Spinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI.Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 μL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically.Spinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Rats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group.Human bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI. In spinal cord injury (SCI) cellular degeneration and the disruption of connections between the brain and the body cause paralysis and the loss of sensory and autonomic functions. Worldwide, the incidence of SCI ranges from 13 to 163 per million people per year, depending on the country .Spinal cord injury triggers local and systemic secondary mechanisms resulting in a chronic inflammatory state, which is mainly responsible for extensive cell death . These mA promising strategy to cure neurodegenerative pathologies is based on the application of stem cells –9. In thThe objective of the present study was to assess the safety and therapeutic benefits of acute intrathecal injection of the novel bmSC preparation in SCI-lesioned rats and to compare it with acute intraperitoneal injection of MP. So far, one other study has been published with these bmSC as a treatment of SCI . In thesHospital Nacional de Parapléjicos reviewed experimental protocol, surgical procedures and post-operational care (163CEEA/2017), which were subsequently approved by the Consejería de Agricultura y Ganadería de Castilla-la Mancha . During the acclimatization period, six to eight weeks old male Wistar rats , were kept in pairs under standard housing conditions  with ad libitum access to food and water. Following surgery, animals were kept in separate cages. A total of 26 animals entered the study . Fifteen minutes before surgery, rats were weighed and received the analgesic buprenorfine 0.05 mg/kg (Buprex 0.03 mg/mL). After induction of anesthesia, the fur on the back was shaved. The animals were then taped to the operating table, their body temperature being maintained on a heat pad with a rectal thermometer. Corneal dehydration was prevented with ophthalmic ointment .Surgery was performed with the help of an operating microscope (Leica). After skin incision and dissection of the muscle layers covering the vertebrae, serrate muscles were spread with a retractor , and the spinal cord was exposed by laminectomy of vertebrae T9-T11 without damaging the dura mater . To suspend the spinal cord for applying the contusion injury, the dorsal spinal processes T7 and T12 were fixed with the clamps of the impactor device . The impactor rod was positioned centrally at T9/T10 over the spinal cord midline, and the contusion was applied by pressing it against the dorsal surface of the tissue. When a force of 2 N was reached, the rod was immediately retracted (zero dwell time). The device was calibrated before every experiment. We checked the procedure visually (hematoma) and by monitoring the IH displacement/time and force/time plots. In two cases, where the impactor rod hit a bone, the laminectomy was extended and the contusion injury repeated.Following this procedure, the rats were released from the IH clamps. The wound was covered with subcutaneous fat tissue from the same animal. Overlying muscles were re-apposed and sutured, and the skin was closed with a non-interrupted intradermal suture (resorbable thread 4.0) and disinfected with iodine. Animals were then disconnected from anesthesia and received 2 × 2.5 mL isotonic saline s.c. and antibiotic treatment marbofloxacine 5 mg/kg .Following surgery, rats were housed individually to prevent biting at skin sutures. Throughout the study, we performed daily overall health assessments, including inspection of the animals’ well-being, body weight, urodynamic assessments and routine checks to detect urinary tract infections. For the first 3 days animals received two daily s.c. injections of buprenorfine 0.05 mg/kg for pain relief. Subcutaneous injections of 5 mg/kg marbofloxacine were given at the day of surgery, at 2 and 4 days post operation (dpo). Postsurgical care also included food pellets soaked in water and a water bottle with longer tube. The bladders were checked twice daily and voided manually until the rats were urinating spontaneously. The volume of retained urine was recorded. In case of urinary infection, the animals were treated with marbofloxacine 5 mg/kg s.c. every 48 h until the urine was clear and without blood. Euthanasia at the end of the study was induced by i.p. injection of 100 mg/kg of sodium pentobarbital .Animals were pseudo-randomly assigned to four experimental groups, which all received the same SCI but differed in the treatment procedure Fig. . Group 16 viable cells.Bone marrow-derived cells for SCI treatment were prepared at the Neuroplast facility, Geleen, Netherlands, under GMP conditions. The cells were not expanded by cultivation . Recruitment of volunteers for bone marrow collection, procedures and documentation were approved by the ethics committee of Maastricht University Medical Center (METC 13–2-032). From two donors  50 mL bone marrow was collected. Clotting was prevented by EDTA adjuvants. The fresh bone marrow was immediately processed, using automated Ficoll density gradient centrifugation to remove the erythrocytes and reactive proteins. Subsequently, B-cells (CD20+), T-cells (CD3+), monocytes (CD14+) and natural killer cells (CD56+) were removed using antibody-based cell sorting with magnetic beads . The viability and cell type composition of each batch was analyzed with flow cytometry . For the present study, cells were cryoprotected with DMSO, frozen in liquid nitrogen, shipped on dry ice to Toledo, Spain, and then stored in liquid nitrogen until use. Cell viability was again determined after thawing, i.e. immediately before application in vivo . On average, cisterna magna injections contained 2.3 +/− 0.5 × 10For cisterna magna injections of bmSC or saline, 1.5 h after SCI, animals were re-anesthetized with ketamine 50 mg/kg (Ketolar 50 mg/mL. i.p.) combined with xylacine 5 mg/kg  and one i.p. injection of atropine 0.04 mg/kg. The head and neck of anesthetized rats were shaved, and the animals were positioned in a stereotactic frame (Kopf) with the neck flexed to 70° at the atlanto-occipital joint. Ophthalmic ointment was applied, and the skin was superficially disinfected with 70% ethanol.Simultaneously, the bmSC were prepared for injection: For the treatment of two rats, one batch containing 1 mL of frozen cell suspension was thawed in a 37 °C water bath, spun down, washed with saline, centrifuged and resuspended in 210 μL saline. From this, 10 μL was removed for cytometric counting of cell numbers and determination of cell viability. The remaining 200 μL cell suspension was kept on ice until the rats were ready for receiving the injections. The setup for slow injection into the cisterna magna consisted of an electric syringe pump and a sterile 1 mL plastic syringe connected to a Fogarty arterial embolectomy catheter 0.67 mm, fixed to the stereotactic device. A steel canula 23G 0.6 mm was used to penetrate the atlanto-occipital membrane before inserting the catheter.When the anesthetized rat was in place, the atlanto-occipital membrane was accessed by midline anterior-posterior incisions of skin and muscles, which were separated and fixed laterally. Syringe and catheter, previously flushed with sterile saline, were loaded with cell suspension (bmSC treatment) or saline (vehicle treatment) and placed in the holder of the microliter pump. Under microscopic control, the membrane was then punctured and access to the cisterna magna confirmed by observing the appearance of clear cerebrospinal fluid. The catheter was inserted and its content slowly infused (100 μL/3 min) before retracting the catheter. Finally, the muscle and skin were sutured, wiped with Betadine, and the animal was placed in its cage, receiving post-operative care as described for SCI.Rats belonging to the MP group received in total five i.p. injections of 30 mg/kg MP, given at 1 h after SCI and subsequently one every 8 h. Lyophilized MP was reconstituted to 20 mg/mL just prior to injection and kept at 4 °C for the remaining applications. Rats belonging to the second control group were treated with the same volume of saline, 150 μL/100 g, injected i.p. at the same times.Recovery of limb movements was evaluated using the Basso/Beattie/Bresnahan (BBB) locomotor function test  for 5 miThe Rotarod test , which rBefore SCI and at the end of the 9 week observation period, tactile allodynia/hyperalgesia was tested manually using a kit of von Frey filaments with a range of different diameters. For this, rats were placed individually in small cages with a wire mesh bottom. To deliver a constant force, a filament with specific diameter was pressed perpendicularly to the plantar surface of the hind paw until it buckled and held for 2–5 s. A response was considered positive when the animal exhibited any nocifensive behavior such as brisk withdrawal or licking of the paw . Both hiTen weeks after SCI the rats were sacrificed with an overdose of sodium pentobarbital followed by transcardial perfusion with phosphate buffered saline (PBS) and 4% paraformaldehyde/PBS. Spinal cords were prepared, post-fixed for 1 h, then transferred to PBS and stored at 4 °C. For histological processing, 18 mm long spinal cord segments that included the lesion site were dissected, dehydrated, embedded in paraffin and cut in 3 μm transverse sections using a Leica RM2265 microtome. Sections separated by 250 μm were mounted on polylysine-coated glass slides (Superfrost Plus) and stored at 4 °C. To assess the extension of the lesion, the complete series of spinal cord sections of all rats were rehydrated, stained with hematoxylin/eosin (H&E), dehydrated again and cover slipped with Histomount (Merck).Prior to immunohistochemical staining, rehydrated sections were incubated for 30 min at 90 °C (water bath) in 10 mM Na citrate/0.05% Tween 20, pH 6.0, for antigen retrieval. Standard procedure included blocking 1 h at RT with 5% normal goat serum/0.05% Tween 20 in Tris-buffered saline (TBS-T), incubation with primary antibodies for 12 h at 4 °C in a humidified chamber and 2 h incubation with fluorescence-labeled secondary antibodies at RT. Nuclei were stained with 10 μg/mL Hoechst-33342 for 15 min at RT. Sections were cover slipped with Mowiol/DAPCO or ImmuMount (Thermoscientific). We used the following primary antibodies, usually in a double staining protocol in the dilutions indicated in parenthesis:Rabbit anti-GFAP, polyclonal , rabbit anti-caspase-3/activated , rat anti-MBP, polyclonal , mouse anti-Smi32, monoclonal , mouse anti-β (III) tubulin, monoclonal , mouse anti-CD68, monoclonal , mouse anti-NeuN, monoclonal , guinea pig anti-Iba1, polyclonal , mouse anti-human mitochondria, monoclonal, fluorescence-labeled with Cy3 . Secondary antibodies were labeled with fluorescent dyes: Goat anti-guinea pig IgG, Alexa-488 , goat anti-rabbit IgG, TRITC , goat anti-mouse