Tomohiro Kanda

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer

Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.

Sequencing and bioinformatics-based analyses of the microRNA transcriptome in hepatitis B-related hepatocellular carcinoma.

MicroRNAs (miRNAs) participate in crucial biological processes, and it is now evident that miRNA alterations are involved in the progression of human cancers. Recent studies on miRNA profiling performed with cloning suggest that sequencing is useful for the detection of novel miRNAs, modifications, and precise compositions and that miRNA expression levels calculated by clone count are reproducible. Here we focus on sequencing of miRNA to obtain a comprehensive profile and characterization of these transcriptomes as they relate to human liver. Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 314000 miRNAs from HCC and more than 268000 from ANL for registered human miRNAs. Computational bioinformatics identified 7 novel miRNAs with high conservation, 15 novel opposite miRNAs, and 3 novel antisense miRNAs. Moreover sequencing can detect miRNA modifications including adenosine-to-inosine editing in miR-376 families. Expression profiling using clone count analysis was used to identify miRNAs that are expressed aberrantly in liver cancer including miR-122, miR-21, and miR-34a. Furthermore, sequencing-based miRNA clustering, but not individual miRNA, detects high risk patients who have high potentials for early tumor recurrence after liver surgery (P = 0.006), and which is the only significant variable among pathological and clinical and variables (P = 0,022). We believe that the combination of sequencing and bioinformatics will accelerate the discovery of novel miRNAs and biomarkers involved in human liver cancer.

Long‐term results of elective hepatectomy for the treatment of ruptured hepatocellular carcinoma

BACKGROUND/PURPOSE According to the General rules for the clinical and pathological study of primary liver cancer, compiled by the Liver Cancer Study Group of Japan, ruptured hepatocellular carcinoma (HCC) is classified as stage 4, even if the tumor is small and solitary. We examined the long-term results of elective hepatectomy for the treatment of ruptured HCC. METHODS A first hepatectomy was performed without operative death in 193 patients with HCC. Ten patients had ruptured HCC (ruptured group) and 183 patients had nonruptured HCC (nonruptured group). The extension of HCC was macroscopically classified as stage 1 in 23 patients, stage 2 in 71, stage 3 in 53, and stage 4 in 46. RESULTS Cumulative survival rates in the ruptured group at 1, 5, and 10 years were 90.0%, 67.5%, and 20.3%, respectively. The cumulative survival rate was lower in patients with stage 4 disease in the nonruptured group than that in patients in the ruptured group (P < 0.05). Cumulative survival rates did not differ significantly between patients in the ruptured group and those with stage 2 or stage 3 disease. CONCLUSIONS Survival rates after elective hepatectomy in patients with ruptured HCC are good, even if the disease is classified as stage 4.

Silicon drain with channels along the sides for internal biliary stenting of hepaticojejunostomy in hepatic hilar malignancies.

Background:  We compared two types of stents in patients who underwent surgery for hepatic hilar malignancies.

Serum procalcitonin concentration within 2 days postoperatively accurately predicts outcome after liver resection

Abstract Background: Relatively high mortality and morbidity rates are reported after liver resection (LR). However, the early predictors of complications after LR are not clear. This study was performed to clarify the usefulness of procalcitonin (PCT) for the early prediction of complications after elective LR. Methods: This observational study included 72 consecutive patients who underwent elective LR from December 2015 to March 2017. Patients were categorized into two groups: those with and without postoperative complications (Clavien-Dindo grade ≥II). The values of postoperative inflammatory markers (white blood cell [WBC] count, C-reactive protein [CRP] and PCT) were compared between the two groups. Results: CRP and PCT were significantly higher in patients with than without complications; however, the WBC count showed no difference within 5 days postoperatively. The maximum area under the receiver operating characteristic curves within 2 days after LR using the WBC count, CRP and PCT were 0.608, 0.697 and 0.860, respectively, PCT had the best predictive ability in the early postoperative period. The PCT level peaked within 2 days postoperatively in 61 patients (85%). The maximum PCT level within 2 days postoperatively (PCT1−2) was significantly higher in patients with than without complications (0.52 vs. 0.19 ng/mL, p<0.001). A cutoff PCT1−2 level of 0.35 ng/mL achieved 80% sensitivity and 83% specificity. In patients without complications, there was no difference in PCT1−2 even when the surgical procedure differed (p=0.935). Conclusions: PCT1−2 is an early predictive marker after LR and can be similarly used regardless of the LR procedure.

