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Background & Aims: Tacrolimus has been associated with recurrence of primary biliary cholangitis (PBC) after liver transplantation (LT), which in turn may reduce survival. This study aimed to assess the association between the type of calcineurin inhibitor used and long-term outcomes following LT in patients with PBC. Methods: Survival analyses were used to assess the association between immunosuppressive drugs and graft or patient survival among adult patients with PBC in the European Liver Transplant Registry. Patients who received a donation after brain death graft between 1990 and 2021 with at least 1 year of event-free follow-up were included. Results: In total, 3,175 patients with PBC were followed for a median duration of 11.4 years (IQR 5.9–17.9) after LT. Tacrolimus (Tac) was registered in 2,056 (64.8%) and cyclosporin in 819 (25.8%) patients. Following adjustment for recipient age, recipient sex, donor age, and year of LT, Tac was not associated with higher risk of graft loss (adjusted hazard ratio [aHR] 1.07, 95% CI 0.92-1.25, p = 0.402) or death (aHR 1.06, 95% CI 0.90-1.24, p = 0.473) over cyclosporin. In this model, maintenance mycophenolate mofetil (MMF) was associated with a lower risk of graft loss (aHR 0.72, 95% CI 0.60-0.87, p

Abstract Background Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. Methods We describe an integrated whole‐exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. Results We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke‐associated Asian iCCAs. Conclusions By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

This paper presents the design and prototype of a constant volume (isochoric) vessel that can be used for the preservation of large organs in a supercooled state. This prototype is a preliminary version of a more advanced design. The device consists of a cooling bath operated by a mechanical vapor compression refrigeration unit and an isochoric chamber made of stainless steel. The preservation of organs using supercooling technology in an isochoric chamber requires a continuous temperature and pressure monitoring. While the device was initially designed for pig liver experiments, its innovative design and preservation capabilities suggest potential applications for preserving other organs as well. The isochoric reactor may be used to accommodate a variety of organ types, opening the door for further research into its multi-organ preservation capabilities. All the design details are presented in this study with the purpose of encouraging researchers in the field to build their own devices, and by this to improve the design. We chose to design the device for isochoric supercooling as the method of preservation to avoid the ice formation.

Circulating cell-free DNA (ccfDNA) quantity correlates with the clinical characteristics and prognosis of various cancer types. We investigated whether ccfDNA levels and the neutrophil-to-lymphocyte ratio (NLR) have prognostic value in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral blood was collected from 82 patients with PDAC prior to any diagnostic procedure or the administration of chemotherapy. Plasma DNA was isolated, and ccfDNA concentration and NLR were determined. We found that ccfDNA levels were correlated with age and tumor burden. Moreover, higher values of NLR (≥3.31) were linked with worse overall survival (OS) (4 vs. 10 months; log rank p = 0.011), and an elevated ccfDNA concentration (≥25.79 ng/mL) was strongly associated with shorter OS (4 vs. 8 months; log rank p = 0.009). According to the results of the multivariable Cox regression analysis, the baseline concentration of ccfDNA was an independent prognostic factor for OS (HR 0.45, 95% CI 0.21–0.97, p = 0.041). Furthermore, the combination of ccfDNA levels with NLR greatly enhanced the prognostic accuracy of PDAC patients. Our study demonstrates that ccfDNA concentration and NLR are independent predictors of survival in PDAC. Subsequent studies should validate this combination as a prognostic indicator in PDAC patients and assess its utility for guiding therapeutic decisions.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal cancer globally. Over 90% of HCC cases arise in the context of liver cirrhosis, and the severity of the underlying liver disease or advanced tumor stage at diagnosis significantly limits treatment options. Early diagnosis is crucial, and all guidelines stress the importance of screening protocols for HCC early detection as a public health objective. As serum biomarkers are not optimal for early diagnosis, liquid biopsy has emerged as a promising tool for diagnosis, prognostication, and patients’ stratification for personalized therapy in various solid tumors, including HCC. While circulating tumor cells (CTCs) are better suited for personalized therapy and prognosis, cell-free DNA (cfDNA) and extracellular vesicle-based technologies show potential for early diagnosis, HCC screening, and surveillance protocols. Evaluating the added value of liquid biopsy genetic and epigenetic biomarkers for HCC screening is a key goal in translational research. Somatic mutations commonly found in HCC can be investigated in cfDNA and plasma exosomes as genetic biomarkers. Unique methylation patterns in cfDNA or cfDNA fragmentome features have been suggested as innovative tools for early HCC detection. Likewise, extracellular vesicle cargo biomarkers such as miRNAs and long non-coding RNAs may serve as potential biomarkers for early HCC detection. This review will explore recent findings on the utility of liquid biopsy for early HCC diagnosis. Combining liquid biopsy methods with traditional serological biomarkers could improve the overall diagnostic accuracy for early HCC detection.

The study of anastomoses in liver transplantation represents a complex and special subject considering the fact that a variability of methods have been described since the first liver transplant in 1963, made by Thomas Starzl. In liver transplantation, graft implantation includes 4 anastomoses: cavo-cava, portal, arterial and biliary. The Gordon patch represents the plasty performed between the aortic patch of the celiac trunk and the aortic patch of the superior mesenteric artery. It only applies to anatomical variants of the hepatic artery, namely when the common hepatic artery arises from the celiac trunk and the right hepatic artery arises from superior mesenteric artery. The present article describes the aspect of the surgical techniques through the experience at the Digestive Diseases and Liver Transplantation Center, Fundeni Clinical Institute, Bucharest, Romania.

Abstract Background Insulin producing cells generated by liver cell transdifferentiation, could serve as an attractive source for regenerative medicine. The present study assesses the relationship between DNA methylation pTFs induced liver to pancreas transdifferentiation. Results The transdifferentiation process is associated with DNA demethylation, mainly at gene regulatory sites, and with increased expression of these genes. Active inhibition of DNA methylation promotes the pancreatic transcription factor-induced transdifferentiation process, supporting a causal role for DNA demethylation in this process. Conclusions Transdifferentiation is associated with global DNA hypomethylation, and with increased expression of specific demethylated genes. A combination of epigenetic modulators may be used to increase chromatin accessibility of the pancreatic transcription factors, thus promoting the efficiency of the developmental process.

