Publisher Correction: RNA-Seq transcriptome profiling in three liver regeneration models in rats: comparative analysis of partial hepatectomy, ALLPS, and PVL.

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RNA-Seq transcriptome profiling in three liver regeneration models in rats: comparative analysis of partial hepatectomy, ALLPS, and PVL.

The liver is a unique organ that has a phenomenal capacity to regenerate after injury. Different surgical procedures, including partial hepatectomy (PH), intraoperative portal vein ligation (PVL), and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) show clinically distinct recovery patterns and regeneration. The observable clinical differences likely mirror some underlying variations in the patterns of gene activation and regeneration pathways. In this study, we provided a comprehensive comparative transcriptomic analysis of gene regulation in regenerating rat livers temporally spaced at 24 h and 96 h after PH, PVL, and ALPPS. The time-dependent factors appear to be the most important determinant of post-injury alterations of gene expression in liver regeneration. Gene expression profile after ALPPS showed more similar expression pattern to the PH than the PVL at the early phase of the regeneration. Early transcriptomic changes and predicted upstream regulators that were found in all three procedures included cell cycle associated genes (E2F1, CCND1, FOXM1, TP53, and RB1), transcription factors (Myc, E2F1, TBX2, FOXM1), DNA replication regulators (CDKN1A, EZH2, RRM2), G1/S-transition regulators (CCNB1, CCND1, RABL6), cytokines and growth factors (CSF2, IL-6, TNF, HGF, VEGF, and EGF), ATM and p53 signaling pathways. The functional pathway, upstream, and network analyses revealed both unique and overlapping molecular mechanisms and pathways for each surgical procedure. Identification of molecular signatures and regenerative signaling pathways for each surgical procedure further our understanding of key regulators of liver regeneration as well as patient populations that are likely to benefit from each procedure.

Validation of the peak bilirubin criterion for outcome after partial hepatectomy.

INTRODUCTION: Postoperative liver failure (PLF) is a dreaded complication after partial hepatectomy. The peak bilirubin criterion (>7.0 mg/dL or ≥120 µmol/L) is used to define PLF. This study aimed to validate the peak bilirubin criterion as postoperative risk indicator for 90-day liver-related mortality. METHODS: Characteristics of 956 consecutive patients who underwent partial hepatectomy at the Maastricht University Medical Centre or Royal Free London between 2005 and 2012 were analyzed by uni- and multivariable analyses with odds ratios (OR) and 95% confidence intervals (95%CI). RESULTS: Thirty-five patients (3.7%) met the postoperative peak bilirubin criterion at median day 19 with a median bilirubin level of 183 [121-588] µmol/L. Sensitivity and specificity for liver-related mortality after major hepatectomy were 41.2% and 94.6%, respectively. The positive predictive value was 22.6%. Predictors of liver-related mortality were the peak bilirubin criterion (p < 0.001, OR = 15.9 [95%CI 5.2-48.7]), moderate-severe steatosis and fibrosis (p = 0.013, OR = 8.5 [95%CI 1.6-46.6]), ASA 3-4 (p = 0.047, OR = 3.0 [95%CI 1.0-8.8]) and age (p = 0.044, OR = 1.1 [95%CI 1.0-1.1]). CONCLUSION: The peak bilirubin criterion has a low sensitivity and positive predictive value for 90-day liver-related mortality after major hepatectomy.

Effect of toll-like receptor 7 and 9 targeted therapy to prevent the development of hepatocellular carcinoma.

BACKGROUND & AIMS: Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll-like receptors play a crucial role in immunity against microbial pathogens and recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC. METHODS: Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG-ODN respectively) and inhibition with a specific antagonist (IRS-954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)-induced model of HCC. RESULTS: TLR7 and TLR9 expression was up-regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS-954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001). CONCLUSION: The data suggest that inhibiting TLR7 and TLR9 with IRS-954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.

The flavonoid monoHER prevents monocrotaline-induced hepatic sinusoidal injury in rats.

