Early detection of pancreatic cancer by comprehensive serum miRNA sequencing with automated machine learning.

BACKGROUND: Pancreatic cancer is often diagnosed at advanced stages, and early-stage diagnosis of pancreatic cancer is difficult because of nonspecific symptoms and lack of available biomarkers. METHODS: We performed comprehensive serum miRNA sequencing of 212 pancreatic cancer patient samples from 14 hospitals and 213 non-cancerous healthy control samples. We randomly classified the pancreatic cancer and control samples into two cohorts: a training cohort (N = 185) and a validation cohort (N = 240). We created ensemble models that combined automated machine learning with 100 highly expressed miRNAs and their combination with CA19-9 and validated the performance of the models in the independent validation cohort. RESULTS: The diagnostic model with the combination of the 100 highly expressed miRNAs and CA19-9 could discriminate pancreatic cancer from non-cancer healthy control with high accuracy (area under the curve (AUC), 0.99; sensitivity, 90%; specificity, 98%). We validated high diagnostic accuracy in an independent asymptomatic early-stage (stage 0-I) pancreatic cancer cohort (AUC:0.97; sensitivity, 67%; specificity, 98%). CONCLUSIONS: We demonstrate that the 100 highly expressed miRNAs and their combination with CA19-9 could be biomarkers for the specific and early detection of pancreatic cancer.

Imported Hepatitis E Virus Genotype 1: A Rare Case of Acute Hepatitis Managed with Steroid Pulse Therapy.

A 26-year-old Indian man who had arrived in Japan 24 days prior presented to our hospital with abdominal pain and a fever. A blood test revealed marked hepatic dysfunction, and imaging tests confirmed a diagnosis of acute hepatitis. The patient's liver function and coagulability deteriorated, and his general condition was poor. Given the possibility of fulminant hepatic failure, we initiated steroid pulse therapy. Following the initiation of steroid therapy, the patient's liver function and subjective symptoms rapidly improved. Testing revealed positive findings for IgA-hepatitis E virus, and a genetic analysis of hepatitis E identified genotype 1, which is not endemic to Japan, leading to a definitive diagnosis of imported hepatitis E infection from India. The successful response to steroid therapy highlights the potential benefit of this approach in managing severe cases of acute hepatitis E, a rare occurrence in Japan. This case underscores the importance of considering hepatitis E infection in individuals with a recent travel history to regions with high prevalence and the potential benefits of steroid therapy in managing severe cases of acute hepatitis E.

Primary Peritonitis due to Group A Streptococcus Successfully Treated with Intraperitoneal Drainage.

A 42-year-old woman was admitted to our hospital because of lower abdominal pain and diarrhea. Although the initial symptoms and imaging findings were similar to those of acute enteritis, blood and ascites cultures led to the diagnosis of primary peritonitis caused by group A Streptococcus. In many cases, the disease rapidly deteriorates, and laparotomy is performed for the early diagnosis and to reduce the number of bacteria in the abdominal cavity. In the present case, intraperitoneal drainage was effective for avoiding surgery. We suggest that intraperitoneal drainage is effective for the treatment of this disease.

Inflammatory Pseudo-tumor of the Liver Accompanied by Eosinophilia.

A 28-year-old woman was referred to our hospital for liver dysfunction and neck pain. Blood tests revealed elevated liver enzymes and eosinophilia. Ultrasonography, computed tomography, and magnetic resonance imaging showed a mass lesion near the hepatic hilus. The tumor was considered to be an inflammatory pseudo-tumor or malignancy. A liver-mass biopsy was performed and led to a diagnosis of inflammatory pseudo-tumor. In the present case, a markedly elevated eosinophil count was a characteristic clinical feature, and the patient underwent steroid therapy. Treatment resulted in a reduced eosinophil count, improved neck symptoms, and disappearance of the inflammatory pseudo-tumor.

Late-onset type-2 autoimmune pancreatitis with two mass lesions diagnosed by endoscopic ultrasound-guided fine-needle aspiration.