In the 1990s a sampling team from the Surgery Department of Saint Luc Clinic in Louvain Brussels, published a study showing that single aortic infusion is as safe as classic, standard infusion (aortic and portal) but the attempt was not fully accepted. Also, some recent studies recommend the safe use of single aortic infusions in the case of multiple organ sampling from the brain-dead donor. In order to test the effectiveness of this method we made a retrospective study between 2017 and 2019 at the Center for General Surgery and Liver Transplantation of the Fundeni Clinical Institute, Bucharest, Romania. First of all, we performed a comparative analysis between the single aortic sampling group and the group in which we used the standard sampling. We also analyzed the impact of singular aortic sampling on cases with anatomical arterial variants.

This study was motivated by the increasing interest in finding ways to preserve organs in a supercooled state for transplantation. Previous research with small volumes suggests that the isochoric (constant volume) thermodynamic state enhances the stability of supercooled solutions.The primary objective of this study was to investigate the feasibility of storing a large organ, such as the pig liver, in a metastable isochoric supercooled state for clinically relevant durations. To achieve this, we designed a new isochoric technology that employs a system consisting of two domains separated by an interior boundary that can transfer heat and pressure, but not mass. The liver is preserved in one of these domains in a solution with an intracellular composition, which is in osmotic equilibrium with the liver. Pressure is used to monitor the thermodynamic state of the isochoric chamber. In this feasibility study, two pig livers were preserved in the device in an isochoric supercooled state at -2°C. The experiments were terminated voluntarily, one after 24 h and the other after 48 h of supercooling preservation. Pressure measurements indicated that the livers did not freeze during the isochoric supercooling preservation. This is the first proof that organs as large as the pig liver can remain supercooled for extended periods of time in an isotonic solution in an isochoric system, despite an increased probability of ice nucleation with larger volumes. To serve as controls and to test the ability of pressure monitoring to detect freezing in the isochoric chamber, an experiment was designed in which two pig livers were frozen at -2°C for 24 h and the pressure monitored. Histological examination with H&E stains revealed that the supercooled liver maintained a normal appearance, even after 48 h of supercooling, while tissues in livers frozen to -2°C were severely disrupted by freezing after 24 h.

Post liver transplantation (LT) fibrosis has a negative impact on graft function. Cytokine production in the host immune response after LT may contribute to the variable CYP3A-dependent immunosuppressive drug disposition, with subsequent impact on liver fibrogenesis, together with host-related factors. We aimed to investigate whether the cytochrome P4503A5*3 (CYP3A5*3) or TBX21 genotypes impact post-LT liver fibrogenesis. Furthermore, the impact of immunosuppressants on cellular apoptosis has been evaluated using human hepatocytes harvested from cirrhotic explanted livers. We have enrolled 98 LT recipients that were followed for occurrence of liver fibrosis for at least 12 months. There was a statistically significant higher trough level of TAC in patients with homozygous CC-TBX21 genotype (7.83 ± 2.84 ng/ml) vs. 5.66 ± 2.16 ng/ml in patients without this genotype (p = 0.009). The following variables were identified as risk factors for fibrosis ≥2: donor age (p = 0.02), neutrophil to lymphocyte ratio (p = 0.04) and TBX21 genotype CC (p = 0.009). In the cell culture model cytometry analysis has indicated the lowest apoptotic cells percentage in human cirrhotic hepatocytes cultures treated with mycophenolate mofetil (MMF) (5%) and TAC + MMF (2%) whereas the highest apoptosis percentage was registered for the TAC alone (11%). The gene expression results are concordant to cytometry study results, indicating the lowest apoptotic effect for MMF and MMF + TAC immunosuppressive regimens. The allele 1993C of the SNP rs4794067 may predispose to the development of late significant fibrosis of the liver graft. MMF-based regimens have a favourable anti-apoptotic profile in vitro, supporting its use in case of LT recipients at high risk for liver graft fibrosis.

Therapeutic decision-making for advanced GIST liver metastases is challenging due to limited clinical evidence. This case study aims to demonstrate the survival benefit of resection in non-responsive cases. A 40-year-old male presented with abdominal pain, weight loss, altered general status, massive hepatomegaly, and intermittent melaena. He was diagnosed with stage IV GIST with the primary tumor in the ileal loop and multiple gigantic synchronous bilobar liver metastases. Despite 31 months of tyrosine-kinase inhibitor therapy post-primary tumor resection, the disease remained unresponsive. The patient was admitted to our tertiary center with significant hepatomegaly. A two-stage debulking liver resection was performed after a multidisciplinary team decision. The first operation debulked the left hemiliver through a non-anatomical ultrasound-guided resection of segments 2, 3, and 4. The second operation (7 weeks later) debulked the right hemiliver through a right posterior sectionectomy involving segments 5 and 8. Despite receiving a second line of tyrosine-kinase inhibitor therapy after surgery, the disease progressed both within and outside the liver. However, the patient survived for 55 months, with a postoperative survival benefit of 25 months. In conclusion, this case emphasizes the significant survival benefit achieved through a complex two-stage debulking liver resection for giant liver metastases, even in cases where systemic therapy fails.

This technical paper introduces a novel organ preservation system based on isochoric (constant volume) supercooling. The system is designed to enhance the stability of the metastable supercooling state, offering potential long-term preservation of large biological organs at subfreezing temperatures without the need for cryoprotectant additives. Detailed technical designs and usage protocols are provided for researchers interested in exploring this field. The paper also presents a control system based on the thermodynamics of isochoric freezing, utilizing pressure monitoring for process control. Sham experiments were performed using whole pig liver sourced from a local food supplier to evaluate the system’s ability to sustain supercooling without ice nucleation for extended periods. The results demonstrated sustained supercooling without ice nucleation in pig liver tissue for 24 and 48 h. These findings suggest the potential of this technology for large-volume, cryoprotectant-free organ preservation with real-time control over the preservation process. The simplicity of the isochoric supercooling device and the design details provided in the paper are expected to serve as encouragement for other researchers in the field to pursue further research on isochoric supercooling. However, final evidence that these preserved organs can be successfully transplanted is still lacking.