BACKGROUND: Sinusoidal obstruction syndrome (SOS) occurs in 50-70% of patients after oxaliplatin treatment for hepatic colorectal metastasis. SOS is associated with portal hypertension and is caused by oxidative damage to endothelial cells and matrix metalloproteinase (MMP) induction. We studied the effect of a flavonoid (monoHER) on SOS prevention. METHODS: A monocrotaline (MTC) SOS model was used in rats, with pre-treatment of monoHER. We studied hepatocellular damage and MMP expression. The potential inhibition of oxaliplatin cytotoxicity by monoHER was tested in vitro in colorectal cancer cell lines. RESULTS: MonoHER ameliorated the increase in portal pressure after MCT (72 hr: 7.3 ± 2.7 mmHg vs. 11.4 ± 3.0 mmHg, P = 0.016 MCT + monoHER vs. MCT, P < 0.01). MonoHER prevented hepatocellular damage (ALT: 48 hr 42.2 ± 3.1 IU/L vs. 253.4 ± 171.7 IU/L, P = 0.034; 72 hr: 46.2 ± 4.3 IU/L vs. 311.9 ± 163.6 IU/L, MCT + monoHER vs. MCT, P < 0.01). The liver damage score was lower in the monoHER group (72 hr: 4.8 ± 3.6 vs. 10.3 ± 0.5, MCT-monoHER vs. MCT, P < 0.01) associated with less inflammatory cell infiltration. Livers of MCT treated rats had higher expression of MMP-9 when compared to monoHER pairs at 24 hr (P = 0.016) and 72 hr (P < 0.001). MonoHER had no effect on in vitro proliferation of colorectal cancer cells when used either alone or in combination with oxaliplatin. CONCLUSIONS: MonoHER prevented MCT induced portal hypertension and hepatic injury in rats.

Use of hepatocyte and stem cells for treatment of post-resectional liver failure: are we there yet?

Post-operative liver failure following extensive resections for liver tumours is a rare but significant complication. The only effective treatment is liver transplantation (LT); however, there is a debate about its use given the high mortality compared with the outcomes of LT for chronic liver diseases. Cell therapy has emerged as a possible alternative to LT especially as endogenous hepatocyte proliferation is likely inhibited in the setting of prior chemo/radiotherapy. Both hepatocyte and stem cell transplantations have shown promising results in the experimental setting; however, there are few reports on their clinical application. This review identifies the potential stem cell sources in the body, and highlights the triggering factors that lead to their mobilization and integration in liver regeneration following major liver resections.

R0 but not R1/R2 resection is associated with better survival than palliative photodynamic therapy in biliary tract cancer.

BACKGROUND: There is a need for better management strategies to improve the survival and quality of life in patients with biliary tract cancer (BTC). AIM: To assess prognostic factors for survival in a large, non-selective cohort of patients with BTC. METHOD: We compared outcomes in 321 patients with a final diagnosis of BTC (cholangiocarcinoma n = 237, gallbladder cancer n = 84) seen in a tertiary referral cancer centre between 1998 and 2007. Survival according to disease stage and treatment category was compared using log-rank testing. Cox's regression analysis was used to determine independent prognostic factors. RESULTS: Eighty-nine (28%) patients underwent a surgical intervention with curative intent, of whom 38% had R0 resections. Among the 321 patients, 34% were given chemo- and/or radiotherapy, 14% were palliated with photodynamic therapy (PDT) and 37% with biliary drainage procedures alone. The overall median survival was 9 months (3-year survival, 14%). R0-resective surgery conferred the most favourable outcome (3-year survival, 57%). Although patients palliated with PDT had more advanced clinical T-stages, their survival was similar to those treated with attempted curative surgery but who had positive resection margins. On multivariable analysis, treatment modality, serum carbohydrate-associated antigen 19-9, distant metastases and vascular involvement were independent prognostic indicators of survival. CONCLUSION: In this large UK series of BTC, palliative PDT resulted in survival similar to those with curatively intended R1/R2 resections. Surgery conferred a survival advantage only in patients with R0 resection margins, emphasising the need for accurate pre-operative staging.