A 65-year-old man with ulcerative colitis presented with aggravated diabetes. Computed tomography showed two masses in the body and tail of the pancreas. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was performed, with histopathological findings suggesting autoimmune pancreatitis (AIP). Type-2 AIP was suspected, and administration of prednisolone was initiated. The pancreatic masses had disappeared after the treatment. In this case, EUS-FNA was effective for the diagnosis of type-2 AIP. The two-lesion mass formation observed here is a rare presentation of the disease. In patients with a history of ulcerative colitis, the possibility of late-onset type-2 AIP should be kept in mind.

Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although rigorous efforts identified the presence of 'cancer stem cells (CSCs)' in PDAC and molecular markers for them, stem cell dynamics in vivo have not been clearly demonstrated. Here we focused on Doublecortin-like kinase 1 (Dclk1), known as a CSC marker of PDAC. Using genetic lineage tracing with a dual-recombinase system and live imaging, we showed that Dclk1+ tumor cells continuously provided progeny cells within pancreatic intraepithelial neoplasia, primary and metastatic PDAC, and PDAC-derived spheroids in vivo and in vitro. Furthermore, genes associated with CSC and epithelial mesenchymal transition were enriched in mouse Dclk1+ and human DCLK1-high PDAC cells. Thus, we provided direct functional evidence for the stem cell activity of Dclk1+ cells in vivo, revealing the essential roles of Dclk1+ cells in expansion of pancreatic neoplasia in all progressive stages.

Lesser Omental Panniculitis.

A 44-year-old woman presented to our hospital with abdominal pain. Abdominal ultrasonography and computed tomography showed a mass-like change in the lesser omentum between the liver and stomach. Esophagogastroduodenoscopy revealed a submucosal tumor-like change, and endoscopic ultrasonography (EUS) revealed that the mass was located outside of the stomach wall. We performed EUS fine-needle aspiration and diagnosed panniculitis of the lesser omentum. Based on these findings, we suggest that mass-like lesions in the lesser omentum and submucosal tumor-like changes in the anterior wall on the lesser curvature side of the stomach be evaluated for the possibility of panniculitis of the lesser omentum.

Development of Disseminated Tuberculosis with Intestinal Involvement due to Adalimumab Administration Despite Latent Tuberculosis Treatment.

Treatment of latent tuberculosis infection (LTBI) reduces the probability of reactivation of tuberculosis associated with anti-tumor necrosis factor (TNF) α inhibitors, but no chemoprophylaxis is completely protective. We herein report a woman with rheumatoid arthritis who developed disseminated tuberculosis with intestinal involvement during adalimumab administration despite LTBI treatment. Tuberculosis reactivation was not detected in sputum or urine but was detected from the terminal ileal mucosa. Detection of intestinal tuberculosis is rare in patients being treated with anti-TNFα therapy after LTBI treatment. As anti-TNFα inhibitors have become more common, the rate of reactivation of tuberculosis, including intestinal tuberculosis, has increased in patients being treated for LTBI.

Arid1a is essential for intestinal stem cells through Sox9 regulation.

