To our knowledge, these are the first empirical estimates of tumour growth in the absence of treatment in a wild population. Through this animal-cancer system our findings may enhance understanding of how tumour properties interact with growth dynamics in other types of cancer.A pressing and unresolved topic in cancer research is how tumours grow in the absence of treatment. Despite advances in cancer biology, therapeutic and diagnostic technologies, there is limited knowledge regarding the fundamental growth and developmental patterns in solid tumours. In this ten year study, we estimated growth curves in Tasmanian devil facial tumours, a clonal transmissible cancer, in males and females with two different karyotypes  and facial locations , using established differential equation models and model selection. Logistic growth was the most parsimonious model for diploid, tetraploid and mucosal tumours, with less model certainty for dermal tumours. Estimates of daily proportional tumour growth rate per day (95% Bayesian CIs) varied with ploidy and location . Final tumour size (cm Furthermore, the control of tumour development and growth rate depends on dynamic interactions and feedback processes with the host immune system, cell-mediated mechanisms and molecular evolutionary events5. Thus, understanding the mechanistic dynamics of the tumour and the host and how they influence tumour growth is a major challenge. Mathematical models of tumour growth rates have been mostly focused on understanding the quantitative theory of increase in tumour volume6, tumour response to radiation therapies and modelling the growth and evolution of subpopulations of cells9. In a recent study, Gerlee10 reviewed the historical development of tumour growth laws, from the early view of exponential growth to more complex models that take into account structural and biological attributes of tumour cells. However, most studies have been based on quantitative theory and have not used empirical data on tumour growth rates. Providing empirical data from naturally occurring neoplastic diseases in the absence of treatment has been until now a difficult task. In that sense, cancers in wildlife can be regarded as useful study systems to understand mechanistic and quantitative attributes of tumour cells. Wildlife cancers, and particularly transmissible cancers, have been increasingly recognised as a new threat to conservation and biodiversity12. In most cancers the tumour dies and disappears within its hosts. However, in transmissible cancers the tumour cells are the infectious agent and are transmitted as a clonal cell line across hosts. Studying the developmental progression of tumours in transmissible cancers provides a unique opportunity for understanding growth rate theory and has useful applications for understanding tumour development in human and non-human cancers.The propagation of cells in neoplastic diseases is driven by deregulation of control in cell proliferation, as well as several of the energetic and metabolic steps necessary to fuel cell growth and division11. This is because most cancers in wildlife are difficult to study as their clinical signs are usually imperceptible  and in many occasions affected animals die unseen or are preyed upon before clinical signs appear11. The emerging view of cancers, from an evolutionary and ecological perspective, is that they are a collection of heterogeneous cells that evolve in tumour microenvironments with complex ecological processes13. Hence, evolutionary theory and mathematical modelling are becoming important tools to understand the ecology of cancers and factors associated with the stimulus and suppression of tumour growth rates15. Establishing tumour growth curves for transmissible cancers in wildlife may help to determine optimal time periods for transmission, predict disease progression and survival after infection. In addition, assessing tumour growth dynamics and cancer progression may be used in more complex models for estimating the likelihood of metastasis.Evaluating the ecological significance of oncogenic phenomena, their epidemiology and prevalence in the wild is a challenging taskSarcophilus harrisii)17. The tumour has a neuroendocrine origin18 and is transmitted as a clonal cell-line by direct inoculation of live tumour cells when animals bite each other20. Since its emergence two decades ago, DFTD has evolved into several karyotypic sublineages21 and new evidence suggests that diploid tumours are associated with higher transmission rate, prevalence and population effects than tetraploid tumours22. Tetraploidy has been found to reduce cell proliferation and tumour growth rates24. Devil facial tumours provide a unique opportunity to empirically evaluate tumour growth theory in the wild. Unlike most cancers, DFTD is not subject to treatment so repeat measures can be made across differing ploidy and facial locations. Furthermore, understanding how tumour growth rates vary between different karyotypic lineages may provide a framework to scale results from experiments in model organisms up to humans, as well as providing realistic growth trajectories to parameterise numerical models.Tasmanian devil facial tumour disease (DFTD) is a rare transmissible cancer threatening its unique host, the Tasmanian devil  varied with ploidy and location  but not significantly with sex   also varied . Tumours with no karyotype data available were regarded as unknown ploidy and tumours that were located between both dermal and mucosal tissues were regarded as unknown location. We only used data for which tumour ploidy was known, regardless of location, to model tetraploid vs diploid growth rates, and combined all data for which tumour location was available, regardless of ploidy, to test the effect of tumour location  on growth rates. Figure To understand the effects of cancer treatments on tumour growth dynamics, it is essential to assess patterns of tumour growth and to mechanistically model them in the absence of treatment. Our study is, to the best of our knowledge, the first attempt to determine realistic patterns of tumour growth and mechanistically model growth in a natural and untreated population based on empirical evidence. This result is possible due to the combination of a long-term detailed dataset  and a flexible modelling approach. We identified variation in growth rates and potential carrying capacities (the expected maximum volume) across the four distinct tumour characteristics in both males and females. A logistic model of tumour growth was the best supported in our study system for tetraploid, diploid and mucosal tumours. Exponential growth was the most supported model for dermal tumours. This suggests that for tumours outside of the oral cavity where growth is not limited by physical constraints such as bones and tissue, as is the case of tumours inside the oral cavity, there is no threshold for tumour growth within the life expectancy of infected devils.25. Likewise, an increasing number of statistical approaches in growth rate theory have attempted to capture the biological and mathematical complexities underlying growth rates27. Considering these complexities in neoplasms, understanding growth rates is essential to predict the effects of therapies. Though some types of cancers are caused by infectious agents such as viruses29, only in rare circumstances do they become directly transmissible from one host to another. While there are only few cases known in nature: DFTD, the canine transmissible venereal tumour affecting domestic dogs30, and the recently found leukaemia type of cancer found in soft shelled clam31, there is also evidence that malignant sarcomas can be accidentally transplanted from patient to surgeon32 and from mother to foetus33. These cases highlight the capacity of cancer cells to escape their host and become transmissible. Given the nature of DFTD, a clonal transmissible cancer acting as an obligate parasitic cancer cell line, we acknowledge that caution should be taken when extrapolating our results to other cancers, in both humans and non-human animals. Nonetheless, our model based on empirical data from a wild population may be useful for parameterising mathematical models and further understanding growth rate theory in neoplasms.Our findings may extend to tumour growth in other organisms, and contribute to an increased appreciation of how effective cancer therapies are against background tumour growth. However, cancers are highly heterogeneous and differences in tumour growth rates across species and histiogenesis are expected to occur. Numerous studies and models have demonstrated high variability in tumour growth in response to treatmentOur devil—DFTD system is useful for studying tumour growth because individuals can be captured on multiple occasions and their tumours measured repeatedly and these wild animals are not subject to treatment. Furthermore, because DFTD is a clonal transmissible cancer of neuroendocrine origin, , we can model growth rate in a system that allows capturing intrinsic growth properties over time. On one hand, this clonal cancerous cell-line of neuroendocrine origin allows minimising the numerous biological complexities behind growth rates in cancers from different origins. On the other hand, particular caution should be taken when comparing growth behaviour in other cancers in model organisms.We acknowledge that substantive intraspecific variation in rates of tumour growth and carrying capacities may influence model parsimony. The differences we observed between individuals occurred irrespective of their sex. The statistical model used to fit the mathematical tumour growth models to the field data is necessarily exploratory due to the complex processes involved. A next step to clarify this complexity might involve a hierarchical Bayes approach, however, this would require a much larger dataset. Such a model could assume, for example, that the amount of time between tumour implantation and first observation follows a given continuous distribution. In particular, an Empirical Bayes approach could be used to estimate a prior for this distribution.22. This suggested that tetraploid tumours could have slower growth rates than diploid tumours, thereby offering a selective advantage by increasing the survival and reproductive output of infected individuals. These new results, however, tend to suggest the opposite effect. Diploids have a lower growth rate based on Bayesian credible intervals, though this difference was not statistically different based on a permutation test  as the only tetraploid group, with lack of other differences most likely due to low sample size. The lack of significant differences in growth rates between males and females  may help to capture the relationship between growth functions and cell biological parameters over time. Our models may offer insights into the natural patterns of tumour growth in the absence of any type of treatment, and therefore provide widely applicable base-line information for mechanistic tumour growth models to better understand the effects of cancer therapies. While our study provides insight into overall patterns of tumour growth, we acknowledge that other genetically controlled mechanisms such as apoptosis, autophagy or necrosis can also influence growth. Likewise, other intrinsic and extrinsic factors in our study population such as levels of stress in infected individuals, physiological and hormonal changes associated with reproduction that may result in immunosuppression and co-infection with other pathogens could also have an impact on tumour growth. Nevertheless, our study provides the first assessment of patterns of natural tumour growth, and therefore a basis from which to understand these additional complexities.Our