Expression of Selected miRNAs in Undifferentiated Carcinoma with Osteoclast-like Giant Cells (UCOGC) of the Pancreas: Comparison with Poorly Differentiated Pancreatic Ductal Adenocarcinoma.

Undifferentiated carcinoma with osteoclast-like giant cells (UCOGC) of the pancreas represents a rare subtype of pancreatic ductal adenocarcinoma (PDAC). Despite a distinct morphology and specific clinical behavior, UCOGCs exhibit unexpected similarities in regard to DNA mutational profiles with conventional PDAC. Treating pancreatic ductal adenocarcinoma is particularly challenging, with limited prospects for cure. As with many other malignant neoplasms, the exploration of microRNAs (miRNAs, miRs) in regulating the biological characteristics of pancreatic cancer is undergoing extensive investigation to enhance tumor diagnostics and unveil the therapeutic possibilities. Herein, we evaluated the expression of miR-21, -96, -148a, -155, -196a, -210, and -217 in UCOGCs and poorly differentiated (grade 3, G3) PDACs. The expression of miR-21, miR-155, and miR-210 in both UCOGCs and G3 PDACs was significantly upregulated compared to the levels in normal tissue, while the levels of miR-148a and miR-217 were downregulated. We did not find any significant differences between cancerous and normal tissues for the expression of miR-96 and miR-196a in G3 PDACs, whereas miR-196a was slightly, but significantly, downregulated in UCOGCs. On the other hand, we have not observed significant differences in the expression of the majority of miRNAs between UCOGC and G3 PDAC, with the exception of miR-155. UCOGC samples demonstrated lower mean levels of miR-155 in comparison with those in G3 PDACs.

The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR.

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with ß-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and ß-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

Sesquiterpenes Are Agonists of the Pregnane X Receptor but Do Not Induce the Expression of Phase I Drug-Metabolizing Enzymes in the Human Liver.

Sesquiterpenes, the main components of plant essential oils, are bioactive compounds with numerous health-beneficial activities. Sesquiterpenes can interact with concomitantly administered drugs due to the modulation of drug-metabolizing enzymes (DMEs). The aim of this study was to evaluate the modulatory effects of six sesquiterpenes (farnesol, cis-nerolidol, trans-nerolidol, α-humulene, ß-caryophyllene, and caryophyllene oxide) on the expression of four phase I DMEs (cytochrome P450 3A4 and 2C, carbonyl reductase 1, and aldo-keto reductase 1C) at both the mRNA and protein levels. For this purpose, human precision-cut liver slices (PCLS) prepared from 10 patients and transfected HepG2 cells were used. Western blotting, quantitative real-time PCR and reporter gene assays were employed in the analyses. In the reporter gene assays, all sesquiterpenes significantly induced cytochrome P450 3A4 expression via pregnane X receptor interaction. However in PCLS, their effects on the expression of all the tested DMEs at the mRNA and protein levels were mild or none. High inter-individual variabilities in the basal levels as well as in modulatory efficacy of the tested sesquiterpenes were observed, indicating a high probability of marked differences in the effects of these compounds among the general population. Nevertheless, it seems unlikely that the studied sesquiterpenes would remarkably influence the bioavailability and efficacy of concomitantly administered drugs.

Carbonyl Reduction of Flubendazole in the Human Liver: Strict Stereospecificity, Sex Difference, Low Risk of Drug Interactions.

Flubendazole (FLU), an anthelmintic drug of benzimidazole type, is now considered a promising anti-cancer agent due to its tubulin binding ability and low system toxicity. The present study was aimed at determining more information about FLU reduction in human liver, because this information has been insufficient until now. Subcellular fractions from the liver of 12 human patients (6 male and 6 female patients) were used to study the stereospecificity, cellular localization, coenzyme preference, enzyme kinetics, and possible inter-individual or sex differences in FLU reduction. In addition, the risk of FLU interaction with other drugs was evaluated. Our study showed that FLU is predominantly reduced in cytosol, and the reduced nicotinamide adenine dinucleotide phosphate (NADPH) coenzyme is preferred. The strict stereospecificity of FLU carbonyl reduction was proven, and carbonyl reductase 1 was identified as the main enzyme of FLU reduction in the human liver. A higher reduction of FLU and a higher level of carbonyl reductase 1 protein were found in male patients than in female patients, but overall inter-individual variability was relatively low. Hepatic intrinsic clearance of FLU is very low, and FLU had no effect on doxorubicin carbonyl reduction in the liver and in cancer cells. All these results fill the gaps in the knowledge of FLU metabolism in human.

