The clinical efficacy of the third Military Medical University formula (TMMU formula) for fluid resuscitation stage was evaluated to improve the treatment level of adult patients with extensive burns during the shock stage.  Retrospective analysis of the data of 55 patients undergoing fluid resuscitation according to the TMMU formula within six hours after burn injury. The following indicators were collected: (1) demographic and injury information; (2) fluid resuscitation information; (3) efficiency information, including cardiovascular function, liver function, renal function, coagulation function evaluation indicators, blood concentration, and average urine output index.  (1) In the first and second 24 hours after injury, the median fluid rehydration coefficient was 1.68 ml/kg·(%) TBSA and 1.15 ml/kg·(%) TBSA, the median ratio of crystal to colloid was 2.24 and 1.67, and the median urine output index was 0.75 ml/kg·h and 1.05 ml/kg·h, respectively. (2) The actual fluid volume during patient resuscitation is higher than the formula calculated volume, and this difference is more obvious in patients with burn area ≥80%. (3) In the second 24 hours, the value of the actual total fluid volume minus the formula total volume in the group with crystal to colloid ratio ≤2 was significantly lower than that in the ratio >2 group. (4) At 24 and 48 hours after injury, the cardiovascular function, liver function, renal function, and coagulation function were better than those before fluid resuscitation.  Early application of the TMMU formula for fluid resuscitation in adult patients with extensive burns is safe and effective, but the actual input volume often exceeds the volume calculated by the formula, especially in the second 24 hours after burn injury and in patients with larger burn areas. Increasing the colloid input volume can help reduce the total amount of fluid used for resuscitation. Burn shock is the main early complication of severe large-area burns. Active and effective shock prevention and treatment is essential to the successful treatment of patients with large-area burns . Fluid rThe Parkland formula is widely used in Western countries, and the TMMU formula is widely used in China. All formulas mentioned previously use burn area and body weight to predict the amount of fluid replacement and do not consider the impact of burn depth and other aspects . In clinThis study included the clinical data of early adult patients with extensive burns who were admitted to the author's hospital from January 2015 to December 2019 .(1) Inclusion Criteria. The inclusion criteria were as follows: patients with second- or third-degree burn area ≥50% total body surface area (TBSA).(2) Exclusion Criteria. The exclusion criteria were as follows:combined injury ,patients aged <18 years,delayed resuscitation (patients admitted to the authors' department to receive fluid resuscitation outside 6 hours after injury),patients burned by electrical contact with deep tissue muscle necrosis,patients who gave up treatment within 48 hours for personal reasons,patients who lost medical records.This study followed the principles outlined in the Declaration of Helsinki and was approved by the Ethics Committee of Southwest Hospital, Third Military Medical University  . This study was retrospective, and patients' informed consent was exempted.Liquid Recovery. After admission, all patients underwent fluid resuscitation in accordance with the principle of the TMMU formula. In the first 24 hours after injury, the amount of fluid was II° and III° burn area × weight (kg) × 1.5 ml ＋ water 2000 ml, the ratio of crystal to colloid fluid (RCC) was 2 : 1, half of the total fluid was injected in the first eight hours after injury, and the remaining fluid was injected in the last 16 hours. In the second 24 hours after injury, the crystal and colloid fluid amounts were half of those in the first 24 hours, and the water content remained unchanged. The crystal fluids were mainly Ringer lactate solution and 5% sodium bicarbonate solution, the colloid fluid was the same type of plasma , and the water fluid was 5% glucose solution.Liquid Volume Adjustment. The patient's heart rate, respiration, blood pressure, and peripheral blood oxygen saturation were monitored, the patient's hourly intake and outflow were recorded, the rate and volume of fluid rehydration were adjusted according to the patient's hourly changes in urine volume, and the urine volume was maintained at 0.5–1 mL/kg/h.Demographic and injury information, including sex, age, weight, injury factors, time to admission after burn, total burn area (TBA), and third-degree burn area, was collected.Fluid resuscitation information, including the actual input volume and formula predicted input volume of crystals, colloid, water, and total fluids during the first and second 24 hours after burn injury, was collected. The fluid rehydration coefficient (FRC) and RCC were calculated for the first and second 24 hours after injury /(weight × TBA)).Efficiency information, including heart rate (HR), hemoglobin (HB), hematocrit (HCT), mean arterial pressure (MAP), plasma osmotic pressure (POP), serum creatinine, blood urea nitrogen (BUN), lactic acid, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin and activated partial thromboplastin time (APTT) at the time of admission and at 24 hours and 48 hours after burn injury, was collected. The average urine output index (UI) was calculated for the first and second 24 hours after injury ).U test. P < 0.05 was considered statistically significant.All data were analyzed using SPSS 24.0 software . Due to the abnormal distribution, the results were reported as medians and interquartile ranges (IQRs), and nonparametric tests were employed. Paired data were compared by Wilcoxon signed rank test. Unpaired data were compared by the Mann–Whitney A total of 55 patients with early extensive burns were included in this study. The patients included 46 males (83.6%) and 9 females (16.4%), with a ratio of 5.1 : 1.0. There were 49 patients (89.1%) with third-degree burns, and the second-degree burn area ranged from 0 to 95% TBSA, with the median being 27.0  TBSA. The duration of admission ranged from 107 to 358 minutes, with the median being 242  minutes. The patients' ages ranged from 21 to 75 years, with the median being 45.0  years. The TBA ranged from 51% to 95% TBSA, with the median being 72.0  TBSA. The weight ranged from 42.5 to 85.0 kg, with the median being 63.0  kg. The causes of injury were flames in 44 patients (80.0%), scalds in seven patients (12.7%), arc burns in two patients, and chemical burns in two patients (3.6%). During antishock therapy, none of the 55 patients received vasoactive drugs, and none developed complications such as heart failure, abdominal compartment syndrome, and MODS.P < 0.05), while the actual colloid fluid volume was not significantly different from the formula-predicted volume (P > 0.05). In the second 24 hours after injury, the actual crystal, colloid, water, and total fluid volumes were all significantly greater than the formula-predicted volumes (P < 0.05)  TBSA, and the median RCC was 2.24. In the second 24 hours after injury, the median FRC was 1.15 ml/kg (%) TBSA, and the median RCC was 1.67 . In the  < 0.05) .a ≥ 80% group according to the burn area. In the first 24 hours after injury, the actual amount of total fluid volume in both groups was greater than the formula-predicted volume, and the actual total fluid volume in the ≥80% group was significantly greater than the formula-predicted volume (P < 0.05). However, there was no significant difference between the actual crystal and colloid volume and the formula-predicted volume, as well as the actual amount of total fluid volume and the formula-predicted volume in the 50% to 79% group (P < 0.05) (Patients were divided into a 50% to 79% group and 9% group . In the  < 0.05) .The RCC in the TMMU formula is 2. The patients were divided into two groups: RCC ≤ 2 group and RCC > 2 group. The results showed that, in the first 24 hours after injury, there was no statistical significance between the actual total fluid volume and the formula total fluid volume in the two groups. However, in the second 24 hours after injury, the difference value between the actual total fluid volume and the formula predicted volume in the RCC ≤ 2 group was significantly less than that in the RCC > 2 group; that is, more colloid input could reduce the total fluid volume in the second 24 hours after injury .P < 0.05). The patients' serum creatinine, BUN, lactic acid, ALT, and AST levels at 24 hours and 48 hours after injury were significantly lower than those at admission, and the liver and kidney function was significantly improved, and the difference was statistically significant (P < 0.05). At these three time points, the HR, MAP, and POP were all within the normal range. At 24 hours and 48 hours after injury, the APTT was not significantly different compared with those before rehydration (P > 0.05). At 24 hours and 48 hours after injury, the albumin was significantly lower than that at admission, and the difference was statistically significant (P < 0.05) (Tables All patients underwent fluid resuscitation according to the formula and passed through the burn shock period smoothly. In the first and second 24 hours after injury, the median UI was 0.75 ml/kg/h and 1.05 ml/kg/h, respectively. Further analysis of the three time points, namely, at admission, 24 hours, and 48 hours after injury, showed that the patients' HB and HCT levels at 24 hours and 48 hours after injury were significantly lower than those at admission, and the blood concentration was significantly improved, and the difference was statistically significant  Tables  and 7.Large-area severe burns are difficult to treat and are a focus of burn treatment. In the early stage, shock is caused by increased vascular permeability and extravasation of fluid , 10. TheThe formula for fluid replacement is of guiding significance to the fluid resuscitation of burn patients, but it cannot be mechanically replicated without various in the actual treatment process. In the current rehydration formula, patients' burn area and weight are the decisive factors of rehydration volume. In addition, factors such as burn depth and inhalation injury also play an important role in the loss of fluid in patients with large-area burns. Burn scholars have conducted a number of studies and proposed a number of new schemes for fluid resuscitation , 15. HowThere has always been a controversy about the components of the rehydration formula, which mainly include the crystal resuscitation scheme represented by the Parkland formula and the crystal colloid scheme, in which crystals and colloids are alternately supplemented. The Parkland formula is widely used in Europe and the United States, while the approach in China basically involves a crystal colloid scheme. As mentioned earlier, the actual volume delivered in the shock phase is generally greater than the formula-predicted volume, especially for burn patients who are administered only crystal fluid for resuscitation. The more severe the burn is, the greater the amount of fluid infusion is required. In the crystal scheme, excessive crystalloid input is prone to fluid creep, resulting in pulmonary edema, abdominal compartment syndrome, and other complications , 16. In In this study, we confirm that early application of the TMMU formula in adult patients with severe large-area burns is safe and effective. However, the actual fluid rehydration volume often exceeds the formula-predicted volume in clinical practice, especially in the second 24 hours after injury and in patients with larger burn areas; these results indicate that increasing the colloid input is beneficial to reducing the total amount of fluid rehydration administered. Of course, this study is a retrospective analysis, and its conclusions still need to be further verified by large-sample, multicenter clinical studies.