framework allows fitting observable data from tumours of different karyotypes and locations to growth equations. Although we made simplifying assumptions to reflect the existing quantitative knowledge of tumour growthIn conclusion, we have provided the first empirical estimates of tumour growth rates in a natural and untreated population of hosts. Our results may help to understand how tumours grow in other organisms, and more importantly, will help to parameterise numerical models of growth rates and predict potential effects of therapeutic treatment in solid tumours.22) where the population has been sampled with a capture-mark-recapture framework at three month intervals since the beginning of the epidemic outbreak in 2006. Devils were trapped in custom made pipe traps (constructed from 30 cm diameter PVC pipe) and transferred into a hessian sack where they were handled, sexed and examined for DFTD status . While being handled, the devil’s eyes were covered with the sack to calm the animal and allow examination. Wild Tasmanian devils have a freezing response to handling (unresponsive with no resistance to examination) and therefore no sedation is required. All fieldwork (with the exception of one trapping session) was undertaken by the same investigator . Tumours were measured using SPI precision callipers and tumour volume was estimated by converting maximum tumour length, width and depth of each tumour into cubic centimetres. Tumour karyotype  are given in Table V = volume in cm3 and t = time in days.We use five models previously defined by Gerlee34. We assumed a lognormal error distribution in tumour volume to approximate measurement error, random variations in growth rate over time and between individual tumours. This approach also has the advantage that it is relatively computationally efficient and easy to implement. A particular issue with the available data is that it provides no context for the initial condition of each tumour. Though we know at what dates a particular tumour was measured and its volume at those dates, it was not possible to establish when it was first implanted in the individual and its initial volume – there is no consistent baseline across all tumours. We therefore include an extra parameter in the model for each tumour describing at which time t0 relative to its first observation that it reached an arbitrary small volume (V0 = 0.125 cm3), and optimise over this parameter in order to find the maximum likelihood for each tumour. The value of V0 is entirely cosmetic and has been set as an estimate of the minimum visible tumour size to provide reasonable visualisation of values for time since tumour detection  to select the most parsimonious model V0 = 0.25 cm3, a V0 = 0.25 cm3, a V0 = 0.25 cm3, ai, measured for the jth time, has volume Vi,j measured at time ti,j relative to the first measurement , is given an “initial” time ti,0 described above, and the given growth model V is based on parameters θ, the likelihood of the parameters θ together with the initial times ti,0 for all tumours can be described as:x | µ, σ2) represents the probability density of the normal distribution with mean µ and variance σ2 at value x, and ε represents the best-fit error rate. The maximum likelihood is then calculated by optimising over the free parameters θ, ti,0 and ε – the latter two can be optimised independently so dimensionality is not an issue.Where each tumour Supplementary Information


Colorectal cancer (CRC) is a highly heterogeneous disease, with pathologically similar cancers having completely different responses to treatment and patient survival. Intra-tumour heterogeneity  has recently been demonstrated to be an important factor in the development and behaviour of cancer cells and can be used to determine response to anticancer therapy.Patients with resected CRC had DNA extracted from eight defined tumour areas which were analysed for two genetic mutations (BRAF and KRAS) and one epigenetic trait (CpG island methylator phenotype/CIMP). Normal adjacent tissue was studied as control.p = 0.85), but 6 of the 12 patients (50%) demonstrated at least one heterogeneous area within the tumour.Twelve patients with CRC were included. Intra-tumoural heterogeneity for KRAS mutation was seen in 2 patients (17%). There was no statistical evidence of CIMP status heterogeneity (Intra-tumoural heterogeneity for both genetic and epigenetic factors in CRC is more prevalent than previously thought in Stage II and Stage III CRC. This study provides new insight into epigenetic heterogeneity of CRC and supports the development of a more targeted biopsy strategy to support expansion of personalised treatment. Intra-tumour heterogeneity is defined as the distinct morphological and phenotypic differences within a tumour. This includes cellular morphology, gene expression, proliferation, metabolism, motility and metastatic potential . It has KRAS, p53 and LOH in the 5q locus, and 18q locus. They found that 67% of the analysed tumours had tumour heterogeneity in at least 1 gene locus confirming significant tumour heterogeneity in advanced CRC, and recommending a different approach of tumour sampling.There is no consensus around what part, or how many samples of a CRC primary tumour should be sampled in order to identify potentially prognostic molecular or histopathological characteristics of a tumour. Baisse et al.  performeThe aim of this study was to establish whether a significant level of genetic or epigenetic heterogeneity could be demonstrated in patients undergoing surgery for colorectal cancer. An assessment could therefore be made on the adequacy of pre-operative biopsies in representing the whole tumour in terms of the genetic and epigenetic fingerprint, as there are currently no guidelines to support this.Twelve patients having elective surgery for colorectal cancer between January and June 2014 were randomly selected in this prospective study. Two pathologists reviewed the samples and rejected them if they were too small (<5 cm), as this would have restricted the ability to sample multiple areas for analysis. None of the patients had neoadjuvant treatment (radiotherapy or chemotherapy), as this may have altered the genetic or epigenetic profile of the tumour. The decision not to give neoadjuvant therapy was made by the regional multidisciplinary team (MDT) and was not influenced by the study. This decision was based on the pre-operative staging of the tumour. There were no other exclusion criteria, and patients were randomly selected. The tumour sample demographics, site of tumour, clinical and pathological stage, type of surgery, type and duration of adjuvant therapy, as well as overall and disease-free survival were recorded. Ethical approval for this study was granted by South West Wales REC (Project Ref No.:11/WA/0256).Two pathologists from Singleton and Morriston Hospitals, Swansea, sampled the resection specimen in a two-dimensional template configuration Fig. .Fig. 1SpThis resulted in eight areas of the primary tumour being sampled , as well as one sample of normal surrounding colonic tissue and of any metastatic lymph nodes.Formalin-fixed paraffin-embedded (FFPE) colorectal cancer specimens were utilised for this study. Several representative 5-µm sections of the biopsy were cut and mounted unstained onto glass slides, and DNA from these tissues was obtained using the MasterPure Complete DNA and RNA purification kit .The quantity and quality of DNA were measured at absorbance between 230 and 320 nm using spectrophotometry . DNA quantity was calculated by multiplying the measured concentration following spectrophotometry at 260 nm with the dilution factor. DNA was diluted to a working concentration of 20 ng/μl. Purity was further analysed by calculating the absorbance at 260 nm to absorbance at 280 nm ratio.CIMP-High if ≥2/3 group 1 markers methylatedCIMP-Intermediate if <2/3 group 1 but ≥3/5 group 2 methylatedCIMP-Low if <2/3 group 1 and <3/5 group 2 methylated.MSP was accomplished by performing bisulphite conversion of genomic DNA . The PCR products were resolved using gel electrophoresis on a 30% polyacrylamide gel. Depending on the methylation status of each CpG island, each patient could be classified as one of three epigenotypes: CIMP-High, Intermediate or Low using a two-panel approach , 8. The Pyrosequencing analysis was performed upon the clinical specimens of this research project in collaboration with the Leeds Cancer Research UK Centre . Pyrosequencing conditions used were as previously published by this group . This suggests that the 12 tumour samples are generally homogenous in terms of CIMP classification.Figure Two patients had metastatic lymph nodes resected at the index operation (patients 4 and 7). The tumour and lymph node characteristics are shown in Table KRAS mutations across the tumour (patients 5 and 11). When these areas are compared to the CIMP status of those tumours, the KRAS mutation is seen in areas of hypermethylation. Figure p value since it represents the chance of seeing such agreement conditional on the null hypothesis of no association.As previously mentioned, two patients had heterogeneous p = 0.009) to this event. There is therefore a significant association between the two variables, KRAS mutation and CIMP status.In patient 5, there is one area within the tumour and normal tissue proximal to the tumour that is both exclusively CIMP-L and KRAS-wt. The probability of this occurring spontaneously is 2.8% (1/9 × 1/8). In patient 11, the chance that the CIMP-L area will be in a KRAS-wt area is 33% (3/9). Given the situation, we have observed in both patients and assuming the link was random, we can assign a p-value of only around 1% , and unfortunately, they failed to adequately explain which part of the resection sample was tested. This is a drawback of this study, as it would be expected that a homogenous result was seen if the same area of the tumour was sampled in the biopsy and resection specimens.More recently, Fadhil et al.  highlighThe results from this work suggest that the majority of colorectal cancers are largely homogenous in terms of KRAS, BRAF and CIMP status. Currently, only pre-operative biopsies for KRAS status have any bearing on treatment, as the decision to use EGFR-inhibitors is based on this result. In our sample, 2 of 6 patients with a KRAS mutation (33%) had some degree of heterogeneity found in the sample; therefore, a recommendation can be made that that at least two biopsies from different parts of the tumour be taken to take the heterogeneity into consideration.KRAS mutation and BRAF wild type. It was not possible to formally classify the patients into a definite Jass Grouping [Previous work by Jass  attempteGrouping , as no dFrom the relationship seen between the genetic and epigenetic factors, we can postulate that the epigenetic changes have occurred later on than the KRAS mutation, as there was a strong association between the unmethylated areas with KRAS-wt. This phenomenon was only seen in two patients, and larger numbers would be needed to expand on this theory.The large amount of methylation-specific PCR needed  limited the amount of patients in this study to 12. It is possible that we did not have a representative sample of the general population because of this. Other limitations included the necessity of selecting patients with advanced local disease (pT3/4) that were not selected for neoadjuvant therapy. The guidelines state that there is no evidence to support pre-operative chemotherapy versus surgery alone in patients with locally advanced colon cancer; however, in the patients with rectal cancer, chemoradiotherapy before surgery is recommended for patients with high-risk locally advanced rectal cancer to allow tumour response and shrinkage . Of the TP53 and PIC3CA) and microsatellite instability may have provided further insight into the cause of intra-tumour heterogeneity.The group of CIMP panel markers that were selected also limited the results, and it is possible that this is not a true representation of the CIMP status of the whole genome. Recent work has established that single-cell genome-wide bisulphite sequencing is a valid method of measuring DNA methylation in mice . This teThis pilot study involving 12 patients exploring intra-tumour heterogeneity of colorectal cancer has demonstrated relative homogeneity of genetic factors (BRAF and KRAS mutations), as well as epigenetic homogeneity (in terms of DNA methylation). It has analysed data from a relatively small number of patients, but importantly, the location of the biopsies in the tumour was decided according to a defined template, which investigated mutations based on tumour topography. It has also revealed interesting patterns of association of CIMP and KRAS/BRAF mutation in keeping with the work of Jass . This woClinicians should be aware that at least two pre-operative biopsies be taken to avoid mis-sampling when assessing KRAS status. This number may well increase in future if the CIMP status is found to be clinically significant as a prognostic marker.Current research is focusing on both KRAS and BRAF as predictive and diagnostic biomarkers in patients with metastatic disease treated with anti-EGFR therapies, such as panitumumab and cetuximab –27. As t


