The long-term aim of developing laser based particle acceleration towards clinical application requires not only substantial technological progress, but also the radiobiological characterization of the resulting ultra-short and ultra-intensive particle beam pulses. After comprehensive cell studies a mouse ear tumour model was established allowing for the penetration of low energy protons (~20 MeV) currently available at laser driven accelerators. The model was successfully applied for a first tumour growth delay study with laser driven electrons, whereby the need of improvements crop out.To optimise the mouse ear tumour model with respect to a stable, high take rate and a lower number of secondary tumours, Matrigel was introduced for tumour cell injection. Different concentrations of two human tumour cell lines  and Matrigel were evaluated for stable tumour growth and fulfilling the allocation criteria for irradiation experiments. The originally applied cell injection with PBS was performed for comparison and to assess the long-term stability of the model. Finally, the optimum suspension of cells and Matrigel was applied to determine applicable dose ranges for tumour growth delay studies by 200 kV X-ray irradiation.3. As disclosed by immunofluorescence analysis these small tumours already interact with the surrounding tissue and activate endothelial cells to form vessels. The formation of delimited, solid tumours at irradiation size was shown by standard H&E staining and a realistic dose range for inducing tumour growth delay without permanent tumour control was obtained for both tumour entities.Both human tumour models showed a high take rate and exponential tumour growth starting at a volume of ~10 mmThe already established mouse ear tumour model was successfully upgraded now providing stable tumour growth with high take rate for two tumour entities  that are of interest for future irradiation experiments at experimental accelerators. Current state-of-the-art treatment of cancer patients most often includes radiotherapy with high energy photons or electrons that are delivered by compact clinical electron linear accelerators. Besides, a small but increasing number of patients are treated with ions (protons and heavier ions)  that are10 Gy/min) particle beam pulses [As one potential alternative the acceleration of ions on micrometre scale by high-intensity laser systems was investigated during the last decade . The necm pulses ,3. Up tom pulses –8 preparThe recently established mouse ear tumour model  providesIn the present study, the mouse ear tumour model should be improved by applying Matrigel (MG) for tumour cell injection. MG supports and stabilises the tumour take without changing the histological appearance  and shouad libitum. To further suppress their immune response, mice were whole-body irradiated with 4 Gy  three days before cell injection. Anaesthesia was generally performed by 10 mg/kg Xylazin  and 100 mg/kg Ketamin Experiments were performed using 7-to-14-week-old female and male NMRI (nu/nu) mice continuously purchased from the breeding facility at Oncoray—National Center for Radiation Research in Oncology in Dresden. The animal facility and the experiments were approved according to the German animal welfare regulations and to the local ethics committee . Animal housing provided 12 h light– 12 h dark cycle, constant temperature of 26–27°C and relative humidity of 45–60% with maximal 10 mice per cage of Euro Standard Type III. Mice were fed with commercial laboratory animal diet for nude mice and water 2 in humidified atmosphere.Tumour growth studies were carried out with the undifferentiated human squamous cell carcinoma (HNSCC) FaDu ,13 and t6 FaDu cells resolved in PBS [3–1*106 cells in pure (9.7 mg/ml) or diluted MG . The growth of LN229 tumours was surveyed for suspensions of 1*102–1*105 cells in pure MG and of 1*105 cells in PBS again scheduling 18 mice per group, 90 mice altogether. Tumour growth was continuously checked by calliper measurements and corresponding tumour volumes were calculated by the formula of a rotational ellipsoid π/6 x a x b2, where a is the longest and b the shorter tumour axis perpendicular to a. Tumour origin was routinely checked by microsatellite analysis and standard H&E staining.Subcutaneous tumour growing was induced by injecting 25–30 μl of the different tumour cell suspensions in the middle of the right ears of anaesthetised mice ,15 by usd in PBS  or of 1*The 200 kV X-ray tube including filters, dosimetry and setup for mouse irradiation are described in detail by Brüchner et al. . BrieflyThe dose homogeneity within the irradiation field and the dose rate of 0.975 Gy/min at tumour position were determined with GafChromic EBT-2 and EBT-3 films  calibrated for 200 kV X-rays against two Farmer ionization chambers  that were readout with a Unidos electrometer (PTW).a axes) and fulfil the allocation criteria (see 3.4) the animals were randomly allocated to the untreated control and the different treatment groups . The dose values were specified in accordance to preceding experiments with FaDu tumours on mouse ear (unpublished); whereas for LN229 similar radiosensitivity and comparable dose values were expected from tumour control studies. Mice of the “0 Gy” groups were anaesthetised and positioned in mouse setup boxes, like those of the other dose groups, but sham irradiated.The optimum concentrations of cells and MG (see 3.1) determined in the first part of the experiments were used for injection in those animals (138 per entity) scheduled for irradiation experiments and histological studies. Again, the animals were continuously checked for tumour growth firstly by eye and after reaching a size of around 2 mm by calliper measurement. When the tumours reached a diameter of 2.5–3.5 mm , the perfusion marker Hoechst 33342 (Sigma Aldrich) and CD31 as marker for the vascular endothelium . The staining and scanning processes of tumour sections have been described before , 17; posTumour diameters were measured two to three times a week during the follow ups of 120 days for FaDu and of 180 days for LN229, respectively. The animals were sacrificed when the tumour reached the diameter of 7–8 mm, at the end of follow up or when they appeared to suffer. To assess the different injection media and cell concentrations, the tumour take rates, potential allocation rates and allocation times were compared. Tumour volume doubling times (VDT) were calculated in the range of exponential tumour growth.V5) was calculated by subtracting the average time the untreated control tumours need to achieve the fivefold relative tumour volume from the average time required by irradiated tumours of the particular dose groups. Animals showing permanent local tumour control during follow up or developing nearby secondary tumours were excluded from analysis.For the 200 kV X-ray irradiation experiment relative tumour volumes were calculated with respect to the tumour volume at allocation day and the times required to achieve a certain relative volume were averaged for all animals of an experimental group. The radiation induced tumour growth delay  considering values of p < 0.05 as statistically significant. Graphical representation of take rates and tumour growth curves was realised by the software OriginPro 8G .6 cells in undiluted MG the high take rate of almost ~100% was stable over three staggered injections, which is a noticeable improvement compared to the variable take rate of 60–90% found before [Starting with the HNSCC FaDu, the tumour growth after injection of different suspensions of MG and tumour cells in the right mouse ear was evaluated to find the optimum mixture of cells and MG that result in a high take rate and stable tumour growth. The results of this optimization process are resumed in d before . Referri3 Tumour volume of ~10 mmReddish/red tumour indicating sufficient perfusion and a vital tumourPosition in the middle of mouse ear and uniform shape, ideally round or ellipticOne single tumour in the target area, no doublets or second tumour on or close to the earApplying the above mentioned criteria, the different suspensions for tumour cell injection were assessed with respect to the subsequent (see 3.5.), but also upcoming irradiation experiments . As alre5 cells in undiluted MG was specified as optimum for subsequent radiobiological studies, since for HNSCC FaDu and for LN229 glioblastoma a high and stable take rate and an acceptable allocation time linked with a good allocation rate could be achieved. Of course, the take rate obtained after injection of 106 FaDu tumour cells was also very high, but the very fast tumour growing of this group demand for prompt treatment and argues against its application at experimental accelerators where a delay cannot be excluded. Beyond take rate, MG was also introduced to reduce outflow of cells and with it the growth of secondary tumours. However, since the tumours were not treated during the optimisation process, the follow up was rather short and related to this a low rate of secondary tumour induction was seen. To be more realistic, the secondary tumour incidence for the two tumour models was therefore assessed during the irradiation campaign.To sum up, the injection of 10The application of any tumour model for radiobiological tumour growth delay studies require the definition of a reasonable dose range that result in a significant growth delay compared to untreated tumours, but not in permanent tumour control. To study the dose dependent radiation response FaDu and LN229 tumour cells were each inoculated in 108 animals, from which 95–100% developed a tumour confirming the optimised injection parameters and the stability of the model. Accordingly, allocation times of 14.5 ± 0.8 days and of 23.4 ± 1.0 days, similar to that measured before, were found for FaDu and LN229, respectively. The corresponding allocation rates vary between 74% for FaDu and 89% for LN229 being noticeable higher than in the optimisation experiments  and the V5 in the present study was however not affected by this phenomenon. Taking all facts together, drop-outs occurred at all steps from inoculation to analysis, esp. for FaDu, but due to the high take rate the total number of animals to be scheduled for growth delay experiments could still be reduced.Despite of these improvements one drawback of the FaDu mouse ear tumour model, namely the high rate of secondary tumours, was not abolished after optimisation. Like in the previous study of Oppelt et al. , where PV5 of 6.8 ± 2.3 days and of 16.5 ± 2.3 days were measured after irradiation with 3.8 Gy and 7.9 Gy (V5 is at the limit of significance (p = 0.056), whereas the GDV3 and GDV7 are significantly different. The GDV5 of FaDu and LN229 are summarized in Comparing the tumour growth curves of the untreated controls and the sham irradiated groups, a significant difference was neither found for FaDu  nor for d 7.9 Gy , respectd 7.9 Gy  and a lod 7.9 Gy . The appThe present study reveals not only the optimised injection parameters that result in a high take rate and in stable tumour growth, but also some aspects that should be taken into account by using the model for future experiments. The first aspect is the injection of tumour cells, which should be done by a trained person to assure a low failure rate, i.e. by accidental ear piercing, and there with a high allocation rate.The second aspect is the definition of doses for irradiation experiments or drug testing, which should be considered carefully, since for FaDu the risk of secondary tumours and for LN229 the tendency of tumour scabbing will increase for longer follow up times, respectively. Moreover, the application of the mouse ear tumour model for tumour growth delay studies is limited to maximum doses of 8 Gy for FaDu and ~14 Gy for LN229. Above these values the determination of tumour control instead of growth delay should be applied for radiobiological characterisation.5 cells for tumour cell injection. The increased and now stable take rate of > 95% allows the application of a lower number of animals by retaining the same statistical significance. A constant allocation time and high allocation rate enable the scheduling of irradiation campaigns at experimental accelerators with limited beam time and reduced beam reliability (i.e. increased beam loss). Moreover, it could be shown that the FaDu tumour model was stable over more than one year, which is one essential prerequisite for long-term irradiation experiments and experiment replication. For the LN229 tumour model the stability of tumour growth parameters was already confirmed over months and will be further proven in upcoming experiments.The mouse ear tumour model was considerably improved for FaDu by the application of MG instead of PBS and by an optimised, reduced number of 10Beside these radiation independent properties of the model, a reasonable dose range for future radiobiological growth delay studies was determined for both the HNSCC FaDu and the GBM LN229. Taking into account the different relative biological effectiveness between 200 kV X-rays used for the dose definition and any other radiation quality, the model is now prepared, e.g. for the study at laser driven proton beams, but also for other applications with low penetrating radiations like the investigation of micro beam treatment . The maiS1 TableTumour volumes (Vol) and corresponding sem (standard error of the mean) in dependence on time (days) after inoculation of HNSCC FaDu tumour cells in the right mouse ear. For injection the tumor cells were suspended either in pure (9.7 mg/ml) or in diluted  Matrigel. The number of animals per group is given in brackets.(DOCX)Click here for additional data file.S2 TableGroup mean tumour volumes (Vol) and standard error of the means (sem) in dependence on time (days) after injection of LN229 tumour cells in the right mouse ear. The tumor cells were suspended in pure (9.7 mg/ml) Matrigel and the number of animals per group is given in brackets.(DOCX)Click here for additional data file.S3 Table6 cells in PBS and (b) of 1*105 cells in Matrigel for the different temporally separated experiment campaigns. The number of animals is given in brackets.Long-term stability of the FaDu mouse ear model shown by the averaged tumour growth curves measured as respective tumour volume increase (± sem) after inoculation (a) of 1*10(DOCX)Click here for additional data file.S4 Table5 cells in MG for four temporally different cohorts. The corresponding numbers of animals per cohort are given brackets.Average tumour volumes (± SEM) measured for LN229 tumours after inoculation of 1*10(DOCX)Click here for additional data file.S5 TableDose dependent tumour growth data for the mouse ear tumour models of HNSCC FaDu (a) and the glioblastoma LN229 (b). The average times (± SEM) to achieve a certain relative volume increase are given as mean values over all animals in the group; the number of animals per group are given in brackets.(DOCX)Click here for additional data file.


