Finally, future research questions that will help bring the use of local hyperthermia as systemic immunotherapy closer to clinical application are discussed.Local tumour hyperthermia for cancer treatment is currently used either for ablation purposes as an alternative to surgery or less frequently, in combination with chemotherapy and/or radiation therapy to enhance the effects of those traditional therapies. As it has become apparent that activating the immune system is crucial to successfully treat metastatic cancer, the potential of boosting anti-tumour immunity by heating tumours has become a growing area of cancer research. After reviewing the history of hyperthermia therapy for cancer and introducing methods for inducing local hyperthermia, this review describes different mechanisms by which heating tumours can elicit anti-tumour immune responses, including tumour cell damage, tumour surface molecule changes, heat shock proteins, exosomes, direct effects on immune cells, and changes in the tumour vasculature. We then go over  Hyperthermia therapy is a treatment approach in which the temperature of a particular area of the body or the whole body is heated above normal temperatures to achieve therapeutic effects. There are three basic hyperthermia categories, local, regional and whole body. When local hyperthermia is used for cancer treatment, heat is applied to a solid tumour. The heating temperature can be as high as 80 °C when the purpose is to completely ablate the tumour , or can Currently most cancer patients die not from the primary tumour, but from metastatic disease, and the only approach to treating unrecognised metastases is chemotherapy. Immune-based cancer therapies are on the cusp of becoming part of the standard of care for many cancers because the immune system can actively seek and eliminate occult metastatic disease that may often survive chemotherapy. Recent studies indicate that many cytotoxic chemotherapeutic drugs mediate their effects in part by stimulating anti-tumour immunity . With thThe use of heat for cancer treatment goes back to Ancient Egypt. According to the Edwin Smith Papyrus, a record of scientific approaches to medicine in Ancient Egypt, an Egyptian polymath named Imhotep burned off masses growing on the breast with a heated poker around 2600 BC. Certainly by 2000 BC, local destruction of tumours using cautery had become a widely used method for cancer therapy . In AnciIt had also been historically noted that infections were occasionally associated with cancer remission. In 1866 Busch in Germany reported that a malignant sarcoma on his patient underwent complete remission within two years after experiencing erysipelas infection , and thiSince Coley’s patients developed fever that persisted throughout the treatment , the potIn 1913, William Mayo documented some directly relevant clinical observations concerning the impact of local tumour hyperthermia [Despite Mayo’s inspiring finding, the potential of locally heating tumours before surgery in order to stimulate anti-tumour immune responses had no further reports until recently. While there are multiple reasons for this, it is at least partly due to lack of technologies to maintain tumours at a specific temperature in a uniform manner for a sustained time period and to measure temperatures precisely during the treatment . CurrentThere are several methods for inducing local hyperthermia. One historical method is to expose tumours to external lights such as an infrared light or to submerge the tumour site in a water bath. These strategies are suitable for tumours that are easily accessible, for example skin tumours, but not for tumours on internal organs, and even for skin tumours precise temperature and spatial control is difficult. An actively utilised method is to insert metallic probes into the tumour and deliver energies that preferentially deposit heat in the metal probe, such as microwaves or radiowaves, to raise the tumour temperature. This method enables rapid and large increases in the temperature and is therefore good for ablation purposes. However, it tends to form a significant temperature gradient across the tumour  and thisde novo synthesis and polymerisation of DNA are more temperature sensitive due to denaturation and aggregation of synthetases and polymerases [Temperatures in the range of 39–45 °C can arrest cell proliferation and kill cells. The effects are dependent on a combination of temperature and time of exposure to that temperature that together are referred to as thermal dose . When ceymerases , and thiymerases .Originally, the purpose of local hyperthermia therapy was simply to burn off cancer cells to get rid of them, similar to that of surgery. Since the positive correlation between the thermal dose and cytotoxicity is known historically ,32, it win vitro at 39.5 °C for 6 h increased surface expression of MICA, an NKG2D ligand, but not MHC class I [in vitro at 43 °C for 30 min had increased surface MHC class I levels [+ T cells. Increased lysis of tumour cells by NK cells and CD8+ T cells within the heated tumour can further improve anti-tumour immune responses, for instance by creating a more inflammatory cytokine milieu.Hyperthermia can increase the visibility of tumour cells to the immune system. Repasky’s group showed that heating tumour cells  class I , making  class I . KobayasI levels , which a+ T cells via MHC class I and thus elicit tumour-specific CD8+ T cell responses [+ T cell-dependent manner [The topic that has been studied most extensively in the context of hyperthermia-induced anti-tumour immunity is the role of heat shock proteins (HSPs). HSPs are a heterogeneous group of molecular chaperones with various functions that are up-regulated when cells are stressed in a variety of manners, including heat exposure . HSPs aresponses . By propt manner , this anin situ local hyperthermia, it should be noted that the overall outcome is influenced by the sum of all the effects by different HSPs. The amount of HSP production is dependent on the HSP subgroup, heating temperature and cell type. In the case of Hsp70 in the B16F10 mouse melanoma cell line, for example, Hsp70 release starts at 41 °C, reaches maximum at 43 °C and is completely diminished at 45 °C [While many HSPs are considered generally immunostimulatory ,43,48,49at 45 °C . As for at 45 °C . These pBesides heat, many kinds of physiological and chemical stress induce HSP expression. One of the most striking findings in recent cancer therapy research is that the efficacy of many chemotherapy drugs that have been used historically to kill cancer cells was actually relying on immune activation by chemotherapy-induced immunogenic cell stress and death . In fact+ T cell responses in mice [ex vivo systems [Exosomes, small membrane vesicles of 30–100 nm in diameter, are normally released by cells and have roles in intercellular communication . Tumour- in mice  and huma systems ,61.+ T cell responses in human HLA transgenic mice [+ and CD8+ T cells [Tumour cells experiencing stress, such as hypoxia and heat, also release increased amounts of exosomes . The strnic mice . Chemoki T cells .+ T cells by blocking the NKG2D receptor [On the other hand, immunosuppressive properties of tumour-derived exosomes are also known. For example, some exosomes of tumour origin express death ligands such as FasL and TRAIL, triggering apoptosis of activated T cells ,68. Exosreceptor . There areceptor ,70. Althin vitro studies by Repasky’s group, heating human NK cells at 39.5 °C for 6 h does not alter the surface expression level of NKG2D but results in NKG2D clustering as seen on NK cells activated with IL-2, leading to better lysis activity [+ T cells in vitro at 39.5 °C for 6 h enhances antigen-specific IFN-γ production and target tumour cell killing ability [in vitro culture at 39.5–40 °C for 2–3 h is also reported [When tumours are treated with local hyperthermia, tumour-infiltrating immune cells will also be exposed to heat. Fever is a natural immune stimulatory mechanism associated with fighting infections, so it is not surprising that fever level hyperthermia has stimulatory effects on leukocytes. According to activity . They al ability . Culturi ability ,73. Actireported . Almost reported , highligIn situ local hyperthermia increases the permeability of tumour vasculature. Dewhirst’s group showed that heating rat tumours at 42 °C for 1 h using a water bath increases the diameter of arterioles entering tumours by 35%, resulting in better tumour perfusion [2 [Lastly, changes in the vasculature within the tumour may help immune cell mobilisation. erfusion . Others usion [2  or betteusion [2 , but ove+ effector/memory T cell trafficking into the tumour, both of which are recapitulated by intravenously administering IL-6 instead of giving whole body hyperthermia. Our lab has shown that locally heating tumours at 43 °C for 30 min increases the intratumoural concentration of IL-6 [Immune cell trafficking may be further improved through changes in the vasculature adhesion molecules. Evans’ group showed that whole body hyperthermia of 39.5 °C for 5 h in mice increased intratumoural IL-6 trans-signalling . This in of IL-6 . TherefoIf the impact of local tumour hyperthermia is not stimulation of systemic immune response, then ablative thermal doses applied to the local tumour would be preferable to milder doses since it would eliminate the local tumour completely. With all the evidence that locally heating tumours would improve local anti-tumour immunity, it is important to determine whether the heat induces sufficient systemic immune responses to reject or at least retard the growth of untreated tumours. In fact, a variety of animal studies demonstrate the ability of hyperthermia of one tumour to affect the growth of other tumours not exposed to heat. Most researchers utilise subcutaneous or dermal tumour models for experimental feasibility. While many successful studies heat tumours at 42–45 °C, some studies show hyperthermia at ablation temperatures is also effective in eliciting anti-tumour immunity . The mos+ T cells and CD8+ T cells in both the heated and unheated tumours [Groups led by Kobayashi, Honda and Jimbow, the pioneers of the field of nanomaterial-induced local hyperthermia for immunotherapy against cancer, apply an alternating magnetic field to induce heat production by nano-sized liposomes containing iron oxide that are directly injected into the tumour. Upon heating of T-9 rat glioma on one flank for 30 min (approximately 42–45 °C throughout the heating process), not only the heated tumour, but also the unheated T-9 tumour implanted on the contralateral flank of the same rat completely disappeared. This coincided with increased infiltration of NK cells, CD4 tumours , indicatThey also showed that mice whose primary subcutaneous B16 melanoma was heated at 43 °C for 30 min have splenocytes with increased cytotoxicity specifically against B16 cells and show better resistance against secondary B16 rechallenge on the other flank compared to naïve mice . Althoug+ T cells in dLN, and conferred resistance against rechallenge with B16 tumours given on both the primary tumour side and the contralateral side [+ T cells, (but resistance was induced in mice that lacked either NK cells or IL-12), demonstrating that the anti-tumour efficacy is mediated by CD8+ T cells. Neither CD8+ T cell activation nor rechallenge resistance occurred when the primary tumour was heated at 45 °C instead of 43 °C, again suggesting the existence