In clinical practice, carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 are the most common markers measured before and after surgery for gastric cancer (GC). However, which pre- or post-operative combined tumour markers (CEA and CA19-9) have more prognostic value remains unclear.Consecutive patients undergoing a resection for GC at the Fujian Medical University Union Hospital were included as a discovery database between January 2011 and December 2014. The prognostic impact of pre- and post-operative tumour markers was evaluated using Kaplan–Meier log-rank survival analysis and multivariable Cox regression analysis. The results were then externally validated.P < 0.001). Multivariable analysis revealed that the number of pre-operative positive tumour markers was an independent prognostic factor (P < 0.05). For patients with abnormal pre-operative tumour markers, normalisation of tumour markers after surgery is an independent prognostic protective factor (hazard ratio (HR) = 0.618; 95% confidence interval (CI) = 0.414–0.921), and patients with both positive post-operative tumour markers had double the risk of overall death . Similar results were observed in the internal validation and external validation cohorts.A total of 735 and 400 patients were identified in the discovery cohort and in the validation cohort, respectively. Overall survival rates decreased in a stepwise manner in association with the number of pre- and post-operative positive tumour markers (both Pre-operative tumour markers have a better discriminatory ability for post-operative survival in GC patients than post-operative tumour markers, and the normalisation of tumour markers after surgery was associated with better survival. Thus, by integrating other significant prognostic factors, such as tumour markers, the individual prognosis of patients can be better assessed.3 In clinical practice, carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 are the most commonly used markers for the early diagnosis and monitoring of GC. These markers have been confirmed to be associated with prognosis and recurrence of GC after surgery.5 However, previous studies mainly focused on the pre-operative level of CEA and CA19-9.8 There are rare and conflicting studies evaluating the prognostic value of post-operative tumour markers.10 Therefore, the purpose of this study was to determine whether pre- or post-operative combined tumour markers (CEA and CA19-9) are more prognostic. Specifically, we asked whether patients with pre-operative positive tumour markers that normalise after gastrectomy had a better survival than patients with post-operative positive tumour markers.Gastric cancer (GC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide.7 our inclusion criteria were as follows: (1) a histologically confirmed adenocarcinoma of the stomach; (2) no evidence of tumours invading the adjacent organs , paraaortic lymph node enlargement or distant metastasis demonstrated by abdominal computed tomography and/or abdominal ultrasound and posteroanterior chest radiographs; and (3) a D1/D1 + /D2 lymphadenectomy with a curative R0 resection. The exclusion criteria were as follows: (1) metastatic disease (n = 58), (2) neoadjuvant chemotherapy (n = 75), (3) malignant disease of other organs (n = 100) and (4) lack of pre- and post-operative tumour marker (CEA and CA19-9) data (n = 920). Finally, 735 patients were included in the study as the discovery cohort  was recommended to the majority of patients with advanced GC.13 Additional external validation was performed using a dataset from Affiliated Hospital of Qinghai University between January 2012 and December 2016, which satisfied the aforementioned inclusion and exclusion criteria. Finally, 400 patients were included as the validation cohort.Patients who consecutively underwent curative resection for stage I to III GC from January 2011 to December 2014 at Fujian Medical University Union Hospital (FMUUH) were enrolled in this study. As described previously,7 pre-operative tumour markers were measured within 1 week before gastrectomy. Post-operative tumour markers were measured in the serum sample obtained at the first visit, between 1 and 2 months after gastrectomy.14 A cut-off value of 5 ng/ml was used to establish positivity for CEA, which was determined by previous study.16 The cut-off value of 37 U/ml was used to establish positivity for CA19-9.16 Therefore, patients were grouped according to the number of positive tumour markers as follows: (1) patients with 0 positive tumour markers (negative for both CEA and CA19-9); (2) patients with 1 positive tumour marker (either positive for CEA or CA19-9); (3) patients with 2 positive tumour markers (positive for both CEA and CA19-9). The clinical characteristics and survival rates were analysed according to the number of positive tumour markers. Patients with 0 positive tumour markers were defined as normal tumour marker group, and patients with 1 or 2 positive tumour markers were defined as abnormal tumour marker group. Additionally, the normalisation of tumour markers was defined as the change from abnormal tumour marker group before surgery to normal tumour marker group after operation.As described previously,17 Overall survival (OS) was defined as the time from surgery to death from any cause or to the time of censoring on the date of the last follow-up. Cancer-specific survival (CSS) was calculated from the date of surgery to the date of death from GC.18Post-operative follow-up evaluations were performed every 3 months for 2 years and then every 6 months from years 3 to 5. The final follow-up evaluation was conducted in December 2018. Most routine follow-up appointments included a physical examination, laboratory testing , chest radiography and abdominopelvic ultrasonography or computed tomography, along with an annual endoscopic examination as described previously.χ2 test or Fisher’s exact test, whereas differences in continuous variables between groups were analysed using the Mann–Whitney U test. Survival curves of different groups were estimated and compared by Kaplan–Meier method and log-rank test. The Cox proportional hazards regression model was applied to perform univariable and multivariable analyses. First, for all patients in discovery cohort (FMUUH), pre- and post-operative tumour markers with traditional clinicopathological variables were included in the univariable and multivariable analyses, and identified that pre-operative tumour markers were independent prognostic factors of survival. Second, for patients with positive tumour markers before surgery, post-operative tumour marker response and the number of positive tumour markers after surgery with traditional clinicopathological variables was included in the univariable analyses. The variables associated with survival with P < 0.05 and post-operative tumour marker response were included in the multivariable analysis 1. The variables associated with survival with P < 0.05 and the number of positive tumour markers was included in the multivariable analysis 2. The time-dependent receiver-operating characteristic (t-ROC) curves were used to compare the prognostic abilities of the pre- and post-operative tumour markers.19 Lastly, internal validation was performed by simple bootstrapping, applying resampling with replacement 10,000 times.20 Moreover, we validated our findings in an independent cohort. All tests were two sided with the significance level set at P < 0.05. All data were analysed using SPSS, ver. 22.0  and R ver. 3.5.2 (R Project). The R packages “timeROC” were used for calculation of the time-dependent ROC analysis.Categorical variables were compared using the In total, 1135 patients were identified in the study. Of these patients, 735 were included in the discovery cohort and 400 in the validation cohort. For the discovery and validation cohorts, the median time to follow-up was 57.0 and 47.0 months, respectively, and the 5-year OS rates were 66.8% and 68.3%, respectively. Furthermore, the 5-year CSS of the discovery cohort and the validation cohort was 68.8% and 72.7%.2. Based on the TNM staging system, 81 (11.0%), 220 (29.9%) and 434 (59.1%) of the patients had stage I, II and III disease, respectively. Additionally, 463 (63.0%) patients received adjuvant chemotherapy after surgery. Compared with the discovery cohort, patients in the validation cohort were significantly associated with younger age, increased BMI, smaller tumour size and pathological findings, such as differentiated histological type, an earlier TNM stage, and vascular and perineural invasion : 54–67 years) and a mean body mass index (BMI) of 21.7 kg/mP < 0.001), male sex (P < 0.001), proximal tumour location (P = 0.004), larger tumour size (P < 0.001) and a more advanced TNM stage (including T stage and N stage) . Similarly, an increased number of positive tumour markers after surgery was also associated with older age (P = 0.003), larger tumour size (P = 0.010) and a more advanced TNM stage (including T stage and N stage)  patients with no positive tumour markers, 189 (25.7%) patients with one and 34 (4.6%) with two positive tumour markers. After gastrectomy, 591 (80.4%) patients had no positive tumour markers, 131 (17.8%) patients had one and 13 (1.8%) had two positive tumour markers. Table n = 0: 73.6%, n = 1: 53.5%, n = 2: 37.8%, P < 0.001, Fig. n = 0: 69.8%, n = 1: 55.7%, n = 2: 38.5%, P < 0.001, Fig. P < 0.05, Table n = 1: hazard ratio (HR) = 1.401, P = 0.022; n = 2: HR = 1.850, P < 0.001), but not the number of post-operative positive tumour markers , 48 patients (9.4%) had abnormal tumour markers after surgery. The survival rate of patients with normal tumour markers after surgery was similar to that of patients with abnormal tumour markers after surgery , but not with other clinicopathological data , but both were worse than that of patients with normal pre-operative tumour markers , and patients with two positive tumour markers after surgery had a more than 2-fold increased risk of overall death compared with that of patients with normalised post-operative tumour markers  patients had normalised post-operative tumour markers, and 96 (43.0%) patients had positive tumour markers after surgery. The normalisation of tumour markers after surgery was only associated with N stage , but not the number of post-operative positive tumour markers  patients with no positive tumour markers before surgery, 85 (21.3%) patients with one, and 15 (3.8%) with two positive tumour markers before surgery. After the operation, 344 (86.0%) patients had no positive tumour markers, 49 (12.3%) patients had one and 7 (1.8%) had two positive tumour markers. In the univariable analysis, an increase in the number of pre- and post-operative positive tumour markers was associated with shorter OS  had normalised post-operative tumour markers and 36 (36.0%) had non-normalised post-operative tumour markers. Multivariable analysis revealed that normalisation of tumour markers after surgery was an independent prognostic factor for OS  has been widely concerned in liquid biopsies. Recently, many studies have investigated the use of ctDNA for prognostic value in gastrointestinal cancers.P < 0.05), suggesting that patients with a higher risk had post-operative tumour marker testing. The timing of post-operative tumour marker measurement was not controlled, although it was limited to between 1 and 2 months after surgery. Second, there were significant differences of clinicopathological features between discovery and validation cohorts. However, the prognostic values of tumour markers were consistent in the two cohorts. Lastly, in the early period, CA72-4 was not the recommended tumour marker for routine testing pre-operatively in China.31 Therefore, the data of CA72-4 in our study are incomplete, which may affect the prognosis of GC. Despite these limitations, our study demonstrated that the prognostic value of pre-operative CEA and CA19-9 exceeds that of post-operative CEA and CA19-9 and validates our findings externally in an independent cohort. Routine measurement of post-operative tumour markers is warranted for patients with pre-operative positive tumour markers.This study has several limitations. First, as a retrospective study, it may have been subject to selection bias. For example, patients who had post-operative tumour markers measured were more likely to have stage III disease (59.9% vs. 46.9%, Supplementary files