Background: The purposes of the study were to determine the variations in hepatic arterial supply, to delineate the optimal methods of arterial anastomoses and reconstructions in liver transplantation and to analyse the incidence of arterial complications. Methods: The surgical anatomy of the extrahepatic arterial vascularization was investigated retrospectively in 209 donors and patients who underwent liver transplantation at Fundeni Clinical Institute (Bucharest, Romania) from January 1, 2015 to December 31, 2017. The vascular anatomy of the hepatic grafts was classified according to Michels’ description and other rare variations. Results: Anatomical variants of the classical pattern were detected in 26.3% of the livers (n = 55). The most common variant was a replaced right hepatic artery arising from the superior mesenteric artery (n = 17; 8.13%), followed by a common hepatic artery from superior mesenteric artery (n = 6; 2.87%). Arterial reconstructions were reported in 97 cases (45.5%). In recipients, used sites were intermediate: common hepatic artery (CHA) in 73.8% (n = 158), distal: proper hepatic artery (PHA) or common hepatic artery/gastro-duodenal artery bifurcation (CHA/GDA bifurcation) in 16.4% (n = 35) and proximal: coeliac trunk-splenic artery-aorta (CT–SA–A) in 9.3% (n = 20) of patients. Most common reconstructions were short graft artery (CT) on the recipient CHA (n = 33, 34.02%) and long graft artery: complex reconstruction between CT and superior mesenteric artery (SMA) - accessory right hepatic artery (RHA) from SMA on CHA (n = 12, 12.37%) and right hepatic graft artery on PHA or CHA/GDA bifurcation (n = 16, 16.49%). Conclusion: The information about the different hepatic arterial patterns, as well as establishing specific methods for arterial anastomoses and reconstructions is important in the determination of better outcomes.

PURPOSE Cholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries. MATERIALS AND METHODS In this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non–Ov-associated CCA tumors. RESULTS Compared with non–fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers. CONCLUSION FGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

Gastrointestinal stromal tumors (GISTs) are rare neoplasms, colorectal location being met in less than 5% of cases. Knowledge about this site related particularities are limited. The aim of this study is to present our experience with colorectal GISTs between 2005 and 2018 from the clinical, morphological, and immunohistochemical perspectives, with emphasis on prognostic factors. From a total of 203 gastrointestinal stromal tumors registered, 12 were colorectal (6%). The number of colonic tumors surpassed that of the rectum (9 : 3) and on the right side were registered more cases than on the left side (6/3). 9 were primary tumors and 3 were recurrences. Men and women were represented equally and the age range was between 22 and 76. Tumor dimensions varied between 0.5 and 14 cm. Microscopically, spindle cell type was dominant. Mitotic rate was variable between 1 and 115/50HPFs. Accordingly, for primary tumors progression risks were assigned (low risk: 2 cases, intermediate risk: 3 cases and high risk: 4 cases). All GISTs were CD117 and DOG1 positive. Four of the patients died of the disease.

Recurrent or de novo non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) following liver transplantation (LT) is a frequent event being increasingly recognized over the last decade, but the influence of recurrent NASH on graft and patient outcomes is not yet established. Taking into consideration the long term survival of liver transplanted patients and long term complications with associated morbidity and mortality, it is important to define and minimize risk factors for recurrent NAFLD/NASH. Metabolic syndrome, obesity, dyslipidemia, diabetes mellitus are life style risk factors that can be potentially modified by various interventions and thus, decrease the risk of recurrent NAFLD/NASH. On the other hand, genetic factors like recipient and/or donor PNPLA3, TM6SF2, GCKR, MBOAT7 or ADIPOQ gene polymorphisms proved to be risk factors for recurrent NASH. Personalized interventions to influence the different metabolic disorders occurring after LT in order to minimize the risks, as well as genetic screening of donors and recipients should be performed pre-LT in order to achieve diagnosis and treatment as early as possible.

Background and Objectives: In the early period after liver transplantation, patients are exposed to a high rate of complications and several scores are currently available to predict adverse postoperative outcomes. However, an ideal, universally accepted and validated score to predict adverse events in liver transplant recipients with hepatitis C is lacking. Therefore, we aimed to establish and validate a machine learning (ML) model to predict short-term outcomes of hepatitis C patients who underwent liver transplantation. Materials and Methods: We conducted a retrospective observational two-center cohort study involving hepatitis C patients who underwent liver transplantation. Based on clinical and laboratory parameters, the dataset was used to train a deep-learning model for predicting short-term postoperative complications (within one month following liver transplantation). Adverse events prediction in the postoperative setting was the primary study outcome. Results: A total of 90 liver transplant recipients with hepatitis C were enrolled in the present study, 80 patients in the training cohort and ten in the validation cohort, respectively. The age range of the participants was 12–68 years, 51 (56,7%) were male, and 39 (43.3%) were female. Throughout the 85 training epochs, the model achieved a very good performance, with the accuracy ranging between 99.76% and 100%. After testing the model on the validation set, the deep-learning classifier confirmed the performance in predicting postoperative complications, achieving an accuracy of 100% on unseen data. Conclusions: We successfully developed a ML model to predict postoperative complications following liver transplantation in hepatitis C patients. The model demonstrated an excellent performance for accurate adverse event prediction. Consequently, the present study constitutes the foundation for careful and non-invasive identification of high-risk patients who might benefit from a more intensive postoperative monitoring strategy.

Background and Objectives: Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the leading cause of cancer-related mortality. It arises and progresses against fibrotic or cirrhotic backgrounds mainly due to infection with hepatitis viruses B (HBV) or C (HCV) or non-viral causes that lead to chronic inflammation and genomic changes. A better understanding of molecular and immune mechanisms in HCC subtypes is needed. Materials and Methods: To identify transcriptional changes in primary HCC tumors with or without hepatitis viral etiology, we analyzed the transcriptomes of 24 patients by next-generation sequencing. Results: We identified common and unique differentially expressed genes for each etiological tumor group and analyzed the expression of SLC, ATP binding cassette, cytochrome 450, cancer testis, and heat shock protein genes. Metascape functional enrichment analysis showed mainly upregulated cell-cycle pathways in HBV and HCV and upregulated cell response to stress in non-viral infection. GeneWalk analysis identified regulator, hub, and moonlighting genes and highlighted CCNB1, ACTN2, BRCA1, IGF1, CDK1, AURKA, AURKB, and TOP2A in the HCV group and HSF1, HSPA1A, HSP90AA1, HSPB1, HSPA5, PTK2, and AURKB in the group without viral infection as hub genes. Immune infiltrate analysis showed that T cell, cytotoxic, and natural killer cell markers were significantly more highly expressed in HCV than in non-viral tumors. Genes associated with monocyte activation had the highest expression levels in HBV, while high expression of genes involved in primary adaptive immune response and complement receptor activity characterized tumors without viral infection. Conclusions: Our comprehensive study underlines the high degree of complexity of immune profiles in the analyzed groups, which adds to the heterogeneous HCC genomic landscape. The biomarkers identified in each HCC group might serve as therapeutic targets.