Successful treatment of limy bile syndrome extending to the common bile duct by laparoscopic cholecystectomy and common bile duct exploration: A case report and literature review.

Limy bile syndrome extending to the common bile duct (CBD) is a rare condition that lacks a standardized treatment. Laparoscopic cholecystectomy with laparoscopic choledocholithotomy by CBD exploration is preferred because it preserves the function of the sphincter of the Vaters papilla and allows treatment of both lesions. A 37‐year‐old man who was receiving entecavir for chronic hepatitis B developed right upper quadrant pain. Abdominal ultrasonography revealed a calcified shadow in the gallbladder and CBD. Abdominal imaging revealed a liquid‐like material identified by a calcified shadow in two phases separated by a fluid‐fluid level. Abdominal and 3‐D drip infusion cholangiography CT showed stones in the gallbladder and CBD with limy bile. The patient underwent laparoscopic cholecystectomy and choledocholithotomy. Intraoperatively, white–yellow‐colored bile and stones were drained from the CBD. A C‐tube was placed. Postoperatively, remnant stones and radiopaque materials were absent. The stones comprised of >95% calcium carbonate.

Evaluation of positive ductal margins of biliary tract cancer in intraoperative histological examination

At present the only method available to confirm microscopic infiltration of cancer into ductal margins during surgery, is intraoperative histological examination. In the present study, the status of the surgical margins and postoperative course were evaluated to determine any correlation between remnant carcinoma and postoperative survival. All consecutive patients who underwent resection for biliary tract cancer between January 2004 and May 2012 were identified from a database. Positive margin cases were divided into two groups, invasive carcinoma and carcinoma in situ (CIS). Immunohistochemical staining targeting Ki67 and p53 for positive margins was performed. Cases of major vessel invasion were significantly increased in the positive group compared with the negative group. The recurrence rate was significantly lower in the CIS group compared with the invasive group. The survival rate was significantly increased in the CIS group compared with the invasive group. The expression levels of p53 and Ki67 were significantly increased in the invasive group compared with the CIS group. No statistical correlations were observed between the expression of p53 or Ki67 and the survival or recurrence of disease. In the positive group, resected margin status was the principal factor associated with recurrence-free survival according to Cox-regression analysis. In conclusion, the status of the resected margins in the positive group was the most important factor for postoperative survival and recurrence in cholangiocarcinoma, not immunohistochemical staining targeting Ki67 and p53.

Cecal Volvulus Following Elective Laparoscopic Cholecystectomy: A Case Report

Cecal volvulus is characterized by torsion of the cecum around its own mesentery. However, cecal volvulus rarely develops soon after elective laparoscopic cholecystectomy. We report on a case of cecal volvulus that developed in a 54-year-old women 1 day after elective laparoscopic cholecystectomy and was successfully treated via colonoscopic decompression. The symptoms gradually improved in conjunction with recovery from postoperative ileus. Whether the incidence of volvulus has increased with the use of laparoscopic procedures, including laparoscopic cholecystectomy, has yet to be determined. Considering the current trend toward minimally invasive surgery, cecal volvulus should be considered in patients who have postoperative abdominal pain and distention.

A Case of Cavernous Hemangioma of the Gallbladder Treated With Single-Incision Laparoscopic Cholecystectomy

Although hepatic hemangiomas are common, gallbladder (GB) hemangiomas are extremely rare. We present a case of a cavernous GB hemangioma, which was detected during routine ultrasonography screening. The 51-year-old female patient was asymptomatic and had no relevant medical history. The preoperative imaging findings, including those of endoscopic ultrasonography, suggested that malignancy was highly unlikely in this submucosal tumor. Thus, we performed a single-incision laparoscopic cholecystectomy as a diagnostic and curative procedure. The postoperative pathological examination confirmed a GB hemangioma. In this case, the preoperative diagnosis was quite difficult, especially because of this rare presentation of a GB hemangioma. This is probably why the definitive diagnosis is established postoperatively in most previously reported cases of GB hemangioma.