Survival rates in children with extraocular tumour extension can be improved with a combination of chemotherapy, surgery, and radiotherapy.In high-income countries, the survival rate of children with retinoblastoma has improved over the years: from 5% to >95%. This improvement is a result of early diagnosis and intervention by specialist retinoblastoma teams. However, in low- and middle-income countries, the survival rate continues to remain low, with 39% mortality rates in Asia and 70% in Africa.While leucocoria (a white reflex) is a common presenting sign of retinoblastoma in most high-income countries, an enlarged eye or proptosis with extraocular tumour extension remains the most common presenting sign in low- and middle-income countries.Perform a general examination and examine both eyes thoroughly, preferably under anaesthesia. Since bone marrow and cerebrospinal fluid are two potential sites for tumour spread, bone marrow aspiration and cerebrospinal fluid analysis are recommended before starting treatment. If there is enlargement of regional lymph nodes, a fine needle aspiration biopsy can be undertaken to determine if the tumour has spread to the nodes. A magnetic resonance imaging or computed tomography of the orbit should be done to ascertain the extent of the disease.In this article, we describe the treatment protocol we have used in our clinic since the year 2000. Treatment includes several cycles of high-dose systemic chemotherapy, followed by surgery (enucleation or exenteration), and then external beam radiotherapy to the orbit. Whenever there is an overt extraocular tumour extension, including gross optic nerve extension or extrascleral tumour extension, high-dose systemic chemotherapy should be given every 3 weeks as the primary treatment. Primary enucleation or orbital exenteration should be avoided until after the tumour shrinks so that surgery is more successful without leaving behind tumour residue. Various combinations of chemotherapy have been reported in the literature. We currently use the combination of vincristine, etoposide, and carboplatin, and have found it to be effective .High-dose systemic chemotherapy is given until the extraocular component of the tumour regresses. An average of 6 cycles of high-dose systemic chemotherapy results in complete regression of the extraocular tumour component in 95% cases, thus avoiding the need for orbital exenteration . Repeat In eyes with regressed extraocular tumour, secondary enucleation with placement of an implant is performed. In eyes with residual extraocular tumour despite a maximum of 9 cycles of high-dose systemic chemotherapy, secondary orbital exenteration is recommended.Six weeks after surgery, external beam radiation (45 to 50 Gy) is delivered to the orbit. The radiation field should include the regional lymph nodes if the patient had regional lymph node involvement at presentation.Post-radiation, high-dose systemic chemotherapy is continued to complete a total of 12 doses of chemotherapy. In cases with stage IV disease at presentation with central nervous system involvement, additional intrathecal chemotherapy is recommended.Using this treatment protocol, in our recent analysis of 20 patients with stage III disease who were compliant with treatment, 17 patients survived and were doing well at a median follow-up duration of 77 months.In summary, early diagnosis and appropriate treatment of retinoblastoma is crucial to improve the chances of life, globe, and vision salvage. In patients with delayed presentation including extraocular extension of retinoblastoma but without systemic or central nervous system metastases, multimodal treatment in specialist centres improves the chances of survival. The compliance to treatment plays a very important role in determining the likelihood of survival.


Grant 2014–37 (http://www.ec.js.edu.cn/) from Jiangsu Education Department was received by Affiliated Hospital of Stomatology, Nanjing Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The publisher apologizes for the errors.There are errors in the Funding section. The correct funding information is as follows: Grant 81370025 (


Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery.G12D/+LSL-Kras; R172H/+LSL-Trp53; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation.Gemcitabine metabolites were analysed in Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2′,2′-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5′-nucleotidases  were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%.Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine. Pancreatic ductal adenocarcinoma (PDAC) and liver metastases are characterised by pronounced tumour stroma consisting of activated cancer-associated fibroblasts (CAFs), and abundant extracellular matrix components.Depletion of stromal components by various pharmacological approaches has resulted in increased intratumoural gemcitabine levels suggesting that the tumour stroma may act as physical barrier for drug delivery.Depletion of CAFs has been shown to promote tumour growth and spread.Primary human and murine pancreatic tumours show an increased number of α-smooth muscle actin-positive fibroblasts compared with matched liver metastases. Murine primary tumours accumulate significantly more gemcitabine metabolites.In CAFs and pancreatic stellate cells (PSCs), significantly higher intracellular concentrations of gemcitabine metabolites were identified, in comparison to tumour cell lines derived from primary murine PDAC and metastases.Lower expression of key inactivating enzymes, such as hydrolytic cytosolic 5′-nucleotidases in CAFs and PSCs increase the intracellular concentration of gemcitabine when compared with cancer cells and may serve as targets for stromal reprogramming.CAFs and PSCs entrap active gemcitabine intracellularly and may thus limit the availability of the drug for cancer cells.Targeting the metabolic machinery of fibroblasts may enhance local availability of gemcitabine in the tumour without interfering with the tumour-restraining properties of CAFs.Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most aggressive solid tumours with increasing incidence worldwide.1119F NMR) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Both methods cannot be compared directly as 19F NMR is unable to distinguish all gemcitabine metabolites, and is less sensitive.Indeed, subsequent preclinical investigations introduced alternative strategies to successfully deplete stromal fibrosis and relieve vessel compression. For instance, degradation of hyaluronan and collagen, as well as reduced fibrosis following vitamin-D receptor activation in pancreatic stellate cells (PSCs) resulted in increased drug delivery and response to gemcitabine treatment in various genetically engineered mouse models (GEMMs).G12D/+; LSL-Trp53R172H/+; Pdx-1-CreLSLKras (KPC) tumours over time using LC-MS/MS as the most sensitive detection method for gemcitabine metabolites. Whereas serum concentrations of the gemcitabine prodrug 2′,2′-difluorodeoxycytidine (dFdC) peaked 1 hour after intraperitoneal injection, intratumoural accumulation of the activated gemcitabine metabolite 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) reached maximum levels only after 2 hours and coincided with the maximum induction of apoptotic cell death as evidenced by cleaved caspase-3 immunohistochemistry.28In order to overcome these limitations, we recently analysed the pharmacokinetic and pharmacodynamic profile of gemcitabine in Using this standardised protocol, we here revisit the pharmacokinetic profile of gemcitabine in murine PDAC in vitro and vivo, and dissect the stromal and the neoplastic compartment of primary tumours and matched liver metastases. Our findings offer an alternative explanation for the clinical failure of gemcitabine in stroma-rich PDAC.For details, see online supplementary material and methods.10.1136/gutjnl-2016-311954.supp1supplementary material and methodsPDAC tumour samples with matched liver metastases were obtained from formalin-fixed and paraffin-embedded (FFPE) tissue blocks collected for clinical purposes at Karolinska Institute. The PDAC diagnosis was confirmed by a staff pathologist at Karolinska Institute. Ethical approval was obtained from Karolinska Institutional Review Board (EPN D-No. 2014/2147-31/1). Tissue microarrays with PDAC (n=50) were assembled from representative FFPE archival tissue blocks that had been sampled from pancreatoduodenectomy specimens at Oslo University Hospital, Norway. Each tumour was represented by two 1.0 mm cores. Permission for the study was obtained from the Regional Committee for Medical and Health Research Ethics for Southern Norway (REK nr. S-05081).For pharmacokinetic studies, gemcitabine was administered at 100 mg/kg by intraperitoneal injections. Tissues were harvested 2 hours after gemcitabine administration.Fresh frozen tumour samples were processed and analysed using LC-MS/MS as previously described.Statistical analysis was carried out using GraphPad Prism V.6.05 (GraphPad Software). The Mann-Whitney non-parametric U test was used unless indicated otherwise, and results are presented as mean±SE; p<0.05 was considered to be statistically significant.To determine whether overall survival in KPC mice is associated with intratumoural concentrations of gemcitabine, in particular the active, cytotoxic metabolite, we re-analysed n=10 KPC bulk tumours from a previously published preclinical trialWe reasoned that either stromal composition or vascularisation of the different tumour tissues may account for the significantly different gemcitabine concentrations. To this end, we systematically assessed and compared the desmoplastic reaction of primary KPC tumours and matched liver metastases (n=8) by biochemical and immunohistochemical methods. Immunohistochemistry for α-smooth muscle actin (α-SMA), collagen and SPARC provided clear evidence that desmoplasia, in particular fibroblast density, was reduced in liver metastases compared with matched primary tumours A–C. WestTo validate our findings in human specimen, we analysed a small cohort of patients with pancreatic cancer (n=11) with matched primary tumours and liver metastases. Although both tissues showed a clear desmoplastic reaction with heterogeneous distribution throughout the lesions, α-SMA-positive area was markedly reduced in all liver metastases compared with matched primary