Craniopharyngioma is a rare brain tumour. Despite being histologically benign, it behaves aggressively and is often difficult to manage. Descriptive epidemiological data on the tumour is lacking in sub-Saharan Africa, and there is none for Zimbabwe. The tumour usually has a cystic component that has been raising interest in the past decade. Few studies have looked at the biochemical composition thereof. This study aims to give a landscape view of craniopharyngiomas (CPs) in Zimbabwe and then profile the biochemical properties of the cystic component of paediatric adamantinomatous craniopharyngioma.A prospective cohort study was done in Zimbabwe over a 2-year period to study the epidemiological distribution of craniopharyngioma and examine the biochemical composition of adamantinomatous craniopharyngioma cystic fluid in the paediatric population. Fifteen patients were recruited who had craniopharyngiomas, and of those, nine paediatric adamantinomatous craniopharyngiomas had fluid analysed for biochemical components. SPSS statistical package was used to analyse the data. Descriptive statistics were used for epidemiological data.The incidence of CP was calculated to be 0.53 per million person-years. Incidence among the paediatric population 0–14 years was 1.2 per 100,000 person-years. Several biological components were found to be elevated significantly compared to serum and cerebral spinal fluid (CSF). These are sodium, potassium, urea, alkaline phosphatase, phosphate, magnesium, albumin, gamma-glutamyl transferase, calcium, low-density lipids, and glucose.The incidence of CP in Zimbabwe is similar to the rest of the world. Some biochemical components have been noted to be markedly elevated in the cystic fluid and were mirroring serum rather than CSF in concentration. Craniopharyngioma (CP) is a benign, brain tumour that commonly occurs in the suprasellar region of the brain. It has a worldwide incidence of 0.5–2 cases per million persons per year. This represents 1%–4% of tumours in the paediatric population. In the United States alone, around 338 cases occur per annum. There have been no ethnicity or gender differences noted. Epidemiological data for this tumour are difficult, not only because the tumour is rare but also because most cancer registries compute data on malignant tumours omit CP although it is borderline malignant. Furthermore, in the international classification for the disease of oncology, CP is classified under one code even though we now know that it represents dual pathologies. Adamantinomatous craniopharyngioma occurs predominantly in the paediatric population with a peak age of 5–14 years and is of embryonal aetiology , and if there was any doubt, then the diagnosis was not made. Otherwise, a histological result was the gold standard.The study did not influence or affect the standard management of craniopharyngioma which included resection, debulking, and Ommaya shunt insertion depending on the decision of the managing surgeon and the particular patient.All patients presenting to the three main teaching hospitals in the country (only state institutions offering neurosurgery services at the time of the study) with a diagnosis of craniopharyngioma were considered for study. Liaison with all neurosurgeons in the country (eight at that time) facilitate access to those patients from the private institutions (no CP patients were seen in the private sector during the period of study). Hence, all patients seen in the country with craniopharyngiomas could be captured.Ethical approval was obtained from the Medical Research Council of Zimbabwe (MRCZ/A/1854). Informed consent was obtained from the participants or their guardians; assent was obtained for all children above the age of 7 years.The patients (or caregivers) were interviewed. Past hospital documentation that included imaging and histology was reviewed. Demographic, history, examinations, investigations findings, and management information were captured. Patients with missing data would be excluded.Plasma and sera were obtained from blood collected in well-labelled ethylenediamine tetraacetic acid and coagulant-free blood collection tubes, respectively. Serum was tested for biochemical components.CSF was collected during the insertion of a ventriculoperitoneal shunt when this was indicated for the treatment of the patient. After normal shunt insertion during the confirmation of the shunt working, some CSF fluid is allowed to drop out of the peritoneal end of the shunt.Cystic fluid was collected, from the Ommaya reservoir, during scheduled drainage of the fluid or it was collected during surgical resection of the tumour.The specimens were sent to a clinical pathology laboratory for biochemical analysis: urea and electrolytes, calcium, magnesium, phosphate, and liver function tests. All lipid measurements were performed in the same central laboratory. Serum concentrations of total cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), cholesterol, and triglycerides were also measured.The clinical management of the patients’ ailments was per hospital protocol, and the study did not interfere with their standard management.t-test or one-way ANOVA with Bonferroni multiple comparison tests were used. For non-parametric data, a Mann–Whitney or Kruskal–Wallis test along with a Dunn’s multiple comparison test was used. Comparisons of proportions were performed using the Fisher exact and Chi-square tests. Significance was accepted with p values of <0.05. Data were expressed as median (90% confidence limits) as appropriate. Incidence was calculated using the formula: Data were then captured into a software package SPSS  for analysis. Descriptive statistics were used to summarise the patients’ demographical and clinical data. To determine statistical significance for parametrically distributed data, a Of a total of 15 patients with craniopharyngioma, 9 patients were diagnosed with paediatric adamantinomatous craniopharyngioma and were recruited for fluid analysis in the study . The numOver the 2-year study period, a total of 15 patients were diagnosed. The Zimbabwe population was estimated at 14 million; hence, the incidence of craniopharyngioma was calculated to be 0.53 per million person-years. Incidence among the paediatric population 0–14 years was 1.2 per 100,000 person-years, which translates to five children in the 0–14 years age group are expected to present for the first time with craniopharyngioma annually in Zimbabwe.The following biochemical components were elevated in CP cystic fluid in comparison to the serum normal values: sodium, potassium, urea, alkaline phosphatase, phosphate, magnesium, albumin, gamma-glutamyl transferase (GGT), calcium, low-density lipoproteins, and glucose . In addiThis paper presents for the first time the incidence of craniopharyngiomas in the paediatric population in a sub-Saharan country, Zimbabwe. It was calculated to be 0.53 per million person-years and in accordance with global estimates. However, the incidence among the 0–14-year age group is twofold higher than in other countries, with five children expected to present with CP every year. This is very worrisome given that Zimbabwe is a low-income country with relatively low health services. It is plausible that this elevation may be that the actual incidence of CP tumours in sub-Saharan Africa is high and also have high mortality (preventing them from getting into adulthood). A follow-up study to determine 5- and 10-year survival of these patients is urgently required to confirm these data.Our study showed a male preponderance of 2:1. While American and Finland data showed no gender preference, a large hospital-based study in the United Kingdom reported a male preponderance with a ratio of 1.3–1.6 in the paediatric population . We noteStaphylococcus aureus and Salmonella (31).We report that CP cystic fluid had elements found in both CSF and serum, though the quantity was significantly elevated in the cystic fluid. This finding is significant as it is evident that the fluid is actively secreted by tumour components as opposed to past studies which suggested that the fluid was transudate. We report elevated urea and electrolytes compared to the serum values of the same participants. The level of sodium was reported to be twice as high as in the serum of CP patients. Sodium is reported to have antimicrobial properties, particularly in lactic acid-producing bacteria such as lmonella –20. Addilmonella –13. All lmonella –31.The CP cystic fluid is also termed machinery oil fluid particularly because of its elevated oil levels. Low-density lipoproteins (LDL) were 100 times elevated in CP cystic fluid compared to serum. In our study, cholesterol was 100 times elevated in the CP compared to serum. This finding can be potentially useful in spectrometry to diagnose CP in the absence of histology , 35. In To the best of our knowledge, amylase is being reported for the first time in craniopharyngioma cystic fluid in this study. Studies on the biochemical composition of the cystic fluid are scanty, and there have been reports of lipids, proteins, glucose, urea, creatinine, sodium, calcium, magnesium, phosphorous, chlorine, ALT, AST, and GGT, but none on amylase , 34, 35.The study strength is that the catchment area encompassed the entire country and we managed to confirm most of the diagnoses histologically. Some of the limitations of the study are that some patients, particularly those who seek help in traditional medicine or foreign countries, could have been missed. More specimens would increase the strength of the study; however, craniopharyngioma is classified as a rare disease, hence the small participant size , 2.Our study demonstrates for the first time the prevalence of CP in a sub-Saharan country, Zimbabwe, to be 0.53 per million person-years. This study demonstrates higher concentrations of sodium, potassium, urea, bilirubin, creatinine, cholesterol, and low-density lipoproteins in CP fluid compared to serum; this better understanding of the cystic fluid composition has a bearing on the further development of targeted diagnostics and therapeutics in this difficult to manage tumour.