Diabetes mellitus (DM) is a complex metabolic disease with many specifically related complications. Early diagnosis of this disease could prevent the progression to overt disease and its related complications. There are several limitations to using existing biomarkers, and between 24% and 62% of people with diabetes remain undiagnosed and untreated, suggesting a large gap in current diagnostic practices. Early detection of the percentage of insulin-producing cells preceding loss of function would allow for effective therapeutic interventions that could delay or slow down the onset of diabetes. MicroRNAs (miRNAs) could be used for early diagnosis, as well as for following the progression and the severity of the disease, due to the fact of their pancreatic specific expression and stability in various body fluids. Thus, many studies have focused on the identification and validation of such groups or “signatures of miRNAs” that may prove useful in diagnosing or treating patients. Here, we summarize the findings on miRNAs as biomarkers in diabetes and those associated with direct cellular reprogramming strategies, as well as the relevance of miRNAs that act as a bidirectional switch for cell therapy of damaged pancreatic tissue and the studies that have measured and tracked miRNAs as biomarkers in insulin resistance are addressed.

Liver resection for malignant tumors should respect oncological margins while ensuring safety and improving the quality of life, therefore tumor staging, underlying liver disease and performance status should all be attentively assessed in the decision process. The concept of parenchyma-sparing liver surgery is nowadays used as an alternative to major hepatectomies to address deeply located lesions with intricate topography by means of complex multiplanar parenchyma-sparing liver resections, preferably under the guidance of intraoperative ultrasound. Regenerative liver surgery evolved as a liver growth induction method to increase resectability by stimulating the hypertrophy of the parenchyma intended to remain after resection (referred to as future liver remnant), achievable by portal vein embolization and liver venous deprivation as interventional approaches, and portal vein ligation and associating liver partition and portal vein ligation for staged hepatectomy as surgical techniques. Interestingly, although both strategies have the same conceptual origin, they eventually became caught in the never-ending parenchyma-sparing liver surgery vs. regenerative liver surgery debate. However, these strategies are both valid and must both be mastered and used to increase resectability. In our opinion, we consider parenchyma-sparing liver surgery along with techniques of complex liver resection and intraoperative ultrasound guidance the preferred strategy to treat liver tumors. In addition, liver volume-manipulating regenerative surgery should be employed when resectability needs to be extended beyond the possibilities of parenchyma-sparing liver surgery.

Abstract Background The expression of microRNAs (miRNAs) is a promising prognostic and diagnostic tool in hepatocellular carcinoma (HCC). Here we performed small RNA sequencing (sRNA-seq) of tissue, serum and serum exosomes to investigate changes in miRNA expression between the different sample types and correlated the expression with clinical parameters. We also performed gene expression arrays on tumor and normal tissue. Results Paired tissue, serum and serum exosomes sequencing revealed consistent positive correlation of miR-21 between serum exosomes and tumor tissue, indicating that miR-21 could be exported from tissue to circulation via exosomes. We found that let-7 miRNAs are generally upregulated in serum exosomes compared to whole serum, indicating that these miRNAs could be preferentially loaded into exosomes. Comparing serum from HCC patients with serum from healthy individuals revealed a global increase of miRNAs in serum from HCC patients, including an almost 4-fold increase of several miRNAs, including the liver-specific miR-122. When correlating miRNA expression with clinical parameters we detected significant association between hepatitis B virus (HBV) infection and miR-122 in serum as well as several serum and tissue-miRNAs that correlated with surgery type. We found that miR-141 and miR-146 correlated with cirrhosis in tumor tissue and normal tissue, respectively. Finally, high expression of miR-21 in tumors were associated with poor survival. Focusing on gene expression we found several significant messenger RNAs (mRNAs) between tumor and normal tissue and a Gene Ontology (GO) analysis revealed that these changes were mainly related to cell cycle and metabolism. Further, we detected mRNAs that correlated with cirrhosis and HBV infection in tissue. Finally, GO analysis of predicted targets for miRNAs down-regulated in tumor found that these were enriched for functions related to collagen synthesis. Conclusions Our combined data point to altered miRNA and mRNA expression contributing to both generally impaired lipid metabolism and increased cell proliferation and a miRNA-driven increase in collagen synthesis in HCC. Our results further indicate a correlation in miRNA expression between exosomes, serum, and tissue samples suggesting export from tumors via exosomes. This correlation could provide a basis for a more tumor-specific miRNA profile in serum.

Abstract Background Insulin-dependent diabetes is a multifactorial disorder that could be theoretically cured by functional pancreatic islets and insulin-producing cell (IPC) implantation. Regenerative medicine approaches include the potential for growing tissues and organs in the laboratory and transplanting them when the body cannot heal itself. However, several obstacles remain to be overcome in order to bring regenerative medicine approach for diabetes closer to its clinical implementation; the cells generated in vitro are typically of heterogenic and immature nature and the site of implantation should be readily vascularized for the implanted cells to survive in vivo. The present study addresses these two limitations by analyzing the effect of co-implanting IPCs with vasculature promoting cells in an accessible site such as subcutaneous. Secondly, it analyzes the effects of reconstituting the in vivo environment in vitro on the maturation and function of insulin-producing cells. Methods IPCs that are generated by the transdifferentiation of human liver cells are exposed to the paracrine effects of endothelial colony-forming cells (ECFCs) and human bone marrow mesenchymal stem cells (MSCs), which are the “building blocks” of the blood vessels. The role of the vasculature on IPC function is analyzed upon subcutaneous implantation in vivo in immune-deficient rodents. The paracrine effects of vasculature on IPC maturation are analyzed in culture. Results Co-implantation of MSCs and ECFCs with IPCs led to doubling the survival rates and a threefold increase in insulin production, in vivo. ECFC and MSC co-culture as well as conditioned media of co-cultures resulted in a significant increased expression of pancreatic-specific genes and an increase in glucose-regulated insulin secretion, compared with IPCs alone. Mechanistically, we demonstrate that ECFC and MSC co-culture increases the expression of CTGF and ACTIVINβα, which play a key role in pancreatic differentiation. Conclusions Vasculature is an important player in generating regenerative medicine approaches for diabetes. Vasculature displays a paracrine effect on the maturation of insulin-producing cells and their survival upon implantation. The reconstitution of the in vivo niche is expected to promote the liver-to-pancreas transdifferentiation and bringing this cell therapy approach closer to its clinical implementation.