tumours A, B.Since the highest concentrations of gemcitabine were detected in primary pancreatic tumours, our results suggested that the stromal microenvironment may promote, rather than impede gemcitabine accumulation in bulk tissue.G12D (see online 50 of tumour cells) significantly increased tumour cell viability by 40%–80% in two KPC cell lines compared with CAF-conditioned media with fresh gemcitabine, suggesting a gemcitabine scavenging effect of CAFs in vitro  and metastatic foci (n=4) from four different KPC mice. To dissect the stromal and neoplastic compartment, we further generated primary CAF lines (n=2) from murine pancreatic tumours as well as PSCs (n=3) from healthy B6 mice showing typical spindle-shaped morphology, and strong expression of α-SMA, SPARC and fibronectin . The verin vitro E, F.Gemcitabine activation and inactivation is a complex multistep process that is driven by a number of enzymes and cellular transporters . Gemcitabine was administered at 100 mg/kg intraperitoneally every 3–4 days until end point. To assess gemcitabine metabolising proteins in murine and human PDAC, we performed immunohistochemistry of gemcitabine-treated KPC mice  and patient samples . Nt5c1A was strongly expressed in all murine and human tumour cells, whereas the tumour microenvironment was largely devoid of Nt5c1A expression E. Cda an50 for gemcitabine (20–30 nM) in primary murine tumour cell lines (KPC n=2), tumour cells derived from metastatic foci (KPCm n=2) and fibroblasts (CAF n=2) ) compared with fibroblasts   or gemcitabine (n=8) for 9 days.Despite the fact that gemcitabine is a highly active drug in vitro, monotherapy fails to show meaningful antitumour effects in patients with PDAC and genetically engineered mice. Apart from biochemical signalling loops within the tumour microenvironment,28These considerations prompted us to reassess drug delivery in pancreatic cancer with a particular focus on the predominant cell types, that is, stromal cells and tumour cells. Our data show that gemcitabine accumulated in fibroblast-rich KPC tumours more effectively than in liver metastases displaying less desmoplasia and CAFs. This result was surprising as we had expected to see similar or higher levels of drug in liver metastases, which are surrounded by the well-perfused liver. In vitro, fibroblasts revealed a drug scavenging effect by metabolising gemcitabine thus entrapping significant amounts of dFdCTP that are not available for tumour cells anymore. Assuming that gemcitabine scavenging is more pronounced in bulk KPC tumours with strong desmoplasia, we investigated the effects of gemcitabine treatment on the frequency of liver metastases. On gemcitabine treatment, the number of all metastases was reduced by more than half, and this effect was particularly pronounced in small, only microscopically visible metastases. The effective killing of metastases and circulating tumour cells on gemcitabine has been recently proposed in a preclinical study. Metastatic lesions of all size were shown to have significantly higher apoptotic rates than primary tumours, and the metastatic burden was robustly reduced following long-term treatment with gemcitabine and nab-paclitaxel.The reduced content of α-SMA fibroblasts in murine and human liver metastases compared with matched primary tumours is intriguing. Although the small set of matched human samples appear to confirm our murine data, confirmation from a larger set of human samples is required. Our observation is in contrast to previously published data that describe comparable stromal components in liver metastases and primary tumours.Mechanistically, we found a number of gemcitabine metabolising enzymes with low expression in stromal cells that would explain the increased amount of gemcitabine and its activated metabolite. The two main gemcitabine inactivating enzymes are Cda and Dctd, and both act at different nodes during gemcitabine metabolism. Whereas Cda quickly inactivates gemcitabine to dFdU, Dctd metabolises dFdCMP to dFdUMP preventing further activation of dFdCMP to dFdCTP. To the best of our knowledge, differential expression of Nt5cAs in pancreatic cancer cells and fibroblasts has not been reported before. Since Nt5cAs catalyse the hydrolysis from dFdCMP to gemcitabine, it seems like the most suitable target for reprogramming the tumour stroma may be by decreasing dFdCTP concentrations in CAFs.Furthermore, our results may also provide an explanation why the extent of desmoplasia was often reported to be negatively correlated with patient survival.Although fibroblast-depletion approaches may appear attractive for patients with PDAC to increase the intratumoural availability of gemcitabine for cancer cells, several studies recently reported that pharmacological and genetic depletion of activated fibroblasts results in accelerated pancreatic tumour growth, increased invasiveness, stemness and immune-modulation.5In conclusion, multiple studies suggest that impaired drug delivery is a major reason for the failure of therapeutic agents in pancreatic cancer. With our study, we add another layer of complexity showing that fibroblast drug scavenging increases intratumoural gemcitabine accumulation entrapping active gemcitabine within stromal cells thus making it unavailable for tumour cells. However, we cannot exclude the possibility that increasing drug delivery by modulating vessel patency and density might still have beneficial therapeutic effects in PDAC. Our data provide an alternative explanation for the failure of gemcitabine in PDAC and challenge the paradigm of a biophysical stroma barrier for gemcitabine delivery. Targeting the metabolic programme in CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine in PDAC.


Conjugation of Cy5.5 to MALAT1 ASO was accomplished using standard NHS (N-hydroxysuccinimide) ester procedures, and the labelled MALAT1 ASO was purified with a Glen-Pak DNA Purification Cartridge and reversed-phase high performance liquid chromatography (HPLC). The in vitro cellular uptake results showed that the percentage of cell binding increased with an increasing final concentration and increased with increasing incubation time for the MHCC-LM3 tumour cell flow cytometry analyses. in vivo optical imaging exhibited 5’ (Cy5.5)-MALAT1 ASO uptake in the tumour with a maximum at 30 min p.i. that slowly washed out over time. High contrast to normal tissue was gradually observed from 4 h to 48 h p.i. Tumour-to-normal ratios of fluorescence intensities were plotted as a function of time. The in vivo competition assay showed little uptake of the probe into the tumours at any time point, indicating effective competition, selectivity of probe binding and retention by tumours in vivo. Our proposed Cy5.5 labelling of MALAT1 ASO can serve as a potent optical probe for in vivo imaging of tumour expressing MALAT1. Importantly, the successful development of optical probes provides a basis for specific molecular diagnoses in the field of lncRNAs.With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs (lncRNAs). The objectives of this study were to develop a novel lncRNA MALAT1 near-infrared optical probe, to evaluate the characteristics of this optical imaging probe  Based on these studies, we hypothesised that MALAT1 is a potential target for a specific molecular diagnosis.With the advent of next-generation sequencing technology, there is rapidly increasing interest in long noncoding RNAs (lncRNAs). Despite the outdated opinion that lncRNAs may be mere transcriptional noise, evidence suggests that lncRNAs play a vital role in regulating gene expression, cell metabolism, tumour development and progression . The lonin vitro . These a[in vivo . For exa[in vivo  reportedin vivo optical imaging has advantages of not requiring ionising radiation, low cost, and easily generated optical imaging probes [in vivo studies is becoming an important field for cancer research [Functional and non-invasive molecular imaging techniques, such as optical imaging, nuclear imaging, and magnetic resonance imaging (MRI), are gradually being incorporated into every aspect of cancer management. Of these, g probes . Additiog probes , 11. Theresearch .in vitro and determined whether it can be used for the imaging of MALAT1 expression in malignant tumours in vivo.The purpose of the study was to explore the feasibility of development of molecular probes for lncRNA. This study reports the method for preparing molecular optical imaging probes  for specific targeting of overexpressing MALAT1 tumours cells. We evaluated the characteristics of this optical imaging probe − for 5' (Cy5.5)-MALAT1 ASO , and m/z=6149 (M+H)− for MALAT1 ASO. Elution times on the HPLC column were 16.61 min for 5’ (Cy5.5)-MALAT1 ASO, and 16.28 min for MALAT1 ASO.The schematic molecular structure of the 5’ (Cy5.5)-MALAT1 ASO conjugate is shown in Figure in vitro were performed. The binding specificity of 5’ (Cy5.5)-MALAT1 ASO to tumour cells was determined using fluorescence activated cell sorter (FACS) studies. FACS revealed that the percentage of cell binding (M2) increased with the increasing final concentrations . These results suggest that a positive correlation exists between the uptake and final concentration of the 5’ (Cy5.5)-MALAT1 ASO from 20 nM to 250 nM (r=0.91). Figure in vitro binding between different concentrations of 5’ (Cy5.5)-MALAT1 ASO and MHCC-LM3 cells observed using a fluorescence microscope.To determine whether the new imaging probes result in specific cellular uptake, uptake studies in vitro binding of 5’ (Cy5.5)-MALAT1 ASO to MHCC-LM3 cells at different times is shown in Figure To investigate the kinetics and intracellular uptake of the 5’ (Cy5.5)-MALAT1 ASO, MHCC-LM3 cells were incubated at 37°C with 5’ (Cy5.5)-MALAT1 ASO at a final concentration of 100 nM at different time. FACS revealed that the percentage of cell binding (M2) increased with increasing incubation times , and a positive correlation existed between the uptake and increasing incubation time from 30 min to 8 h (r=0.97). in vitro cellular distribution study showed that 5’ (Cy5.5)-MALAT1 ASO is specific and selective for MHCC-LM3 cells using a fluorescence microscope, and the accumulation of 5’ (Cy5.5)-MALAT1 ASO is retained in the nucleus -MALAT1 ASO probes are shown in Figure in vivo selectivity of probe binding is retained in the tumours, we also performed an in vivo competition assay. These