Molecular diagnosis of pancreatobiliary tract cancer by detecting mutations and methylation changes in bile samples.

Background: Patients with pancreatobiliary tract cancer usually have a poor clinical outcome, with a 5-year overall survival rate below 20%. This is mainly associated with the late diagnosis. In addition, the standard-of-care for patients with malignant biliary stenosis involves a major surgery, the Whipple procedure. An accurate preoperative diagnosis, including differentiation from benign diseases, is critical to avoid unnecessary treatment. Here we developed BileScreen, a sensitive detection modality for the diagnosis of pancreatobiliary tract cancer based on massively parallel sequencing mutation and methylation changes in bile samples. Methods: A total of 338 patients, from five hospitals in China, with pancreatobiliary system disorders were enrolled in this study between November 2018 and October 2020, and 259 were included for the analysis of BileScreen. We profiled 23 gene mutations and 44 genes with methylation modifications in parallel from bile samples, and set up a model for the detection of malignancy based on multi-level biomarkers. Findings: We applied the BileScreen assay in a training cohort (n = 104) to set up the model and algorithm. The model was further evaluated in a validation cohort (n = 105), resulting in 92% sensitivity and 98% specificity. The performance of BileScreen was further assessed in a prospective test cohort (n = 50) of patients diagnosed with suspicious or negative pathology by endoscopic retrograde cholangiopancreatography and were confirmed in follow-up. BileScreen yielded 90% sensitivity and 80% specificity, and outcompeted serum carbohydrate antigen 19-9 in detecting pancreatobiliary tract cancer in all three cohorts, especially in terms of specificity. Interpretation: Taken together, BileScreen has the ability to interrogate mutations and methylation changes in bile samples in parallel, thus rendering it a potentially sensitive detection method to help in the diagnosis of pancreatobiliary tract cancer in a safe, convenient and less-invasive manner. Funding: This study was supported by the Capital's Funds for Health Improvement and Research (2020-2-4025 to S.H.), the National Natural Science Foundation of China (81972311 to H.Z.), CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-12M-4-002 to H.Z.), the CAMS Innovation Fund for Medical Sciences(CIFMS) (2021-I2M-1-066 to CJQ), the Non-profit Central Research Institution Fund of Chinese Academy of Medical Sciences (2019PT310026 to H.Z.) and Sanming Project of Medicine in Shenzhen (SZSM202011010 to H.Z.).

Characteristics of bile microbiota in cholelithiasis, perihilar cholangiocarcinoma, distal cholangiocarcinoma, and pancreatic cancer.

Significant proof suggests an essential role played by the bile microbiota in biliary diseases. This study retrospectively analyzed the differences in biliary microbes among patients with perihilar cholangiocarcinoma (pCCA), distal cholangiocarcinoma (dCCA), pancreatic cancer (PC), and cholelithiasis (CH). Bile samples were obtained from 53 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP), and the bile microbiota was analyzed through 16S rRNA gene analysis and next-generation sequencing. Based on the results of linear discriminant analysis effect size (LEfSe), the top three biomarkers for pCCA at the genus level were Pseudomonas, Sphingomonas, and Halomonas; for dCCA were Streptococcus, Prevotella, and Halomonas; and for PC were Pseudomonas, Chloroplast, and Acinetobacter. The top five genera in the pCCA, dCCA, and PC groups showed predictive values with areas under the receiver operating characteristic curves of 91.56%, 95.56%, and 96.59%, respectively. The PICRUSt2 analysis outcomes displayed the diversities of fifteen pathways between the CH and pCCA groups, 22 pathways between the CH and dCCA groups, and eighteen pathways between the CH and PC groups. As this pilot study identified specific microbial bile markers for patients with CH, pCCA, dCCA, and PC, the clinical implications are vast. Further study focusing on distinct bacterial populations in bile will help differentiate biliary diseases.

The Effect of Preoperative Biliary Drainage with or without Pancreatic Stenting on Complications after Pancreatoduodenectomy: A Retrospective Cohort Study.