IgG, Alexa-594 , goat anti-mouse IgG, Alexa-488 , and goat anti-rat IgG, Alexa-488 (1/500).Sections stained with H&E were photographed with a stereology microscope (Olympus BX61) using 4x and 10x objectives. For quantitative evaluation we determined the maximal anterior-posterior extension of the tissue lesion and the tissue loss in the lesion center of each rat. The latter was calculated by comparing the remaining tissue area in transverse spinal cord sections to corresponding sections of a rat without SCI.Immunohistochemical staining was evaluated using a Leica epifluorescence microscope. Following visual inspection, objectives and exposure times were selected to account for different signal intensities obtained with different antibodies. Exposure conditions were held constant for quantitative evaluation with GFAP (5x objective), CD68 (10x), Iba1, Smi32, MBP (20x) and activated caspase-3 (40x). Photographs were analyzed using Fuji Image-J, applying the same brightness/contrast adjustments and threshold values for each marker.integrated density in regions of interest (ROI) in the glial scar around the lesion center; Iba-1: in the white matter in sections anterior and posterior of the lesion and in the lesion center; Smi32 and CD68: in the dorsal columns and in the ventrolateral white matter in sections anterior and posterior of the lesion. Signal intensities were normalized to values found in spinal cord sections from non-injured rats. For evaluation of apoptosis we counted cell nuclei that were IR for activated caspase-3 and expressed data as percentage of all nuclei in the ROI, which were located in dorsal and ventral grey matter, anterior and posterior of the lesion center  was measured as p < 0.05 as statistically significant.Unless stated otherwise in the figure legends, data are presented as mean values +/− standard error of the mean (SEM). Non-parametric data are represented in a box and whiskers graph. The statistical analysis of weight and behavioral data , performed with GraphPad Prism v5 software, consisted of two-factor ANOVA, followed by post-hoc Tukey tests. For histological data, the differences among means were analyzed with unpaired Student’s t-test, considering The general health condition of the animals was not compromised. No adverse effects such as sickness behavior or tissue reaction to the bmSC were observed. Unexpectedly, one (bmSC-treated) rat was found dead after 29 dpo and one (MP treated) after 50 dpo. Pathological inspection of these animals did not show any significant abnormality. In five cases, biting at hind limbs occurred, but no animal had to be sacrificed due to sickness behavior or urinary infection.p < 0.001). Post-hoc testing revealed that bmSC-treated rats initially lost significantly more weight than MP and vehicle-treated controls, but that they later on gained significantly more body weight compared to those groups .Following SCI surgery, body weight in all rats typically fell by 10–15% during the first 4 days and subsequently recovered with an average weight gain of about 8% per week during the first 5 weeks of the study and then slowing declining to 2% per week . This was, however, the case in three animals of the NaCl control group and one rat of the MP treatment group Fig. c.p > 0.05). The lesion centers, characterized by a fluid-filled cavity and scar formation, had an average size of 1.4 mm (SD = 1.1 mm) also without significant effect of treatment.At the end of the study, the spinal cords of all animals were investigated with histology. Hematoxylin/eosin staining of transverse sections revealed extensive tissue damage caused by the contusion injury Fig. . At the At 2 dpo, 23 successfully operated animals scored < 2 in the BBB locomotor function scale , demonstrating a reasonable degree of reliability of the SCI rat model in our hands. Scoring differences between two blinded independent investigators were low (0–1), and in case of differences, the mean score of the two evaluators was recorded.p < 0.001), and a significant interaction effect was found between the groups and the treatment over time  = 5.75, p < 0.001). Importantly, rats treated with bmSC had significantly better motor function (BBB scores) compared to MP-treated rats at 4 days (p = 0.015), 7 days (p = 0.029), 2 weeks (p = 0.008), 3 weeks (p = 0.005), 4 weeks (p = 0.009) and 5 weeks (p = 0.015) after surgery  was able use their hindlimbs to maintain balance on the rotating bar. Spontaneous recovery caused a significant increase in Rotarod score during the first 4 weeks in all experimental groups , indicating that the injected cells might have reduced activation of microglia or macrophages.Microglia and macrophages were stained with antibodies against Iba1, again using sections including the lesion site, anterior and posterior of this region Fig. a-g. In tNon-phosphorylated neurofilaments are associated with their disassembled state in neuronal cell somata. In mature axons, in contrast, neurofilaments are heavily phosphorylated. Since this depends on myelin signals, the presence of non-phosphorylated neurofilaments in fiber tracts is indicative of demyelination and axonal damage , 22. We p < 0.05). The strong expression of non-phosphorylated neurofilaments in ventral and lateral fiber tracts was not significantly different between treatments , which is present in activated microglia and macrophages . We founApoptosis was evaluated using an antibody against activated caspase-3, which at 10 weeks after SCI was clearly identified in cell nuclei  was injected into the cisterna magna. These allogeneic implants were not rejected and significantly improved the recovery of motor functions compared to MP treatment. The mean BBB score of bmSC treated rats after 9 weeks was 10.8 compared to 6.8 after MP intervention. Differences between bmSC and saline injections (score of 9.3) were smaller and did not reach significance Figs. . On the To date, more than 25 studies have been published using bone marrow-derived stem cells to treat SCI in rats , 25, 26.beneficial effects of MP administration were obtained in 34% of the studies, no effects in 58%, and mixed results in 8%. The results were inconsistent both among and within species, even when attempts were made to detect any patterns in the results through subgroup analyses” . Despite these limitations, the implants were not rejected, the treated animals showed no sickness behavior and a better recovery of body weight than control groups , adult stem cells are easily isolated from blood, bone marrow or adipose tissue. In contrast to iPSC , 33 they1) Following standard extraction of bone marrow from the iliac crest of human donors, we are using a novel procedure to prepare bmSC, which is based exclusively on the elimination of macrophages and lymphocytes without manipulation or expansion in vitro. This procedure allows implantation within 48 h after harvesting of the cells. In most of the previous studies bmSC were expanded to large numbers before use , 8 and t2) Cell implants that are intended for the therapy of CNS pathologies first need to reach their target tissues. While contusion SCI initially disrupts the blood-spinal cord barrier, this is restored by endogenous repair processes. Therefore, systemic applications of cells, such as by intravenous injection , 40, may3) Thus, the time of intervention is another crucial parameter when considering stem cell therapy of SCI. By far the most animal experiments have been carried out in the acute phase, and with bmSC this seems to be justified because their main benefit is expected to be neuroprotection by modulating the immediate inflammatory response , 45. OurThis raises the question regarding the mechanisms by which the injected bmSC were effective in our experiments. Increasing evidence suggests that extracellular vehicles (EVs) are important players in mediating the therapeutic effects of therapeutically applied stem cells , 46, 47.The formation of fibrotic and glial scar is a major impediment to axonal regeneration after SCI. While reports with bmSC have claimed to reduce this , 13, 15,Were there continuing effects in the chronic stage? The behavioral data show that almost all improvement in sensory-motor performance of the rats occurred within the first three weeks and that the therapeutic benefit of bmSC treatment also occurred in this period Fig. . Using aUsing acute intervention with bmSC we were able to improve the natural recovery process within the first 10 weeks after lesion compared with corticosteroid treatment without adverse effects due to a possible immunological rejection. By reaching these objectives, the results of this study confirmed the beneficial effects of stem cells that were obtained earlier using immune-compromised rats and balloon compression SCI .In this and many other studies implanted stem cells could not be identified in the tissue when this was attempted in the chronic stages after SCI. Their failure to survive may be attributed to a hostile microenvironment created by the lesion , 51. It In the past, even the most promising results of preclinical studies with rodents could not be translated to clinical therapies of SCI or any other neurodegenerative disease. Depending on the physiological question and the risk of treatment, additional studies with non-human primates may therefore be necessary before a clinical trial is justified . Since lIn a clinical trial, bmSC would be prepared from the same patient and implanted without cryopreservation and within 48 h after injury. Since the potential risk for the patient is minimal we do not see the necessity of an intermediate study with large mammals or non-human primates, especially since differences in the motor systems between primate species are also not negligible . ExaggerAdditional file 1 Fig. S1. Evaluation of cellular apoptosis. Spinal cord sections were processed with double IHC against activated caspase-3/NeuN (neurons) and activated caspase-3/Iba1 (microglía/macrophages) and combined with DAPI nuclear staining. a Drawing of spinal cord and transverse sections indicating the ROIs for evaluation in the grey matter (40 x objective). b Low power photograph of the dorsal horn of a rat with SCI, demonstrating the distribution of apoptotic nuclei (pink). Annotation indicates outline of grey matter, ROI, counted cell nuclei and scale bar = 100 μm.Additional file 2 Fig. S2. Motor recovery as revealed with the Rotarod test. Motor performance in the rotarod stepping test is expressed in the amount of time [sec] that the animals maintained themselves on the rotating bar (mean ± SEM); SCI + bmSC: treatment with human bone marrow-derived stem cells; SCI + MP: injections of methyl prednisolone; SCI control: injections of NaCl. Before SCI, all animals reached the maximum time of 300 s. The first evaluation was performed at 4 dpo. Differences between groups were not significant. Rats that did not attempt to hold on to the bar and therefore received a score of zero sec were not excluded from the statistical evaluation.


