Eugenia Pareja

Expanded criteria for liver transplantation in patients with cirrhosis and hepatocellular carcinoma

Orthotopic liver transplantation (OLT) selection for patients with hepatocellular carcinoma (HCC) is a matter of debate. The Milan criteria (MC) have been largely adopted by the international community. The main aim of this study was to evaluate the survival rates and recurrence probabilities of a new proposal for criteria (up to 3 tumors, each no larger than 5 cm, and a cumulative tumor burden ≤ 10 cm). Patients with cirrhosis and HCC included on the waiting list (WL) from 1991 to 2006 were retrospectively analyzed. Outcomes in patients who had tumors within and beyond the MC were compared. The survival analysis was done (1) with the intention‐to‐treat principle and (2) among transplanted patients. A total of 281 patients were included in WL. Twenty‐four cases did not undergo OLT (a dropout rate of 8.5%); all but 1 case had tumors within the MC. Of the 257 transplanted patients, 26 had tumors beyond the MC in the pre‐OLT evaluation. Based on the intention‐to‐treat analysis, the 5‐year survival was 56% versus 66% in patients who had tumors within and beyond the MC, respectively (P = 0.487). Among transplanted patients, the 5‐year survival was 62% versus 69%, respectively (P = 0.734). Through multivariate analysis, microvascular invasion was an independent prognostic factor of poor survival (P = 0.004). The recurrence probabilities at 1 and 5 years were 7% versus 12% and 14% versus 28% in patients with tumors within and beyond the MC, respectively (P = 0.063). The multivariate analysis demonstrated that both poorly differentiated tumors (P < 0.001) and microvascular invasion (P < 0.001) increased the risk of recurrence. The expansion to up to 3 nodules, each up to 5 cm, and a cumulative tumor burden ≤ 10 cm did not result in a reduction of survival in comparison with patients who had tumors within the MC. Liver Transpl 14:1449–1460, 2008.

Lessons learned from anatomic variants of the hepatic artery in 1,081 transplanted livers

The aim of this study is to contribute our experience to the knowledge of the anatomic variations of the hepatic arterial supply. The surgical anatomy of the extrahepatic arterial vascularization was investigated prospectively in 1,081 donor cadaveric livers, transplanted at La Fe University Hospital from January 1991 to August 2004. The vascular anatomy of the hepatic grafts was classified according to Michels description (Am J Surg 1966;112:337‐347) plus 2 variations. Anatomical variants of the classical pattern were detected in 30% of the livers (n = 320). The most common variant was a replaced left artery arising from the left gastric artery (9.7%) followed by a replaced right hepatic artery arising from the superior mesenteric artery (7.8%). In conclusion, the information about the different hepatic arterial patterns can help in reducing the risks of iatrogenic complications, which in turn may result in better outcomes not only following surgical interventions but also in the context of radiological treatments. Liver Transpl 13:1401–1404, 2007.

Effect of Calcineurin Inhibitors in the Outcome of Liver Transplantation in Hepatitis C Virus-Positive Recipients

Background. There is a paucity of good studies evaluating the impact of calcineurin inhibitors on posttransplantation outcome in hepatitis C virus (HCV)-infected liver transplant (LT) recipients. Methods. We sought to determine whether there are differences on posttransplantation survival and histologic recurrence in HCV-LT recipients based on initial immunosuppression (IS) by conducting a prospective study comparing tacrolimus (Tac) versus cyclosporine-based IS in patients undergoing LT between 2001 and 2007. Protocol liver biopsies were performed. Results. Baseline characteristics (demographics, liver function at LT, genotype distribution, donor, surgery, and IS except for the type of calcineurin inhibitor) did not differ between groups. Severe disease (defined as bridging fibrosis, cirrhosis, cholestatic hepatitis, or allograft loss or death because of recurrent disease in the first year) was present in 67 of 253 (26.5%) and was equally distributed in the CsA and Tac groups (27% vs. 26%; P=0.68). Two thirds of protocol biopsies performed at 1 year showed some fibrosis without differences between CsA and Tac groups (75% vs. 70%). Advanced fibrosis (bridging fibrosis and cirrhosis) was diagnosed in 30% CsA and 24.5% Tac patients (P=NS). No differences in survival at 1 and 7 years were observed (83% and 67% vs. 78% and 64%, respectively, P=0.4). In summary, in patients undergoing LT for HCV-related liver disease, posttransplantation outcome is not related to the calcineurin inhibitor used.

Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM ClNH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.

Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver

Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

Influence of Preservation Solution on the Isolation and Culture of Human Hepatocytes from Liver Grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 ± 41% vs. 79 ± 14%, p < 0.05), yield of cells (4.0 ± 5.2 × 106 vs. 8.2 ± 5.6 × 106 cells/g, p < 0.05), and protein content at 24 h of culture (0.6 ± 0.6 vs. 1.2 ± 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.