Good agreement of results demonstrates the capabilities of the developed numerical model to predict and analyze the motion and deformation of RBCs in complex geometries. The proposed model (methodology) will help to elucidate different rheological and hematological related pathologies and finally to design better treatment strategies.Due to the important biological role of red blood cells (RBCs) in vertebrates, the analysis of reshaping and dynamics of RBCs motion is a critical issue in physiology and biomechanics. In this paper the behavior of RBCs within the immature capillary plexus during embryonic development of zebrafish has been analyzed. Relying on the fact that zebrafish embryos are small and optically transparent, it is possible to image the blood flow. In this way the anatomy of blood vessels is monitored along with the circulation throughout their development. Numerical simulations were performed using a specific numerical model that combines fluid flow simulation, modeling of the interaction of individual RBCs immersed in blood plasma with the surrounding fluid and modeling the deformation of individual cells. The results of numerical simulations are in accordance with the  Danio rerio), has great advantages over other species for studying vascular development. First of all, the dimension of zebrafish is very small, since an adult zebrafish can grow to 4 cm on average. Zebrafish are very fertile and lay a large number of eggs. The reproduction is external, outside the mother's body. Optical analysis of this species is also very easy. Due to its small dimensions, the embryos can survive with only a small amount of oxygen that they receive by passive diffusion. All the interior organs, such as eyes, brain, heart, inner ear are developed within the first 3 days post fertilization. The cardiovascular system is one of the first systems that is formed during the embryonic development of zebrafish. After only 1 day post fertilization, the zebrafish is sufficiently developed for the blood to start circulating. Detailed explanation of the formation of all major blood vessels in zebrafish can be found in the literature  that differentiate into erythrocytes, lymphocytes, and platelets. It is also called adult hematopoiesis and it has the capability of self-renewal and produces all mature hematopoietic lineages  were maintained in a facility with the system water at 28.5°C with 14 h light: 10 h darkness circadian rhythm. An endothelial specific reporter transgenic line Tg(fli1a:eGFP)y7 was acquired from aquatic resource program  and used for collecting in vivo experimental data. The transgenic embryos were attained from natural spawning with a 2:1 ratio  and staged according to the standard conditions  method was used to simulate fluid flow. This method was successfully applied to modeling the motion of solid bodies through a fluid domain  represents the probability of a particle to be located within an element in space dx around point x, in moment in time t, where x denotes the particle position vector. When the LB method is implemented, the equation that represents the entire numerical scheme is most commonly separated into two steps—a collision step and a propagation step. Two values of distribution function are defined—The basic quantity in the LB method is the distribution function The mentioned steps can be described using following equations:Collision step:Propagation step:These two steps are repeated in a series of iterations, whereas each of these two steps must be applied to all particles, i.e., nodes of the mesh, before the next step starts.Fi represents the discretized external force term, ρ represents the fluid density, u represents the fluid velocity, ξi represent vectors defining the abscissae of the lattice structure and index i represents the component of the distribution function that is calculated. The abscissae for the three-dimensional isothermal flow of incompressible fluid used in this paper (denoted by D3Q27) are shown in Figure i = 1, …, 27) of the distribution function are calculated and these components are used to precisely define the possible directions of motion of fictional fluid particles in three-dimensional space.In the above equation, τ represents the relaxation time, The equilibrium distribution function is defined using the following equation:g represents the external force field.The discretized external force term is defined as:Macroscopic quantities necessary to describe the fluid flow at the macroscale level, such as density, pressure, velocity, are evaluated in terms of the calculated components of distribution function.Density is calculated as:The expression for velocity is given by:cs represents a constant related to the LB method. For the D3Q27 lattice structure that is used in this paper, this constant is equal to Pressure is calculated in terms of the fluid density, in the considered node of the mesh, and this relation is given by:RBCs or erythrocytes are highly differentiated cells that contain a cellular membrane that surrounds the inner structure of the cell. In this study, an approximation is introduced, that assumes that the entire internal structure can be represented as an incompressible Newtonian fluid, because it is considered that the influence of the membrane is crucial for the deformation of the entire cell. The cellular membrane is composed of two layers of lipids and a thin skeleton of interconnected proteins , the tensor invariants are used, because this is more appropriate for the numerical calculation of the reaction force caused by the change of surface strain, which is calculated using the finite element method  presented by Peskin  was usedSince the discretization of the RBC membrane and fluid domain do not exactly overlap, the influence of various points from each mesh has to be taken into consideration when calculating quantities relevant for the simulation. Thus, an interpolation scheme has to be applied.Dijk is used to define the Dirac function at a specific point of the fluid domain, indexes i, j and k denote the currently considered point in the fluid mesh, l = 1, 2, …, G are the coordinates of the l-th point of the mesh that is used to model the RBC membrane and G is the number of points in this mesh.Interpolation is performed using the Dirac delta function, that is approximated as follows:r) is defined in the literature  and r is the distance between the considered points in the fluid and the mesh representing the membrane of the RBC.The value of function δδ(rFl(t) is the force with which the l-th point of the mesh representing the membrane of the RBC opposes the effect of the surrounding fluid. Calculation of this force is explained in detail in Section Modeling the Deformation of the RBC.As already mentioned, in fluid flow simulations the effect of the immersed object is introduced through an external force, that acts on the fluid surrounding the membrane. Due to the applied interpolation scheme, the reaction force at one point of the RBC membrane is transferred to several points of the fluid mesh. This force can be expressed as:The velocity of all points in the mesh representing the membrane of the RBC is interpolated over the surrounding points in fluid mesh, and it is calculated using the following equation:Applying the Euler Forward method on Equation (16), the new positions of points in the mesh representing the membrane of the RBC are obtained:This way, the deformation of the RBC membrane at every time step is calculated. Using the obtained deformation, the reaction forces due to the new deformation of the membrane are calculated. These forces cause a change in the external force field in the fluid domain, thus causing an effect of the immersed object to the fluid. Fluid on the other hand, again causes new deformation of the RBC membrane. This process is repeated in each iteration, until the defined condition is satisfied or the defined number of iterations is reached.In this paper it is considered that the RBC of the zebrafish has an elliptic shape, according to the previous investigations of the zebrafish cardiovascular system  and the intussusceptive pillars are modeled using the Bounce-back approach . This paper is also supported by the Swiss National Science Foundation, SCOPES project (JRP: IZ73Z0_152454/1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NFKB2 mutation in common variable immunodeficiency and isolated adrenocorticotropic hormone deficiency: A case report and review of literature”, which appeared in Volume 95, Issue 40 of Medicine, one of the references in Table [6] should have appeared as Lindsley et al[3].In the article, “


