Next-Generation Sequencing Analysis of Pancreatic Cancer Using Residual Liquid Cytology Specimens from Endoscopic Ultrasound-Guided Fine-Needle Biopsy: A Prospective Comparative Study with Tissue Specimens.

This study evaluated the feasibility and clinical utility of liquid-based cytology (LBC) specimens via endoscopic ultrasound-guided fine-needle biopsy (EUS-FNB) for next-generation sequencing (NGS) of pancreatic cancer (PC). We prospectively evaluated the performance of DNA extraction and NGS using EUS-FNB samples obtained from PC. Thirty-three consecutive patients with PC who underwent EUS-FNB at our hospital were enrolled. DNA samples were obtained from 96.8% of the patients. When stratified with a variant allele frequency (VAF) > 10% tumor burden, the NGS success rate was 76.7% (n = 23) in formalin-fixed paraffin-embedded (FFPE), 83.3% (n = 25) in LBC, and 76.7% (n = 23) in frozen samples. The overall NGS success rate was 86.7% (n = 26) using FFPE, LBC, or frozen samples. The detection rates for the main mutated genes were as follows: 86.7% for KRAS, 73.3% for TP53, 66.7% for CDKN2A, 36.7% for SMAD4, and 16.7% for ARID1A. LBC had the highest median value of VAF (23.5%) for KRAS and TP53. PC mutation analysis using NGS was successfully performed using LBC compared with FFPE and frozen samples. This approach provides an alternative and affordable source of molecular testing materials.

Comparison of recurrent biliary obstruction with the use of metal and plastic stents in EUS-guided biliary drainage: A propensity score-matched analysis.

Background and Objectives: In transpapillary biliary drainage, metal stents (MSs) exhibit a lower incidence of a biliary obstruction than plastic stents (PSs). However, few studies have compared recurrent biliary obstruction (RBO) when MSs and PSs are used in EUS-guided hepaticogastrostomy (EUS-HGS) and choledochoduodenostomy (EUS-CDS). We retrospectively evaluated the RBO for both stents in each procedure. Patients and Methods: : Between November 2012 and December 2020, 85 and 53 patients who underwent EUS-HGS and EUS-CDS for unresectable malignant biliary obstruction, respectively, were enrolled. Factors associated with RBO were assessed. Clinical outcomes were compared between the MS and PS groups using propensity score matching. Results: : The clinical success rate and procedure-related adverse events were similar in the MS and PS groups. Multivariate analysis identified the use of PS as a factor associated with RBO (EUS-HGS, P = 0.03; EUS-CDS, P = 0.02). After matching, the median time to RBO in EUS-HGS (MS: 313; PS: 125 days; P = 0.01) in the MS group was longer than that in the PS group. The cumulative incidence of RBO at 1, 3, and 6 months in the MS group was significantly lower than that in the PS group for EUS-HGS (MS: 4.0%, 8.2%, and 8.2%; PS: 12.4%, 24.9%, and 39.5%, respectively, P = 0.01). Conclusions: : MS exhibited a lower rate of RBO than PS for EUS-HGS and EUS-CDS.

Sequential evaluation of MUC promoter methylation using next-generation sequencing-based custom-made panels in liquid-based cytology specimens of pancreatic cancer.

BACKGROUND: As liquid-based cytology (LBC) specimens preserve high-quality DNA, clinical sequencing of LBC specimens using next-generation sequencing (NGS) is becoming a common strategy. This study aimed to evaluate the feasibility of NGS-based custom-made panels for evaluating MUC promoter methylation in LBC specimens. METHODS: Thirty-one patients with pancreatic cancer were enrolled in the study. Cancer tissue samples were obtained using endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNA/FNB). LBC, formalin-fixed paraffin-embedded (FFPE), and fresh frozen specimens were prepared for DNA extraction after pathological diagnosis. These specimens were then subjected to NGS analysis using custom-made cancer gene screening and methylation panels comprising 28 cancer-related genes and 13 gene promoter regions, including MUC1, MUC2, and MUC4. RESULTS: The success rate of NGS using the cancer gene panel was comparable among the LBC, FFPE, and frozen specimens, and the presence of cancer cell-derived somatic mutations in each specimen was confirmed. The specimens were then tested using a methylation panel that revealed the sequential methylation status of CpG islands located in the promoter regions of MUC genes. The methylation status results obtained from LBC specimens were almost comparable with those from FFPE and frozen specimens. CONCLUSIONS: MUC and other gene methylation analyses using an NGS-based panel were successfully performed in residual LBC specimens obtained by EUS-FNA/FNB. Therefore, this approach provides an alternative source of molecular tests for gene mutations and methylation, especially in the pancreatic cancers, which are often unresectable and unsuitable for obtaining FFPE specimens.