To construct a machine learning algorithm model of lymph node metastasis (LNM) in patients with poorly differentiated-type intramucosal gastric cancer. 1169 patients with postoperative gastric cancer were divided into a training group and a test group at a ratio of 7:3. The model for lymph node metastasis was established with python machine learning. The Gbdt algorithm in the machine learning results finds that number of resected nodes, lymphovascular invasion and tumor size are the primary 3 factors that account for the weight of LNM. Effect of the LNM model of PDC gastric cancer patients in the training group: Among the 7 algorithm models, the highest accuracy rate was that of GBDT (0.955); The AUC values for the 7 algorithms were, from high to low, XGB (0.881), RF (0.802), GBDT (0.798), LR (0.778), XGB + LR (0.739), RF + LR (0.691) and GBDT + LR (0.626). Results of the LNM model of PDC gastric cancer patients in test group : Among the 7 algorithmic models, XGB had the highest accuracy rate (0.952); Among the 7 algorithms, the AUC values, from high to low, were GBDT (0.788), RF (0.765), XGB (0.762), LR (0.750), RF + LR (0.678), GBDT + LR (0.650) and XGB + LR (0.619). Single machine learning algorithm can predict LNM in poorly differentiated-type intramucosal gastric cancer, but fusion algorithm can not improve the effect of machine learning in predicting LNM. With the development of endoscopic techniques, improved diagnostics and the global popularization of gastric cancer screening, the early gastric cancer (EGC) detection rate increases every year, especially in Japan and Korea3. EGC can be treated with endoscopic resection, D1 or D2 radical surgical resection, as well as other medical auxiliary treatments according to tumor stage4. The indications and effects of the various treatments vary. EGC only considers the depth of focal infiltration; it does not consider lymph node metastasis, an important factor in choosing an EGC treatment regimen. Therefore, it is necessary to accurately stage EGC patients prior to surgery to select a reasonable treatment option. Studies have shown that EGC with lymph node metastasis (LNM), the number of lymph node metastases, and lymph node metastasis in different regions, have important effects on EGC treatment and prognosis5. Therefore, for over 80% of patients with EGC, radical surgery on D1 or D2 increases unnecessary lymph node dissection. It also increases the trauma caused by surgery, and affects patient recovery. In recent years, the development of endoscopic mucosal dissection and endoscopic mucosal resection has brought new developments to EGC treatment. There is now less trauma and quick postoperative recovery. Thus, patients can avoid the heavy trauma and long recovery time caused by laparotomy or endoscopic surgery. However, it is important to accurately judge lymph node metastasis before surgery6.Gastric cancer is the world's fourth most common neoplastic disease, and the second most fatal tumor-related disease7; Additionally, machine learning can predict hospital acquired pneumonia in patients with schizophrenia8; Machine learning techniques can also predict 5-year survival in patients with chondrosarcoma9.In recent years, many studies have reported on machine learning in medicine. For example, using large preoperative data to develop and validate machine learning algorithms can predict hospital stay and patient-specific hospital costs after primary total hip arthroplasty12. This study assesses clinicopathological factors for predicting LNM in intramucosal PDC. It also develops and validates a risk model for predicting LNM using machine learning to provide a basis for the treatment of poorly differentiated-type intramucosal gastric cancer.However, few studies have investigated the prediction of LNM in early poorly differentiated early gastric cancerhttps://www.ebi.ac.uk/biostudies/studies?query=S-EPMC4881979), accession numbers: S-EPMC4881979. We prospectively analyzed data from patients diagnosed with PDC who had undergone radical gastrorectal resection and lymph node dissection. Patients included in the study were confirmed as having pure poor differentiated-type T1 (tumor invasion confined to mucosa or submucosa) gastric cancers. The tumors were classified histologically according to the World Health Organization’s Classification of Tumors13.There were no human involved in this study. And this is only a secondary data analysis study using public databases. Data are available from the BioStudies (public) database , gender, tumor depth, age, presence of ulcer, tumor size, location of tumor, general appearance, number of resected nodules and presence of LNM. Tumors were staged according to the Seventh Edition of the American Joint Committee on Cancer Staging (7th Edition)Logistic regression (LR) is a broad classification machine algorithm that can predict the probability of future results, whereas "regression" is actually a classification. Accurate, logistic regression is a dichotomous classification algorithm.Random forest (RF) is a supervised learning algorithm. It is trained with the "bagging" method. The bagging method combines multiple models, and can be more effective than a single model. Thus, it can increase the overall effect.XGB generates multiple regression trees based on features, and each regression tree learns the corresponding residuals, and the sum of the residuals is the predicted value of the sample.GBDT is an integrated learning method that uses gradients as input to later trees to learn multiple trees. The combination of multiple trees can then generate a comprehensive learner with strong generalizability.https://cran.r-project.org/bin/windows/base/old/3.4.3/), and machine learning modeling was performed with python, version 3.6.5 (https://www.python.org/downloads/release/python-365/). Pearson’s correlation analysis was calculated, and the machine learning algorithm was performed with the following algorithms: XGB, RF, GBDT, LR, XGB + LR, RF + LR and GBDT + LR. 70% of the data was divided into training groups for development, and 30% were verified by the test groups. When missing values were dichotomous, the number of digits was used, and multiple imputation was used for continuous variables. MSE refers to mean squared error.Statistical analysis was conducted in R, version 3.4.3  of them. The age of the lymph node metastasis and non-metastasis groups did not statistically vary between the training and test groups ; The AUC values for the 7 algorithms were, from high to low, XGB (0.881), RF 0.802), GBDT (0.798), LR (0.778), XGB + LR 0.739), RF + LR (0.691) and GBDT + LR (0.626). Among the 7 algorithms, GBDT’s MSE was the lowest (0.045) and LR was the highest (0.054) ; Among the 7 algorithms, the AUC values, from high to low, were GBDT (0.788), RF (0.765), XGB (0.762), LR (0.750), RF + LR (0.678), GBDT + LR (0.650) and XGB + LR (0.619). XGB had the lowest MSE 0.048)  primary gastric adenocarcinoma, and between 9.4 and 16.1% for early (T1) primary gastric adenocarcinoma12. Our findings suggest that 5.2% of patients with poorly differentiated-type intramucosal gastric cancer develop lymph node metastases. This is consistent with previous findings. Furthermore, the results of this study indicate that the Gbdt machine learning algorithm yields the first 3 factors that account for the weight of lymph node metastasis: number of resected nodes, lymphovascular invasion and tumor size. At the same time, single machine learning algorithm can predict LNM in poorly differentiated-type intramucosal gastric cancer, but fusion algorithm can not improve the effect of machine learning in predicting LNM.At present, research has focused on minimally invasive surgery that can maintain postoperative patient survival rates. The goal is to minimize surgical injury with safe and effective operating procedures, so that patients can enjoy higher quality of life18. A large sample study in the United States showed that tumor stage, pathological type, and tumor size are independent predictors of LNMin early gastric cancer19. Chen et al. have concluded that tumor diameter ≥ 3 cm, whether it is pathological or low-differentiation type, whether it is mixed adenocarcinoma or signet ring cell carcinoma, tumor infiltration into the submucosa, and vascular invasion are independent risk factors for LNM20. Our results