of an optimal temperature range for anti-tumour immune activation even within this narrow temperature range. This highlights the importance of precisely controlling the tumour temperature in a uniform manner, which can be done relatively easily in nanomaterial-mediated hyperthermia [Using solid iron oxide-based nanoparticles and an excisable dermal B16 tumour model, our lab compared mice whose primary tumours were heated and excised and mice whose primary tumours were unheated and excised [rthermia  as we al+ T cells and NK cells in the spleen, and splenocytes from treated mice showed higher cytotoxicity against B16 and NK-sensitive YAC-1 target cells than the untreated controls. Chen et al. heated subcutaneous Lewis Lung tumours at 42–43 °C for 1 h three times using microwaves [+ and CD8+ T cells into the tumour and that the recruitment was dependent on TLR4 expressed by tumour cells and DCs. Although these two studies did not show whether the immunological changes were sufficient to affect tumour growth of unheated tumours, the results at least tell us that heating tumours at 42–43 °C using microwave methods is also able to elicit anti-tumour immune responses.Some studies using non-nanomaterial heating methods also produced valuable results. Kubes et al. heated subcutaneous B16 tumours at 42 °C for 7 min three times by applying microwaves . This trcrowaves . They shden Brok et al. transferred either splenocytes or serum from donor mice whose subcutaneous B16-ova tumours were heated twice for 80 sec by radiofrequency ablation (reached 75–80 °C) into naïve host mice [+ T cells was combined with this hyperthermia therapy, the transferred CD8+ T cells expanded better in the spleen and dLN and the contralateral tumour grew slower than in adoptive transfer only mice. This is an encouraging example that shows hyperthermia therapy is able to cooperate with immunotherapy even when hyperthermia therapy itself is not potent enough to retard tumour growth.An interesting study by Bear et al. utilised gold nanoshells that generate heat upon exposure to near infrared radiation . Heating+ T cells, and DCs, and (3) by improving immune-cell trafficking between the tumour and lymphoid organs. Local tumour hyperthermia at 42–45 °C in mice induces tumour-specific resistance against rechallenge in a CD8+ T cell-dependent manner. This efficacy is sensitive to small temperature differences, which means that there is a narrow optimal temperature range at least for a poorly immunogenic tumour. Since locally ablating tumours also generates splenocyte-mediated tumour specific resistance, an optimal temperature range may exist at higher temperatures and associated thermal doses as well. Thorough comparison of different temperatures/thermal doses is needed to fully understand what temperatures are most suitable for immune stimulation. Following identification of thermal dose optima, the underlying mechanisms that differentiate the responses to different thermal doses can be explored.It is exciting to see the many promising results from the preclinical studies described above. To summarise, locally heating tumours at 39–45 °C can elicit anti-tumour immune responses (1) by enabling tumour cells to stimulate the immune system through increased surface expression of MICA or MHC class I and release of HSPs and/or exosomes, (2) by directly activating intra-tumoural immune cells such as NK cells, CD8To make local hyperthermia therapy a more powerful immunological approach for clinical application, it is critical to optimise the treatment as much as possible. Hyperthermia treatment protocols in reported studies differ in numerous factors including not only heating temperatures but also heating methods and duration, treatment timing and intervals, cancer types and stages, and therapeutic readout. It is important to understand how each parameter influences immune activation and the therapeutic outcome and to rigorously compare various protocols with only one variable at a time. This will eventually enable adjustment of each parameter to maximise the treatment efficacy according to the individual clinical situation.One of the questions needing further exploration is whether local hyperthermia cooperates with other clinically used therapies. It is likely that local hyperthermia therapy amplifies effects by other immunotherapies, such as checkpoint blockade and adoptive T cell therapy as already indicated by some studies ,88. Sincin situ enhances anti-tumour immunity will help optimise the therapy for clinical application. Although there is still much room for improvement, emerging evidence demonstrates the potential of local hyperthermia therapy as a powerful tool to support other emerging approaches to cancer immunotherapy.Further understanding the detailed biological and immunological mechanisms of how heating tumours Funding for this study came from a Dartmouth Center of Nanotechnology Excellence National Institutes of Health grant 1 U54 CA151662, a Center for Molecular, Cellular, and Translational Immunological Research National Institute of General Medical Sciences grant P20 RR15639, a Norris Cotton Cancer Support grant P30 CA023108, and Dartmouth Immunobiology of Myeloid and Lymphoid Cells grant 5T32AI007363-22. The authors alone are responsible for the content and writing of the paper.


We have employed the Lie group analysis for the simplification of the nonlinear differential system and computed the absolute invariants explicitly. An efficient numerical technique namely the shooting method has been employed for the constructions of solutions. Dual solutions are computed for velocity profile of an upper-convected Maxwell (UCM) fluid flow. Plots reflecting the impact of dual solutions for the variations of Deborah number, Hartman number, wall mass transfer are presented and analyzed. Streamlines are also plotted for the wall mass transfer effects when suction and blowing situations are considered. The non-Newtonian fluids at present are acknowledged more appropriate for scientific and technological applications than the materials obeying the Newtonian law of viscosity. However, there exists no single constitutive equation by which all the non-Newtonian materials can be described. Researchers and applied mathematicians have proposed a variety of constitutive equations in view of their rheological properties describing the non-Newtonian behavior. Such constitutive equations give rise to several complexities in the momentum equations which arises various challenges for researchers to model the problems in mathematical form and to evaluate/analyze the computed solutions mathematically and physically. The classifications of non-Newtonian fluids in general are classified into three types known as the differential, rate and integral. Available information on the topic indicates that much attention of the researchers has been accorded to the flows of second, third and fourth order fluids i.e. the subclasses of differential type materials. Undoubtedly these subclasses predicts the normal stress, shear thickening and thinning features but cannot describe relaxation time effect which many polymers show at low molecular weight. The relaxation time effect can be examined using Maxwell model (the simplest subclass of rate type fluid). The literature for flows of Maxwell fluid is quite sizeable. Few recent studies and several references on rheology of Maxwell fluid can be seen in the investigations –15.As per our knowledge no attempt has been made for the Lie group analysis and the development of dual solutions for magnetohydrodynamic (MHD) –21 flow B0 is applied along the ρ represented the density, ν the kinematic viscosity, σ the electrical conductivity and C1 > 0 is shrinking constant.Let us consider an upper-convected Maxwell (UCM) fluid flowing over a permeable shrinking sheet in a two-dimensional steady and incompressible field. The uniform magnetic field of strength By using the following dimensionless quantitiesM and the Deborah number β are defined as follows:Eqs –3 take tψ is introduced such asThe stream function Making use of Eq , law of In order to proceed for the Lie group analysis we considerx,y,ψ,u,v,θ,ϕ) transform into the coordinates  whereas Eqs , whereas no such value is given initially. In order to compute the desired result we select an initial guess and then applied the fourth-order Runge-Kutta method to approximate the value up to the desired accuracy of 10−5.It is noted that in order to compute the solution of the above differential equation as an initial value problem, we require the value of S ≥ 1.8018  exists only when the wall mass transfer satisfies the inequality S ≥ 1.7264 whereas for Maxwell fluid model (β > 0.0) the inequality reduces to S ≥ 1.6. Moreover dual nature of the solutions for both hydrodynamic and hydromagnetic flow situations are presented and found quite different. It is observed that the value of skin friction increase rapidly for the first solution whereas it decreases for the second solution but the variations in first solution is quite significant when compared with the other. Physically we can say that the increase in Deborah number β results into a decrease in a vorticity generation of a Maxwell fluid. Since the vorticity generation due to shrinking for Maxwell fluid is not as much as of Newtonian fluid therefore the boundary layer separation for Maxwell fluid is delayed and the similar solution exists for lesser amount of wall mass transfer. Moreover it is also noted that for the Newtonian fluid flow, both solutions are closed to each other and nearly parallel to x-axis which shows the linear behavior of the skin friction whereas for the non-Newtonian fluid, the variations in both solutions are quite significant and found quite opposite. Figs β on the velocity fields f′ and f respectively. It is observed that the dual solutions for velocity against different values of β exist. It is observed that the non-Newtonian fluid behaves much like liquids  for smaller value of Deborah number however for larger values of Deborah number, fluid transforms from liquids to the position of becoming a viscoelastic solid . Thus the flow retards by increasing Deborah number which is quite in agreement with the β for the first solution whereas from second solution, the oscillatory effects and cross over is found. The effects of wall mass suction (due to the permeability of the surface) on the flow are demonstrated in the Figs S and both solutions show quite opposite behavior. The magnitude of first solution enhances with an increasing in the suction rate whereas the magnitude of second solution decreases. Figs M on the velocity. It is observed that dual nature of solutions exists for different values of M. It is also illustrated that the first solution is dominant in comparison to the second solution (see Figs This part of the paper corresponds to some graphical observations so the one can analyze the physical insight of the problem. Figs 8018 see . Moreovesee Figs  and 11. The main results of the present analysis are mentioned below:Larger values of Deborah number β results in to steady flow of non-Newtonian fluid with lessor amount of wall mass transfer.Momentum boundary layer decreases with Deborah number in both first and second solutions.Wall mass transfer has opposite effects on the viscous boundary layer in first and second solutions.Magnetic field retards the rheology and the dynamics of the flow field.Suction and blowing phenomena show the opposite behavior on the streamlines.