Background: The aim of this study was to investigate the impact of postoperative complications on the long-term outcomes of patients who had undergone simultaneous resection (SR) of colorectal cancer and synchronous liver metastases (SCLMs). Methods: We conducted a single-institution survival cohort study in patients with SR, collecting clinical, pathological, and postoperative complication data. The impact of these variables on overall survival (OS) and disease-free survival (DFS) was compared by log rank test. Multivariate Cox regression analysis identified independent prognostic factors. Results: Out of 243 patients, 122 (50.2%) developed postoperative complications: 54 (22.2%) major complications (Clavien–Dindo grade III–V), 86 (35.3%) septic complications, 59 (24.2%) hepatic complications. Median comprehensive complication index (CCI) was 8.70. Twelve (4.9%) patients died postoperatively. The 3- and 5-year OS and DFS rates were 60.7%, 39.5% and 28%, 21.5%, respectively. Neither overall postoperative complications nor major and septic complications or CCI had a significant impact on OS or DFS. Multivariate analysis identified the N2 stage as an independent prognostic of poor OS, while N2 stage and four or more SCLMs were independent predictors for poor DFS. Conclusion: N2 stage and four or more SCLMs impacted OS and/or DFS, while CCI, presence, type, or grade of postoperative complications had no significant impact on long-term outcomes.

Pancreatic adenocarcinoma (PDAC) has low survival rates worldwide due to its tendency to be detected late and its characteristic desmoplastic reaction, which slows the use of targeted therapies. As such, the discovery of new connections between genes and the clinicopathological parameters contribute to the search for new biomarkers or targets for therapy. Transient receptor potential (TRP) channels are promising tools for cancer therapy or markers for PDAC. Therefore, in this study, we selected several genes encoding TRP proteins previously reported in cellular models, namely, Transient Receptor Potential Cation Channel Subfamily V Member 6 (TRPV6), Transient receptor potential ankyrin 1 (TRPA1), and Transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8), as well as the TRPM8 Channel Associated Factor 1 (TCAF1) and TRPM8 Channel Associated Factor 2 (TCAF2) proteins, as regulatory factors. We analyzed the expression levels of tumors from patients enrolled in public datasets and confirmed the results with a validation cohort of PDAC patients enrolled in the Clinical Institute Fundeni, Romania. We found significantly higher expression levels of TRPA1, TRPM8, and TCAF1/F2 in tumoral tissues compared to normal tissues, but lower expression levels of TRPV6, suggesting that TRP channels have either tumor-suppressive or oncogenic roles. The expression levels were correlated with the tumoral stages and are related to the genes involved in calcium homeostasis (Calbindin 1 or S100A4) or to proteins participating in metastasis (PTPN1). We conclude that the selected TRP proteins provide new insights in the search for targets and biomarkers needed for therapeutic strategies for PDAC treatment.

Background and objectives. In colorectal cancers, the embryologic origin of the primary tumor determines important molecular dissimilarities between right-sided (RS) and left-sided (LS) carcinomas. Although important prognostic differences have been revealed between RS- and LS-patients with resected colorectal liver metastases (CLMs), it is still unclear if this observation depends on the RAS mutational status. To refine the impact of primary tumor location (PTL) on the long-term outcomes of patients with resected CLMs, the rates of overall survival (OS), relapse-free survival (RFS) and survival after recurrence (SAR) were compared between RS- vs. LS-patients, according to their RAS status. Material and Methods. All patients with known RAS status, operated until December 2019, were selected from a prospectively maintained database, including all patients who underwent hepatectomy for histologically-proven CLMs. A log-rank test was used to compare survival rates between the RS- vs. LS-group, in RAS-mut and RAS-wt patients, respectively. A multivariate analysis was performed to assess if PTL was independently associated with OS, RFS or SAR. Results. In 53 patients with RAS-mut CLMs, the OS, RFS and SAR rates were not significantly different (p = 0.753, 0.945 and 0.973, respectively) between the RS and LS group. In 89 patients with RAS-wt CLMs, the OS and SAR rates were significantly higher (p = 0.007 and 0.001, respectively) in the LS group vs. RS group, while RFS rates were similar (p = 0.438). The multivariate analysis performed in RAS-wt patients revealed that RS primary (p = 0.009), extrahepatic metastases (p = 0.001), N-positive (p = 0.014), age higher than 65 (p = 0.002) and preoperative chemotherapy (p = 0.004) were independently associated with worse OS, while RS location (p < 0.001) and N-positive (p = 0.007) were independent prognostic factors for poor SAR. Conclusions. After resection of CLMs, PTL had no impact on long-term outcomes in RAS-mut patients, while in RAS-wt patients, the RS primary was independently associated with worse OS and SAR.

Hepatocellular carcinoma (HCC) is a significant health problem. Globally is the second most common cause of cancer-associated death and the fifth most frequent neoplasm. The main risk factors for the onset of HCC are well recognized, including the presence of cirrhosis, chronic hepatitis C and hepatitis B infections and heavy alcohol consumption. In an early stage disease, there are potentially curative therapies, such as surgical resection, transplantation and loco-regional procedures. However, at the time of diagnosis, a large number of patients present an advanced stage disease, according to the Barcelona Clinic Liver Cancer (BCLC) classification. Background. Sorafenib chemotherapy is the first-line therapy for patients with hepatocellular carcinoma (HCC) in an advanced stage. The aim of this study was to evaluate prognostic factors of survival in HCC patients treated with sorafenib, in real-life clinical practice. Methods. We perform an retrospective, non-randomized study and we analyzed 162 patients with HCC who were treated with sorafenib 800 mg/day in Oncology Department of Fundeni Clinical Institute between 2009 and 2016. Results. Mortality in our patients group was more than 80%, with survival rate about 22 months and a median survival rate 13 months. The patients with liver cirrhosis has a severe evolution compared with those who has hepatitis. We found a good survival rate for HCV infected patients compared with HVB or VHB +VHD etiology. BCLC and Child-Pugh classification have an important role in overall survival.