corroborate this view.Many clinical pathological factors related to LNM in early gastric cancer have been studied21. Pokala et al. concluded that early intramucosal gastric cancer with tumor diameter < 4 cm has a low risk of LNM, and can be locally resected22. This is consistent with the results of our study. Our results corroborate this view.The Japanese gastric cancer assistance group noted that the LNM rate was low for tumors > 2 cm in diameter, patients with no ulcers, tumors ≤ 3 cm in diameter, and differentiated intramucosal cancers with ulcers. This could serve as an absolute indication for ESD24. Studies have shown a high rate of LNM in undifferentiated early gastric cancer25. As the tumor grows, the invasion deepens and the LNM rate increases. The LNM rate has been shown to be associated with lymphangitic tumor thrombus26. Female patients with early gastric cancer are more likely to develop lymph node metastases than males. This is presumably related to endogenous estrogen levels27. Another study has shown that low differentiation, infiltration into the submucosa, large tumors, and venous or lymphatic invasion are independent risk factors for LNM28. These findings are also corroborated by our findings.Submucosal cancers have a higher rate of LNM than intramucosal cancers. Furthermore, they may be rich in capillaries in the submucosa of the gastric wall, which are usucaptible to cancer cell invasionAt present, the main problem of machine learning method in medical practice is the lack of application scenarios and related clinical data. At present, a large number of published machine learning articles only use simple machine learning algorithms. In this study, we also use the machine learning fusion algorithm. However, the results of the test set fusion machine learning algorithm are not ideal. This also proves that when the machine learning algorithm is applied in medical clinic, it should pay attention to the application scenarios and the collection of relevant data.This study has several limitations. Firstly, it only used routine hematoxylin and eosin staining. Therefore, accurate diagnosis of lymph node micrometastases was difficult. For example, lymph node micrometastasis may be a key causative factor in recurrent gastric cancer treatment. Furthermore, this study included only data on tumor characteristics; no data on patient-related tumor genes were collected. This may have contributed to the lack of optimal predictive results. Because different regions, different races and different treatment schemes may cause different incidence of lymphatic metastasis, and the rate of lymph node metastasis in intramucosal gastric adenocarcinoma is low in this study and previous studies.However, these will not affect the prediction results of machine learning in this study. However, more multi-center and forward-looking research is needed in the future.Single machine learning algorithm can predict LNM in poorly differentiated-type intramucosal gastric cancer, but fusion algorithm can not improve the effect of machine learning in predicting LNM. This may provide guidance for personalized treatment of such patients.


Kidney transplantation is the best treatment option for end stage renal disease (ESRD), but graft rejection is still a big obstacle that occurs in spite of immunosuppressive therapy. B cells are considered as the major reason for renal graft rejection because of antibody production. Due to their roles in B cell function, we intended to evaluate the B cell activating factor (BAFF) and its receptors including BAFF receptor (BAFF-R), B cell maturation antigen (BCMA), and transmembrane activator and cyclophilin ligand interactor (TACI) in renal transplant patients. + B cells were analyzed by flow cytometry, the serum level of BAFF was analyzed by ELISA, and mRNA expressions of BAFF and BAFF receptors  were measured using quantitative real-time PCR.  The study included 40 kidney allograft patients with cAMR, 40 stable kidney allograft patients, and 8 healthy volunteers with normal kidney function. The percentage and absolute number of CD19 The percentage and the absolute number of B cells decreased significantly in stable and cAMR patients compared to healthy individuals. The serum level and gene expression of BAFF, as well as the mRNA level of BCMA, were increased significantly in both cAMR and stable patients compared to healthy volunteers. There was an overexpression of TACI mRNA in cAMR patients compared to stable patients.  Both soluble protein and mRNA transcript of BAFF increased in transplant recipients. However, BAFF neither at the serum level nor at the mRNA transcript level cannot be a good biomarker for the prediction of cAMR. In addition, expression of TACI, compared to other receptors of BAFF, confers a potential to be used in distinguishing cAMR and stable kidney transplant patients. According to the statistics provided by the global observatory on donation and transplantation (GODT), more than 85,000 kidney transplantations have been done worldwide up to 2016 . AlthougThere are several methods for recognition of antibody-mediated rejection (AMR), include of measuring the serum level of donor-specific antibodies (DSAs) and staining of biopsied tissue for C4d as a complement fragment that has precipitated following antibody activity. However, the serum levels of DSAs and C4d depositions on the graft are not ideal biomarkers . The serγ, and contribute to T cell activation. Also, IL-17 that can be generated by B cells stimulates endothelial, epithelial, and fibroblast cells to produce chemokines and cytokines, leading to the recruitment of neutrophils and establishment of inflammatory conditions. Moreover, by producing cytokines like tumor necrosis factor- (TNF-) α, B cells directly promote inflammation, endothelial and epithelial cell injuries, as well as inflammatory renal disorders that cause allograft rejection [Several mechanisms have been attributed to B cells during graft rejection. The most important function of B cells is producing antibodies against donor human leukocyte antigen (HLA) and non-HLA antigens. These antibodies contribute to complement fixation and cell lysis, antibody-dependent cellular cytotoxicity (ADCC) by NK cells, increasing the thickness of endothelial and smooth muscle cells, activating the platelet, developing inflammatory conditions, thrombosis, and overall reducing of graft function , 12. Morejection . B cellsejection . TherefoκB. Afterward, TRAFs bind to TRAF binding sites and leading to the release of NF-κB inducing kinase (NIK). The inhibitor of κB kinase 1 (IKK1) inhibits NF-κB in a normal situation, but when BAFF stimulates its receptor, the released NIK phosphorylated the IKK1. The phosphorylated IKK1 is removed from NF-κB, and then, NF-κB is activated and moves to the nucleus to carry out its activities. But the TACI signaling pathway is different and is contributed with NF-κB, activator protein (AP)-1, and nuclear factor of activated T-cells (NF-AT). The consequences of the BAFF signaling are included germinal center formation, B cell differentiation and survival, and IgE and IgG class switching, as well as plasma cell survival [B cell-activating factor (BAFF), also known as TNFSF13B, BLyS, TALL-1, and CD257, is a cytokine that belongs to the TNF superfamily members . BAFF issurvival . Therefo2). Patients with T cell-mediated rejection or cAMR patients that their rejection was not approved by biopsy were excluded from the study. The remaining 40 patients (with a mean post-transplantation time of 69.59 months) had stable graft function without any symptoms of graft rejection, active infection, allergy, and autoimmunity (with mean creatinine concentration: 1.23 mg/dl and mean eGFR: 74.76 ml/min/1.73 m2). Additionally, eight healthy volunteers with normal kidney function and no history of kidney diseases were enrolled in the study as the healthy control group (with mean creatinine concentration: 0.81 mg/dl and mean eGFR: 107 ml/min/1.73 m2). The patient's demographic data and their baseline characteristics are summarized in Tables Eighty kidney transplant subjects  were enrolled in this study recruited from a multicenter collaboration of Labbafinejad, Shariati, and Sina hospitals, Tehran, Iran. Of these 80 subjects, 40 patients (with a mean post-transplantation time of 72.55 months) were diagnosed as cAMR through laboratory findings (urea and creatinine levels) and biopsy analysis  which have high creatinine concentration (mean: 3.30 mg/dl) and low estimated glomerular filtration rate  was used for isolating the peripheral blood mononuclear cells (PBMCs) from the whole blood. Isolated PBMCs cryopreserved in a media including 10% dimethyl sulfoxide  and 90% fetal bovine serum  and then stored in liquid nitrogen tank until use.