The topological length The evolving shape of material fluid lines in a flow underlies the quantitative prediction of the dissipation and material transport in many industrial and natural processes. However, collecting quantitative data on this dynamics remains an experimental challenge in particular in turbulent flows. Indeed the deformation of a fluid line, induced by its successive stretching and folding, can be difficult to determine because such description ultimately relies on often inaccessible multi-particle information. Here we report laboratory measurements in two-dimensional turbulence that offer an alternative topological viewpoint on this issue. This approach characterizes the dynamics of a  Lagrangian description, the trajectory-based representation of hydrodynamics. Describing and characterizing Lagrangian properties of fluid turbulence is important for a better understanding of many natural and industrial processes, including turbulent mixing, the distribution of plankton in the ocean, or the spreading of pollutants in the atmosphere235389More than a century ago, O. Reynolds showed that watching the dynamics of coloured fluid lines in a flow was a powerful way to uncover the turbulent fabric of the underlying fluid motionperiodic flows61112121314111213The merger of ideas from Lagrangian hydrodynamics with those of dynamical systems has been a key route to unraveling the complexity of chaotic advection in braids made of Lagrangian trajectories. This approach is capable of capturing the deformation of fluid elements using topological considerations and a limited number of Lagrangian trajectories. The method is suitable for studying two-dimensional (2D) flows. So far, the potential of the braid method has been rarely investigated experimentally56142930Recent advances in laboratory modeling of turbulent flows, the development of experimental particle tracking techniques, as well as the availability of new mathematical methods have made it possible to extend the investigation to non-periodic and turbulent flows315161718192042223246142627Here, we report new experimental measurements of topological braids in 2D turbulent flows. Experiments have been carried out in a broad range of the turbulence kinetic energy by using both electromagnetically forced and Faraday wave driven 2D turbulence. The topological “length” J across the fluid cell36B is generated by placing a matrix of magnetic dipoles underneath the cell. The Lorenz J × B force produces local vortices at the forcing wave number The experiments are carried out in two different experimental setups used to produce homogeneous two-dimensional (2D) turbulent flows. First, we take advantage of the remarkable similarity between the horizontal motion of particles on the surface of a fluid perturbed by Faraday waves and the fluid motion in 2D turbulence171819Eulerian velocity field is measured by using particle image velocimetry (PIV) techniques. 2s−2 and forcing scales To visualize the horizontal fluid motion, the liquid-air interface is seeded with 50 μm diameter particles. The Lagrangian trajectories of hundreds of particles in the horizontal x-y plane17The turbulent fluid motion is also characterized here by using PTV which allows us to measure simultaneously the t being the third coordinate, x-y-t trajectories are projected onto the x-t (or y-t) plane, physical braid made of over- and under-crossings of strands. The crossings are the key topological information upon which the braid description hinges. The crossings of trajectories in 2D turbulence are qualitatively illustrated in In this approach, 2D trajectories are viewed as 3D strands, with time topological braid which is really the sequence of crossings of the trajectories previously described.- the topological loop which is like a fluid ribbon entangled within the braid.- the braiding factor (see details on the computation of Methods). In the course of time, each crossing along the braid distorts the loop and forces it to stretch or coil around the strands The topological braid is based only on the relative position of tracer particles and as such it does not require geometrical information such as the actual distance between strands . The top2s−2 and for various forcing scales We measure the temporal evolution of the topological length 2s−2. The initial loop is randomly entangled in the braid; as a consequence, at To further characterize this complexity, we measure the braid entropy In these experiments, Recently the concept of chaotic advection was further enriched by considering topological chaos614The main results of this paper appear in A recent study of  see . QuantitAlthough such a connection between single and multi-particle descriptors might sound surprising, it may originate from the uncorrelated motion of the particles that compose the braid. Indeed, we emphasize that the transition to the exponential growth regime of les  see . At thesMuch interest lies in determining Lagrangian tenets of turbulence irreversibility that would complement the Kolmogorov energy flux relations formulated in the Eulerian frame3233342SO4 water solution, SG = 1.03) is placed on top of a 4 mm thick layer of heavier (specific gravity SG = 1.8) non-conducting fluid (FC-3283). The fluid cell has a square section of 300 × 300 mm2. A matrix of 30 × 30 magnetic dipoles spaced in a checkerboard fashion 10 mm apart is placed under the bottom of the fluid cell producing spatially varying vertical magnetic field In these experiments, turbulence is generated using two different methods. In the first, 2D turbulence is generated electromagnetically in stratified layers of fluidIn the second setup, Faraday surface waves are used to generate 2D turbulence192s−2  and a 16 bit resolution using the Andor Neo sCMOS camera. The flows are characterized using both particle image velocimetry (PIV) and particle tracking velocimetry (PTV) techniques. We use PIV to compute the Eulerian energy spectra of the flows shown in 172s−2 and integral characteristic timescale The flows are visualized by placing 50In the course of time, each crossing along the braid distorts the topological loop and forces it to get more and more entangled in the impenetrable strands of the braid, see . It has braidlab library14D and the braiding factor To compute the topological braids made of fluid tracers trajectories and their corresponding braiding factor How to cite this article: Francois, N. et al. Braid Entropy of Two-Dimensional Turbulence. Sci. Rep.5, 18564; doi: 10.1038/srep18564 (2015).


Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded. Synovial fluid has been widely used for research, diagnosis, and treatment of joint diseases, such as osteoarthritis (OA). Although β-actin is extensively used as loading control in western blot , it is nA1AT, a 52-kDa protease inhibitor, is synthesized in the endoplasmic reticulum of the liver cells, released to blood, and diffused to lung epithelial cells , which iIn this study, we identified and confirmed that A1AT is abundant and relatively constant in OA and normal synovial fluid by Mass Spectrometry, western blot and ELISA respectively. Since there is no established loading control for western blot with human synovial fluid samples, we proposed that A1AT may be a good candidate for internal control in human synovial fluid studies.The study was approved by the Institutional Review Board at Rhode Island Hospital of the US and Shanxi medical University of China, and informed consent was obtained from each donor.OA synovial fluid was obtained during patient OA knee joint replacement . Normal control synovial fluid was obtained from healthy volunteers and the contralateral uninjured knee of patients undergoing unilateral ACL reconstruction (ACLR) .Patients who had inflammatory joint disease, acute major trauma, malignant tumors, or abnormal renal and liver function were excluded from the study. Patients who took corticosteroid treatment within the 3 months preceding surgery were also excluded from the study.A volume of 0.5–5 ml of synovial fluid was aspirated from the knee joint just before total knee replacement or arthroscopy. The synovial fluid was immediately centrifuged at 2,000 g for 10 minutes to remove cells and debris, and the supernatants were aliquoted and rapidly frozen at −80°C until analysis.Equal amount of synovial fluid samples from OA patients and normal controls were diluted by 10 times (1:10 dilution) with lysis buffer containing protease inhibitor  and electrophoresed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The gel was prefixed in 50% MeOH, 10% HoAC, 40% H2O for 30 minutes and then stained with 0.25% Coomassie Brilliant blue R-250  in the above solution for 4 hours. The gel was detained in 5% MeOH, 7.5% HoAC, 87.5% H2O until background was clear. The detained gel was stored in 7% HoAc and a photograph was taken using Canon camera .After 1:10 dilution, equal amount of synovial fluid samples from OA patients and normal controls were separated using SDS-PAGE under reducing conditions, as previously reported . Once prOA synovial fluid (N=10) and normal control synovial fluid (N=11) was diluted by 5000 times (1:5000 dilution), and analyzed by human A1AT ELISA (enzymelinked immunosorbent assay) kit . Samples were prepared in triplicates, and test was repeated 3 times.All data are analyzed using SPSS 13.0 software . Student’s T test was used for paired group comparisons. P values less than 0.05 were considered statistically significant.Human synovial fluid samples obtained from OA patients and control group were diluted and separated on a SDS-PAGE gel as described above. On the SDS-PAGE gel, the thickest band was located between 75 kDa and 50 kDa using Coomassie Blue staining and was consistently present in each synovial fluid sample among OA patients and normal controls, suggesting a highly abundant protein . SequencAs a good internal control for western blot, A1AT levels should be constant in OA and normal synovial fluids. To test this possibility, western blot and ELISA were performed to determine A1AT levels in OA and normal samples. The differences of Ihh, IL-1b, TNF-α, Fibronectin, and MMP-13 contents between OA and normal synovial fluid samples were detected by western blot while A1AT levels detected on the same membrane remained constant in all samples . In addiPrevious study has demonstrated that A1AT, a serine protease inhibitor synthesized in the liver, is highly abundant in both blood serum and synovial fluid. Using the quantitative radial immunodiffusion method, Swedlund et al found that A1AT contents in normal and OA synovial fluid are very close ,12. In tThere is not a well-established loading control for western blot with synovial fluid samples. Many studies present western results without loading control –16, whilAlthough A1AT level is increased in response to inflammatory conditions, such as rheumatoid arthritis , there iIn summary, our finding is the first study to show that A1AT can be used as a loading control in western blot for human synovial fluid studies. With a good loading control, western results with synovial fluid samples will be more convincing.