Efficacy of endoscopic ultrasonography in evaluating tumor thickness in patients with superficial pharyngeal carcinoma: A pilot study.

OBJECTIVES: A histopathological tumor thickness of ≥1000 µm has been reported as one of many risk factors for recurrent lymph node metastasis in superficial pharyngeal cancer (SPC). However, methods for assessing this risk factor preoperatively have not yet been established. Hence, the current study aimed to evaluate the efficacy of endoscopic ultrasonography (EUS) in measuring tumor thickness preoperatively in patients with SPC. METHODS: This single-center retrospective study included 44 consecutive patients with 47 lesions who underwent endoscopic submucosal dissection (ESD). Prior to surgery, EUS examination was performed while under general anesthesia. Further, microvascular irregularity in the target lesion was evaluated using the Japan Esophageal Society (JES) magnification endoscopic classification system. RESULTS: A significant correlation was noted between histopathological and EUS tumor thickness (Spearman's correlation r == 0.879, p < 0.001). In tumors ≥1000 µm thick on histopathology, the cutoff value for EUS tumor thickness was 2.6 mm, and the following values were obtained: sensitivity, 100%; specificity, 81.8%; positive predictive value (PPV), 70%; negative predictive value (NPV), 100%; and accuracy, 87.2%. In B2 lesions ≥1000-µm thick, the following values were obtained: sensitivity, 85.7%; specificity, 90.9%; PPV, 80%; NPV, 93.8%; and accuracy, 89.4%. The diagnostic accuracy rate of combined EUS and the JES magnifying endoscopic classification system was 95.7%. CONCLUSIONS: Tumor thickness assessed using EUS was effective in diagnosing histopathological tumor thickness of ≥1000 µm. The combined use of EUS and the JES magnifying endoscopic classification system may be useful for assessing preoperative risk factors for lymph node metastasis in SPC.

A case of immunoglobulin G4-related sclerosing mesenteritis without other organ involvement.

A 64-year-old man presented to our hospital with abdominal pain and 4-5 episodes of watery diarrhea per day for 2 months. Abdominal ultrasound examination revealed a mass in the peritoneal cavity, and computed tomography showed a 13.4 cm mass in the mesentery and a 3 cm mass in the mesocolon. The patient underwent laparoscopic partial resection for diagnosis. Microscopically, abundant fibrosis and numerous immunoglobulin (Ig) G4-positive plasma cells were observed. The serum level of IgG4 was 665 mg/dl postoperatively. These findings suggested that the lesion was consistent with IgG4-related sclerosing mesenteritis. Oral steroids resulted in rapid disappearance of symptoms and a decrease in masses. Recently, sclerosing mesenteritis are reported as IgG4-related disease or mimicking IgG4-related disease but multiple lesions rarely occur in the same organ. We report a case of IgG4-related sclerosing mesenteritis with multiple lesions without involvement of other organs, such as the pancreas and salivary glands.

Acetaldehyde breath test as a cancer risk marker in patients with esophageal and hypopharyngeal squamous cell carcinoma.