The model of ideal fluid flow around a cylindrical obstacle exhibits a long-established physical picture, where originally straight streamlines are deflected over the whole space by the obstacle. Inspired by transformation optics and metamaterials, recent theories have proposed the concept of fluid cloaking, which is able to recover the straight streamlines, as if the obstacle did not exist. However, such a cloak, similar to all previous transformation-optics-based devices, relies on complex metamaterials with inhomogeneous parameters and is difficult to implement. Here we deploy the theory of scattering cancellation and report on the experimental realization of a fluid-flow cloak without metamaterials. This cloak is realized by engineering the geometry of the fluid channel, which effectively cancels the dipole-like scattering of the obstacle. The cloaking effect is demonstrated through the direct observation of recovered straight streamlines in the fluid flow. Our work sheds new light on conventional fluid control and may find application in microfluidic devices. By judiciously engineering the geometry of the fluid channel, an `invisibility' cloak able to make an object undetectable from fluid flow is realized experimentally. Ideal fluid flow around a cylinder is a fundamental problem discussed in many textbooks on fluid mechanics . Being iRecently, with the inspiring development of transformation optics and metamaterials, substantial interest has arisen in constructing invisibility cloaking devices that are able to hide an object from external physical fields. These kinds of transformation-optics cloaking devices were firstly proposed and realized for electromagnetic waves , and theScattering cancellation is another powerful approach for cloak design. Originally proposed for plasmonic particles in quasistatic electric fields , it has Here we apply the approach of scattering cancellation to fluid control, and construct a fluid-flow cloak that is capable of hiding a cylindrical obstacle without disturbing the straight external streamlines . The flU is the speed of the stream that flows uniformly in the Let us start with Fig. mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimThis similarity to electromagnetics implies that it is possible to construct a fluid cloak by applying scattering cancellation. Indeed, we can consider a cloak as shown in Fig. l cloaks ,34,35 thHowever, we still have two problems to tackle. Firstly, Equation  requiresmentclass2pt{minimTo tackle the problems mentioned above, we consider the creeping flow with a low Reynold number  a reference sample without any obstacle, (ii) an obstacle sample without a cloak and (iii) an obstacle sample with a cloak. The movie provided in Figure y-variations. The y-variations, as shown in Fig. y-variation is relatively large compared to the simulation as it is limited by the resolution in the measurement.The results for the obstacle sample with the cloak, which demonstrate the realization of fluid cloaking, are shown in Fig. xy plane around the obstacle, in fact lowered their heights in the z-direction; as a result, they disappeared in the video. After finishing the detour, the dyes in these streamlines returned to the initial heights when they left the cloaking shell region, and thus reappeared in the video.Interestingly, in Fig. y-coordinates (y − yref) in the absence and presence of the fluid-flow cloak, respectively. It can be seen from the y-coordinates in Fig. y-coordinates of streamline fronts were all flat, as shown in Fig. y-coordinates, it is difficult to tell the existence of the obstacle that was cloaked.It is worth emphasizing that, although straight streamlines can be recovered by the cloak as if the obstacle did not exist, it does not mean the streamlines are completely unaffected. This can be demonstrated by showing the time evolution of the streamline fronts. Using the homogeneous fluid flow as the reference, we extracted the positions  of the streamline fronts at different moments and subtracted the measurement results with and without the cloak by those of the 4 reference points in Fig. x-coordinates. As shown in Fig. x-direction, the measured curves of x-coordinates were less smooth compared to those of y-coordinates, but the relative positions, namely the time delay, among x-coordinates, were still apparent.However, the existence of the obstacle can still be revealed by monitoring the time evolution of x-coordinates for the obstacle sample with the cloak. Figure Now we check the time evolution of In contrast to the optical cloak, there is no overall size limit to the fluid-flow cloak as the fluid flow is always governed by the Laplace equation, which is independent of the size. In other words, the proposed approach can be applied to cloaking an object of arbitrary size and shape in a fluid channel. The only prerequisite of our proposal is that the height of the fluid channel is smaller than the radius of the obstacle as well as the width of the fluid chamber, in order to achieve the Hele-Shaw flow. This requirement makes our approach useful for microchannels where the height of the fluid channel is intrinsically small.In summary, our work demonstrates an innovative approach to realizing a fluid-flow cloak that can hide a cylindrical obstacle in a narrow fluid channel. In contrast to previous designs employing complex metamaterials, our fluid cloak adopts the scattering cancellation approach and is realized by merely adjusting the geometry of the fluid channel. Such a simple solution is applicable to any fluid flow that can be approximated as an ideal fluid flow. The proposed fluid-flow cloak can serve as a new strategy in fluid-flow control, especially in the application of microfluidic channels, where a Hele-Shaw flow takes effect. We envision that such fluid-flow cloaks can be used to minimize the distortion caused by devices  that have to be in contact with the fluid.The samples were manufactured through 3D printing. A piece of glass plate was placed on top of the samples to enclose the flow channel. The indicators, the glycerin dyed with black acrylic paint, were injected manually through four evenly distributed thin rubber tubes to visualize the streamlines. Apart from that, the background fluid was dyed with white acrylic paint to improve the visualization of the streamlines. The experiments were recorded by a camera from the top. The simulations were conducted in COMSOL Multiphysics.nwab205_Supplemental_FilesClick here for additional data file.


Primary hepatic neuroendocrine tumours (PHNET) are extremely rare. Currently, no evidence-based guidelines are available for PHNET treatment, especially for unresectable tumours.We present the case of a 43-year-old man who was admitted to our hospital with complaints of backache for more than 1 month. The imaging examination showed a 5.5×5.3 cm lesion in the liver and no extrahepatic lesions, which was confirmed as a grade 2 PHNET by the pathological results and exclusion of non-hepatic origins. A multidisciplinary team (MDT) consultation revealed that the lesion was an unresectable primary hepatic neuroendocrine tumour (uPHNET) but could be potentially treated by conversion surgery. The patient was initially administered four cycles of chemotherapy with temozolomide, 5-fluorouracil, and ondansetron, and was evaluated as stable disease (SD) according to the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1). Because of the limited clinical benefit of chemotherapy, the patient subsequently underwent transcatheter arterial chemoembolisation (TACE) treatment, which reduced the tumour size and converted uPHNET to resectable tumours. A complete response (CR) was achieved after surgery, and the patient has been disease-free.This case was reported by a patient with uPHNET who benefited from the pre-operative TACE, providing a potentially effective management strategy for refractory tumours. Neuroendocrine tumours (NETs) are rare tumours that originate from the diffuse neuroendocrine cell system, most commonly in the gastrointestinal tract, followed by the lung, and usually metastasize to the liver , 2. PrimA 43-year-old man visited our hospital in May 2019, presenting with backache for over 1 month. The relevant medical history included a smoking history of 20 packs/years. No alcohol consumption or history of diabetes or hepatitis. A physical examination revealed no abnormalities.The patient underwent abdominal contrast-enhanced computed tomography (CT) scan. The CT showed a 5.5×5.3 cm solid mass in the liver with a close relationship to the left portal vein and had no extrahepatic lesions (His preoperative laboratory data were as follows: direct bilirubin (DBIL) 6.0 umol/L , indirect bilirubin (IBIL) 13.0 umol/L , triglyceride (TG) 2.57 mmol/L , high-density lipoprotein cholesterol (HDL-CHO) 0.86 mmol/L , and low-density lipoprotein cholesterol (LDL-CHO) 3.62 mmol/L . Serum levels of tumour markers such as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were within normal ranges.The patient underwent oesophagogastroduodenoscopy and chest CT examination to identify extrahepatic lesions, which revealed normal findings. Because of the lack of obvious characteristics of the preoperative diagnosis, the patient underwent an ultrasound-guided core biopsy needle of the liver lesion 1 week after admission. Microscopic examination of the biopsy specimen (diameter: 0.3 cm) from the intrahepatic mass revealed tumour cells with abundant eosinophilic cytoplasm and uniform round nuclei. Immunohistochemical staining revealed that the tumour cells were positive for cytokeratin AE1/AE3, cytokeratin 18, synaptophysin (Syno), and chromogranin A (ChrA). Ki67 index was approximately 5%. A final diagnosis of NET (grade 2) was established.We recommended a PET-CT scan for systemic evaluation to rule out metastatic neuroendocrine carcinoma. One month later, the patient underwent somatostatin receptor scintigraphy  at an outside hospital . The results revealed high somatostatin expression inside the liver, and no abnormalities were found outside the liver, confirming the PHNET diagnosis.Considering the extent of the tumour lesions in both the right and left hepatic lobes, as well as the left portal vein, a multidisciplinary team (MDT) consultation showed that the lesion was uPHNET but could be potentially treated by conversion surgery.To downsize tumours and convert uPHNET to resectable tumours, the patient was initially administered four cycles of chemotherapy with temozolomide, 5-fluorouracil, and ondansetron on July 16, 2019. MRI re-examination was performed after chemotherapy on October 9, 2019, revealing no change in the tumour size, which was evaluated as stable disease (SD) according to RECIST 1.1   and suitable polyvinyl alcohol particles. The procedure was performed by an experienced vascular surgeon. After surgery, the CT results showed visible lipiodol deposition and necrosis of a few tumour masses, but