Linker Threonine-phosphorylated Smad2/3 Is a Biomarker of Colorectal Neoplastic Stem-like Cells that Correlates With Carcinogenesis.

BACKGROUND/AIM: This study analysed threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) expression and investigated whether pSmad2/3L-Thr is related to the transition from human colorectal adenoma (CRA) to carcinoma (CRC). MATERIALS AND METHODS: Immunofluorescent staining was performed forß-catenin, p53, CDK4, Ki67, Sox9, aldehyde dehydrogenase (ALDH) 1, and pSmad2/3L-Thr. RESULTS: We analysed specimens of diffuse p53-positive CRCs arising from p53-negative CRAs. Percentage of p53, nuclear ß-catenin, Ki67, CDK4, and pSmad2/3L-Thr-positive cells at the site of CRCs was significantly higher than that at the site of CRAs. At the site of normal colorectal mucosae, few epithelial cells were stained positively for pSmad2/3L-Thr. At the site of CRCs, pSmad2/3L-Thr-positive cells showed co-localization with p53, nuclear ß-catenin, and ALDH1. At any site, pSmad2/3L-Thr-positive cells showed co-localization with CDK4. CONCLUSION: pSmad2/3L-Thr correlates with human CRC carcinogenesis, and pSmad2/3L-Thr-positive cells show human colorectal stem cell-like and cancer stem cell characteristics.

Development of Severe Acute Pancreatitis Following Uncovered Metallic Stent Placement: A Rare Case Report.

Self-expandable metallic stents (SEMSs) are widely used for malignant biliary stricture (MBS). Acute pancreatitis is an early complication following SEMS placement. In the present case, the patient developed severe acute pancreatitis after SEMS placement for MBS because of metastatic lymph nodes. Endoscopic retrograde cholangiopancreatography, endoscopic sphincterotomy and an endoscopic nasobiliary drainage tube placement were performed. After seven days, an uncovered SEMS was placed; however, severe acute pancreatitis occurred, and the SEMS was drawn out emergently. In SEMS placement for patients with MBS caused by non-pancreatic cancer, SEMS should be selected carefully while considering each patient's case.

Specific Smad2/3 Linker Phosphorylation Indicates Esophageal Non-neoplastic and Neoplastic Stem-Like Cells and Neoplastic Development.

BACKGROUND: There is little known about stem cells in human non-neoplastic and neoplastic esophageal epithelia. We have demonstrated expression of linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr), suggesting presence of stem-like cells in mouse esophageal epithelium, and identified presence of pSmad2/3L-Thr-positive cells that might function as cancer stem cells in mouse model of colorectal carcinoma. AIMS: We explore whether pSmad2/3L-Thr can be used as a biomarker for stem cells of human esophageal epithelia and/or neoplasms. METHODS: We have used esophageal tissues from inpatients undergoing endoscopic submucosal dissection and performed double immunofluorescent staining of pSmad2/3L-Thr and Ki67, CDK4, p63, Sox2, CK14, p53, ALDH1, CD44 or D2-40 after which the sections were stained with hematoxylin and eosin. RESULTS: pSmad2/3L-Thr-positive cells showed immunohistochemical co-localization with CDK4, p63, CD44 and Sox2 in the basal and parabasal layers of non-neoplastic esophageal epithelia. In esophageal neoplasms, they showed immunohistochemical co-localization with p53, CDK4, ALDH1 and CD44. There was a significant increase in the percentage of pSmad2/3L-Thr-positive cells in the p53-positive neoplastic cell population with development of esophageal neoplasia. pSmad2/3L-Thr-positive cells localized to the lower section of low-grade intraepithelial neoplasia and were observed up to the upper section in carcinoma in situ. In invasive squamous cell carcinoma, they were scattered throughout the tumor with disappearance of polarity and were found in intraepithelial primary lesions and sites of submucosal and vessel invasion. CONCLUSIONS: We determined significant expression of pSmad2/3L-Thr in human esophageal non-neoplastic and neoplastic epithelia, indicating that these are epithelial stem-like cells and cancer stem cells, respectively, that correlate with developing esophageal neoplasms.