Patients with inactive acetaldehyde dehydrogenase 2 (ALDH2) are at high risk for esophageal squamous cell carcinoma (ESCC) and hypopharyngeal squamous cell carcinoma (HPSCC). The acetaldehyde breath test (ABT) may demonstrate ALDH2 gene polymorphisms. We evaluated the usefulness of the ABT in patients with ESCC and HPSCC. The squamous cell carcinoma (SCC) group consisted of 100 patients who were treated with endoscopic submucosal dissection (ESD) for ESCC or HPSCC, and the control group (HC) consisted of 275 healthy subjects. The SCC group comprised the "single subgroup" (n = 63), in which a single lesion was initially treated with ESD, and the "multiple subgroup" (n = 31), in which multiple lesions were initially treated with ESD. First, we compared the groups' risk factors for carcinogenesis and measured the acetaldehyde-to-ethanol (A/E) ratio. Then we tested the groups' differences in the abovementioned carcinogenic risk factors. We found that the proportion of individuals in the SCC group with inactive ALDH2 (A/E ratio ≥ 23.3) was significantly higher than that in the HC group (p = 0.035), as was the A/E ratio (p < 0.001). Also, the proportion of individuals with inactive ALDH2 in the multiple subgroup was significantly higher than that in single subgroup (p = 0.015), as was the A/E ratio (p = 0.008). In conclusion, ABT may be a potential screening tool for detecting people at risk of ESCC and HPSCC. In addition, it could be a useful tool in detecting patients at risk of multiple or double carcinomas among patients with ESCC and HPSCC. Trial registration: Trial Registration number: UMIN000040615 [https://rctportal.niph.go.jp/en/detail?trial_id=UMIN000040615], Data of Registration: 01 46 June 2020, retrospectively registered.

Increased Gene Copy Number of DEFA1A3 Is Associated With the Severity of Ulcerative Colitis.

INTRODUCTION: DEFA1A3 encodes human neutrophil peptides (HNPs) 1-3 and has multiple copy number variations (CNVs). HNPs are associated with innate immunity. Ulcerative colitis (UC), a chronic inflammatory gastrointestinal disorder, is a life-threatening condition, and predictive markers of UC severity are needed. This study investigated the relationship between DEFA1A3 CNV and UC severity. METHODS: This study enrolled 165 patients with UC. The relationship between DEFA1A3 CNV and disease severity was analyzed based on Mayo score, patient characteristics, and treatment methods. In addition, serum and stimulated neutrophil-derived HNP concentrations were also measured in patients with high and low DEFA1A3 CNV. RESULTS: DEFA1A3 CNV was significantly correlated with Mayo score and white blood cell count (R = 0.46, P < 0.0001; R = 0.29, P = 0.003, respectively), and only high copy numbers of DEFA1A3 were independent factors for severe UC (P < 0.001, odds ratio: 1.88, 95% confidence interval, 1.34-2.61). The number of severe UC patients with high DEFA1A3 CNV was significantly greater than those with low CNV. We confirmed the associations between DEFA1A3 and UC severity using a validation cohort. In addition, the HNP concentration in high-copy number patients was significantly higher after neutrophil stimulation than that in low-copy number patients. DISCUSSION: This study demonstrated that there is a correlation between DEFA1A3 copy number and severity in patients with UC. In addition, neutrophils from UC patients with higher DEFA1A3 CNV had high reactivity of secretion of HNPs after stimulation. DEFA1A3 CNV may be a novel severity marker and a potential therapeutic target for UC.

Prognostic significance of extracellular volume fraction with equilibrium contrast-enhanced computed tomography for pancreatic neuroendocrine neoplasms.

BACKGROUND: /Objectives: Identifying reliable pretreatment imaging biomarkers for pancreatic neuroendocrine neoplasm (PanNEN) is a key imperative. Extracellular volume (ECV) fraction quantified with equilibrium contrast-enhanced CT can be easily integrated into routine examinations. This study aimed to determine whether ECV fraction with equilibrium contrast-enhanced computed tomography (CECT) could predict long-term outcomes in patients with PanNEN. METHODS: This study was a retrospective observational study of 80 patients pathologically diagnosed with PanNEN at a single institution. ECV fraction of the primary lesion was calculated using region-of-interest measurement within PanNEN and the aorta on unenhanced and equilibrium CECT. The impact of clinical factors and tumor ECV fraction on progression-free survival (PFS) and overall survival (OS) was assessed with univariate and multivariate analyses using Cox proportional hazards models. The correlation between WHO classification and tumor ECV fraction was evaluated using Kendall rank correlation coefficients. RESULTS: PFS and OS rates were estimated as 93.4% and 94.6%, 78.7% and 86.2%, 78.7% and 77.0%, and 78.7% and 66.6% at 1, 3, 5, and 10 years, respectively. Multivariate analysis revealed that Union for International Cancer Control (UICC) stage (hazard ratio [HR] = 3.95, P = 0.003), WHO classification (HR = 12.27, P = 0.003), and tumor ECV fraction (HR = 11.93, P = 0.039) were independent predictors of PFS. Patient age (HR = 1.11, P < 0.001), UICC stage (HR = 3.14, P = 0.001), and tumor ECV fraction (HR = 5.27, P = 0.024) were independent significant variables for predicting OS. Tumor ECV fraction had a weak inverse relationship with WHO classification (P = 0.045, τ = -0.178). CONCLUSIONS: ECV fraction determined by equilibrium CECT and UICC stage may predict survival in patients with PanNEN.