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen.

Monoterpenes R-pulegone (PUL) and R-menthofuran (MF), abundant in the Lamiaceae family, are frequently used in herb and food products. Although their hepatotoxicity was shown in rodent species, information about their effects in human liver has been limited. The aim of our study was to test the effects of PUL, MF and acetaminophen (APAP, as a reference compound) on cell viability and microRNA (miRNA) expression in human precision-cut liver slices. Slices from five patients were used to follow up on the inter-individual variability. PUL was toxic in all liver samples (the half-maximal effective concentration was 4.0 µg/mg of tissue), while MF and surprisingly APAP only in two and three liver samples, respectively. PUL also changed miRNA expression more significantly than MF and APAP. The most pronounced effect was a marked decrease of miR-155-5p expression caused by PUL even in non-toxic concentrations in all five liver samples. Our results showed that PUL is much more toxic than MF and APAP in human liver and that miR-155-5p could be a good marker of PUL early hepatotoxicity. Marked inter-individual variabilities in all our results demonstrate the high probability of significant differences in the hepatotoxicity of tested compounds among people.

The metabolism of flubendazole in human liver and cancer cell lines.

Flubendazole (FLU), a benzimidazole anthelmintic drug widely used in veterinary medicine, has been approved for the treatment of gut-residing nematodes in humans. In addition, FLU is now considered a promising anti-cancer agent. Despite this, information about biotransformation of this compound in human is lacking. Moreover, there is no information regarding whether cancer cells are able to metabolize FLU in order to deactivate it. For these reasons, the present study was designed to identify all metabolites of Phase I and Phase II of FLU in human liver and in various cancer cells using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. Precision-cut human liver slices and 9 cell lines of different origin (breast, colon, oral cavity) were used as in vitro model systems. Our study showed that FLU with a reduced carbonyl group (FLUR) is the only FLU metabolite formed in the human liver. All human cancer cell lines were able to form FLUR. In addition, methylated FLUR was detected in breast cells MCF7 and intestinal SW480 cells. The accumulation of FLU and its reduction to FLUR markedly differed among cells. The extent of FLU reduction was in a good correlation with the detected expression level of carbonyl reductase 1. In most cases, FLU entered in a higher amount and was reduced to a lesser extent in proliferating (metastatic) cells than in differentiated (non-cancerous, non-metastatic) ones. These results support the promising potential of FLU in anti-cancer therapy.

Distribution of metastases in mesorectum is unpredictable: Metastases do not respect tumor localization even in small non-circumferential rectal cancers.

INTRODUCTION: Low anterior resection with total mesorectal excision (TME) is the gold standard for surgical treatment of rectal carcinoma. The radicality of this procedure is negatively counterbalanced by morbidity, lethality, and numerous other complications. Local excision would appear to be an attractive alternative, but its radicality is disputable due to risk of undetected metastasis to the mesorectum. The study aimed to determine the location of mesorectal metastases with respect to circumferentially - located tumors in patients with tumors involving less than one-third of the rectal circumference. MATERIALS AND METHODS: Resected specimens from patients with tumors smaller than one-third of the circumference were divided into: Sector A - tumorous, and Sector B - nontumorous. Group A was created by the pathologist cutting part of the rectal wall with the adjacent mesorectum, as though imitating a full-thickness excision. RESULTS: The study comprised 35 patients with a mean age of 66 years, of which 23 were men (65.7%) and 12 were women (34.2%). Tumors were predominantly (y)pT1-T2; a total of 799 lymph nodes and 5 tumor satellites were examined. Six patients (17.1%) were identified as stage (y)pN+. A total of 3 positive findings (lymph node metastasis or satellites) were detected in 3 patients (8.5%) in tumorous Sector A; and 8 positive findings were detected in 4 patients (11.4%) in non-tumorous Sector B. CONCLUSION: Rectal carcinoma involving one-third of the rectal circumference metastasizes discontinuously, and spreads into parts of the mesorectum beyond the tumor area.

Impact of postoperative complications on clinical and economic consequences in pancreatic surgery.

