Qualification of tumour mutational burden by targeted next-generation sequencing as a biomarker in hepatocellular carcinoma.

BACKGROUND & AIMS: Tumour mutational burden (TMB) predicts improved response and survival to immunotherapy. In this pilot study, we optimized targeted next-generation sequencing (tNGS) to estimate TMB in hepatocellular carcinoma (HCC). METHODS: We sequenced 48 non-paired samples (21 fresh-frozen [FF] and 27 paraffin-embedded [FFPE]), among which 11 FFPE samples were pretreated with uracil-DNA glycosylase (UDG). Thirty samples satisfied post-sequencing quality control. High/low TMB was defined by median number of mutations/Mb (Mut/Mb), across different minimum allele frequency (MAF) thresholds (≥0.05, ≥0.1 and ≥0.2). RESULTS: Eligible patients (n = 29) were cirrhotic (84%) with TNM stage I-II HCC (75%). FFPE samples had higher TMB (median 958.39 vs 2.51 Mut/Mb, P < .0001), estimated deamination counts (median 1335.50 vs 0, P < .0001) and C > T transitions at CpG sites (median 60.3% vs 9.1%, P = .002) compared to FF. UDG-treated samples had lower TMB (median 4019.92 vs 353 Mut/Mb, P = .041) and deamination counts (median 6393.5 vs 328.5, P = .041) vs untreated FFPE. At 0.2 MAF threshold with UDG treatment, median TMB was 5.48 (range 1.68-16.07) and did not correlate with salient pathologic features of HCC, including survival. CONCLUSION: While tNGS on fresh HCC samples appears to be the optimal source of tumour DNA, the low median TMB values observed may limit the role of TMB as a predictor of response to immunotherapy in HCC.

Biglycan expression and clinical outcome in patients with pancreatic adenocarcinoma.

Conflicting results have been reported on the role of extracellular matrix (ECM) proteins in pancreatic cancer. Preclinical studies suggest that the overexpression of biglycan (proteoglycan-I, PG-I), a leucine-rich protein of the ECM, may induce growth arrest of pancreatic cancer cells. The aim of this study was to assess the prognostic role of biglycan expression in pancreatic cancer. We also evaluated MIB-1 and COX-2 expressions (as potential markers of growth and aggressiveness) to better characterize the biology of the tumors. The classical pathological parameters (grading, desmoplasia, perineural, or vascular invasion) as well as molecular determinants of prognosis were examined. MIB-1 (a proliferative index associated with prognosis in most tumors), COX-2, and PG-I expressions were detected by immunohistochemistry and immunofluorescence on tissue samples from 53 patients with pancreatic cancer and reviewed by two independent pathologists. To verify PG-I expression, three rabbit sera (LF104, LF112, and LF121 from NIH, Bethesda, MA, US) were tested. Logrank test and Cox's model were applied for statistical analysis. Out of 53 patients, 40 had stage III and IV pancreatic cancer. Fourteen patients did not express any of the PG-I epitopes. The patients who expressed at least two PG-I epitopes had shorter survival compared to those with single epitope or lacking any expression (28 vs 44 weeks, P = 0.0021). The MIB-1 higher expression predicted shorter survival (25 vs 41 weeks, P = 0.0059). The other parameters were not associated with clinical outcome. Multivariate analyses confirmed PG-I expression and MIB-1 as independent negative prognostic factors. Patients who presented PG-I expression in the ECM had the worse prognosis compared to those who did not. Our results are not in contrast with the hypothesis that ECM proteins are a potential barrier to metastatic spread in localized pancreatic cancer. Rather, we underline the complexity of tumor-stroma interactions in the advanced stage of cancer and the need of further study.

Integrated phenotyping of the anti-cancer immune response in HIV-associated hepatocellular carcinoma.

