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1.  Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing 
PLoS Medicine  2014;11(4):e1001628.
Gary Peltz, Jeffrey Glenn, and colleagues report that a pre-clinical mouse toxicology model can detect liver toxicity of a drug that caused liver failure in several early clinical trial participants in 1993.
Please see later in the article for the Editors' Summary
Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.
Methods and Findings
Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.
FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants.
Please see later in the article for the Editors' Summary
Editors' Summary
Before new drugs are approved for clinical use, they undergo extensive preclinical (laboratory-based) and clinical testing. In the preclinical studies, scientists investigate the causes of diseases, identify potential new drugs, and test promising drug candidates in animals. Animal testing is performed to determine whether the new drug is likely to work, and to screen for drug-induced toxicity. In preclinical toxicology studies, new drugs are given to two or more animal species to find out whether the drug has any short- or long-term toxic effects such as damage to the liver (hepatotoxicity). Drugs that pass these animal tests enter clinical trials. Phase I clinical trials test new drugs in a handful of healthy volunteers or patients to evaluate their safety and to identify possible side effects. In phase II trials, a larger group of patients receives the new drug to evaluate its safety further and to get an initial idea of its effectiveness. Finally, in phase III trials, very large groups of patients are randomly assigned to receive the new drug or an established treatment for their disease. These randomized controlled trials provide detailed information about the effectiveness and safety of a candidate drug, and must be completed before a drug can be approved for clinical use.
Why Was This Study Done?
Since animals are not perfect models for people, candidate drugs can cause toxicities in clinical trials that were not predicted by preclinical toxicology testing performed using animal species. For example, in 1993, 15 participants in a phase II trial were given a nucleoside analogue called fialuridine to treat hepatitis B virus infection (nucleoside analogues often have antiviral activity). Seven participants developed liver failure and lactic acidosis (buildup of lactic acid in the blood). Analysis of liver tissue from the affected participants revealed steatosis (fatty degeneration), intracellular fat droplets, and swollen mitochondria (these organelles are the powerhouses of the cell). Five participants subsequently died, and two had to have a liver transplant. In preclinical toxicology testing in mice, rats, dogs, and primates, there had been no indications that fialuridine would be hepatotoxic in people. It now seems that the expression of a nucleoside transporter in the mitochondria of humans but not of other animals may underlie the human-specific mitochondrial toxicity and hepatotoxicity of fialuridine. With several other nucleoside analogues in development, a better screening tool for human-specific mitochondrial toxicity is needed. In this study, the researchers investigate whether fialuridine toxicity can be detected in TK-NOG mice with chimeric (humanized) livers. TK-NOG mice are immunodeficient mice that have been genetically engineered so that human liver cells (hepatocytes) transplanted into these animals establish a long-lived mature “human organ.”
What Did the Researchers Do and Find?
The researchers treated chimeric (with transplanted human liver cells) and control (without transplanted human liver cells) TK-NOG mice with several doses of fialuridine. After treatment with the highest dose (1,600-fold above the dose used in the phase II trial) for four days, the chimeric mice developed liver failure and lactic acidosis. Moreover, steatosis and lipid and mitochondrial abnormalities developed in the regions of their livers that contained human hepatocytes but not in regions that contained mouse hepatocytes. Notably, the control mice had not developed liver toxicity after 14 days of treatment with the highest dose of drug. Liver toxicity was also easily detectable in chimeric mice that had been treated for 14 days with a fialuridine dose only 10-fold above that used in the human trial. Treatment with another nucleoside analogue that does not cause liver toxicity in people did not cause liver toxicity in the chimeric mice.
What Do These Findings Mean?
These findings show that fialuridine-induced liver toxicity can be readily detected using TK-NOG mice that have humanized livers at drug doses only 10-fold higher than those that caused liver failure in the phase II trial. Although the liver toxicity developed much more quickly in these mice than in the human trial participants, the clinical features, laboratory abnormalities, and structural changes seen in the fialuridine-treated chimeric TK-NOG mice closely mirrored those seen in fialuridine-treated people. The use of TK-NOG mice containing humanized livers in toxicology testing will not reveal whether drugs have human-specific toxicities outside the liver. Since they are highly immunocompromised, chimeric TK-NOG mice cannot be used to detect immune-mediated drug toxicities. Nevertheless, these findings suggest that the use of chimeric mice in toxicology studies could help improve the safety of candidate drugs that are tested in humans.