studies showed little uptake of the probe in the tumours at any time points, indicating effective competition and selectivity of probe binding and retention by the tumours in vivo -MALAT1 ASO probe uptake, which is significantly lower in the tumour (p=0.002), suggesting the targeted specificity of the 5’(Cy5.5)-MALAT1 ASO probe. The kidneys showed increased uptake of the 5’ (Cy5.5)-MALAT1 ASO probe, which was identical to the imaging in vivo. The contrast ratios of tumour to normal organs for non-blocked and blocked groups were calculated and presented in Figure Furthermore, excised organs were evaluated ex vivo and quantified using ROI that encompassed the entire organ. The 5’ (Cy5.5)-MALAT1 ASO was predominantly taken up by the MHCC-LM3 tumour at 24 h p.i Figure , as seenLncRNAs are a group of RNAs that do not encode proteins, and the lengths of these molecules are greater than 200 nucleotides . Recent MALAT1 is also an independent prognostic marker for poor outcomes and patient survival in several cancer types, including stage I non-small cell lung cancer and hepatocellular carcinoma , 23. Ma in vivo, exhibiting significant anticancer effects. ASO, which is relatively smaller than siRNAs, give a higher rate of delivery into the nucleus where most lncRNAs are confined [MALAT1 has many characteristics that make it attractive as a biomarker and therapeutic targets , 26. MALconfined  observedconfined  reportedin vivo demarcation of tumours, to conjugate with the MALAT1 ASO [Considering the above factors, we hypothesised that MALAT1 is a potential molecular target and that fluorophore-conjugated MALAT1 ASO would generate a novel molecular probe for the optical imaging of tumours. To create the probe, we selected the Cy5.5 dye, which is used as a promising contrast agent for the LAT1 ASO , 29. Thein vitro and in vivo. in vitro cellular uptake experiments with 5’ (Cy5.5)-MALAT1 ASO showed that it can selectively bind to and be taken up by MHCC-LM3 cells, and this retention is time-dependent. FACS analyses revealed that the percentage of cell binding (M2) increased with increasing incubation times from 30 min to 8 h, with a positive correlation existing between uptake and increasing incubation time (r=0.97). By contrast, 5’ (Cy5.5)-MALAT1 ASO bound to MHCC-LM3 cells in a dose-dependent manner, and there was increased uptake of MALAT1 antisense oligonucleotides by MHCC-LM3 cells with increasing concentrations, ranging from 20 nM to 250 nM. In addition, the intracellular distribution of 5’ (Cy5.5)-MALAT1 ASO was also examined, and the results showed that 5’ (Cy5.5)-MALAT1 ASO was specifically retained in the nucleus, which is identical to a previous study [The present study identified a near-infrared molecular imaging probe for cells over-expressing MALAT1 that allows for non-invasive specific detection of tumours us study .in vivo. The 5’ (Cy5.5)-MALAT1 ASO uptake in the tumour showed a maximum at 30 min p.i. and slowly washed out over time. High contrast to background tissue was gradually observed from 4 h to 48 h p.i. The normal tissue demonstrated rapid probe uptake. However, probe washout in the tumour is much slower than in normal tissue, leading to excellent tumour-to-normal tissue contrast at later time points. 5’ (Cy5.5)-MALAT1 ASO exhibited high contrast tumour-to tissue ratios for the imaging of dissected tissues and organs, which were consistent with the in vivo imaging findings.The whole-body fluorescent images of the MHCC-LM3 cell xenograft mouse model presented in Figure in vivo tumour imaging, high specificity binding is an important property [in vivo, competition studies were conducted by injecting unlabelled MALAT1 ASO before injecting 5’ (Cy5.5)-MALAT1 ASO. Significantly reduced tumour uptake of 5’ (Cy5.5)-MALAT1 ASO was observed for the blocked group at all times p.i. (P < 0.05), indicating that it is a target-specific probe. In the blocked experiments, signals from the mouse kidney were gradually increased, reaching a peak 24 h after administration, and then gradually decreasing, suggesting that the probe is mainly excreted from the urinary tract.For the development of new molecular probes for property . To furtin vivo imaging of tumours expressing MALAT1. More importantly, the successful development of optical probes provides for specific molecular diagnoses in the field of lncRNAs, which deserve further study.In summary, our results provide evidence that Cy5.5 labelling of MALAT1 ASO is a convenient approach and that these novel optical probes can serve as specific optical probes for the Single strand 20-base DNA sequences were modified as phosphorothioates , and the base sequence used was as follows: GGGAGTTACTTGCCAACTTG (MALAT1 ASO) . The Cy5* 250 mm). For the HPLC linear gradient, the following buffers were used: A, 2% acetonitrile in 0.1 M TEAA, pH 7; B, 50% acetonitrile in 0.1 M TEAA, pH 7. The column was eluted at 1 mL/min at room temperature. Mass spectra were obtained on a Q-Tof premier-UPLC system equipped with an electrospray interface (ESI) . The product was collected, lyophilised, and stored in the dark at -20°C until use.The HPLC system was equipped with a quaternary pump, variable wavelength detector, vacuum degasser, and ChemStation for system control and chromatography analysis. HPLC was performed using a C18 column  bearing 5’-aminoalkyl linkers was dissolved in 15 μL of 0.1 M NaHCOthe dark . Then, 42 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum with 100 U/mL penicillin and 100 μg/mL streptomycin.The human MHCC-LM3 cell line, which was selected for its high MALAT1 expression , was pur6 cells per well) and incubated at 37°C, 50 mL/L CO2 for 24 h, until the cell density reached 80%. In one experiment, MHCC-LM3 cells were incubated for 4 h at 37°C with 150 μL medium (with serum) per well containing different final concentrations  of 5’(Cy5.5)-MALAT1 ASO using Lipofectamine 2000  in accordance with the manufacturer’s procedure. In another experiment, MHCC-LM3 cells were incubated for different times  at 37°C with a total volume of 150 μL and final concentrations of 100 nM 5’(Cy5.5)-MALAT1 ASO. After incubation of MHCC-LM3 cells with 5’ (Cy5.5)-MALAT1 ASO, the percent transfected cells were screened and sorted by FACS. These experiments were repeated three times, and parallel wells were included in each experiment.MHCC-LM3 cells were detached with trypsin and then counted and plated in 6-well flat-bottomed culture plates in DMEM with 10% foetal bovine serum -MALAT1 ASO, the cells were washed twice in 0.1% Tween-20 2× SSC at 56°C for 2 min each, followed by one wash in 0.1% Tween-20 2× SSC at room temperature for 2 min. 4’,6- Diamidino-2-phenylindole  was used to stain the cell nucleus, and fluorescence images were collected using an Olympus AX70 fluorescence microscope.In addition, an 7 MHCC-LM3 cells resuspended in 100 μL of PBS into the right axilla, and the cells were allowed to grow 3–5 weeks until tumours were 200–500 mm3 in volume. The mice were maintained using a standard diet, bedding, and environment, with free access to food and drinking water.All animal work was performed in accordance with and approved by the Zhejiang University Institutional Animal Care and Use Committee guidelines. BALB/c nu/nu mice  were used in this study. BALB/c nu/nu mice with MHCC-LM3 cell tumours were randomly divided into 3 groups of 5 mice each. The MHCC-LM3 cell xenograft model was generated by subcutaneous injection of 1.0*10In vivo fluorescence imaging was performed using a filter set with an excitation of 658 nm and emission of 719 nm on a Clairvivo OPT plus . Identical illumination settings  were used for acquiring all images. Fluorescence images were acquired at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h after injection of 5’ (Cy5.5)-MALAT1 ASO using 1 s exposure time, anesthetised with 2% isoflurane. The non-blocked mice (n=5) received 100 nmol of 5’(Cy5.5)-MALAT1 ASO injected into each mouse via tail vein and were subjected to optical imaging at various time points post-injection. For the blocked group of mice (n=5), unlabelled MALAT1 ASO (100 nmol) was first injected, and 100 nmol 5’ (Cy5.5)-MALAT1 ASO was injected 30 min later via tail vein. Ellipsoid regions of interest (ROI) of equal area were drawn on the tumour and on the contralateral sites of the left axilla, and the fluorescent intensity was quantified (C/mm2).in vivo imaging experiments, the mouse was sacrificed by cervical dislocation under isoflurane anaesthesia. The tumours and major organs or tissues  were collected for ex vivo imaging of the biodistribution of the fluorescent probe, and the fluorescence intensity for each sample was reported (C/mm2).At the end of the t test. A p value less than 0.05 was considered statistically significant. Analyses were performed using SAS v8.0 .Data are presented as the means ± SD (standard deviation) of n independent measurements. Statistical analysis was performed using Student’s