Background & Aims: HIV-seropositivity shortens survival in patients with hepatocellular carcinoma (HCC). Although risk factors for HCC including HCV infection can influence T cell phenotype, it is unknown whether HIV can influence functional characteristics of the T cell infiltrate. Methods: From the Liver Cancer in HIV biorepository, we derived 129 samples of transplanted (76%) or resected (20%) HCC in eight European and North American centres. We profiled intra- and peritumoural tissue to evaluate regulatory CD4+/FOXP3+ and immune-exhausted CD8+/PD1+ T cells in HIV+ (n = 66) and HIV- (n = 63) samples. We performed targeted transcriptomics and T-cell receptor sequencing in a restricted subset of samples evaluated in relationship with HIV status. We correlated immunopathologic features with patients' characteristics including markers of HIV infection. Results: Of the 66 HIV+ patients, 83% were HCV coinfected with an undetectable HIV viral load (51%) and a median blood CD4+ cell count of 430 cells/mm3 (range 15-908). Patients who were HIV+ were compared with HIV- controls with similar staging characteristics including Barcelona Clinic Liver Cancer (BCLC) stage A-B (86% vs. 83%, p = 0.16), <3 nodules (90% vs. 83%, p = 0.3) and median alpha-foetoprotein values (10.9 vs. 12.8 ng/ml, p = 0.72). HIV+ samples had higher PD-L1 expression rates in tumour tissue (51% vs. 8% p <0.0001) and displayed denser intratumoural CD4+/FOXP3+ (p <0.0001), CD8+/PD1+ (p <0.0001), with lower total peritumoural CD4+ (p <0.0001) and higher peritumoural CD8+/PD1+ (p <0.0001). Gene set analysis revealed HIV+ cases to have evidence of dysregulated adaptive and innate immunity. Tumour-infiltrating lymphocyte clonality was not influenced by HIV status. Conclusions: HIV-associated HCC harbours a profoundly immune-exhausted tumour microenvironment, warranting prospective testing of immunotherapy in this treatment-deprived patient population. Impact and Implications: Hepatocellular carcinoma is a non-AIDS defining malignancy characterised by poor survival. The programmed cell death (PD-1) pathway governs antiviral and anticancer immune exhaustion and is a therapeutic target in HCC. This study highlights how HIV infection is associated with significantly higher PD-L1 expression in HCC cells and in the surrounding microenvironment, leading to changes in cytotoxic and regulatory T cell function and dysregulation of proinflammatory pathways. Taken together, our results suggest dysfunctional T cell immunity as a mechanism of worse outcome in these patients and suggest clinical testing of checkpoint inhibitors in HIV-associated HCC.

Association between vitamin D receptor genetic polymorphisms and acute cellular rejection in liver-transplanted patients.

Vitamin D receptor (VDR) polymorphisms may confer susceptibility to immunologically mediated liver diseases. We aimed to verify whether recipient VDR polymorphisms might affect the incidence of acute cellular rejection (ACR) of the graft after liver transplantation (LT). Two hundred and fifty-one liver-transplanted patients surviving at least 1month were studied. ACR in the first post-LT year was graded according to the Banff score. Recipients genotyping for VDR polymorphic sites (FokI C>T, BsmI G>A, ApaI T>G, TaqI T>C) was performed. A significant difference was found between patients with and without ACR episodes in allele frequencies of BsmI (G: 0.660 vs. 0.545, P=0.017) and TaqI (T: 0.667 vs. 0.543, P=0.010). Patients carrying the G-*-T/G-*-T diplotypes of the BsmI G>A, ApaI T>G and TaqI T>C experienced more frequently ACR: 33/79 Vs 42/172, P=0.005. Carriage of G-*-T/G-*-T diplotypes was an independent predictor of ACR (OR 2.41, P=0.006), with CMV reactivation (OR 2.34, P=0.033) and HCV aetiology (OR 1.86, P=0.036). In conclusion, recipient VDR polymorphic loci are strongly associated with ACR occurrence during the first year after LT. The knowledge of VDR genetic polymorphisms may be helpful in identifying recipients at higher risk of ACR and in selecting them for a more aggressive immunosuppressive therapy.

Early diagnosis and treatment of postoperative endoscopic recurrence of Crohn's disease: partial benefit by infliximab--a pilot study.

