended version of a previously described dynamic stochastic simulation model that contains three hospitals of 693 beds, each with an extramural population of 220,000 subjects Individuals are also subdivided into “frequent” patients and “occasional” patients, distinguished by hospitalization rates of once per year (frequent) and once per ten years  . Patients from either group can be admitted to both non-ICU and ICU wards and the mortality rate during hospitalization is the same for both groups. As a result, on average, 50% of the hospital population consists of “frequent” patients. In this study we use the “frequent” group as a high-risk population for MRSA carriage, and one of the possible screening strategies includes screening of “frequent” patients. All patients are either carrier of MRSA or uncolonized and susceptible for colonization and 1% of the colonized patients is 10 times more infectious .ARInfection control interventions are not based on the true colonization status, but on the available documentation of the colonization status only. Patients either (1) have documented carriage, (2) are not suspected of MRSA colonization (but could still be colonized), or (3) are suspected of colonization, e.g., after documented carriage during previous hospitalization or because of risk factors for MRSA carriage. Throughout this paper the latter patient category will be labelled as “flagged” patients. Importantly, we assume that the pathogen predominantly spreads in hospitals through cross-transmission and that there is hardly any spread in the community. Transmission occurs primarily between patients and HCW in the same ward, but occurs also, at a much lower rate, between wards. Transmission parameters are chosen such that the per admission reproduction number MRSA blood stream infections may impact LOS, as was shown, by de Kraker et al. The microbiological screening method is, in all simulations, a rapid diagnostic test with turnaround time of 1 day and sensitivity and specificity of 93% and 96%, respectively We consider four different S&I strategies that are compared to a do-nothing scenario without any active screening at admission. In all four S&I strategies patients identified as MRSA-carrier will be “flagged” as such. The flagged status will be removed when such a patient has a negative conventional culture. The following S&I strategies are considered:S&I of flagged patients at hospital admissionS&I of all patients at ICU admission and flagged patients at hospital admissionS&I of “frequent” patients and flagged patients at hospital admissionS&I of all patients at hospital admission .In all scenarios we assume that 12% of the admission screenings that should be performed according to the strategy are missed. In each scenario patients documented as carrier will be treated in isolation, which reduces the likelihood of transmission by 100% (perfect isolation), 50%, 25% or 10%, with 25% as default value Screening of “frequent” patients is not a strategy that is currently applied. Yet, since previous hospitalization is associated with MRSA colonization Although no limits to isolation capacity are assumed, we keep track of the number of patients in isolation to determine the volume of isolation capacity needed. The daily probability to develop an infection for a colonized patient is set at 0.7% and 0.2% in ICU and non-ICU wards, respectively, with sensitivity analysis ranges of (0.14%–1.4%) and (0.1%–0.3%) respectively. This implies that on average 3% and 1.4% of all patients in ICU and non-ICU wards will develop an infection in the do-nothing scenario  [27], [2We estimated the costs of the different S&I strategies for a hospital using a 3% inflation rate per year. The analysis was performed from a hospital perspective and costs are reported in Euros using the price level of 2010. The default incremental costs from a hospital perspective of these infections (including a costs of prolonged due to MRSA-infection length of stay) were €30,000 in ICU and €1,000 in non-ICU wards, with ranges for sensitivity analysis of  and  respectively T). The 90% credibility intervals denote the uncertainty due to the inherent stochasticity of the dynamics of MRSA .For every S&I strategy and set of costs we determined  in 10 years time at the costs of €845.000, €909.000, €949.000 and €972.000 . The cosWhether a strategy will become cost-saving from the hospital perspective, as compared to the do-nothing scenario, critically depends on the isolation efficacy and the costs per infection averted. With our default efficacy of isolation of 25%, only two strategies are expected to be cost-saving within 10 years: screening of flagged and ICU patients and screening of flagged patients only. The expected total gain in 10 years time is estimated to be €420.000 and €850.000 respectively . When efT – become lower than in the do-nothing scenario , all four strategies will have lower daily costs as compared to the do-nothing scenario within 10 years (T<10 years) for high values of isolation efficacy. With isolation efficacies of 25% or 10% only S&I of flagged and S&I of flagged and ICU patients will reach T within 10 years in the considered range of q. With decreasing values of q, the time T increases and at some critical value of q, the number of infections prevented by a strategy becomes too low to compensate for the costs of the intervention. This critical value of q depends on the strategy and the isolation efficacy .The value of Changing the discount rate to either 2% or 4% hardly influenced the results.in silico conditions. Although universal screening at hospital admission leads to the fastest decline in both the hospital-wide and ICU prevalence of MRSA, it also requires the highest investment costs and the longest time till return of investment. In our analyses, screening all patients at ICU admission and those previously detected with MRSA  or screening of flagged patients only were almost equally cost-saving in a 10 years period and were both associated with the fastest return of investment. These strategies should, therefore, be seriously considered by hospitals that aim to control the nosocomial spread of MRSA.Using a dynamic stochastic simulation model, we have evaluated four intervention scenarios to control the spread of MRSA under comparable Our findings are complimentary to those of two other modelling studies on screening for carriage with antibiotic-resistant bacteria in hospitalized patients. In one study, Hubben and co-workers compared the effects of PCR-based and chromogenic screening tests Yet, this is an important aspect of the dynamics of nosocomial MRSA as it explains why control measures may have not only a direct, almost instantaneous, effect on the prevalence