PURPOSE: Patients who develop complications consume a disproportionately large share of available resources in surgery; therefore the attention of healthcare funders focuses on the economic impact of complications. The main objective of this work was to assess the clinical and economic impact of postoperative complications in pancreatic surgery, and furthermore to assess risk factors for increased costs. METHODS: In all, 161 consecutive patients underwent pancreatic resection. The costs of the treatment were determined and analyzed. RESULTS: The overall morbidity rate was 53.4%, and the in-hospital mortality rate was 3.7%. The median of costs for all patients without complication was 3,963 Euro, whereas the median of costs for patients with at least one complication was significantly increased at 10,670 Euro (P < 0.001). In multivariate analysis American Society of Anesthesiologists ≥ 3 (P = 0.006), multivisceral resection (P < 0.001) and any complication (P < 0.001) were independently associated with increased costs. CONCLUSION: Postoperative complications are associated with an increase in mortality, length of hospital stay, and hospital costs. The treatment costs increase with the severity of the postoperative complications. Those factors that are known to increase the treatment costs in pancreatic resection should be considered when planning patients for surgery.

Intra-abdominal drainage following pancreatic resection: A systematic review.

AIM: To study all the aspects of drain management in pancreatic surgery. METHODS: We conducted a systematic review according to the PRISMA guidelines. We searched the Cochrane Central Registry of Controlled Trials, EMBASE, Web of Science, and PubMed (MEDLINE) for relevant articles on drain management in pancreatic surgery. The reference lists of relevant studies were screened to retrieve any further studies. We included all articles that reported clinical studies on human subjects with elective pancreatic resection and that compared various strategies of intra-abdominal drain management, such as drain vs no drain, selective drain use, early vs late drain extraction, and the use of different types of drains. RESULTS: A total of 19 studies concerned with drain management in pancreatic surgery involving 4194 patients were selected for this systematic review. We included studies analyzing the outcomes of pancreatic resection with and without intra-abdominal drains, studies comparing early vs late drain removal and studies analyzing different types of drains. The majority of the studies reporting equal or superior results for pancreatic resection without drains were retrospective and observational with significant selection bias. One recent randomized trial reported higher postoperative morbidity and mortality with routine omission of intra-abdominal drains. With respect to the timing of drain removal, all of the included studies reported superior results with early drain removal. Regarding the various types of drains, there is insufficient evidence to determine which type of drain is more suitable following pancreatic resection. CONCLUSION: The prophylactic use of drains remains controversial. When drains are used, early removal is recommended. Further trials comparing types of drains are ongoing.

DRAPA trial--closed-suction drains versus closed gravity drains in pancreatic surgery: study protocol for a randomized controlled trial.

BACKGROUND: The morbidity of pancreatic resection remains high, with pancreatic fistula being the most common cause. The important question is whether any postoperative treatment adjustment may prevent the development of clinically significant postoperative pancreatic fistulae. Recent studies have shown that intraabdominal drains and manipulation using them are of great importance. Although authors of a few retrospective reports have described good results of pancreatic resection without the use of intraabdominal drains, a recent prospective randomized trial showed that routine elimination of drains in pancreaticoduodenectomy is associated with poor outcome. An important issue arises as to which type of drain is most suitable for pancreatic resection. Two types of surgical drains exist: open drains and closed drains. Open drains are considered obsolete nowadays because of frequent retrograde infection. Closed drains include two types: passive gravity drains and closed-suction drains. Closed-suction drains are more effective, as they remove fluid from the abdominal cavity under light pressure. However, some surgeons believe that closed-suction drains represent a potential hazard to patients and that negative pressure might increase the risk of pancreatic fistulae. Nobody has yet specifically dealt with the question of which kind of drainage is most appropriate in pancreatic surgery. METHODS/DESIGN: The aim of the DRAins in PAncreatic surgery (DRAPA) trial is to compare the closed-suction drain versus the closed passive gravity drain in pancreatic resection. DRAPA is a dual-centre, prospective, randomized controlled trial. The primary endpoint is the rate of postoperative pancreatic fistula; the secondary endpoint is postoperative morbidity with follow-up of 3 months. DISCUSSION: No study to date has compared different types of drains in pancreatic surgery. This study is designed to answer the question whether any particular type of drain might lower the rate of postoperative pancreatic fistula or other complications. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01988519. Registered 13 November 2013.

Surgical technique in distal pancreatectomy: a systematic review of randomized trials.