BACKGROUND: Current data indicate that infliximab-given immediately after surgery-may be very effective in preventing postsurgical recurrence of Crohn's disease. However, it is unknown whether a similar benefit would result from early diagnosis and treatment, rather than prevention of endoscopic recurrence. AIMS: The primary outcome of this study was to clarify whether infliximab, given after diagnosis of postoperative endoscopic recurrence of Crohn's diseases (Rutgeerts score ≥ 2) can induce endoscopic remission (score <2) at 54 weeks. The secondary outcomes were improvement in the endoscopic score and clinical recurrence at 54 weeks. METHODS: In this prospective open label multicenter pilot study 43 patients with ileocolonic Crohn's disease subjected to curative surgery underwent colonoscopy 6 months after surgery. Patients with endoscopic recurrence (Rutgeerts score ≥2) were treated with either mesalamine 800 mg tid or infliximab 5 mg/kg bw on a maintenance basis. Colonoscopy was performed after 54 weeks of therapy. RESULTS: A total of 24/43 patients were diagnosed with endoscopic recurrence at 6 months. Thirteen were treated with infliximab and 11 with mesalamine. None of the 11 mesalamine-treated patients had endoscopic remission at 54 weeks. Two had clinical recurrence at 8 and 9 months. Fifty-four percent of patients treated with infliximab had endoscopic remission at 54 weeks (P = 0.01) while 69% had an improvement in the endoscopic score. None had clinical recurrence. CONCLUSIONS: Treatment of postsurgical endoscopic lesions by infliximab appears superior to mesalamine. However, a sizeable proportion of patients did not fully benefit from this strategy.

Focal hepatic lesions in acid sphingomyelinase deficiency: Differential diagnosis between foamy macrophages aggregates and malignancy.

Acid sphingomyelinase deficiency (ASMD) is a rare metabolic disorder due to biallelic mutation in the SMPD1 gene. The defect leads to the accumulation of sphingomyelin within the cells of the reticulo-endothelial system, particularly in the spleen, liver, lungs, and bone marrow causing hepato-splenomegaly, lung disease and hematological abnormalities. At present, data on abdominal imaging in ASMD are limited. Here we describe the characteristics of focal liver lesions observed in a 30 years old female. During the Magnetic Resonance follow up an increase in number and size of the lesions, showing T1 hypointensity and T2 hyperintensity with contrast enhancement, was observed. Contrast enhanced ultrasound evidenced rapid wash-in and steady isoecogenicity without appreciable wash-out at 80 seconds. The main lesion was biopsied to rule out the presence of a hepatocellular carcinoma, and showed to be a benign foamy macrophages aggregate. In this report, we discuss the possible pathogenesis of focal hepatic lesions in ASMD and their differential diagnosis.

Trans-arterial chemoembolization as a loco-regional inducer of immunogenic cell death in hepatocellular carcinoma: implications for immunotherapy.

BACKGROUND: Modulation of adaptive immunity may underscore the efficacy of trans-arterial chemoembolization (TACE). We evaluated the influence of TACE on T-cell function by phenotypic lymphocyte characterization in samples of patients undergoing surgery with (T+) or without (T-) prior-TACE treatment. METHODS: We profiled intratumoral (IT), peritumoral (PT) and non-tumoral (NT) background tissue to evaluate regulatory CD4+/FOXP3+ (T-reg) and immune-exhausted CD8+/PD-1+ T-cells across T+ (n=58) and T- (n=61). We performed targeted transcriptomics and T-cell receptor sequencing in a restricted subset of samples (n=24) evaluated in relationship with the expression of actionable drivers of anti-cancer immunity including PD-L1, indoleamine 2,3 dehydrogenase (IDO-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), Lag-3, Tim-3 and CD163. RESULTS: We analyzed 119 patients resected (n=25, 21%) or transplanted (n=94, 79%) for Child-Pugh A (n=65, 55%) and Barcelona Clinic Liver Cancer stage A (n=92, 77%) hepatocellular carcinoma. T+ samples displayed lower IT CD4+/FOXP3+ (p=0.006), CD8+ (p=0.002) and CD8+/PD-1+ and NT CD8+/PD-1+ (p<0.001) compared with T-. Lower IT (p=0.005) and NT CD4+/FOXP3+ (p=0.03) predicted for improved recurrence-free survival. In a subset of samples (n=24), transcriptomic analysis revealed upregulation of a pro-inflammatory response in T+. T+ samples were enriched for IRF2 expression (p=0.01), an interferon-regulated transcription factor implicated in cancer immune-evasion. T-cell clonality and expression of PD-L1, IDO-1, CTLA-4, Lag-3, Tim-3 and CD163 was similar in T+ versus T-. CONCLUSIONS: TACE is associated with lower IT density of immune-exhausted effector cytotoxic and T-regs, with significant upregulation of pro-inflammatory pathways. This highlights the pleiotropic effects of TACE in modulating the tumor microenvironment and strengthens the rationale for developing immunotherapy alongside TACE.