of the nosocomial MRSA in the hospital, but also an indirect effect due to interruption of the so-called feedback loop; when less patients acquire colonization during hospitalization, less patients will be colonized upon readmission to the hospital  contacts between patients and HCWs, allowing HCWs to act as transmission vectors of MRSA. Moreover, antibiotic selective pressure is higher in ICUs than in non-ICU wards, which may increase the likelihood that a HCW will pick up a pathogen during a physical patient contact and that another patient will be successfully colonized after being contacted by a temporarily contaminated HCW. Finally, the severity of disease of critically ill patients in ICU wards makes them more susceptible to acquire colonization with MRSA than patients in non-ICU wards. Several studies indeed support the potential effects of ICU-screening on hospital-wide resistance levels With regard to the costs of interventions, our analyses were most sensitive to the costs associated with an ICU-acquired infection caused by MRSA. Many studies have quantified the costs of ICU-acquired bacteremia and ventilator-associated pneumonia One of the simplification of the model is that patients should be colonized with MRSA before they are at risk of getting an infection with MRSA, i.e., we did not explicitly incorporate that some patients may acquire MRSA infection directly without being colonized first, i.e., due to invasive medical procedures. A slight increase in the daily probability for colonized patients to acquire an infection would lead to the same ratio of colonized and infected patients. Therefore, our sensitivity analysis on the daily probability for colonized patients to acquire an infection can also be interpreted as a proxy for a sensitivity analysis to the parameter which determines how often patients acquire an infection without being colonized.We also assumed that the rates of conventional microbiological cultures performed for clinical reasons are independent of screening on admission (0.03 and 0.3 per patient day in non-ICU and ICU wards). We have assumed that a clinical suspicion of infection is the main reason for obtaining clinical cultures, and that screening for MRSA-carriage on admission reduces the frequency of obtaining clinical cultures in case of a clinical suspicion of infection.Our model contains many parameters and some parameter values are unknown, whereas others may differ between hospitals and countries. We have based our values on data from the literature and from our own hospital, where possible. To fully capture the effects of parameter uncertainty we would have considered to perform a probabilistic sensitivity analysis (PSA) for all parameters simultaneously, as was performed by Robotham et al. We did not include decolonization of detected carriers as a measure to control MRSA. Naturally, adding this measure (if successful at low costs) would increase intervention effects and would make the duration till return of investments shorter. Although persistently colonized HCWs were included as potential sources for MRSA transmission, we did not include screening and decolonization of them as intervention measure. This intervention measure would - in most settings – only slightly enhance the control of MRSA transmission, at the cost of significant expenses due to the necessity to replace colonized HCWs.The (cost)-efficacy of admission screening strategies critically depends on the effectiveness of the infection prevention measures taken when a carrier of MRSA is detected. If these measures are not very effective, it may not be wise to invest lots of efforts in detecting carriers. The effectiveness of barrier precautions has been sufficiently high in the Netherlands and the Scandinavian countries to prevent high prevalence levels of MRSA. However, it is still debated whether patient isolation prevents transmission at all In conclusion, our study demonstrates marked and robust differences in the costs and effects of different infection control measures for MRSA. Because of the central role of ICU wards in patient flow in hospitals, the vulnerability of ICU patients to infections caused by MRSA and the high costs associated with these infections targeted infection control measures in ICU wards are likely to be the most effective and cost-saving from a hospital perspective.Figure S1Number of infections prevented in ICUs and the cost of the intervention during the first 5 years after implementation in a high-endemicity settings . Isolation efficacy was 100% (A), 50% (B), 25% (C) and 10% (D). The credibility intervals denote the uncertainty due to the inherent stochasticity of the dynamics of MRSA and contain 90% of our simulation results. The dots correspond to the means after 1,2,.. years.(TIF)Click here for additional data file.Figure S2Univariate sensitivity analysis of the total costs during the first 10 years after implementation of the intervention when the isolation efficacy is 100%. The black line corresponds to the mean costs for the default parameter (see eter see  and the eter see .(TIF)Click here for additional data file.Figure S3Univariate sensitivity analysis of the total costs during the first 10 years after implementation of the intervention when the isolation efficacy is 50%. The black line corresponds to the mean costs for the default parameter (see eter see  and the eter see .(TIF)Click here for additional data file.Figure S4Univariate sensitivity analysis of the total costs during the first 10 years after implementation of the intervention when the isolation efficacy is 10%. The black line corresponds to the mean costs for the default parameter (see eter see  and the eter see .(TIF)Click here for additional data file.Figure S5Admission prevalence of MRSA and the annual attributable mortality rates as function of the time since start of the intervention. The upper graphs denote the dynamics of the MRSA prevalence at admission for different values of the isolation efficacy . The lower graphs depict the dynamics of the annual attributable mortality rates. Both the admission prevalence and the attributable mortality e decrease due to the so-called feedback loop. Interventions in hospital start at time 0 and the lines for negative time correspond to the “do-nothing” scenario. Efficacy of patient isolation varies from left to right from 100%, 50%, 25% to 10%. The lines denote the mean of 1000 simulations; the coloured shaded areas denote the 90% credibility intervals due to stochasticity. All parameter values are at the default-value.(TIF)Click here for additional data file.Text S1Explanation of the calculation of the time T. Abstract in Dutch. Abstract in Russian.(DOC)Click here for additional data file.