We analysed the haemodynamics of indocyanine green (ICG) in mouse organs and tumours and evaluated responses to anti-angiogenic agents in an allograft tumour mouse model by photoacoustic imaging.Thirty-six male mice  were used. Real-time photoacoustic imaging of organs and tumours after intravenous injection of ICG was conducted in mice until 10 min after ICG injection. ICG distribution in tumour tissues was assessed by immunohistochemical staining and observation of ICG-derived fluorescence. Vascular permeability changes induced by the vascular endothelial growth factor (VEGF)-blocking agent VEGF-trap on tumour photoacoustic signals were studied.p < 0.010). In tumours, the photoacoustic signal increased within 30 s after ICG injection in a dose-dependent manner (r2 = 0.899) and then decreased gradually. ICG was found to extravasate in tumour tissues. In VEGF-trap-treated mice, the photoacoustic signal in the tumour decreased at the early phase before inhibition of tumour growth was detected .The photoacoustic signals in salivary glands and tumours after intravenous injection of iCG  were significantly increased compared with those in the liver, kidney, and great vessel (0.234 ± 0.043, 0.204 ± 0.058 and 0.127 ± 0.040, respectively; Photoacoustic imaging with ICG administration demonstrated extravasation of ICG in mouse organs and tumours, indicating the potential for early detection of changes in vascular permeability during cancer therapy. The photoacoustic signal of indocyanine green was increased in tumoursIndocyanine green was found to extravasate in tumour tissuesThe photoacoustic signal was derived from extravasated indocyanine greenIn VEGF-trap-treated mice, photoacoustic signals decreased before tumour shrinkagePhotoacoustic imaging is an emerging non-invasive optical technology that proved to have potential for cellular and molecular specific visualisation at clinically relevant depths of various organs . As appaIndocyanine green (ICG) is a fluorescent cyanine dye used as a medical diagnostic agent for monitoring cardiac and liver function , 6. ICG In this study, the potential of ICG-enhanced photoacoustic imaging was evaluated using organs and tumour allografts from mice. Moreover, the applicability of photoacoustic imaging coupled with ICG for evaluating changes in tumour vascular permeability associated with anti-angiogenic therapy was examined. The purpose of this study was to analyse the haemodynamics of ICG in mouse organs and tumours and evaluate the tumour response to anti-angiogenic agents by photoacoustic imaging.All procedures were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the of the Ministry of Education, Culture, Sports, Science and Technology of Japan and approved by the Committee on Animal Experimentation of Kanazawa University.Photoacoustic imaging was carried out with a Vevo LAZR small-animal ultrasound and photoacoustic imaging system . This machine was composed of an ultrasound system equipped with a laser pulse system and animal measurement box Fig. . To acquTo investigate the reliability and quantitative capacity of photoacoustic imaging, in vitro experiments were performed. Three different concentrations of ICG  and saline as a control were imaged on cotton buds and measured using the photoacoustic imaging system.c-Brd/BrdCrCrl albino  male mice  were used. Hair at the examination site was removed with hair removal cream one day before the photoacoustic imaging procedure.For in vivo experiments, C57BL6/J and B6N-Tyr7 cells suspended in 100 μL were injected subcutaneously into the backs of the mice under general anaesthesia with pentobarbital . Subcutaneous tumours were imaged with a photoacoustic imaging system ten days after tumour implantation, when the tumours had reached about 1 cm in maximum diameter.A Lewis lung carcinoma (LLC) cell allograft model was used. Briefly, LLC cells were cultured in minimum essential medium supplemented with 10% foetal bovine serum. At 70–80% confluence, the tumour cells were trypsinised and 1.0 × 102) was also calculated.For anti-vascular endothelial growth factor (VEGF) antibody treatment, mice were injected intraperitoneally with VEGF-trap  at a concentration of 5 mg/kg ten days after tumour implantation. Photoacoustic imaging was performed three days after VEGF-trap injection. Tumour volumes were monitored every two days after tumour implantation for 20 days  and fixed on a platform. Each target organ was observed by B-mode ultrasound and a favourable position at maximum area coverage was chosen. Before injection of ICG, pre-contrast photoacoustic imaging was performed for 1 min. Next, ICG was injected via the tail vein using a 27-G butterfly needle attached to a disposable microsyringe at an initial rate of 0.02 ml/s using an automatic injector . Signals of photoacoustic imaging were obtained under the following conditions: 780 nm excitation light; 42-dB photoacoustic gain; 22-dB two-dimensional gain and 5 frames/s. To reduce the effects of respiratory motion, we used a respiratory gate. Photoacoustic signals in the liver, kidney, salivary gland, inferior vena cava and LLC implanted in the skin of mice were collected during the 10-min period after intravenous ICG injection (5 mg/ml) and peak photoacoustic signals were calculated. For semi-quantitative analysis of tumours after ICG injection, three different concentrations of ICG  were administered and photoacoustic signals were acquired. For experiments involving VEGF-trap treatment, 5 mg/ml ICG was administered and peak photoacoustic signals were calculated. Photoacoustic signals were acquired during the 10-min period until the signal reached a plateau.To visualise the extravasation of ICG in tumour tissues, ICG was injected via the tail vein; 30 s later, vessels were washed with saline under cardiac puncture and drained from the superior vena cava. Tumour tissues were embedded in Optical Cutting Temperature compound . Cryosections (10 μm) were stained with anti-CD31 antibodies  overnight at 4 °C and incubated with Alexa Fluor 488-conjugated secondary antibodies  for 1 h at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole . Samples were imaged using an inverted microscope  equipped with an ORCA-Flash4.0 digital CMOS camera (Hamamatsu Photonics), configured with a ICG-filter set  or with confocal laser microscopy, as previously described .p values < 0.050 were considered as statistically significant.Statistical analysis and graphical display of data were performed using GraphPad software . All values are reported as mean ± standard deviation (SD). One-way analysis of variance with post-hoc Tukey test was used to compare photoacoustic signals among groups. Correlation analysis between photoacoustic signals and ICG concentrations in vitro experiments and between photoacoustic signals in tumours and ICG concentrations in the animal experiment was performed using Pearson’s correlation analysis. Two-tailed unpaired t-tests were used to compare photoacoustic signals and tumour volumes between the control and anti-VEGF therapy groups. Results with 2 = 0.970; Fig. Cotton swabs presoaked with three different concentrations of ICG provided dose-dependent increases in photoacoustic signals Fig. , demonstIn all examined organs photoacoustic signals rapidly increased after ICG injection and peaked within approximately 30 s, followed by gradual decay Fig. . The qua2 = 0.899; Fig. B-mode photoacoustic imaging showed increases in photoacoustic signals diffusely within tumours after ICG injection Fig. a. The phOn histopathological examination, extravascular leakage of ICG was observed in the interstitial space outside the vascular lumen lined by CD31-positive vascular endothelium in tumours Fig. . ExtravaA single administration of VEGF-trap dramatically decreased ICG-photoacoustic signals on day 13 compared with saline administration Fig. , Table 4This study showed that photoacoustic imaging with ICG administration could be used to evaluate ICG extravasation in tumours and facilitate the early detection of changes in vascular permeability during cancer therapy. The use of the photoacoustic imaging system enabled safe and real-time image acquisition to convey underlying pathologic information for the target tissue without radiation exposure.Some experimental studies and preclinical studies using photoacoustic imaging have been reported, including non-invasive imaging of the breast, sentinel lymph nodes and skin –16. HoweIn our study, we clarified that ICG can be used as a contrast agent for non-invasive dynamic photoacoustic imaging in a mouse model. Importantly, transient ICG signals in photoacoustic imaging are thought to originate from the extravascular space. Of the various organs tested in this study, photoacoustic signals from tumours and salivary glands showed the strongest peak just after injection. In contrast, the inferior vena cava, including the large intravascular space with flowing blood, did not show any clear increase in ICG signals. This suggested that the ICG signal observed during the early phase after administration was mainly derived from the extravascular space, not the intravascular space with our photoacoustic settings. This observation was supported by data showing that salivary glands containing hyperpermeable fenestrated capillaries revealed a higher photoacoustic signal intensity than other normal organs .ICG diffuses into the systemic vasculature and the ICG bound to albumin is then exclusively metabolised and cleared by the liver at a half-life of about 5 min . HoweverThe objective tumour response has been used as an important endpoint for assessment of the therapeutic effects of cancer therapy. With molecular targeted drugs, including VEGF inhibitors, tumour shrinkage is delayed and tumour cells may disappear without any reduction in the tumour volume. When using molecular targeted drugs, downregulation of VEGF, normalisation of the tumour vasculature, pruning of excess vessels and reduction of interstitial fluid pressure and vessel permeability occur in the tumour consecutively –29. In tIn conclusion, we found that photoacoustic imaging with ICG administration demonstrated extravasation of ICG in mouse organs and tumours, indicating the potential for early detection of changes in vascular permeability during cancer therapy.


Intravenous administration of fluids is an essential part of critical care. While some fluid administration is likely beneficial, there is increasing observational evidence that the development of fluid overload is associated with increased mortality. There are no randomised trials to confirm this association in patients with acute kidney injury. We aim to perform a pilot trial to test the feasibility of forced fluid removal compared to standard care in patients with acute kidney injury and severe fluid overload, the FFAKI trial.Then FFAKI trial is a pilot, multicentre, randomised clinical trial recruiting adult intensive care patients with acute kidney injury and fluid overload, defined as more than 10% of ideal bodyweight. Patients are randomised with concealed allocation to either standard care or forced fluid removal with a therapeutic target of negative net fluid balance ≥1 mL/kg/h. The safety of fluid removal is continually evaluated according to predefined criteria of hypoperfusion: lactate ≥4 mmol/L, mean arterial pressure <50 mmHg or mottling beyond the edge of the kneecaps. If patients fulfil one of these criteria, fluid removal is suspended until hypoperfusion has resolved. The primary outcome measure is fluid balance at 5 days after randomisation and secondary outcomes include mean daily fluid balance, fluid balance at discharge from the intensive care unit, time to neutral fluid balance, number of serious adverse reactions and number of protocol violations. All patients are followed for 90 days.The FFAKI trial started in October 2015 and, when completed, will provide data to evaluate whether a large trial of forced fluid removal in critically ill patients is feasible. Our primary outcome will show if the experimental intervention leads to a clinically relevant difference in fluid balance, which could prove beneficial in intensive care patients with acute kidney injury.EudraCT, identifier: 2015-001701-13. Registered on 19 September 2015;NCT02458157. Registered on 21 May 2015;ClinicalTrials.gov, identifier: Danish Ethics Committee, identifier: H-15009589H. Registered on 22 September 2015; Danish Health and Medicines Authority, identifier: 2015070013. Registered on 11 August 2015.The online version of this article (doi:10.1186/s13063-017-1935-2) contains supplementary material, which is available to authorized users. I2 = 62%). Mean positive daily fluid balance was also shown to be associated with death with an OR of 1.16 ; however, there was significant heterogeneity amongst the studies .Administration of isotonic crystalloid solutions is a common intervention in critically ill patients. In some cases this leads to the accumulation of fluids and development of fluid overload, defined as a positive fluid balance corresponding to 10% or more of total bodyweight. The cut-off at 10% was first utilised in a paediatric observational study by Gillespie et al. . GrowingThe observed association between fluid overload and outcome may in part be mediated by the development of interstitial oedema leading to deranged organ architecture, increased diffusion distances for oxygen and metabolites and increased interstitial pressure .A study in healthy volunteers demonstrated