Postoperative acute kidney injury, respiratory dysfunction, and infections occurred in 14.0%, 19.7% and 28.1% of cases, respectively. The median LOS in ICU was 20 h and the hospital-LOS was 10 days. Positive intraoperative fluid balance (2.4 ± 1.0 L) resulted in a significant increase of TBW (1.4 ± 2.4 L) and of ECW (1.4 ± 1.2 L). Intraoperative fluid balance significantly correlated with TBW change  and with AFO change . A significant correlation was found between pre- and postoperative AFO and RFO on one hand, and ICU-LOS on the other. BIA may be a useful tool for the perioperative assessment of volume status.Water retention and intercompartmental redistribution occur frequently in association with adverse postoperative outcomes, yet the available strategies for non-invasive assessment are limited. One such approach for evaluating body water composition in various circumstances is bio-electrical impedance analysis (BIA). This study aims to appraise the usefulness of the Body Composition Monitor  in assessing body fluid composition and intercompartmental shifts before and after open major abdominal surgery. This prospective, clinician blinded observational study enrolled all the patients scheduled consecutively for elective major open abdominal surgery during a 1-year period starting from January 1st, 2016. BIA parameters—total body water (TBW), extracellular water (ECW), intracellular water (ICW), absolute fluid overload (AFO), and relative fluid overload (RFO) were measured before and after surgery. The results were compared with fluid balance and outcome parameters such as organ dysfunction, ICU-and hospital length of stay (-LOS). The study population included 71 patients aged 60.2 ± 12 of whom 60.6% men and with a BMI of 26.3 ± 5.1 kg/m While intraoperative fluid administration is common with patients undergoing surgery, views differ with regard to the type, timing, and volume of the actual intraoperative fluid therapy –3. IntraTraditionally, significant amounts of fluid would be infused during the perioperative period , 6 beforAvoiding too much or too little intervention in the perioperative period is a long-recognized goal, but guidance is currently vague: exercise caution with regard to dosage, type, rate, duration, and timing of fluid administration.The most common early occurrence following major abdominal surgery is fluid overload . Water eRegarding the best method for assessing volume status, there is still ongoing debate. Clinical signs are a poor and late indicator of fluid volume and intercompartmental distribution. For intravascular space evaluation, there are several recommended strategies using non-invasive, minimally invasive or invasive monitoring, but few that address extravascular water. Bio-electrical impedance analysis (BIA) assesses body composition and estimates total and extracellular water volumes based on the tissue’s capacity to conduct electrical impulses. BIA is currently recommended for appraising fluid status in hemodialysis patients. Some data regarding BIA use in critically ill patients , sepsis In this study, we evaluate the usefulness of the Body Composition Monitor device  for the assessment of body fluid composition and intercompartmental shifts before and after open major abdominal surgery. We compare BIA results with intraoperative fluid balance and with outcome parameters: postoperative organ dysfunction, ICU- and hospital length of stay (LOS).We conducted a pilot prospective, clinician blinded, observational study at the “Sf. Spiridon” Emergency University Hospital Iasi, Romania for a 1-year period starting January 1st, 2016.The study was approved by the Research Ethics Committee of the “Sf. Spiridon” Emergency University Hospital Iasi . Before enrolling, all the patients signed an informed consent which included written information about the nature of the study, its aim, the expected advantages and possible risks, and also data protection assurances.All the patients meeting the inclusion criteria were enrolled consecutively during the aforementioned period. The inclusion criteria were: (1) age > 18 years old, (2) open major abdominal surgery, (3) elective surgery. Exclusion criteria were also applied: (1) patient’s refusal, (2) laparoscopic surgery, (3) emergency surgery, (4) conditions that interfered with making accurate BIA measurements: limb amputation, metallic cardiac or joint prostheses, cardiac pacemakers or stents, decompensated cirrhosis.The principle on which bio-impedance is based is that different tissues have different conductive and resistive properties when a small electric current is applied at different frequencies. The impedance of the body consists of two components: (1) resistance, which is proportional to fluid volume, and (2) reactance, meaning the reciprocal of the capacitance of cell membranes. At a low frequency (5 kHz), impedance indicates extracellular fluid because current does not flow through cell membranes. At frequencies above 100–200 kHz, the current will penetrate the cell membrane and impedance will reflect both extracellular and intracellular fluids .Extracellular and intracellular resistance values are obtained based on the Cole model  while TBThe BIA device also displays absolute fluid overload (AFO), the difference between normal, expected ECW and the actual, measured ECW, expressed in liters, as well as relative fluid overload (RFO), absolute fluid overload/extracellular water ratio (AFO/ECW), expressed in percentages. A negative AFO points to the patient’s underhydration while a positive one indicates overhydration. Based on RFO values, a patient’s hydration status was classified into three categories: dehydrated (RFO < − 10%), normohydrated (− 10% ≤ RFO ≤+ 15%), hyperhydrated (RFO > 15%).In our research, BIA was assessed using the Body Composition Monitor  according to the manufacturer’s recommended technique. First, the patient was placed in supine position on the hospital bed without touching metal objects, with the limbs in slight abduction and palms placed flat on the bed surface. Then, after the skin was cleaned with alcohol, 4 non-recyclable electrodes were attached: 2 on one hand  and 2 on the ipsilateral foot . Measurements were performed by one of our two trained physicians.BIA was performed preoperatively within 1 h before surgery. Data regarding the patient’s body weight and height, gender and age were entered into the BIA device.® Delta Draeger multiparameter monitor). General anaesthesia was induced with propofol 1–2 mg/kg, fentanyl 2–3 µg/kg, and atracurium 0.5 mg/kg. Airways were secured with tracheal intubation and anaesthesia was maintained with 2% sevoflurane in 50% air/oxygen mixture, fentanyl, and atracurium intermittent boluses according to clinical judgement. All patients had volume-controlled ventilation with a tidal volume of 6–8 mL/kg and PEEP 5 cm H2O. All patients had a urinary catheter inserted after induction and their urinary outputs were accurately monitored.Anesthesia was managed according to hospital protocol. All patients were premedicated with 10 mg of diazepam and they fasted overnight for 6–8 h. In the operating room, all patients were monitored before/during anaesthesia using three lead electrocardiography, non-invasive blood pressure measured every 3–5 min, peripheral pulse oximetry, central temperature and end-tidal carbon dioxide concentration. Some of the patients required additional invasive haemodynamic monitoring . In case of a blood loss of more than 500 mL, the hospital protocol for intraoperative fluid administration recommends colloids to be 1/3 of the given volume.The decisions regarding the volume and timing of intraoperative fluid administration were made by the anaesthesiologist, who was unaware of BIA measurements in order for the results to reflect current clinical practice. The clinician could choose between crystalloids  and colloids (hydroxyethyl starch 130/0.4 VoluvenThe second BIA assessment was performed within the first postoperative hour in the Post-Anesthesia Care Unit or the Intensive Care Unit.Fluid management in the postoperative period was recommended by the attending physician (the anaesthesiologist or the surgeon) who acted according to routine clinical practice and was equally unaware of BIA measurements.The following baseline data were recorded for all patients: gender, age, anthropometric data , patient diagnosis and surgical intervention, American Society of Anesthesiologists (ASA) risk classification, co-morbidities , preoperative and intraoperative hemodynamic parameters , intraoperative blood loss, urine output, and hemodynamic support (inotropic and/or vasoactive drugs). Intraoperative blood loss was assessed by measuring blood in suction bottles, blood-soaked mops and gauze pieces. Information regarding the volume and type of fluid administered during surgery and in the first two postoperative days was also collected.The intraoperative fluid balance was calculated by subtracting the losses  from the infused volumes . In the postoperative period, we recorded the fluid balance on the 1st and 2nd days. The daily fluid balance was calculated from the patient’s medical records as the difference between fluid intake  and fluid output . The cumulative fluid balance (CFB) was also calculated as the sum total of the fluid balances recorded intraoperatively as well as on the 1st and 2nd postoperative days. The cumulative fluid overload (CFO) was calculated by dividing the CFB by the preoperative weight of each patient, and the result was expressed as a percentage. With regard to CFO, we divided