Low-dose maintenance therapy with infliximab prevents postsurgical recurrence of Crohn's disease.

BACKGROUND & AIMS: Infliximab might prevent postsurgical recurrence of Crohn's disease. However, it is unclear whether long-term therapy is necessary and whether alternative strategies could be applied to minimize potential side effects and reduce the costs of treatment. METHODS: We performed a prospective cohort study in 12 consecutive patients, treated immediately after surgery with maintenance infliximab (5 mg/kg), who did not have clinical or endoscopic evidence of disease recurrence after 24 months; they were followed up for an additional year. Infliximab treatment was then discontinued; patients with disease recurrence, based on endoscopy (Rutgeerts score, >or=2), were given lower doses of infliximab (starting with 1 mg/kg) to re-establish mucosal integrity. Surrogate markers of disease activity (fecal calprotectin [FC], C-reactive protein, and erythrocyte sedimentation rate) were assessed after each infliximab dose. RESULTS: None of the patients had clinical or endoscopic recurrence of Crohn's disease 3 years after surgery. However, discontinuation of infliximab caused endoscopic recurrence after 4 months in 10 of 12 patients (83%). All 10 patients then were treated again with infliximab, which, at a dose of 3 mg/kg every 8 weeks, restored and maintained mucosal integrity for 1 year. Among the surrogate markers, FC levels correlated with endoscopic scores (Wald test, P < .0001). CONCLUSIONS: Long-term maintenance therapy with infliximab is required to maintain mucosal integrity in patients after surgery for Crohn's disease. However, a dose of 3 mg/kg (a 40% reduction from the standard dose) was sufficient to avoid disease recurrence, determined by endoscopy, in all patients at 1 year. FC levels correlate with mucosal status at different infliximab doses.

Steatosis of the hepatic graft as a risk factor for post-transplant biliary complications.

BACKGROUND: Despite recent advances in organ preservation, immunosuppression, and surgical techniques, the biliary tree is still considered the Achilles' heel of liver transplantation. The aim of this study is to retrospectively analyze the incidence of biliary complications and identify risk factors that might predispose to the development of biliary problems. METHODS: From January 2004 to December 2007, 117 consecutive liver transplantations were retrospectively analyzed for the development of biliary complications by the review of medical records. Patients were divided into group 1 with biliary complications (n = 43) and group 2 without biliary complications (n = 74). RESULTS: The overall biliary complication rate was 36.8% (leakage 6% and stricture 30.8%). Univariate analysis indicated that significant predictors of biliary complications were the time interval between portal and arterial reperfusion (p = 0.037) and macrovacuolar steatosis of the graft > 25% (p = 0.004). Stepwise logistic regression model demonstrated that a macrosteatosis of the graft > 25% (OR = 5.21 CI 95% [1.79-15.15], p = 0.002) was the only independent risk factor predicting biliary complications after liver transplantation. No differences in patient's and graft's survival were noted between the two groups. CONCLUSION: According to our experience, transplanting a liver with > 25% of steatosis is a risk factor for the development of biliary complication.