that infusion of 2 L crystalloid leads to renal swelling . Renal iGiven the observational nature of the available data, there is a high risk of confounding by indication and a causal relationship cannot be established before randomised data are available . We aim The FFAKI trial is a multicentre, randomised, site-stratified, clinical pilot trial with adequate computer generation of the allocation sequence with permuted blocks of varying size and allocation concealment. Randomisation is performed using sequentially numbered, opaque, sealed envelopes.The trial statistician is blinded but due to the nature of the intervention it is not possible to blind patients or caregivers.The trial is initiated at three separate centres in Denmark. Each centre has one or two primary investigators with expert knowledge in the FFAKI intervention and protocol. Participants in the FFAKI trial will receive protocol-specific treatment during their entire ICU stay. Therefore, every caregiver at participating centres receives training in the protocol by either the principal investigator or one of the primary investigators. It is expected that the primary centre  will include 20–30 patients and each secondary centre will include 10–15 patients.The trial protocol was written according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Statement . A populAll patients admitted to the ICU at the participating centres will be eligible for screening according to the following:Age 18 years or olderAKI defined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria Renal Recovery Score (RRS) ≤60%Fluid overload defined as a positive fluid balance of at least 10% of ideal body weightAble to undergo randomisation within 12 h of fulfilling the other inclusion criteriaKnown allergy to furosemide or sulphonamides2 or chronic renal replacement therapy)Known prehospitalisation advanced chronic kidney disease (estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2) >80% and positive end-expiratory pressure (PEEP) >10 cmH2O)Severe hypoxic respiratory failure Severe dysnatraemia (plasma concentrations <120 or >155 mmol/L)Hepatic comaMentally disabled undergoing forced treatmentPregnancy/breastfeedingLack of commitment for ongoing life support including renal replacement therapy (RRT)Lack of informed consentPatients who fulfil all of the inclusion criteria and none of the exclusion criteria will be enrolled in the trial and allocated to either the experimental treatment  or standard care. Based upon own observational data we expect that roughly 10% of adult patients admitted for more than 24 h will be available for inclusion in the FFAKI trial  <50 mmHg or mottling beyond the edge of the kneecaps).The therapeutic effect is evaluated at three daily summary points on the ICU observation charts , while safety variables are evaluated continuously.The first choice for fluid removal is diuretic therapy with furosemide which is continued for a minimum of 8 h. If the therapeutic goal cannot be achieved and maintained by diuretic therapy it is replaced by fluid removal with continuous renal replacement therapy (CRRT).Mottling beyond the edge of the kneecaps Hypotension (MAP <50 mmHg) resistant to inotropes and vasopressorsPlasma lactate ≥4 mmol/L During the entire trial, the physiologic response to fluid removal is monitored with three variables indicating inadequate circulation. These are:Mottling and MAP are monitored continuously and lactate is routinely measured four to six times each day and on clinical indication.Pause fluid removalGive a crystalloid fluid bolus of 250–500 mLRe-evaluate circulatory status within 30 minRepeat fluid therapy and revaluation until adequate circulation  have been maintained for minimum 1 hRestart fluid removal in 25% reduced dose for a minimum of 4 h before evaluating effectIf one or more signs of inadequate circulation are present the resuscitation algorithm is started:Fluid removal is continued until the patient achieves neutral cumulative fluid balance (±1000 mL), which is then attempted maintained for the entire duration of ICU admission.+ >6 mmol/L)Hyperkalaemia (p-KSevere metabolic acidosis attributable to AKI (pH <7.25 and standard base excess (SBE) below −10 mmol/L) resistant to intravenously (IV) administered bicarbonate infusion2/FiO2 < 13 kPa and bilateral infiltrates/oedema on the chest X-raySevere respiratory failure with PaOAside from these absolute indications, RRT may be provided in case of progressive azotaemia and blood urea (BUN) >25 mmol/LIn the control group receiving standard care, there are no protocolled interventions. Fluid administration and removal is done at the discretion of the treating clinicians. Use of dialysis is encouraged only in the presence of severe disturbances in fluid, electrolyte and acid-base balance:All data are collected in a paper-based Case Research Form (CRF) and participants are followed for a total of 90 days. The primary outcome of the FFAKI trial is cumulative fluid balance 5 days after randomisation. All outcome variables are shown in Table The FFAKI trial is externally monitored according to Good Clinical Practice (GCP) (EU-Directive-2001/20) guidelines including monitoring of consents and source data by external staff.Protocol adhesion is monitored by the GCP guidelines on the first five patients in both the intervention and control arm at each centre and protocol violations are registered in the CRF of all patients. In case of protocol violations the principle investigator will initiate re-education of participating caregivers.β value of 0.80 and a two-sided α value of 0.05. The sample size estimation is based upon observational data of patients fulfilling the FFAKI inclusion criteria admitted at our ICU in 2012 and 2013  7.4 L).The main objective of the FFAKI trial is to determine whether the trial intervention is feasible. To capture this we have decided to examine whether the trial intervention leads to a clinically relevant difference in fluid balance after 5 days. This is treated as an interim outcome and in accordance with the newly published Consolidated Standards of Reporting Trials (CONSORT) extension to randomised pilot and feasibility trials , we usedTwo complimentary analyses of the primary outcome will be performed in order to account for attrition due to death. In the first analysis the subject-specific fluid balances are modelled in a ‘linear random-effects model’ unconditional on survival status. Difference in fluid balance 5 days after randomisation will be assessed using a Wald test. In the second analysis we will estimate the ‘survival average causal effect’ of the intervention using principal stratification and include a sensitivity analysis to assess the influence of possible violations of assumptions. In both models missing data due to dropout will be handled using ‘inverse probability weighting’.t test. Nonparametric data will be reported as median (interquartile range (IQR)) and compared using the Mann-Whitney U test.All parametric data will be presented as mean (SD) and compared using Student’s Patient physiology might not allow early and forced fluid removalPatients who develop AKI and fluid overload are characterised by severe critical illness, often with failure of multiple organ systems and they receive a multitude of interventions including mechanical ventilation, fluid therapy, antibiotics, vasopressors, inotropes and dialysis. The underlying condition further compromises the physiology of the patient leading to leaky capillaries and loss of intravascular albumin . Fluid r2.The Hawthorne effect might alter the administration of IV fluids leading to reduced incidence of fluid overload and abolish the need for forced fluid removalFluid overload is an iatrogenic condition that arises as a consequence of the current practice of fluid therapy in critical illness. When a trial of forced fluid removal is initiated there will be an inevitable increased focus on the indications for fluid administration and removal. This produces a risk of a Hawthorne effect leading to less fluid administered by clinicians which would reduce the incidence of fluid overload in the given patient population and abolish the need for a trial of this nature3.Heterogeneity of the patient population could dilute any potential effect of the intervention leading to reduced power and the need for a very large sample sizeThe baseline chance of recovering renal function following AKI in the ICU varies with age, gender and the severity of kidney injury. Patients who have a high chance of recovering with the current standard of care will have very little potential benefit of participating in a trial of new therapeutic interventions. Furthermore, these patients could dilute the potential effect of a given intervention and lead to a loss in power and need for larger sample sizes. We have recently developed a model to predict the chance of recovering renal function following AKI in the ICU, which is called the ‘Renal Recovery Score’ (RRS) (not yet published). In the development and validation of the model we found that the 20% of patients with the highest chance of recovering renal function had a RRS value of >64% and an 80–85% frequency of recovery. To selectively include patients with a moderate to high risk of persistent renal injury we will be using a RRS value of <60% as part of the inclusion criteria4.Lack of blindingDue to the nature of the intervention it is not possible to blind participants, caregivers administering the intervention or caregivers administering co-interventions. This introduces a risk of bias which we will evaluate with fluid therapy and dialysis data in both treatment arms. To avoid overly aggressive fluid removal in the control group we discourage the use of dialysis unless specifically indicated according to previously described criteria5.Lack of protocol adherenceForced fluid removal in critically ill patients with continued need for vasopressors and inotropes might seem controversial to some clinicians and it is uncertain whether our intervention can be performed with the acceptance of the treating clinicians. Therefore, we register the frequency of protocol violations defined as the use of dialysis outside the recommended indications in the control group, and cessation of fluid removal before fulfilment of safety criteria or achievement of neutral fluid balance in the experimental groupThe FFAKI trial consists of a complex intervention that alters the core of current therapy for critically ill patients. Furthermore, the intervention is designed to treat an iatrogenic condition (fluid overload). To our knowledge there are no previous implementations of forced fluid removal in a randomised clinical design and we have identified several key areas in which a trial of forced fluid removal might prove unfeasible:By performing a pilot trial with a primary outcome of fluid balance 5 days after randomisation we will be able to evaluate whether the intervention is feasible. Furthermore we will know if it leads to a clinically relevant difference in fluid balance which would indicate that implementation of this protocol in a larger sample size would provide analysable results regarding a causal relationship between fluid removal and patient-centred outcomes of renal recovery and survival.The FFAKI trial was initiated in October 2015 and recruitment is currently ongoing at three different sites.


There are errors in the Funding section. The grant recipient’s name should be “Yuqin Yao” instead of “Yuqing Yao.” The correct funding statement is: This work was supported by National Science Foundation of China, grant 81402564 to Yuqin Yao; National Natural Science Foundation of China, grant 81773375 to Yuqin Yao; and National High Technology Research and Development Program of China, grant 2015AA020904 to Yuqin Yao.