patients into two groups: normally hydrated (NH) in case of CFO < 5% and fluid overloaded (FO) if CFO ≥ 5%. Standard hematological and biochemical values  were recorded preoperatively and then daily during the postoperative ICU hospitalization period.2/FiO2) < 300, reintubation or difficult weaning of mechanical ventilation. To diagnose infection, we used the Centers for Disease Control 2008 guidelines [All patients were monitored during their hospital stay for timely detection of organ dysfunction or complications. Acute kidney injury was defined according to the KDIGO criteria . Respiraidelines . We alsot test. The relationship between variables was assessed using the Pearson correlation coefficient.Continuous variables are reported as mean ± SD, median and inter-quartile range or as frequency percentages, as appropriate. The Shapiro–Wilk test was conducted to appraise the distribution of continuous variables. Logarithmic transformation was performed for variables with non-parametric distribution. Pre- and postoperative comparisons among patients were made using the paired All analyses were undertaken using Stata SE software, version 12 . A two-tailed p < 0.05 was considered significant.Of the 164 patients with elective major open abdominal surgery performed during the aforementioned period, 71 met all the inclusion criteria and were enrolled in the study.2, and 43 (60.6%) were male. The median duration of anaesthesia was 180 (140–250) min. The majority of patients were ASA 3 (54.9%) and the most frequent type of surgery involved the gastrointestinal tract  was 3.7 ± 1.5 L, equivalent to 15.2 ± 5.5 mL/kg/h of surgery. The median blood loss was 0.6 (0.1–1.1) L and the mean urinary output was 0.5 ± 0.4 L, resulting in a positive mean intraoperative fluid balance of 2.4 ± 1.0 L. The postoperative fluid balance was positive on both days following the intervention, but more so on the 1st day (Table Postoperative AKI occurred in 10 cases (14.0%), respiratory dysfunction ensued in 14 cases (19.7%), and developed 20 patients (28.1%) developed infections. The median length of stay in ICU was 20 h (ranging from 1 to 528 h) and hospital LOS was 10 days (4-37). 19 patients (26.7%) had an ICU stay longer than 2 days. Four patients (5.6%) died while in hospital.The preoperative BIA parameters were TBW 35.9 ± 7.6 L, ECW 16.3 ± 3.5 L, ICW 19.5 ± 4.4 L, ECW/ICW 0.85 ± 0.11, AFO 0.2 ± 1.4 L and RFO 3 (− 3.5 to 7)%. Before the surgery, ten patients (14.1%) were dehydrated (RFO < − 10%) and two (2.8%) were overhydrated (RFO > 15%). We also calculated the ratios between TBW, ICW, ECW, and the patient’s preoperative body weights, and the mean values were 49% (TBW), 26% (ICW), and 23% (ECW).The postoperative BIA parameters were TBW 37.3 ± 7.6 L, ECW 17.7 ± 3.8 L, ICW 19.4 ± 4.2 L, ECW/ICW 0.93 ± 0.12, AFO 1.3 ± 1.5 L and RFO 8 (2 to 12) %. The postoperative status was dehydration (RFO < − 10%) in just one patient (1.4%) and overhydration (RFO > 15%) in 22 patients (31%).Except for ICW, all other BIA parameters increased significantly during the postoperative period: TBW 1.4 ± 2.4 L (p < 0.001), ECW 1.4 ± 1.2 L (p < 0.001), AFO 1.1 ± 1 L (p < 0.001) and RFO 5 (2 to 10)% (p < 0.001). ICW decreased non-significantly (− 0.1 ± 2.2 L) (p = 0.76)   as well as with pre- and postoperative RFO   had a CFO < 5%  and 22 (31%) had CFO ≥ 5% (fluid overload—FO group). The baseline characteristics of both groups are presented in Table Except for ICW, all other BIA parameters  significantly increased postoperatively for both FO and NH groups (p < 0.001) Table .More statistically significant differences were found in the FO group versus the NH group regarding respiratory dysfunction: 11 patients (50%) from the FO group versus 3 patients (6.1%) from the NH group (p < 0.001). Last but not least, in relation to the patients’ length of stay in ICU, 13 patients (59%) from the FO group versus 6 patients (10.2%) from the NH group were required to spend two or more days there (p < 0.001).To our knowledge, this is the first prospective, clinician blinded, observational study which investigates the role of bio-electrical impedance in the assessment of body fluid composition and intercompartmental fluid shifts before and after open major abdominal surgery.Major abdominal surgery represents a traumatic insult associated with metabolic and hormonal disturbances which may ultimately induce inflammation, catabolism, and water and salt retention . Volume The mean postoperative TBW and ECW increased significantly, and so did the ECW/ICW ratio from preoperative 0.85 to postoperative 0.93. This pattern is commonly seen in critically ill patients and in late stages of liver cirrhosis, heart failure, and kidney failure . FurtherUnlike clinical examination, BIA is an objective technique that can provide numerical, credible results even in the case of patients for whom clinical assessment alone fails to identify abnormalities in fluid status. BIA has, at least theoretically, the capacity to identify both hyperhydrated and dehydrated patients. This evaluation has the advantage of early identification of hyperhydrated patients prior to the clinical occurrence of edema. BIA was validated by isotope dilution methods , the golThe results of our study suggest that BIA can be used to monitor compartments volume during the perioperative period. A positive 2.4 L mean intra-operative fluid balance was detected by BIA assessment as a 1.4 L increase in TBW. The difference may be explained by insensitive losses  which were not included in the fluid balance calculation. A similar result was reached by Ernstbrunner et al. who used BIA to assess perioperative volume status in patients undergoing gynecological surgery and showed that a mean positive intraoperative fluid balance of 1.6 L resulted in a TBW increase of only 1 L and an average ECW expansion by 0.8 L . At the According to our analysis, intraoperative fluid balance significantly correlated with perioperative change in TBW and AFO, but did not reach statistical significance for ECW change. Using the same BIA monitor, Ernstbrunner et al. identified a stronger correlation between intraoperative fluid balance and changes in both TBW and ECW. The discrepancies between our findings and Ernstbrunner et al. can be explained by the differences between the two studies in terms of type of surgery , patient characteristics , blood loss (median blood loss 0.6 L versus significant blood loss excluded), intraoperative volume repletion strategies  and intraoperative insensitive losses .Physiologically, water accounts for 50–60% of body weight and is unevenly distributed between the different compartments: intracellular water amounts to 33–40% of body weight and extracellular water represents 17–20% of body weight . In our When making decisions regarding type and dose of fluid administration, one must always bear in mind that fluids are drugs nonetheless and, as such, they should be prescribed with caution considering their indications and contraindications. Given that a positive intraoperative fluid balance is common, a strategy aiming for a negative postoperative fluid balance may play an important role in preventing postoperative complications. In our study, patients in the NH group had a negative fluid balance on the 2nd postoperative day and a lower complication rate, while the FO group featured positive fluid balance and more organ dysfunctions.At present, the accepted definition of fluid overload is a 10% increase in body weight , 19, 25.During anaesthesia and surgery, patients often develop hypotension. Despite anaesthetics-related decreased peripheral vascular resistance and/or myocardial depression, patients often receive aggressive volume therapy. Such excessive fluid administration increases the interstitial space volume, impairing tissue oxygenation and healing, and contributing to organ dysfunction. These effects become more prominent in encapsulated organs, where small increases in interstitial volume result in significant increases in interstitial pressure and a significant reduction in capillary blood flow. Therefore, fluid overload is harmful to the kidney function, for instance . IncreasDespite the fact that we did not find any significant correlation between any pre- or postoperative BIA parameters and the risk of postoperative complications , a significant correlation was noted between the pre- and postoperative AFO, RFO and the ICU-LOS. This may be explained, on one hand, by the fact that longer ICU-LOS may be related to mild/moderate organ dysfunction, which did not reach the severity threshold of complications. On the other hand, AFO and RFO are more refined parameters of BIA measurement and may better express subclinical correlations between intraoperative water volume, distribution and mild to moderate organ dysfunction.The impact of perioperative fluid overload on morbidity and mortality has been described in several clinical studies , 5–8. DaBIA is a non-invasive, inexpensive technique with limited inter-observer variation. It performs well for both healthy subjects and chronic patients with major but subtle water distribution disturbances . In suchThe accuracy of BCM measurement may be altered by electrical interference; therefore, BCM is not recommended with ICU patients. Dewitte et al. showed that the BCM device is safe and feasible with ICU patients, while data reproducibility was very good, whether with or without ICU monitoring and mechanical ventilation .Our study is subject to a number of limitations. Firstly, since ours is a single-center study with a relatively small number of patients, our findings need to be corroborated with those by other, larger or multi-centric studies. Secondly, the study was a non-interventional, observational pilot study, and as such, no cause-effect relationship can be assumed. Also, the patents were not classified according to the accepted definition of fluid overload—a 10% increase in body weight—and we used a 5% threshold in order to investigate if BIA monitoring may help with the early detection of fluid accumulation. Moreover, this study included patients with open major abdominal surgery, of whom only 26.7% had an ICU stay ≥ 2 days. Finally, the postoperative BIA measurements were performed only once during the first hour. Extending BIA fluid monitoring in the postoperative period might have allowed for a more in-depth assessment of fluid compartment status and shifts, as well as correlation with patient outcomes.In conclusion, BIA is a simple, non-invasive, low-cost bedside technique which may prove for the perioperative assessment of volume status in open major abdominal surgery. For instance, fluid overloaded patients are more likely to require longer ICU hospitalization, and BIA may be used to identify such situations. Further studies are required to evaluate whether BIA-guided perioperative fluid administration may improve the postoperative volume status and clinical outcomes in patients undergoing major open abdominal surgery.