Predicting clinical response to anticancer drugs remains a major challenge in cancer treatment. Emerging reports indicate that the tumour microenvironment and heterogeneity can limit the predictive power of current biomarker-guided strategies for chemotherapy. Here we report the engineering of personalized tumour ecosystems that contextually conserve the tumour heterogeneity, and phenocopy the tumour microenvironment using tumour explants maintained in defined tumour grade-matched matrix support and autologous patient serum. The functional response of tumour ecosystems, engineered from 109 patients, to anticancer drugs, together with the corresponding clinical outcomes, is used to train a machine learning algorithm; the learned model is then applied to predict the clinical response in an independent validation group of 55 patients, where we achieve 100% sensitivity in predictions while keeping specificity in a desired high range. The tumour ecosystem and algorithm, together termed the CANScript technology, can emerge as a powerful platform for enabling personalized medicine. Efficacy of anticancer treatments vary across patients, imposing a need for personalized approaches. Here the authors show that responsiveness to chemotherapy can be predicted using tumour explant cultures in a patient-matched microenvironment, coupled with a machine-learning algorithm. KRAS, but provide clinical benefit in only 10–20% of selected patients16The ability to predict patient tumour response to cytotoxic or target defined therapeutic agents remains a holy grail. While molecular and genetic profiling is driving the evolution of subtype-specific personalized therapy134in vitro and ex vivo preclinical approaches that employ cell lines and spheroids312ex vivo organotypic tumour models are all limited by their inability to capture the full biological approximation of the native tumour, resulting in poor mapping to clinical outcomes192021Emerging evidence implicates intratumoral heterogeneity, both hierarchical and stochastic, in the variability of response to chemotherapy, which is not captured by the existing cancer cell biomarker-based approaches. Genetic and epigenetic distinctions within clonal populations could critically determine whether a particular drug combination will benefit a patient or result in resistance89101112910141617ex vivo tumour ecosystem, where thin tumour sections with conserved cellular and microenvironmental heterogeneity and architecture were cultured in tissue culture wells coated with grade-matched tumour matrix support in the presence of autologous serum (AS) containing endogenous ligands. The integration of the tumour ecosystems with a novel machine learning algorithm formed the CANScript platform, which reliably predicted the therapeutic efficacy of targeted and cytotoxic drugs in patients with head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). The robustness of this platform in predicting clinical response could potentially be useful for personalizing cancer treatment.To create a clinically relevant predictive model, here we engineered an ex vivo setting. Indeed, three-dimensional (3D) matrix support is emerging as a critical factor that dynamically determines the fate of tumours in terms of integrity, survival, metastasis and response to chemotherapy23240) proliferation (Ki-67) state compared with the corresponding unmatched counterparts and no matrix controls. As expected, high-grade tumours did exhibit a greater capacity to preserve the proliferation profile even in low-grade TMP. Low-grade tumours in high-grade matrix performed poorly (0 baseline). As shown in 0 baseline. While gelatin coating was no better than non-coated condition, collagen partially supported tumour proliferation, tumour area and phosphorylation of ERK1/2 but not cell viability. Interestingly, Matrigel, a widely used murine tumour-derived matrix, resulted in increased cell viability, tumour area and phospho-ERK but not in proliferation  using neutralizing antibodies to EGFR, which is consistent with the aberrant activation of EGFR pathway in a majority of HNSCC and other cancers of epithelial origin2772 h control, suggesting that despite the predominant role of EGFR in some individual tumours additional constitutive mechanisms exist that might contribute to minimal maintenance of these tumours  control, was significantly inferior to AS. Similar pattern was observed for Ki-67 (0 baseline) showed that a bulk of the proteins in RTK cascades that were upregulated are largely conserved in the tumour explants cultured in 2% AS  obtained from treatment naïve patients  with AS and recombinant EGF. As shown in or Ki-67 . Taken tin 2% AS .0) phenotypes better than T72 h control or EGF+TMP(0 baseline) tumour and significantly enhanced compared with control explants cultured without AS and TMP, or with either AS or TMP alone. The pattern of augmentation of Ki-67 upon AS+TMP was found to be consistent and significant (0) and serially sectioned tumour explants cultured under different conditions. Indeed, a high degree of conserved global transcriptomic profile consistent with the primary tumour was observed only in the case of the CANScript platform that integrated both TMP and AS, while supplementing the explant cultures with either AS or TMP(+EGF) alone resulted in distinct transcriptomic signatures  and angiogenesis  signatures  were contextually integrated in the explant system. Immunohistochemistry (IHC) labelling was used to evaluate a number of static and dynamic phenotypic markers associated with functional heterogeneity of tumour microenvironment. Profiling for CD68 (marker for immune component)r EGF+TMP. Similarr EGF+TMP. Furthernificant . Togethegnatures . Concurrgnatures . To furtgnatures  was analgnatures . It is ignatures . Taken tThe conservation of patient tumour heterogeneity in the CANScript tumour ecosystem prompted us to explore the possibilities of using this as a preclinical tool to predict anticancer drug response. To assess this, we first compared drug response in human tumour-derived xenotransplants (HTX) and in matched CANScripts explants . Primary HNSCC tissues were propagated in severe combined immunodeficiency mice up to second passage (P2-HTX). Since response and resistance to a particular drug combination can be intrinsically controlled by deregulation at the genetic and epigenetic levels,113637ex vivo to in vivo correlation in response to a general cytotoxic drug combination that we observed in HNSCC samples encouraged us to further validate the predictive ability of CANScript for targeted therapeutics. For this purpose we used HTXs generated from HNSCCs harbouring wild-type or mutant KRAS. Consistent with the results observed earlier with cytotoxics, a positive response in the CANScript explants with cetuximab , minimal tumour growth inhibition was evident in vivo  between CANScript explants outcomes and in vivo HTX responses  for the 70 HNSCC patients and cetuximab+FOLFIRI for the 39 CRC patients. The functional read-outs from these CANScripts, quantified in terms of viability, histopathology, proliferation and apoptosis, together with the observed clinical response in the matched patients, classified as progressive disease/non-response (NR), partial response (PR) or complete response (CR) based on PERCIST guidelines , while the remaining 10 presented with progressive disease. Interestingly, the CANScript platform predicted two CRs, two PRs and nine NRs, with only one actual NR case being wrongly predicted as CR. As shown in In the next step, the learned model was refined to classify the predicted responders into partial and complete responders (PR and CR), by selecting a threshold that maximized PR versus CR prediction accuracy on the training set. Following this, scores between 19.1 and 55.14 were classified as PR, and those >55.14 as CR. As can be seen in KRAS status, only 1 exhibited CR and 12 exhibited PR, and the remaining 39 presented with progressive disease. While the use of more than one biomarkers, for example, the use of wild-type KRAS and BRAF to select patients eligible for cetuximabWhile biomarker driven personalized cancer therapy has emerged as a powerful concept, the mere presence of a biomarker in a cancer cell may not translate into clinical efficacy167A key attribute of the CANScript platform is its ability to capture the intratumoral heterogeneity to a greater degree than achieved by biomarker-based selection of cancer cells. Cancer stem cells, stromal cells such as intra and peritumoral immune cells, and vascular components can further add to the heterogeneity and contribute towards tumour survival, progression and metastasis151734101134Interestingly, short-term cultivation of primary explants of human tumours had been explored previously, for example, growing the specimens in plasma clots2025444546471516172125202730505153545556ex vivo to in vivo functional correlation data clearly show the benefit of using CANScript technology as a surrogate of animal modelling. In addition, the minimal amount of tissue required to establish the CANScripts means multiple explants per tumour biopsy, which allows us to better capture the impact of intratumoral heterogeneity on outcome.The ability to predict outcomes is not only attractive from a clinical perspective, but also has major implications on preclinical cancer research, where the focus has been to develop assays that can bridge the translational gap. While animal models have been used as the front line in predicting efficacy, the predictive value of these models is debatable, a consequence of using cell lines cultured over years that are no longer representative of the original tumour. Furthermore, transgenic murine models may recapitulate a specific cancer pathway, but fail to capture the true heterogeneity that is characteristic of human tumours. For example, we observed in our study that while EGFR generally plays a critical role in HNSCC, additional driver mechanisms such EphB4, AKT, ERK1/2, Tie2, VEGFR2, cAbl, FGFR1, HER3 and IR are activated. Indeed, such stochastic heterogeneity has been implicated in the induction of adaptive resistance. There is therefore a resurgence in the use of early-passage patient-derived xenografts for predicting clinical responses. Consistent with these recent studies, we observed a good concordance in terms of histopathology and gene expression between the tumour biopsy (P0) and the 2ndpassage xenografts (P2-HTX). However, we did observe unique mutations between the P0 biopsy and the P2-HTX xenografts. It is possible that these differences between P0 and P2-HTX arise due to intratumour heterogeneity at the time of implantationA powerful feature of the CANScript platform is its use of a novel machine learning approach that is tailored to make accurate predictions particularly for potential responders. Specifically, the algorithm operates in two stages: it first employs the recently proposed SVMpAUC-based learning algorithm to distinguish between responders and non-responders in a way that maximizes sensitivity (fraction of responders predicted as responders). Indeed, the learned model in our case achieved 100% sensitivity on the test set while keeping specificity in an acceptably high range. In the second stage, the algorithm learns an additional threshold to separate responder predictions into complete responder and partial responder predictions. This approach was found to be superior to the performance of a standard, widely used support vector ordinal regression algorithm that directly aims to make predictions in the three categories and does not explicitly incorporate the need for high sensitivity. Interestingly, studies have correlated complete pathological response to the long-term progression-free survival6g for 15 min. All sera samples were aliquoted and stored at −80 °C for further