Burn injury is a leading cause of unintentional death and injury in children, with the majority being minor (less than 10%). However, a significant number of children sustain burns greater than 15% total body surface area (TBSA), leading to the initiation of the systemic inflammatory response syndrome. These patients require IV fluid resuscitation to prevent burn shock and death. Prompt resuscitation is critical in pediatric patients due to their small circulating blood volumes. Delays in resuscitation can result in increased complications and increased mortality. The basic principles of resuscitation are the same in adults and children, with several key differences. The unique physiologic needs of children must be adequately addressed during resuscitation to optimize outcomes. In this review, we will discuss the history of fluid resuscitation, current resuscitation practices, and future directions of resuscitation for the pediatric burn population. Burn injury is a leading cause of unintentional death and injury in children until 14 years of age (as high as the third most common cause in children ages 5 to 9) . While mFluid resuscitation as a treatment for burn injury is thought to date back to the eighteenth century when Gerard van Swieten administered fluid via enema to rehydrate burn victims . The ideIn November, 1942, Oliver Cope and Francis Moore utilized this knowledge and improved upon it as they treated the victims of the Cocoanut Grove nightclub fire. Cope and Moore recognized the relationship between the amount of fluid resuscitation required and the size of the burn. Their formula for IV fluid resuscitation used equal parts of plasma and saline and prescribed 150 ml of fluid for each 1% TBSA burn plus maintenance fluids during the first 24 h following injury . Half ofIn contrast with two figure formulae, single figure formulae do not consider the maintenance needs separate from the burn resuscitation fluids. They combine all of their fluid needs into a single formula. Gelin was the first to propose a single figure formula. He proposed three levels of fluid resuscitation. Patients with burns less than 30% TBSA received 2 ml/kg/%TBSA burn in the first 24 h, those with 30–60% TBSA received 2.5 ml/kg/%TBSA burn in the first 24 h, and those with greater than 60% TBSA received 3 ml/kg/%TBSA burn of low molecular weight dextran. Single figure formulae have become the cornerstone of adult burn fluid resuscitation. The most frequently used formula in current burn care is the Parkland formula, developed by Baxter and Shires following a series of studies on dogs , 17. TheThe initial resuscitation formulas that were developed for adult burn-injured patients were initially used in burn-injured children with “proportionally smaller quantities” . This waThe principles of fluid resuscitation are similar for children and adults, but there are specific differences as well. In general, Ringer’s lactate solution should be started in patients of all age groups. Due to their limited glycogen stores, infants are at risk of hypoglycemia if a glucose source is not provided as part of their resuscitation. Their blood glucose levels should be monitored closely, and a source of 5% dextrose is provided as well. As stated earlier, the volume of fluid required per percent burn is higher in children due to their increased baseline BSA. Therefore, pediatric burn resuscitation formulas are always two figure formulae that calculate an estimated fluid resuscitation (EFR) and add maintenance fluids (MFs) with or without dextrose depending on the child’s age and size. The cutoff for using adult formulae depends on the source, but it is generally felt to be somewhere between 30 and 50 kg.2 BSA in the 48 h following injury . In younger children, their formula for resuscitation was much more complex. Younger children received the same 4 ml/kg/%TBSA burn + 1500 ml/m2 total BSA over the first 24 h, but the composition of the fluid changed every 8 h. In the first 8 h, the fluid was lactated Ringer’s with 50 mEq of sodium bicarbonate. The second 8 h was lactated Ringer’s alone, and the third 8 h was lactated Ringer’s plus 12.5 g of 25% albumin per liter. The team at Shriners Hospitals for Children in Galveston developed a formula that uses only BSA [2 BSA burn as a resuscitation fluid and 2000 ml/m2 total BSA as a maintenance fluid. As with the previously described adult formulas, half is given over the first 8 h and the remainder is given over the next 16 h. The fluid utilized in this formula is lactated Ringer’s solution with 12.5 g of 25% albumin per liter plus 5% dextrose as needed.In the current practice, there are two main formulas that are utilized in pediatric burn patients: the Cincinnati Formula and the Galveston Formula. Shriners Hospitals for Children in Cincinnati developed their own formula which is similar to the Parkland formula but adds in a maintenance fluid calculation based on BSA . In oldeonly BSA . The GalThere has not been a head-to-head comparison of the two commonly used pediatric formulas. One group has attempted to model the use of the two different formulas over a range of patient weights and burn sizes and compare these to the physiologic norms of children, but has not examined the use of the formulas in actual practice . They foWhile a lot of research has gone into the creation of resuscitation formulas, it is critical to remember that they are merely estimates of the amount of fluid that will be required for a given burn-injured patient. Resuscitation formulas should be used to initiate therapy. However, the IV fluid resuscitation rate should be reevaluated on an hourly basis and IV rate adjusted accordingly. Both over-and under-resuscitation are equally problematic in pediatric burn-injured patients. Under-resuscitation leads to burn shock, suboptimal tissue perfusion, end-organ failure, and death . The conIn order to ensure that patients are not being either over- or under-resuscitated, IV fluid rates need to be adjusted based on urinary output (UO). Hourly UO continues to be the most commonly used endpoint in guiding the administration of resuscitation fluids . In chilIn modern burn units, there are far more sophisticated methods to measure response to burn resuscitation than UO. Many of these monitoring tools are able to assess the moment-to-moment physiological state of the patient. For example, abnormal admission arterial lactate levels and base excess values correlate with the magnitude of injury and are now recognized as markers of global poor perfusion and uncompensated shock . FailureNovel methods of measuring physiologic parameters are being developed and utilized in both pediatric and adult burn patients. In adult patients, standard Parkland resuscitation was compared to resuscitation guided by invasive thermodilution (TDD) measurements in patients with greater than 20% TBSA burn . This stDue to the fact that cardiac output decreases during the first 24 to 36 h after major burn injury, children with burns greater than 40% TBSA should have advanced monitoring including central venous pressures so that their response to fluids can be adequately assessed. Children receiving IV fluid rates twice that were predicted by the Parkland formula, with continued inadequate urine output, are likely to have either heart failure or other complications of over-resuscitation, including abdominal compartment syndrome or pleural effusion .In general, the treatment for hypotension in pediatric burn-injured patients is fluid resuscitation. However, even proper fluid resuscitation of burn shock may not achieve complete normalization of physiologic variables due to the fact that burn injury leads to continued cellular and hormonal changes in the patient . In thesThe use of colloids in burn resuscitation is an area where considerable controversy exists. Plasma proteins are important in maintaining oncotic pressure to balance the outward hydrostatic pressure. The need to administer large volumes of crystalloid fluids during burn resuscitation to prevent burn shock leads to a decrease in plasma protein concentration and worsens extravascular egress of fluid and edema formation. Replacing plasma proteins using colloids  could in theory mitigate this effect. It was this theory that led to the use of colloids in the early burn resuscitation formulas such as those developed by Cope and Moore  and EvanThe use of colloid at all in initial burn resuscitation was called into question by radioisotope studies conducted by Baxter and Shires  and PruiThere was initially a lot of interest in artificial colloids (hydroxyethyl starch (HES) and dextrans) as possible colloids to use as adjuncts to burn resuscitation in order to limit the amount of crystalloid that is given and prevent over-resuscitation and its complications. In particular, HES has been studied as an adjunct to fluid resuscitation, although none of these studies have been done in a pediatric burn population. A randomized study of 26 patients was done comparing a pure crystalloid resuscitation to one that substituted one third of the crystalloid volume with 6% HES . They foThe large-scale studies needed to determine the efficacy and potential pitfalls of using albumin in children have also not been conducted at this time, but there are a few smaller studies that suggest that albumin might be beneficial in pediatric burn patients. Faraklas et al.  retrospep < 0.001) and had twice the mortality rate  as their counterparts who received standard crystalloid resuscitation [Hypertonic saline has been suggested as a burn resuscitation fluid because it helps to correct the extracellular sodium deficit which is an important component of burn shock . Hyperoscitation . Despitecitation . While hAs has already been discussed, the treatment of major burn injury includes the administration of crystalloid solutions to correct hypovolemia and to restore peripheral perfusion. This post-burn volume replacement increases oxygen delivery to previously ischemic tissue, leading to ischemia-related tissue injury and the production of oxygen free radicals. Due to its antioxidant properties, there has been a significant amount of interest in the use of high-dose vitamin C in burn resuscitation to ameliorate the effects of hypovolemic and ischemic tissue damage. High-dose vitamin C is thought to protect against membrane lipid peroxidation to limit capillary leak and is a potent oxygen-derived free radical scavenger . MatsudaThere have been two prospective human trials evaluating the use of high-dose vitamin C in burn resuscitation of adults. Mann et al.  conducteWhile the initial data on high-dose vitamin C infusions seems positive in its ability to reduce fluid resuscitation volumes and subsequent edema, there are a number of concerns associated with its use that have become apparent. One concern is the osmotic diuresis that occurs due to high-dose vitamin C. The fear is that patients may become dehydrated if fluids are titrated down too aggressively in light of this diuresis elevating UO values. There is some data to support this concern. When treated with high-dose vitamin C, there is an increase in UO despite a significant decrease in fluid volume , 51. AddIn burn resuscitation, it is important to remember that one is always dealing with a shock state and are often walking the line between class II and class III shock. The practitioner’s level of discomfort with a patient in the shock state and our desire to eliminate it as quickly as possible often leads to over-resuscitation and its associated complications. In an effort to deal with practitioner discomfort and standardize resuscitation as much as possible, computerized systems have been developed. Closed-loop resuscitation is a fully automated system that leads to continuous adjustment of the fluid infusion rate based on a computer-controlled algorithm. The device adjusts the infusion rate to achieve a defined physiological endpoint . A study by Hoskins et al.  comparedMost physicians are not comfortable with a completely closed-loop system. This led to the development of the Computerized Decision Support System (CDSS) . This syResuscitation of burned children has improved markedly over the years. Adequate fluid resuscitation is essential to optimizing the survival of burned children. Although multiple regimens and fluids are available for resuscitation, alteration of fluid infusion rate guided by clinical endpoints is the mainstay of therapy. In the future, closed-loop resuscitation methods may improve pediatric burn outcomes.