Brussels, BelgiumInformations:Phone: +3225378701registration@co-mana.comE-mail: valvesymposium.comSite: Chicago, United StatesInformations: Office of Continuing Medical EducationPhone: 312-503-8533Fax: 312-503-4531cme@northwestern.eduE-mail: www.cme.northwestern.eduSite: Philadelphia, United StatesInformations: Eva JonesPhone: 1-702-508-5200: 8033atherosclerosis@insightconferences.comE-mail: http://atherosclerosis.conferenceseries.comSite: Singapore, SingaporeInformations: Course Secretariatnhccme@nhcs.com.sgE-mail: Cambridge, United KingdomInformations: Matthew KirkbyPhone: 01223750020matthew.kirkby@zingconferences.comE-mail: Toronto, CanadaInformations: Jessie AlisonPhone: 7025089022E-mail: hypertension@conferenceseries.comhttp://hypertension.conferenceseries.com/Site: Beijing, ChinaInformations: Meetings at AATSPhone: 978-927-8330meetings@aats.orgE-mail: http://aats.org/valvebeijing/Site: Seoul, South KoreaInformations: Pyowon Parkpwpark@skku.eduE-mail: http://www.valveforum.orgSite: Istanbul, TurkeyInformations: Randi Wilsoneacta@ics.dkE-mail: http://www.eacta.org/echocourses/eacta-echo-course-2016Site: Cape Town, South AfricaInformations: Helene UysPhone: +27 31 303 9852Fax: +27 31 303 9529info@wscts2016.co.zaE-mail: www.wscts2016.co.za/Site: Philadelphia, United StatesInformations: Ansheia SpencePhone: 215-898-6400penncme@mail.med.upenn.eduE-mail: Cape Town, South AfricaInformations: Prof. Charles Yankah and Dr. Willie KoenPhone: +49-172-3020143cyankah@web.de, wkoen@iafrica.comE-mail: www.pascats.comSite: Newcastle upon Tyne, United KingdomInformations: Anna Burleyanna.burley@aesculap-academy.comE-mail: www.aesculap-academia.co.ukSite: Homburg, GermanyInformations: Jessica KuenstlerPhone: +4961829466636Fax: +4961829466644j.kuenstler@kelcon.deE-mail: Orlando, United StatesInformations: Patti DeshaiesPhone: 919-681-6370patricia.deshaies@duke.eduE-mail: Washington, United StatesInformations: Mowahib VermillionPhone: 703-992-9948info@facts-care.orgE-mail: http://www.facts-care.org/Site: Anaheim, United StatesInformations: Martha GomezPhone: (323) 361-4941Fax: (323) 361-3668mgomez@chla.usc.eduE-mail: www.chla.org/event/cme-eventsSite: Chicago, United StatesInformations: Office of Continuing Medical EducationPhone: 312-503-8533Fax: 312-503-4531cme@northwestern.eduE-mail: Singapore, SingaporeInformations: Secretariat, the Academiasims.sssc@singhealth.com.sgE-mail: http://www.singhealthacademy.edu.sg/sdc2016Site: Birmingham, United KingdomInformations: Lorraine RichardsonPhone: +447711132946Fax: 01296 733 823lorrainerichardson1@btinternet.comE-mail: www.birminghamreviewcourse.co.ukSite: Philadelphia, United StatesInformations: Ms. Micah HollidayPhone: 215-590-5263Fax: 215-590-4342hollidaydm@email.chop.eduE-mail: www.chop.edu/aaoca-2016Site: Washington, United StatesInformations: Amy Copelandscreening@lungcanceralliance.orgE-mail: Baltimore, United StatesInformations: Laura MedekPhone: (312) 202-5839lmedek@sts.orgE-mail: Chicago, United StatesInformations: Office of Continuing Medical EducationPhone: 312-503-8533Fax: 312-503-4531cme@northwestern.eduE-mail:


A 53 year-old man, with untreated lower urinary tract symptoms for two years was catheterized for acute retention of urine. He had an enlarged, boggy non tender prostate.The transrectal ultrasound (TRUS) revealed a fluid filled cystic mass arising from the prostate and 200cc of hemorrhagic fluid was aspirated. There was no growth on culture of fluid and Xpert® MTB/RIF along with three acid fast bacillus smears was negative for tuberculosis. Cytology was not performed on the aspirated fluid. Magnetic resonance imaging revealed prostate volume of 52cc and a 94x77mm cystic and solid lesion  with semBased on the locally advanced nature of the disease, he was initiated on neoadjuvant Leuprolide acetate 22.5mg subcutaneously  and he voided successfully without a catheter on follow-up. At three months, his PSA was 21.9ng/mL with marked reduction in the size of the cystic lesion (50x40mm) . He receHemorrhagic pelvic cyst remains a rare presentation of carcinoma prostate -5. With


Vegetables are an important part of a healthy diet because they help prevent several chronic diseases. Mothers of preschoolers reported difficulty getting their young children to eat vegetables, and many did not know how to cook child-pleasing recipes.Mommio.The cooking habits of mothers of preschoolers, their perceptions of recipes designed for their children, and the involvement of their children in food preparation were assessed to inform a food parenting video game called A cross-sectional survey design was used. Eligibility criteria included mothers of 3- to 5-year-old children who reported difficulty getting their children to eat vegetables. Participants completed a demographic questionnaire with questions about their food preparation practices. They were asked to select up to 4 of the 10 provided recipes they wanted to try and to prepare and report back on their experiences.Mommio’s in-game recipe box were evaluated at least once and some up to 5 times with a total of 85 evaluations. This well-educated, mostly employed, sample of 27 mothers of preschoolers preferred simple, quick recipes. They ate primarily at home, made dinners from scratch, and indicated that the 46 recipes were generally simple, quick, and easy to prepare. Involvement in preparation enhanced their child’s acceptance of the food. Prior food and preparation preferences influenced the children’s acceptance of the dish at the ensuing meal.Most (46) of the 50 recipes included in The high rate of home recipe preparation indicated that including a recipe selection and preparation component in a food parenting video game could be attractive and may enhance effectiveness. Mothers reported that the recipes provided were generally easy to prepare, tasted good, and the instructions were easy to understand, suggesting they could be helpful to the mothers when playing a vegetable parenting game. Some mothers reported that involving their children in recipe preparation influenced their children’s willingness to eat the vegetables. The highest rated recipes are being included in the game, and mothers will be encouraged to involve their children in recipe preparation. Vegetables are an important part of a healthy diet because they help prevent several chronic diseases ,2. As chMommio, a first-person role-playing mobile three-dimensional (3D) video game currently under development [Mommio featured an in-game “recipe box” containing a limited number of vegetable recipes [Mommio, some studies have indicated a high frequency of eating meals away from home, thereby possibly negating the value of a recipe feature [elopment ,12, is belopment . Intende recipes . Partici recipes . Althoug feature .Interactive media, including video games, have been shown to advance parenting skills . AlthougMommio), and (3) whether mothers would report that involving their preschool children in food preparation would increase their children’s consumption of vegetables. Participants also evaluated recipes designed for possible inclusion in the game.This study assessed the following: (1) the cooking habits of mothers of preschoolers who had difficulties getting their children to eat vegetables, (2) the acceptability and usefulness of potential recipes in a vegetable parenting game  most frequently consumed by 3- to 5-year-olds was generated from the National Health and Nutrition Examination Survey. Recipes that included these and many other nutritious vegetables were collected primarily from our prior projects and public websites -25. GiveMommio quest, Kiddio either tastes the vegetable to signify a victory or runs out of the room, indicating a loss.A 3D representation of a home and a grocery store allows a child character (nicknamed “Kiddio”), a dog, and a male adult character to roam the house. The nonplayer characters are controlled by artificial intelligence algorithms. The player assumes the role and point of view of the child’s mother. Player exploration  is permitted and expected. The player primarily interacts with Kiddio, a preschool-aged character, who prompts the player to act by saying “I’m hungry.” As shown in Mommio video game logo. To ensure most recipes were tested, the last 4 participants to join the study were asked to include recipes not selected by previous participants. Testers were obtained for 46 of the 50 recipes. An overall recipe rating scale was employed: “Considering your entire experience of preparing, serving, and tasting the recipe, how would you rate this recipe?” with the following 5 response options: “Hated it,” “Disliked it,” Didn’t hate or like it,” “Liked it,” or “Loved it.”Each participant first completed a demographic and screening questionnaire that included the questions listed in Quantitative data were analyzed using the statistical analysis system . Descriptive statistics were used to calculate frequencies and percentages. Answers to open-ended questions were coded using thematic analysis  accordinMommio game (11 participants), were selected from the Children’s Nutrition Research Center’s research volunteer list (11 participants), or were recruited via online posts and flyers (5 participants). The sample was well educated (70% had graduated from college), married (78%), employed (78%), worked full-time outside the home (63%), and had a household income over US $60,000/year  made lunches for their children on weekends only and reported diverse frequencies of preparing snacks for their children but provided dinner or supper at home most days of the week 59%; . Most moA subsample of 20 mothers evaluated a total of 46 recipes. Each recipe was evaluated at least once and up to 5 times with a total of 85 evaluations of the 46 recipes. The recipes were well received. A total of 13 recipes received a mean recipe rating of 5 (“Loved it”), with another 13 receiving a 4 or higher rating (“Liked It”). Of these top 26 recipes, 20 recipes were rated as “very easy to prepare” and another 8 recipes rated as “somewhat easy.” A diverse variety of vegetables were rated at the higher end of the mean recipe rating scale. The same diversity was true for the vegetables at the lower end of the scale, suggesting no one or two vegetables dominated the positive or negative ratings. Furthermore, 4 recipes were not selected for testing by at least one mother. No obvious pattern can be discerned as to why these were not selected, except possibly for “spiciness” in the title of one recipe. Neither the number of ingredients nor the reported ease of preparation appeared to be associated with the recipe ratings. As would be expected, the taste of the recipe was related to the mean overall recipe rating, and the instructions were generally easily understood.Less than half of the parents (45%) had prepared a similar dish before. When asked how likely it was that they would make each dish, 46% of the parents reported that the dishes would definitely be made and 35% reported that they might be made. In addition, 61% of the parents indicated they had all of the ingredients at home needed to prepare the recipes. Moreover, 74% of the parents reported that the recipe instructions were “very easy to follow with few ingredients” and easy to prepare (87%):My family loves chick peas and hummus; it is pricey to buy it already made. I always wanted to learn how to make it, but I thought it was difficult, but this recipe proved me wrong.Many reported that the ingredients were inexpensive:Peppers have great benefits and usually they are on sale.Most parents (81%) liked the finished recipes. Parents mentioned that they enjoyed learning new ways to prepare vegetables:A different way of preparing veggies is always nice.Other parents appreciated the new take on old recipes:It was easy and something similar to what I have made before, just without lemons.Multiple parents evaluated the recipes according