use.Tumours samples were collected by core biopsy at the beginning of treatment and at the time of surgical removal for deserving patients (for patient detail see 3) and digested with dispase (Stem cell Technologies Inc. ), and subsequently the cells were separated using a sieveg for 15 min and the supernatant was discarded to retain the pellet. The pellet was incubated in 2 M Urea buffer (0.15 M sodium chloride and 0.05 M Tris pH 7.4) and stirred for 1 h at 50 °C. The complex extracted proteins were solubilised in Urea bufferg for 20 min and resuspended in the 2 M Urea buffer, aliquoted and stored at −80 °C. In addition, extracted protein samples were run at denaturing conditions in the presence of standard molecular weight ladder. When the run was complete, the gel was transferred into a suitable staining tray and fixed in a solution containing formaldehyde in a shaker for 2 h. The gel was washed three times with 1 × wash solution once in every 5 min. The gel was incubated with sensitizing solution containing sodium-thiosulphate for 2 min with gentle shaking and visualized using silver staining.Surgically removed fresh tumour tissues were dissected into small sections (~1–2 mm−1 in 50 mM ammonium bicarbonate buffer. The pH of protein samples was adjusted to ~8.5. The samples (50 μl) were reduced with 10 mM DTT at 56 °C for 45 min, incubated at 95 °C for 5 min and then allowed to cool. Alkylation was carried out by using 55 mM final concentration of iodoacetamide in the dark. Trypsin (13 ng μl−1) was added at a ratio of 1:30 and enzyme/sample mixture was mixed well. Tubes with sample were placed into thermostat and incubated at 55 °C for 2 h and then 37 °C overnight. Digested samples were subjected for SpeedVac at 30 °C for 2–3 h. 5% formic acid was added for adjusting the pH to 3. The samples were either subjected directly to MS analysis or stored at −20 °C.The protein mixture was dissolved at the concentration of 1 μg ml−1 bovine serum albumin (BSA) or β-gal. One micro liter of this was injected on column. Hence, the area of BSA/ β-gal was used for normalization. A separate Mascot run was performed with carboxymethylation as a dynamic modification to obtain area of BSA or β-gal protein. Area of the respective protein is normalized to the control area of respective sample. Reserpine  was used as a working standard. Digested peptides were subjected to analysis by injecting into nano LCMS/MS−1. Eluted peptides from the column were ionized using ESI source with ion spray voltage 2250 V and temperature 120 °C. Ionized peptides were analysed by one full MS scan and four consecutive product ion scans of the four most intense peaks, using rolling collision energy. An Information Dependant Acquisition (IDA) experiment was used to specify the criteria for selecting each parent ion for fragmentation, which included selection of ions in m/z range: >400 and <1600, of charge state of +2 to +5, exclusion of former target ions for 30 s, accumulation time of 1 s for a full scan and 2 s for MS/MS. The data generated by the Analyst software were stored in a.wiff format. The machine generated data files were analysed using ProteinPilot version 4.0 software with a combined NCBI Human Database , Paragon Algorithm and Proteome Discoverer1.3 software. All searches were performed with tryptic specificity allowing two missed cleavages. Trypsin and keratin entries were retained in the list generated. During the analysis, in the search parameters modification of cysteine by idoacetamide and biological modifications programmed in algorithm were allowed. Mass tolerance for precursor ion and fragment ions were set to 100 p.p.m. and 0.2 Da, respectively. In Paragon Algorithm, protein score was calculated on the basis of percentage confidence level of the peptides identified. Protein score of minimum 0.47 (fit incorrect rate is 0%) corresponding to a confidence level >66% were used. To rule out false discoveries, we carried out a False Discovery Rate (FDR) analysisSample was vacuum dried and reconstituted in 12 μl of 0.1% formic acid containing 12.5 fmol μlFor peptide and protein identification, peak lists were correlated with the human protein database656667−1) unless mentioned otherwise. To visualize the coat, the matrix was incubated with anti-Collagen1 antibody at a dilution of 1:50  for 1 h at room temperature. After four washes in PBS, slides were incubated with Alexa Fluor 555  for additional 45 min at room temperature in dark. Slides were washed with PBS and finally mounted with Vecta-Shield DAPI (Vector laboratories. H-1200) to confirm the absence of nuclear contamination in premixed TMP cocktail. Images were visualized under immunofluorescence microscopy setting using red and blue filters  and images were captured with DFC 425C (Leica) camera.TMP cocktails were prepared, based on the relative abundance of key components obtained from LCMS/MS analysis of HNSCC and CRC patient tumour tissues using human proteins as shown in 1) for 4 h, washed twice and were fixed in 10% buffered formalin for 10 min , washed in PBS and dehydrated with 70 and 100% ethanol for 5 min each. Immediately before imaging, the slides were coated with gold and the images were captured using a Cambridge scanning electron microscope with EDAX attachment.Electron microscope compatible cover slips  were coated with freshly prepared TMP cocktails (100 μg mln=3) were treated with either anti-EGF neutralizing antibody  or with TPF (for HNSCC) or with cetuximab+FOLFIRI (for CRC) or with dimethylsulphoxide  for 72 h. The final concentration of DMSO was kept ≥0.01%. Media with drugs were changed every 24-h interval. A portion of each tumour slice was used for cell viability (assessed by WST) and remaining tumours were fixed in 10% buffered formalin and embedded in paraffin. The paraffin-embedded tumours were used for histological (hematoxylin and eosin stain) and IHC analysis including proliferation and cell death.Tumour tissues were sectioned into ~300 μm slices using McIlwain tissue chopper (TedPella). These tumour sections were randomized and cultured in 48-well flat bottom plates coated with stage and grade-matched TMP with RPMI medium supplemented with 2% AS, 8% FBS (Life Technologies. 10270-106), 1 × Insulin-Transferrin-Selenium , 1 × GlutaMAX (Life Technologies. 35050-061) and 1 × penicillin, streptomycin and amphotericin B (Life Technologies. 15140-122). Tumour slices (3) and implanted subcutaneously onto the flanks (both sides) in immune compromised 5–6 weeks old, female severe combined immunodeficiency  mice. Tumour bearing mice (at the time of commencement of treatment maximum tumour size was restricted to 100–150 mm3) were treated with vehicle  or concurrent regimen of TPF  or single agent cetuximab (4 mg kg−1) for 3–4 weeks. Tumour volume was calculated using the following formula, Tumour volume (mm3)=(π/6) LWH; where L=length (mm), W=width (mm) and H=height (mm). All mice studies and experimental protocols were approved by the institutional animal ethics committee.Freshly isolated primary human tumours were washed in normal saline and cut into a small pieces , i=1,...,n (here n=109), where xi is a feature vector containing the four functional read-outs for the i-th patient and yi is 1 if the i-th patient is a responder and −1 otherwise, the SVMpAUC algorithm learns a weight vector w maximizing (a concave lower bound on) the partial area under the ROC curve  up to a specified false positive rate β (here β=0.25), defined as follows70We learned a model for predicting patient responses as NR/PR/CR in two stages. At the first stage, PR and CR labels were grouped together into a single responder (R) category, and the recently proposed SVMpAUC algorithmSβ contains indices j of the top β fraction of non-responders in the training set, ranked according to scores w.xj. This produced a weight vector w assigning coefficients of 0.2977, 0.5562, 0.0073 and 0.1388 to the viability, histology, proliferation and apoptosis read-outs, respectively. Together with a threshold of 19.1 corresponding to (approximately) β=0.25 false positive rate on the training set, this yielded an initial NR/R prediction model. In the second stage, the above model was further refined to classify the predicted responders as PR and CR; this was done by selecting a threshold (55.14) that maximized PR/CR classification accuracy on the training set.Where t-test, linear regression and Spearman coefficient of correlation was analysed using GraphPad Prism version 5 for Windows, GraphPad Software.One way analysis of variance and Student’s ex vivo, in vivo, microarray, IHC and LCMS experiments and analysed the data. A.P. analysed the assay data. N.H. and S.A. were involved in algorithm development. B.M., S.T., P.R., M.S., S.A., S.S., P.M. wrote the manuscript. R.S., G.K.B., A.M.S., M.A.K. contributed to the clinical aspects of the study. G.B., P.H. conducted exome experiments and data analyses. R.B. and M.L. provided critical inputs for exome and pathology data and results. P.K.M. supervised the study.B.M., P.R., S.A., S.S. and P.K.M. were involved in the design of experiments. B.M., B.U, S.T., M.D., N.B., D.D.P. B.U.S., A.T. conducted How to cite this article: Majumder, B. et al. Predicting clinical response to anticancer drugs using an ex vivo platform that captures tumour heterogeneity. Nat. Commun. 6:6169 doi: 10.1038/ncomms7169 (2014).Supplementary Figures 1-10, Supplementary Tables 1-3, Supplementary Methods and Supplementary References


The versatility of ECGT allows for response-based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti-tumour response for successfully affecting not only the treated tumours, but also non-treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.Electroporation improves the anti-tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation-mediated chemotherapeutics with interleukin 12 (IL-12) plasmid DNA produces a strong yet safe anti-tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL-12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL-12 gene therapy for long-term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro-chemo-gene therapy (ECGT) with IL-12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression  Combining the cytotoxicity of chemotherapy with the anti-tumour immune responses of immunomodulatory therapy inhibits tumour growth in multiple types of cancer and even large, refractory tumours via EP Electroporation is a physical method for transfecting agents, such as chemotherapeutics and plasmid DNA (pDNA), into cells. In EP, brief electric pulses induce transient pores in the cell membrane and transport the agents into the cytosol As a result of the high toxicity of delivering the recombinant cytokine, gene therapy with interleukin 12 (IL12) pDNA is another treatment strategy that can benefit from EP-mediated delivery to accessible tumour nodules . Several Phase I trials using this exact treatment in melanoma and other histotypes have proven the safety, and Phase II studies are currently in progress to validate the efficacy. Additionally, the anti-tumour immune response induced by intratumoural treatment with IL12 pDNA can inhibit non-treated metastatic tumours A previous report presented the case results of five canine subjects that received only one intratumoural ECGT treatment cycle with IL-12 pDNA plus bleomycin To this end, this study was designed to allow for repeated treatment cycles, additional tumour histotypes and another chemotherapeutic agent (gemcitabine). The results from these studies will demonstrate the versatility, safety and efficacy of EP-mediated intratumoural IL12 pDNA treatments with and without chemotherapeutics.Over the course of 3 years, a total of 13 evaluable canines with naturally occurring neoplasms were enrolled in this study. The subjects' details are listed in TableAll animal handling and