Covering Post-Endoscopic Submucosal Dissection Ulcers in Miniature Swine with Hexanoyl (Hx:C6) Group-Modified Alkaline-Treated Gelatin Porous Film (HAG) Induces Proper Healing by Decreasing Inflammation and Fibrosis.

BACKGROUND AND AIMS: Hexanoyl (Hx:C6) group-modified alkaline-treated gelatin porous film (HAG) is a newly developed degradable hydrogel characterized by strong adhesiveness and high affinity for vascular endothelial growth factor (VEGF). The aim of this study was to clarify the effect of HAG sheets on the healing process of post-endoscopic submucosal dissection (ESD) porcine gastric artificial ulcers. METHODS: (1) To evaluate the adhesiveness of HAG sheets over time, we performed ESD to create 1 artificial ulcer and covered the lesion with 1 HAG sheet using 1 miniature swine. We observed 2 ulcers by endoscopic and microscopic examinations. (2) To examine the effect of HAG sheets on post-ESD ulcer healing, we performed ESD using 5 miniature swine. The artificial ulcers were covered with HAG sheets, or left uncovered after ESD (day 0), followed by macroscopic and microscopic examinations. On days 7 and 14, we observed 2 ulcers by endoscopic examinations. On day 14, the animals were sacrificed, and histological examination was performed on the 3 stomachs that could be extirpated. RESULTS: (1) On day 7, adhesion of HAG sheets was observed. (2) Gastric ulcer area on day 7 was significantly larger in the covered ulcers than in the non-covered ulcers (p = 0.046). On day 14, although there was no significant difference in ulcer area irrespective of covering (p = 0.357), the covered ulcers tended to repair less fold convergence than non-covered ulcers. The covered ulcer sheets significantly decreased inflammatory cell infiltration (p = 0.011), but significantly increased the abundance of macrophages (p = 0.033), in submucosal layers. Also, the abundance of alpha-smooth muscle actin-positive cells in submucosal layers of the covered ulcers was significantly reduced (p = 0.044), leading to a decrease in collagen accumulation. In addition, fibrosis and atrophy of the muscularis propria were significantly lower for covered ulcers than for non-covered ulcers. Furthermore, microvessels and VEGF-positive cells were significantly more abundant in the submucosal layers of the covered ulcers (p < 0.001 and p = 0.024, respectively). CONCLUSIONS: HAG sheets induced post-ESD ulcer healing with less submucosal inflammation and muscularis propria injury and have the potential to decrease excess scarring.

Significance of Linked Color Imaging for Predicting the Risk of Clinical Relapse in Ulcerative Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with unknown etiology. Recently, mucosal healing has emerged as an important therapeutic endpoint in UC. Linked color imaging (LCI) is a novel endoscopic system that enhances the color differences of the gastrointestinal mucosa. Our previous study emphasized the redness and yellowness of the lesion using LCI observation, which was useful for the evaluation of histological mucosal activity in UC. In this study, we aimed to evaluate the correlation between LCI observation and clinical relapse rate in UC patients. We retrospectively analyzed UC patients who underwent total colonoscopy between August 2016 and October 2018 at our facility with Mayo endoscopic scores of 0 or 1. We assessed the correlation between orange-like color lesion (defined as LCI-scarlet color lesions) and clinical relapse rate (requiring additional treatment for UC) during the 1-year follow-up period. Fifty-eight patients (22 female, 36 male; median age at diagnosis, 47.2 (18-80) years) who underwent colonoscopy were analyzed. During the 1-year follow-up period, clinical relapse was observed in 12 patients (20.1%) among which ten patients (83.3%) had an LCI-scarlet color lesions recognized by LCI. By contrast, 29 patients (63%) had no LCI-scarlet color lesions in the clinical remission group (n = 46). There was a significant difference in LCI-scarlet color between the clinical relapse and remission groups, remaining significantly associated with clinical relapse. LCI findings, including an orange-like color lesion, have diagnostic implications for predicting the risk of clinical relapse in UC during the 1-year follow-up period.