some active lesions remained. TACE was possibly beneficial for patients in clinical evaluation. Therefore, the patient subsequently received three TACE treatments with lipiodol embolism (8 ml) and suitable polyvinyl alcohol particles for the same tumour lesion after the first TACE. CT re-examination or MRI results revealed further reduction in tumour size and marked tumour necrosis. Partial response (PR) status was evaluated according to the RECIST 1.1 criteria. Meanwhile, liver function and tumour marker levels were within normal ranges during the treatment and follow-up periods.Imaging results showed that the lesion had shrunk to an ideal size for surgery. Approximately 1 month later , the patient underwent left hepatic lobectomy, caudate lobectomy, and cholecystectomy and had an uneventful postoperative course. The resected specimen revealed a solid tumour measuring 3.2 cm × 3.0 × 2.8 cm, and the cut surface of the tumour was gray-yellow in colour with large regions of necrosis. Immunohistochemical staining revealed that the tumour cells were positive for AE1/AE3, CK18, Syno, and ChrA, with a Ki67 index of 5%  among all liver tumours . Due to 8% among PHNET is extremely difficult to diagnose precisely from imaging studies which display slight or significant arterial enhancement of the tumour parenchyma in the arterial phase followed by washout in the portal and/or late phases . Some caMoreover, tumour serum markers, such as CEA, AFP, and carbohydrate antigen19-9 (CA19-9), and inflammatory indices (erythrocyte sedimentation rate and C-reactive protein), are generally normal. Recently, Li et al. reported a case of PHNET in a patient with chronic hepatitis C and highly elevated AFP .A definite diagnosis of PHNET principally results from pathological examination and exclusion of other primary tumour sites , 4, 6. UThere were no significant differences in the pathological features of PHNETs and NETs in the digestive system. The tumour cells may be arranged in solid nests, ribbons, strips, and glandular tubes and may be accompanied by different degrees of blood sinus formation and interstitial fibrous tissue hyperplasia . The cytHowever, a definitive diagnosis of a liver mass requires the exclusion of metastatic NET. The pancreas is a common primary site for NETs . RecentlCurrently, no evidence-based guidelines are available for the treatment of PHNETs. Complete surgical tumour resection with negative margins is considered the optimal scheme , 6, 8. AHowever, our study had the main limitation of being a single case report with insufficient evidence to support the benefits of the treatment. The mechanisms of the biological behaviours of PHNETs and appropriate therapeutic strategies have not been clearly elucidated owing to the tumour rarity.In conclusion, the present case provides a potentially effective management strategy for uPHNET, which completely regressed with combined pre-operative TACE and surgery. The rare case showed that pre-operative TACE could be an appropriate approach to shrink refractory tumours. Further studies are required to confirm the effectiveness of this method.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XYZ, HRY, and HZL performed image acquisition and completed the manuscript. All authors contributed to the article and approved the submitted version.National Natural Science Foundation of China (30970839&31170957).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic– discrete element method (CFD‐DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional groups of microorganisms in the biofilm, enables the study of the contribution of EPS toward biofilm resistance to fluid shear stress. Furthermore, the stress–strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm. Our predicted emergent viscoelastic properties of biofilms were consistent with relevant experimental measurements. Deformation and detachment of biofilms with different extracellular polymeric substance volume ratios  under the same flow conditions. Bacteria are then irreversibly attached to the surface and develop cell–cell cohesion. Matured biofilms are embedded in extracellular polymeric substances (EPS) which are produced by bacteria themselves  and protein  which is an open‐source tool for individual‐based modeling of microbial communities  which provides a flexible interface between large‐scale atomic/molecular massively parallel simulator (LAMMPS)  of biofilm was developed by coupling the computational fluid dynamics approach (CFD) with the discrete element method (DEM). This model was implemented on NUFEB (23). Cyclic boundary conditions were also applied to the front and back walls to reduce computational effort. A no‐slip boundary condition was adopted on the bottom wall where the fluid velocity is zero. The details of cohesion among all the functional groups are discussed in Section The processes of biofilm growth and biofilm deformation were decoupled in this study, that is, fluid flow was applied to a pregrown biofilm. The pregrown biofilm was “grown” under static conditions for 5.3 days using the NUFEB individual based model which was described in Jayathilake, et al. . The kin2.1EPS/g CODS) which corresponds to EPS/biofilm ratio of 20%–51% here. To investigate the biofilm deformation and detachment events, the biofilm with 46% EPS was subjected to inlet flow velocity between 0.1 and 0.4 m/s  for a duration of 40 ms. In the next simulation, the inlet fluid velocity was kept at 0.3 m/s to study the effect of EPS production on biofilm deformation and detachment. The detachment rate coefficient, which is defined as the ratio of the volume of detached biofilm clusters to the total volume of preformed biofilm, was calculated during the initial 14 ms . Cluster detachment from the biofilm was defined as erosion if the particle number of the cluster was less than 1000 and sloughing if the particle number of the detached cluster exceeded 1000. The EPS amount, the mean and maximum heights, the roughness and porosity of different biofilms are summarized in Supporting Information  were selected to measure the deformation angle thus to obtain the averaged shear strain  and then stopped immediately. Afterward, the biofilm was allowed to relax for 17 ms (unloading cycle). During the loading period, the fluid shear stress was increased by increasing the fluid velocity from 0 m/s at a constant acceleration. For the biofilm with 46% EPS, deformation‐recovery tests were carried out by exposing the biofilm to the ramping flow with different accelerations: 20, 30, and 40 m/sl Figure . The shen Figure .2.3v⃗i is the velocity of the particle i; mi is the particle mass; f⃗c,i is the contact force among collided particles . U⃗s and U⃗fare particle velocity and fluid velocity, respectively. F⃗fp is the fluid–particle interaction force. ∇p is the pressure gradient, R⃗ is the stress tensor consisting of viscous stress and Reynolds stress, only viscous stress was computed since the Reynolds number is small here (3.75–15). The Eulerian field ϵs, U⃗s, and F⃗fp are calculated by averaging the Lagrangian information of particles . In this study, five different values of cohesive strength were used for the interactions of bacterial cells with bacterial cells, bacteria cells with EPS agents, bacteria cells with particle‐wall, EPS agents with the particle‐wall, EPS agents with EPS agents. Since EPS plays a significant role on binding the bacterial cells, the cohesive strength driven by EPS was assumed to be three orders of magnitude larger than that for bacteria  microcolonies moved downstream by rolling in a flow cell , the biofilm clusters could easily detach from the biofilm matrix (erosion‐dominated) at high frequency, accompanied by the escape of single bacterial cells Figure . This maThe detachment frequency of biofilm decreased with the increase in EPS amount. Figure The local detachment, such as erosion and sloughing, could be significantly captured within the time of 14 ms. However, biofilm removal occurred after this period when the inlet fluid velocity was greater than 0.2 m/s. Therefore, the detachment rate coefficient, which is defined as the ratio of the volume of detached biofilm cluster to the total volume of biofilm per millisecond, was calculated during the initial detachment period (14 ms) and adopted to describe the biofilms detachment behavior under the range of hydrodynamic conditions. Each simulation was run for three replicates and the average results were calculated. As displayed in Figure 3.32 and reached the maximum value (0.06 m/s) at 3 ms. The maximal deformation angle was captured at the same time  subjected to the ramping fluid velocity at a constant acceleration of 40 m/sFigure Bacillus subtilis . The result (p < 0.05) suggests that the change of shear modulus with EPS amount has a statistically significant difference.In general, all the simulated biofilms exhibited some strain stiffening effect followed by strain softening at larger strains, which is due to the viscoelastic properties and the change in the microstructure of biofilms during the deformation Figure . This isAfter the fluid was stopped, the stress on the biofilms decayed exponentially and the deformation recovered slowly which is a common feature for viscoelastic materials , the detachment rate increased with inlet fluid velocity . When the inlet flow velocity was below 0.1 m/s, the biofilm deformed along the fluid direction with sparse erosion. Biofilm sloughing occurred when the inlet flow velocity increased to 0.2 m/s. When the inlet fluid velocity reached 0.3 and 0.4 m/s, the detachment events were dominated by sloughing and the remainder biofilm layer was removed in a rolling motion.P. aeruginosa and Staphylococcus aureus reported in literature , the biofilm easily detached from the surface and dispersion of individual cells was observed. In these cases, the limited amount of EPS was incapable of protecting the biofilm bacteria from shear stress. Biofilm detachment frequency decreased with the increase of EPS amount. The biofilms were stiffer at higher loading rate, which is a typical characteristic for viscoelastic materials. Such viscoelastic features of biofilms also led to the hysteresis loop (energy dissipation), which was predicted by the stress–strain curves in our simulations and experimental measurements (Rupp et al., Yuqing Xia, Pahala G. Jayathilake, Paul Stoodley, and Jinju Chen designed the research. Yuqing Xia performed the simulation work and acquired the data. Yuqing Xia and Jinju Chen did data analysis. Pahala G. Jayathilake, and Bowen Li contributed to data analysis. Yuqing Xia, Pahala G. Jayathilake, and Jinju Chen prepared the original draft. Jinju Chen and Pahala G. Jayathilake provided the overall guidance of the work. All the authors contributed to the writing of the manuscript, revised it, and approved the final version.The authors declare no conflict of interest.Supporting information.Click here for additional data file.