BACKGROUND: The necessity of preoperative biliary drainage (PBD) prior to pancreaticoduodenectomy (PD) is still controversial. However, in some settings, PBD with endoscopic retrograde cholangiopancreatography (ERCP) procedure is recommended as a preferred management. Meanwhile, pancreatic duct stenting in the drainage procedure is rarely performed for selected indications, and its associated complications after PD remain quite unknown. METHODS: A retrospective observational longitudinal cohort study was performed on patients who underwent PBD and PD from a prospectively maintained database at the National Cancer Center from March of 2015 to July of 2019. Patients who underwent biliary stenting alone, biliary and pancreatic stenting, were distributed into two study cohort groups, and their records were scrutinized for the incidence of postoperative complications. RESULTS: A total of 83 patients who underwent successful PD after biliary drainage were identified. 29 patients underwent nasobiliary drainage (ENBD)/plastic or metal bile duct stenting (BS) and pancreatic duct stenting (PS group), and 54 patients underwent only ENBD/BS, without pancreatic duct stenting (NPS group). No differences were found between the two groups with respect to in-hospital time, overall complication rate, respective rate of serious (grade 3 or higher) complication rate, bile anastomotic leakage, bleeding, abdominal infection, surgical wound infection, organ dysfunction, and pancreatic anastomotic leakage. Postoperative gastrointestinal dysfunction rates differed significantly, which occurred in 3 (5.56%) cases in the NPS group, compared with 6 (20.7%) cases in the PS group (P = 0.06). In the univariate and multivariate regression model analysis, pancreatic duct stenting was correlated with higher rates of gastrointestinal dysfunction [odds ratio (OR) = 4.25, P = 0.0472]. CONCLUSION: Our data suggested that PBD and pancreatic duct stenting prior to pancreatoduodenectomy would increase the risk of postoperative delayed gastric emptying, while the overall incidence of postoperative complications and other complications, such as pancreatic leakage and bile duct leakage, showed no statistical difference.

Comment on "Updated meta-analysis of pancreatic stent placement in preventing post-endoscopic retrograde cholangiopancreatography pancreatitis".

I read with interest an article "Updated meta-analysis of pancreatic stent placement in preventing post-endoscopic retrograde cholangiopancreatography pancreatitis" by Fan and colleagues in World J Gastroenterol 2015; 21(24): 7577-7583. Although I appreciate their work, I have found problems with the data extracted and analyzed by the authors, and will give my comment in this letter. It would be valuable if the authors could provide an accurate estimation of their extracted data.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor.

The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

Influence of surface modification and static pressure on microdialysis protein extraction efficiency.

There is growing interest in using microdialysis (MD) for monitoring larger and more complex molecules such as neuropeptides and proteins. This promotes the use of MD membranes with molecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of the membrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure. In the meantime, despite the large pore size, studies have shown that membrane biofouling still lead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextran and Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency (EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests, using as MD sample a protein standard with similar concentrations as in human cerebral spinal fluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysate fractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for total protein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. The FR results suggested that the surface modified catheters were less sensitive to the pressure and provide higher precision, and provided a FR closer to 100%. The surface modification did not show a significant effect on the protein EE. The average total protein EE of surface modified catheters was slightly higher than that of the native ones. The MS EE data of individual proteins showed a clear trend of complex response in EE with pressure.

Cerebral microdialysis for protein biomarker monitoring in the neurointensive care setting - a technical approach.