Fluid management is a cornerstone in the treatment of burns and, thus, many different formulas were tested for their ability to match the fluid requirements for an adequate resuscitation. Thereof, the Parkland-Baxter formula, first introduced in 1968, is still widely used since then. Though using nearly the same formula to start off, the definition of normovolemia and how to determine the volume status of burn patients has changed dramatically over years. In first instance, the invention of the transpulmonary thermodilution (TTD) enabled an early goal directed fluid therapy with acceptable invasiveness. Furthermore, the introduction of point of care ultrasound (POCUS) has triggered more individualized schemes of fluid therapy. This article explores the historical developments in the field of burn resuscitation, presenting different options to determine the fluid requirements without missing the red flags for hyper- or hypovolemia. Furthermore, the increasing rate of co-morbidities in burn patients calls for a more sophisticated fluid management adjusting the fluid therapy to the actual necessities very closely. Therefore, formulas might be used as a starting point, but further fluid therapy should be adjusted to the actual need of every single patient. Taking the developments in the field of individualized therapies in intensive care in general into account, fluid management in burn resuscitation will also be individualized in the near future. Burn patients in the initial resuscitation phase typically require large volumes to restore adequate perfusion pressure and prevent organ failure. To estimate the actual fluid requirements, Baxter and Shires introduced the Parkland formula in 1968 and thus enabled a better outcome of burn patients [Apart from the Parkland formula, many other formulas have been proposed to estimate the necessary resuscitation volume more closely, e.g., Evans formula  or the mThough hypovolemia was initially the predominant cause for mortality of burn shock  and stilBeside an incline in pulmonary function because of lung edema, the abdominal compartment syndrome (ACS) is one of the most devastating complications with a mortality of over 80% in burn patients ,14. Due Still, the discussion about the most suitable method to estimate the adequate resuscitation volumes and how to keep the golden middle of normovolemia is going on. This review will provide an overview of different analyzing methods and parameters to assess fluid requirements. The question of how and when to use colloids or which kind of resuscitation fluid should be used will not be covered by this review. We rather focus on the evolution from a goal-directed resuscitation with the use of different parameters to the most recent development of an individualized resuscitation taking individual organ function into account.To avoid under- and over-resuscitation, different parameters were analyzed in the past to guide fluid management. This “goal-directed” therapy was discussed early in the history of burn resuscitation. As early as 1968, Baxter and Shires used not only urinary output but also a pulmonary catheter (PAC) in the clinical part of their study to measure the cardiac output as an additional parameter ,18,19,20Therefore, a vital point for sensible goal-directed fluid therapy is which parameters to choose and how to combine them. The different parameters to guide fluid therapy are discussed in this section.The most widely used parameters are urinary output and vital signs such as blood pressure or mean arterial blood pressure (MAP) and heart rate since they are measurable with minimal effort and in most circumstances and locations worldwide.Additionally, Baxter and Shires used urine output (UO) as target parameter in their formula-based resuscitation of burn patients. In the following years, urine output remained the leading parameter for goal-directed resuscitation. Though UO can be used easily as an indicator for sufficient resuscitation, it is not a reliable parameter if adequate tissue perfusion and oxygen delivery are the ultimate goals. Furthermore, oliguria or anuria may also be signs for multiple pathologies—not only hypovolemia. In the aforementioned abdominal compartment syndrome, for example, oliguria is a common sign with rising IAP. In fact, UO should not be used as sole parameter to guide resuscitation. However, still it is one of the most popular parameters and still recommended by the American Burn Association . Dries aOf note, serum lactate and arterial base deficit are reliable markers of tissue perfusion. Both parameters indicate uncompensated shock and cell death also in burn patients . HoweverStatic parameters, e.g., the central venous pressure (CVP) were often used in the past, though the volumetric status is influenced by many dynamic processes such as changing intrathoracic pressure during in- and expiration with alterations of blood flow and thus changing stroke volume. Therefore, static parameters in general show low reliability to reflect the actual volumetric status of the patient . MultiplAccordingly, the sole determination of the diameter of the inferior vena cava (IVC) is not reliable enough and cut-off values depend on the patients’ height. However, the changes in IVC diameter during in- and expiration—i.e., the respiratory variation—proved an adequate tool that can be easily determined via ultrasound bedside ,30 and wIn the last few years, the transpulmonal thermodilution (TTD) has gained popularity as a useful method to determine volumetric parameters as well as cardiac output (CO) and systemic vascular resistance (SVR). The TTD technique  uses volume boluses of 10–20 mL of ice cold saline and measures the time between the injection site  and an arterial catheter . Therefore, it is less invasive than the formerly used pulmonary artery catheter (PAC), which has been replaced by the TTD-technique. TTD enables the calculation of volumetric parameters such as the global enddiastolic volume (GEDV) or intrathoracic blood volume (ITBV) which reliably reflect the actual preload .The increasing pulmonary edema with ongoing resuscitation is represented by the extra-vascular lung water (ELW). Branski et al. could show that an increasing mortality is linked to increased ELW-values . This maAs a drawback, the TTD- technique and arterial pressure wave analysis presumes a normal cardiac function without cardiac valve dysfunction and excludes patients with arrhythmia. Additionally, a pre-existing history of pulmonary diseases influences the calculation and interpretation of the ELW.In general, TTD and arterial pressure wave analysis show their greatest reliability not in single measurements, but in detecting changes in the course of ongoing resuscitation.2 and an hourly urine output of at least 0.5 mL/kg. Arlati and his group showed reduced resuscitation volumes in the first 12 h post-burn, i.e., 3.2 mL/kg/%TBSA, compared to the estimated Parkland formula (4.6 mL/kg/%TBSA). The permissive hypovolemia group even showed an optimized lactate clearance compared to the control group. ITBV as preload parameter ranged between 650 and 750 mL/m2 and, therefore, significantly below normal values (900 mL/m2). Despite the size of the study (n = 12), Arlati could show significant decreased multiple organ dysfunction scores (MODS) in the permissive hypovolemia group.Initially, goal-directed therapy was used to achieve normal or supra-normal parameters. However, when normal or even supra-normal values of preload parameters (GEDV and ITBV) are targeted, resuscitation volumes estimated by the Parkland formula are exceeded regularly ,35,36,372, ITBV > 600 mL/m2 and rapid lactate clearance. The mean volume given was 4.05 mL/kg/%TBSA confirming the results of Charles Baxter. Of note, urine output was not used as resuscitation parameter and the authors reported no correlation between CI and urine output. However, in some cases with a UO of >0.5 mL/kg/h, severe hypovolemia with elevated lactate levels were found and vice versa. Hence, both studies demonstrated the CI to be superior as parameter for goal-directed therapy compared to the widely used urine output or preload parameter alone.In a larger study by Sanchez et al. (n = 132), the cardiac index was also used as primary goal to guide burn fluid therapy . Goals wIn contrast to the goal-directed therapy, which uses a preset goal for every patient, the individualized approach considers co-morbidities and defines the goals in concordance to the individual organ function of every patient. As a consequence of increasing life expectancy, the average age as well as the incidence of co-morbidities has been rising significantly over the last few decades. Whereas occupational health and safety increased over the last years and occupation associated burns are decreasing likewise, the percentage of elderly and morbid patients rises continuously in developed countries .In the course of burn resuscitation, cardiac function is a limiting factor for fluid administration. Thus, it is important to understand the pathophysiologic response to fluid challenges. In short, the myocardium is able to optimize its contractility in a certain range, i.e., within the steep part of the Frank–Starling curve. In this optimum range the myocardium is stretched by adequate preload without over-stretching in case of volume overload . UnfortuThe transpulmonary thermodilution (TTD) technique as mentioned above can also be used to analyze the arterial pressure wave . The variation of the pulse pressure variation (PPV) and stroke volume variation (SVV) reliably predict the fluid responsiveness with a sensitivity of 80% . FurtherThe use of ultrasound as point of care diagnostic is now widely used. The most intriguing points are its rapid availability and non-invasive use. Additionally, the broad spectrum of applications ranges from identification of different causes of shock (hypovolemia versus pulmonary embolism etc.), examination of potential complications or combined injuries , lung ultrasound, cardiac function, volumetric status and fluid responsiveness . Lung ulAs mentioned above, the diameter of the inferior vena cava (IVC) is an easily assessed but unreliable parameter for the actual volumetric status. However, the respiratory variation of the IVC—i.e., the dynamic changes in diameter during the respiratory cycle—reliably reflects the volumetric status . The resThe cut-off point for the calculated IVC variability lies at 12% and, thus, measurements over 12% are predictive of fluid responsiveness .Though the variation is influenced by ventilator settings and respiration efforts in spontaneously breathing patients , a recenFocused cardiac ultrasound includes measurement of contractility of the left and right ventricle, stroke volume, valve dysfunction as well as respiratory changes (stroke volume variation) or changes after fluid challenges ,52. TherWhereas overt hypovolemia can be determined quickly by visual assessment of the left ventricle , estimation of contractility and fluid responsiveness makes further measurements necessary. Herein, ejection fraction (EF) and stroke volume should be assessed as parameters for left ventricle contractility. The stroke volume (measured as velocity time index of the left ventricular outflow tract) is used to derive cardiac output  and measRecently, the contractility