Intravenous fluid administration is an essential part of perioperative care for children receiving a kidney transplant. There is a paucity of evidence to guide optimal perioperative fluid management. This study aimed to identify the volume of perioperative fluids administered across 5 UK paediatric kidney transplant centres and explore associations between fluid volume administered, graft function, and fluid-related adverse events.st January 2020 and 31st December 2021 were included.Data were collected from five UK paediatric kidney transplant centres on perioperative fluid volumes administered, and incidence of pulmonary oedema, systemic hypertension, and requirement for intensive care support. Children < 18 years of age who received a kidney-only transplant between 1p < 0.001). Association between urine volume post-transplant and day 7 eGFR was also negative (p < 0.001). Adverse events were frequent but no significant difference was found in the fluid volume administered to those who developed an adverse event, vs those who did not.Complete data from 102 children were analysed. The median total volume of fluid administered in 72 h was 377 ml/kg (IQR 149 ml/kg) with a high degree of variability. A negative relationship between total fluid volume administered and day 7 eGFR was noted (This study describes a high degree of variability in perioperative fluid volumes administered to children receiving kidney transplants. Both fluid volume and urine output were negatively associated with short-term graft function. These data contrast traditional interpretation of high urine output as a marker of graft health, and highlight the need for prospective clinical trials to optimise perioperative fluid administration for this group.A higher resolution version of the Graphical abstract is available as Supplementary informationThe online version contains supplementary material available at 10.1007/s00467-022-05690-3. Kidney transplantation is the treatment of choice for children with kidney failure . Kidney Ensuring adequate perfusion of a transplant kidney is important as intravascular volume depletion brings risk of delayed graft function and acute tubular injury. Delayed graft function in adult recipients is associated with reduced graft survival . Whilst Whilst inadequate fluid administration may impair graft perfusion, superfluous fluid intake increases the risk of fluid overload with consequential respiratory compromise and hypertension. Respiratory compromise may result in unexpected paediatric intensive care admission and the need for ventilatory support. Systemic hypertension may result in end-organ damage to sensitive vascular beds in the brain and lungs for example. It lies with the healthcare professional team to determine the appropriate volume of fluid to administer to children following kidney transplant.Current fluid management practices in the immediate postoperative phase following kidney transplantation in children have not been described in the literature. Differing strategies are known to be used, including insensible losses plus ml for ml replacement of urine output and fixed target fluid administration. This study aims to describe the volumes of fluid administered perioperatively across 5 large UK paediatric kidney transplant centres and explore if there are associations between the fluid volume administered with overall graft health or the incidence of adverse events including pulmonary oedema, hypertension, and requirement for intensive care support.Anonymised perioperative data for children under 18 years of age transplanted between 1 January 2020 and 31 December 2021 in five UK paediatric nephrology transplant centres was collated retrospectively. Those with incomplete data regarding fluid intake and urine output in the first 3 postoperative days were excluded from the analysis.Data were fully anonymised at individual hospital sites and ethical principles adhered to throughout. The study received local ethical approval, and the requirement for individual participant consent was waived.st January 2020 and 31st December 2021. Anonymised data were collected by the lead clinician at each hospital via a standardised spreadsheet.Participants were identified by a lead clinician at each hospital and included all children who had received a kidney transplant between 1th centile for age and height on two or more consecutive readings [Systemic hypertension was defined as systolic blood pressure above the 95U) were used for statistical comparisons. A P value < 0.05 was considered statistically significant.Continuous data are summarised as median (interquartile range) or median (range). Non-parametric tests . Potential confounders including diuretic dose, use of inotropes, type of transplant, and age were evaluated using non-parametric tests as appropriate.All statistical analyses were performed using SPSS Statistics for Windows version 28.A total of 113 patients were identified from 5 transplant centres. Eleven were excluded due to incomplete data; thus, 102 were included in the final analysis. Forty-two (41%) were female; the median age at transplant was 12.5  years. Fifty-six (55%) were living donor and 46 (45%) deceased donor transplants. A range of ethnicities were represented .The median volume of fluid administered intraoperatively was 66 ml/kg (IQR 52 ml/kg) Fig. .Fig. 1VoThe daily fluid volume administered decreased from a median of 148 ml/kg (range 23–786 ml/kg) on day 1 to a median of 70 ml/kg (range 9–194 ml/kg) on day 3 postoperatively .Children weighing less than 20 kg received a median of 507 ml/kg total fluid volume, compared with 356 ml/kg for children ≥ 20 kg (p < 0.001).Intraoperative fluid volumes were significantly greater for children < 20 kg .Postoperative fluid administered (excluding intraoperative fluid) was also greater for children under 20 kg  children received furosemide intraoperatively with a median dose administered of 1.5 mg/kg (IQR 1.1). Twenty-nine children (28%) were given mannitol at a median dose of 500 mg/kg (IQR 156). Postoperatively furosemide was given to 39 (38%) children with a median dose of 1 (IQR 1.1) mg/kg.Inotropes were administered intraoperatively in 68 (67%) children and postoperatively in 39 (38%) children.All grafts survived.2. The median total urine output over the whole operative period was 255 ml/kg (IQR 156 ml/kg). The total fluid volume administered exceeded urine output by 131 ml/kg  .The median estimated glomerular filtration rate at day 7 was 84 (IQR 57.6) ml/min/1.73 m2 (IQR 60) at day 7, compared with 70 ml/min/1.73 m2 (IQR 42.7) in those ≥ 20 kg (p < 0.001).Children weighing < 20 kg had median eGFR 135 ml/min/1.73 mp < 0.001); children who received higher fluid volumes had lower day 7 eGFR. A similar negative relationship between the total amount of urine output and the day 7 eGFR was found (p < 0.001) , however, did not explain the negative relationship between eGFR and fluid volume.A negative relationship between total fluid administered and day 7 eGFR was observed (01) Fig. . NeitherThree children experienced delayed graft function. All three received deceased donor kidneys. Two were > 20 kg with an unknown aetiology to their kidney failure; the third was < 20 kg with an underlying diagnosis of CAKUT. None received inotropic support but all three received intraoperative and postoperative diuretics. All experienced hypertension and two were admitted to PICU. All three received less than the median volume of fluid seen in the whole cohort (377 ml/kg) with the < 20 kg child receiving 283 ml/kg in total, and the > 20 kg children receiving 362 ml/kg and 238 ml/kg respectively.Systemic hypertension was the predominant adverse event in 53 52%) children. Oxygen requirement and confirmed pulmonary oedema affected 31 (30%) and 14 (14%) children respectively. Nine (9%) patients had an unexpected admission to paediatric intensive care (PICU)  with a negative skew, suggesting that increasing volumes of fluid occur with decreasing day 7 eGFR. This observation was not related to diuretic dose or inotrope administration. This result contrasts findings of the 2021 retrospective analysis by Porn-Feldman et al. [Notably, we have found a significant difference between the total amount of fluid given per kg and day 7 eGFR  participants, and restriction to five kidney transplant centres. Notwithstanding these limitations, complete data from a defined 2-year study period on 102 children clearly illustrates the current variation in practice and questions the validity of urine output as a marker of short-term graft health.In conclusion, there is a substantial variability in the volume of fluid administered to children receiving kidney transplants in current UK practice. A negative association between fluid volume administered and short-term graft function was also observed. It also calls into question the value of using urine output as a key marker of graft function. The retrospective nature of this data precludes the determination of causal relationships, but it does emphatically highlight the need for high-quality prospective clinical trials to optimise perioperative fluid administration in children receiving kidney transplants.Graphical Abstract(PDF 190 kb)


Emerging evidence indicates that immunogenicity plays an important role in intrahepatic cholangiocarcinoma (ICC). Herein, we systematically evaluated the clinical relevance of immunogenicity in ICC.Highly immunogenic ICCs identified in the public dataset and the Cancer Immunome Atlas (TCIA) were assessed to determine the prognostic impact of immunogenicity in ICC and key components after curative resection. We also investigated the clinical relevance of the immune milieu in ICC.