Additional Information
Please access these websites via the online version of this summary at
The US Food and Drug Administration, the body that approves drugs for clinical use in the US, provides an overview for patients about the drug development process from the laboratory to the clinic
The UK Medicines and Healthcare Products Regulatory Agency (MHRA) provides more detailed information for patients and the public about the drug development process, including a section on preclinical research, which includes information on animal testing
The US National Institutes of Health provides information about clinical trials, including personal stories from people who have taken part in clinical trials
The UK National Health Service Choices website has information for patients about clinical trials and medical research, including personal stories about participation in clinical trials
Understanding Animal Research is a UK advocacy group that provides information about the importance of animal research to the public, teachers, scientists, journalists, and policy makers
Wikipedia has a page on animal testing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC3988005  PMID: 24736310
2.  Delayed ethanol elimination and enhanced susceptibility to ethanol-induced hepatosteatosis after liver resection 
World Journal of Gastroenterology : WJG  2014;20(48):18249-18259.
AIM: To investigate ethanol-induced hepatic steatosis after liver resection and the mechanisms behind it.
METHODS: First, the preliminary examination was performed on 6 sham-operated (Sham) and 30 partial hepatectomy (PH) male Wistar rats (8-wk-old) to evaluate the recovery of the liver weight and liver function after liver resection. PH rats were sacrificed at the indicated time points (4, 8, and 12 h; 1, 3, and 7 d) after PH. Second, the time point for the beginning of the chronic ethanol exposure (1 wk after sham- or PH-operation) was determined based on the results of the preliminary examination. Finally, pair-feeding was performed with a controlled diet or with a 5-g/dL ethanol liquid diet for 28 d in another 35 age-matched male Wistar rats with a one-week recovery after undergoing a sham- (n = 15) or PH-operation (n = 20) to evaluate the ethanol-induced liver injury after liver resection. Hepatic steatosis, liver function, fatty acid synthase (Fas) gene expression level, the expression of lipid metabolism-associated enzyme regulator genes [sterol regulatory element binding protein (Srebp)-1 and peroxisome proliferator-activated receptor (Ppar)-α], the mediators that alter lipid metabolism [plasminogen activator (Pai)-1 gene expression level and tumor necrosis factor (Tnf)-α production], and hepatic class-1 alcohol dehydrogenase (Adh1)-associated ethanol elimination were investigated in the 4 groups based on histological, immunohistochemical, biochemical, Western blotting, reverse transcriptase chain reaction, and blood ethanol concentration analyses. The relevant gene expression levels, liver weight, and liver function were assessed before and 1 wk after surgery to determine the subject’s recovery from the liver resection using the rats that had been subjected to the preliminary examination.
RESULTS: In the PH rats, ethanol induced marked hepatic steatosis with impaired liver functioning, as evidenced by the accumulation of fatty droplets within the hepatocytes, the higher increases in their hepatic triglyceride and blood alanine aminotransferase and blood aspartate aminotransferase levels after the 28-d pair-feeding period. The Sham-ethanol rats, not the PH-ethanol rats, demonstrated the up-regulation of Srebp-1 and the down-regulation of Ppar-α mRNA expression levels after the 28-d pair-feeding period. The 28-d ethanol administration induced the up-regulation of Pai-1 gene expression level and an overproduction of TNF-α in the Sham and the PH rats; however, the effect was more significant in the PH rats. The PH-ethanol rats (n = 4) showed higher residual blood ethanol concentrations than did the Sham-ethanol rats (n = 6) after a 5-h fast (0.66 ± 0.4 mg/mL vs 0.2 ± 0.1 mg/mL, P < 0.05); these effects manifested without up-regulation of Adh1 gene expression, which was present in the Sham-ethanol group after the 28-d pair-feeding period. One week after the liver resection, the liver weight, function, the gene expression levels of Fas, Srebp-1, Ppar-α, Pai-1 and Tnf-α recovered; however, the Adh1 gene expression did not recover in rats.