+ cells were considered as total B cells population  and FBS (10%), and then incubated at 37°C for 15 minutes. Subsequently, phosphate-buffered saline (PBS) and FBS (0.1%) were utilized as a washing buffer to wash out the cells. Then, the cells were suspended in PBS and were stained with anti-human CD19 FITC mAb  according to the manufacture's protocol. CD19pulation . The B cThe serum of the coagulated whole blood samples was isolated after centrifuging and then stored at -70°C until use. Soluble BAFF levels were measured in the serum samples by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's recommended procedures . Values were reported as picograms per milliliter (pg/ml).β-actin, and GAPDH, were checked out, and GAPDH was chosen to normalize the mRNA expression of genes. The alteration in the expression level of each gene was calculated by the comparative Ct method as fold change using the 2-∆∆Ct formula.RNA was extracted from the whole blood samples by RNA extraction kit , and RNA quality was determined using the NanoDrop ND-1000 spectrophotometer  by measuring the absorbance at 260/280 and 260/230 nm. Extracted RNAs were reverse transcribed to complementary DNA (cDNA) by reverse transcription system kit . Quantitative real-time PCR was performed using SYBR green master mix and primers . Three hK independent samples  test employed for comparing the number and percentage of CD19+ B cells and lymphocytes, as well as mRNA expression levels between three groups. Correlation analysis was conducted to find any relation among the numerical variables. Nonparametric data were shown by the median ± interquartile range (IQR), and parametric data were presented as mean ± standard deviation (SD). P values ≤ 0.05 were considered statistically significant.The data analysis was performed by the SPSS software . To determine the normality of scale data distribution, the Kolmogorov-Smirnov test was used. One-way ANOVA was performed to compare the BAFF serum levels between three study groups. Tables P < 0.001) and cAMR patients  compared to healthy individuals (36.41 ± 5.62%)  and cAMR patients  in comparison to healthy subjects (2631.5 ± 836.84/mm3), while no significant difference was observed between stable and cAMR patients  compared to healthy individuals (5.77 ± 1.21%)  in comparison to healthy subjects (179.36 ± 80.15/mm3), and no significant difference was observed in percentage and absolute number of CD19+ B cells between stable and cAMR patients  ; howeverpatients . Also, s± 1.21%) , and alspatients .P = 0.038) and cAMR patients  compared to healthy individuals (1931.2 ± 666.7 pg/ml). However, there was not any significant difference between cAMR patients compared to stable group (P = 0.006) higher than cAMR patients in the Tac group  and cAMR patients  compared to healthy individuals . On the other hand, no statistically significant difference was observed between stable and cAMR patients with respect to the mRNA expression of BAFF (P = 0.041) higher than STS stable patients . The same results were seen for cAMR patients; LTS cAMR patients  showed significant (P = 0.039) overexpression of BAFF mRNA compared to STS cAMR patients   . But no Our results showed that there was not any significant difference in the BAFF-R mRNA expression level between both cAMR patients and healthy individuals, as well as stable patients and healthy control group. And no statistically significant difference was detected between cAMR and stable patients (supplementary figure P = 0.02) and cAMR patients  showed significantly higher BCMA mRNA expression level compared to healthy individuals . Nonetheless, there was not statistically significant difference between stable and cAMR patients  in the cAMR patients  compared to the stable patients  (P = 0.029) higher than STS stable patients . The LTS cAMR patients  also showed significant (P = 0.04) overexpression of TACI mRNA expression level compare to LTS stable patients   . Moreove = 1.90) , but drur = 0.26, P = 0.01) with blood urea level of patients. On the other hand, the BAFF mRNA expression did not have any correlation with urea, creatinine, eGFR, and BMI. However, we detected that the BAFF mRNA expression was positively correlated significantly with age  and years after transplantation  . It was observed that the BAFF serum level had a significant positive correlation (= 0.002) . Moreove= 0.002) .r = −0.22, P = 0.03) and a significant positive correlation  were observed between mRNA expression of TACI with eGFR and blood urea level, respectively. However, no correlation was detected between TACI mRNA expression level and creatinine, years post-transplantation, age, and BMI . The BAFF serum level and the mRNA expression of BAFF, BAFF-R, TACI, and BCMA were compared between stable transplanted patients with living or deceased graft source and also cAMR patients which received their graft from living or deceased ones. The results showed that there was not statically significant difference in BAFF serum level  and the −/−) have a prolonged graft survival compared to wild type mice [A study on cardiac allograft mice indicated that mice with deletion of BAFF cytokine gene or mutant BAFF-R  in kidney transplantation, and the results of these studies are conflicting. Our results showed that TACI is the only receptor that was increased in cAMR patients compared to stable recipients. This observation may stem from the fact that TACI is a common receptor for both BAFF and APRIL cytokines and binds to BAFF with high affinity. Also, the TACI signaling pathway is different from two other receptors. TACI has an extra ligand-binding site, which can be the cause of its different results compared to BAFF-R and BCMA. Our results showed that the TACI expression was increased in cAMR patients compared to stable patients, and this increased TACI remains high over time. When we investigated the TACI expression changes in short- and long-term survival patients, the same results were shown for both time points. It means that same as STS cAMR patients which had more TACI compared to STS stable patients, LTS cAMR patients showed more TACI in comparison to LTS stable patients too . Also, wBy investigating 143 transplanted patients at three time points include the time of renal dysfunction, the time of anti-HLA antibody appearance, and the time of DSA development, Thibault-Espitia et al. reported no correlation between serum and mRNA expression levels of BAFF, same as our results. But this study reported that both serum and mRNA expression levels of BAFF correlated negatively with TACI and BAFF-R transcripts, while they did not have any correlation with BCMA. In addition, they indicated that patients with upregulated BAFF-R expression were more prone to developing renal dysfunction, and also, patients with low BAFF transcripts and high soluble BAFF levels had a higher risk to develop DSAs . Again, Overall, in spite of lower B cell count in transplanted patients because of lymphocyte depleting immunosuppressive drugs, their BAFF level is increased. Both soluble protein and mRNA transcript of BAFF increased in transplant recipients; as well, BAFF transcripts had a positive correlation with years post-transplantation and increased over the years after transplantation in both stable and cAMR patients. However, BAFF neither at the serum level nor at the mRNA transcript level cannot be a good biomarker for prediction of cAMR. In the case of receptors, TACI as a common receptor for both APRIL and BAFF is more important compared to other receptors, because it has a higher expression level in cAMR patients compare to stable patients, and it may distinguish cAMR and stable patients, even years after transplantation, because both STS and LTS cAMR patients have increased TACI compared to STS and LTS stable patients. Nonetheless, further studies are still required to disclose the clear involvement of TACI in the context of kidney transplantation.