treatments followed National Institute of Health (NIH) guidelines and were approved by the Institutional Animal Care and Use Committee at the University of Texas MD Anderson Cancer Center. Because of the lack of a commercially available canine IL12 pDNA, all previous veterinary studies in canines with IL12 pDNA have used either human or feline IL-12 pDNA constructs via callipers or a small millimetre ruler, as necessary for tumours in the oral cavity of small breed canines. At this point, biopsies from the tumour area were collected (after measurement and prior to treatment). With the IL12 pDNA and the chemotherapeutic diluted as described, the pDNA with or without chemotherapeutic was injected directly into the tumour tissue. For bleomycin the dose was 100 μl (1 unit) per centimetre, and the gemcitabine doses were increased in a dose escalation manner from 0.5 to 10 mg/cm (data not shown). None of these doses induced an adverse event and the optimal effective dose was determined to be 2 mg/cm of diameter. If the largest tumour diameter was greater than 1 cm, the tumour received an injection of the pDNA and chemotherapeutic for each centimetre . Within 5 min. of the injections, a 6-needle electrode connected to a BTX 830 pulse generator was placed around the injection site and two 20-msec., 350 V/cm electric pulses with a 100-msec. interval between pulses were delivered Prior to each treatment, a physical exam of the subject and an interview with the subjects' owner were performed. Anaesthesia was induced with intravenous propofol and maintained on isoflurane in oxygen. The details of the anaesthesia and other general veterinary practices were performed as per standard operating procedures as previously described via millimetre ruler or callipers. Two tumour diameters, the longest diameter (a) and the diameter perpendicular to the longest diameter (b), were measured, and the tumour volume (V) was calculated with the following formula: V + (π/8)*(a*b2). To analyse tumour growth in individual treatment cycles, the tumour volumes were normalized by the volume at the beginning of that cycle, so all day 0 values are 100%. Four (4) responses were calculated: complete response (CR), complete disappearance of all measurable nodules for at least 21 days with histopathological confirmation; partial response (PR), greater than 20% reduction from initial tumour volume prior to treatment cycle; progressive disease (PD), greater than 20% increase from initial tumour volume prior to treatment cycle; and stable disease (SD), neither significant volume increase for PD nor reduction for PR. These data were analysed via multiple T-tests at each time-point. Because of missing time-points, two-way ANOVA was not possible. Survival data were obtained by data mining the electronic medical record database at GCVS. Only patients with complete medical records from the day of cancer diagnosis until death or censored were included. Patients that received surgical interventions on the primary tumour were excluded as to only analyse standard, non-surgical treatments. The difference between survival curves was analysed with the Mantel-Cox test. Statistical analyses were performed with GraphPad Prism version 6.03 for Windows At each visit, the tumour volume of all accessible tumours was measured n + 9), limbs (n + 7), and nasal plenum (n + 3). Only one patient had a distant metastatic nodule (SCC/PC subject). Nine of the thirteen subjects received previous treatment with radiation (n + 4), surgery (n + 3), or both surgery and radiation (n + 2). For all subjects that received previous treatment, disease progression resumed for at least 3 weeks prior to initiating any treatments in this trial. In these 19 nodules, a total of 58 treatment cycles were performed. Because of subject non-compliance, only 53 clinical responses could be determined. Details for the clinical responses are displayed in TableA total of 13 evaluable subjects were enrolled in this study, and the details for the subject population are listed in Tabletion n + . For allThe low number of patients and the heterogeneity among spontaneous neoplasms obfuscates statistical analyses; however, comparing the tumour volume changes within each individual treatment cycle (21–28 days) as separate curves allows multiple different analyses.Bleomycin and cisplatin are two chemotherapeutics that benefit from EP The therapeutic benefits of ECGT with IL12 pDNA and bleomycin or gemcitabine were compared in multiple histotypes. Indeed, ECGT with bleomycin or gemcitabine are similarly effective in SCC and PC and equally ineffective for treating sarcomas. In oral and cutaneous SCC and PC, ECGT with IL12 pDNA plus either bleomycin or gemcitabine reduced tumour volume by 21 days after the first treatment of that cycle Fig. and B. NTo ensure that the gene component of the ECGT treatment is conferring a benefit, some patients received EGT with only IL12 pDNA. In cutaneous and oral SCC, EGT and ECGT treatments equally regressed tumour volume by 25% through day 14; however, by day 21, tumour growth resumed in those receiving IL12 pDNA only while tumour volume continued to decrease in those patients receiving ECGT Fig.. On the Three SCC lesions on the immune-privileged nasal planum received treatment with ECGT, but these treatments were incapable of inhibiting tumour growth. Compared to the 27% average regression from ECGT treatment in oral and cutaneous SCC, nasal planum SCC lesions did not respond to ECGT treatments and tumour volume increased almost 75% in 21 days Fig..i.e. nasal planum) indicates that direct cytotoxicity by ECT is not sufficient to induce the robust anti-tumour response seen in ECGT.In one patient with SCC lesions, a metastatic SCC lesion was identified in the pre-scapular lymph node ipsilateral to the tumour-bearing forelimb 21 days after the first treatment cycle began. This tumour did not receive any intratumoural treatments, yet the volume of this lesion regressed steadily for nearly 120 days after the only treatment cycle performed in the forelimb SCC lesions Fig.. On day i.e. the smaller the tumour, the more effective the therapeutic. The efficacy of these treatments based on tumour size was determined by pooling all treatment cycles from responding histotypes and then separating into groups based on initial tumour diameters < or >3 cm. In IL12-only EP-mediated treatments, there are no differences in response compared to ECGT with tumour volumes being reduced by at least 27% on day 14 after both treatments; however, tumour growth returned in the IL12-only treatment group to the initial volume by day 21 , the first treatment cycle of ECGT, with gemcitabine or bleomycin resulted in an average decrease to 63% of initial tumour volume after only 21 days . Sadly, the same dose level of rIL12 that appeared safe in the Phase I trials caused severe side effects including death, and the therapeutic responses seen in murine models were not repeated in humans i.e. treated) and non-target (i.e. non-treated) spontaneous melanoma nodules, as well as other tumour histotypes Like the recombinant protein studies, gene therapy with IL12 pDNA is consistently shown to effectively treat multiple tumour histotypes in rodent models and induce an anti-tumour immune response capable of affecting non-treated, metastatic nodules Since EP is not specific for only pDNA, a nearly innumerable amount of agents including chemotherapeutics can be delivered to tumours, as well as almost any other tissue via inhibition of cytadine deaminase, a gemcitabine metabolizing enzyme With this system, almost any chemotherapeutic with poor membrane permeability that requires internalization could become more effective if delivered in conjunction with EP. One such agent is gemcitabine, a pyrimidine analogue which inhibits DNA synthesis but has difficulty entering cells to induce tumour cell death. Indeed, the efficacy of gemcitabine for treating pancreatic cancer can be improved with the co-administration of Nanoparticle albumin-bound (nab)-paclitaxel. The nab-paclitaxel created increased intracellular concentrations of gemcitabine Recently, the European Union approved ECT, EP with single chemotherapeutic agents, based on the success of clinical trials. However, as was seen in murine models Although both IL12 pDNA and chemotherapeutic treatments with EP have been shown to be effective, the synergistic power of combining these treatments may be able to overcome the above listed shortcomings of either treatment alone. In a murine study, intratumoural ECGT with IL12 pDNA and bleomycin was much more effective compared to either treatment alone for inhibiting primary and metastatic tumour growth, preventing tumour-recurrence, and extending survival through the induction of IFNg, inhibition of angiogenesis and increase in CTL activity To this end, 13 evaluable subjects with SCC, AA, or sarcomas received multiple treatment cycles of ECGT with IL12 and gemcitabine or bleomycin. In these studies, there were zero instances of severe adverse events at any dose level, and the only recurring minor adverse events were transient bleeding after treatment and ulceration because of necrosis of the tumour tissue, typically exacerbated by the canines' pre-dilection for licking the wound. Also, there were no instances of toxicity found in urinalysis or blood profiles . This laPatients with very large, recurrent or multiple tumours generally require multiple treatment cycles to manage tumour growth. In several such patients, the safety of this EP-mediated system allowed for the safe administration of multiple cycles, and the versatility of EP-mediated treatments permitted response-based modifications to the treatments to reach multiple therapeutic endpoints within each patient while prolonging and improving the quality of life.One SCC subject and two AA subjects  and S4 rIn addition, these ECGT treatments are capable of quickly reducing tumour volume in large oral and cutaneous SCC lesions Fig.. These tOne of the benefits to including the IL12 pDNA is the induction of a robust yet specific antitumour response All of the data presented thus far has demonstrated that ECGT with IL12 and bleomycin or gemcitabine is safe, well-tolerated, effective and versatile, but the effects have yet to be compared to standard therapies. For SCC and most other cancers, surgery is the best option if available and would not detract from the quality of life. In canines, surgery  confers a 1-year survival of 95% and a median survival time of 474 days. On the other hand, the 1-year survival rate for invasive SCC with surgery is only 10%. To identify the benefit of ECGT compared to standard, non-surgical treatment options, the survival time of subjects with complete medical records treated with radiation or chemotherapy at GCVS, the host of this trial, were compared to all evaluable subjects treated with at least 1 cycle of ECGT in this trial. The median survival for subjects receiving standard therapy was only 97 days compared to 287 days for ECGT, a 3-fold extension of survival. Likewise, the 1-year survival was nearly 25% for ECGT compare to only 17% for standard therapy Fig.. So, surThese exciting results highlight the versatility, safety, tolerability and efficacy of repeated administration of IL12 pDNA, chemotherapy and EP. ECGT with IL12 pDNA plus bleomycin or gemcitabine can safely and effectively diminish target and non-target lesions in SCC, PC and AA, but not sarcomas. In oral and cutaneous SCC, these ECGT treatments can eradicate large and small tumours or significantly reduce the volume to improve the quality of life and possibly enhance the efficacy of subsequent surgery, radiation or other therapies. In addition, these results further show the potential of immunomodulatory cancer treatments and will help to finally turn this potential into a reality.