Objective. To evaluate the efficacy of double Fallopian tube sperm perfusion in comparison with single-sperm perfusion in patients with nontubal subfertility undergoing controlled ovarian stimulation. Study Design. Sixty-six patients undergoing standard ovarian stimulation regimen were randomized to receive either single-sperm perfusion group 1 (n = 33) or double-sperm perfusion group 2 (n = 33). The same insemination method was performed in subsequent cycles if the patient does not become pregnant in the first one. A maximum of three cycles was performed. Fallopian tube sperm perfusion was carried out with pediatric Foleys catheter, which prevents reflux of sperm suspension. Semen was prepared by a classical swim-up technique. Results. A total of 133 cycles performed 68 single FSP cycles and 65 FSP cycles. There were group, 19 clinical pregnancies (29.2% per cycle) of which 16 ongoing pregnancies (24.6% per cycle) were obtained. These differences were statistically significant. The prevalence of multiple pregnancies, abortions, and ectopic pregnancies was similar in both groups. Conclusion. The results of this study indicate that there is a significant improvement of pregnancy rates in patients with nontubal subfertility when treated with double-sperm perfusion after controlled ovarian stimulation in comparison with single-sperm perfusion. Double-sperm perfusion is simple, easy to perform, inexpensive, and convenient for the patients with nontubal subfertility before adoption of other methods of assisted reproduction. Artificial insemination in conjunction with ovarian stimulation is usually offered to infertile couples when the woman has patent Fallopian tubes prior to other assisted reproductive methods . OvarianThere is firm evidence that FSP gives rise to higher pregnancy rates than standard IUI in couples with subfertility and therefore should be advised in these couples , 9 as shThe objective of this study was to evaluate the efficacy of FSP either by single-sperm perfusion or double-sperm perfusion in patients with nontubal subfertility undergoing ovarian stimulation. Patients with non-tubal subfertility attending the Infertility Unit at the Department of Obstetrics and Gynecology Mansoura University Hospital were invited to participate in the Patients had to fulfill the following inclusion criteria (1) age of women <35 years, (2) duration of infertility >2 years, (3) normal hysteroscopy and laparoscopic finding with regular uterine cavity and patent tubes, and (4) normal semen parameters according to the WHO standard positive postcoital test. Every patient was extensively counseled and gave informed consent prior to participating in the study. All patients underwent ovarian stimulation with standard protocols were for ovarian stimulation with Merional (IBSA) starting on cycle day 3 baseline, and transvaginal scanning was performed, and when no ovarian cyst 150 IU of FSH was titrated according to ovarian response and monitored, and by transvaginal scanning when the follicle was >18 mm in diameter and when there were not more than 3 follicles >16 mm in diameter, 10000 I choriomon (IBSA) was given intramuscular in the first treatment cycle, and patients were randomized on day of HCG administration according to computer-generated randomization list, to undergo single or double FSP. The same insemination method was performed in subsequent cycles if they did not become pregnant in the first one; a maximum of three cycles was performed; single FSP was performed 36 hours after HCG administration, whereas double FSP was performed 18 and 42 hours after HCG semen was collected by masturbation after 4 days of ejaculatory abstinence and was allowed to liquefy at 37°C for 20 minutes before processing semen was prepared by classical swim-up technique using Ham and then centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 0.5 mL of the medium, then 1 mL of the medium was gently layered over the specimen. The tube was inclinated at angle 45 and incubated for 60 minutes at 37°C. It was then returned to the upright position, and the upper most 0.5 mL was removed and resuspended in 4.5 mL of Ham's F10. The total number of motile spermatozoa in the inseminate I was assessed; Fallopian tube sperm perfusion was performed using pediatric Foley's catheter. The luteal phase was supported by the furthest doses of 200 mg daily progesterone (prontogest). Pregnancy test was performed 2 days from the missed period. A transvaginal ultrasound was arranged to confirm the intrauterine pregnancy and to determine the number of gestational sacs after a positive pregnancy test. Only clinical ongoing pregnancies were considered in this study; clinical pregnancy is diagnosed by presence of fatal heart beat on ultrasound examination or products of conception of abortions. t-test; categorical data were compared using the Fishers exact or chi-square test where appropriate differences were considered statistically significant at P < 0.05; statistics were done using SPSS software program version 9. Continuous data were expressed as mean ± SD, and statistical significance between the two groups was determined using the Student's A total of 66 consecutive eligible patients underwent 133 stimulated cycles and proceeded FSP. Thirty-three patients were assigned to receive single FSP and 33 double FSP. Sixty-eight single FSP cycles and 65 double FSP cycles were completed. There were no differences between the two groups with regard to the age of women, duration of infertility, and body mass index (BMI) . As regaRepeated insemination during the previously periovulatory period may improve the chance of pregnancy by increasing the number of spermatozoa inseminated enhancing the fertilizing capacity of spermatozoa to fertilize oocytes that are released over a period of at least several hours . SilverbIn the previous studies, the pregnancy rate per cycle achieved in couples with unexplained infertility after ovarian stimulation when FSP was used has been reported to be in the range of 8.6–40% , 10, 14.Regarding double FSP, our results indicated that both clinical and ongoing pregnancy rates per cycle  were significantly higher than those with single FSP . Pregnancy rate per patient was also significantly higher in the double FSP group. Based on medline search of studies published in the last 15 years as well as thorough review of the references cited in these identified published series and of the literature, our study is the first to evaluate the efficacy of doubles in the treatment of nontubalfertility, and accordingly no similar studies were found for comparison; however, the same principle of totally injecting large inseminate volume, used in our study by double FSP, has been recently evaluated by another method called intrauterine tuboperitoneal insemination, in which 10 mL of the inseminate was used. This method was found to give significantly higher pregnancy rates than single FSP (29.4 versus 17.6). The better results of double FSP may be explained by that repeated insemination achieves the presence of at the site of higher concentration of motile spermarzoa around the oocytes. At the site of fertilization as well as in the pouch of Dougals furthermore, repeated insemination under pressure may be necessary for achievement of perfusion spill in normal tubes or tubes with minimal adhesions. This pressure is also necessary, in midfollicular phase, and before ovulation, the endometrial glandular lumen of the Fallopian tube is narrowed by some quantities of gland by partial tubal luminal obstruction due to the presence of tubal ostium membranes. This has shown to be useful procedure in patients with subfertility and may be attempted in such patients before moving on towards much more expensive methods of assisted reproductive techniques.The results of this study indicated that there is a significant improvement of pregnancy rates in patients with non-tubal subfertility when treated with double FSP after controlled ovarian stimulation in comparison with single FSP. Double FSP method is simple, easy to perform, inexpensive, and convenient for the patients. Trails of this method are useful before the adoption of other expensive assisted reproductive techniques. However, its efficacy regarding other factors as endometriosis or abnormal semen parameter remains to be investigated.