Circulating CYFRA 21-1 is a Specific Diagnostic and Prognostic Biomarker in Biliary Tract Cancer.

BACKGROUND: Biliary tract cancer (BTC) has a poor prognosis, in part related to difficulties in diagnosis. Cytokeratin 19 (CK19) is a constituent of the intermediate filament proteins of epithelial cells. CK19 fragments (CYFRA 21-1) are rarely identified in the blood of healthy individuals. We assessed the utility of CYFRA 21-1 as a diagnostic and prognostic marker of BTC. METHODS: Blood was prospectively collected from patients with benign biliary disease (n = 39), primary sclerosing cholangitis (n = 19), PSC-related cholangiocarcinoma (n = 6) and sporadic BTC (n = 60). CYFRA 21-1 levels were measured in duplicate by ELISA. RESULTS: CYFRA 21-1 (≥ 1.5 ng/mL) had a sensitivity of 56% and specificity of 88%, compared with figures of 79% and 78% for CA 19-9 (≥ 37U/mL). Using a higher cut-off of 3 ng/mL, CYFRA 21-1 had a sensitivity of 30% and specificity of 97%. Combination of CYFRA 21-1 (≥ 1.5 ng/mL) and CA 19-9 (≥ 37 U/mL) resulted in sensitivity and specificity of 45% and 96%. In contrast to CA 19-9, CYFRA 21-1 (≥ 3.0 ng/mL) alone was a strong predictor of prognosis (median survival 2 months vs 10 months, p = 0.001). CONCLUSION: Elevated circulating CYFRA 21-1 is a specific, but less sensitive diagnostic marker than CA 19-9, predicts a poor outcome and may act as a surrogate marker of circulating tumor cells in BTC. Further prospective studies of its utility in assessing operability and response to chemotherapy are needed.

Presentation and management of post-treatment relapse in autoimmune pancreatitis/immunoglobulin G4-associated cholangitis.

BACKGROUND & AIMS: Autoimmune pancreatitis (AIP) is a multisystem disorder that often has extrapancreatic manifestations such as immunoglobulin G4-associated cholangitis (IAC). Patients respond rapidly to steroids but can relapse after therapy. We assessed the clinical management of relapse in a group of patients with AIP/IAC. METHODS: We performed a prospective study of patients diagnosed with AIP from 2004-2007 who received steroids. Treatment outcome was defined clinically, radiologically, and biochemically as response to steroids, remission after steroids, failure to wean steroids, and relapse. Steroids +/- azathioprine (AZA) were used to treat patients who failed, relapsed, or could not be weaned from steroids. RESULTS: Twenty-eight patients with AIP were studied; 23 (82%) had IAC. All patients responded within 6 weeks to prednisolone therapy. Twenty-three patients achieved remission after a median of 5 months of treatment (range, 1.5-17 months), whereas 5 patients (18%) could not be weaned because of a disease flare. Of the patients who achieved remission, 8 of 23 (35%) subsequently relapsed. Overall, 13 of 23 patients (57%) with AIP/IAC relapsed, compared with 0 of the 5 with isolated AIP (P = .04, Fisher exact test). Steroids were increased/restarted in all patients who relapsed; 10 also received AZA. Remission was achieved and maintained in 7 patients; they remain on AZA monotherapy at a median of 14 months (range, 1-27 months). CONCLUSIONS: Relapse or failure to wean steroids occurred in 46% of patients with AIP. Patients with IAC are at particularly high risk of relapse. AZA appears to be effective in patients with post-treatment relapse or who cannot be weaned from steroids. To view this article's video abstract, go to the AGA's YouTube Channel.

FR-167653, a selective p38 MAPK inhibitor, exerts salutary effect on liver cirrhosis through downregulation of Runx2.