to the time needed for preparation or expressed a concern about long preparation time:It was simple and did not take a lot of time to cook.The recipe was very easy to follow. It just takes a lot of time.The preparation took longer than I expected.One of the mothers commented on the need for only basic equipment:I really liked this recipe. It was different and very easy. Love that I only had to use one pot.Alternatively, another mother commented on the food preparation skills needed to prepare one of the recipes:It was extremely difficult to cut the squash. It was hard and difficult to control or hold still. The deseeding was like trying to clean out a pumpkin—very messy.Some parents said they liked that the recipes were a healthier version of some foods they already consumed:I liked it, it was easy to make. It’s a healthier substitution for any dip...It is a great recipe. I usually hate the salty pizzas from stores although we have to eat them once in a while. The recipe is simple and I can add salt as little as I want.The 3- to 5-year-old children were involved in preparing 51% of the recipes. One of the participants stated:It was easy to do with the kids, very few ingredients all in one baking dish. They enjoyed the experience.Participants indicated that children helped by washing vegetables; adding ingredients to the blender, bowls, or pots; or measuring or mixing ingredients. Some participants commented on how children’s involvement in the preparation of the recipes influenced their willingness to try the foods:She was very proud to serve what she made and she made her siblings excited about helping with meals as well....did not want to try them but since she helped making the recipe and the 3-year-old ate all of his, she went ahead and ate them once I gave her ranch yogurt dressing to dip them in.Approximately 88% of the children tasted the finished recipes and 73% ate them. Prior food preferences were mentioned as an important factor when children decided to try their food:He doesn’t dislike broccoli so it wasn’t hard to ask him to try.They hate the taste of pepper, so I was not able to make them eat even a bite.Some mothers mentioned how the method of preparation influenced their children’s consumption of the recipe:Both of my children enjoyed the recipe more than I expected. The small cubes of potatoes really helped them eat it better.My child had a hard time handling the corn and eating it off the cob.The final presentation of the recipe was also relevant:It was a very different pasta recipe for him, he loved that it had corn and beans and it is very colorful.He didn’t want to try it at all, maybe because it looked boringSpicy flavors were not popular among 3- to 5-year-old children:For my little people, cayenne pepper is not a spice they prefer, so I think it’s an odd seasoning in this recipe. I even cut it in half and still had complaints about being too spicy...Another mother mentioned:Kids were afraid to try at first because they thought it would be spicy.Only 40% of the participants modified the different recipes when evaluating them. These modifications were made because of the family food preferences. They commented:We don’t like the taste of parsley so I used a purple leaf...don’t know the name of it but it is my 3-year-old’s favorite vegetable growing in our backyard.We added olives. We always add olives to the pizza. My kids insist.When asked if they would modify the recipe before they make it again, 54% said they would:I would add something to make it more flavorful. The red peppers were good and the lemon juice, but even after adding salt and pepper I would have added more spices or other veggies to make it have a more flavorful taste.The small potatoes I bought for the recipe inspired me. I want to try to make them tastier.This sample of 27 well-educated and mostly employed mothers of preschoolers regularly cooked at home, unlike most families in the United States . AlthougMommio. According to open-ended questionnaire responses, mothers preferred recipes that included basic ingredients and little equipment, similar to a review about recipe development for limited-resources audiences [Of the 50 recipes offered, 46 were tested by at least one mother. A total of 41 recipes received scores above the median possible recipe score (2.5), with 13 receiving the highest rating (5) and 18 receiving the next highest rating (4-4.5). This suggests that an ample number of tasty, easy-to-understand and prepare recipes are available for incorporating into udiences . MothersSome participants 15%) reported that the recipes were healthier versions of foods they currently ate. However, mothers’ primary perceived barrier to preparing the recipes was the time required. In 2015 in the United States, 59% of all women and 70% of mothers with children under the age of 18 years were employed outside their home , similar% reporteOlder children (aged 6-10 years) who participated in the food preparation process ate significantly more salad 42 g (76%) than the ones where only the parent prepared, likely because of enhanced feelings of autonomy and pride . MothersThe mothers in this study mentioned that the way ingredients were presented was an important factor in a kid friendly recipe. The need to cut food into 0.5-inch chunks to avoid choking hazards  was wellResearch has indicated that preexisting child food preferences were a major determinant of intake . In thisMommio was designed for mothers, and therefore, fathers were not included in this sample. No effort was made to deliberately include mothers with high school education or less, a demographic known to lack confidence in preparing healthy meals [As the game’s name suggests, hy meals . Future We found that the high rate of home recipe preparation indicated that including a recipe selection and preparation component in a food parenting video game could be attractive and may enhance effectiveness. Mothers reported that the recipes provided were generally easy to prepare, tasted good, and the instructions were easy to understand, suggesting they could be helpful to the mothers when playing a vegetable parenting game. Some mothers reported that involving their children in recipe preparation influenced their children’s willingness to eat the vegetables. The highest rated recipes are being included in the game, and mothers will be encouraged to involve their children in recipe preparation.


The following grant is missing from the Funding section: Gates Foundation (OPP1066118).The publisher apologizes for the error.


Fluid infusions are given to children to treat cardiovascular compromise from shock in locations across the globe, where resources vary widely. The most common cause of cardiovascular compromise is septic shock. In low and middle income countries severe malnutrition complicates sepsis largely from malaria, pneumonia, and diarrheal diseases, leading to interesting treatment conundrums. The high mortality rate of about 40% in children with severe malnutrition and shock from diarrheal diseases results from the confluence of clinical factors, inequity, socio-economic and cultural context resulting in late presentation, increased vulnerability, and sub-optimal care . Fluid rIn response to these uncertainties, Obonyo and colleagues investigated the cardiovascular response to two fluid infusion regimes (bolus and continuous infusion over 5 h) in severely malnourished children with hypovolemic shock due to diarrheal diseases with dehydration . The autSeptic shock is a complex pathophysiological derangement; its expression depends on microbial agent virulence, systemic inflammation, endothelial and microcirculation disruption, primary and secondary immune derangements, and coagulation and parenchymal tissue insults , 6. EndoPoor outcomes in children with septic shock in resource-poor settings are likely due to multi-organ dysfunction of late sepsis and insults to the myocardium where intensive care support is limited. Robust cardiac contractility relies on the myocardial milieu where substrates such as magnesium, calcium, acid base balance, hemoglobin, temperature, and energy stores are normal. Levels of these substrates are commonly abnormal in critically ill children. Thus, the inability to monitor or treat electrolyte disturbances and acid base may result in higher mortality rates due to cardiovascular collapse , 10. WheThe optimal approach to fluid administration in both high and low income countries is not yet settled, with no rigorous study conducted in high income countries. The FEAST trial, the largest and most rigorous attempt to address approaches to fluid administration was done in low and middle income settings in Africa. Validated approaches to administration of fluid in septic shock are sorely needed in view of the myriad etiologies and pathophysiologic perturbations and the recent body of evidence pointing to positive fluid balance as a predictor of poor outcomes in critically ill children even when intensive care is available.Fluid resuscitation in sepsis has endured a long history of success, starting with initial experiences with cholera. What is still unclear is how much fluid should be infused and how fast to replenish intravascular volume deficits in children with shock and complex co-morbidities. We need to challenge our present dogma regarding approaches to fluid resuscitation. For instance, we need to further explore the role of increased lactate in septic shock because direct tissue oximetry has failed to show hypoxia and tissue partial pressure of oxygen may be elevated . OliguriWhile impractical in most low and middle income settings, an in-depth understanding of the pathophysiology and response to fluid in shock will be best obtained using multimodal monitoring including clinical examination and invasive central venous pressure and echocardiographic monitoring . IdeallyScientific evidence thrives by unearthing facts and burying opinions. Thus, we need randomized clinical trials if we are to further our understanding of fluid therapies. Each child with sepsis is unique and factors such as their genetic predisposition, the social economic context, the inciting agent, and the trajectory of the septic episode must be considered in enrolment to understand and transition from generic to personalized and ultimately precision therapies. Challenging the status quo is never easy, but it may be time to rewrite history.