A 68-year-old female had a three-week history of severe low back pain radiating down the posterior left buttocks and left leg exacerbated by standing and walking. Lumbar spine MRI revealed cystic mass with similar intensity to cerebrospinal fluid located on dorsolateral left side of the sacral spinal canal inferior to the S1 pedicle. There was compression of left exiting S1 and traversing S2 nerve roots. Neurosurgery consult was requested to evaluate the cystic mass in the sacral spinal canal. After clinical evaluation, an unusually located synovial cyst was thought possible. Cyst contents were heterogeneous, suggestive of small hemorrhage and acute clinical history seemed reasonable. Left S1 and partial left S2 hemilaminectomy was performed and an epidural, partially hemorrhagic cyst was removed. There was no obvious connection to the ipsilateral L5-S1 facet joint. Pathology revealed synovial cyst, and the patient's leg pain was improved postoperatively. This synovial cyst was unusual as it had no connection with the facet joint intraoperatively and its location in the sacral canal was uncommon. A 68-year-old female with a long history of intermittent back pain had a relatively acute onset of severe low back pain radiating down the posterior left buttocks and left leg for three weeks. She denied any trauma or other possible inciting factors. The pain had been getting progressively worse, and she also had developed numbness in the left S1 and S2 dermatomal distributions. An epidural steroid injection performed at a local facility provided partial relief and lasted for approximately 24 hours. The pain was described as intense, electrical pain different from any pain she had ever felt in terms of distribution and severity. Due to the exacerbating factors including standing and walking, thus, she presented to clinic in a wheelchair and preferred to lay in the fetal position. She had presented to the emergency department two days prior to neurosurgery consultation due to continued worsening of pain and numbness.A thorough neurological examination was negative except for a positive straight leg raise on the left.Lumbar spine X-ray did not reveal any abnormal alignment or transitional anatomy . Lumbar The patient was taken to surgery for a left S1 and partial left S2 hemilaminectomy. An epidural, partially hemorrhagic cyst inferior to the left S1 pedicle was dissected off the dura . The cysWe present a case of a lumbar synovial cyst that on imaging and at surgery did not appear in proximity or continuous with the ipsilateral L5-S1 facet joint. This is rare, as these cysts are often found to be continuous with the facet joint of origin at surgery. The cyst resulted in adjacent nerve root compression and following cyst resection there was improvement in the radicular symptoms. The literature on unusually located lumbar synovial cysts is reviewed.Lumbar synovial (or juxtafacet) cysts are a common cause of radicular pain due to their proximity to the exiting and traversing nerve roots. Low back pain is often related to degenerative changes in the lumbar facet joints. Many of these epidural cysts are found at the L4-5 level, presumably due to the greater degree of motion at this level, and to a lesser extent at the L5-S1 and L3-4 levels . SynoviaAs stated above, while many lumbar synovial cysts are straightforward in presentation, appearance, and location, some are not. Métellus and colleagues  reportedWhile lumbar synovial cysts often demonstrate continuity with the facet joint that may only be appreciated intraoperatively and not on preoperative imaging, they can also be remotely located. In our patient, the synovial cyst was found in the sacral spinal canal and distinctly separate from the facet joint. It is important to remember that epidural masses in the lumbosacral spine that present and image similar to synovial cysts may in fact be synovial cysts. Despite unusual locations of some of these synovial cysts, surgical resection remains a preferred treatment option.


Chemoradiotherapy is the standard of care for patients with oesophageal cancer unsuitable for surgery due to the presence of co-morbidity or extent of disease, and is a standard treatment option for patients with squamous cell carcinoma of the oesophagus. Modern regimens of chemoradiotherapy can lead to significant long-term survival. However the majority of patients will die of their disease, most commonly with local progression/recurrence of their tumours. Cetuximab may overcome one of the principal mechanisms of tumour radio-resistance, namely tumour repopulation, in patients treated with chemoradiotherapy.The purpose of this research is first to determine whether the addition of cetuximab to definitive chemoradiotherapy for treatment of patients with non-metastatic carcinoma of the oesophagus is active (in terms of failure-free rate), safe, and feasible within the context of a multi-centre randomised controlled trial in the UK. If the first stage is successful then the trial will continue to accrue sufficient patients to establish whether the addition of cetuximab to the standard treatment improves overall survival.SCOPE1 is a two arm, open, randomised multicentre Phase II/III trial. Eligible patients will have histologically confirmed carcinoma of the oesophagus and have been chosen to receive definitive chemoradiotherapy by an accredited multidisciplinary team including a specialist Upper GI surgeon. 420 patients will be randomised to receive definitive chemoradiotherapy with or without cetuximab using a 1:1 allocation ratio.During Phase II of the study, the trial will assess safety (toxicity), activity (failure-free rate) and feasibility (recruitment rate and protocol dose modifications/delays) in 90 patients in the experimental arm. If the experimental arm is found to be active, safe, and feasible by the Independent Data Monitoring Committee then recruitment will continue into Phase III. This second stage will recruit a further 120 patients into each arm and compare the overall survival of both groups.All patients randomised into Phase II will contribute to the Phase III comparison of overall survival. In addition to overall survival, Phase III of the study will also assess toxicity, health related quality of life and cost effectiveness. A detailed radiotherapy protocol and quality assurance procedure has been incorporated into this trial.ISRCTN47718479ISRCTN:  Worldwide, oesophageal cancer is the eighth most common cancer, responsible for an estimated 482,300 new cases and 406,800 deaths in 2008 and is the fifth highest in mortality rate among tumour sites . In the Surgery has long been, and remains, the cornerstone for cure of oesophageal cancer and is considered for all patients with potentially resectable oesophageal cancer who are fit for surgery and have no evidence of distant disease. Approximately 23% of patients survive 5 years with the most commonly used surgical treatment strategy . The mosIn a pivotal study , US InteIn the UK, most experience has been gained in those patients, both with SCC and AC, deemed unsuitable for surgery due to the presence of co-morbidity or extent of disease ,13. DespConcurrent CRT regimens have been based upon cisplatin and 5FU. Both have good single agent activity in oesophageal malignant disease and are amongst the best radio-sensitisers in tumour models ,15. The 5FU has historically been given as a continuous infusion throughout treatment. Capecitabine (Xeloda™), an oral fluoropyrimidine, sequentially converted to 5FU via 3 enzymes located in liver and tumour tissue, mimics the effect of continuous infusional 5FU. Capecitabine has been shown to be at least as effective as infusional 5FU in advanced oesophago-gastric cancer  and the The majority of patients who relapse do so within the previously irradiated area ,10,19. TBonner et al  reportedIn patients with metastatic colorectal cancer, cetuximab has shown significant activity in combination with chemotherapy in patients who have relapsed on the same chemotherapy regimen given alone . EXPERT-Anti-EGFR therapies have been combined with CRT in the treatment of thoracic malignancies. 