Long-term model of colitis-associated colorectal cancer suggests tumor spread mechanism and nature of cancer stem cells.

Although chemical-induced animal models of colorectal cancer (CRC) suggest a lot about the disease, more efforts are required to establish metastasis models. Azoxymethane (AOM) and dextran sodium sulfate (DSS)-treated (AOM/DSS) Crl:CD-1 mice were sacrificed after 10 or 20 weeks in our previous study, and most colon tumors exhibited intramucosal adenocarcinomas. Our observations were extended until 30 weeks to study a colitis-associated advanced CRC mouse model, and explore whether linker threonine-phosphorylated Smad2/3 (pSmad2/3L-Thr) immunostaining-positive cells were involved in the progressive course of colitis-associated CRC as cancer stem cells. AOM/DSS mice were sacrificed at 10, 20 and 30 weeks after AOM administration. Following the histopathological analysis, immunohistochemical staining was performed for the following markers: CD34, podoplanin, ß-catenin, E-cadherin, Ki67, Bmi1 and pSmad2/3L-Thr. Compared with AOM/DSS mice at 10 and 20 weeks, submucosal tumor infiltration and tumor invasion into vessels were markedly increased at 30 weeks. In the parts of colon tumors from AOM/DSS mice, particularly in mice at 30 weeks, the positive signal of E-cadherin was clearly reduced in the cell membranes. The percentage of Ki67-positive tumor cells in mucosal areas of AOM/DSS mice was higher than that in the sites of submucosal infiltration. In mucosal areas of colon tumors, pSmad2/3L-Thr-positive cells were scattered among tumor cells. At sites of submucosal infiltration and vessel invasion of these tumors, pSmad2/3L-Thr-positive cells were also observed among tumor cells. In colon tumors from AOM/DSS mice at 30 weeks, the percentage of pSmad2/3L-Thr-positive cells among the nuclear ß-catenin-positive tumor cells was higher than that among the cytoplasmic ß-catenin-positive tumor cells. For both non-neoplastic and neoplastic epithelial cells, pSmad2/3L-Thr-positive cells exhibited immunohistochemical co-localization with Bmi1. The present study developed an advanced CRC mouse model that exhibited tumor infiltration into the submucosa and invasion into vessels. The present study re-confirmed the theory that pSmad2/3L-Thr-positive cells may be cancer stem cells.

Recent advances of endoscopic retrograde cholangiopancreatography using balloon assisted endoscopy for pancreaticobiliary diseases in patients with surgically altered anatomy: Therapeutic strategy and management of difficult cases.

Endoscopic retrograde cholangiopancreatography (ERCP) is an endoscopic modality established for diagnosis and treatment of pancreaticobiliary diseases. However ERCP in patients with surgically altered anatomy (SAA) has been difficult, and more invasive therapies have been primarily selected. The development of balloon assisted endoscopes (BAEs) innovatively facilitated ERCP in such patients. Recent advances of BAEs and other devices greatly contributed to increasing success of ERCP using BAEs (BAE-ERCP). Furthermore, interventions using Endoscopic Ultrasound (EUS-intervention) have been reported to be useful for pancreaticobiliary diseases in patients with SAA, which provide more options for endoscopic therapies and are also expected as a rescue therapy for difficult cases of BAE-ERCP. In order to thoroughly complete endoscopic treatment for pancreaticobiliary diseases with SAA, it is important to standardize the BAE-ERCP procedures based on the features of respective endoscopes and to establish a strategy for endoscopic treatment which includes analysis of BAE-ERCP difficult cases and selection of cases for rescue therapy. In addition, it is essential to be acquainted with the characteristics of possible adverse events of the procedure and to be able to deal with them for safe accomplishment of endoscopic treatment.

Usefulness of half-covered metallic stent placement in preventing acute cholecystitis complication in pancreatic cancer-induced distal biliary stricture.