Despite recent improvements in surgical technique, the morbidity of distal pancreatectomy remains high, with pancreatic fistula being the most significant postoperative complication. A systematic review of randomized controlled trials (RCTs) dealing with surgical techniques in distal pancreatectomy was carried out to summarize up-to-date knowledge on this topic. The Cochrane Central Registry of Controlled Trials, Embase, Web of Science, and Pubmed were searched for relevant articles published from 1990 to December 2013. Ten RCTs were identified and included in the systematic review, with a total of 1286 patients being randomized (samples ranging from 41 to 450). The reviewers were in agreement for application of the eligibility criteria for study selection. It was not possible to carry out meta-analysis of these studies because of the heterogeneity of surgical techniques and approaches, such as varying methods of pancreas transection, reinforcement of the stump with seromuscular patch or pancreaticoenteric anastomosis, sealing with fibrin sealants and pancreatic stent placement. Management of the pancreatic remnant after distal pancreatectomy is still a matter of debate. The results of this systematic review are possibly biased by methodological problems in some of the included studies. New well designed and carefully conducted RCTs must be performed to establish the optimal strategy for pancreatic remnant management after distal pancreatectomy.

Clinical and economic consequences of pancreatic fistula after elective pancreatic resection.

BACKGROUND: Postoperative pancreatic fistula is the main cause of morbidity after pancreatic resection. This study aimed to quantify the clinical and economic consequences of pancreatic fistula in a medium-volume pancreatic surgery center. METHODS: Hospital records from patients who had undergone elective pancreatic resection in our department were identified. Pancreatic fistula was defined according to the International Study Group on Pancreatic Fistula (ISGPF). The consequences of pancreatic fistula were determined by treatment cost, hospital stay, and out-patient follow-up until the pancreatic fistula was completely healed. All costs of the treatment are calculated in Euros. The cost increase index was calculated for pancreatic fistula of grades A, B, and C as multiples of the total cost for the no fistula group. RESULTS: In 54 months, 102 patients underwent elective pancreatic resections. Forty patients (39.2%) developed pancreatic fistula, and 54 patients (52.9%) had one or more complications. The median length of hospital stay for the no fistula, grades A, B, and C fistula groups was 12.5, 14, 20, and 59 days, respectively. The hospital stay of patients with fistula of grades B and C was significantly longer than that of patients with no fistula (P<0.001). The median total cost of the treatment was 4952, 4679, 8239, and 30 820 Euros in the no fistula, grades A, B, and C fistula groups, respectively. CONCLUSIONS: The grading recommended by the ISGPF is useful for comparing the clinical severity of fistula and for analyzing the clinical and economic consequences of pancreatic fistula. Pancreatic fistula prolongs the hospital stay and increases the cost of treatment in proportion to the severity of the fistula.

Pancreatic Castleman disease treated with laparoscopic distal pancreatectomy.

BACKGROUND: Castleman disease is an uncommon lymphoproliferative disorder most frequently occurring in the mediastinum. Abdominal forms are less frequent, with pancreatic localization of the disease in particular being extremely rare. Only seventeen cases have been described in the world literature. METHOD: This report describes an interesting and unusual case of pancreatic Castleman disease treated with laparoscopic resection. RESULTS: A 48-year-old woman presented with epigastric pain. CT scan showed a well-encapsulated mass on the ventral border of the pancreas. Endosonography with fine needle aspiration biopsy was performed. Biopsy showed lymphoid elements and structures of a normal lymph node. The patient was treated with laparoscopic distal pancreatectomy. The pancreas was transected with a Ligasure device and the pancreatic stump was secured with a manual suture. One year after surgery the patient was complaint-free and showed no signs of recurrence of the disease. CONCLUSIONS: Laparoscopic distal pancreatectomy is a feasible and safe method for the treatment of lesions in the body and tail of the pancreas. Transection of the pancreas with a Ligasure device offers the advantages of low bleeding and low risk of pancreatic fistula.