Cerebral microdialysis (MD) was introduced as a neurochemical monitoring method in the early 1990s and is currently widely used for the sampling of low molecular weight molecules, signaling energy crisis, and cellular distress in the neurointensive care (NIC) setting. There is a growing interest in MD for harvesting of intracerebral protein biomarkers of secondary injury mechanisms in acute traumatic and neurovascular brain injury in the NIC community. The initial enthusiasm over the opportunity to sample protein biomarkers with high molecular weight cut-off MD catheters has dampened somewhat with the emerging realization of inherent methodological problems including protein-protein interaction, protein adhesion, and biofouling, causing an unstable in vivo performance (i.e., fluid recovery and extraction efficiency) of the MD catheter. This review will focus on the results of a multidisciplinary collaborative effort, within the Uppsala Berzelii Centre for Neurodiagnostics during the past several years, to study the features of the complex process of high molecular weight cut-off MD for protein biomarkers. This research has led to new methodology showing robust in vivo performance with optimized fluid recovery and improved extraction efficiency, allowing for more accurate biomarker monitoring. In combination with evolving analytical methodology allowing for multiplex biomarker analysis in ultra-small MD samples, a new opportunity opens up for high-resolution temporal mapping of secondary injury cascades, such as neuroinflammation and other cell injury reactions directly in the injured human brain. Such data may provide an important basis for improved characterization of complex injuries, e.g., traumatic and neurovascular brain injury, and help in defining targets and treatment windows for neuroprotective drug development.

Fluorescence imaging of macromolecule transport in high molecular weight cut-off microdialysis.

When microdialysis (MD) membrane exceeds molecular weight cut-off (MWCO) of 100 kDa, the fluid mechanics are in the ultrafiltration regime. Consequently, fluidic mass transport of macromolecules in the perfusate over the membrane may reduce the biological relevance of the sampling and cause an inflammatory response in the test subject. Therefore, a method to investigate the molecular transport of high MWCO MD is presented. An in vitro test chamber was fabricated to facilitate the fluorescent imaging of the MD sampling process, using fluoresceinylisothiocyanate (FITC) dextran and fluorescence microscopy. Qualitative studies on dextran behavior inside and outside the membrane were performed. Semiquantitative results showed clear dextran leakage from both 40 and 250 kDa dextran when 100 kDa MWCO membranes were used. Dextran 40 kDa leaked out with an order of magnitude higher concentration and the leakage pattern resembled more of a convective flow pattern compared with dextran 250 kDa, where the leakage pattern was more diffusion based. No leakage was observed when dextran 500 kDa was used as a colloid osmotic agent. The results in this study suggest that fluorescence imaging could be used as a method for qualitative and semiquantitative molecular transport and fluid dynamics studies of MD membranes and other hollow fiber catheter membranes.

Refined microdialysis method for protein biomarker sampling in acute brain injury in the neurointensive care setting.

There is growing interest in cerebral microdialysis (MD) for sampling of protein biomarkers in neurointensive care (NIC) patients. Published data point to inherent problems with this methodology including protein interaction and biofouling leading to unstable catheter performance. This study tested the in vivo performance of a refined MD method including catheter surface modification, for protein biomarker sampling in a clinically relevant porcine brain injury model. Seven pigs of both sexes (10-12 weeks old; 22.2-27.3 kg) were included. Mean arterial blood pressure, heart rate, intracranial pressure (ICP) and cerebral perfusion pressure was recorded during the stepwise elevation of intracranial pressure by inflation of an epidural balloon catheter with saline (1 mL/20 min) until brain death. One naïve MD catheter and one surface modified with Pluronic F-127 (10 mm membrane, 100 kDa molecular weight cutoff MD catheter) were inserted into the right frontal cortex and perfused with mock CSF with 3% Dextran 500 at a flow rate of 1.0 µL/min and 20 min sample collection. Naïve catheters showed unstable fluid recovery, sensitive to ICP changes, which was significantly stabilized by surface modification. Three of seven naïve catheters failed to deliver a stable fluid recovery. MD levels of glucose, lactate, pyruvate, glutamate, glycerol and urea measured enzymatically showed an expected gradual ischemic and cellular distress response to the intervention without differences between naïve and surface modified catheters. The 17 most common proteins quantified by iTRAQ and nanoflow LC-MS/MS were used as biomarker models. These proteins showed a significantly more homogeneous response to the ICP intervention in surface modified compared to naïve MD catheters with improved extraction efficiency for most of the proteins. The refined MD method appears to improve the accuracy and precision of protein biomarker sampling in the NIC setting.