A case of gallbladder neuroendocrine carcinoma diagnosed preoperatively using somatostatin receptor scintigraphy.

Gallbladder neuroendocrine carcinoma (NEC) is a rare gallbladder tumor. The current report is a case of a patient preoperatively diagnosed with gallbladder NEC using somatostatin receptor scintigraphy (SRS). A 63-year-old man was admitted to our hospital by a family doctor after abdominal ultrasonography revealed thickened walls of the neck of his gallbladder. At Kagoshima University Hospital, CT and MRI of the abdomen and endoscopic ultrasonography confirmed the thickening of the walls of the neck of the gallbladder. However, it did not resemble a typical gallbladder cancer or tumor, such as a neuroendocrine tumor or malignant lymphoma. Positron emission tomography and SRS showed abnormal accumulation at the tumor site. Endoscopic retrograde cholangiopancreatography was performed, adenocarcinoma was suspected based on intra-gallbladder bile cytology, and a cholecystectomy with lymphadenectomy was performed. The postoperative pathological diagnosis was small cell NEC (pT3a, N0, M0, stage II). Immunohistochemistry indicated that the gallbladder tumor cells were positive for synaptophysin, chromogranin A, and cluster of differentiation (CD) 56, and negative for somatostatin receptors (SSTR) 2 and 5. Gene expression assays revealed the expression of all SSTR subtypes (SSTR1-5) in the tumor. Generally, NECs exhibit poor accumulation in SRS, however, the results of the current case suggest that SRS may be useful in the preoperative diagnosis of NEC.

Factors Associated with the Technical Success of Bilateral Endoscopic Metallic Stenting with Partial Stent-In-Stent Placement in Patients with Malignant Hilar Biliary Obstruction.

BACKGROUND: Bilateral biliary drainage decreases the risk of cholangitis, but bilateral endoscopic metallic stenting is technically challenging. AIM: We retrospectively evaluated the factors associated with successful bilateral self-expanding metal stent (SEMS) placement using the partial stent-in-stent (PSIS) method for malignant hilar biliary obstruction and also assessed the safety and efficacy of this technique. METHODS: From April 2010 to February 2016, 47 consecutive patients (mean age, 73.0 ± 8.6 years; 32 males and 15 females) underwent PSIS placement for malignant hilar biliary obstruction in our hospital. The technical success of PSIS, clinical response, and complications were investigated. Factors associated with the technical success of PSIS were assessed. Using a propensity score-matched analysis, we compared the procedure time, clinical response, complications, stent patency, and survival time in 17 matched patients treated with bilateral SEMS placement using a SEMS delivery system of <6.0 or ≥6.0 Fr. RESULTS: The technical success rate was 77%. The clinical response rate was 91%, and the complication rate was 26%. Regarding complications, pancreatitis occurred in 5 patients (11%), cholangitis in 6 (13%), and cholecystitis in 1 (2%). A multiple logistic regression analysis identified the use of a SEMS with a delivery system < 6.0 Fr as a factor associated with technical success (P = 0.033; odds ratio, 10.769; 95% confidence interval, 1.205-96.212). In the 17 matched patients assigned according to the SEMS delivery system size, the procedure time was significantly shorter in those with a delivery system size < 6.0 Fr than in those with ≥6.0 Fr (P < 0.01). There were no significant differences in the clinical response, complication rate, stent patency, or survival time between the two groups. CONCLUSION: Using a delivery system < 6.0 Fr in size helped improve the technical success and reduced the procedure time for the placement of a SEMS by the PSIS method.