Metabolomics discloses donor liver biomarkers associated with early allograft dysfunction

BACKGROUND & AIMS Early allograft dysfunction (EAD) dramatically influences graft and patient outcome after orthotopic liver transplantation and its incidence is strongly determined by donor liver quality. Nevertheless, objective biomarkers, which can assess graft quality and anticipate organ function, are still lacking. This study aims to investigate whether there is a preoperative donor liver metabolomic biosignature associated with EAD. METHODS A comprehensive metabolomic profiling of 124 donor liver biopsies collected before transplantation was performed by mass spectrometry coupled to liquid chromatography. Donor liver grafts were classified into two groups: showing EAD and immediate graft function (IGF). Multivariate data analysis was used to search for the relationship between the metabolomic profiles present in donor livers before transplantation and their function in recipients. RESULTS A set of liver graft dysfunction-associated biomarkers was identified. Key changes include significantly increased levels of bile acids, lysophospholipids, phospholipids, sphingomyelins and histidine metabolism products, all suggestive of disrupted lipid homeostasis and altered histidine pathway. Based on these biomarkers, a predictive EAD model was built and further evaluated by assessing 24 independent donor livers, yielding 91% sensitivity and 82% specificity. The model was also successfully challenged by evaluating donor livers showing primary non-function (n=4). CONCLUSIONS A metabolomic biosignature that accurately differentiates donor livers, which later showed EAD or IGF, has been deciphered. The remarkable metabolomic differences between donor livers before transplant can relate to their different quality. The proposed metabolomic approach may become a clinical tool for donor liver quality assessment and for anticipating graft function before transplant.

Comparative prospective study of two liver graft preservation solutions: University of Wisconsin and Celsior

University of Wisconsin solution (UWS) is the gold standard for graft preservation. Celsior solution (CS) is a new solution not as yet widely used in liver grafts. The aim of this study was to compare the liver function of transplanted grafts stored in these 2 preservation solutions. The primary endpoints were the rates of primary nonfunction (PNF) and primary dysfunction (PDF). We performed a prospective and pseudorandomized study that included 196 patients (representing 104 and 92 livers preserved in UWS and CS, respectively) at La Fe University Hospital (Valencia, Spain) between March 2003 and May 2005. PNF and PDF rates, liver function laboratory parameters, postoperative bleeding, vascular and biliary complications, and patient and graft survival at 3 years were compared for the 2 groups. The 2 groups were similar in terms of donor variables, recipient variables, and surgical techniques. The PNF rates were 2.2% and 1.9% in the CS and UWS groups, respectively (P = not significant), and the PDF rates were 15.2% and 15.5% in the CS and UWS groups, respectively (P = not significant). There were no significant differences in the laboratory parameters for the 2 groups, except for alanine aminotransferase levels in month 3, which were lower in the CS group (P = 0.01). No significant differences were observed in terms of complications. Three‐year patient and graft survival rates were as follows for years 1, 2, and 3: 83%, 80%, and 76% (patient) and 80%, 77%, and 73% (graft) for the UWS group and 83%, 77%, and 70% (patient) and 81%, 73%, and 67% (graft) for the CS group (P = not significant). In conclusion, this study shows that CS is as effective as UWS in liver preservation. Liver Transpl 15:1709–1717, 2009.

Long‐term outcome of ‘long‐term liver transplant survivors’

There are few studies focusing on long‐term complications in liver transplant (LT) recipients. The aim of this study was to define the outcome of LT recipients having survived at least 10 years from LT. Of 323 adult LT done between 1991 and 1997, the 167(52%) alive >10 years post‐LT (baseline time) formed the study population. Long‐term outcome measures included the following: immunosuppression, metabolic complications [obesity, arterial hypertension (AH), diabetes, dislypidemia], cardiovascular events (CVE), chronic renal dysfunction‐CRD, and de novo tumors. Median age at LT was 50 years. Most common indication was postnecrotic cirrhosis (89%), mostly because of HCV (46%). At study‐baseline (10 years post‐LT), 29% were obese and AH, diabetes, dislypidemia, and CRD were present in 75%, 30%, 42%, and 36%, respectively. In most cases, these complications were already present 1 year post‐LT; less than one quarter developed them onward. The 6 year cumulative survival since baseline reached 84% (n = 24 deaths), with most deaths related to recurrent graft diseases (mostly HCV) followed by de novo tumors or CVE. 1, 3, 5 and 10 years cumulative rates of CVE and de novo tumors since baseline were 2%, 5%, 10% and 17%, and 1%, 3%, 6% and 13%, respectively. Chronic renal impairment was independently associated with survival and development of CVE since baseline. The medium‐term survival of ‘long‐term survivors’, i.e. patients alive 10 years after LT is good, but metabolic complications and CRD are common and continue to increase afterwards. Cardiovascular events and de novo tumors increase gradually over time and represent a major cause of late mortality.

Exocrine pancreatic changes following acute attack of biliary pancreatitis

Following the Cambridge and Marseilles Symposia, functional recovery of the pancreas occurs if the primary cause and complications of the disease have been eliminated. However, recent research showed contradictory results, owing to the difference in diagnostic methods and the proportion of patients studied in relation to the etiologic factor and severity of the disease, as well as the differences in the tests utilized. Sixty-three consecutive patients with acute biliary pancreatitis were prospectively studied. Seventeen were men (27%) and 46 were women (73%), with an average age of 62.3 years, 45 were mild cases and 18 were severe. All patients underwent a cholecystectomy. No patient in this series underwent necrosectomy. During the acute phase, severity was evaluated following the Atlanta criteria as well as the existence of necrosis and its percentage by means of dynamic computed tomography (CT). During the follow-up, different tests were used to assess the pancreatic exocrine function, 1 month, 6 months and 1 year after the acute pancreatitis (AP) episode. The possible existence of pancreatic exocrine insufficiency following biliary origin AP as well as whether this possible deficit was related to the severity of the episode was investigated. We found no such insufficiency 1 year after the episode, and no link with the severity of the episode.