Background. The aim of this study was to identify pre-transplant risk factors that lead to tumor recurrence and to evaluate mortality in liver transplanted patients from the single Liver Transplant Center in Romania. Methods. 81 patients who underwent liver transplantation between 2011 and 2015 at the Fundeni Clinical Institute were assessed based on age, sex, type of operation, histopathological staging, pre-transplant bridging therapies, etiology of cirrhosis, San Francisco and Up to seven scores, AFP and CA19-9. Results. Between 2011 and 2015, 81 out of 454 liver transplants were performed for hepatocarcinoma (17.84%). The most frequent etiology was the HVB and HVD co-infection 39.7%. The 1 and 3 years-survival rates were 81.15%, respectively 72.5%. The 1 year and 3 years tumor recurrence rate were 14.49%, respectively 15. Conclusions. We identified as risk factors for 1 year-tumor recurrence the following: pre-transplant Milan, San Francisco and Up to Seven scores, pre-transplant AFP > 200 ng/ml and Edmonson/Steiner staging. Age over 65 at transplant was not correlated to cancer recurrence, 1 year and 3 years mortality rates or postoperative complications.

Gastric cancer has remained in the top five cancers for over ten years, both in terms of incidence and mortality due to the shortage of biomarkers for disease follow-up and effective therapies. Aiming to fill this gap, we performed a bioinformatics assessment on our data and two additional GEO microarray profiles, followed by a deep analysis of the 40 differentially expressed genes identified. PPI network analysis and MCODE plug-in pointed out nine upregulated hub genes coding for proteins from the collagen family (COL12A1, COL5A2, and COL10A1) or involved in the assembly (BGN) or degradation of collagens (CTHRC1), and also associated with cell adhesion (THBS2 and SPP1) and extracellular matrix degradation (FAP, SULF1). Those genes were highly upregulated at the mRNA and protein level, the increase being correlated with pathological T stages. The high expression of BGN (p = 8 × 10−12), THBS2 (p = 1.2 × 10−6), CTHRC1 (p = 1.1 × 10−4), SULF1 (p = 3.8 × 10−4), COL5A1 (p = 1.3 × 10−4), COL10A1 (p = 5.7 × 10−4), COL12A1 (p = 2 × 10−3) correlated with poor overall survival and an immune infiltrate based especially on immunosuppressive M2 macrophages (p-value range 4.82 × 10−7–1.63 × 10−13). Our results emphasize that these genes could be candidate biomarkers for GC progression and prognosis and new therapeutic targets.

Background and purpose Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models.Basic procedures We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib—dosed orally at 5, 10 or 20 mg/kg daily—combined with anti-PD1 antibodies (10 mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer.Main findings Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (Rag1-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10—a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes—in murine HCC and in blood of patients with HCC. Using Cxcr3-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy.Principal conclusions Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8 T-cell infiltration through elevated CXCL10 expression in HCC cells.

Microsurgery represents an important branch in Plastic and Reconstructive Surgery. It involves fine skills which doctors need to repair nerves, vessels and thus being able to perform replantation and transplantation of different types of tissue. After traumatic injuries, a plastic surgeon is capable of either coverage of the exposed noble tissue or can perform the replantation of the amputated limb using microsurgery. This field can be very challenging at the beginning, but very rewarding in the end. The utility microsurgery is quite vast; however, the skills to perform such surgery require a lot of training beforehand. Before doing any replantation or other tissue transplant in humans, it would be recommended that a surgeon should have a basic microsurgical course completed and afterwards several hours of practice in front of the microscope. Last but not least, one should also test the skills acquired in vivo, in order to improve and perform the correct manoeuvres from the beginning. In order to do this, a plastic surgery trainee must therefore have a dedicated laboratory where he/she can practice this art. This place should be quiet, equipped with microscopes and microsurgery instruments and authorized to perform experiments on live animals.

IntroductionLiquid biopsy using circulating microvesicles and exosomes is emerging as a new diagnostic tool that could improve hepatocellular carcinoma (HCC) early diagnosis and screening protocols. Our study aimed to investigate the utility of plasma exosomal miR-21-5p and miR-92-3p for HCC diagnosis during screening protocols.MethodsThe study group included 106 subjects: 48 patients diagnosed with HCC during screening, who underwent a potentially curative treatment (surgical resection or liver transplantation), 38 patients with liver cirrhosis (LC) on the waiting list for liver transplantation, and 20 healthy volunteers. The exosomes were isolated by precipitation with a reagent based on polyethylene glycol and were characterized based on morphological aspects (i.e., diameter); molecular weight; CD63, CD9, and CD81 protein markers; and exosomal miR-21-5p and miR-92a-3p expression levels.ResultsWe first demonstrate that the exosome population isolated with the commercially available Total Exosome Isolation kit respects the same size ranging, morphological, and protein expression aspects compared to the traditional ultracentrifugation technique. The analysis of the expression profile indicates that miR-21-5p was upregulated (p = 0.017), and miR-92a-3p was downregulated (p = 0.0005) in plasma-derived exosomes from HCC subjects, independently from the patient’s characteristics. AUROC for HCC diagnosis based on AFP (alpha-fetoprotein) was 0.72. By integrating AFP and the relative expression of exosomal miR-21-5p and miR-92a-3p in a logistic regression equation for HCC diagnosis, the combined AUROC of the new exosomal miR HCC score was 0.85—significantly better than serum AFP alone (p = 0.0007).ConclusionTogether with serum AFP, plasma exosomal miR-21-5p and miR-92a-3p could be used as potential biomarkers for HCC diagnosis in patients with LC subjected to screening and surveillance.

This study is aimed at investigating tumoral and inflammatory cells and the significance of the prognostic factors of pancreatic ductal adenocarcinoma (PDAC); it is also aimed at determining the role of immunohistochemistry in the diagnosis and prognosis of this neoplasm. Materials and Methods. 230 cases of pancreatic ductal adenocarcinoma were included in the study group; these cases were selected from the archives of the Department of Pathology of the Fundeni Clinical Institute over a ten-year period. Immunohistochemistry was performed using the following antibodies: MUC 1, CD 34, Factor VIII, CD 68, MMP-7, CEA, p21, p53, and Ki 67. Results. There were 133 male (57.8%) and 97 female (42.2%) patients included in this study, with ages between 20 and 81 years old (mean age: 58.2 years) and with tumors located in the pancreatic head (n=196; 85.2%), pancreatic body (n=12; 5.2%), and pancreatic tail (n=20, 8.7%), as well as panpancreatic tumors (n=2; 0.9%). Patients presented with early stages (IA and IB), with low pathologic grade (G1), with small size tumors (less than 1-1.5 cm), with tumors located in the head of the pancreas, (p53: negative; p21: positive; and CD 68: positive in peritumoral tissue), with low nuclear index (Ki 67