Intra-tumour heterogeneity is a common molecular phenomenon in metastatic clear cell renal carcinoma (mRCC), representing the genetic complexity of a tumour with multiple metastatic sites. The present commentary discusses the observed phenomena of phenotypic intra-tumour heterogeneity in mRCC patients treated with the tyrosine kinase inhibitors sunitinib or pazopanib. Here, drug response can be different on the level of each evaluated metastasis in the individual patient. This questions the currently used radiologic staging systems of RECIST criteria and demands for a modification of radiologic response assessment with the consequence of a patient-tailored therapy in the clinical setting.http://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-016-0729-9.Please see related article:  Renal cell carcinoma (RCC) is a multifaceted tumour. The same histopathological subtype, stage and grade in clear cell RCC demonstrates a different tumour behaviour among patients, called inter-tumour heterogeneity (ITH) . ITH is Considerable progress has been made in the treatment of metastatic RCC (mRCC), with an improvement of overall survival following the implementation of anti-angiogenic tyrosine kinase inhibitors (TKIs) since 2006 . Completn = 27 patients with multiple metastases) each metastasis was evaluated based on the principles of RECIST 1.1 to define responding, stable or progressing lesions. A heterogeneous drug response was defined as the deviation of response patterns within one patient, while a homogenous response was defined as all lesions falling within the same response category. Heterogeneous response was detectable in 56% (15/27) of patients and homogenous response in 44%. There was no difference in heterogeneous response in patients who had a suboptimal dosing through dose reductions or those that underwent nephrectomy. Reason for progressions was mainly the appearance of new lesions (67%), while the progression of existing lesions was a rare event (11%); 22% of patients exhibited both.In a recent article, Crusz et al.  hypothesIn clinical practice, the decision to switch or to continue a given systemic therapy is a common challenge, especially in the presence of heterogeneous progression and response patterns. Thus, the identification of cancer types with a respective heterogeneous response pattern is likely to influence clinical decision-making and, therefore, clinical outcome. As shown, a clinical ITH was observed for mRCC upon sunitinib or pazopanib treatment . The occStudies with monoclonal antibodies or cytokines have shown that an increase in total tumour burden, for example, by oedema or inflammation, both of which are defined as progressive disease by WHO and RECIST criteria, is later followed by sustained tumour regression seen as CR, PR and SD. The development of novel criteria, designated as immune-related response criteria, was based on data from ipilimumab clinical trials in melanoma . These cThe aim of targeted therapy is to inhibit the critical proliferation or survival pathways on which the tumour depends. This VEGF-based strategy has significantly improved patient outcomes in mRCC within the last years. The underlying tumour biology demonstrates that the majority of somatic gene mutations are not ubiquitously present within a tumour, but is rather characterised by the existence of subclones derived from a common clonal progenitor, with variations between different regions of the same tumour and metastases . As a cl


Hepatocellular carcinoma (HCC) has one of the poorest survival rates among cancers. Using multi-regional sampling of nine resected HCC with different aetiologies, here we construct phylogenetic relationships of these sectors, showing diverse levels of genetic sharing, spanning early to late diversification. Unlike the variegated pattern found in colorectal cancers, a large proportion of HCC display a clear isolation-by-distance pattern where spatially closer sectors are genetically more similar. Two resected intra-hepatic metastases showed genetic divergence occurring before and after primary tumour diversification, respectively. Metastatic tumours had much higher variability than their primary tumours, suggesting that intra-hepatic metastasis is accompanied by rapid diversification at the distant location. The presence of co-existing mutations offers the possibility of drug repositioning for HCC treatment. Taken together, these insights into intra-tumour heterogeneity allow for a comprehensive understanding of the evolutionary trajectories of HCC and suggest novel avenues for personalized therapy. Hepatocellular carcinoma has one of the poorest survival rates amongst cancers. Here, the authors highlight the intra-tumour heterogeneity of this disease, finding that spatially closer tumour sectors are genetically more similar and that intra-hepatic metastasis is accompanied by rapid genetic diversification. Liver cancer is the fifth most common cancer globally but the second most important cause of cancer deathsUnderstanding the origin and development of intra-tumour heterogeneity (ITH) has the potential to yield important insights for therapeutic target selection and drug development910In this study, using multi-regional sampling of nine resected HCC with different aetiologies, we find that the phylogenetic relationships of tumour sectors from the same patients have diverse levels of genetic sharing, spanning early to late diversification. Distinct to what is found in colorectal cancers where genetic variegation accompanies the development from adenoma to carcinomaWe studied early pathological stages . Through experimental validation, we found that the true positive rate of the somatic variants was high 96%, . In all 15CTNNB1 and TP53  and were shared among all tumour sectors , we identified 65 significantly mutated driver genes for HCCand TP53 . Of thesand TP53 . TP53 wa alleles . Combini sectors . This suCCND1 were found to be amplified in patient 6 and patient 9  across the genome for all the samples 20. The matient 9 . In geneUsing somatic variants called from individual sectors, we constructed the evolutionary relationships for primary tumour sectors (Methods). Mutations that were common to all sectors appeared on the trunk of the tree and mutations that were private to subsets of the samples appeared on branches. Among all cases, the level of differences between tumour sectors varied considerably . To compAcross nine cases, the phylogenetic trees took a variety of forms. For example, patients 1, 4 and 6 showed typical star genealogies with very short internal branches, indicating rapid population expansion in the history of the tumourAcross all phylogenetic trees with sufficient divergence , sectors from one end of the tumour consistently grouped together, with separation of the two clades setting apart from the tumour centre . This suThe phylogenetic analysis was based on the presence and absence of somatic changes across tumour sectors, which only utilized a portion of the available genetic information. Next, we quantified the genetic relationships among populations by measuring allele frequency differences among samples using a classical metric that was derived from population genetics  tend to result in poorer clinical prognosis across several cancer types31EGFR and KIT) can be targeted with existing therapeutics (Methods and EGFR L858R for lung cancer), several of them were on important functional domains  seen in the sectors from metastatic tumours suggested a single point of origin for the metastases . InteresDuring metastases, when a small group of cells (clone) leaves the parental population to form a newly established colony, two major expectations known as the founder effect363738The divergence between the metastatic and primary tumour, represented by the migratory branch , was quiTERT gene integration and one non-coding integration hotspot on chromosome 10 (far from any known gene) were foundTERT gene occurred early in the history of tumour progression and was also found in all tumour samples for patients 1 and 8 (including the metastatic tumour for patient 1). Together, these results suggested that viral integration can be an important early driver for HCCAnother important driver of HCC development is the viral integration. Using a computational procedure to search for paired-end reads where one end maps to the human genome and the other end maps to the HBV genome, we identified viral integrations in four HBV-positive patients from our cohort41In colorectal cancers, genetic lineages often segregate in a variegated pattern where cell populations from the left and right side of the tumour interleave in the phylogenetic relationshipOur study offers the first insights into interesting dynamics in intra-hepatic HCC metastasis. The comparisons between metastatic and primary tumour have so far revealed high genetic divergence across several cancer types36373811The absence of further adaptation at distant metastasis could be a feature specific to the liver, where the organ microenvironment appears to be more homogeneous across the liver. Mechanisms allowing local migration in the primary tumour may overlap with pathways for intra-hepatic migrationThe clear spatial segregation of genetic lineages in HCC is analogous to the annual ring in woody plants and they offer a useful approach for understanding clonal evolution in the context of cancer. As ancestral lineages tend to segregate inside the tumour, spatial sampling will allow the reconstruction of more realistic and clinically relevant pre-clinical models  that reconstitute various levels of genetic transformation . Such a history of tumour evolution may, for instance, be constructed to investigate functional or adaptive differences between ancestral and derived clones . Notwithstanding, spatially separated biopsies may be necessary for sampling even greater genetic variability within the tumour.Owing to limited sample size in patient number, the correlation between clinical phenotypes  and the ITH profiles are still provisional. In addition, the number of intra-hepatic metastases presented here is still small. Further studies with a larger cohort will be needed to further confirm and extend the conclusions of this study. The natural history of tumour progression is often described as belonging to either one of two classical models: either a linear evolution model with sequentially acquired driver mutations (late diversification)All patients had surgical resection performed by the joint Hepato-Pancreato-Biliary Surgery service of the National Cancer Center Singapore and the Singapore General Hospital. Pre-operative diagnoses and staging were performed by multi-phasic radiological scans (computed tomography or magnetic resonance imaging) and diagnoses were confirmed histologically after surgery. All nine HCCs were early-stage tumours (AJCC stages 1 and 2). The study was approved by the Central Institution Review Board of SingHealth of which both National Cancer Center Singapore and SGH were constituent members (CIRB 2012/669/B) and each patient gave informed written consent. Entire resected surgical specimens were retrieved immediately after removal from the surgical field in the operating room and transported on ice in a temperature controlled cooler container to a pathologist at the SingHealth Tissue Repository situated in an adjacent building. Each specimen was then measured and photographed. Normal non-tumour liver tissue at least 2 cm away from the tumour was harvested and labelled as normal liver (control). A single slice was then made in the tumour through the capsule and the cut section of the liver tumour photographed. The tumour was then inspected for necrosis, fibrosis, haemorrhage and cystic changes. Multiple pieces along the long axis of the tumour were then harvested, often at least 1 cm apart and cut into pieces measuring 1 × 0.5 × 0.5 cm. Circumstances such as cystic change, haemorrhage or necrosis would reduce the number of tumour pieces harvested. The numbers of sectors harvested depend on the size of the tumours.All harvested tissues were snap frozen using liquid nitrogen and stored afterwards in a −80 °C freezer. Matching pieces were collected for routine processing and histological examination. Real-time quantitative PCR (Roche COBAS Ampliprep/COBAS TaqMan HBV Test version 2.0) was used to quantify the viral load in the HBV positive patients.DNA was extracted from the normal and tumour tissues independently using Qiagen DNeasy Blood and Tissue kit. After quality check with electrophoresis, DNA were sonicated to shorter fragments using the Covaris system. Following quality check with Agilent 2100 Bioanalyzer, DNA fragments were end-repaired, ligated with sequencing adapters, amplified before WGS or WES by the sequencing platform at the Genome Institute of Singapore. For WES, NimbleGen SeqCap EZ Human Exome Library v3.0 