Liver cirrhosis remains a difficult-to-treat disease with a substantial morbidity and mortality rate. There is an emerging body of data purporting a pivotal role of the activated p38 mitogen-activated protein kinase (MAPK) in the process of cirrhosis. Several anticirrhotic agents have been developed over the past few years, and most of them exert their effects by indirectly inhibiting the p38 pathway. Effect of a selective p38 inhibitor is yet to be reported. In this study, we evaluated the salutary effect of FR-167653 (FR), a selective p38 inhibitor, in a carbon tetrachloride (CCl(4))-induced rat cirrhotic model. Twenty rats were assigned into four groups: Sham, olive oil only; Control, CCl(4) in olive oil; FR50, FR 50 mg/kg/day and CCl(4); and FR100, FR 100 mg/kg/day and CCl(4). FR dose-dependently inhibited activation of p38 and had an ameliorating effect on cirrhosis formation. Significant dose-dependent reduction in alpha-smooth muscle actin immunostaining and hydroxyproline content of the liver was noticed in the FR-treated rats. Also densitometric analysis showed a significant reduction in azan-stained area in the FR-treated rats. These fibrotic changes were observed in the myofibroblasts including the hepatic stellate cells and portal fibroblasts. mRNA expression of runt-related protein 2 (Runx2), a profibrogenic transcription factor, was significantly low in FR-treated livers, indicating that Runx2 might be a key downstream regulator of the p38 pathway. A similar reduction in expression of Smad4 and tissue inhibitor of metalloproteinase-1 was noticed in the FR-treated rats. In conclusion, FR treatment exerted a significant beneficial effect in a CCl(4)-induced rat cirrhotic model. The ameliorating effect of FR could be partially attributable to an inhibition of the Smad4/p38/Runx2 axis in the cirrhotic liver.

Transforming growth factor-beta1 induced hepatocyte apoptosis--a possible mechanism for growth of colorectal liver metastasis.

The mechanisms of colonization and growth of metastatic liver tumors from colorectal cancers remain obscure. Forty-three resected colorectal metastatic liver tumors with surrounding livers were evaluated for apoptotic index (AI), proliferation index (PI), and immunohistochemical expressions of TGF-beta1. TGF-beta receptor II, Fas, and Fas-ligand. All the parameters were significantly higher in the peri-tumoral livers than in the tumors with the exception of PI, which was significantly high in tumors. Enhanced TGF-beta1 expression was noticed at the interface between the metastatic tumor and the adjacent liver parenchyma. The AIs of hepatocytes in the TGF-beta1-positive areas (8.7 +/- 7.5%, n = 43) were significantly higher when compared with those in the TGF-beta1-negative areas (2.4 +/- 4.5%, n = 42) (p < 0.001). However, the same kind of correlation could not be found in metastatic tumors. The enhanced expression of TGF-beta1 and hepatocyte apoptosis in the peri-tumoral liver parenchyma may suggest that TGF-beta1 plays a substantial role in the development of colorectal liver metastasis.

Sequential changes of connexin32 and connexin26 in ischemia-reperfusion of the liver in rats.

Serial changes in expression of hepatic gap junction components, connexin32 and connexin26 expressions during ischemia (60 min)-reperfusion injury of the liver were evaluated by immunofluorescence staining and reverse transcription-polymerase chain reaction in rats. Hepatic tissue calcium content and liver enzymes (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase), were also examined. Connexin expressions were down-regulated during ischemia and steeply increased during the early reperfusion period. This upsurge in connexin was coincided with the augmentation in tissue calcium content level. And the mRNA levels of connexin changed in parallel with the connexin protein level until 60 min after reperfusion. Since it is known that the changes in intracellular Ca(2+) concentration controls the intercellular communication via gap junction, these findings suggest the possibility that gap junction may play a definitive role in reperfusion injury of the liver. Further studies may be necessary to clarify the exact role of connexins in hepatic ischemia-reperfusion injury.

Pattern of recurrence after extended esophagectomy for squamous cell carcinoma of the esophagus.