+/forkhead box P3 (FoxP3)+ tumour-infiltrating lymphocytes (TILs), T-cell immunoglobulin and mucin domain 3 (TIM-3) and human leukocyte antigen-A (HLA-A) in highly immunogenic ICCs. Immunohistochemical analysis of the in-house cohort showed that intratumoral FoxP3+ TILs correlated with CD8+ TILs  and that high FoxP3+/CD8+ ratio (FCR) was an important marker for poor survival . Furthermore, the FCR was higher in tumour-free lymph nodes in ICCs with lymph node metastases than in those without lymph node metastases .Using the Gene Expression Omnibus dataset 89749 and TCIA, we identified CD8FCR should be considered an important biomarker that represents the immune environment of ICC based on its potentially important role in tumour progression, especially lymph node metastasis. The incidence of intrahepatic cholangiocarcinoma (ICC), which accounts for 10–20% of all primary liver cancers, is increasing worldwide . Thus, t+, CD8+ and CD45RO+ tumour-infiltrating lymphocytes (TILs) are observed in around 50% of the tumours [+ TILs in ICCs [Uncovering the role of tumour immunogenicity in many malignancies has led to the development of immunotherapeutic strategies, which are considered a novel treatment option in ICC as well . Tumour  tumours , indicat in ICCs –12, and  in ICCs . Howeverhttps://tcia.at/) based on data from The Cancer Genome Atlas and completed the immunogenic characterisation of 20 solid tumours [Several bioinformatics approaches, including single-cell and bulk RNA sequencing, have enabled the identification of immune cell populations within the immune milieu , 15. Aar tumours .To elucidate the characteristics of highly immunogenic ICCs and the relationship of tumour immunity with tumour biology, we performed a comprehensive analysis of the immune milieu in ICC. First, we characterised highly immunogenic ICCs based on metagene analysis using a publicly available dataset and TCIA. Second, we assessed the prognostic impact of tumour immunity after liver resection in patients with ICCs. Finally, we determined the clinical relevance of the immune milieu in ICC.A total of 61 consecutive patients who underwent curative hepatic resection for ICC between January 1, 1999 and December 31, 2015 in Okayama University Hospital were enrolled in this study. The study samples comprised primary lesions from 61 patients with ICC and corresponding lymph nodes from 47 of the 61 patients. The baseline clinicopathological variables, including age, sex, aetiology, location, macroscopic morphology, differentiation, tumour size, serosal invasion, tumour number, preoperative carbohydrate antigen 19-9 and carcinoembryonic antigen levels and tumour stage are summarised in Table The study comprised an initial discovery phase followed by an independent clinical validation phase. In the discovery phase, we identified highly immunogenic ICCs and deregulated immune components using the publicly available Gene Expression Omnibus (GEO) microarray database (GSE 89749)  and the 2O2 for ten minutes, antigen retrieval was performed with ethylene-diamine-tetraacetic acid buffer (pH 9.0). Thereafter, the slides were incubated with Protein Block Serum-Free Ready-to-Use  for ten minutes followed by anti-CD8a antibody or anti-FoxP3 antibody . Thereafter, the slides were incubated with indicated primary antibodies  using immunohistochemistry. Manual immunostaining was performed using formalin-fixed paraffin-embedded tissues. Briefly, 4 µm-thick sections of representative blocks were deparaffinised and dehydrated using gradient solvent washes. Following endogenous peroxidase blockade using 3% HThe slides were evaluated under the guidance of a pathologist without reference to the patient’s clinical profile. First, we defined intratumoral and stromal TILs as lymphocytes within the tumour nest or in direct contact with tumour cells and within the tumour stroma, respectively, following the recommendations of the International TILs Working Group 2014 . Second,U or Wilcoxon signed-rank test. The correlation was estimated using Spearman’s rank correlation coefficient. OS was calculated from the date of surgical resection to the date of death due to ICC or last follow-up for censored patients. OS was estimated using the Kaplan–Meier method and compared by the log-rank test. All P-values were calculated using two-sided tests, and a P-value of < 0.05 was considered to indicate statistical significance.All statistical analyses were performed using EZR  , which ihttps://tcia.at/) and determined the immunophenoscore of each ICC as an indicator of immunogenicity based on the z-score of four immune components, namely effector cells, suppressor cells, immune checkpoint molecules, and MHC-related proteins. Figure + tumour-infiltrating lymphocytes (TILs) as effector cells, tumour-infiltrating regulatory T cells (Treg) as suppressor cells, TIM-3 as an immune checkpoint molecule and HLA-A, based on the immunophenoscore analysis.To characterise the immune milieu in ICC, we first performed a comprehensive analysis of the ICC immunogenicity using the RNA sequencing data from the publicly available GSE 89749 dataset , which c+ PD-1+ TILs and CD4 + FoxP3+ TIL counts in the stroma and intra-tumour in nine ICC patients selected based on high TILs in hematoxylin and eosin staining  and 2.0% (IQR 0.3–3.6%) of CD8+ TILs, respectively. Because CD8+ PD-1+ TILs, exhausted CD8 TILs, were much less observed compared to CD8+ PD-1− TILs, CD8 could be used as a marker of activated CD8+ TILs in ICC. In addition, stromal and intratumoral FoxP3+ CD4+ TILs in ICC accounted for almost 100% of FoxP3+ TILs, indicating that FoxP3 could be used as a marker of Treg TILs. Based on these results, for further analysis, we used CD8 and FoxP3 as a marker of activated CD8+ and Treg TILs in ICCs, respectively.Not only the immunogenicity of the entire tumour but also the local immune response to the tumour can impact tumour biology such as that observed in breast cancer . To eluc+ and FoxP3+ TILs in the same flied using immunohistochemistry in the clinical validation cohort. The median number of CD8+ TILs was significantly higher in the stroma than in the intratumoral area in the same section , CD8+ TIL counts were significantly correlated with FoxP3+ TIL counts in the intratumoral area ; however, there was a trend of correlation between CD8+ TIL counts and HLA-A expression . These results indicated that FoxP3+ TILs might mainly function as suppressors within the tumour, which is a characteristic of highly immunogenic ICCs.Finally, we assessed the intratumoral expression levels of TIM-3 and HLA-A in ICC using immunohistochemistry. While moderate or intense HLA-A expression was observed in ~66% of the ICCs, 54% of the examined ICCs exhibited absent or weak TIM-3 expression  to classify patients into low and high-FCR groups using the cut-off value generated using X-tile analysis: the cut-off value of FCR was 0.22. Interestingly, the OS of high-FCR ICCs was worse than that of low-FCR ICCs . In this study, both TILs counts were higher in stroma than those in intra-tumour. These results suggest that ICC inhibits CD8+ TILs infiltrating to intra-tumour by inducing Tregs in the stroma.We found that FoxP3ur cells ; therefour cells . Intereseristics , consistltration . Scott e+ and CD8+ TILs, i.e. the FCR, was associated with poor prognosis and lymph node metastasis in ICC. To date, not only the CD8+ and FoxP3+ TIL counts but also the FCR has been reported as prognostic predictors in several malignancies, such as non-small cell lung carcinoma, breast cancer, and gastroenterological malignancies [+ TILs in the tumour immune milieu compared with the number of CD8+ TILs was shown to contribute to poor OS and refractoriness to chemotherapy [+ TIL count in intra-tumour of ICC have poor survival. These results suggest that inhibition of cytotoxicity of CD8+ TILs through FoxP3+ TILs in intra-tumour could more affect the tumour biology than blockade of CD8 + infiltrating to intra-tumour by Treg in the stroma.In the current study, we also found that the ratio between FoxP3gnancies –30. In agnancies , 31. In otherapy , 31. Addotherapy  in agree+ TILs in the metastatic lesion and the corresponding primary tumour in breast cancer have been reported [+ TILs were less accumulated in metastatic sites than in the corresponding primary tumour, and concluded that immune escape could accelerate tumour progression [+ TILs were also less observed in metastatic lymph nodes than in the primary tumour . This result was concordant with the previous reports, and indicated that the tumour cells escape from immune surveillance. In addition, Zhou et al. illustrated that tumour-infiltrating Tregs selectively migrate to tumour-draining lymph nodes [+ TILs were more accumulated in metastatic lymph nodes than in the primary tumour . High FoxP3+ TIL counts were reported in metastatic lymph nodes in other malignancies such as non-small cell lung carcinoma, breast cancer and cervical cancer [+ TILs.To the best of our knowledge, this is the first study showing that FCR is associated with lymph node metastasis in ICC. While there have been no reports concerning about immunogenic meaning of FCR in the metastatic lesion and primary tumours, several studies focusing on CD8reported , 33. Ogigression . In thisph nodes . Indeed,l cancer –38. Intrl cancer  and suggl cancer . Similarl cancer , and ICCl cancer . Our fin+ TILs with immune checkpoint blockade [+ TILs could be a target for therapy in the future.Recent efforts to improve prognosis and OS rates in patients with malignancies have been devoted to the development of therapeutic approaches targeting FoxP3blockade . Althoug+ and FoxP3+ TIL counts remain to be determined similarly to an issue with previous studies. Further studies in larger cohorts are needed to validate the utility of CD8+ and FoxP3+ TIL counts as prognostic and predictive biomarkers in ICC.In the present study, the small sample size due to the rarity of ICC was a major limitation. Additionally, this was a single-centre study. However, the analysis of the immune milieu in the primary tumour as well as in the corresponding lymph nodes was a strength of our study. We also acknowledge that the cut-off CD8+ and FoxP3+ TILs might contribute to ICC progression by forming a premetastatic niche in lymph nodes. These data highlight the critical role the FCR might play in cancer biology and suggest that CD8+ and FoxP3+ TILs might be considered therapeutic targets in ICC.In conclusion, we used complementary analyses to demonstrate the potential utility of intratumoral FCR as a prognostic marker in ICC. Furthermore, this is the first study to reveal that the balance of CD8Supplementary dataBJC-A3339392 checklist


We focused on GFAP, a putative biomarker for astrocytic tumours and glioblastoma multi-formes (GBMs). In silico modelling was paired with experimental measurement of cell GFAP concentrations and used to predict the tumour volumes and identify key parameters which limit detection. The average GBM volumes of 449 patients at Leeds Teaching Hospitals NHS Trust were also measured and used as a benchmark. Our model predicts that the currently proposed GFAP threshold of 0.12 ng ml−1 may not be suitable for early detection of GBMs, but that lower thresholds may be used. We found that the levels of GFAP in the blood are related to tumour characteristics, such as vasculature damage and rate of necrosis, which are biological markers of tumour aggressiveness. We also demonstrate how these models could be used to provide clinical insight.Brain tumours are the biggest cancer killer in those under 40 and reduce life expectancy more than any other cancer. Blood-based liquid biopsies may aid early diagnosis, prediction and prognosis for brain tumours. It remains unclear whether known blood-based biomarkers, such as glial fibrillary acidic protein (GFAP), have the required sensitivity and selectivity. We have developed a novel  CurrentAstrocytomas are the most common type of glioma. One biomarker for astrocytic tumours is glial fibrillary acidic protein (GFAP) –5, which−1 in most astrocytomas (74%), but was detected in most GBMs (89%) [−1 for optimized detection of GBMs [−1 [−1 reaching 11.4 ng ml−1) [−1 has been suggested for distinguishing all grades of glioblastomas from healthy individuals [Prospective studies have investigated using GFAP as a blood-based biomarker for astrocytomas. Serum GFAP was not detected above the analytical sensitivity of 0.08 ng mlst GBMs 8% 9]. Re of GBMs ,9. Altho. Re of G−1 associated with significantly lower survival-free prognosis [Serum GFAP has been linked to prognosis, with GFAP greater than 0.2 ng mlrognosis . Serum Grognosis . They alrognosis ,5,9,10. rognosis ,9–11.in vivo data, especially from the earlier stages of tumour growth, makes quantification challenging. To overcome this, a range of experimental and mathematical modelling techniques have been used to understand tumour growth dynamics [Brain tumour growth is complex and heterogenous, and it remains difficult to quantify. A lack of dynamics –15. Sevedynamics ,15. One dynamics . There hdynamics . Howeverdynamics ,17,18. Tdynamics –21 and tMathematical modelling offers a powerful way of scanning a wide range of different scenarios in a time- and cost-efficient way. It can also offer additional mechanistic understanding and in the future may be used alongside current clinical methods to guide more effective treatment strategies e.g. ,22,23).,23.22,23Mathematical modelling has been used to explore the use of blood-based biomarkers for several different cancers –26. HoweWe developed mathematical models which describe dynamic serum biomarker kinetics in relation to brain tumour growth. In this study, we focused on the CNS-specific intermediate filament protein GFAP ,28. We i. 2. 