Loss of mucosal tolerance to glycoprotein 2 isoform 1 is a potential novel diagnostic biomarker for cholangiocarcinoma.

BACKGROUND: Anti-glycoprotein 2 (anti-GP2) IgA and antineutrophil-cytoplasmic antibodies to proteinase 3 (PR3-ANCA) have been reported as predictive markers of cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis (PSC), but their prevalence in CCA patients without PSC remains unclear. METHODS: This study involved Asian discovery (n = 118) and European validation (n = 38) cohorts of CCA patients without PSC, alongside 49 Asian and 82 European pancreatic ductal adenocarcinoma (PDAC) patients, 21 with benign pancreatic neoplasms (BPN) and 45 with hepatocellular carcinoma (HCC), and 157 healthy controls (HC) from Asia and Europe. We analyzed the prevalence of PR3-ANCA, IgA and IgG against GP21 and GP24, and the CA19-9 levels. RESULTS: Anti-GP21 IgA was the most prevalent in both CCA cohorts (discovery: 55.1 %; validation: 42.1 %) and significantly higher than in other groups except PDAC (all p < 0.05). It demonstrated the best diagnostic performance in distinguishing CCA from disease controls and HC, outperforming tumor markers. No significant correlation was found between anti-GP21 IgA levels and CA19-9 levels. CONCLUSION: Our findings show that anti-GP21 IgA revealing the loss of mucosal tolerance is a potential novel diagnostic biomarker for CCA.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Immune response mechanisms in acute and chronic pancreatitis: strategies for therapeutic intervention.

Acute pancreatitis (AP) is one of the most common inflammatory diseases of the gastrointestinal tract and a steady rising diagnosis for inpatient hospitalization. About one in four patients, who experience an episode of AP, will develop chronic pancreatitis (CP) over time. While the initiating causes of pancreatitis can be complex, they consistently elicit an immune response that significantly determines the severity and course of the disease. Overall, AP is associated with a significant mortality rate of 1-5%, which is caused by either an excessive pro-inflammation, or a strong compensatory inhibition of bacterial defense mechanisms which lead to a severe necrotizing form of pancreatitis. At the time-point of hospitalization the already initiated immune response is the only promising common therapeutic target to treat or prevent a severe disease course. However, the complexity of the immune response requires fine-balanced therapeutic intervention which in addition is limited by the fact that a significant proportion of patients is in danger of development or progress to recurrent and chronic disease. Based on the recent literature we survey the disease-relevant immune mechanisms and evaluate appropriate and promising therapeutic targets for the treatment of acute and chronic pancreatitis.

IRAK3-mediated suppression of pro-inflammatory MyD88/IRAK signaling affects disease severity in acute pancreatitis.

Acute pancreatitis (AP), which is characterized by self-digestion of the pancreas by its own prematurely activated digestive proteases, is a major reason for hospitalization. The autodigestive process causes necrotic cell death of pancreatic acinar cells and the release of damage associated molecular pattern which activate macrophages and drive the secretion of pro-inflammatory cytokines. The MYD88/IRAK signaling pathway plays an important role for the induction of inflammatory responses. Interleukin-1 receptor associated kinase-3 (IRAK3) is a counter-regulator of this pathway. In this study, we investigated the role of MYD88/IRAK using Irak3-/- mice in two experimental animal models of mild and severe AP. IRAK3 is expressed in macrophages as well as pancreatic acinar cells where it restrains NFκB activation. Deletion of IRAK3 enhanced the migration of CCR2+ monocytes into the pancreas and triggered a pro-inflammatory type 1 immune response characterized by significantly increased serum levels of TNFα, IL-6, and IL-12p70. Unexpectedly, in a mild AP model this enhanced pro-inflammatory response resulted in decreased pancreatic damage, whereas in a severe AP model, induced by partial pancreatic duct ligation, the increased pro-inflammatory response drives a severe systemic inflammatory response syndrome (SIRS) and is associated with an increased local and systemic damage. Our results indicate that complex immune regulation mechanism control the course of AP, where moderate pro-inflammation not necessarily associates with increased disease severity but also drives tissue regenerative processes through a more effective clearance of necrotic acinar cells. Only when the pro-inflammation exceeds a certain systemic level, it fuels SIRS and increases disease severity.

Plasma renalase levels are associated with the development of acute pancreatitis.