15 patients with stage III non-small cell lung cancer received gefitinib with 60Gy radiotherapy, given concurrently with carboplatin and escalating doses of paclitaxel. This anti-EGFR small molecular agent was well tolerated even in the cohort receiving full dose chemotherapy, with an impressive response rate of 91% . RTOG 03In oesophageal cancer, cetuximab has been tested in combination with irinotecan, cisplatin and concurrent radiotherapy in Phase II studies: a single centre study  and in the SWOG-SO414 study. In the former study, in resectable tumours, 17 of 39 planned patients had completed the protocol by the ASCO 2006 meeting . Grade 3In a larger series, 37 patients with oesophago-gastric cancer underwent carboplatin AUC = 2, paclitaxel 50 mg/m2, cetuximab 250 mg/m2 weekly concurrently with 50.4Gy radiotherapy. There were no Grade 4 non-haematological toxicities, 20% of patients had Grade 3 oesophagitis. Eighteen of 27 (67%) had no residual disease on endoscopic biopsy after treatment and 7 of 16 (43%) who underwent surgery had a complete pathological response . BuildinSince the start of the SCOPE1 trial, a phase II study of neoadjuvant CRT with oxaliplatin and infusional 5-FU plus cetuximab followed by postoperative docetaxel and cetuximab has completed in patients with operable adenocarcinoma of the oesophagus . It was Other Phase I/II studies of cetuximab in oesophageal cancer are still ongoing: EXCEL , FFCD 0505 , and NCT00425425 (43 patients in Germany). RTOG 0436, a Phase III trial (420 patients) evaluating the addition of cetuximab to paclitaxel, cisplatin, and radiation for patients with oesophageal cancer who are treated without surgery, is still in recruitment. A Swiss Phase III trial  giving CRT with or without cetuximab followed by surgery is also in recruitment.Assessing response to CRT, without surgically removing the oesophagus for histological examination, is notoriously difficult. Endoscopy negative rates following CRT range from 30-80% (mean 61%) -42. AlthThe main study aim of the SCOPE1 trial is to assess the effect on overall survival of adding cetuximab to the standard treatment. We will also examine the effect on toxicity, health related quality of life (HRQL), and cost effectiveness. In this paper we describe the study protocol.2 day 1, week 1 only, then 250 mg/m2 weekly thereafter for a further 11 weeks).SCOPE1 is a two arm, open, randomised Phase II/III trial. It is being run in approximately 50 participating centres throughout the UK. Eligible patients will have histologically confirmed carcinoma of the oesophagus  and have been chosen to receive definitive CRT by an accredited multidisciplinary team (MDT) including a specialist Upper GI surgeon. At randomisation, participants are assigned to either the control arm or the research arm using a 1:1 allocation ratio , activity and feasibility in 90 patients in the experimental arm. Activity will be measured as treatment failure-free rate and feasibility as the number of protocol dose modifications and delays. If toxicity, activity and feasibility are found to be unacceptable recruitment into the trial will stop. If the experimental arm is found to be active, safe and feasible, by the Independent Data Monitoring Committee (IDMC), recruitment will continue into Phase III of the study. Phase III of the study will recruit a further 120 patients into each arm  and compare the overall survival of both groups. The experimental arm of CRT and cetuximab will be compared against the control arm of CRT alone. This second stage of the trial is powered to detect a survival advantage with the addition of cetuximab.All patients randomised into Phase II will contribute to the Phase III comparison of overall survival. In addition to overall survival, Phase III of the study will also assess toxicity, HRQL, and cost effectiveness.Detailed radiotherapy treatment protocols have been notably absent in recent UK Upper GI Cancer trials involving CRT, as have procedures to assess adherence to treatment recommendations. Therefore a detailed radiotherapy protocol and quality assurance procedure ) will be incorporated into this trial.SCOPE1 has been ethically approved by the Research Ethics Committee for Wales and has approval from the Medicines and Health Care Product Regulatory Agency to be conducted in the UK. The Wales Cancer Trials Unit, a Cancer Research UK core funded and National Cancer Research Institute accredited Clinical Trials Unit, is coordinating the trial. Velindre NHS Trust is the sponsor for the trial. A Trial Steering Committee and an Independent Data Monitoring Committee has been set up to monitor the progress and safety of the study. The SCOPE1 Trial Management Group, including clinicians, clinical trial unit staff, patient representatives, nursing and pharmacy representatives, carry out the day-to-day running of the trial.Eligible patients will have been diagnosed with carcinoma of the oesophagus, AC, SCC, or undifferentiated carcinoma, or Siewert Type 1 or 2 tumour of the gastro-oesophageal junction (GOJ) and selected for treatment with definitive CRT by an appropriate specialised MDT. Such patients may enter the trial if they meet all the necessary inclusion and none of the exclusion entry criteria  and an endoscopic ultrasound  should be performed. One of these assessments should be carried out within 4 weeks. Within 1 week prior to randomisation the following screening assessments should be performed: a history and physical examination ; full blood count; serum renal, liver and bone profile (including serum magnesium); glomerular filtration rate; electrocardiogram; and pregnancy test in females of child bearing age.The treatment failure-free rate in patients treated with CRT is approximately 60% at 24 weeks ,42. WithCalculations were performed using the ART package  in StataPatients are randomised centrally by the Wales Cancer Trials Unit using the method of minimisation which includes a random element. Patients are stratified for a number of clinically important stratification factors. The randomisation allocation ratio for control: research arm will be 1:1.The primary outcome measure is treatment failure rate in the research arm. Treatment failure rate will be assessed at 24 weeks (12 weeks after completion of CRT) and is defined as pathologically confirmed (by endoscopic assessment and/or biopsy) residual disease and/or CT scan of thorax and abdomen showing progressive disease according to RECIST criteria. Treatment failure will also include progression or death due to disease before 24 weeks.The secondary outcome measures are:Toxicity: will be scored using the NCI CTCAE v3.0 and RTOG late radiation morbidity scoring criteria at baseline, during treatment and at pre-specified time points on follow-up. Serious adverse events will be monitored "real-time" by the Chief Investigator and TMG.