Impact of static pressure on transmembrane fluid exchange in high molecular weight cut off microdialysis.

With the interest of studying larger biomolecules by microdialysis (MD), this sampling technique has reached into the ultrafiltration region of fluid exchange, where fluid recovery (FR) has a strong dependence on pressure. Hence in this study, we focus on the fluid exchange across the high molecular weight cut off MD membrane under the influence of the static pressure in the sampling environment. A theoretical model is presented for MD with such membranes, where FR has a linear dependence upon the static pressure of the sample. Transmembrane (TM) osmotic pressure difference and MD perfusion rate decide how fast FR increases with increased static pressure. A test chamber for in vitro MD under static pressure was constructed and validated. It can hold four MD probes under controlled pressurized conditions. Comparison showed good agreement between experiment and theory. Moreover, test results showed that the fluid recovery of the test chamber MD can be set accurately via the chamber pressure, which is controlled by sample injection into the chamber at precise rate. This in vitro system is designed for modelling in vivo MD in cerebrospinal fluid and studies with biological samples in this system may be good models for in vivo MD.

Proteomic analysis of apoptotic and oncotic pancreatic acinar AR42J cells treated with caerulein.

This study aims to determine the differentially expressed proteins in the pancreatic acinar cells undergoing apoptosis and oncosis stimulated with caerulein to explore different cell death process of the acinar cell. AR42J cells were treated with caerulein to induce cell model of acute pancreatitis. Cells that were undergoing apoptosis and oncosis were separated by flow cytometry. Then differentially expressed proteins in the two groups of separated cells were detected by shotgun liquid chromatography-tandem mass spectrometry. The results showed that 11 proteins were detected in both apoptosis group and oncosis group, 17 proteins were detected only in apoptosis group and 29 proteins were detected only in oncosis group. KEGG analysis showed that proteins detected only in apoptosis group were significantly enriched in 10 pathways, including ECM-receptor interaction, cell adhesion molecules, and proteins detected only in oncosis group were significantly enriched in three pathways, including endocytosis, base excision repair, and RNA degradation. These proteins we detected are helpful for us to understand the process of cell death in acute pancreatitis and may be useful for changing the death mode of pancreatic acinar cells, thus attenuating the severity of pancreatitis.

Early proteome analysis of rat pancreatic acinar AR42J cells treated with taurolithocholic acid 3-sulfate.

BACKGROUND: Bile acids are the initiating factors of biliary acute pancreatitis. Bile acids can induce the activation of intracellular zymogen, thus leading injury in pancreatic acinar cells. Pathological zymogen activation in pancreatic acinar cells is a common feature of all types of acute pancreatitis. The proteins expressed in pancreatic acinar cells during the activation of zymogen may determine the severity of acute pancreatitis. The present study aims to determine the differentially expressed proteins in taurolithocholic acid 3-sulfate-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. METHODS: Rat pancreatic acinar AR42J cells were treated with taurolithocholic acid 3-sulfate for 20 min. Laser confocal scanning microscopy and flow cytometry were used to detect activated trypsinogen in pancreatic acinar AR42J cells. After the determination of trypsinogen activation, proteome analysis was performed to identify the proteins differentially expressed in taurolithocholic acid 3-sulfate-treated cells and non-treated cells. RESULTS: After treatment with taurolithocholic acid 3-sulfate for 20 min, the activation of trypsinogen in AR42J cells was concurrent with changes in the protein expression profile. Thirty-nine differentially expressed proteins were detected; among these, 23 proteins were up-regulated and 16 proteins were down-regulated. KEGG analysis indicated that these proteins are involved in cellular metabolic pathways, cellular defensive mechanisms, intracellular calcium regulation and cytoskeletal changes. CONCLUSION: The expression of proteins in the pancreatic acinar cell changes at the early stage of biliary acute pancreatitis. These differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism biliary acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.