BACKGROUND/OBJECTIVES: Severe acute pancreatitis is associated with significant morbidity and mortality. Identifying factors that affect the risk of developing severe disease could influence management. Plasma levels of renalase, an anti-inflammatory secretory protein, dramatically decrease in a murine acute pancreatitis model. We assessed this response in hospitalized acute pancreatitis patients to determine if reduced plasma renalase levels occur in humans. METHODS: Plasma samples were prospectively and sequentially collected from patients hospitalized for acute pancreatitis. Two forms of plasma renalase, native (no acid) and acidified, were measured by ELISA and RNLS levels were compared between healthy controls and patients with mild and severe disease (defined as APACHE-II score ≥7) using nonparametric statistical analysis. RESULTS: Control (33) and acute pancreatitis (mild, 230 (76.7%) and severe, 70 (23.3%) patients were studied. Acidified RNLS levels were lower in pancreatitis patients: Control: 10.1 µg/ml, Mild 5.1 µg/ml, Severe 6.0 µg/ml; p < 0.001. Native RNLS levels were increased in AP: Control: 0.4 µg/ml, Mild 0.9 µg g/ml, Severe 1.2 µg/ml p < 0.001; those with severe AP trended to have higher native RNLS levels than those with mild disease (p = 0.056). In patients with severe AP, higher APACHE-II scores at 24 h after admission correlated with lower acid-sensitive RNLS levels on admission (r = -0.31, p = 0.023). CONCLUSION: Low plasma acidified RNLS levels, and increased native RNLS levels are associated with AP. Additional studies should assess the clinical correlation between plasma RNLS levels and AP severity and outcomes.

Activated regulatory T-cells promote duodenal bacterial translocation into necrotic areas in severe acute pancreatitis.

OBJECTIVE: In acute pancreatitis (AP), bacterial translocation and subsequent infection of pancreatic necrosis are the main risk factors for severe disease and late death. Understanding how immunological host defence mechanisms fail to protect the intestinal barrier is of great importance in reducing the mortality risk of the disease. Here, we studied the role of the Treg/Th17 balance for maintaining the intestinal barrier function in a mouse model of severe AP. DESIGN: AP was induced by partial duct ligation in C57Bl/6 or DEREG mice, in which regulatory T-cells (Treg) were depleted by intraperitoneal injection of diphtheria toxin. By flow cytometry, functional suppression assays and transcriptional profiling we analysed Treg activation and characterised T-cells of the lamina propria as well as intraepithelial lymphocytes (IELs) regarding their activation and differentiation. Microbiota composition was examined in intestinal samples as well as in murine and human pancreatic necrosis by 16S rRNA gene sequencing. RESULTS: The prophylactic Treg-depletion enhanced the proinflammatory response in an experimental mouse model of AP but stabilised the intestinal immunological barrier function of Th17 cells and CD8+/γδTCR+ IELs. Treg depleted animals developed less bacterial translocation to the pancreas. Duodenal overgrowth of the facultative pathogenic taxa Escherichia/Shigella which associates with severe disease and infected necrosis was diminished in Treg depleted animals. CONCLUSION: Tregs play a crucial role in the counterbalance against systemic inflammatory response syndrome. In AP, Treg-activation disturbs the duodenal barrier function and permits translocation of commensal bacteria into pancreatic necrosis. Targeting Tregs in AP may help to ameliorate the disease course.

A rare PRSS1 p.S127C mutation is associated with chronic pancreatitis and causes misfolding-induced ER-stress.

BACKGROUND: /Objectives: Sequence variants in several genes have been identified as being associated with an increased inherited risk to develop chronic pancreatitis (CP). In a genetic survey of a CP patient we identified in the PRSS1gene a new c.380C > G sequence variation, giving rise to a non-synonymous p.S127C mutation. Functional studies were performed to analyze the associated pathophysiology of the variant. METHODS: Following generation of an expression vector for the new PRSS1 variant we compared its expression, secretion and catalytic activity with already known PRSS1 risk variants in HEK 293T cells. The intracellular protein accumulation and induction of endoplasmic reticulum (ER)-stress was analyzed. RESULTS: Prediction tool analysis indicated a probably deleterious effect of the p.S127C variant on protein function which was confirmed by detection of a secretion defect in HEK293T cells leading to intracellular protein accumulation. While protein misfolding was associated with reduced trypsin activity, the increased expression of BIP and presence of spliced XBP1 indicated that the p.S127C variant induces ER stress and activates the UPR signaling pathway. CONCLUSIONS: The disease mechanism of the PRSS1 p.S127C variant involves defective protein secretion and the induction of ER-stress due to accumulation of presumably misfolded trypsinogen within the ER. The new variant should be considered disease-causing with an incomplete penetrance. Our results confirm that in addition to dysregulated trypsin-activity or reduced fluid secretion, ER-stress induction is an important trigger for acinar cell damage and the development of recurrent or chronic pancreatic inflammation.