BACKGROUND AND AIM: A self-expandable metallic stent (SEMS) is commonly used for biliary stricture caused by pancreatic cancer. Covered SEMS may obstruct the cystic duct, causing acute cholecystitis. This study aimed to determine the outcomes of using a half-covered SEMS with an offset covered portion for preventing cystic duct obstruction. METHODS: Among 80 patients with half-covered SEMS placement for the treatment of pancreatic cancer-induced distal biliary stricture, 74 were followed up. The half-covered SEMS has a total length of 6 or 7 cm, and the offset covered part was 0.5-4.5 or 0.5-5.5 cm, respectively. Intraductal ultrasonography (IDUS) and endoscopic nasobiliary drainage (ENBD) were performed during the initial endoscopic retrograde cholangiopancreatography (ERCP). IDUS findings and ENBD tube cholangiogram confirmed the cystic duct confluence. SEMS placement was performed on the second ERCP or several weeks after the initial tube stent placement. RESULTS: Half-covered SEMS placement was successful in all patients. However, four (5.4%) patients exhibited early complications, including acute cholecystitis in one patient and stent displacement in another. Over 30 days, cholangitis, tumor growth, and stent displacement occurred in nine (11.3%), five (6.3%), and two (2.5%) patients, respectively. The median stent patency was 71.1 weeks, and the median overall survival in patients with and without chemotherapy was 31.8 and 12.2 weeks, respectively. CONCLUSIONS: With confirmation of the cystic duct confluence, half-covered SEMS placement may become a treatment option for distal biliary stricture caused by pancreatic cancer to prevent acute cholecystitis. Half-covered SEMS patency was comparable with that of covered SEMS.

Evaluation of complications after endoscopic retrograde cholangiopancreatography using a short type double balloon endoscope in patients with altered gastrointestinal anatomy: a single-center retrospective study of 1,576 procedures.

BACKGROUND AND AIM: Endoscopic retrograde cholangiopancreatography (ERCP) using balloon-assisted endoscope such as double-balloon endoscope is even effective for patients with surgically altered anatomy. Yet comprehensive studies on complications of ERCP using balloon-assisted endoscope have not been made. We analyzed the characteristics and the causes of complications of ERCP using double-balloon endoscope (DB-ERCP) procedures and aimed to suggest effective managements. METHODS: A total of 1576 procedures of DB-ERCP in 714 patients with surgically altered gastrointestinal anatomy in our hospital were evaluated retrospectively using a statistic analysis. RESULTS: The overall complication occurrence rate was 5.8%. By type of complications are perforation 3.2%, mucosal laceration 0.5%, hemorrhage 1.0%, pancreatitis 0.6%, respiratory disorder 0.4%, and others 0.2%. By type of surgical reconstruction methods were Roux-en-Y reconstruction with choledocho-jejunal anastomosis 4.2%, Roux-en-Y reconstruction without choledocho-jejunal anastomosis 6.7%, pancreaticoduodenectomy 4.5%, pylorus preserving pancreaticoduodenectomy 4.2%, Billroth II gastrectomy (B-II) 11.6%, and other reconstruction method (others) 7.4%. The contributing factors calculated by a multivariate analysis were B-II (odds ratio: 1.864, 95% confidence interval: 1.001-3.471, P = 0.050) and the presence of naïve papilla (odds ratio: 3.268, 95% confidence interval: 1.426-7.490, P = 0.005). CONCLUSIONS: DB-ERCP is a safe method with a total complication rate of 5.8% that could be considered within an acceptable range. The most common complication was the injury of the digestive tract such as perforation. Affecting risk factors for complications were B-II and the presence of naïve papilla. DB-ERCP procedures should be performed carefully of these factors.

Utility of newly developed short-type double-balloon endoscopy for endoscopic retrograde cholangiography in postoperative patients.