Background. Laparoscopic hysterectomies are increasingly popular; a morcellation device is often used. Although there are some clear benefits, morcellation of tissue does have potential risks. Case Presentation. In this case report we present a 55-year-old woman with an abdominal tumour 4 years after a laparoscopic hysterectomy using a morcellation device. Postoperative histological analysis, compromised by morcellated tissue, showed benign myoma. Because of the benign tumour no follow-up was performed. The patient presented now with an abdominal tumour, and she was scheduled for surgical removal of the tumour. During abdominal surgery the tumour appeared malignant and biopsies were taken. Histological analysis showed leiomyosarcoma, and the patient was referred to a third care centre for further treatment. The patient recovered quickly after abdominal removal of the tumour; however, after 7 months the patient had complaints and a CT scan showed a large intra-abdominal tumour with possible lung metastasis. The patient received palliative chemotherapy and died after 10 months. Conclusion. This case shows that although unexpected after a hysterectomy, a leiomyosarcoma has to be considered in case of a suspect tumour in the lower abdomen. Laparoscopic hysterectomies are increasingly popular because of the short recovery period of patients, causing short hospital stays and reducing healthcare costs –3. In a A 55-year-old woman was referred to our clinic with malaise and an abdominal tumour. In 2009 she had a LASH procedure with use of a morcellation device for bleeding problems caused by a myomatous uterus. At the primary surgery the BMI of the patient was 27.7. As the patient was premenopausal the adnexa remained in situ. Postoperative histological analysis on the morcellated tissue  showed benign myomas, with some mitotic activity and infarct type necrosis. Although the patient received adequate thrombotic prophylaxis by dalteparin, the patient developed postoperatively a deep-venous-thrombosis but recovered quickly after adequate treatment. As the tissue showed benign myomas no follow-up was performed. The patient was referred now with malaise, weight loss, and an abdominal mass. A vaginal ultrasound showed a large tumour (13 cm × 13 cm × 10 cm) most likely attached to the adnexa. There was no free fluid seen intra-abdominally. CA-125 and CEA were analysed to determine the malignancy risk . As the tumour markers were normal, the patient was scheduled for surgical removal of the tumour. During an abdominal surgery procedure 2 normal adnexa were seen, and the tumour appeared malignant and seemed connected to the remaining cervical tissue, the greater omentum, and sigmoid. No other abnormalities were observed intra-abdominally. As the tumour was connected to the omentum and sigmoid, and since the tumour bled easily, a malignancy was suspected and biopsies were taken for further diagnosis and the procedure was ended. A CT scan was performed and showed a large tumour without any evidence for intra-abdominal or thoracal metastasis. Histological analysis of the biopsies showed a smooth muscle tumour with high mitotic rates, tumour necrosis, and nuclear atypia, diagnostic for leiomyosarcoma. The patient was referred to a third care centre for surgical treatment and follow-up. She had abdominal removal of the tumour and showed a quick recovery. Because of the unexpected findings the original uterine tissue was reanalysed. Reanalysis of the morcellated uterine tissue showed features which confirmed those described in the original report. The patient received standard follow-up care, and after 7 months a CT scan was made because of abdominal complaints. The CT scan showed a large intra-abdominal tumour with possible lung metastasis. The patient received palliative chemotherapy and died after 10 months.A recent study showed that there is a small probability of unexpected malignancies in correctly prescreened patients for LASH procedures . In thisBecause of its clear advantages laparoscopic surgery becomes more and more popular and all sorts of technical devices are designed and used. Over years a morcellation device has been developed and has become a widely used tool, as it enables the possibility to reduce the size of tissue intra-abdominally , 15. HowRecently, several statements have been released to discourage the wide spread use of morcellation and only offer this to appropriately screened, low risk women –22. AlthThe effects of morcellation on pathology examination have been studied in a small study in which 10 women were included undergoing total hysterectomy without uterine morcellation; after pathology examination using standard techniques the uteri were deidentified, and all uteri were morcellated . After mThe fact that our patient got symptoms almost 5 years after her LASH, showing a single tumour without any (macroscopic) metastases, could suggest a malignant deterioration of nonmalign uterine cells spread during the morcellation procedure, or it could suggest a malignant deterioration of part of the cervical tissue which was not removed completely at the first surgery.To summarize, the unexpected tumour origin in this case shows that tissue may remain after a macroscopic complete removal of an organ. The leiomyosarcoma in this patient could be a result of malignant deterioration of nonmalign uterine cells spread during the morcellation procedure, or malignant deterioration of remaining cervical tissue. Doctors should consider an unexpected cause in case of a suspect tumour in the lower abdomen.This case report describes a woman with an unexpected leiomyosarcoma 4 years after the removal of the uterus without any macroscopic intra-abdominal metastasis. This case shows that a leiomyosarcoma always has to be considered in case of a suspect tumour in the lower abdomen, even after previous removal of the uterus.


Anastomosing variant of capillary haemangioma is a rare and recently described vascular tumour with a proclivity for the genitourinary tract. Here we present the case of a 64-year-old man with incidental finding of 3.4 cm renal mass on CT who had laparoscopic nephrectomy with a good postoperative recovery. Histopathological diagnosis of anastomosing haemangioma of the kidney was made and the patient was followed up for 10 months without evidence of tumour recurrence. Vascular tumours of the kidney are very rare. Anastomosing haemangioma of the kidney is a subtype of capillary haemangioma. We describe a case of anastomosing haemangioma in a 64-year-old male patient with the CT imaging findings, histopathological features, treatment, and outcome.A 64-year-old male who had suffered a deep vein thrombosis of the left lower limb underwent an ultrasound, which revealed an incidental finding of a 2.5 cm lesion involving the left kidney. His past medical history included type 2 diabetes mellitus and peripheral vascular disease with claudication. He had no haematuria or weight loss.A CT abdomen-pelvis with contrast confirmed a suspicious 3.4 cm left mid pole enhancing renal mass with involvement of the segmental renal vein . There wThe results were discussed in the multidisciplinary team meeting and consequently with the patient and he agreed to proceed to a laparoscopic nephrectomy, which was performed a week later.Grossly, the renal lesion measured 2.3 × 2.4 × 1.8 mm was lobulated and mahogany-brown in colour. Further macroscopic assessment showed a slightly haemorrhagic tumour located in the renal hilum and renal sinus fat, with a portion of the tumour identified within the branch of a renal segmental vein . MicroscOn immunohistochemistry, the tumour was positive with CD34 and CD31. The endothelium of the renal vein branch demonstrated CD34 positivity  and the Anastomosing haemangioma is a morphological variant of capillary haemangioma described by Montgomery and Epstein. The term “anastomosing haemangioma” describes the histological observation of anastomosing pattern of capillary sized vascular channels reminiscent of splenic sinusoids .The incidence of the tumour in male patients suggests a slight male predilection with a reported male to female ratio of 1.8 : 1 . The aveGrossly, anastomosing haemangioma of the kidney is often small sized with an average size of 1.8 cm . The tumThe tumour typically stains positive with endothelial markers CD31 and CD34 and factor VIII-related protein . ContrasWe have presented a case of anastomosing haemangioma of the kidney which is a recently described vascular tumour which runs a benign course. This case is of particular note because of the gross tumour involvement of a segmental branch of the left renal vein which was identified on CT imaging and confirmed microscopically. This case expands the knowledge of this rare tumour and emphasizes the importance of histopathological diagnosis in ruling out more aggressive differential diagnosis which has implications on further management.