Systemic Bile Acids Affect the Severity of Acute Pancreatitis in Mice Depending on Their Hydrophobicity and the Disease Pathogenesis.

Acute pancreatitis (AP) is a major, globally increasing gastrointestinal disease and a biliary origin is the most common cause. However, the effects of bile acids (BAs), given systemically, on the pancreas and on disease severity remains elusive. In this study, we have investigated the roles of different circulating BAs in animal models for AP to elucidate their impact on disease severity and the underlying pathomechanisms. BAs were incubated on isolated acini and AP was induced through repetitive injections of caerulein or L-arginine; pancreatic duct ligation (PDL); or combined biliopancreatic duct ligation (BPDL). Disease severity was assessed using biochemical and histological parameters. Serum cholecystokinin (CCK) concentrations were determined via enzyme immunoassay. The binding of the CCK1 receptor was measured using fluorescence-labeled CCK. In isolated acini, hydrophobic BAs mitigated the damaging effects of CCK. The same BAs further enhanced pancreatitis in L-arginine- and PDL-based pancreatitis, whereas they ameliorated pancreatic damage in the caerulein and BPDL models. Mechanistically, the binding affinity of the CCK1 receptor was significantly reduced by hydrophobic BAs. The hydrophobicity of BAs and the involvement of CCK seem to be relevant in the course of AP. Systemic BAs may affect the severity of AP by interfering with the CCK1 receptor.

Mobilization of CD11b+/Ly6chi monocytes causes multi organ dysfunction syndrome in acute pancreatitis.

Objective: Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life -threatening organ complications, such as respiratory and renal failure, is unknown. Design: Organ dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification. Results: Similar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation. Conclusion: In AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.

Identification of early predictors for infected necrosis in acute pancreatitis.

BACKGROUND: In acute pancreatitis, secondary infection of pancreatic necrosis is a complication that mostly necessitates interventional therapy. A reliable prediction of infected necrotizing pancreatitis would enable an early identification of patients at risk, which however, is not possible yet. METHODS: This study aims to identify parameters that are useful for the prediction of infected necrosis and to develop a prediction model for early detection. We conducted a retrospective analysis from the hospital information and reimbursement data system and screened 705 patients hospitalized with diagnosis of acute pancreatitis who underwent contrast-enhanced computed tomography and additional diagnostic puncture or drainage of necrotic collections. Both clinical and laboratory parameters were analyzed for an association with a microbiologically confirmed infected pancreatic necrosis. A prediction model was developed using a logistic regression analysis with stepwise inclusion of significant variables. The model quality was tested by receiver operating characteristics analysis and compared to single parameters and APACHE II score. RESULTS: We identified a total of 89 patients with necrotizing pancreatitis, diagnosed by computed tomography, who additionally received biopsy or drainage. Out of these, 59 individuals had an infected necrosis. Eleven parameters showed a significant association with an infection including C-reactive protein, albumin, creatinine, and alcoholic etiology, which were independent variables in a predictive model. This model showed an area under the curve of 0.819, a sensitivity of 0.692 (95%-CI [0.547-0.809]), and a specificity of 0.840 (95%-CI [0.631-0.947]), outperforming single laboratory markers and APACHE II score. Even in cases of missing values predictability was reliable. CONCLUSION: A model consisting of a few single blood parameters and etiology of pancreatitis might help for differentiation between infected and non-infected pancreatic necrosis and assist medical therapy in acute necrotizing pancreatitis.

Inhibition of KRAS, MEK and PI3K Demonstrate Synergistic Anti-Tumor Effects in Pancreatic Ductal Adenocarcinoma Cell Lines.