BACKGROUND/AIMS: To further investigate the underlying mechanism of the systemic spread of esophageal squamous cell carcinoma. METHODOLOGY: Out of 151 patients who underwent a curative esophageal resection, 41 (27.1%) developed recurrent esophageal cancer. Nine recurrences (22%) were distant-hematogenous, 17 (41.5%) non-hematogenous, and 15 (36.5%) mixed. Hematogenous deposits accompanied 58.5% of the recurrences. The relation between several clinicopathological factors and the pattern of recurrence was evaluated. RESULTS: Univariate analysis recognized the lack of adjuvant chemoradiation, the tumor location in the lower esophagus and the tumor dedifferentiation as promoting factors for hematogenous recurrence. Poorly differentiated or undifferentiated tumors presented a significantly higher microvessel density than moderately or well differentiated tumors. Tumor differentiation and tumor lower localization were independent predictors of hematogenous recurrence. CONCLUSIONS: Patients with poorly differentiated or undifferentiated tumors, which are located at the lower esophagus and present high microvessel density, should be considered at high risk for hematogenous recurrences after extended esophagectomy.

Expression and prognostic significance of PTEN product protein in patients with esophageal squamous cell carcinoma.

BACKGROUND: PTEN is a candidate tumor-suppressor gene in a variety of malignant tumors. The prognostic importance of PTEN product protein (PTEN) and its correlation with clinicopathologic characteristics have yet to be delineated in patients with esophageal carcinoma. METHODS: Specimens from 97 patients with esophageal squamous cell carcinoma were used for the immunohistochemical evaluation of PTEN expression. RESULTS: PTEN expression was detected in the nucleus in 48 specimens (49.5%). There were statistically significant correlations between nuclear PTEN expression and macroscopic tumor classification, T stage, and American Joint Committee on Cancer (AJCC) stage (P < 0.01), indicating that PTEN expression was down-regulated by advancement of the disease process. There were no statistically significant correlations between nuclear PTEN expression and the intensity and extent of cytoplasmic PTEN expression. The 10-year overall survival rate was significantly better in patients with positive nuclear PTEN expression (n = 48 patients) compared with the rate in patients with negative nuclear PTEN expression (n = 49 patients; P < 0.01). The results of a multivariate analysis of factors that were prognostic for survival showed that AJCC stage (P < 0.05; relative risk, 2.038) and negative nuclear PTEN expression (P < 0.05; relative risk, 1.825) were significant factors indicative of poor survival. CONCLUSIONS: Nuclear PTEN expression may be a favorable biologic marker and a useful prognostic indicator in patients with esophageal squamous cell carcinoma.

Expression and prognostic significance of S100A2 protein in squamous cell carcinoma of the esophagus.

The purpose of this study was to evaluate the expression of S100A2 Ca2+-binding protein and its prognostic significance in the management of squamous cell carcinoma of the esophagus. Changes in cytosolic Ca2+ concentration control a wide range of cellular responses including cellular apoptosis. Intracellular S100 Ca2+-binding proteins are key molecules in transducing Ca2+ signaling. Among these, S100A2 has recently attracted major interest due to its stable expression in normal epithelia and down-regulation in some tumors. As a candidate tumor suppressor, expression of S100A2 has been proposed as a valuable prognostic marker in different tumors. We examined the clinical significance of S100A2 expression in 116 resected specimens of esophageal squamous cell carcinomas (ESCC) using immunohistochemistry. S100A2 was positive in 49 cases (42.2%) and its expression was significantly higher in large (p=0.01) and well differentiated tumors (p=0.013). Lymph node-positive tumors had a lower expression of S100A2 protein in comparison to the corresponding lymph node negative equivalents in each of the T stages, but the difference was statistically significant (p=0.041) only for the T1b tumors. S100A2 status became an independent predictor of patient survival (p=0.026) in lymph node-negative cases but not in node-positive cases. Evaluation of S100A2 protein expression may play an important role in the management of ESCC. The node-negative ESCC patients without S100A2 expression might be a high-risk group with poor survival and will need further attention to design appropriate adjuvant therapy.