2.1n = 483). The average volume at which these GBMs were diagnosed was calculated. Formal ethics approval was granted under the project and license—enhancing understanding and prediction of cancer outcomes with baseline characteristics from routinely collected data (IRAS ID. 277122). Baseline data included patient age, sex and WHO performance status .The neuro-oncology multi-disciplinary meeting records at Leeds Teaching Hospitals NHS Trust between 2014 and 2020 were retrospectively reviewed, and all adult patients (16 years and over) with histologically proven GBMs were included , diffusion weighted, T1 pre- and post-gadolinium sequences (Gd-T1) and a volumetric T1-weighted sequence post-gadolinium. Enhancing tumour volume was estimated following the established protocol for GBMs which is most effective for their irregular shapes . Orthogo. 2.2et al. [We measured the average GFAP of human glioma stem cells (G144 GSCs) and human GBM cells (U251). To quantify the average GFAP per cell, we used a standard protocol to quantify GFAP expression using western blotting and protein quantification as described by Wang et al. .2.The G144 GSCs were gifted by Dr Steve Pollard (University of Edinburgh) and cultured as monolayers in serum-free basal media  with added supplements . The U251 commercial human GBM cells  were cultured in Minimal Essential Media  supplemented with 10% fetal bovine serum  and 1% MEM non-essential amino acid solution . Both G144 and U251 were cultured at 37°C in 5% COg for 3 min. The supernatant was transferred to fresh microtubes and stored at –20°C.Experiments were repeated for three different passages of each cell line and all presented errors are standard errors. Cells were pelleted per passage and each pellet was pipetted vigorously with 100 µl of lysis buffer . The lysate was incubated on ice for 15 min in 1.5 ml microtubes  before being centrifuged using a VWR MICROSTAR 17R at 4°C and 17 000 × icroplate Manager® software.The protein concentrations of the lysates were determined relative to eight known concentrations of bovine serum albumin  using the Pierce™ bicinchoninic acid protein assay kit , following the 96-well plate protocol. The absorbance at 570 nm was measured using an iMark™ Microplate Reader  and the accompanying MThe protein quantities of samples were determined using western blotting. A standard curve was created  and used to determine the protein content of the lysate. This was then combined with the average number of cells in the lysate to calculate the quantity of GFAP per cell. Full details of the western blot protocol can be found in the electronic supplementary material, methods, but briefly lysate samples were run alongside serial dilutions of protein standard  with a molecular weight protein ladder at each end . The gel was placed under a voltage of 150 V for 1 h to separate the proteins using PowerPac™ basic power supply (BioRad).α Tubulin . After incubation with specific secondary antibodies conjugated to peroxidase (Sigma) proteins were visualized by Clarity ECL substrate (BioRad) using the BioRad Chemidoc XRS + system and analysed using Image Lab software (BioRad). Electronic supplementary material, figure S2, shows an example of a western blot used for GFAP quantification. The intensity of the bands, which are proportional to protein levels, was quantified using ImageJ software.The protein was transferred to a membrane and blocking was carried out. Membranes were then immunoblotted with the following antibodies: GFAP Polyclonal Rabbit antibody  and . 2.3in silico model for brain tumour serum biomarker kinetics with three main components TU(t)) and healthy tissue (HUHK), as well as the rate at which the biomarker is degraded (γ). The mass of biomarker in the blood (pQ) is given byThe levels of biomarker in the blood were assumed to depend on the input from the tumour  and TK, HK are the corresponding fractions entering the blood. The concentration of biomarker in the blood is then equal to pV is the volume of blood. The serum GFAP concentration of healthy individuals (HC) is given by where TV(t) is the volume of the tumour at time (t). To model the growth of necrosis, we assume that necrosis occurs after a specific tumour onset volume (Vn0) is reached. This volume corresponds to the onset time Tn0. We assume that necrosis also follows Gompertzian growth [NV(t)) is given byTVmax, nVmax are the tumour and necrotic plateau volumes, TV0 is the initial volume of the tumour (in this case taken as the volume of a single cell) and NR, TR are the initial growth rates. These volumes can be converted into corresponding numbers of cells using the average cellular densities. This Gompertzian growth model has been shown to be a good approximation of brain tumour growth [A range of growth models have been proposed to describe tumour growth ,15. Incln growth  so that r growth ,19,20. H. 2.4In this study, we focus on the CNS-specific intermediate filament protein GFAP. The key components of our dynamic model for GFAP are tumour and necrotic growth, biomarker production and the fraction entering the blood .−1), possibly due to secondary tissue necrosis. However, for astrocytic tumours, it is generally accepted that GFAP is primarily produced via tumour necrosis [Necrosis is a hallmark of tumour progression in astrocytic tumours. During necrosis, GFAP is released into the interstitial fluid (IF) . GFAP hanecrosis –36 and sTU(t)) would take the formNQ is the average mass of GFAP per dying cell, NN is the number of necrotic cells which is related the necrotic volume (NV) and the average necrotic cell density (Nδ) by NN = NδNV, nNmax is the maximum number of necrotic cells which is given by nNmax = nVmaxNδ and Tn0 is the time necrosis starts in the tumour.For a tumour with GFAP produced via necrosis, the tumour production term ((TK) is dependent on anything which affects the transport of biomarker into the bloodstream, as well as the removal or decay of the biomarker from the tumour. The breakdown of the glymphatic system, which is usually responsible for the rapid clearance of GFAP from the brain, may lead to enhanced retention of GFAP in the tumour [Vk0/Tk0) is reached and can be approximated as a Hill function, so that TK(t) is given byKmin and Kmax are the minimum and maximum TK fractions, h is the Hill coefficient and Tk0 is the time at which the BBB is first compromised. We assume that when the BBB is intact (prior to tumour growth), the fraction of GFAP which enters the blood is zero (Kmin = 0) and that TK and TU are related, as necrosis occurs due to hypoxia, which is associated with blood vessel deformation. We therefore assume that changes in TK begin at the same time as necrosis starts so that Tn0 = Tk0.After GFAP has been emitted into the IF, it may enter the blood either via BBB disruption  or via te tumour ,39. Thise tumour . Therefodt) and detection volume (dV). These define the time and volume at which the serum GFAP crosses the GFAP cut-off threshold limit used to identify GBM patients and so give the time and volume at which a tumour would be detected at the current threshold. If the parameters in our model change, the detection time and volume will also change. We define this detection volume change as Vd2 is the detection volume for the second set of parameters and Vd1 is the detection volume for the original set of parameters.If we simulate the dynamic serum GFAP as a function of time or tumour volume for a given set of parameters in our model, then we can obtain the corresponding tumour detection time . This model may be used to obtain a profile for a specific tumour based on its parameters, or to predict global averages based on average parameters for GBMs. We parametrize our model based on the experimental data available. For each parameter, we use the current experimental data to obtain global averages as well as ranges observed across GBMs . We use To assess the impact heterogeneity and parameter changes have on detection, we ran sensitivity analyses. A combination of local and global sensitivity analyses was performed depending on the parameters. Local sensitivity analysis involves varying one parameter while average values are assumed for the remaining parameters. This has the limitation of only exploring first-order effects of the parameters on the model outcome. There are limited experimental data on certain parameters in our model and some of our model parameters are inextricably linked. This led us to run certain parameter combinations globally as discussed below. In the future, given further experimental data it would be possible to also use additional techniques for global sensitivity analysis of all the parameters and further explore the relationship between parameters and their impact on detection.in vivo data of 106 untreated glioblastomas [4 cells ml−1) and necrotic (4800 × 104 cells ml−1) regions of the tumour were used to convert between volumes and cell numbers [The parameters defining Gompertzian growth in GBMs were obtained from lastomas . The hetlastomas ,18. Prev numbers ,45. We u numbers .TVmax, TR), and for each set of these parameters, we derived an associated set of possible necrotic growth parameters  from the range mentioned in the literature  are not well quantified (equation (2.5); Kmax, h). However, the processes involved, such as the perfusion , have been quantified for gliomas using a range of methods (e.g. dynamic perfusion CT and dynamic MRI) [TK that match the experimental data simultaneously and assess these combined affects.The parameters which govern the fraction of GFAP able to enter the bloodstream due to tumour growth (mic MRI) ,43,46,47mic MRI) ,10. WhenHC) [γ) [The remaining parameters   and the CH) [γ) ) are not. 3. 