Kirsten rat sarcoma virus (KRAS) mutations are widespread in pancreatic ductal adenocarcinoma (PDAC) and contribute significantly to tumor initiation, progression, tumor relapse/resistance, and prognosis of patients. Although inhibitors against KRAS mutations have been developed, this therapeutic approach is not routinely used in PDAC patients. We investigated the anti-tumor efficacy of two KRAS inhibitors BI-3406 (KRAS::SOS1 inhibitor) and sotorasib (KRAS G12C inhibitor) alone or in combination with MEK1/2 inhibitor trametinib and/or PI3K inhibitor buparlisib in seven PDAC cell lines. Whole transcriptomic analysis of combined inhibition and control groups were comparatively analyzed to explore the corresponding mechanisms of inhibitor combination. Both KRAS inhibitors and corresponding combinations exhibited cytotoxicity against specific PDAC cell lines. BI-3406 enhance the efficacy of trametinib and buparlisib in BXPC-3, ASPC-1 and MIA PACA-2, but not in CAPAN-1, while sotorasib enhances the efficacy of trametinib and buparlisib only in MIA PACA-2. The whole transcriptomic analysis demonstrates that the two triple-inhibitor combinations exert antitumor effects by affecting related cell functions, such as affecting the immune system, cell adhesion, cell migration, and cytokine binding. As well as directly involved in RAF/MEK/ERK pathway and PI3K/AKT pathway affect cell survival. Our current study confirmed inhibition of KRAS and its downstream pathways as a potential novel therapy for PDAC and provides fundamental data for in vivo evaluations.

Independent Validation and Assay Standardization of Improved Metabolic Biomarker Signature to Differentiate Pancreatic Ductal Adenocarcinoma From Chronic Pancreatitis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma cancer (PDAC) is a highly lethal malignancy requiring efficient detection when the primary tumor is still resectable. We previously developed the MxPancreasScore comprising 9 analytes and serum carbohydrate antigen 19-9 (CA19-9), achieving an accuracy of 90.6%. The necessity for 5 different analytical platforms and multiple analytical runs, however, hindered clinical applicability. We therefore aimed to develop a simpler single-analytical run, single-platform diagnostic signature. METHODS: We evaluated 941 patients (PDAC, 356; chronic pancreatitis [CP], 304; nonpancreatic disease, 281) in 3 multicenter independent tests, and identification (ID) and validation cohort 1 (VD1) and 2 (VD2) were evaluated. Targeted quantitative plasma metabolite analysis was performed on a liquid chromatography-tandem mass spectrometry platform. A machine learning-aided algorithm identified an improved (i-Metabolic) and minimalistic metabolic (m-Metabolic) signatures, and compared them for performance. RESULTS: The i-Metabolic Signature, (12 analytes plus CA19-9) distinguished PDAC from CP with area under the curve (95% confidence interval) of 97.2% (97.1%-97.3%), 93.5% (93.4%-93.7%), and 92.2% (92.1%-92.3%) in the ID, VD1, and VD2 cohorts, respectively. In the VD2 cohort, the m-Metabolic signature (4 analytes plus CA19-9) discriminated PDAC from CP with a sensitivity of 77.3% and specificity of 89.6%, with an overall accuracy of 82.4%. For the subset of 45 patients with PDAC with resectable stages IA-IIB tumors, the sensitivity, specificity, and accuracy were 73.2%, 89.6%, and 82.7%, respectively; for those with detectable CA19-9 >2 U/mL, 81.6%, 88.7%, and 84.5%, respectively; and for those with CA19-9 <37 U/mL, 39.7%, 94.1%, and 76.3%, respectively. CONCLUSIONS: The single-platform, single-run, m-Metabolic signature of just 4 metabolites used in combination with serum CA19-9 levels is an innovative accurate diagnostic tool for PDAC at the time of clinical presentation, warranting further large-scale evaluation.

Malnutrition Is Highly Prevalent in Patients With Chronic Pancreatitis and Characterized by Loss of Skeletal Muscle Mass but Absence of Impaired Physical Function.

Background/Aims: Patients with chronic pancreatitis (CP) have an increased risk of malnutrition, a condition linked to reduced muscle mass and physical performance. We have investigated the risk factors, phenotypic presentation, and health implications associated with malnutrition in CP. Materials and Methods: In a multicenter cross-sectional study we recruited patients with confirmed CP and healthy volunteers as a control group. Malnutrition was diagnosed according to the criteria proposed by the Global Leadership Initiative on Malnutrition. We performed detailed examinations of body composition and physical function as well as testing of routine blood parameters and markers of inflammation. Results: We included 66 patients [mean (±SD) age: 56.0 (±14.5) years; 51 males] and an equal number of age- and sex-matched controls. Moderate malnutrition was diagnosed in 21% (n = 14) and severe malnutrition in 42% (n = 28) of patients. Besides weight loss malnourished patients showed lower fat and skeletal muscle mass compared to both non-malnourished subjects and healthy controls. Only in severe malnutrition, blood parameters reflected elevated inflammation and reduced muscle reserves. Handgrip strength in patients did not differ by nutritional status but there was a significant correlation (rho = 0.705, p < 0.001) with skeletal muscle mass. Although 20 patients (30%) had pathologically reduced skeletal muscle mass, only two individuals (3%) had sarcopenia with concomitantly reduced handgrip strength. Conclusion: Malnutrition is a frequent complication of CP characterized by loss of skeletal muscle mass. As this condition becomes evident only at an advanced stage, regular testing for altered body composition is recommended. Suitable biomarkers and the link between loss of muscle mass and physical function require further investigation. Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT04474743], identifier [NCT04474743].