Long-term survival of a patient after resection of a gastrointestinal stromal tumor arising from the pancreas.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) may arise in any part of the gastrointestinal tract; extra-gastrointestinal locations are extremely rare. Only a few cases of extragastrointestinal stromal tumor arising from the pancreas were reported. None of the reports described a long-term follow-up of the patients. METHOD: This report describes an interesting and unusual case of GIST arising from the pancreas. RESULTS: A 74-year-old female presented with a palpable abdominal mass. CT scan showed a large mass 11 x 8 x 4 cm originating from the tail of the pancreas. Percutaneous biopsy revealed a GIST predominantly with spindle cells, but some parts also contained epitheloid cells. The patient was treated by distal pancreatic resection with splenectomy. Immunohistochemistry of the tumor showed a staining pattern characteristic of GIST. The patient has achieved a long-term survival of five years and six months without any sign of recurrence of the disease. CONCLUSION: This is the first reported case of an extra-gastrointestinal stromal tumor arising from the pancreas treated surgically, with a long-term survival.

Suture-free anastomosis of the colon. Experimental comparison of two cyanoacrylate adhesives.

BACKGROUND: We explored the potential of two cyanoacrylate tissue adhesives for constructing colonic anastomoses. METHOD: The study involved 12 female domestic pigs. The animals were divided into two equal groups. In both groups, the sigmoid colon was transected. An intestinal anastomosis was constructed with a modified circular stapler (all staples were withdrawn) and cyanoacrylate tissue adhesives. Glubran 2 was used in group A and Dermabond was applied in group B. Fourteen days after the first operation, a follow-up surgery was performed in both groups. The glued section of the colon was resected, processed with the standard paraffin technique and stained with haematoxylin-eosin. The finished specimens were examined under light microscopy. Assessments were made for the presence of fibroblasts, neutrophils, giant polynuclear cells, neovascularisation and collagen deposits. Adhesions, anastomotic dehiscence, peri-anastomotic inflammation and intestinal healing were assessed peri-operatively. RESULTS: All anastomoses in group A healed with no signs of pathology. In group B, fibrotic adhesions and stenoses tended to occur in areas surrounding the anastomoses. Histological examinations confirmed increased fibrosis. CONCLUSION: The tissue adhesive Glubran 2 appears to be (under experimental conditions) a promising synthetic adhesive for colonic anastomosis construction; conversely, the tissue adhesive Dermabond was unsuitable for suture-free anastomosis construction.

A novel approach for reinforcing the pancreatic remnant in laparoscopic distal pancreatectomy: an experimental study on a porcine model.

The major postoperative complication after open and laparoscopic distal pancreatectomy is pancreatic fistula. Different operative techniques have been tested to minimize this unpleasant complication. We evaluated a new technique for pancreatic stump reinforcement with synthetic glue after laparoscopic distal pancreatectomy. Ten female domestic pigs were divided into 2 groups; in group A (n=5), the pancreas was transected using an EndoGIA stapler, whereas in group B (n=5), the pancreas was transected using a Ligasure device and the pancreatic stump was reinforced with a hydrogel sealant. The clinical postoperative course was uneventful in all the cases. No differences were observed in pancreatic remnant healing between the groups, and only minor microscopic alterations of the healing process were found in the groups. The technique using Ligasure transection reinforced by the hydrogel sealant seems to be comparable with the standard transection technique using the stapler.

Laparoscopic right hepatectomy--our approach for vascular control: video clip.

The authors describe their technique of laparoscopic right hepatectomy as a short video clip (supplementary video http://links.lww.com/SLE/A4). This approach is based on a detailed anatomic dissection and interruption of hilar structures and hepatic vein before liver transection.

Diverting ileostomy set removable without laparotomy--feasibility study.

We constructed an ileostomy set that creates a diverting ileostomy not requiring surgery for closure. The set has two primary components: a double-balloon drainage catheter and a plastic holder. The plastic holder has a rotary component that is used to tighten a percutaneosly placed lasso-loop stitch and purse the enterotomy after the drainage catheter is removed. We tested the ileostomy set in 10 experimental pigs. The drainage catheter was introduced transcecally into the terminal ileum behind Bauhin's valve, and the balloon was inflated. Three to seven days later the drainage catheter was removed, and the cecotomy was closed by percutaneous tightening of the lasso-loop stitch. One week later each animal underwent relaparotomy, and we examined the abdominal cavity for peritonitis. An ileocecal resection was performed, and specimens were collected and evaluated microscopically to ensure that the catheter had not damaged the intestinal wall. The only change noted in the terminal ileum was submucosa lymphatic tissue hyperplasia. No intestinal leaks occurred, and percutaneous enterotomy closure was successful.