• Feasibility: will be measured through the number of protocol dose modifications and delays.• The primary outcome measure is overall survival (OS). Overall survival is calculated from the date of randomisation to death from any cause. Those patients still alive will be censored at the date last seen.The secondary outcome measures are: toxicity (as above), HRQL, RTTQA and cost effectiveness:HRQL: This aspect of the trial aims determine whether cetuximab in addition to CRT:• ◦ improves generic and disease specific aspects of HRQL following treatment than CRT alone. Specific HRQL domains that are expected to be better in the experimental group are: physical and role function, fatigue, dysphagia and eating restrictions◦ is associated with poorer HRQL during treatment. Specific HRQL domains that are expected to be worse in the experimental arm include: dyspnoea, skin rashes and diarrhoea.Generic domains of HRQL will be assessed with the EORTC core Quality of Life Questionnaire, the EORTC QLQ-C30. This inCost effectiveness: A cost-utility analysis of cetuximab in addition to CRT in oesophageal cancer will be performed from the perspective of the UK National Health Service, but with consideration also given to patient and family related costs. The cost-effectiveness will be considered using two time horizons: within trial and lifetime. In order to undertake the latter a decision-analytic model will be developed to assess the costs and effects over the lifetime of patients. The model will be a Markov model and the transition probabilities will be based on parameter estimates derived from the trial (and other sources from within the literature). The within trial component will involve a comparison of the additional costs associated with the use of cetuximab in the treatment regimen ± any changes in resources utilised elsewhere. Costs of cetuximab will be based on discussions with clinicians and finance staff, while other healthcare resources utilised by patients during the study period will be collected via the healthcare resource utilisation log administered during treatment and at follow-up. At each of the 3-4 weekly visits, patients will be asked to indicate whether they have had any contacts with their GP, practice nurse, community nurse or attended hospital either as an outpatient or in-patient. They will be asked whether the contact was connected to their condition or for any other purpose. The involvement of others in providing transport and/or support will also be logged. In addition, they will be asked to indicate the medication they have been taking during the 3-4 week period. Consultations with healthcare professionals will be costed using published sources of unit costs and healthcare resources utilised will be added to the respective treatment costs in each arm. Quality adjusted life years (QALYs) will be derived from survival data generated during the second stage of the trial and from the EQ-5D scores collected at baseline, during treatment and at follow-up. A patient diary will also be used to collect additional data. These data will then be used to populate the lifetime model.• RTTQA: A detailed QA program will be in place to ensure adherence to the protocol (see RTTQA section below). Major and minor deviations will be collected.• NB: Although activity, safety and feasibility will not be formally assessed in the control arm in Phase II, the randomisation of patients will continue using the same process and on a 1:1 allocation and therefore those patients in Phase II will be included in the analysis of Phase III.Participants will be seen at hospital at randomisation, the end of each treatment cycle, then at Month 6, 9, 12, 16, 20, 24, 36, 48, and 60 after randomisation. Research staff at the hospitals will be expected to complete trial CRFs which record evidence of primary and secondary outcome measures.All analyses will be on an intention-to-treat basis, i.e. all patients randomised will be included, and all patients will be analysed according to their allocated group whatever treatment they received. Both Phase II and Phase III analyses will include:• Descriptive statistics of the patient characteristics within each treatment group• A CONSORT flow diagram of enrolment, intervention allocation, and follow-up• Tables of toxicities at each timepoint • Treatment compliance during each cycle (in terms of proportions of patients with delay/reduction to CRT) within each treatment group. An exploration of predictors of poor compliance will be performed.When 6 month follow up data has been obtained for 83 patients in the research arm then the Stage 1 analysis will be performed. According to the Flemings design, we need to see 58 "failure free" patients out of 83 patients followed up to 6 months in the research arm.The main analysis will compare overall survival between the two groups using unadjusted logrank test. Final analysis will take place when 269 events (deaths) have been reported, which is expected to be approximately 1 year after accrual closes. The secondary outcome of proportions of patients with SAEs will be compared using a chi-squared test.No formal subgroup analyses are planned to look at differences in primary and secondary endpoints between treatment groups within specific groupings based on patient characteristics. However, if any treatment difference is found we will look to see whether it is consistent across patient subgroups  although this analysis will be exploratory in nature.The HRQL data will be scored according to the algorithms described in the EORTC QLQ-C30 scoring manual . All scaThe economic evaluation will assess the differences in mean costs between the trial arms and use with the differences in QALYs to generate a cost per QALY estimate. The EQ-5D allows estimation of quality adjusted life years (QALYs) which will be the main effectiveness measure in the economic analysis. Between group differences will be estimated using the area under the curve method adjusted for differences at baseline. Since cost data are often skewed, bootstrapping methods will be used to produce 95% confidence intervals alongside point estimates. A probabilistic sensitivity analysis will be undertaken and a cost-effectiveness acceptability curve produced. In addition, a series of one-way sensitivity analyses will be undertaken to assess the robustness of the estimate to changes in costs, resources utilised, utility scores and QALYs. In the case of non-dominance, results will be reported in the form of an