The diffusion and perfusion magnetic resonance (MR) images can provide functional information about tumour and enable more sensitive detection of the tumour extent. We aimed to develop a fuzzy feature fusion method for auto-segmentation of gliomas in radiotherapy planning using multi-parametric functional MR images including apparent diffusion coefficient (ADC), fractional anisotropy (FA) and relative cerebral blood volume (rCBV). For each functional modality, one histogram-based fuzzy model was created to transform image volume into a fuzzy feature space. Based on the fuzzy fusion result of the three fuzzy feature spaces, regions with high possibility belonging to tumour were generated automatically. The auto-segmentations of tumour in structural MR images were added in final auto-segmented gross tumour volume (GTV). For evaluation, one radiation oncologist delineated GTVs for nine patients with all modalities. Comparisons between manually delineated and auto-segmented GTVs showed that, the mean volume difference was 8.69% (±5.62%); the mean Dice’s similarity coefficient (DSC) was 0.88 (±0.02); the mean sensitivity and specificity of auto-segmentation was 0.87 (±0.04) and 0.98 (±0.01) respectively. High accuracy and efficiency can be achieved with the new method, which shows potential of utilizing functional multi-parametric MR images for target definition in precision radiation treatment planning for patients with gliomas. However, conventional MRI does not accurately show the actual tumour borders of glial neoplasms because it reflects only anatomic rather than functional properties of the tumour2. And tumour cells could be found in serial biopsies beyond signal intensity changes on T2 MRIs3. In contrast, advanced imaging techniques, such as diffusion and perfusion MRI, provide functional information about the tumour microenvironment and enable more sensitive detection of the tumour extent. For instance, the parameter of apparent diffusion coefficient (ADC) is derived from diffusion-weighted imaging (DWI) and is proved to be correlated reciprocally with tumour cellularity4. Another useful parameter of fractional anisotropy (FA) is calculated from diffusion tensor imaging (DTI) and is sensitive to the changes in fibre bundles. Furthermore, dynamic-susceptibility contrast (DSC) and its derived parameter of relative cerebral blood volume (rCBV) can be used to assess the tumour vascularity.To avoid missing target and spare critical healthy brain tissue around the target volume in radiotherapy treatment planning of gliomas, cancerous tissue involvement must be correctly defined in the gross tumour volume (GTV) delineation. Conventional T1-weighted contrast-enhanced (T1C) and T2-weighted (T2) magnetic resonance imaging (MRI) reveal soft tissue with high contrast and could help improve the accuracy of tumour volume definition5. Additionally, FA values was also used to identify tumour cell infiltration along white matter tracts in high-grade gliomas in order to preserve coverage of the likely routes of dissemination and spare uninvolved brain, generating a better radiotherapy target volume delineation6. Furthermore, the integration of ADC, rCBV and metabolic information of proton MR spectroscopic imaging (1H-MRSI) is able to discriminate infiltrating tumour from surrounding vasogenic oedema or normal tissues for improving the evaluation of glioblastoma extent8.Those diffusion and perfusion parameters have been investigated to define accurate tumour extent and delineate target volume in radiation treatment planning. For instance, DTI-derived FA values was used to detect white-matter abnormalities resulting from tumour infiltration and reduce the size of the planning-target volume (PTV), resulting in escalated doses without an increase in normal tissue complication probability (NTCP) in radiotherapy treatment planning for patients with high-grade gliomas11. The application of automatic segmentation methods might be helpful to solve this problem. Recently, most of the automatic segmentation methods using information integration of multimodal MR images of patients with gliomas focus on deformable models and clustering techniques12. Although methods based on deformable models allow for real-time applications with high computational efficiency, they are sensitive to the initial condition and local functional minimum, which may limit the applications in MR images with inherent noise and vague tumour border15. Compared with deformable models, clustering techniques such as K-mean and Fuzzy C-means (FCM) algorithms treat structures in medical images as patterns and use techniques from pattern recognition fields to perform segmentation13. Thus, clustering techniques have more applications for the medical images where the boundaries of structures or areas of interest are often poorly defined. In a recent study, spatial FCM algorithm was used to improve the accuracy in segmentation of different pathogenic region of glioblastoma multiforme (GBM) in multi-parametric  MRI fusion framework16.As the types of MR modalities involved in studies increase, accurate manual delineation of GTV in radiotherapy treatment planning becomes highly time-consuming and complicated for radiation oncologists, which may lead to intra- and inter-observer variability9.Nevertheless, accurate automatic segmentation of gliomas in multimodal MR images remains a challenging issue for three main reasons: (1) the tumour heterogeneous nature and the difference in image acquisition method make one tumour shows different shape, size and location in different image modality  is presented in this study for automatic segmentation of gliomas in radiation treatment planning. The new method provides each voxel with a possibility value belonging to the tumour which helps in preventing missing high risk regions in GTV definition in radiation treatment planning.In this study, all methods were carried out in accordance with relevant guidelines and regulations. All images were collected and used with the approval of the Ethics Committee of Tianjin University Biomedical Engineering and patients’ written consents. And informed consents were obtained from all of the patients.17. And the FA values would decrease in case of tumour infiltration because the reduction of the directional order of axonal tracts18. For rCBV values, they would increase in regions adjacent to the tumour because the tumour growth and invasion is dependent on developing blood supply19. Therefore, a glial tumour often presents as a relative bright region  in ADC and rCBV map, while a dark region  in FA map. However, these descriptions are very fuzzy and there is no commonly accepted threshold standard of these parametric values for precisely defining the tumour border. Thus, we considered transforming these fuzzy linguistic descriptions into mathematics fuzzy models and treating the definition of tumour extent as fuzzy image processing which understands and represents image features as fuzzy set20. For each modality (ADC/FA/rCBV), we created one mathematics fuzzy model that transforms the image volume into fuzzy feature space in which the fuzzy feature represents the possibility belonging to tumour9. Based on the fuzzy fusion result of the three fuzzy feature spaces, the auto-segmentation was conducted to generate the final result on functional multi-parametric image data.In assessing gliomas with functional multi-parametric images, commonly used descriptions about the appearance of gliomas in images by clinical radiation oncologists were considered. In ADC maps, the majority of image intensity attributable to tumour, necrosis and oedema in peritumoral area are higher than normal-appearing white matterAdditionally, conventional anatomical MRI image data (T1C and T2) was also included to improve the accuracy and reliability of final segmentation. FCM algorithm was used in the tumour segmentation in anatomical MRI image data.The framework of fuzzy fusion and tumour segmentation is shown in Fig. 21. In this study, the membership functions for each parametric value are fuzzy models for extracting fuzzy feature that represents the possibility of each voxel belonging to tumour. There are many methods for generating membership functions such as perception-based method22, heuristic methods23 and histogram-based methods24. For taking full advantage of the statistical probability distributions of multi-parametric values of tumour in functional maps, one histogram-based method was developed in the generation of membership functions which consists of following two steps.Fuzzy feature extraction: In the application of fuzzy set theory, estimation of membership functions from image data is an important step27 were added as samples, making that the total sample size for ADC values 32. The FA values in hypointense region such as solid enhancing tumour mass and tumour-infiltrated tissue can be used to generate samples of tumour18. On FA maps in our clinical datasets of 4 patients, 16 FA values  were measured by placing ROIs of 25 pixels in enhancing tumour and regions adjacent to enhancing tumour. Additionally, 5 FA values were generated in similar locations from 3 published articles28, resulting in that the total sample size for FA values 21. Because the rCBV values in and adjacent to tumour are often higher than in the corresponding contralateral areas, rCBV ratio values = rCBV [tumour]/rCBV [contralateral normal white matter] is usually used in evaluation8. 12 rCBV ratio values  were generated from our clinical datasets of 3 patients, in which the rCBV values were calculated in ROIs placed in solid enhancing tumour, hyperintense non-enhancing peritumoral area and contralateral normal white matter. Moreover, 20 rCBV ratio values were measured in similar locations on rCBV maps from 5 published articles29. Thus, the sample size for rCBV ratio value was 32.In order to avoid bias in sample collection conducted by a single institution, parametric values of tumour from our clinical multi-parametric MRI datasets and related research articles published by different institution were treated as samples for each modality. The clinical image datasets were obtained from 4 patients with biopsy-confirmed gliomas including 2 low-grade and 2 high-grade astrocytomas. Each patient underwent DWI, DTI and DSC imaging, the ADC, FA, rCBV maps were calculated respectively. The ADC values in hyperintense region such as solid enhancing tumour mass, tumour margin, infiltrating tumour, tumour-infiltrated oedema and infiltrated tissue are considered as reasonable representation of tumour. 