of the right ventricle has been focused on since it is crucial for fluid responsiveness. Therefore, the tricuspid annular plane systolic excursion (TAPSE) should be measured as parameter for right ventricular contractility and right ventricle dilatation and flattening of the septum as the “red flags” for fluid administration should not be missed . EchocarAdditionally, these parameters can be re-assessed after a fluid challenge. Therefore, mini-fluid challenges of 100 mL of colloids are sufficient to certify fluid responsiveness by optimizing cardiac output by at least 10% . InsteadIn contrast to single measurements of SVV or cardiac output, the change after volume challenge—nevertheless, via fluid administration or PLR—is independent from ventilator settings, spontaneous breathing or arrhythmia . Thus, tIn 1968, Baxter and Shires proposed 3.5–4.5 mL/kg/% TBSA to estimate fluid requirements in burn patients. Still widely used, this formula seems to match the actual fluid requirements adequately in the majority of patients with moderate burn injuries. However in severely burned patients exceeding 60% TBSA, fluid requirements increase disproportionally. Thus, a formula-based resuscitation will predict fluid requirements more inaccurately with rising TBSA. Cancio et al. showed fluid requirements of 6 mL/kg/%TBSA in burn patients exceeding 80% TBSA .The development of transpulmonary thermodilution (TTD) enabled the measurement of preload parameters (GEDV and ITBV), cardiac output (CO/CI) and ELW as a parameter of increasing lung edema with limited invasiveness compared to the pulmonary artery catheter (PAC). Herein, recent studies have shown that preload parameters should be used with caution to guide resuscitation in the acute burn shock since normal values are only achievable by significant over-resuscitation. In contrast, cardiac output or cardiac index proved to be reliable to guide fluid administration and enable a closer adjustment of resuscitation to the actual fluid requirements of the patient. Hereby, goal directed resuscitation using cardiac output allows for permissive hypovolemia and concurrently optimized oxygen delivery .Baxter and Shires transferred their findings in different animal experiments to the clinical setting . The firTherefore, the goal directed resuscitation should be refined to an individualized resuscitation. Herein, focused echocardiography plays a vital role to assess cardiac function initially and cardiovascular response to fluid administration in the course of resuscitation. Furthermore, the analysis of fluid responsiveness  demonstrates the potential improvement of increased volume administration beforehand and thus avoids the hazards of over-resuscitation . MoreoveThe difference between the goal-directed and individualized approach is an adjustment of fluid therapy to the preset goals in the first case and an adjustment of multiple target parameters to the organ function of the individual patient in the second case. In short, a “one size fits all” approach sets the criteria to be met by the patient, whereas an individualized approach relies on the assessment of cardiac, pulmonary and kidney function in the first step and consecutively defines reliable goals and necessary fluid administration for this patient in the second step. Thus, individualization can be seen as a future development of the goal-directed therapy.As Kevin Chung stated: “The complex nature of the body’s response to burn injury compounded by the variable response to resuscitation likely makes the starting point almost irrelevant.” . Therefo


Starting with the first definition of epigenetics “to understand how the genotypes of evolving organisms can respond to the environment in a more co‐ordinated fashion” originated in the research of Conrad Hal Waddington (1942) until current days, the field of epigenetics has evolved greatly . We are Novel Drugs Targeting the Microenvironment and the Epigenetic Changes in Hematopoietic Malignancies covers recent advancements in our understanding of the role of the microenvironment in hematological malignancies. To this end, response to epigenetic drugs are presented within a cohort of juvenile myelomonocytic leukemia (JMML) cases that were molecularly annotated via targeted next generation sequencing (NSG). Treatment with 5-Azacitydine was well tolerated and had effective results in both de novo JMML and relapsed patients (The Research Topic entitled patients . The autpatients . The hetpatients . The conpatients . In addipatients .6-methyladenosine (m6A) is the most common non-genetic alteration in mRNAs. M6A impacts RNA metabolism and thus, mediates aberrant gene expression seen in disease development  increases the expression of Tal1 (6A binding complex) can bind multiple mRNAs to impair their translation and function ; CCCDI-UEFISCDI, Project No. PN-III-P4-ID-PCCF-2016-0112 within PNCDI III, as well as by awarded for Young Research Teams 2020-2022 (Grant No. PN-III-P1-1.1-TE-2019-0271), as well as by an international collaborative grant of the European Economic Space between Romania and Iceland 2020-2022 (Grant No. 19-COP-0031). GG is funded by K08 HL127269 (GG), R03 HL145226 (GG), P01CA225618 (GG), and P30 CA006973. LQ is funded by a grant from the National Natural Science Foundation of China (Grant No. 81800180) and the National Young Elite Scientists Sponsorship Program by the China Association for Science and Technology (Grant No. 17-JCJQ-QT-032).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.


To immunohistochemically evaluate the association between the presence of cancer-associated fibroblasts (CAFs) and the tumour expression of podoplanin (PDPN) in head and neck squamous cell carcinoma (HNSCC) and their association with clinicopathological variables. A tissue microarray (TMA) with biopsy sections from patients diagnosed with HNSCC was stained with antibodies against the CAFs marker, α-smooth muscle actin (α-SMA), and PDPN. We subsequently evaluated their expression to determine the association between them and with clinicopathological variables including age, primary tumour site, TNM stage, and tumour differentiation grade.P ≤ 0.05). PDPN staining on tumour cells showed low expression in 72% of cases, and it was not associated with any clinicopathological variable or with the presence of CAFs. Positive reaction to α-SMA was observed in the tumour stroma, revealing spindle-shaped cells compatible with CAFs, which showed a high expression in 62% of cases and a significant association with laryngeal carcinomas, advanced clinical stages, and lower tumour differentiation ( The presence of CAFs in the tumour stroma is related to an aggressive phenotype and could increase as the disease progresses, although based on our findings, it would have no relationship, at least directly, with the expression of PDPN. Key words:Cancer-associated fibroblasts, myofibroblasts, head and neck neoplasms, podoplanin, immunohistochemistry. Head and neck cancer (HNC), which is the sixth most common form of cancer in the world, comprises a heterogeneous group of malignant neoplasms, with more than 90% of cases corresponding to squamous cell carcinoma (SCC) originating in the epithelium of the mucosal lining of the oral cavity, oropharynx, larynx, hypopharynx, and nasopharynx ,2. DespiCurrently, the aim is to identify biomarkers that allow us to understand the molecular mechanisms associated with tumour development to investigate therapeutic targets that limit the progression of the disease and improve the patient’s prognosis . One of The study of cancer over the last few decades has focused on the participation of the tumour stroma in its development and progression, since it presents a wide cellular and molecular heterogeneity derived from an active interaction between the neoplasm and the host tissues. It has consequently been defined as the tumour microenvironment . In thisEvidence exists that the tumour cells of oral cavity SCC have the capacity to induce the differentiation of CAFs and that these in turn would induce the expression of PDPN in tumour cells through the secretion of TGF-β, favouring EMT and invasiveness of the carcinoma . This in- SampleWe conducted a retrospective study over a sample of formalin-paraffin fixed tissue sections obtained from biopsies of patients diagnosed with HNSCC, purchased in a tissue microarray format . A TMA comprises 60 tissue sections, of 5 µm thickness, from primary tumours collected from 30 HNSCC patients (two specimens per case) and 21 tissue sections from normal mucosa, serving as control. Of the 30 primary tumours included on the TMA, 16 came from the larynx, 9 came from the oral cavity, and five came from the nasosinusal mucosa. One of the oral cavity primary tumour specimens did not meet the histopathological criteria for the diagnosis of squamous cell carcinoma, and it was excluded from the study.Clinical and histopathological data from the 29 HNSCC patients are presented in Patients ranged from 34 to 81 years (mean 57 years) in age, and 82.7% of them were male. Of the 29 primary tumours analysed, 12 were early stage I/II and 17 advanced stage III/IV; 4 had associated cervical lymph node metastases and 25 were non-metastatic; and 12 were classified as differentiated tumours (G1) and 17 as undifferentiated tumours (G2 or G3).This research was approved by the ethical committee of the University of Talca with the approval code 2017-01-BV-VR on april 2017. Nevertheless, the human tissue samples were acquired in a commercial format by biomax , who declare that all tissue is collected under the highest ethical standards with the donor being informed completely and with their consent. They ensure that follow standard medical care and protect the donors' privacy. They also declare that all human tissues are collected under HIPPA approved protocols, have been tested negative for HIV and Hepatitis B and approved for commercial product development.