Comparison of portal venous and delayed phases of gadolinium-enhanced magnetic resonance imaging study of cirrhotic liver for the detection of contrast washout of hypervascular hepatocellular carcinoma.

OBJECTIVE: To retrospectively compare portal venous phase (PVP) and delayed phase (DP) for the detection of tumor washout at gadobenate dimeglumine-enhanced liver magnetic resonance imaging (MRI) in cirrhotic patients with hypervascular hepatocellular carcinoma (HCC). METHODS: Thirty-three patients with 55 HCCs underwent 1.5-T MRI by means of fat-suppressed T1-weighted sequence obtained before and after gadobenate dimeglumine administration, during early and late arterial phases, PVP (70 seconds), and DP (180 seconds). Detection rates of contrast washout of hypervascular HCCs and tumor-to-liver contrast on PVP and DP were measured and compared. RESULTS: Among 54 hypervascular HCCs, washout was present in 24 (44%) of 54 tumors on PVP and in 44 (82%) of 54 on DP (P < 0.001). In 20 (37%) of 54 tumors, washout was deemed present only on DP. Delayed phase images yielded significantly higher mean tumor-to-liver contrast absolute values compared with PVP images (-24.5 [56.1] vs -9.3 [52.6], P = 0.001). CONCLUSIONS: Delayed phase is superior to PVP for the washout detection of hypervascular HCC at gadobenate dimeglumine-enhanced MRI of cirrhotic liver.

Hepatocellular metastasis recurrence in liver transplant after treatment with direct antiviral agents.

Chronic HCV liver infection is considered one of the main causes of liver cirrhosis and hepatocellular carcinoma (HCC). For a selected group of patients, orthotopic liver transplantation (OLTx) is the most effective option to cure both liver diseases. After liver transplantation, patients may be at risk of viral infection reactivation and HCC recurrence. HCV recurrence on the transplanted organ can lead to graft cirrhosis and therefore the clearance of virus with antiviral therapies has a pivotal role on the prevention of graft damage. Nowadays, direct antiviral agents (DAAs) represent the choice treatment for HCV recurrence in liver transplanted patients, ensuring high eradication rates. We present the case of a liver transplant recipient who developed, 7 years after OLTx and immediately after a DAAs treatment, a subcutaneous abdominal mass with histological characteristics of HCC.

Expression of the interferon-inducible proteins MxA and IFI16 in liver allografts.

AIMS: To test the hypothesis that the activation of the interferon (IFN) system pathways might link hepatitis C virus (HCV) recurrence in the liver allograft with acute cellular rejection. METHODS AND RESULTS: In this retrospective study, allograft biopsy specimens from 28 adult patients (14 HCV+ and 14 HCV-) who had undergone their first liver transplantation were analysed. Eleven biopsy specimens showed acute cellular rejection (Banff rejection activity index score > or =3). Specimens were immunostained for two IFN-inducible proteins, MxA and IFI16, and for CD45. The predominant MxA reactivity pattern was hepatocytic, whereas IFI16 was expressed in both the hepatocellular and inflammatory compartments. Moderate to strong MxA expression in hepatocytes was associated positively with rejection score (P < 0.01), donor's age < or =45 years (P < 0.05) and aspartate aminotransferase levels >40 U/l on the day of biopsy (P < 0.05), and inversely with infiltration of portal triads by IFI16+/CD45+ cells (P < 0.005) and time to progression beyond Ishak stage 2 of recurrent hepatitis C (P < 0.01). On multivariate analysis, MxA expression in hepatocytes was independently associated with allograft rejection and donor's age. CONCLUSIONS: Acute allograft rejection and recurrence of HCV infection in the liver allograft appear to intersect in the IFN system pathways.

MTHFR C677T polymorphism and risk of HCC in patients with liver cirrhosis: role of male gender and alcohol consumption.