To determine the diagnostic performance, cut-off values, and optimal drive frequency range for staging hepatic fibrosis using tomoelastography by multifrequency MR elastography of the liver and spleen.This prospective study consecutively enrolled a total of 61 subjects between June 2014 and April 2017: 45 patients with chronic liver disease and proven stage of fibrosis and 16 healthy volunteers. Tomoelastography was performed at 1.5 T using six drive frequencies from 35 to 60 Hz. Cut-off values and AUC were calculated. Shear wave speed (in m/s) of the liver and spleen was assessed separately and in combination as a surrogate of stiffness.p = 0.03). Full-field-of-view elastograms displayed not only the liver and spleen but also small anatomical structures including the pancreas and major vessels.For compound multifrequency processing of the liver, cut-off and AUC values by fibrosis stage were as follows: F1, 1.52 m/s and 0.89; F2, 1.55 m/s and 0.94; F3, 1.67 m/s and 0.98; and F4, 1.72 m/s and 0.98. Diagnostic performance of the best single drive frequencies  was similar . Combined analysis of the liver and spleen slightly improved performance at 60 Hz in F4 patients  and compound multifrequency processing are equivalent for staging hepatic fibrosis.• Combined assessment of hepatic and splenic stiffness slightly improves diagnostic performance for staging hepatic fibrosis. Detection and treatment of hepatic fibrosis at an early stage can prevent ongoing hepatocellular damage, progression towards cirrhosis, and complications such as portal hypertension and ascites. The combined examination of the liver and spleen has emerged as a promising field of research –4. It haMagnetic resonance elastography (MRE) is a noninvasive imaging technique for staging hepatic fibrosis by assessing mechanical tissue properties –9. It isThe primary aim of this study was to determine the diagnostic performance, cut-off values, and optimal drive frequency range for staging hepatic fibrosis using tomoelastography of the liver in patients with biopsy-proven fibrosis or imaging findings of cirrhosis. As a secondary aim, we investigated whether a combined analysis of the liver and spleen would further improve diagnostic performance.The study was approved by the institutional review board, and written informed consent was obtained from all subjects. In this prospective monocenter study, a total of 61 subjects were consecutively enrolled: 45 patients (18 women) and 16 healthy volunteers (8 women). Patients were examined between June 2014 and April 2017. Inclusion criteria were the presence of chronic liver disease (CLD) and liver biopsy performed or planned within 1 year of enrollment. Patients without biopsy but definite imaging findings of cirrhosis with a nodular liver contour and segmental hypertrophy or atrophy were also included since histological sampling would have been unethical in these cases. Further inclusion and exclusion criteria are shown in Fig. 3 resolution, 2 averages, and 50-Hz motion-encoding gradient frequency. To avoid an increased postprandial hepatic blood flow and stiffness, subjects fasted for at least 4 h [Tomoelastography was performed on a 1.5 T MRI scanner  with a 12-channel phased-array coil. We used a custom-designed piezoelectric driver, fast single-shot 3D wave-field acquisition at drive frequencies of 35 to 60 Hz with 5-Hz increments, as proposed by Hirsch et al [east 4 h , 16. Thehttps://bioqic-apps.charite.de and described in detail by Tzschätzsch et al [. Briefly, it has been demonstrated that complex wave number recovery and amplitude-weighted averaging of multiple harmonic frequencies in a compound map of stiffness outperforms detail resolution and noise robustness of single frequencies. Compound multifrequency processing improves elastograms by reducing areas of low shear wave displacement and wave nodes. Tomoelastography parameters are shear wave speed  and penetration rate . While c relates to stiffness , a relates to damping of shear waves . Full-field-of-view elastograms of the liver and spleen were derived from the same scan. Each slice of the elastograms was generated by compounding 216 images of MMRE raw data . Regions of interests were generated using a systematic approach: (i) The contours of liver and spleen were manually outlined using the magnitude image. (ii) A lower shear wave speed threshold of 1 m/s was applied to reduce boundary effects of major blood vessels and regions of insufficient shear wave excitation. Values above 1 m/s were included in the analysis. Consistent filter settings were used for all subjects. Liver fat content was calculated according to Fischer et al [For image processing, we used a recently introduced tomoelastography pipeline, which is entirely available at ch et al . Brieflyer et al . For pater et al .p ≤ 0.01. Spearman’s rank correlation coefficient (Rs) was calculated for a pairwise comparison of all parameters with the stage of fibrosis. Pearson’s correlation coefficient (Rp) was calculated for hepatic and splenic shear wave speed. A two-sided t test was used to assess the differences between patients and healthy volunteers as well as hepatic and splenic shear wave speed. The level of significance was p ≤ 0.05. Sensitivity, specificity, negative and positive predictive values, area under the receiver operating characteristic curve (AUC) with 95% confidence intervals (CI), and optimized cut-off values using the Youden index were calculated for fibrosis staging, accounting for single drive frequencies as well as compound multifrequency processing. AUC with 95% CI for combined hepatic and splenic parameters was calculated using binary logistic regression and compared with hepatic AUC with a level of significance of p ≤ 0.05, as described by DeLong et al [The Shapiro–Wilk test was used to assess normal distribution with a level of significance of ng et al . Mean AU2 and 24 ± 4 kg/m2 and 3 ± 7% and 3 ± 5%, respectively. Liver fat content was not available for 1 patient. Healthy volunteers had no known history of any liver disease. Patients had the following CLDs: chronic hepatitis B and C , primary sclerosing cholangitis (n = 9), nonalcoholic steatohepatitis (n = 6), autoimmune hepatitis (n = 6), toxic liver disease (n = 5), primary biliary cholangitis (n = 2), Wilson’s disease (n = 2), cryptogenic fibrosis (n = 2), diffuse liver metastases from breast cancer (n = 1), and alcoholic liver disease (n = 1). The mean (± SD) time interval between biopsy and tomoelastography was 69 ± 99 days. Fibrosis stage distribution in the patients included in the analysis (n = 43) based on the reference standard was as follows: F0, n = 1; F1, n = 10; F2, n = 5; F3, n = 9; and F4, n = 18. No correlation was found between age, BMI, liver fat content  and the stage of fibrosis.The flow of subject enrollment is shown in Fig. c) data but not for penetration rate (a) data. Mean c- and a-values of the liver and spleen are listed in Table c-values of compound multifrequency processing are displayed in Fig. c derived from compound multifrequency processing was evident between patients and healthy volunteers ; in contrast, no significant difference was found for mean splenic c . Mean c was significantly higher in the spleen compared with that in the liver for all single drive frequencies  and compound multifrequency processing (p = 0.02). There was a strong significant correlation between hepatic c and the stage of fibrosis . For liver fat content, there was a weak significant correlation with hepatic a ; in contrast, no significant correlation was found for hepatic c .Tomoelastography of the liver and spleen failed for 2 and 5 patients, respectively, due to insufficient shear wave propagation based on technical difficulties with the custom-designed piezoelectric driver setup. The overall technical success rate was 96.7% for the liver and 91.8% for the spleen. Figure Rs ≥ 0.86 with p < 0.001; Table c-values showed an increased mean AUC of 0.97 at 60 Hz; however, statistical significance was only evident for stage F4  of 92% (85–96%) for the liver and 96% (93–98%) for the spleen was found.c, high AUC values suggest an excellent discriminative ability for staging hepatic fibrosis while detail resolution was improved compared with available MRE techniques. Full-field-of-view elastograms show a pixel-wise detail resolution in a tomographic fashion, which replaces the need to superimpose elastograms with conventional morphological images to identify abdominal organs. The best mechanical drive frequencies for the liver in terms of diagnostic performance are 45 Hz, 55 Hz, 60 Hz, and compound multifrequency processing.To our knowledge, this is the first study investigating MRE of both the liver and spleen for staging hepatic fibrosis. We aimed to determine the diagnostic performance, cut-off values, and optimal drive frequency range of tomoelastography for this indication. For hepatic Our results suggest a better diagnostic performance for higher drive frequencies and for staging severe fibrosis (F3) or cirrhosis (F4), which is consistent with the literature , 20. ForOur current results demonstrate that the diagnostic performance of compound multifrequency processing is equivalent to that of higher single drive frequencies and not inferior as reported by Asbach et al . NeverthIt is a stimulating result that the combined analysis of liver and spleen improved diagnostic performance in our study, which is in contrast to the results of an ultrasound elastography study by Leung et al . Howeverc as well as hepatic and splenic a, low AUC values suggest a poor ability or failure to stage hepatic fibrosis. This poor sensitivity of a, as a representation of damping, for staging fibrosis has been shown previously [a with steatosis has been demonstrated recently by a study investigating nonalcoholic fatty liver disease and should be implemented in future studies and data analysis for liver fat quantification [For splenic eviously , 20. Howfication . Our resFuture studies should investigate the significance of tomoelastography for the assessment of focal liver lesions and fibrosis heterogeneity as an additional biomarker besides overall stiffness and compare the diagnostic performance of different MRE setups and image processing pipelines.Our study has some limitations. First, we examined a small number of patients, especially in the F2 group, since liver biopsy is increasingly avoided in clinical routine in favor of noninvasive diagnostic tests. Moreover, we had a deviated population with a larger proportion of subjects in the F0 and F4 group, which can lead to overestimation of diagnostic performance. Second, there was a long interval between tomoelastography and biopsy, which can lead to misclassification. However, a recent meta-analysis suggests a low risk of disease progression bias when the interval is less than 1 year . Third, In conclusion, tomoelastography provides cut-off values with excellent diagnostic accuracy for staging hepatic fibrosis. While diagnostic performance was comparable to that reported for other elastography techniques in prior studies, tomoelastography provided full-field-of-view elastograms of the abdomen with unprecedented pixel-wise detail resolution in a tomographic fashion. Our analysis of single-frequency tomoelastography suggests that scan time can be further reduced in future studies, making tomoelastography easier to implement in clinical routine.