16 samples  were generated from the 4 clinical image datasets (ADC maps) in which the ADC values were calculated in regions of interest (ROIs) with size of 25 pixels placed in non-cystic mass, tumour margins, areas of abnormal MR signal surrounding the enhancing tumours. In addition, 16 ADC values from 5 published articles30. The tumour membership frequency of each fuzzy interval of parametric value was represented using histograms and treated as probability distributions of membership functions for parametric values. Then, the histograms were modelled with a mixture of parameterized functions to generate the membership function. Curve fitting was applied in the definition of the most suitable mixture format of parameterized functions with low fitting error. The membership functions corresponding to ADC, FA and rCBV values are defined with following equations:Fuzzy statistics method was used to calculate the tumour membership frequency of parametric values using the samples of each modality as mentioned aboveour Fig. .Figure 39. The fusion process was conducted on voxel level to generate each voxel value in fused fuzzy feature volume noted as Fuzzy fusion of three fuzzy feature spaces: Isolated diffusion/perfusion parameters only reveal partial information about brain-tumour microenvironments and are insufficient to enable clinicians make crucial decisions throughout the whole treatment. To take advantage of multimodal fuzzy information and compromise the three memberships, the geometric mean operator was used in this study for fuzzy fusion of the three fuzzy feature spacesSegmentation after fusion: To extract a region containing voxels with high possibility belonging to tumour, segmentation was applied on FCM algorithm was applied for segmenting tumour in T1C and T2 MRI image data. The gliomas often show a contrast-enhanced region in T1C image which is much bright than normal-appearing peritumoral white matter and other brain tissues such as grey matter and cerebrospinal fluid (CSF). Thus, T1C images were classified into three clusters, including solid enhancing tumour, normal brain tissues and non-brain image pixels. In T2 images, the hyperintense area often reveals tumour existence because of the tumour-infiltrated oedema of gliomas. This area is bright than normal peritumoral brain tissues. Therefore, three clusters were classified in T2 images which include tumour region, normal brain tissues and non-brain image pixels.Some morphological processing operators were used to fill small holes and disconnect the tumour region with normal tissues in the same cluster. Afterwards, to merge adjacent pixels belonging to the tumour cluster, the region growing algorithm was applied with manually selected seed in the centre of tumour area. The union of region growing results in T1C and T2 images was used as the tumour extent definition in anatomical MR images and noted as The auto-segmentation results from anatomical and functional image data were used to generate the final auto-segmentation result of tumour volume which was formulated as following:2 and slice thickness = 5 mm; DWI images were acquired using spin-echo echo-planar sequence with TR/TE = 4900/85 ms, image matrix = 256 × 256, field of view = 240 × 240 mm2, slice thickness = 6 mm, number of slices = 30 and b-values of 0 and 1000 s/mm2 in three orthogonal directions; DTI images were acquired using spin-echo echo-planar sequence with TR/TE = 6000/85 ms, image matrix = 256 × 256, field of view = 240 × 240 mm2, slice thickness = 6 mm, number of slices = 19, diffusion-sensitizing gradient encoding applied in 25 directions with b value of 1000 s/mm2, and one image set acquired without diffusion-sensitizing gradients; PWI images were acquired by a gradient-echo echo-planar imaging sequence with TR/TE = 2000/40 ms, flip angle = 90°, image matrix = 128 × 128, field of view = 240 × 240 mm2, slice thickness = 6 mm, number of measurements = 60 and number of slices = 17. After the first 10 acquisitions, a bolus of gadopentetate dimeglumine (0.2 mmol/kg) was injected as the contrast agent with a flow rate of 3 ml/s, followed by a 20 ml saline flush. Finally an axial T1C imaging sequence was acquired using spin echo sequence with TR/TE = 520/14 ms, image matrix = 512 × 512, field of view = 240 × 240 mm2 and slice thickness = 5 mm.Imaging and preprocessing: Images for evaluation were acquired from 9 patients who were histopathologically diagnosed with gliomas (6 high-grade and 3 low-grade). The MR images were acquired for each patient on a 1.5 T MR scanner (GE Signa Excite), including T1C, T2, DWI, DTI and perfusion weighted (PWI) images. Whole-brain axial T2 images were acquired using fast spin echo sequence with TR/TE = 3900/92 ms, image matrix = 512 × 512, field of view = 240 × 240 mmMulti-parametric images were generated in a GE medical imaging workstation  by analysing the original DWI, DTI and PWI image sets. The ADC and FA maps were generated from DWI and DTI images on a voxel-level basis respectively after calculating the eigenvalues of diffusion tensor in each voxel. According to the indicator dilution theory for intravascular tracers, voxel-based rCBV maps were created by integrating the area under the concentration-time curve of the contrast agent with the measurement of arterial input function (AIF) to position the ROI on the most visible large contralateral arterial vessel.The 3D dual-modality rigid-body registrations were completed using the automatic image registration tool of MIM 5.2 . Based on the mutual information measure, the software mapped the secondary image sets of T2, ADC, FA and rCBV to the primary image set of T1C respectively.31. The combination result of the two regions was used as GTV on anatomical MR images and noted as −3 mm2/s and 1.11 × 10−3 mm2/s were delineated for high-grade and low-grade gliomas respectively and noted as 31. The delineation process was applied by comparing with contralateral normal brain tissue, and excluding the normal hyperintense region in ADC map such as CSF. For FA maps, compared with the contralateral normal white matter, the hypointense regions with FA value lower than 0.286 and 0.17 were contoured for high-grade and low-grade gliomas respectively and noted as 32. Before delineation on rCBV maps, rCBV ratio values = rCBV[tumour]/rCBV[contralateral normal white matter] were calculated for each voxel in and surrounding the contrast-enhanced tumour area shown in T1C and T2 images. The rCBV[contralateral normal white matter] was obtained by calculating the mean rCBV value in a ROI of 25 pixels placed in the contralateral and symmetrical normal white matter. Subsequently, in accordance with the rCBV ratio values of tumour mass shown in a published study, hyperintense regions with rCBV ratio higher than 5.18 and 2.32 were delineated for high-grade and low-grade gliomas respectively and noted as 33. The necrosis, cystic mass, large vessels and haemorrhage were excluded by referring to the T1C and T2 images. Based on the clinical experience of the observer, the GTV on functional multi-parametric MR image was obtained by integrating the three regions and noted as For evaluating the performance of the proposed framework based on fuzzy feature fusion method for auto-segmentation of gliomas, the GTV delineation on the same MR images was performed by one experienced radiation oncologist who was blinded to the auto-segmentation results. All GTV delineation results were reviewed and validated by another experienced radiation oncologist to minimize uncertainty and assure accuracy of the delineation. For each patient, GTVs were contoured on anatomical and functional multi-parametric MR image data respectively and combined to generate final result. On T1C and T2 images, the contrast-enhanced and hyperintense region was outlined and noted as 11. Additionally, sensitivity and specificity were calculated to evaluate the performance of auto-segmentation.The manual delineation results were used as the ground truth. Then, the quantitatively comparisons between manually delineated GTV (11:34:The volume difference between 11.Apparently, DSC values range from 0 to 1, in which 0 means total disunity of two volumes and 1 means the two volumes are equal and total unity in shape. The evaluations of sensitivity and specificity were conducted as following:The comparison of volumes between manually delineated GTV and automatically segmented GTV is summarized in Table Table High concordances between the contours of Our study has shown that the new fuzzy feature fusion method provides an effective and reliable way to use multi-modality diffusion and perfusion MR images in radiotherapy treatment planning of gliomas. The volumes and shape of GTV defined with this new method are satisfying for all 9 patients compared with the ground truth. Moreover, the mean sensitivity and specificity of the auto-segmentation is 0.87 and 0.98 respectively, revealing the high efficiency and reliability of the auto-segmentation frame work based on the new fuzzy feature fusion method.35. Furthermore, FA values do not carry sufficient information for differential diagnosis of low-grade and high-grade gliomas because there may be tumours that have a higher growth potential but still do not disrupt the organization of white matter fibres in the process of their local invasion, leading to no detectable reduction in FA values36. For rCBV value, it is just a single parameter of tumour behaviour. In addition to tumour vascularity, criteria such as mitotic activity, necrosis and nuclear pleomorphism are important in histological grading of gliomas37. Thus, there is not sufficient theoretical support from reported studies for constructing membership function for low-grade and high-grade gliomas respectively. For each modality, the fuzzy model created in this study can be used to define regions with high possibility belonging to tumour and differentiate tumour region from normal tissue.In the fuzzy feature extraction process, membership functions corresponding to ADC, FA and rCBV values were defined with mixed sample values from low-grade and high-grade gliomas, instead of generating membership functions for low-grade and high-grade gliomas respectively. Because isolated diffusion/perfusion parameters only reveal partial information about brain-tumour microenvironments and are insufficient to differentiate low-grade from high-grade gliomas. For example, ADC values provide only information about the movement of water molecules and may also be affected by various factors such as the density and composition of tumour cells, oedema, tumour necrosis, etc., which might result in inability to discriminate gliomas accuratelyet al. presented a fuzzy information fusion frame work and an average probability of correct detection 96% and an average probability of false detection 5% were obtained through studies of T1-weighted, T2-weighted and proton density images of four patients9. This perception-based method can generate fuzzy membership function with the prior knowledge about tumours described by experts, but lacks a general principle such as maximum likelihood to estimate probability density of input features21. Compared with this perception-based method, our study explored a histogram-based method to the generation of membership functions, which can provide information regarding the probability distribution of parametric values. Furthermore, the fusion method proposed by Dou et al. aimed at combination of structural MR images, which may not be suitable for functional multi-parametric MR images. Recently, FCM algorithm was used in the semi-automatic segmentation method to detect different regions of glioblastoma with the combination of information provided by T2 images, ADC and rCBV maps in a study proposed by Fathi Kazerooni et al.16. The results show over 80% sensitivity, specificity and dice score in the differentiation of various tumorous regions. However, the shapes of the fuzzy membership functions would be affected by the presence of noise in parametric MR maps21. Thus, pre-processing is an important step to deal with image noise before segmentation because the parametric maps are often with low resolution and high noise due to the long-time acquisition and calculation process. Different from this study, our new fuzzy fusion method employs membership functions to interpret parametric values with possibility belonging to tumour, instead of just treating them as image intensity and segmenting into separate clusters. As the results of evaluation indicate, the proposed method yields more than 0.85 of mean DSC, sensitivity and specificity in segmentation of gliomas, which is higher than the study mentioned above.Several auto-segmentation methods based on fuzzy theory are proposed to employ multi-modality MR images for the tumour extent definition of gliomas. Based on knowledge-based technique and fuzzy segmentation, Dou High accuracy in automatic delineation can be achieved with the proposed method which shows promising potential of utilizing functional multi-parametric images for GTV definition in precision radiation treatment planning of gliomas.