Background Percutaneous image-guided tissue ablation (IGA) plays a growing role in the clinical management of solid malignancies. Electroporation is used for IGA in several modalities: irreversible electroporation (IRE), and reversible electroporation with chemotoxic drugs, called electrochemotherapy (ECT). It was shown that the combination of electrolysis and electroporation—E2—affords tissue ablation with greater efficiency, that is, lower voltages, lower energy and shorter procedure times than IRE and without the need for chemotoxic additives as in ECT. Methods A new E2 waveform was designed that delivers optimal doses of electroporation and electrolysis in a single waveform. A series of experiments were performed in the liver of pigs to evaluate E2 in the context of clinical applications. The goal was to find initial parameter boundaries in terms of electrical field, pulse duration and charge as well as tissue behavior to enable real time tissue ablation of clinically relevant volumes. Results Histological results show that a single several hundred millisecond long E2 waveform can ablate large volume of tissue at relatively low voltages while preserving the integrity of large blood vessels and lumen structures in the ablation zone without the use of chemotoxic drugs or paralyzing drugs during anesthesia. This could translate clinically into much shorter treatment times and ease of use compared to other techniques that are currently applied.

Background and Objectives: GISTs are the most frequent type of mesenchymal neoplasm of the digestive tract. The prognosis is mainly determined by tumor dimensions, mitotic rate and location, but other less well-documented factors can influence evolution and survival. The immune microenvironment and checkpoint molecule expression were proven to impact the prognosis in different types of cancer. The aim of this study was to determine PD-L1 expression in GISTs and to evaluate the level of intratumoral immune infiltration in relation to prognostic variables and survival. Materials and Methods: Sixty-five GISTs diagnosed in the same institution between 2015 and 2018 were immunohistochemically tested for PD-L1 and evaluated using CPS. Immune cells were emphasized, with CD3, CD4, CD8, CD20 and CD68 antibodies and quantified. All data were processed using statistical tools. Results: The median age was 61 years (range, 28–78) and 36 patients (55.4%) were males. The location of the tumors was predominantly gastric (46%), followed by the small bowel (17%) and colorectal (6%). In addition, 11% were EGISTs and 20% were secondary tumors (11% metastases and 9% local recurrences). PD-L1 had a variable expression in tumor and inflammatory cells, with a CPS ranging from 0 to 100. Moreover, 64.6% of cases were PD-L1 positive with no significant differences among categories of variables, such as the age and the sex of the patient, tumor location, the primary or secondary character of the tumor, dimensions, mitotic rate, the risk of disease progression and tumor cell type. Immune cells had a variable distribution throughout the tumors. CD3+ lymphocytes were the most frequent type. CD20+ cells were identified in a larger number in tumors ≤5 cm (p = 0.038). PD-L1-positive tumors had a higher number of immune cells, particularly CD3+, CD20+ and CD68+, in comparison to PD-L1-negative ones (p = 0.032, p = 0.051, p = 0.008). Epithelioid and mixed cell-type tumors had a higher number of CD68+ cells. Survival was not influenced by PD-L1 expression; instead, it was decreased in multifocal tumors (p = 0.0001) and in cases with Ki67 ≥ 50% (p = 0.008). Conclusions: PD-L1-positive expression and the presence of different immune cell types, in variable quantities, can contribute to a better understanding of the complex interactions between tumor cells and the microenvironment, with a possible therapeutic role in GISTs.

Background and Objectives: Although many of the neuroendocrine neoplasms (NEN) have a typically prolonged natural history compared with other gastrointestinal tract cancers, at least 40% of patients develop liver metastases. This study aims to identify whether liver resection improves the overall survival of patients with liver metastases from NEN. Materials and Methods: We conducted a retrospective study at “Fundeni” Clinical Institute over a time period of 15 years; we thereby identified a series of 93 patients treated for NEN with liver metastases, which we further divided into 2 groups as follows: A (45 patients) had been subjected to liver resection complemented by systemic therapies, and B (48 patients) underwent systemic therapy alone. To reduce the patient selection bias we performed at first a propensity score matching. This was followed by a bootstrapping selection with Jackknife error correction, with the purpose of getting a statistically illustrative sample. Results: The overall survival of the matched virtual cohort under study was 41 months (95% CI 37–45). Group A virtual matched patients showed a higher survival rate (52 mo., 95% CI: 45–59) than B (31 mo., 95% CI: 27–35), (p < 0.001, Log-Rank test). Upon multivariate analysis, seven independent factors were identified to have an influence on survival: location (midgut) and primary tumor grading (G3), absence of concomitant LM, number (2–4), location (unilobar), grading (G3) of LM, and 25–50% hepatic involvement at the time of the metastatic disease diagnosis. Conclusions: Hepatic resection is nowadays the main treatment providing potential cure and prolonged survival, for patients with NEN when integrated in a multimodal strategy based on systemic therapy.

BackgroundThe combination of electroporation with electrolysis (E2) has previously been introduced as a novel tissue ablation technique. E2 allows the utilization of a wide parameter range and may therefore be a suitable technology for development of tissue-specific application protocols. Previous studies have implied that it is possible to achieve big lesions in liver in a very short time. The goal of this study was to test a variety of electrode configurations for the E2 application to ablate large tissue volumes.Materials and methods27 lesions were performed in healthy porcine liver of five female pigs. Four, two and bipolar electrode-arrays were used to deliver various E2 treatment protocols. Liver was harvested approx. 20h after treatment and examined with H&E and Masson's trichrome staining, and via TUNEL staining for selective specimen.ResultsAll animals survived the treatments without complications. With four electrodes, a lesion of up to 35x35x35mm volume can be achieved in less than 30s. The prototype bipolar electrode created lesions of 50x18x18mm volume in less than 10s. Parameters for two-electrode ablations with large exposures encompassing large veins were found to be good in terms of vessel preservation, but not optimal to reliably close the gap between the electrodes.ConclusionThis study demonstrates the ability to produce large lesions in liver within seconds at lower limits of the E2 parameter space at different electrode configurations. The applicability of E2 for single electrode ablations was demonstrated with bipolar electrodes. Parameters for large 4-electrode ablation volumes were found suitable, while parameters for two electrodes still need optimization. However, since the parameter space of E2 is large, it is possible that for all electrode geometries optimal waveforms and application protocols for specific tissues will emerge with continuing research.