incremental cost effectiveness ratio (ICER) which shows the extra cost of producing one extra QALY. A cost-effectiveness acceptability curve (CEAC) will be used to calculate 95% confidence intervals for the incremental cost effectiveness ratios.The RTTQA for SCOPE1 consists of clinical oncologists, dosimetrists, trial management, medical physicist and research fellows. It is part of the NCRI RTTQA group.An educational CD ROM is being distributed to all investigators. This CD ROM includes three example cases, each of which consists of a clinical summary, CT slice data, outlines and Radiotherapy plan information. GUINESS software is provided for viewing the CT, outline and planning data. A radiotherapy treatment planning and delivery protocol has been written and is supplied in hard copy and electronically on the CD ROM.All clinical and radiation oncologists participating in the trial are required to outline a test case . All planning departments are required to plan this test case. This would usually be carried out on the outline completed by the Principal Investigator. A Plan Assessment Form (PAF) is completed that compares the planned test case with the delivery protocol for major and minor deviations. This, together with the completed plan, is assessed centrally by the SCOPE1 RTTQA team. All centres and Investigators must have a 'Pass' for the planned test case before entering patients into the trial.On trial, centres are required to provide a PAF and a full dataset of images, structures, plan and doses, for each patient. The first clinical case from each investigator, a 10% sample and those with noted major deviations (on the PAF) from the protocol will be assessed centrally by the SCOPE1 RTTQA in real time.This Cancer Research UK funded trial will define the most effective CRT regimen in terms of overall survival for patients with oesophageal cancer unsuitable for surgery due to the presence of co-morbidity or extent of disease. The Phase II/III design allows an early assessment of activity and toxicity of cetuximab in this context before progressing to the definitive Phase III endpoint. This provides an early stopping opportunity whilst also allowing trial momentum to be maintained. This efficiency saves years compared to running concurrent Phase II and Phase III trials by having one rather than two funding and regulatory approval applications and centre set up processes. This trial builds in health economic and HRQL measures which provide a well-rounded assessment of the impact of the experimental regimen. It also has a RTTQA component which standardises radiotherapy delivery within the trial.SWG has received research funding from Roche and Merck. Otherwise none of the authors have any competing interests apart from receiving free drug from Merck for this trial.TDC was responsible for the research question. GOG, TDC, IG, SG, JNS, CH, LSN were responsible for the design of the trial and contributed to the writing of the study protocol. GOG is the Scientific Director and TSM the Clinical Director of the Wales Cancer trials Unit and has overall responsibility for running the trial at the WCTU. RAM is the Trial Manager for the trial. CJP is the Health Economist for the trial and participated in the study design. JMB designed the HRQL aspects of the study. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/466/prepub


Markers that can discriminate between indolent and aggressive prostate tumours are needed. We studied gene methylation in non-neoplastic tissue adjacent to prostate tumour (NTAT) in association with prostate cancer mortality.From two cohorts of consecutive prostate cancer patients diagnosed at one pathology ward in Turin, Italy, we selected 157 patients with available NTAT and followed them up for more than 14 years. We obtained DNA from NTAT in paraffin-embedded prostate tumour tissues and used probe real-time PCR to analyse methylation of the glutathione S-transferase (GSTP1) and adenomatous polyposis coli (APC) gene promoters.Prevalence of APC and GSTP1 methylation in the NTAT was between 40 and 45%. It was associated with methylation in prostate tumour tissue for the same two genes as well as with a high Gleason score. The hazard ratio (HR) of prostate cancer mortality was 2.38  for APC methylation, and 2.92 (1.49–5.74) for GSTP1 methylation in NTAT. It changed to 1.91 (1.03–3.56) and 1.60 (0.80–3.19) after adjusting for Gleason score and methylation in prostate tumour tissue. Comparison of 2 vs. 0 methylated genes in NTAT revealed a HR of 4.30 (2.00–9.22), which decreased to 2.40 (1.15–5.01) after adjustment. Results were stronger in the first 5 years of follow-up .Changes in gene methylation are an early event in prostate carcinogenesis and may play a role in cancer progression. Gene methylation in NTAT is a possible prognostic marker to be evaluated in clinical studies. There is presently no consensus on whether radical prostatectomy or active surveillance should be offered as the treatment of choice, especially when it comes to patients with a low risk prostate cancer In this paper we investigated if methylation changes in the non-neoplastic tissue adjacent to prostate tumour (NTAT) are associated with prostate cancer prognosis. Prostate tumours are often multifocal, and a number of studies observed field cancerisation  in prostate cancer. Studies comparing NTAT with benign prostate tissue and/or cancer tissue identified alterations in gene expression The study is nested in two cohorts of in total 459 consecutive prostate cancer patients who underwent biopsy, radical prostatectomy or transurethral resection of the prostate (TURP) between 1982 and 1988, or between 1993 and 1996, at the pathology ward of the San Giovanni Battista Hospital, Turin, Italy. Details of these two cohorts have been published previously a-priori patients from the 1980s whose diagnostic slides included mainly neoplastic tissue and were associated with considerable technical problems in isolating NTAT without contamination from prostate tumour tissue. All other tissue sources, namely TURPs (n = 11) and radical prostatectomies (n = 23) from the 1980s, and biopsies (n = 164), TURPs (n = 45) and radical prostatectomies (n = 34) from the 1990s, were included in the present evaluation. The uropathologist identified and highlighted one area of NTAT on each diagnostic slide. Areas of prostatic intraepithelial neoplasia were excluded. If more than one area of NTAT was present, the pathologist chose the farthest from the tumour. If there was more than one area that met this criterion, the largest area was chosen. The selected portions of NTAT had at least 1.5 mm of distance from tumour cells, in line with previous studies on epigenetic field effect in prostate cancer Diagnostic slides from patients in the two cohorts were analysed to identify NTAT. We excluded At the end of this process, NTAT was identified in 157 patients, all of whom were included in the present study. The selection process from the original 459 patients acted differently depending on the source of the tumour tissue and on the cohort (1980 or 1990 cohorts). Specifically, we selected 91% of the original patients who