Inactivating mutations of Arid1a, a subunit of the Switch/sucrose nonfermentable chromatin remodeling complex, have been reported in multiple human cancers. Intestinal deletion of Arid1a has been reported to induce colorectal cancer in mice; however, its functional role in intestinal homeostasis remains unclear. We investigated the functional role of Arid1a in intestinal homeostasis in mice. We found that intestinal deletion of Arid1a results in loss of intestinal stem cells (ISCs), decreased Paneth and goblet cells, disorganized crypt-villous structures, and increased apoptosis in adult mice. Spheroids did not develop from intestinal epithelial cells deficient for Arid1a Lineage-tracing experiments revealed that Arid1a deletion in Lgr5+ ISCs leads to impaired self-renewal of Lgr5+ ISCs but does not perturb intestinal homeostasis. The Wnt signaling pathway, including Wnt agonists, receptors, and target genes, was strikingly down-regulated in Arid1a-deficient intestines. We found that Arid1a directly binds to the Sox9 promoter to support its expression. Remarkably, overexpression of Sox9 in intestinal epithelial cells abrogated the above phenotypes, although Sox9 overexpression in intestinal epithelial cells did not restore the expression levels of Wnt agonist and receptor genes. Furthermore, Sox9 overexpression permitted development of spheroids from Arid1a-deficient intestinal epithelial cells. In addition, deletion of Arid1a concomitant with Sox9 overexpression in Lgr5+ ISCs restores self-renewal in Arid1a-deleted Lgr5+ ISCs. These results indicate that Arid1a is indispensable for the maintenance of ISCs and intestinal homeostasis in mice. Mechanistically, this is mainly mediated by Sox9. Our data provide insights into the molecular mechanisms underlying maintenance of ISCs and intestinal homeostasis.

Nardilysin inhibits pancreatitis and suppresses pancreatic ductal adenocarcinoma initiation in mice.

OBJECTIVE: Nardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA). DESIGN: We analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC. RESULTS: We found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D -driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA. CONCLUSION: Nrdc inhibits pancreatitis and suppresses PDA initiation in mice.

The BRG1/SOX9 axis is critical for acinar cell-derived pancreatic tumorigenesis.

Chromatin remodeler Brahma related gene 1 (BRG1) is silenced in approximately 10% of human pancreatic ductal adenocarcinomas (PDAs). We previously showed that BRG1 inhibits the formation of intraductal pancreatic mucinous neoplasm (IPMN) and that IPMN-derived PDA originated from ductal cells. However, the role of BRG1 in pancreatic intraepithelial neoplasia-derived (PanIN-derived) PDA that originated from acinar cells remains elusive. Here, we found that exclusive elimination of Brg1 in acinar cells of Ptf1a-CreER; KrasG12D; Brg1fl/fl mice impaired the formation of acinar-to-ductal metaplasia (ADM) and PanIN independently of p53 mutation, while PDA formation was inhibited in the presence of p53 mutation. BRG1 bound to regions of the Sox9 promoter to regulate its expression and was critical for recruitment of upstream regulators, including PDX1, to the Sox9 promoter and enhancer in acinar cells. SOX9 expression was downregulated in BRG1-depleted ADMs/PanINs. Notably, Sox9 overexpression canceled this PanIN-attenuated phenotype in KBC mice. Furthermore, Brg1 deletion in established PanIN by using a dual recombinase system resulted in regression of the lesions in mice. Finally, BRG1 expression correlated with SOX9 expression in human PDAs. In summary, BRG1 is critical for PanIN initiation and progression through positive regulation of SOX9. Thus, the BRG1/SOX9 axis is a potential target for PanIN-derived PDA.

Nardilysin controls intestinal tumorigenesis through HDAC1/p53-dependent transcriptional regulation.

Colon cancer is a complex disease affected by a combination of genetic and epigenetic factors. Here we demonstrate that nardilysin (N-arginine dibasic convertase; NRDC), a metalloendopeptidase of the M16 family, regulates intestinal tumorigenesis via its nuclear functions. NRDC is highly expressed in human colorectal cancers. Deletion of the Nrdc gene in ApcMin mice crucially suppressed intestinal tumor development. In ApcMin mice, epithelial cell-specific deletion of Nrdc recapitulated the tumor suppression observed in Nrdc-null mice. Moreover, epithelial cell-specific overexpression of Nrdc significantly enhanced tumor formation in ApcMin mice. Notably, epithelial NRDC controlled cell apoptosis in a gene dosage-dependent manner. In human colon cancer cells, nuclear NRDC directly associated with HDAC1, and controlled both acetylation and stabilization of p53, with alterations of p53 target apoptotic factors. These findings demonstrate that NRDC is critically involved in intestinal tumorigenesis through its epigenetic regulatory function, and targeting NRDC may lead to a novel prevention or therapeutic strategy against colon cancer.