Cholangiocarcinoma (CCA) is the primary cancer of the bile duct system. The role of bile duct tissue microbiomes in CCA tumorigenesis is unestablished. To address this, sixty primary CCA tumors and matched normals, from both liver fluke (Opisthorchis viverrini) associated (OVa, n = 28) and non-O. viverrini associated (non-OVa, n = 32) cancers, were profiled using high-throughput 16S rRNA sequencing. A distinct, tissue-specific microbiome dominated by the bacterial families Dietziaceae, Pseudomonadaceae and Oxalobacteraceae was observed in bile duct tissues. Systemic perturbation of the microbiome was noted in tumor and paired normal samples (vs non-cancer normals) for several bacterial families with a significant increase in Stenotrophomonas species distinguishing tumors vs paired normals. Comparison of parasite associated (OVa) vs non-associated (non-OVa) groups identified enrichment for specific enteric bacteria (Bifidobacteriaceae, Enterobacteriaceae and Enterococcaceae). One of the enriched families, Bifidobacteriaceae, was found to be dominant in the O. viverrini microbiome, providing a mechanistic link to the parasite. Functional analysis and comparison of CCA microbiomes revealed higher potential for producing bile acids and ammonia in OVa tissues, linking the altered microbiota to carcinogenesis. These results define how the unique microbial communities resident in the bile duct, parasitic infections and the tissue microenvironment can influence each other, and contribute to cancer.

Background/aim: Major hepatectomies are widely used in curative-intent surgery for perihilar cholangiocarcinoma, but morbidity rates are high. The aim of the study is to explore potential predictors for clinically relevant complications after major hepatectomies for perihilar cholangiocarcinoma. Methods: Seventy patients were included. Univariate and multivariate analyses were performed for risk factors of morbidities using the International Study Group of Liver Surgery definitions. Results: Severe morbidity rate was 36.5%. Clinically relevant posthepatectomy liver failure, bile leak, and hemorrhage rates were 24%, 22%, and 8.5%, respectively. A neutrophil-to-lymphocyte ratio > 3.3 is an independent prognostic factor for severe complications (hazard ratio = 1.258; 95% confidence interval 1.008–1.570; p = 0.042) while the number of blood units > 3 is an independent prognostic factor for clinically relevant liver failure (hazard ratio = 1.254; 95% confidence interval 1.082–1.452; p = 0.003). Biliary drainage and portal vein resection were not statistically correlated with any postoperative complication (p ≥ 0.101). Significantly higher bilirubinemia levels were observed in patients with postoperative hemorrhage (p = 0.023). Conclusion: Clinically relevant morbidity rates after major hepatectomies for perihilar cholangiocarcinoma are high. Liver failure represents the main complication and is correlated with the number of transfused blood units. A patient with increased bilirubinemia appears to have a high risk for postoperative hemorrhage. Biliary drainage and portal vein resection does not appear to have a detrimental effect on morbidities. Neutrophil-to-lymphocyte ratio is a novel independent predictor for severe morbidity after major hepatectomies for perihilar cholangiocarcinoma and may contribute to better and informed decision-making.

Ex-vivo freshly surgical removed pancreatic ductal adenocarcinoma (PDAC) specimens were assessed using pCLE and then processed for paraffin embeding and histopathological diagnostic in an endeavour to find putative image analysis algorithms that might recognise adenocarcinoma. Methods: Twelve patients diagnosed with PDAC on endoscopic ultrasound and FNA confirmation underwent surgery. Removed samples were sprayed with acriflavine as contrast agent, underwent pCLE with an experimental probe and compared with previous recordings of normal pancreatic tissue. Subsequently, all samples were subjected to cross-sectional histopathology, including surgical resection margins for controls. pCLE records, as well as corespondant cytokeratin-targeted immunohistochemistry images were processed using the same morphological classifiers in the Image ProPlus AMS image analysis software. Specific morphometric classifiers were automatically generated on all images: Area, Hole Area (HA), Perimeter, Roundness, Integrated Optical Density (IOD), Fractal Dimension (FD), Ferret max (Fmax), Ferret mean (Fmean), Heterogeneity and Clumpiness. Results: After histopathological confirmation of adenocarcinoma areas, we have found that the same morphological classifiers could clearly differentiate between tumor and non-tumor areas on both pathology and correspondand pCLE (area, roundness, IOD, ferret and heterogeneity (p < 0.001), perimeter and hole area (p < 0.05). Conclusions: This pilot study proves that classical morphometrical classifiers can clearly differentiate adenocarcimoma on pCLE data, and the implementation in a live image-analysis algorithm might help in improving the specificity of pCLE in vivo diagnostic.

Aim. To explore the pattern of the first recurrence and impact on long-term survival after curative intent surgery for perihilar cholangiocarcinomas (PHC). Materials and Methods. Patients with curative intent surgery for PHC between 1996 and 2017 were analyzed. Survival times were estimated using the Kaplan-Meier method. Comparisons were made with the log-rank test. Results. A number of 139 patients were included. The median overall survival was 26 months. A recurrence was observed in 86 patients (61.9%), during a median follow-up time of 89 months. The median disease-free survival was 21 months with 1-, 3-, 5-, and 10-year estimated recurrence rates of 38%, 60%, 69%, and 77%, respectively. A number of 57 patients (41%) developed distant only recurrence, while 26 patients (18.7%) presented local and distant recurrences. An isolated local recurrence was observed in 3 patients (2.2%). The median overall survival was 15 months for patients with local recurrence, 15 months for patients with liver metastases, and 17 months for patients with peritoneal carcinomatosis (p=0.903) as the first recurrence. Conclusion. Curative intent surgery for PHC is associated with high recurrence rates. Most patients will develop distant metastases, while an isolated local recurrence is uncommon. The pattern of recurrence does not appear to have a significant impact on survivals.

Wilson’s disease is a rare autosomal recessive inherited disorder of copper metabolism, which results in copper accumulation in several tissues, especially with liver injury and failure. Orthotopic liver transplantation (OLT) can be lifesaving for patients with hepatic complications of Wilson’s disease- fulminant liver failure or unresponsiveness to medical therapy in chronic liver disease, with or without neurological manifestations. We report the case of a 19-year-old woman receiving a liver transplant for acute liver failure caused by Wilson’s disease, who developed headache as the only symptom of a serious neurological complication after transplantation. The clinical course and neuroimaging demonstrating aneurismal subarachnoid hemorrhage are presented. The neurological complications and the difficulties of medical management in an immunosuppressed patient are discussed. Headache in a recently transplanted patient for acute liver failure may be a sign of a serious cerebral complication, subarachnoid hemorrhage. Early recognition and proper management may prevent life-threatening neurologic complications but chronic immunosuppression may impede a favourable outcome.