We aimed to reveal clinicopathological features and explore survival-related factors of colorectal signet ring cell carcinoma (SRCC). A population-based study was carried out to investigate colorectal SRCC by using data extracted from the surveillance, epidemiology and end results (SEER) database between 2004 and 2015. In total, 3,278 patients with colorectal SRCC were identified, with a median age of 63 (12–103) years old. The lesions of most patients (60.49%) were located in the cecum–transverse colon. In addition, 81.27% patients had advanced clinical stage (stage III/IV), and 76.69% patients had high pathological grade. The 3–, 5–year cancer‐specific survival and overall survival rate was 35.76%, 29.32% and 32.32%, 25.14%. Multivariate analysis revealed that primary site in cecum–transverse colon, married, received surgery, lymph node dissections ≥ 4 regional lymph nodes were independent favorable prognostic. Meanwhile, aged ≥ 65 years, higher grade, tumor size ˃5 cm and advanced AJCC stage were associated with poor prognosis. Patient age, tumor grade, marital status, tumor size, primary tumor location, AJCC stage, surgery and number of dissected lymph node had significant correlation with prognosis of colorectal SRCC. Recently accumulative attention has been paid to colorectal signet ring cell carcinoma (SRCC), initially proposed by Saphir and Laufman in 19512. Stomach is considered as the most common site for primary SRCC, while colorectal SRCC is less frequent3. In addition, colorectal SRCC is a very rare and special type of all CRCs, which has its unique clinicopathological characteristics and prognosis8.Colorectal cancer (CRC) is still a main global health burden, ranking the second of all types of malignancies in terms of cancer-associated mortality10. Moreover, SRCC has been considered as an independent prognostic indicator for poor outcome based on the AJCC stage system11. However, the currently limited understanding of colorectal SRCC is mainly based on case reports as well as small case series, without complete elucidation on the clinicopathological features and prognosis12.Various researches have shown the origination of SRCC from undifferentiated stem cells of colorectal mucosa, thus, rapid growth, poor differentiation, diffuse infiltration as well as high metastatic rate are generally observed13, covers nearly 30% of population in the USA from different geographic regions14, which provides valuable data to investigate rare malignancies17. Therefore, we performed a retrospective analysis of patients with colorectal SRCC registered in the SEER database to summarize clinical characteristics and survival and to delineate features influencing prognosis.The National Institutes of Health (NIH)’ s Surveillance, Epidemiology and End Results (SEER) database, the most authoritative and largest cancer dataset in North AmericaIn order to obtain relevant data from the database, we signed the SEER Research Data Agreement (No. 19817-Nov2018) and further searched for data according to the approved guidelines. The extracted data were publicly available and de-identified, and the data analysis was considered as non-human subjects by Office for Human Research Protection, therefore, no approval was required from institutional review board.SEER*State v8.3.6 tool was used to select qualified subjects. Colorectal SRCC patients who were diagnosed from January 1, 2004 to December 31, 2015 were selected from the Incidence-SEER 18 Registries Custom Data , released August 2019, based on the November 2018 submission. The inclusion criteria included the following: (1) it should be primary colorectal SRCC patients; (2) SRCC diagnosed in line with the International Classification of Disease for Oncology, Third Edition . The exclusion criteria were listed in the following: (1) Patients had multiple primary tumors; (2) Patients with reported diagnosis source from autopsy or death certificate or only clinically diagnosed; (3) Patients did not have some important clinicopathological information, including: AJCC stage and surgical style; (4) Patients had no prognostic data. The remaining qualified populations were included.18. Year of diagnosis was equally divided into 2004–2007, 2008–2011, 2012–2015, which was referred to the previous papers20. The grouping of the age21 and tumor size22 also refers to the published studies. In addition, the staging of cancer is based on the 6th edition of AJCC stage system, which adapted to patients in the SEER database with a diagnosis time of 2004–201523.We analyzed the patients’ characteristics according to the following factors: year of diagnosis ; insured status ; marital status ; gender ; race ; primary site; grade ; tumor size ; AJCC stage ; surgery, and lymph node dissection .The widowed or single (never married or having a domestic partner) or divorced or separated patients were classified as unmarried. The primary tumor site was classified as cecum–transverse colon , descending colon–sigmoid (including the splenic flexure and descending and sigmoid colons), multiple, rectum and unknownThe endpoint of this study was cancer‐specific survival (CSS) and overall survival (OS). CSS was defined as the period from diagnosis to death attributed to colorectal SRCC. OS was defined as the period from diagnosis to death from any cause.P values ≤ 0.1 in univariate analysis, they were incorporated into multivariate Cox proportional hazard analysis. SPSS software version 19.0  was adopted for statistical analysis, and GraphPad Prism 5 was adopted for plotting survival curves. A two-sided P < 0.05 was suggestive of statistical significance.Kaplan–Meier (K–M) method was employed for univariate analysis and stratified analysis, followed by log-rank test to determine the differences of OS and CSS. Of note, if variables had Based on the inclusion and exclusion criteria, 3,278 eligible subjects were included. The flowchart of patient selection was displayed in Fig. P < 0.0001). The 5–year CSS and OS rate in subjects with stage I were 73.66% and 65.63%; stage II: 69.81% and 57.72%; stage III: 37.31% and 31.94%; stage IV: 4.80% and 4.24%, indicating inferior outcome in those with stages III or IV.The median survival time of all included patients was 16.0 months (0–155 months). The 1–, 3– and 5–year CSS rate was 62.66%, 35.76%, and 29.32%, respectively . Additionally, multivariate analysis further demonstrated that married status ; primary site in cecum–transverse colon (P = 0.001) and surgery  were independent prognostic indicators for better survival. Meanwhile, higher grade , tumor size ˃5 cm  and advanced AJCC stage  could independently predict worse prognosis. Similar outcomes were observed in multivariate analysis on OS. Besides, it found that aged ≥ 65 years  and lymph node dissection ≥ 4  were also independent prognostic indicators of OS 28. These findings are consistent with our study. Moreover, we found that the median age was 63 years old in patients with colorectal SRCC, and 1537 (46.89%) patients were old patients. Our results should be more reliable in this large population-based study while most previous researches on colorectal SRCC were only based on single institution.According to the WHO definition, colorectal SRCC is a special type of CRC with prominent intracytoplasmic mucin in over 50% of tumor cells20. Ramya et al. have revealed that the median OS was 18.6 months by involving 206 subjects with colonic SRCC7. In addition, the stage-specific 5-year survival rate was declined with advanced stage. The above results are basically consistent with our results, while the median survival time of our study is shorter (16.0 months).Colorectal SRCC is independently related to increased mortality risks7. Tumor location may be another independent prognostic indicator29. These two prognostic factors are both found in our study. In addition, we found that pathological grade and tumor size were independent prognostic factors of colorectal SRCC.Multiple studies have evaluated the stage-associated in colorectal SRCC, with consistent outcomes indicating better survival in early-stage SRCC12. Additionally, a large population-based study has evaluated the significance of adjuvant chemotherapy by extracting relevant data from nationwide population-based Netherlands Cancer Registry, which enrolled 1972 subjects with colorectal SRCC from 1989 to 2010. In this study, better survival outcome was detected in stage III colon SRCC patients receiving adjuvant chemotherapy than those without6. Tao Shi et al. also found an association between chemotherapy and better survival outcomes in colorectal SRCC patients suffering from distant metastasis21. In contrast, the role of radiotherapy in colorectal SRCC is less studied which has not been evaluated alone or combined with chemotherapy in SRCC histology to date. Nevertheless, neoadjuvant chemoradiotherapy could give rise to good therapeutic response in rectal SRCC population30. This study further emphasized the importance of operation in colorectal SRCC, including the number of dissected lymph node, and regional lymph node dissection ≥ 4 significantly improved the prognosis of patients.In terms of the optimal therapeutic regimens, the multidisciplinary management is required for colorectal SRCC. To be specific, surgical intervention is vitally involved in treating localized tumors17. Furthermore, not all data are available from the SEER database, such as molecular-genetic profiles. Thirdly, as the radiotherapy and chemotherapy variables have a relatively low sensitivity, we didn't include the data of radiotherapy and chemotherapy to avoid reporting misleading results. Finally, we could not determine the therapeutic response or recurrence rate according to the data extracted from SEER. Thus, further investigations are warranted in the future.To our knowledge, the current research is the largest population-based study to explore prognostic indicators in colorectal SRCC in the past decade. In total, 3,278 colorectal SRCC patients were analyzed after retrieving from SEER database. SEER database renders the assessment of a large cross-section of cancer patients and provides long-term follow-up information without inherent institutional biases. Meanwhile, there are certain limitations in our research. Firstly, as a retrospective research based on SEER database, the intrinsic selection biases exists in this studyIn conclusion, we analyzed clinicopathological features as well as survival of colorectal SRCC patients using a population-based approach. Consequently, patient age, tumor grade, marital status, tumor size, primary tumor location, AJCC stage, surgery and dissected lymph node had significant correlation with prognosis of colorectal SRCC. Our study is the largest population-based research to investigate clinicopathological features and survival of patients with colorectal SRCC. Hopefully, our findings are of great significance for the management and future prospective studies for colorectal SRCC.