Residents work at variable times and are often unable to attend all scheduled educational sessions. Therefore, new asynchronistic approaches to learning are essential in ensuring exposure to a comprehensive education. Social media tools may be especially useful, because they are accessed at times convenient for the learner.Assess if the use of Twitter for medical education impacts the attitude and behavior of residents toward using social media for medical education.Preintervention and postintervention surveys. Internal medicine resident physicians were surveyed before the launch of a residency-specific Twitter webpage on August 1, 2013, and again 135 days later, to determine their use of the Twitter application and web page, as well as other social media for medical education.Residents at an internal medicine urban academic training program.All residents within our training program were administered web-based surveys. The surveys assessed resident views and their frequency of use of social media for medical education purposes, and consisted of 10 Likert scale questions. Each answer consisted of a datapoint on a 1–5 scale . The final survey question was open-ended and asked for general comments.p<0.001 for the change). The percentage of residents using the application at least once a week also increased from 11.4 to 60.0% (p<0.001). Almost all residents (38 of 40) felt that social media could be useful as a medical education tool, which slightly increased from 30 out of 35 in the preintervention survey (p=0.01).Thirty-five of 50 residents (70%) completed the presurvey and 40 (80%) participated in the postsurvey. At baseline, 34 out of 35 residents used social media and nine specifically used Twitter. Twenty-seven (77%) used social media for medical education; however, only three used Twitter for educational purposes. After the establishment of the Twitter page, the percentage of residents using social media for educational purposes increased , and 22 used Twitter for this purpose (Residents believe social media could be used for medical education. After we launched a Twitter page for medical education, there was a significant increase in the use and frequency of Twitter for resident medical education over the ensuing 6 months. Further research should be performed to see if social media can impact overall medical knowledge and patient care, and whether longer term use is maintained. Online applications and websites where users contribute, retrieve, and explore content generated by fellow users are popular means to share and communicate information. As an example, Facebook, the most utilized social media application, is used by more than 1.19 billion users every month and has 727 million daily users –10.Social media may be especially useful for enhancing medical education. Restrictions on resident work hours may limit their ability to participate in in-person conferences and educational sessions. Social media can be accessed geographically and temporally in asynchronous manners, allows for easily searchable and stored content, and encourages interactivity  – and thBuoyed by the success of the podcasts, residents sought additional tools to enhance their medical education. We then chose Twitter, an online social networking service, which allows the exchange of information in ‘tweets’, messages shorter than 140 characters . ReportsWe launched the Social Media and Resident Teaching for Medical Education initiative at Johns Hopkins Bayview Medical Center, a tertiary care academic institution located in Baltimore, Maryland. On August 1, 2013, we introduced a Twitter page, titled ‘Hopkins Bayview IM @TEACHBayview’. This page listed a series of Twitter messages . Each ‘tweet’ can contain up to 140 characters and can include photos, videos, and links which, for instance, can reference an online article. In addition, the page included ‘hashtags’, which are keywords preceded by the # character . Each haThe material posted in a Twitter message serves as a summary of one educational lesson, and is categorized into one of six types (as represented by a hashtag). The six hashtags used by @TEACHBayview are detailed in Two weeks prior to the Twitter page launch, we invited all 50 internal medicine residents to complete a voluntary baseline survey, which queried the quantity and type of social media used by residents for medical education purposes. Specifically, the survey used Likert scales to quantify resident attitudes toward social media use for medical education , and addressed the frequency of social media use (including Twitter) for medical education . Finally, free response questions were asked about resident attitudes toward the ‘Hopkins Bayview IM @TEACHBayview’ Twitter page.t-tests to determine statistical significance in changes. Where appropriate, scores are shown as mean (±standard deviation). Qualitative responses are shown in a separate table.To assess the impact of the Twitter page on resident behavior and attitudes toward social media use for medical education we repeated the survey 135 days after launch of the Twitter page. Furthermore, we also reviewed the use of other types of social media by residents before and after the implementation of our Twitter page. Pre- and post-survey results were analyzed using Student p=0.19)). In the presurvey, most used Facebook (31 residents), YouTube (24 residents), and podcasts (17 residents). In contrast, Twitter was used by only nine residents. After the launch of the Hopkins Bayview Twitter page, Facebook and YouTube were still the most frequently used social media . However, Twitter use increased from 9 residents to 23 (p<0.01) and became the third most used social media application.Of the 50 residents, 35 residents participated in the presurvey and 40 in the postsurvey. Residents extensively used social media (34 of 35 in the presurvey (97.1%) and 37 of 40 in the postsurvey (92.5%) (p=0.28), which was not statistically different. For frequency of use, postintervention surveys revealed that 21 residents used social media up to twice a week and 10 residents used social media daily. Nine residents continued to rarely use (less than once a month) social media for medical education. As with use of social media, the frequency of using social media for medical education at least once a week was not statistically significant: 22 of 35 residents versus 31 of 40 .Specific to medicine, 27 of the 35 residents in the presurvey used social media tools to enhance their education , with pop<0.001). In addition, there were also statistically significant increases in using other social media for medical education: Facebook , YouTube , and weblogs . However, podcast use did not change (20 residents before and after the surveys). Also, six residents continued to not use social media for medical education purposes .After our Twitter page was launched, postsurveys showed it was the most popular source for medical education, with 22 of the 40 residents using it for that purpose, compared to 3 of the 35 residents prior to the intervention (p<0.001)  .p=0.01)). In open-ended questions, resident views on Twitter are listed in Resident views also changed, with more favorable views on social media for medical education . Also, we did not obtain demographic data, which was also the case in other publications studying social media , 18, 19.In conclusion, using Twitter to convey and discuss medical concepts has a significantly beneficial impact on internal medicine residents. Residents are favorably inclined to use the application and welcome greater use of social media to supplement their medical education. However, it is unclear whether social media, or Twitter itself, truly impacts resident learning or promotes better patient care. Future studies should address these issues.The authors have not received any funding or benefits from industry or elsewhere to conduct this study.


A 64-year-old male presented to the emergency department (ED) with near syncope and worsening left flank and shoulder pain. He had undergone a difficult colonoscopy two days prior due to a tortuous colon. Initial vital signs were normal. He looked uncomfortable and had significant left upper quadrant abdominal tenderness with guarding. Thirty minutes after ED arrival, his blood pressure dropped to 73/59 mmHg, requiring aggressive fluid resuscitation. Bedside focused assessment with sonography in trauma (FAST) exam demonstrated free fluid in the abdomen with mixed echogenicity of the spleen, suggestive of splenic injury. Computed tomography (CT) demonstrated a large subcapsular splenic hematoma with active extravasation and surrounding intraperitoneal free fluid , Video. Colonoscopy has been associated with spontaneous intra-abdominal organ injury. Jammal et al described a case of subcapsular liver hematoma associated with colonoscopy.


Herpesviridae, varicella zoster virus (VZV) is named for the 2 main diseases (chickenpox and herpes zoster [shingles]) it causes. Varicella (varus (“pimple”) or varius (“speckled”). Herpes zoster derives from the Greek terms herpein (“to creep”) and zoster (“belt”). Not until the twentieth century was VZV recognized as the cause of both these diseases.A member of the family aricella  may be a


In addition to MR imaging, PET imaging of O-(2-[18F]Fluorethyl)-L-Tyrosine (FET) uptake provides information on brain tumour extent and metabolic activity. Similarly, MRS has been shown to be of value for distinguishing high- from low-grade gliomas. Based on 2D spatially resolved MRSI, an overlap between 18FET uptake and the choline/N-acetyl-aspartate (Cho/NAA) ratio of more than 75 % has been reported.To measure spatial correlation of 18FET-PET with 3D spatially resolved MRSI in patients with gliomas.3 each).14 patients (46±16 y) with gliomas (WHO grade II-IV) were examined by simultaneous 18FET-PET-3D-MRSI measurements which covered the whole brain . The data were analysed with respect to the congruency of the suspicious tissue delineated by the FET uptake and the Cho/NAA ratio. Locations of individual maxima and distances in between were determined. The congruency of the tumour was assessed using Dice’s coefficients for assumed same tumour volume. All comparisons were carried out at the spatial resolution of the whole brain spectroscopic image  mm distant from each other. The average level of congruency between the tumour volumes delineated in the FET uptake and MRSI data was (33±25)%. Metabolically active tumour tissue, as depicted by FET uptake, is represented to a low extent by the choline/N-acetyl-aspartate ratio measured by spatially resolved 3D MRSI, which is in contrast to previous findings. Both modalities may reflect independent physiological properties of gliomas.