BACKGROUND: A single nucleotide polymorphism (SNP) C677T in the methylenetetrahydrofolate reductase (MTHFR) gene has been identified. The TT or CT genotypes show a marked reduction of the enzyme activity; this causes higher homocysteine levels and alterations of folate metabolism. Folate metabolism is essential for DNA synthesis and methylation, crucial steps in carcinogenesis. In this paper, we investigated whether the MTHFR C677T SNP could influence the occurrence of hepatocellular carcinoma (HCC) in a cohort of patients transplanted for end stage liver disease of different etiologies. METHODS: Two hundred and twelve consecutive patients who underwent liver transplantation for end stage liver disease due to hepatitis B or C, alcoholic liver disease, and other causes were studied. Two hundred and thirty-six blood donors served as controls. Focal hepatic lesions were searched in the sectioned explanted livers. The presence of the MTHFR C677T SNP was determined via polymerase chain reaction amplification. RESULTS: Among the 65 patients with HCC, 22 had the CC genotype, 30 the CT, and 13 the TT genotype. Only in patients with alcoholic liver disease was a significant association detected between the TT genotype and the presence of liver cancer (6/17 vs. 5/46, p < 0.05). At stepwise logistic regression analysis the independent selected predictors of HCC were found: age at transplantation >55 years (p < 0.001) and the association among male gender, alcoholic liver disease, and MTHFR TT genotype (p = 0.002). CONCLUSIONS: The present study suggests that male TT carriers with alcoholic cirrhosis bear an increased risk of developing HCC.

Occult hepatitis B virus infection in liver transplant recipients with recurrent hepatitis C: relationship with donor age and fibrosis progression.

Liver transplantation (OLT) recipients who receive a graft from donors positive for hepatitis B virus (HBV) anti-core antibodies may develop overt "de novo" HBV infection. The study was undertaken to explore how often HBV infection may remain occult after OLT for hepatitis C, and whether it may represent a factor of graft fibrosis progression. We studied 30 consecutive patients transplanted for hepatitis C liver disease. Specimens from the native liver and from the graft were searched for occult HBV infection (O-HBV). In the native liver, 8/30 patients had detectable O-HBV; during the follow-up, O-HBV infection was demonstrated in 14 graft specimens. Graft O-HBV was associated with older donor age (> or =50 yr; 8/9 vs. 6/21, p < 0.005). Recipients with graft O-HBV and no O-HBV in the native liver who received their grafts from donors aged >40 yr had faster fibrosis progression than recipients with no post-transplant O-HBV, whose grafts came from donors aged >40 yr and recipients whose grafts came from donors aged < or =40 yr (4/7 vs. 1/7 vs. 2/16, p < 0.05). In OLT recipients, O-HBV is more likely to occur when grafts are obtained from aged donors and may affect the rate of fibrosis progression because of recurrent hepatitis C.

[Lack of prognostic predictive value of hepatic agobiopsy in a group of patients operated of hepatic resection for hepatocellular carcinoma]. / Mancanza di valore prognostico predittivo dell'agobiopsia epatica sull'outcome di una serie consecutiva di pazienti resecati per epatocarcinoma.

AIM: To determine prior to surgery whether a fine needle biopsy is able to define a hepatocellular carcinoma grading, or not. MATERIAL OF STUDY: Thirty patients, who all underwent liver resection for HCC. In every case a fine needle biopsy of the neoplasm was taken prior to surgery, and after the operation a complete microscopic assessment of tumor grade according to Edmondson and Steiner classification was taken. RESULTS: We found no correlation between fine needle biopsy grading and post surgical one. We also found no correlation between fine needle grading and other relevant elements, alpha-fetoprotein levels and number of neoplastic nodules. DISCUSSION: Considering the small number of patients in the study, fine needle biopsy seems to be unfit to determine HCC grading before surgery, this is probably due to the different levels of neoplastic differentiation present into every single nodule and to the characteristics of Edmondson and Steiner classification. In Literature there is at least one study, similar to ours, showing problems in the correct attribution of grading level using this classification. Other authors consider the possibility to modify the scale from a 4-levels one to a 3-levels one. CONCLUSIONS: In spite of these discouraging results, and with a strict follow up monitoring any tumor seeding, we think fine needle biopsy is still fundamental for controversial cases, and for new studies on hepatocellular carcinoma, like those over vascular invasion or the molecular profile of the neoplasm.