CONCLUSION: Desensitization to post-hepatectomy ethanol treatment and slow recovery from PH in Adh1 gene expression enhanced the susceptibility to ethanol-induced hepatic steatosis after PH in rats.
PMCID: PMC4277962  PMID: 25561792
Ethanol; Hepatic class-1 alcohol dehydrogenase; Ethanol elimination; Hepatic steatosis; Liver function; Liver resection
3.  Impact of Future Remnant Liver Volume on Post-Hepatectomy Regeneration in Non-Cirrhotic Livers 
Frontiers in Surgery  2014;1:10.
Objective: The purpose of the study is to detect if some parameters can be considered as predictors of liver regeneration in two different patient populations composed of in living donors for adult to adult living donor liver transplant and patients with hepatic malignancies within a single institution.
Summary Background Data: Preoperative multi-detector computed tomography volumetry is an essential tool to assess the volume of the remnant liver.
Methods: A retrospective analysis from an ongoing clinical study on 100 liver resections, between 2004 and 2010. Seventy patients were right lobe living donors for liver transplantation and 30 patients were resected for treatment of tumors. Pre-surgical factors such as age, weight, height, body mass index (BMI), original liver volume, future remnant liver volume (FRLV), spleen volume, liver function tests, creatinine, platelet count, steatosis, portal vein embolization, and number of resected segments were analyzed to evidence potential markers for liver regeneration.
Results: Follow-up period did not influence the amount of liver regenerated: the linear regression evidenced that there is no correlation between percentage of liver regeneration and time of follow-up (p = 0.88). The pre-surgical variables that resulted markers of liver regeneration include higher preoperative values of BMI (p = 0.01), bilirubin (p = 0.04), glucose (p = 0.05), and gamma-glutamyl transpeptidase (p = 0.014); the most important association was revealed regarding the lower FRLV (p < 0.0001) and percentage of liver regeneration. The stepwise regression revealed a strong impact of FRLV (p < 0.0001) on the other predictor variables.
Conclusion: Liver regeneration follows similar pathway in living donor and in patients resected for cancer. Small FRLV tends to regenerate more and faster, confirming that a larger resections may lead to a greater promotion of liver regeneration in patients with optimal conditions in terms of body habitus, preoperative liver function tests, and glucose level.
PMCID: PMC4286982  PMID: 25593935
living related liver transplantation; regeneration; liver resection; portal vein embolization
4.  Effect of preoperative chemotherapy on postoperative liver regeneration following hepatic resection as estimated by liver volume 
In order to analyze postoperative liver regeneration following hepatic resection after chemotherapy, we retrospectively investigated the differences in liver regeneration by comparing changes of residual liver volume in three groups: a living liver donor group and two groups of patients with colorectal liver metastases who did and did not undergo preoperative chemotherapy.
This study included 32 patients who had at least segmental anatomical hepatic resection. Residual liver volume, early postoperative liver volume, and late postoperative liver volume were calculated to study the changes over time. From the histopathological analysis of chemotherapy-induced liver disorders, the effect on liver regeneration according to the histopathology of noncancerous liver tissue was also compared between the two colorectal cancer groups using Kleiner’s score for steatohepatitis grading {Hepatology, 41(6):1313–1321, 2005} and sinusoidal obstruction syndrome (SOS) grading for sinusoidal obstructions {Ann Oncol, 15(3):460–466, 2004}.
Assuming a preoperative liver volume of 100%, mean late postoperative liver volumes in the three groups (the living liver donor group and the colorectal cancer groups with or without chemotherapy) were 91.1%, 80.8%, and 81.3%, respectively, with about the same rate of liver regeneration among the three groups. Histopathological analysis revealed no correlation between either the Kleiner’s scores or the SOS grading and liver regeneration.
As estimated by liver volume, the level of liver regeneration was the same in normal livers, tumor-bearing livers, and post-chemotherapy tumor-bearing livers. Liver regeneration was not adversely affected by the extent to which steatosis or sinusoidal dilatation was induced in noncancerous tissue by chemotherapy in patients scheduled for surgery.