Epidemiological Factors Associated With Intraductal Papillary Mucinous Neoplasm of the Pancreas: A Dual Center Case-Control Study.

OBJECTIVE: The present study aimed to identify epidemiological factors associated with the development of intraductal papillary mucinous neoplasms (IPMN) of the pancreas comparing patients after IPMN resection with population-based controls. METHODS: Preoperative data of 811 patients undergoing pancreatic resection for IPMN were matched in a 1:1 ratio with a random sample of volunteers from the Study of Health in Pomerania, which showed no pancreatic cyst greater than 2 mm in magnetic resonance cholangiopancreaticography. RESULTS: A total of 811 controls with a mean age of 61.9 years (standard deviation, 8.4 years) were matched to cases with a mean age of 66.1 years (standard deviation, 9.3 years). A previous history of pancreatitis, endocrine pancreatic insufficiency was significantly more frequent in IPMN patients compared with controls (P = 0.001). Moreover, adjusted data revealed that urogenital cancer (P = 0.034), colorectal cancer (P = 0.021), as well as first-degree family history of colorectal cancer (P = 0.001) were significantly more frequent in IPMN patients. CONCLUSIONS: A history of urogenital and colorectal cancer often coincides with IPMN, which have an indication for surgery and are associated with preoperative episodes of pancreatitis and with endocrine insufficiency. Prospective studies are needed to investigate the role of these factors in IPMN development.

The Inhibitory Response to PI3K/AKT Pathway Inhibitors MK-2206 and Buparlisib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines.

The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus (KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.

Inhibitory Response to CK II Inhibitor Silmitasertib and CDKs Inhibitor Dinaciclib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines.

Casein kinase II (CK2) and cyclin-dependent kinases (CDKs) frequently interact within multiple pathways in pancreatic ductal adenocarcinoma (PDAC). Application of CK2- and CDK-inhibitors have been considered as a therapeutic option, but are currently not part of routine chemotherapy regimens. We investigated ten PDAC cell lines exposed to increasing concentrations of silmitasertib and dinaciclib. Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated, and bioinformatic clustering was used to classify cell lines into sensitive groups based on their response to inhibitors. Furthermore, whole exome sequencing (WES) and RNA sequencing (RNA-Seq) was conducted to assess recurrent mutations and the expression profile of inhibitor targets and genes frequently mutated in PDAC, respectively. Dinaciclib and silmitasertib demonstrated pronounced and limited cell line specific effects in cell death induction, respectively. WES revealed no genomic variants causing changes in the primary structure of the corresponding inhibitor target proteins. RNA-Seq demonstrated that the expression of all inhibitor target genes was higher in the PDAC cell lines compared to non-neoplastic pancreatic tissue. The observed differences in PDAC cell line sensitivity to silmitasertib or dinaciclib did not depend on target gene expression or the identified gene variants. For the PDAC hotspot genes kirsten rat sarcoma virus (KRAS) and tumor protein p53 (TP53), three and eight variants were identified, respectively. In conclusion, both inhibitors demonstrated in vitro efficacy on the PDAC cell lines. However, aberrations and expression of inhibitor target genes did not appear to affect the efficacy of the corresponding inhibitors. In addition, specific aberrations in TP53 and KRAS affected the efficacy of both inhibitors.

HLA-DRB1∗16 and -DQB1∗05 alleles are strongly associated with autoimmune pancreatitis in a cohort of hundred patients.