- Immunohistochemistryen blocked with 1% bovine serum albumen for 10 min. Next, sections were incubated with anti-α-SMA  and anti-PDPN  primary mouse antibodies for 1 h at 37 °C and washed in phosphate-buffered saline (PBS) three times for 2 min. Sections were incubated with secondary biotinylated antibody at room temperature for 30 min in accordance with the manufacturer’s instructions . All slides were then exposed to avidin-biotin complex and diaminobenzidine tetrahydrochloride  and counterstained with Harris haematoxylin. Internal positive controls were used for each antibody: smooth muscle cells of blood vessels for α-SMA and lymphatic endothelial cells of lymphatic vessel for PDPN. For negative control, the primary antibody was substituted with phosphate-buffered saline.The TMA were dewaxed in xylene and rehydrated in graded alcohols. After a rinse in tap water, sections were immersed in a 3% hydrogen peroxide solution for 10 min. Slides were placed on a steamer at 95 °C for 30 min in Tris-EDTA buffer solution (pH 9.0) for antigen retrieval and th- Immunohistochemical analysisThe IHC-stained TMA sections were observed under a light microscope  and the images for the analysis were obtained through an integrated digital camera . Evaluation of immunostaining for both antigens was performed by two observers independently (V.R-V. and B.V.) who were blinded to the clinical information pertaining to the subjects. Cases with interobserver discrepancies were minimal and reassessed together until a consensus was reached.Cancer-associated fibroblasts were defined as spindle-shaped fibroblasts on tumour stroma expressing α-SMA as described previously . The preFor scoring the immunoreactivity of PDPN, five representative fields at the tumour invasive front of each case were selected under 400x magnification and evaluated on ImageJ 1.47q (NIH®) software, following a previously described method . Membran- Statistical analysisP ≤ 0.05. The statistical tests were performed using SPSS software, version 23.0.0.0 .The Spearman correlation coefficient was used to test the association between the staining scores of α-SMA and PDPN. The association between the expression degree of each antigen and between each antigen and the clinicopathological variables was made by the chi-square test. All differences were considered significant if - Immunohistochemical findingsAll cases showed a positive reaction for α-SMA staining in stromal cells, which showed a cytoplasmic staining pattern that allowed us to observe spindle-like cells, whose morphology is compatible with CAFs. As summarized in In most cases, we observed a low degree of PDPN expression 72%, , with th- Relationship between α-SMA, PDPN, and clinicopathological variablesp ≤ 0.05); notably, 76% of patients in stages III–IV or who present undifferentiated tumours have a high α-SMA expression degree. In addition, laryngeal SCC showed a higher α-SMA expression degree compared with oral cavity and nasosinusal SCC . These findings are graphed on Fig. p = 0.49).Fig. Currently, the management and treatment of HNSCC depends mainly on clinical aspects, where the TNM classification system is highlighted and complemented with histopathological information specific to the tumour and nodal involvement. Despite advances in the study of pathogenesis and treatment, the survival of patients diagnosed with this type of cancer has not improved significantly in recent years ,18. AlthIn the tumour microenvironment of the HNSCC there is a marked presence of CAFs, which participate in processes that promote tumour progression, such as lymphangiogenesis, angiogenesis, modulation of the immune system, and promote the proliferation and migration of tumour cells ,19-21, cin vitro and in vivo , associated with advanced clinical stages  and a lower degree of tumour differentiation. Previous studies have shown the influence of CAFs on the development of SCCHN, associating the expression of α-SMA with aggressive anaplastic tumours, advanced clinical stages, regional lymph node involvement, and recurrence of the disease ,23. More in vivo ,25. AddiAmong the possible mechanisms involved in the interaction between the tumour microenvironment and carcinoma, it has been established that CAFs would induce the expression of proteins that favour the EMT of tumour cells such as PDPN, which has been outlined in recent years as a possible biomarker of tumour progression . PDPN isMost of the studies that evaluate the immunohistochemical expression of PDPN and its relationship with clinicopathological variables have been carried out on oral SCC, where a greater expression of PDPN is associated with low survival and worse prognosis ,17. In ain vivo studies have shown that CAFs induce the expression of PDPN in oral SCC cell lines whose activation is associated with the formation of invadopodia and degradation of the extracellular matrix by activating the insulin-like growth factor-II mRNA binding protein-3 (IMP-3) pathway, causing the tumour cells to acquire an invasive phenotype with migratory capacity, favouring EMT and metastasis (In this research we could not demonstrate any significant association between the presence of CAFs and the tumour expression of PDPN. However, recent studies have shown that oral SCC tumour cells induce the differentiation of CAFs through the production and release of TGF-β and, in turn, CAFs through the secretion of the same factor, which induces both the differentiation of new CAFs and the expression of PDPN in tumour cells . In vitrtastasis , which iThe limitations of the study were the small sample, the use of TMA acquired in commercial format where there is no certainty that the sections obtained are representative of the original biopsy and the lack of functional tests confirming that CAFs induce tumor expression of PDPN. On the other hand, the commercial provider of TMA does not deliver all the clinical and histopathological information necessary to establish an adequate association of the markers evaluated with the prognosis of the patients, such as recurrence, survival or follow-up data after diagnosis. Despite this limitations, there are no studies to date evaluating the relationship between CAFs and the tumour expression of PDPN in different forms of HNSCC, although this relationship has already been demonstrated in other types of cancer ,33. BaseIn conclusion, our investigation showed that a greater presence of CAFs is associated with laryngeal SCC and advanced stages of the disease, possibly because they induce a more aggressive phenotype of the carcinoma, although this would not be related, at least directly, with PDPN expression by the tumour.


Gazer-Theta is a software model checking toolchain including various analyses for state reachability. The frontend, namely Gazer, supports C programs through an LLVM-based transformation and optimization pipeline. Gazer includes an integrated bounded model checker (BMC) and can also employ the Theta backend, a generic verification framework based on abstraction-refinement (CEGAR). On SV-COMP 2021, a portfolio of BMC, explicit-value analysis, and predicate abstraction is applied sequentially in this order.


The study of magnetohydrodynamic flow of a nanoparticle suspension under the influence of varied dimensionless parameters has been the focus of research in contemporary times. This work models the effect of magnetic field, heat generation and absorption parameter in a steady, laminar, two-dimensional boundary layer flow of a nanofluid over a permeable stretching sheet at a given surface temperature and partial slip. The highly nonlinear governing equations are solved numerically using similarity transformations with suitable boundary conditions and converted to ordinary differential equations. A computational model is setup using FORTRAN, where a relevant Adam’s predictor–corrector method is employed to solve the equations. The impact of the dimensionless parameters, including the Brownian motion, thermophoresis, magnetic field, heat generation and absorption parameters, on the velocity, temperature and nanoparticle concentration of fluid flow are analysed systematically. The study of magnetohydrodynamic problems, such as nanofluid flow over a permeable stretching sheet, has recently become relevant due to potential applications in various fields of science, such as metallurgy and chemical engineering processes with industrial applications which include glass fibre, paper production, hot rolling, metal spinning, wire drawing, etc. Research involving two dimensional boundary layer laminar flow was initiated by Sakiadis , who latThe study of nanofluids  is important mainly due to the fact that they can be used to enhance the thermal conductivity and convective heat transfer performance of base fluids such as water, ethylene, glycol, etc. This takes place due to the intense and rigorous distribution of nanoparticle Brownian motion within the base fluid, thus enhancing the uniformity, conductance and properties which have paved the way for substantial research in this field.Sheet stretching involves analysis using both linear and nonlinear equations  as reflected in the numerical study by Rana and Bhargava . This stKeeping in mind the prior investigations involving steady boundary layer flow and heat transfer of a nanofluid through a permeable stretching surface in the presence of partial slip, the present research incorporates the magnetic field effect and heat generation/absorption coefficient into the velocity, temperature and concentration profiles. Given that the end product is proportional to the heat transfer rate, the most frequent topic of boundary layer flow is the heat exchange phenomenon which has applicability to real life problems. Here, the elucidation of extrinsic forces acting on the flow with regard to partial differential equations has been reduced to ordinary differential equations with distinct boundary conditions, and the results were interpreted graphically and are corroborated by the literature.A two-dimensional, laminar, nonlinear steady-state boundary layer flow of a nanofluid across a permeable stretching surface was considered for study, as shown in x and y axes, normal to the surface. Fluid flow occurs when y ≥ 0, which is triggered when the sheet is stretched out of the slit occurring at the origin (when x = y = 0). The flow speed at any arbitrary point on the plate is assumed to be proportional to the power of its distance from the slit, considering boundary layer flow approximations at a distance of x units from the farthest edge. Considering a fixed origin, the sheet velocity is represented by uw(x) = axn, where n is the stretching parameter, a is the constant and x is the coordinate along the stretching surface.Tw and Cw represent the sheet temperature and the sheet nanoparticle fraction, respectively. The pressure and external forces are neglected and the nanoparticle fraction is assumed to be constant across the stretching surface. T∞ and C∞ denote ambient temperature and the associated nanoparticle fraction where Tw > T∞ and Cw > C∞. This physical model has significant applications in modern nanotechnology and thermal manufacturing in various industries.The basic nanofluid governing equations in Cartesian form are given in the following where which is subjected to the following boundary conditionsandm denotes the thermal diffusivity, and τ denotes the ratio between effective heat capacities of the nanoparticle material and the base fluid. In Equations 1–4, u, and v denote the velocity components along x and y axes, respectively, ρf is the base fluid density, a is a positive constant, DB is the Brownian diffusion coefficient, DT denotes the thermodiffusion coefficient, c is the volumetric expansion coefficient, ρp is the particle density, σ denotes the nanofluid electrical conductivity, B0 denotes the magnetic induction, vw denotes the suction/injection velocity and Q0 (Q) denotes the heat generation (absorption) coefficient.