[Prevention of post-surgical recurrence of Crohn's disease. A perspective]. / La prevenzione della recidiva post-chirurgica nel morbo di Crohn: uno sguardo al futuro.

Crohn's disease often leads to stricture formation, occlusion and surgery. Unfortunately the disease tends to recur often. Traditional medications seem very little effective for recurrence prevention. By contrast, biologics appear extremely promising and potentially capable of changing radically the management of this disease.

Gastrointestinal Perforations in Adult Whole-Liver Transplant Patients: Clinical, Radiologic, and Histopathologic Analysis.

BACKGROUND: Gastrointestinal perforation (GIP) is a rare complication after adult liver transplant (LT) associated with high morbidity and mortality. Limited data are available about clinical risk factors and underlying pathogenic mechanisms. METHODS: The retrospective study included all GIP cases from a consecutive cohort of 361 LT recipients during the period 2005-2017. Clinical variables were investigated as potential risk factors for GIP, and radiologic and histopathologic evaluations were undertaken to identify any causative mechanism. RESULTS: A total of 22 patients developed at least 1 episode of GIP (prevalence 6.1%) at a median time of 18.5 [interquartile range, 12.5-28.5] days after LT. The perforations occurred in the small bowel (63.6%), transverse colon (27.3%), right colon (22.7%), left colon (9.1%), and stomach (9.1%). A total of 27.3% of patients developed multiple sites of GIP, and in 31% GIP recurred after curative surgery. The 30-day mortality rate after relaparotomy was 40%. A history of previous abdominal surgery (odds ratio, 2.5) and early post-LT relaparotomy due to other complications (odds ratio, 2.6) were significant risk factors for GIP. No thromboembolic or steno-occlusive complications of any splanchnic vessel were detected at computed tomography scan, while histopathology examination on perforated gastrointestinal segments excluded cytomegalovirus infection, graft-vs-host disease, and inflammatory bowel disease. In all the cases, ischemic necrosis with aspecific microangiopathy and microembolization were the pathologic features detected. CONCLUSIONS: GIP is a severe complication after LT with frequent multiple gastrointestinal involvement and recurrence after curative surgery. The pathologic underlying mechanism is usually microvascular ischemia. Clinical risk factors are history of previous abdominal surgery and early post-LT relaparotomy.

Hyaluronic acid inhibition by 4-methylumbelliferone reduces the expression of cancer stem cells markers during hepatocarcinogenesis.

Hyaluronic acid (HA) is a glycosaminoglycan of extracellular matrix related to cell surface which interacts with various cell types. To understand the role of HA during hepatocarcinogenesis, we assessed the effect of the inhibition of HA deposition and its association with heterogeneous hepatocellular carcinoma (HCC) cells. In this study, we used transgenic mice C57BL/6J-Tg(Alb1HBV)44Bri/J (HBV-TG) and normal C57BL/6 J (WT) for in vivo study, while HCC cells Huh7 and JHH6 as in vitro models. Both models were treated with an HA inhibitor 4-methylumbelliferone (4MU). We observed that 4MU treatments in animal model down-regulated the mRNA expressions of HA-related genes Has3 and Hyal2 only in HBV-TG but not in normal WT. As observed in vivo, in HCC cell lines, the HAS2 mRNA expression was down-regulated in Huh7 while HAS3 in JHH6, both with or without the presence of extrinsic HA. Interestingly, in both models, the expressions of various cancer stem cells (CD44, CD90, CD133, and EpCAM) were also decreased. Further, histological analysis showed that 4MU treatment with dose 25 mg/kg/day reduced fibrosis, inflammation, and steatosis in vivo, in addition to be pro-apoptotic. We concluded that the inhibition of HA reduced the expressions of HA-related genes and stem cells markers in both models, indicating a possible modulation of cells-to-cells and cells-to-matrix interaction.