underwent a TURP or a radical prostatectomy in either of the two cohorts, 33% of those who underwent a biopsy in the 1990 cohort and, consistently with our a-priori criteria, none of those who underwent a biopsy in the 1980 cohort. This selection, being at baseline (i.e. before the occurrence of the outcome), is unlikely to have introduced bias in the associational estimates .Four-five (10 µm thick) sequential sections were cut from the paraffin-embedded tissue of the 157 selected diagnostic slides. The slices overlapped exactly with the slides on which the uropathologist highlighted the selected NTAT area. They were manually dissected, removing the portion of NTAT with a disposable thin blade, which was changed between each subject. DNA was then successfully extracted for all study subjects.The study was approved by the Ethical Committee of the San Giovanni Battista Hospital - CTO/CRF/Maria Adelaide Hospital of Turin . Anonymized data on gene methylation are available upon request to qualified researchers for the purpose of academic, non-commercial research.The commercial QIAamp ® DNA FFPE Tissue Kit  was used for the extraction and purification of genomic DNA. The adequacy of DNA was checked using the PCR amplification of the β-globin housekeeping gene The genomic DNA samples, including positive controls for methylated and unmethylated status, underwent bisulfite modification using Epitect Bisulfite Kit .2O to obtain a final volume of 25 µl. The reaction was carried out in a thermocycler  with the following PCR conditions: 1.30 minutes at 95°C, 15 seconds at 95°C, 1 minute at a primer-specific annealing temperature (60°C) for 50 cycles, and 10 minutes at 72°C. The universal methylated DNA CpGenome  was used as a positive control. Negative controls were included in all reactions.After bisulfite modification, a PCR assay with specific probes to determine the methylation status of APC and GSTP1 promoters was used. This assay allows detection of methylated cytosine with high sensitivity .The effect of NTAT methylation in each of the two genes on cumulative mortality from prostate cancer was investigated taking into account competing risks, and differences in mortality were evaluated with the Gray’s test Since prognostic markers are needed mainly for low- and intermediate-risk tumours, and at the time of diagnosis before deciding about prostatectomy, we conducted two subgroup analyses restricted to: i) study subjects with a Gleason score of 7 or less or ii) subjects who underwent biopsy. We also assessed the association of methylation in NTAT with short-term mortality by conducting separate analyses restricted to the first 5 years of follow-up.Selected characteristics of the 157 study subjects are reported in The prevalence methylation in NTAT was 43.9% for APC and 42% for GSTP1. As summarised in As shown in We found a strong association between presence of APC and GSTP1 methylation in NTAT and long-term mortality and 5-year mortality from prostate cancer. The association remained after adjustment for Gleason score and in analyses restricted to study subjects with a Gleason score of seven or less and in patients who underwent biopsy.These results support the hypothesis that DNA hypermethylation is an early event in carcinogenesis that can be detected before the tumour becomes morphologically evident; they also support the notion of field cancerisation in prostate cancer and its role in prostate cancer progression. The fact that the pattern of methylation is similar in NTAT and in prostate tumour tissue suggests that the alterations found in these tissues are the consequence of similar mechanisms, including a lateral expansion or influence of the tumour or diffuse effects of the same carcinogens.We studied methylation in GSTP1 and APC because of their involvement in crucial pivotal cell control pathways. GSTP1 is a “caretaker” gene, preventing genomic damage mediated by carcinogens or oxidants. Defects in “caretaker” genes (i.e. hypermethylation) may favour susceptibility to cancer development. Moreover GSTP1 may interfere with growth or survival signalling pathways by acting as a tumour suppressor gene In our study, we used mortality from prostate cancer as the outcome  and we had a very long follow-up time. We included both study subjects with a low-risk of prostate cancer, and those with more aggressive prostate cancer. The source of prostate tumour tissue included biopsies, TURPs and radical prostatectomies.a-priori that contamination did occur in some samples, its frequency was at most, limited, which is demonstrated by the fact that the association between methylation in NTAT and prostate cancer mortality remained practically unchanged after adjustment for methylation in prostate tumour tissue. Had the presence of methylation in NTAT been due to contamination from methylation in prostate tumour tissue, the association between methylation in NTAT and mortality from prostate cancer would have been strongly reduced after adjustment.The prevalence of methylation in NTAT that we found is similar or higher to that detected in previous studies, which, however, display considerable inter-study variation The main limitation of our study is the lack of clinical and pathological information other than the Gleason score. In addition we studied a population with, on average, a higher mortality from prostate cancer than typical contemporary patient series. These limitations hamper the possibility of assessing the actual additional prognostic value of methylation in NTAT compared with presently used nomograms. Our study is thus able to suggest a possible role of methytlation in NTAT in prostate cancer progression, although the possibility for clinical translation of this finding requires further investigation in a better defined clinical setting. We also lacked information on treatment at enrolment, implying that our cohort includes a heterogeneous group of patients, which further hampers direct clinical translation of our findings. However, the fact that the source of tumour tissue  was not an effect modifier suggests that our findings on methylation in NTAT are not explained by confounding from patients’ heterogeneities and/or initial treatment.a priori on the basis of substantial evidence of their potential role in prostate carcinogenesis, it is likely that additional prognostic information could be obtained by the study of a larger number of genes.The magnitude of the association between methylation in NTAT and mortality from prostate cancer that we found is stronger than that usually found for methylation in prostate tumour tissue In conclusion, our findings of frequent APC and GSTP1 methylation in NTAT and their association with mortality from prostate cancer support the notion that changes in gene methylation are an early event in prostate carcinogenesis and play a role in cancer progression. The strength of the association with mortality from prostate cancer and the fact that the association remains in patients with a Gleason score below 8 suggest that the methylation pattern in NTAT could be a possible prognostic marker for prostate cancer to be tested in future clinical studies.