BACKGROUND AND AIM: The utility of conventional short-type double-balloon endoscopy (DBE) for pancreatobiliary disease in patients with postoperative anatomy had been widely acknowledged and accepted. However, some technical difficulties yet remained. In an attempt to solve these problems, the new short-type DBE (N-short DBE) was uniquely designed for therapeutic endoscopic retrograde cholangiography (ERC) in postoperative patients. The aim of this study was to evaluate the usefulness of N-short DBE for ERC in postoperative patients. METHODS: From August 2015 to April 2016, ERC using N-short DBE (DB-ERC) was performed in 100 postoperative patients (112 procedures). We retrospectively studied the success rate of reaching the blind end, the median time to reach the blind end, the diagnostic success rate, the therapeutic success rate, the median time to complete ERC-related interventions, the overall success rate, the median time to complete DB-ERC, and adverse events. RESULTS: The success rate of reaching the blind end was 99.1%. The median time to reach the blind end was 10 min (interquartile range [IQR], 6-19 min). The diagnostic success rate was 98.2%. The therapeutic success rate was 100%. The median time to complete ERC-related interventions was 36 min (IQR, 22-62 min). The overall DB-ERC success rate was 97.3%. The median time to complete DB-ERC was 54 min (IQR, 37-73 min). The occurrence of adverse events was 2.7%. CONCLUSIONS: The N-short DBE for ERC in postoperative patients is useful and safe. DB-ERC is promising therapeutic modality in such patients and should be selected as the first-line policy.

Smad2/3 linker phosphorylation is a possible marker of cancer stem cells and correlates with carcinogenesis in a mouse model of colitis-associated colorectal cancer.

BACKGROUND: Epithelial cells affected by somatic mutations undergo transition from a tumour-suppressive to a carcinogenic Smad pathway during sporadic colorectal carcinogenesis, and the specific linker threonine phosphorylation of Smad2/3 in colon epithelial cells indicates stem-like cells. This study extends previous observations to a model of colitis-associated colorectal cancer. METHODS: After Crl:CD-1 mice received an administration of azoxymethane [AOM], the mice were given dextran sodium sulfate [DSS] for 7 days. AOM/DSS-treated mice [AOM/DSS mice] were killed at 10 or 20 weeks. After macroscopic observations, a histopathological analysis was conducted. Immunohistochemical staining was performed using the avidin-biotin immunoperoxidase method [pSmad3C-Ser, pSmad3L-Ser, c-Myc] and immunofluorescent methods [Ki67, ß-catenin, CDK4, cyclin D1, Sox9, pSmad2/3L-Thr]. RESULTS: The colons from AOM/DSS mice were shorter than those from control mice. The number of colon tumours at Week 20 was higher than at Week 10. The inflammation scores for AOM/DSS mice were greater than those for control mice. Immunostaining-positive cells (staining by Ki67, ß-catenin [nuclear and cytoplasmic], cyclin D1, and Sox9) were diffusely distributed in colon tumours. The percentage of pSmad3L-Ser-positive cells in colon tumours was higher than in sites of pre-neoplastic colitis, and that in sites of pre-neoplastic colitis was higher than in control mice. pSmad2/3L-Thr-positive cells were sparsely detected around crypt bases in non-neoplastic colon epithelia and at the tops of tumours, and immunohistochemical co-localisation of pSmad2/3L-Thr with Ki67 was not observed. Immunohistochemical co-localisation of pSmad2/3L-Thr with ß-catenin and CDK4 was observed. CONCLUSIONS: pSmad3L-Ser signalling is an early event in colitis-associated colorectal cancer, and pSmad2/3L-Thr immunostaining-positive cells might be cancer stem cells.

Intrahepatic cholangiocarcinoma diagnosed via endoscopic retrograde cholangiopancreatography with a short double-balloon enteroscope.

Endoscopic retrograde cholangiopancreatography (ERCP) using a double-balloon enteroscope (DBE) in patients with bowel reconstruction due to a previous abdominal surgery is now widely accepted. In particular, a short DBE, which has a 2.8-mm working channel and 152-cm working length, is useful for ERCP because of its good rotational and straightening ability and the availability of various conventional ERCP accessories through the working channel. Herein we report a case of intrahepatic cholangiocarcinoma via ERCP with a short DBE. This is the first report in which the pre-cutting and the brush cytological examination were performed successfully under a DBE to diagnose intrahepatic cholangiocarcinoma pathologically. The short DBE allowed us to perform all diagnostic and therapeutic procedures accepted in conventional ERCP in patients with surgically altered anatomies.