3.1We model the dynamic changes in serum GFAP observed with tumour growth for a ‘typical’ GBM. This was achieved by using population averages to parametrize the model. This allowed us to obtain average values, for example, of detection volume. However, we note that GBMs are incredibly heterogenous which we examine in the next section. We combine previously published average parameters with our own experimental data  (see MatNQ) was determined invitro. We found that the average mass of GFAP per G144 glioma stem cell was 1.7 × 10−4 ± 0.3 × 10−4 ng and per U251 GBM cell was 3.1 × 10−4 ± 0.8 × 10−4 ng.The quantity of GFAP in a single cell (−1) and corresponding detection tumour detection time (dt) and volume (dV) are also shown. The levels of GFAP only start to increase above the baseline level of healthy patients after the onset of necrosis and after the fraction entering the blood increases. Therefore, the tumour volume at which these changes occur  at a −1 [−1 ,5,9,10. To compare these average detection volumes with the volume at which GBMs are currently detected, the average GBM volumes for 449 patients at Leeds Teaching Hospitals NHS Trust were measured. −1), serum GFAP cannot be used to detect GBMs earlier than current methods. However, by lowering this threshold, for example, to the current analytical sensitivity and suggested cut-off for glioblastomas (0.08 ng ml−1), it may be possible to use serum GFAP for early detection of GBMs.Tumour size and volume were available for 449 patients—the median maximum enhancing tumour diameter was 4.2 cm (range 0.5–8.8 cm) and the median enhancing tumour volume was 23.6 ml (0.06–186 ml). Our results therefore suggest that using the critical cut-off threshold for GBMs (0.12 ng ml. 3.2DV) at the threshold cut-off limit of 0.12 ng ml−1. For a given patient, the detection volume will depend on that patient's baseline parameters  and on the fundamental characteristics of that patient's tumour .There is large heterogeneity in the growth and behaviour of different GBMs, leading to analogous variations in the tumour detection volume (DV) and therefore assess its impact. Full descriptions of the different sensitivity analysis techniques performed can be found in the Material and methods. For the parameters defining the fraction of GFAP entering the blood VD) across the possible (TK) parameter combinations varied from 20 to 58 ml giving a change in detection volume of Sensitivity analysis can be used to examine the effect that changing a parameter will have on the detection volume , necrotic growth (NV) and GFAP per cell (NQ)) influence the detection volume at the critical detection cut-off limit of 0.12 ng ml−1. The tumour and necrotic growth are closely related. In order to satisfy the condition that TV > NV, we only used corresponding tumour and necrotic growth parameter combinations (ranges listed in dV) is related to the ratio between tumour and necrotic growth. GFAP is produced as a consequence of necrotic growth not tumour growth and although the two are related it is possible to have a tumour which grows faster without having faster necrotic growth. As shown in figure 7a, if this occurs, the tumour will grow more before detection and be detected at a larger volume.NR) and the volume at which necrosis and the increase in the fraction entering the blood occur (RN = 0.013 cells day−1) and average changes (RN = 0.005) and late changes (It can be seen in CH) and degradation rate (γ) are not tumour dependant but still affect the detection volume. Running simultaneous sensitivity analysis on both these parameters for an ‘average tumour’ (d = 20 ml). This means that baseline differences across patients could have a dramatic impact on detection. As seen in The patient's baseline healthy concentration , for all tumours, the levels of GFAP initially follow the same trend, increasing with volume. Also, if a given tumour is not detectable until it is larger, this will be because the volumes associated with necrosis and the damage to the vasculature all occurred at larger volumes and/or that the rates of these were slower. The exception to this is tumours that exhibit very low GFAP expression. Therefore, even though serum GFAP is not always a reliable predictor of tumour volume, it is, beyond this exception, a good measure of BBB breakdown and necrosis. Both of which are key indicators of tumour severity –52.. 3.3TK and TU which give rise to serum GFAP levels above the current cut-off limit  then detection requires high production rates TU > 2000 ng day−1. Assuming an average quantity of GFAP per cell, this is equivalent to 3 × 106 cells day−1 dying or a volumetric death rate of 0.04 ml day−1. Different parameter combinations can explain such a fast rate, but all combinations would require rapid death of a significant volume. On the other hand, if the BBB is severely compromised (TK = 0.4), then significantly lower production rates, TU > 700 ng day−1, are required for detection. This would be equivalent to 9 × 105 cells day−1 dying or a volumetric death rate of 0.02 ml day−1. This equates to a far slower death of a much smaller volume. Previous studies [Zero values of  studies ,48 sugge. 4in silico model to further understand the use of blood-based biomarkers for brain tumours. We focused on the putative biomarker GFAP and primarily on its use as a blood-based biomarker for GBMs.We developed a novel −1 cannot be used for earlier detection of GBMs. However, for tumours with early onset of characteristics associated with poor prognosis (necrosis and vasculature damage), serum GFAP may be used for earlier detection. If the limit was lowered to the current analytical sensitivity and suggested critical threshold for all glioblastomas (0.08 ng ml−1), the average GBM would be detected at 17 ml—resulting in earlier detection. Lowering the detection limit comes at a cost to specificity; at the limit of 0.08 ng ml−1, a reduction in the specificity to other tumours was observed, but the specificity to healthy patients remains high (92.4%). This limit should therefore be explored further experimentally. As improvements are made to the testing of serum GFAP, this limit may be lowered and so earlier detection of GBMs using serum GFAP is likely.We found that the relationship between serum GFAP kinetics and tumour growth strongly depends on the tumour. We used our model to assess the use of GFAP as a blood-based biomarker for the earlier detection of GBMs. We found that on average the currently suggested critical cut-off serum GFAP detection limit for GBMs of 0.12 ng mlThere are three key requirements for elevated serum GFAP levels in brain tumour patients . First, GFAP must be present in affected cells. Second, there must be a mechanism for GFAP release (e.g. necrosis) and finally there must be a means for GFAP to pass into the blood. If not, then serum GFAP will not be detected. This sets a fundamental limit on early detection.Our model predicted that in all cases where GFAP is detected at the current cut-off limit the patient would benefit from further diagnosis. In most cases, the GFAP levels scale with tumour severity and so several different cut-off limits may be implemented. These results match prospective experimental studies which have shown that GFAP levels scale with patient prognosis ,9.Our model predicts that the exception to GFAP levels scaling with tumour growth and severity occurs late in GBM growth when levels may begin to decrease. It has been shown previously that GFAP expression is influenced by astrocytoma grade . GFAP poNR) and the tumour volume at which necrosis first occurs and that the BBB begins to break down (Vn0/Vk0). The WHO has classified glial tumours according to characteristics, such as necrosis and vascular perfusion, as well as genetic features [Our model was used to show that the two parameters which have the largest impact on the volume at which a specific GBM would be detectable are the rate of necrosis (features ,50,54. OIt may be possible to account for some patient heterogeneity and improve the accuracy of current assays by taking dynamic measurements. We have shown that parameters which are dependent on the patient rather than tumour characteristics, such as the patient's healthy baseline GFAP serum levels and the GFAP decay rate, also impact detection. Taking dynamic measurements could significantly improve the ROC allowing us to lower the critical detection limits. This could be especially powerful at the earlier stages of necrosis onset when the increase in necrosis is rapid and GFAP expression heterogeneity is lower ,28,48. TIn the future, it may be possible to quantify heterogeneities and incorporate errors into our models and predictions for GFAP blood levels for different tumours. We have shown how mathematical modelling can be used to explore different scenarios. For example, we have shown how a specific patient's parameters may be used to predict best- and worst-case scenarios. Mathematical modelling can be combined with GFAP liquid biopsy results alongside other clinical data, such as scans, histological data and molecular profiling, to provide clinical insight. Integrating liquid biopsy with other clinical techniques could allow early detection and diagnosis for personalized precision treatment ,56. To bWe propose that serum GFAP levels may be especially useful in diagnosing early GBMs when used alongside other diagnostic methods. For example, our modelling showed that if imaging and histology fail to reveal necrosis, but the GFAP is very high, this may suggest several scenarios that clinicians need to consider. First, necrosis may have just begun, but be rapid. Second, the tumour may be causing peripheral brain damage resulting in GFAP release and damage to the BBB. Finally, there may be extensive vasculature damage, which could be confirmed, for example, via dynamic contrast-enhancing MRI ,60,61.It may also be possible to pair our models with data on tumour-induced brain deformation and damage ,63 to asIn the future, with a better understanding of the processes which govern biomarker transport through brain tumours and into the bloodstream, it will be possible to incorporate tumour heterogeneity and transport dynamics into more complex and realistic models. To build a more accurate model, further quantification of processes, such as the breakdown of the BBB, would be required.We have presented a generalized form of our model and framework to allow it to be adapted to any blood-based biomarker for a range of brain tumours. Our model describes how to build a model for any biomarker using information on biomarker production via the brain tumour and healthy tissue. The key model components for biomarker production via a brain tumour are tumour growth, biomarker production and the fraction of biomarker that can enter the bloodstream. These can be applied to any brain tumour, for any mechanisms of biomarker production using whichever models are most appropriate. The baseline level of serum biomarker can be obtained for any biomarker by measuring control groups. We have focused on GFAP as it is currently the serum biomarker with the most experimental data allowing model parametrization, but as more biomarkers and experimental data emerge, our model can be easily adapted and parametrized for a range of biomarkers.in silico models may be used to further understand the current limitations, uses and strategies for blood-based biomarkers for brain tumours. We have also shown how experimental work and clinical data can be used to enhance the model's relevance. The mathematical techniques we have developed could also be used more generally in the development and clinical interpretation of liquid biopsies for brain tumours.We are at the early stages of determining how blood-based biomarkers may be used to diagnose and monitor brain tumours. We have shown how simple