was used for exome capture before sequencing. Illumina sequencing platform was used for both WGS and WES (Earlier samples were done using WES and subsequent samples were processed using WGS).http://broadinstitute.github.io/picard/), sequence data then follow through base quality recalibration and realignment using the Genome Analysis Tool Kitq<0.1 from et al.https://github.com/cancerit/ascatNgs). To match the WES data to the WGS in comparing the ITH profiles  with the Burrows–Wheeler Alignerhttp://www.tumorportal.org/ . For mutation signatures in patient 9, we first compiled the mutation pattern across all sites and then projected the mutation pattern as a combination of 30 known signatures using a linear model similar to previous studies (http://cancer.sanger.ac.uk/cosmic/signatures)To test whether HCC driver mutations tend to be of higher frequency than the background mutations, we used a N, we will randomly sample n (n>=1 and n<N) sectors and count the total number of detected somatic mutation for that subsample. For each n, we subsampled multiple times and computed the mean number of detectable somatic changes at that n (denoted as Xn). Using the mean value of detected somatic change at n=1  as the baseline, we calculated the fold increase in detectable somatic variant at each n . To rank the trajectories observed in x=2, that is, randomly sample two sectors) was ranked across cases. For patient 2 and 3 (WES), we used the downsampled data sets to calculate these values .The upward trajectories were drawn by randomly sampling a subset of sectors from the total collection of sectors for each patient. For example, if the number of sectors for a tumour is Using the presence and absence of individual somatic changes, we calculated the hamming distances between individual samples from each patient. With a distance matrix between all sectors, neighbour-joining algorithm implemented in PAUPUsing the allele frequencies extracted from the sequencing output  were also attempted  found in two previous studies41http://www.fda.gov/drugs/scienceresearch/researchareas/pharmacogenetics/ucm083378.htm) and from http://www.genome.jp/kegg/drug/br08341.html.A list of drugs and their associated targets were curated from the FDA website (The raw data (WGS and WES) have been deposited in European Nucleotide Archive under accession code EGAS00001001603. The authors declare that all data supporting the findings of this study are available within the article and its How to cite this article: Zhai, W. et al. The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma. Nat. Commun.8, 14565 doi: 10.1038/ncomms14565 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary ReferencesExperimental validation of the somatic mutationsSignificantly mutated driver genesThe mutation list for the nine patients


Dear editor,As corresponding author I ask for retraction of our article Finelli et al. (2016[Carmine Finelli, MD PhD


The aim of this study was to evaluate the concordance between the metabolic tumour volume (MTV) measured on FDG PET/CT and the anatomical tumour volume (ATV) measured on T2-weighted magnetic resonance imaging (T2W-MRI); and compared with the functional tumour volume (FTV) measured on diffusion-weighted MRI (DW-MRI) in cervical cancer, taking the T2W-ATV as gold standard.Consecutive newly diagnosed cervical cancer patients who underwent FDG PET/CT and DW-MRI were retrospectively reviewed from June 2013 to July 2017.20–80, with intervals of 5%.Volumes of interest was inserted to the focal hypermetabolic activity corresponding to the cervical tumour on FDG PET/CT with automated tumour contouring and manual adjustment, based on SUV 20%–80% thresholds of the maximum SUV (SUVmax) to define the MTVTumour areas were manually delineated on T2W-MRI and multiplied by slice thickness to calculate the ATV.. Diffusion restricted areas was extracted from b0 and ADC map using K-means clustering to determine the FTV(semi-automated).FTV were derived by manually delineating tumour area on ADC map, multiplied by the slice thickness to determine the FTVThe ATVs, FTVs and the MTVs at different thresholds were compared using the mean and correlated using Pearson’s product-moment correlation.30 . This was less than the differences between ATV and FTV(semi-automated)  and FTV . The correlation of MTV30 with ATV was excellent  and better than that of the FTVs.Twenty-nine patients were evaluated (median age 52 years). Paired difference of mean between ATV and MTV was the closest and not statistically significant at MTV30 was the only parameter investigated with no statistically significant difference with ATV, had the least absolute difference from ATV, and showed excellent positive correlation with ATV, suggesting its superiority as a functional imaging modality when compared with DW-MRI and supporting its use as a surrogate for ATV for radiotherapy tumour contouring.Our study demonstrated that MTV Precise determination of cervical tumour boundary is important in radiotherapy to deliver the highest possible radiation dose to cancerous tissues while minimizing that to surrounding healthy tissues.Given its superior soft tissue contrast, MRI is the modality of choice for the anatomical delineation of tumour outline and local tumour extent, especially in determining whether parametrial invasion is present to differentiate early from advanced stage disease. Despite excellent spatial resolution, delineation of tumour extent can be limited using conventional T2-weighted (T2W) sequences in certain scenarios, e.g. isointense tumours and diffusely infiltrative lesions, in assessing response of tumours to therapy and, in particular, in differentiating residual or recurrent disease from post-treatment fibrosis due to the overlap of morphological appearances .18F–fluoro-deoxyglucose positron emission tomography integrated with computed tomography (FDG PET/CT) have been demonstrated to provide information for more precise definition of radiation target  factor of 2 Table . Image aTumour areas were manually delineated on T2W images in sagittal and oblique axial planes and multiplied by the slice thickness to calculate the sagittal and oblique axial tumour volumes. Two reviewers, EL  and AL (5-year experience in MRI), separately placed the ROIs on the T2W images in the sagittal and oblique axial planes, respectively. The volumes were averaged between the two reviewers to determine the ATV.b represents the mean signal intensity with the diffusion gradient, b, S0 is the mean signal intensity when b = 0 s/mm2. VOIs were manually drawn by two reviewers, EL and AL, for each lesion. The first set of VOIs were strict manual delineations of the tumour by both reviewers and excluded the surrounding normal tissue based on the hypointense signal of the tumour on the ADC map with cross reference to the axial T2W images. FTV by the two reviewers was then calculated using these VOIs multiplied by slice thickness. The volumes were averaged between the two reviewers to determine the FTV. The second set of VOIs was drawn by the same two reviewers, EL and AL, to include all of the tumour and did not require exclusion of surrounding normal tissue. Volumetric k-means clustering was then used to automatically separate voxels in the tumour volume into three groups based on S0 and ADC values. These groups were defined as: solid tumour mass with high cellularity having intermediate ADC and intermediate S0 intensities; normal tissue with low cellularity or cystic tissues having high ADC [0 intensities; fat and fibrotic tissues having low ADC low S0 intensities. A study by Gong et al. [0 images and ADC, is a promising method for reliable delineation of heterogeneous tumours in patients with metastatic gastrointestinal stromal tumours. FTV(semi-automated) was calculated by discarding the fat and fibrotic cluster and the normal tissue cluster, leaving the solid tumour mass cluster. Parametric map generation and semi-automatic functional volume segmentation were performed using in-house programs using MATLAB  .Averaged DW signal was used to generate the ADC maps using the Levenberg-Marquardt fitting algorithm under the mono-exponential model described by the function:high ADC , 24 and g et al.  has showp-value < 0.05 was considered statistically significant.The ATVs measured on T2W images, FTVs on DW-MRI and the MTVs at different thresholds on FDG PET/CT were compared. The ATVs, FTVs and MTVs were correlated using Pearson’s product-moment correlation. R version 3.4.1  was used for statistical analysis. A two-tailed A total of 29 patients were evaluated with median age of 52 years (range 27–76 years). Further clinicopathological characteristics were tabulated in Table 2/s. The mean ATVs measured in sagittal and oblique axial planes are 51.7 and 59.3 cm3 respectively.Mean SUVmax of the cervical tumours was 9.2, range 3.3–16.7. Mean ADC of the cervical tumours was 0.934 +/− 0.120 mm30, mathematically represented by mean ATV – mean MTV30, was −2.9 cm3, −5.2%, p = 0.301. This difference was not statistically significant and was the closest to ATV compared with all other FTVs and MTVs measured at other SUVmax thresholds, including the differences between ATV and FTV(semi-automated) (mean ATV – mean FTV(semi-automated) = 25.1 cm3, 45.1%, p < 0.001) and between ATV and FTV  = 11.2 cm3, 20.1%, p = 0.001). The means of the ATV, FTV(semi-automated), FTV and MTV20 to MTV80  and the differences of their means with ATV are shown in Table The paired difference of mean between ATV and MTV20–50 with ATV were excellent , and those of MTV30–40 were better than that with FTV(semi-automated) and FTV. There was a gradual decline in correlation of MTVs with ATV as the percentage threshold of SUVmax increased (Table The correlations of MTV(semi-automated) and FTV, the MTV30 had the least absolute difference from ATV and was the only parameter investigated which did not show a statistically significant difference from ATV. In addition, MTV30 showed excellent positive correlation with ATV.Our study demonstrated that among all metabolic threshold levels and FTVMRI is indispensable in the local disease assessment of cervical cancer. The ability of combined functional volume assessment and local disease extent using MRI alone could present as a promising imaging algorithm for patients with cervical cancer. However, the evidence to support this is limited in the current literature and most studies have used the manual segmentation method based on DW-MRI images , 27.0 and ADC is a promising method for reliable delineation of heterogeneous tumours in patients with metastatic gastrointestinal stromal tumours [b-value and acquisition method for DW-MRI images, and sensitivity to signal-to-noise ratio [The choice of imaging modality used in tumour contouring or segmentation technique can result in varying derived tumour volume , 28, 29. tumours . The rel tumours  and regi tumours  methods se ratio .FDG PET/CT has the advantage of identifying the metabolic activity and providing information on tumour biology. It is increasingly recognized as a useful tool for directing RT planning during intensity-modulated radiation therapy, volumetric-modulated arc treatment  and imagVarious tumour segmentation techniques using FDG PET/CT exist: manual contouring, which consists of visual assessment for determining tumour outline; thresholding, which uses a minimum SUV value to identify target; and gradient edge detection, in which tumour delineation is based on the changes in signal across a given area . As SUV Volume concordance between FDG PET/CT, and T2W and DW-MRI imaging in cervical cancer has been previously observed , 27, 37,30, concordant with previous literature [30 did. Moreover, contouring based on FDG PET/CT can be performed in a semi-automated fashion and this feature is readily available on standard workstation, which is easy to use, providing the merit of reducing the time required for processing, and potentially also improving inter-observer agreement, as shown previously by studies on tumour delineation for rectal and lung cancers [In our study, the mean of differences between ATV and MTV was the smallest with MTVterature . Althoug cancers –41.30, having the least absolute difference from ATV, being the only parameter investigated with no statistically significant difference from ATV, and having an excellent positive correlation with ATV supported its use as a surrogate for ATV for radiotherapy tumour contouring and dose escalation; with the benefit of having metabolic information available for characterizing the biological features of the tumour and optimizing the use of individualized, conformal and biologically effective radiation therapy.Furthermore, MTV30 being the best correlate to ATV.In conclusion, MTV delineation on FDG PET/CT appears promising and superior as a functional imaging modality when compared with DW-MRI in tumour contouring with MTV