ARID1A Maintains Differentiation of Pancreatic Ductal Cells and Inhibits Development of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: The ARID1A gene encodes a protein that is part of the large adenosine triphosphate (ATP)-dependent chromatin remodeling complex SWI/SNF and is frequently mutated in human pancreatic ductal adenocarcinomas (PDACs). We investigated the functions of ARID1A during formation of PDACs in mice. METHODS: We performed studies with Ptf1a-Cre;KrasG12D mice, which express activated Kras in the pancreas and develop pancreatic intraepithelial neoplasias (PanINs), as well as those with disruption of Aird1a (Ptf1a-Cre;KrasG12D;Arid1af/f mice) or disruption of Brg1 (encodes a catalytic ATPase of the SWI/SNF complex) (Ptf1a-Cre;KrasG12D; Brg1f/fmice). Pancreatic ductal cells (PDCs) were isolated from Arid1af/f mice and from Arid1af/f;SOX9OE mice, which overexpress human SOX9 upon infection with an adenovirus-expressing Cre recombinase. Pancreatic tissues were collected from all mice and analyzed by histology and immunohistochemistry; cells were isolated and grown in 2-dimensional and 3-dimensional cultures. We performed microarray analyses to compare gene expression patterns in intraductal papillary mucinous neoplasms (IPMNs) from the different strains of mice. We obtained 58 samples of IPMNs and 44 samples of PDACs from patients who underwent pancreatectomy in Japan and analyzed them by immunohistochemistry. RESULTS: Ptf1a-Cre;KrasG12D mice developed PanINs, whereas Ptf1a-Cre;KrasG12D;Arid1af/f mice developed IPMNs and PDACs; IPMNs originated from PDCs. ARID1A-deficient IPMNs did not express SOX9. ARID1A-deficient PDCs had reduced expression of SOX9 and dedifferentiated in culture. Overexpression of SOX9 in these cells allowed them to differentiate and prevented dilation of ducts. Among mice with pancreatic expression of activated Kras, those with disruption of Arid1a developed fewer PDACs from IPMNs than mice with disruption of Brg1. ARID1A-deficient IPMNs had reduced activity of the mTOR pathway. Human IPMN and PDAC specimens had reduced levels of ARID1A, SOX9, and phosphorylated S6 (a marker of mTOR pathway activation). Levels of ARID1A correlated with levels of SOX9 and phosphorylated S6. CONCLUSIONS: ARID1A regulates expression of SOX9, activation of the mTOR pathway, and differentiation of PDCs. ARID1A inhibits formation of PDACs from IPMNs in mice with pancreatic expression of activated KRAS and is down-regulated in IPMN and PDAC tissues from patients.

Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach.

Chronic inflammation contributes to a wide variety of human disorders. In the stomach, longstanding gastritis often results in structural alterations in the gastric mucosa, including metaplastic changes and gastric cancers. Therefore, it is important to elucidate factors that are involved in gastric inflammation. Nardilysin (N-arginine dibasic convertase; Nrdc) is a metalloendopeptidase of the M16 family that promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of a disintegrin and metalloproteinase (ADAM) proteins. Here, we have demonstrated that Nrdc crucially regulates gastric inflammation caused by Helicobacter felis infection or forced expression of prostaglandin E2 in K19-C2mE mice. Metaplastic changes following gastric inflammation were suppressed by the deletion of Nrdc. Furthremore, the deletion of Nrdc significantly suppressed N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis in the murine stomach. These data may lead to a global therapeutic approach against various gastric disorders by targeting Nrdc.

Deletion of nardilysin prevents the development of steatohepatitis and liver fibrotic changes.

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1-/- mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1-/- mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1-/- mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1-/- mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1-/- mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1-/- mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1-/- mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1-/- mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1-/- mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.