Terlipressin, a long-acting synthetic analogue of vasopressin has been used in the adult population for various indications including hepatorenal syndrome (HRS-AKI), esophageal variceal hemorrhage (EVH) and shock, but its use in pediatrics is still limited to individualized cases and data on safety and efficacy is scant.We reviewed the patient records of children with liver disease and Acute Kidney Injury requiring terlipressin admitted to the Paediatric Intensive Care Unit (PICU) of King’s College Hospital, London from January 2010–December 2017, with special emphasis on its effect on renal parameters and adverse event profile.p = 0.386) and increase in urine output (UO), especially in the hepatorenal syndrome subgroup (HRS-AKI). We found minimal evidence of gastrointestinal side effects including feeding intolerance and vasoconstrictive side effects including cyanosis / ischaemia of extremities.Twenty-one terlipressin administration records in a total of 16 patients (median) (IQR) 10 years (6.1–14.4) were included. The drug was initially given as a bolus dose in all cases, followed by either bolus or infusion with median dosage being 5.2 (3.8–6.7) mcg/kg/hour. After administration, a sustained increase of mean arterial pressure was observed. There was an improvement in serum creatinine (Cr) (at 24 h; Terlipressin was found to be safe in critically sick children with liver disease with positive impact on renal parameters which might be taken as a surrogate marker of HRS reversal, though effects on outcomes are difficult to ascertain. It is important to be aware of all its side-effects and actively watch for them. Future prospective studies are warranted to validate these findings. Terlipressin (triglycyl lysine vasopressin), is a long-acting synthetic analogue of vasopressin. The distribution half-life of terlipressin is 8 min and it gets eliminated in 50 min , 2. VasoUse of terlipressin in children has been limited to vasodilatory shock , 13. ExpA retrospective chart review was conducted using pre-recorded, patient-centered data to study the impact of terlipressin on renal parameters and adverse event profile. We reviewed the patient records of children, admitted to PICU, King’s College Hospital, London who required terlipressin between 2010 and 2017. Data was extracted from electronic patient records (Electronic patient record (EPR), clinical Information system (Metavision) and electronic drug charts (EPMA).The inclusion criterion for the study was the use of terlipressin during the period of stay in PICU for pediatric patients admitted with liver disease. These records have been expressed as ‘patient administration records’ for this study. If terlipressin usage was interspersed by more than 15 days, it was taken as two separate entries, even if it was used in the same patient during same admission.Patients were classified as having hepatorenal syndrome, if they satisfied the acute kidney injury (HRS-AKI) criteria as per the International Ascites Club definition 2012 , 16. NinThe indications of terlipressin in non-HRS AKI included refractory vasodilatory shock (refractory to norepinephrine) and massive upper gastrointestinal bleed. Terlipressin was then administered either as an infusion or bolus, based on PICU consultant’s decision. Terlipressin was continued for 7–10 days based on the response  and was discontinued in case of any adverse effects or no improvement in serum creatinine/urine output after 4 days of starting terlipressin. We doubled the dose after 48 h if there was no response.Categorical data was presented as counts and percentage, and analyzed with help of chi square test. Skewed distribution was described as median and interquartile ranges. For parametric tests, t test (paired or unpaired) and for non-parametric tests, Wilcoxon signed rank test and Mann Whitney U test were used as appropriate.We extracted data for 21 terlipressin administration records, used in 16 patients with liver disease, who were administered terlipressin between 2010 and 2017. These patients had a median age of 10 years (6.1–14.4 years). All patients were treated in PICU  or continued as boluses (13 instances). All patients had chronic liver disease. Of the 16 patients, 10 had acute on chronic liver failure (ACLF) with multiple organ failures and 6 patients had decompensated liver disease with ascites, bleeding varices and portal hypertension. Etiologies for hepatic impairment included genetic causes , metabolic causes and biliary atresia Table .Table 2All patients included in the analysis satisfied the AKI definition of increased serum creatinine above the baseline. The impact on lactate and mean arterial pressure (MAP) are described in Fig. p = 0.45) and 3.4(0.51–11.6) at 6 h(p = 0.688). There was no significant effect on serum sodium as well  This is shown in Table There were 3 adverse events observed following administration. These included 2 incidents of digital ischemia (blanching or blueness of extremities) and one incident of feeding intolerance. In one instance, the drug had to be discontinued because of adverse effect. No significant effect on lactate levels was observed (Baseline - 3.3(0.7–11.23), it remained constant, 3.48(1.1–10.6) at 1 h at the start of drug use.The average duration of bolus use was 40 h (0–60), with median of 5 boluses (range: 1–44) received. The median dose (IQR) used was 5.2(3.8–6.7 mcg/kg/hr). In HRS-AKI we used terlipressin at the dose of 5.2 (4.5–6.4) (IQR) mcg/kg/hr.Of the 16 patients, 7 patients died, mortality of 47.5%. Of these, 2 patients had HRS-AKI, giving a mortality of 28.5% to this group. There was no statistical difference in urine output, creatinine and mean arterial pressure, between the survivors and non-survivors. Multiple causes contributed to this mortality - gastrointestinal bleeding (in 4 patients), multiple organ dysfunction syndrome (MODS) (in 6 patients) and sepsis (in 2 patients).Terlipressin is a powerful V1(vasopressin) receptor agonist . At our Splanchnic arterial vasodilation is considered important in the pathogenesis of HRS-AKI. Use of vasoconstrictive drugs results in splanchnic arteriolar vasoconstriction with decreased portal pressure, thereby resulting in redistribution of blood flow, thereby improving renal blood flow –20. VasoAdult studies have demonstrated improvement in renal parameters. When terlipressin is used in hepatorenal syndrome, reversal of HRS defined as decrease in serum creatinine by 50% baseline to less than 1.5 mg/dl is demonstrated in 45–55% cases , 11, 20.There is a paucity of literature on pediatric terlipressin usage. We could only find a single case series, where the average unique dose of 15 to 20mcg/kg 4-hourly was used. All of the patients in this case series received terlipressin as a continuous infusion at 30 mcg/kg/day . In our Most of the side-effects of terlipressin are due to its severe vasoconstrictive properties. In our study, we found minimal evidence of gastrointestinal side effects (including feeding intolerance) and vasoconstrictive side effects including cyanosis of extremities.There have been many adult case reports of severe side effects after terlipressin usage, including severe skin necrosis (after extravasation of low dose vasopressin) , 24 gastThough previous studies have reported metabolic side effects such as hyponatremia and features of microvascular ischemia with lactic-acidosis, this was not observed in our study , 25.This is a single center retrospective study of patients, hence, it carries the limitations of risk of bias and lack of complete records due to retrospective nature of data collection. In addition, the vital parameters, were recorded with a focus on first 24 h of starting the drug. The biggest challenge is retrospectively classifying patients with HRS AKI versus those with shock, though proven to be non-pre-renal. HRS in paediatrics is not very well defined. It is very difficult to prove the efficacy in terms of bridging to transplant. Does improvement in biochemical parameters equate to improved survival, cannot be answered by our data. It is also limited by the small sample size and use of terlipressin in those patients who were on renal replacement therapy. A prospective, adequately powered, placebo controlled, multicenter trial may be beneficial in validating these results.To conclude, our experience of using terlipressin in critically ill children with liver disease shows that terlipressin is safe and might be useful in decreasing serum creatinine especially in children with HRS-AKI. Whether reduction in serum creatinine and increase in urine output translates to improved clinical outcomes both in terms of reversal of HRS and improved survival (with or without transplant) remains to be seen. An important conundrum which needs to be answered is whether prolonged terlipressin usage can be used to as a bridge to liver transplantation in this cohort of sick liver patients. It is important to be aware of the side-effect profile and expectantly manage them. Future research is needed to validate the findings of this study.