αus in l is the slip length constant.which is proportional to the local sheet stress, and The similarity transformations to solve the governing equations are as follows:By substituting the similarity transformations in The transformed boundary conditions of The working rules for the boundary conditions are:y = 0: v = ±vw, whereAt at η = 0:f(η) = Fw whereTherefore, y = 0: u = uw + us where uw = axn, u = axnf´(η) whereat u = uw + us we get:substituting the above values into f = Fw, f´ = 1 + ξpf´´, θ = 1, ϕ = 1 when y → ∞: u → 0, T → T∞, C → C∞ henceforth as u → 0, f´(η) → 0, as T → T∞, θ(η) → 0, as C → C∞, ϕ(η) → 0Therefore, at η = 0: f´ → 0, θ → 0, ϕ → 0Therefore, at η → ∞: The prime (´) denotes differentiation with respect to η, and the physical parameters involved in the equations are defined as follows:The abbreviations used for the calculations are summarized in f and second order in θ and ϕ to a system of first-order differential equations, in order to simplify the problem as follows.The nonlinear and coupled partial differential equations, represented by Equations 10–12, are solved by using the Adam’s predictor–corrector method which is the most efficient technique in numerical analysis used to solve distinctive problems related to heat transfer, fluid mechanics, and electrical systems. The first step involves reducing the nonlinear differential equations of third order in These equations were solved independently using a FORTRAN program which converts each one of them using different variables to first order as follows.f as:f are:The boundary conditions with respect to η for y3, i.e., y3(0) = α. Hence, y1(0) = Fw, y2(0) = 1 + ξpα, y3(0) = α where α needs to be obtained by using Newton’s method to satisfy y2(η∞) → 0.We assume an initial guess value as α for The boundary conditions with respect to η for θ and ϕ are as follows:The values of α and β, which are the unknowns with respect to θ and ϕ, are obtained by starting with an initial guess and then correcting the values using Newton’s method to satisfy the end conditions as follows:h = 0.01 and η = 4 at the maximum value. The solution is obtained for different parameters used and convergence is achieved with a change in a single parameter. The criteria for convergence are the approximation up to three significant digits. The results derived through computation are plotted as graphs for a clear picture of the numerical problem , increases with an increase in the slip parameter, ξ, which further leads to an increase in the fluid temperature, thus intensifying the thermal boundary layer thickness . An incrThe nanoparticle concentration distribution, ϕ(η), increases with an increase in the slip parameter, ξ, at a given constant surface temperature. An increase in the slip parameter causes friction at the surface which, in turn, generates a frictional force allowing more fluid to flow passed the stretching sheet. This causes an increase in concentration distribution of the fluid as shown in n, has a significant influence on the fluid flow velocity component, f´(η). n, the velocity gradient decreases, thus depleting the thickness of the momentum boundary layer. This is due to the fact that, with the sheet being stretched, there is a retarding force in the fluid in which the fluid particles have severe impact on the velocity component leading to its reduction as we move away from the boundary layer.The nonlinear stretching parameter, n, the temperature gradient, θ(η), increases leading to an increase in the thermal boundary layer thickness. As the value of the stretching parameter increases, the convection process in the particles of the fluid flow intensifies, thus leading to a rapid increase in the temperature gradient which, in turn, is responsible for an increase in the thermal boundary layer thickness , increases slightly but the effect observed is not significant, which is noticed at higher values of n. At lower values of the stretching parameter, there is a negligible change in the fluid concentration of the fluid, which is independent of stretch. On the other hand, there is a significant increase in the stretching parameter at extremely high values that does not seem to affect the fluid particle concentration . When Nb increases, it leads to an increase in θ(η) which causes an increase in the thermal boundary layer thickness of the fluid flow. Higher Brownian motion is responsible for fast movement of fluid particles which, in turn, induces either an increase in acceleration or random acceleration levels resulting in additional energy among particles and, consequently, an increase in the temperature gradient and thermal boundary layer thickness .An increase in the Brownian motion parameter, Nb, results in a decrease in the concentration gradient of the fluid, ϕ(η), due to the fact that the particles move from a high to a low concentration region. As the movement of the fluid particles intensifies with an increase in the Brownian motion, the particles start moving rapidly from regions of higher to regions of lower concentration since the random acceleration decreases the concentration gradient of the fluid flow .With an increase in the thermophoresis parameter, Nt, the temperature gradient of the fluid, θ(η), also increases leading to an enhancement in the thermal boundary layer thickness. Due to the presence of a temperature gradient, different particles of the fluid exhibit different responses to the change in the thermophoresis parameter. As Nt increases, the particles start moving rapidly which causes an elevation in the kinetic energy of the system, resulting in an increase in the temperature distribution and in the boundary layer thickness .A small increase in thermophoresis parameter, Nt, causes a massive increase in the concentration distribution of the fluid flow, ϕ(η), which converges to zero at the boundary layer. A small change in the thermophoresis parameter leads to rapid motion in the fluid particles creating excess heat energy and leading to a massive increase in the concentration distribution. Therefore, M, leads to a decrease in the streamwise velocity component, f´(η), which, in turn, reduces the velocity boundary layer thickness. The external magnetic field has a massive effect on the velocity profile of an electrically conducting fluid, which causes a considerable amount of resistance to its motion and a driving force, called the Lorentz force, which reduces the fluid velocity , increases and so does the thickness of the thermal boundary layer. This is due to the fact that with an external magnetic field being employed, the temperature of the fluid increases, resulting in the rapid movement of the fluid particles which increases both the thermal energy and the boundary layer thickness and reduces the heat transfer from the sheet , which is depicted in M values , is negligible and, therefore, it does not affect the fluid flow velocity. The value of the heat generation/absorption coefficient being positive demonstrates that the heat generated does not have much impact on the velocity gradient of the fluid as the fluid particles move at the same velocity , which increases with an increase in the value of Q as shown in The heat generation/absorption coefficient, Q, does not have much of an effect on the concentration distribution, ϕ(η), as the concentration of the fluid remains unaltered with the change of heat in the fluid. The heat generation coefficient is responsible for changing the heat gradient of the fluid flow, which has a negligible influence on the concentration levels of the fluid particles  and ϕ(η). The external magnetic field was found to have an enormous influence on the velocity gradient of the fluid which decreases with an increase in M due to a drag-like force, or resistance, that is developed by the fluid. Another observation was that the temperature gradient considerably increases with an increase in M, thus enhancing the thermal boundary layer thickness of the fluid as the external magnetic field does not have much of an effect on the concentration gradient. It was also shown that an increase in the heat generation/absorption coefficient, Q, results in a significant increase in the temperature profile without changing the velocity or the concentration of the fluid. With Q being positive, there is heat generation across the flow, which enhances the thermal boundary layer thickness.In this work, it was found that an increase in the slip parameter, ξ, significantly reduces the fluid streamwise velocity gradient which, in turn, reduces the boundary layer thickness. On the other hand, an increase in ξ causes an increase in both the temperature θ(η) and concentration gradients ϕ(η). It was also found that an increase in the stretching parameter


Metastasis refers to the progressive dissemination of primary tumour cells and their colonization of other tissues and is associated with most cancer‐related mortalities. The disproportional and systematic distribution pattern of distant metastasis in different cancers has been well documented, as is termed metastatic organotropism, a process orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. Extracellular vesicles (EVs), nanosized cell‐derived membrane‐bound particles known to mediate intercellular communication, are now considered crucial in organ‐specific metastasis. Here, we review and summarize recent findings regarding EV‐associated organotropic metastasis as well as some of the general mechanisms by which EVs contribute to this important process in cancer and provide a future perspective on this emerging topic. We highlight studies that demonstrate a role of tumour‐derived EVs in organotropic metastasis via pre‐metastatic niche modulation. The bioactive cargo carried by EVs is of diagnostic and prognostic values, and counteracting the functions of such EVs may be a novel therapeutic strategy targeting metastasis. Further investigations are warranted to better understand the functions and mechanisms of EVs in organotropic metastasis and accelerate the relevant clinical translation. Extracellular vesicles (EVs) mediate organotropic metastasis mainly through pre‐metastatic niche modulation. Primary tumour cells disseminate EVs, which can travel to distant metastatic sites and modulate resident or stroma cells in the pre‐metastatic microenvironment through various pathways and prepare a favourable pre‐metastatic niche. At first, abnormally proliferating tumour cells start to invade surrounding tissues, such as basement membrane, which is frequently accompanied by epithelial to mesenchymal transition and marked by decreased E‐cadherin expression and enhanced N‐cadherin expression.1.2Metastasis can happen via various routes, including the lymphatic system, haematogenous routes, transcoelomic spreads, and iatrogenic transplantation. The site‐specific metastases of transcoelomic spreads and iatrogenic transplantation are largely associated with anatomical features. Transcoelomic metastasis occurs when tumour cells disseminate across body cavities such as pericardial, pleural, peritoneal, or subarachnoid spaces.1.3In recent years, EVs have been increasingly recognized as a key player in the tumour microenvironment, with diverse EV cargoes thought to contribute to metastasis and organotropism. Abundant in a variety of human biofluids, from bloodExosomes are endosomal in origin and formed by the fusion of multivesicular bodies (MVBs) with plasma membrane. MVBs contain intraluminal vesicles and they are subsequently released into the extracellular lumen when these MVBs fuse with the plasma membrane.1.4EV signalling is an important mode of intercellular communication, given the myriad of bioactive molecules, including DNA, RNA, and proteins, which are carried and transferred by EVs. Tumour metastasis and progression are not an exception to this. EVs play a pivotal role in communicating between tumour cells and other cells in the tumour microenvironment; via EVs, highly metastatic tumour cells are able to transfer biomolecules to less malignant cells.90K and releasing this VEGF, thus increasing the efficacy of bevacizumab.EV signalling is