BACKGROUND/OBJECTIVES: Autoimmune diseases are often associated with human leukocyte antigen (HLA) haplotypes, indicating that changes in major histocompatibility complex (MHC)-dependent self-peptide or antigen presentation contribute to autoimmunity. In our study, we aimed to investigate HLA alleles in a large European cohort of autoimmune pancreatitis (AIP) patients. METHODS: Hundred patients with AIP, diagnosed and classified according to the International Consensus Diagnostic Criteria (ICDC), were prospectively enrolled in the study. Forty-four patients with chronic pancreatitis (CP) and 254 healthy subjects served as control groups. DNA was isolated from blood samples and two-digit HLA typing was performed with sequence-specific primer (SSP-) PCR. HLA allele association strength to AIP was calculated as odds ratio. RESULTS: We uncovered a strong enrichment of HLA-DQB1 homozygosity in type 1 and type 2 AIP patients. Moreover, a significantly increased incidence of the HLA-DRB1∗16 and HLA-DQB1∗05 alleles and a concomitant lack of the HLA-DRB1∗13 allele was detected in AIP type 1 and type 2 patients. In contrast, the HLA-DQB1∗02 allele was underrepresented in the 'not otherwise specified' (NOS) AIP subtype. We detected no significant difference in the HLA-DRB3, HLA-DRB4 and HLA-DRB5 allele frequency in our cohort. CONCLUSIONS: Although AIP type 1 and type 2 are characterized by distinct histopathological characteristics, both subtypes are associated with the same HLA alleles, indicating that the disease might rely on similar immunogenic mechanisms. However, AIP NOS represented another subclass of AIP.

Genetic Analysis of the ATG16L1 c.898A>G (p.T300A) Variant in Acute and Chronic Pancreatitis.

OBJECTIVES: Human and animal studies suggest an important role of autophagy in the pathogenesis of pancreatitis. ATG16L1 (autophagy-related 16 like 1) is part of a protein complex that is involved in the formation of autophagosomes. The c.898A > G (p.T300A) variant of ATG16L1 is associated with Crohn disease. In this study, we analyzed ATG16L1 c.898A > G (p.T300A) for an association with pancreatitis. METHODS: We genotyped 777 patients and 551 control subjects of German origin by melting curve analysis using fluorescence resonance energy transfer probes. The patient group included 429 patients with nonalcoholic chronic pancreatitis (CP), 141 patients with alcoholic CP, and 207 patients with acute pancreatitis (AP). We classified AP by severity according to the Atlanta symposium 1992. RESULTS: Allele and genotype frequencies of ATG16L1 c.898A > G (p.T300A) did not differ significantly between patients and controls (G allele frequencies: nonalcoholic CP, 49.9%; alcoholic CP, 48.2%; AP, 49.5%; controls, 52.7%). We found no significant association with the severity of AP either. CONCLUSIONS: Our data do not support a role of ATG16L1 c.898A > G (p.T300A) in the pathogenesis of AP or CP or an influence on the severity of AP.

Whole-exome Sequencing Identifies SLC52A1 and ZNF106 Variants as Novel Genetic Risk Factors for (Early) Multiple-organ Failure in Acute Pancreatitis.

OBJECTIVE: The aim of this study was to identify genetic variants associated with early multiple organ failure (MOF) in acute pancreatitis. SUMMARY BACKGROUND DATA: MOF is a life-threatening complication of acute pancreatitis, and risk factors are largely unknown, especially in early persistent MOF. Genetic risk factors are thought to enhance severity in complex diseases such as acute pancreatitis. METHODS: A 2-phase study design was conducted. First, we exome sequenced 9 acute pancreatitis patients with early persistent MOF and 9 case-matched patients with mild edematous pancreatitis (phenotypic extremes) from our initial Dutch cohort of 387 patients. Secondly, 48 candidate variants that were overrepresented in MOF patients and 10 additional variants known from literature were genotyped in a replication cohort of 286 Dutch and German patients. RESULTS: Exome sequencing resulted in 161,696 genetic variants, of which the 38,333 non-synonymous variants were selected for downstream analyses. Of these, 153 variants were overrepresented in patients with multiple-organ failure, as compared with patients with mild acute pancreatitis. In total, 58 candidate variants were genotyped in the joined Dutch and German replication cohort. We found the rs12440118 variant of ZNF106 to be overrepresented in patients with MOF (minor allele frequency 20.4% vs 11.6%, Padj=0.026). Additionally, SLC52A1 rs346821 was found to be overrepresented (minor allele frequency 48.0% vs 42.4%, Padj= 0.003) in early MOF. None of the variants known from literature were associated.Conclusions: This study indicates that SLC52A1, a riboflavin plasma membrane transporter, and ZNF106, a zinc finger protein, may be involved in disease progression toward (early) MOF in acute pancreatitis.