Cola Dissolution Therapy via Ileus Tube Was Effective for Ileus Secondary to Small Bowel Obstruction Induced by an Enterolith.

An 87-year-old bedridden woman developed intestinal obstruction caused by an enterolith or bezoar. Since the patient refused surgery, we administered 1,000 mL/day of cola via an ileus tube to dissolve the stone. Occlusion of the small intestine disappeared on day 6. The excreted stones contained calcium phosphate, which is typical of enteroliths. We later confirmed that the retrieved stones could be dissolved in cola (Coca-Cola®, pH 1.9) as well as 0.10 and 0.010 mol/L hydrochloric acid (pH 1.0 and 2.0, respectively) and food-grade vinegar (pH 2.6). These findings suggest that the enteroliths were dissolved by an acid-base reaction.

Usefulness of septal thickness measurement on endoscopic ultrasound as a predictor of malignancy of branched-duct and mixed-type intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND AND AIM: Septal thickness (ST) can predict a malignant branch-duct (BD) and mixed-type intraductal papillary mucinous neoplasm (IPMN) of the pancreas, but its cut-off value has not been established. The aim of the present study was to determine the optimal ST cut-off value to predict malignancy using endoscopic ultrasound (EUS). METHODS: We retrospectively identified 200 patients with IPMN, including 132 with BD- and mixed-IPMN, who underwent surgical resection between 1989 and 2017. ST was defined as the septum or lesion wall with the maximum diameter in BD- and mixed-IPMN. The possibility of ST as a malignant predictor was examined, as well as the diagnostic ability of ST combined with mural nodule (MN) height for malignant IPMN. RESULTS: Among the 132 IPMN patients, pathological diagnosis was benign in 81 (61.4%) and malignant in 51 (38.6%). Area under the curve for the diagnosis of malignancy using ST was 0.74 for pathological specimens, 0.70 for EUS and 0.56 for computed tomography. Multivariate analysis showed that the odds ratios for ST ≥2.5 mm and MN height ≥5 mm were 3.51 [95% confidence interval (CI), 1.55-7.97, P = 0.003] and 3.36 (95% CI, 1.52-7.45, P = 0.003), respectively. CONCLUSIONS: Septal thickness was an independent predictive factor similar to MN height for malignant IPMN in a multivariate analysis. The ST on EUS appeared to be the thickness of a fibrotic septum associated with the malignant transformation of IPMN. An ST cut-off value of 2.5 mm might provide an accurate prediction of malignant IPMN.

Outcomes of EUS-FNA in patients receiving antithrombotic therapy.

Background and study aims To investigate bleeding risk and thromboembolic risk in patients receiving antithrombotic therapy who underwent endoscopic ultrasound-guided fine-needls aspiration (EUS-FNA). Patients and methods A single-center retrospective study of 908 consecutive patients undergoing EUS-FNA for pancreatic and non-pancreatic lesions patients between March 2013 and March 2017 was performed. Antithrombotic management was classified into three groups: continuous, discontinuation, and heparin replacement. Results A total of 114 patients (12.6 %) were on antithrombotic drugs and 794 (84.6 %) were not. There were six cases of significant bleeding (0.7 %) four in the antithrombotic group (0.4 %) and two (0.2 %) in the non-antithrombotic group, (odds ratio, 9.59; 95 % confidence interval, 2.12 - 43.1; P  = 0.006). Of the four cases in the antithrombotic group, two were on continuous treatment, one was on discontinuation treatment and one was on heparin replacement. All cases of non-significant bleeding occurred in the non-antithrombotic group (3 peri-tumoral hematomas, 1 submucosal hematoma, and 1 intraluminal bleed). The sole thromboembolic event (0.9 %) was a cerebral infarction in the antithrombotic group in a patient on thienopyridine who switched to aspirin before the procedure. Conclusions There was a slight increase in risk of bleeding in patients receiving antithrombotic therapy especially postoperative bleeding; however, there were no cases of severe bleeding was seen and only one case of cerebral infarction which occurred in a high-risk thromboembolic patients. We concluded that EUS-FNA in a safe procedure for patients on antithrombotics, even when antithrombotic therapy is not discontinued during EUS-FNA.