also capable of inducing the changes of tumour cell characteristics and behaviours, such as drug resistance. Challagundla and colleaguesEVs may also play a role in immunosuppression allowing unchecked proliferation of tumours and subsequently, their metastasis. EV uptake by immune cell lineages dampens immune‐cell populations, reducing immune surveillance necessary for the body to recognize and kill tumour cells. A study by Wen et alEVs are also responsible for reprogramming cellular metabolism, which is essential in providing energy for dissemination and proliferation of cancer cells, especially at distant sites where vasculature and nutrients are lacking.These respective findings demonstrate that EV signalling plays an important role in cancer initiation and progression. Further discoveries pertaining to organ‐specific metastasis will be detailed in coming sections.23 pathway in fibroblasts allowing their differentiation into myofibroblastic phenotypes.In the context of cancer metastasis and organotropism, EVs are best known to function by modulating the pre‐metastatic niche formation. Paget's ‘seed and soil’ hypothesis has established the conceptual architecture for organotropic metastasis studies.Furthermore, it is not merely tumour‐derived EVs which are capable of inducing metastasis—EVs derived from other cell types, which interact with the primary tumour cells, can further enhance the metastatic potential of certain tumours. Janowska‐Wieczorek et alThus, EVs are responsible for tuning the distant microenvironment into a piece of favourable ‘soil’ for the ‘seeds’ tumour cells to grow.2.1S100 and promote lung metastasis while blocking lung‐tropic EVs surface integrins using HYD‐1 peptides can inhibit the uptake by lung resident cells and reduce lung metastasis. This is consistent with our findings that tumour‐derived, microRNA‐125b‐containing EVs are preferentially taken up by pulmonary resident fibroblasts and transform them into activated phenotypes, and thus promote lung metastasis.Ch25h protects the lung from pre‐metastatic niche formation via ablating tumour‐derived EVs education, thus reducing melanoma lung metastasis. Surprisingly, reserpine, the classic anti‐hypertensive beta blocker, reduced lung metastasis by refusing tumour‐derived EVs education, which appears to be a potential novel therapeutic target against lung metastasis.Lung is a frequent metastatic site for breast, colorectal, and pancreas cancers as well as melanoma. Hoshino et alOne of the most characteristic features of the lung is its abundance in capillaries, providing the physiological territories for tumours cells to arrest, adhere, extravasate, and grow.+CCR2+ monocytes on which the pro‐metastatic property of annexin A6‐enriched EVs relies. Alarmingly, breast cancer patients undergoing chemotherapy showed higher levels of annexin A6 in circulating EVs as compared to pre‐treatment conditions, implying the potential risks of chemotherapy‐related metastasis. Moreover, Liu et alIn addition to pulmonary vasculature and capillaries, immune cells such as monocytes and neutrophils are documented to dynamically participate in EV‐mediated pulmonary tropism as well. Keklikoglouv et al2.2Brain metastasis is mostly observed in breast, kidney, lung cancers, and melanoma. The distinctive biophysiological property that separates brain from other metastatic sites is the blood‐brain barrier (BBB), which is a highly selective border controlling substance permeability. The BBB consists of continuous capillary wells of endothelium cells joint by tight junction with astrocyte end feet ensheathing and pericytes embedded in the basement membrane.Tumour cells and the brain pre‐metastatic microenvironment can have reciprocal crosstalk via EVs. On the one hand, tumour cells use EVs as messengers to modulate the brain pre‐metastatic niche. Rodrigues et al2.3Tumour cells from melanoma, prostate, lung, and breast cancers can metastasize to bone. One unique feature of the bone microenvironment is the distinct composition and dynamics of bone‐resorbing osteoclasts, bone‐forming osteoblasts, and the bone‐residing osteocytes.Tumour‐derived EVs also alter the pre‐metastatic bone microenvironment though inducing phenotype‐specific differentiation. A recent study by Hashimoto and colleagues2.4S100 and contribute to pre‐metastatic niche formation. Furthermore, blocking integrin αVβ5 on EVs using either RGD peptide or anti‐integrin αvβ5 antibody significantly diminishes EVs uptake by Kupffer cells and formation of liver pre‐metastatic niche. Shao and colleaguesLiver metastasis is primarily described in lung, breast, and gastrointestinal cancers. One of the most studied liver resident cell types that interact with tumour‐derived EVs are Kupffer cells. Kupffer cells are specialized macrophages residing in liver sinusoids next to endothelial cells.On top of Kupffer cells, liver stromal cells are also finetuned by tumour‐derived EVs. Zhang et al3The transport and delivery of the bioactive cargo is fundamental to EV‐mediated intercellular communication and has motivated a number of translational studies examining and exploiting EVs for metastasis‐associated diagnostic, prognostic, and potentially therapeutic applications.From the perspective of diagnostic and prognostic studies, the secretion of EVs by donor cells and uptake by recipient cells have been widely observed in a broad spectrum of human diseases, including cancers,The overall EV count can provide prognostic value for cancer patients. A recent study by Nanou et alDetection of tumour‐derived EVs can also be done through identification of their cargo. Using EV‐associated proteins as biomarkers is one method of detecting EVs in patients. Yoshioka et al developed ExoScreen‐1 SERS peaks of the EVs from prostate, lung, liver and colonic cancer cell lines were double that in normal cells. These changes were apparent from EVs taken from prostate cancer patients’ plasma as well, showing promise in cancer diagnostics via EV monitoring.Another method was devised by Dong and colleaguesSpecific EV proteins associated with certain cancers have been identified as possible biomarkers for cancer diagnosis. Ji et al showed that integrin beta‐like 1 (ITGBL1) was highly expressed in primary CRC and metastatic sites in comparison to normal tissues, and its high levels were also detectable within plasma EVs of CRC patients.Other techniques rely on identification and detection of the nucleic acids contained within tumour‐derived EVs. Since Kahlert et al showed that tumour‐derived EVs carry KRAS and P53 mutations from to their parental cancer cell genomic mutations,In some cases, the associations we can make between certain genetic fragments and cancers may better characterize patients’ condition and hence affect their treatment plan. Bryant et al4From a therapeutic point of view, EVs are emerging as a novel drug delivery system thanks to their inherent biocompatibility and biosafety, in contrast to other synthetic nanoparticles.While EVs can be utilized as a novel drug delivery system, there is also the recognition that native EVs, especially those which are tumour‐derived, can hasten cancer progression and metastasis. Hence, there have been several strategies developed to counter the effects of these EVs: inhibiting secretion of tumour‐derived EVs; removing circulating tumour‐derived EVs from circulation; preventing uptake of tumour‐derived EVs at specific sites or organs; and utilizing EVs from other sources as adjunctive cancer therapy Table .As discussed throughout this paper, tumour‐derived EVs are responsible for cancer progression and metastasis by allowing crosstalk between tumour cells and other cell types. Inhibiting tumours from secreting these malicious EVs may hinder disease progression. Im et al identified sulfisoxazole (SFX) as capable of diminishing small EV (sEV) biogenesis and secretion through the ESCRT‐dependent mechanism.An alternative therapeutic approach aimed at EV function inhibition is by blocking the uptake of tumour‐derived EVs at their target sites and thus halting their downstream actions. Nishida‐Aoki et al utilized anti‐CD9 and anti‐CD63 antibodies to inhibit general EV uptake across multiple organs in mice.Tumour‐derived EVs have been discussed with regards to the formation of the pre‐metastatic niche; removing these EVs from the patient's circulation is a relatively intuitive method to prevent the onset of metastasis. Xie et alEVs from other sources may also be used to improve the efficacy of cancer vaccines. For instance, dendritic cell (DC)‐derived EVs (DC‐EVs) have been used as maintenance immunotherapy. Via direct and indirect routes of stimulation, DC‐EVs and other antigen presenting cell (APC)‐derived EVs stimulate T‐cell responses.In brief, researchers are utilizing EVs as promising therapeutic agents against cancer—either taking advantage of their unique properties, or actively countering their role in tumour progression and metastasis. The above section represents a recent and novel classification of the various studies done in this field. Further research will uncover more potential therapeutic applications of EVs in cancer therapy.5To summarize, organotropic metastasis involves the convergence of many complex biological interactions and processes, orchestrated by a combination of anatomical, pathophysiological, genetic and biochemical factors. In this context, tumour‐derived EVs function, largely, through pre‐metastatic niche establishment via intercellular communication with stromal cells within the niche. Primary tumour cells disseminate EVs which can travel to distant metastatic locations even prior to the final arrival of tumour cells. Bioactive cargo transported by EVs plays an essential part in metastasis and organotropism with EVs surface proteins frequently identified to be essential for recognition and internalization by recipient cells and EV‐enclosed microRNAs vital for signalling initiation in recipient cells. Upon uptake at distant sites, EVs modulate cells in the pre‐metastatic microenvironment through many pathways such as promotion of pro‐inflammatory genes expression and cytokines secretion, direction of phenotype‐specific induction or differentiation, and recruitment of specific cell types. The pre‐metastatic education by EVs turns the hostile or neutral microenvironment at distance into a piece of favourable and fertile ‘soil’ for the ‘seeding’ metastatic tumour cells to reside in and grow. Of note, the reciprocal crosstalk via EVs has been gradually recognized.With many of the current studies, we are gaining an increasingly improved understanding of how EVs promote metastasis and organotropism. The translation of this knowledge can be highly beneficial to cancer management in the clinic Figure . From a MTNL is a cofounder, advisor and shareholder of Carmine Therapeutics, a red blood cell extracellular vesicle company. MTNL and AG are among the co‐inventors of the US provisional patent US 63/000,468 "Methods of delivering agents to immune cells". Other authors declare no conflict of interest.MZ, JYAC, LX and DXZ searched the literature and drafted the manuscript. JYAC and LX visualized the artworks with the conceptual framework from MZ and DXZ. DXZ, AG and MTNL provided guidance and advice and edited the manuscript. All authors read and approved the final manuscript.