PMCID: PMC3621216  PMID: 23497123
5.  Outcomes of simultaneous and delayed resections of synchronous colorectal liver metastases 
Canadian Journal of Surgery  2009;52(6):E241-E244.
The optimal strategy for the treatment of synchronous colorectal liver metastases has not been established yet. In this study, we present the outcomes and survival rates of the patients who underwent simultaneous or delayed resections.
We performed a retrospective analysis of liver resections in our institution between 1997 and 2006.
Among the 89 patients presenting with synchronous colorectal liver metastases, 28 underwent simultaneous and 61 underwent delayed resection. Age, sex and localization of the primary tumour were similar in the 2 groups. Duration of surgery and hospital stay were longer in the simultaneous resection group, and blood loss was also greater in this group. However, these factors did not influence the frequency of complications, which did not differ between the groups. When we included data from initial colectomy, these differences were either not significant or in favour of synchronous resection. In the delayed resection group, colon resection was performed in different hospitals. The 1-, 3- and 5-year survival rates were 78%, 70% and 45%, respectively, in the simultaneous and 88%, 55% and 38%, respectively, in the delayed resection groups.
In select patients, the risk of simultaneous resection of synchronous colorectal liver metastases is comparable to delayed resection, and increases in blood loss and operating time associated with simultaneous resections do not have a negative influence on long-term outcome. Positive outcomes of simultaneous liver resections in our study could be a result of good patient selection or experience with oncological liver surgery.
PMCID: PMC2792389  PMID: 20011158
6.  Low immediate postoperative platelet count is associated with hepatic insufficiency after hepatectomy 
World Journal of Gastroenterology : WJG  2014;20(33):11871-11877.
AIM: To investigate the relationship between low immediate postoperative platelet count and perioperative outcome after liver resection in patients with hepatocellular carcinoma (HCC).
METHODS: In a cohort of 565 consecutive hepatitis B-related HCC patients who underwent major liver resection, the characteristics and clinical outcomes after liver resection were compared between patients with immediate postoperative platelet count < 100 × 109/L and patients with platelet count ≥ 100 × 109/L. Risk factors for postoperative hepatic insufficiency were evaluated by multivariate analysis.
RESULTS: Patients with a low immediate postoperative platelet count (< 100 × 109/L) had more grade III-V complications (20.5% vs 12.4%, P = 0.016), and higher rates of postoperative liver failure (6.8% vs 2.6%, P = 0.02), hepatic insufficiency (31.5% vs 21.2%, P < 0.001) and mortality (6.8% vs 0.5%, P < 0.001), compared to patients with a platelet count ≥ 100 × 109/L. The alanine aminotransferase levels on postoperative days 3 and 5, and bilirubin on postoperative days 1, 3 and 5 were higher in patients with immediate postoperative low platelet count. Multivariate analysis revealed that immediate postoperative low platelet count, rather than preoperative low platelet count, was a significant independent risk factor for hepatic insufficiency.
CONCLUSION: A low immediate postoperative platelet count is an independent risk factor for hepatic insufficiency. Platelets can mediate liver regeneration in the cirrhotic liver.
PMCID: PMC4155380  PMID: 25206294
Thrombocytopenia; Hepatic insufficiency; Hepatocellular carcinoma; Hepatectomy; Hepatitis B
7.  Promotion of hepatic metastases by liver resection in the rat. 
British Journal of Cancer  1992;65(6):794-797.
In the early period following radical hepatectomy for hepatoma, recurrences in the remaining liver are frequently found. In regenerating liver, implantation and growth of tumour cells released into the portal system during surgical treatment might be promoted. We examined the relationship between liver regeneration and the formation of metastases following hepatic resection. Intraportal injections of rat ascites containing hepatoma AH130 cells at a concentration of 1 x 10(5) cells 0.2 ml-1 were made at various periods following two thirds liver resection in rats. Tumour cell injections immediately at 24 h after surgery resulted in an increased number of hepatic metastases compared with control animals. Tumour cell injections 2 weeks after hepatectomy, however, had no significant difference in effect compared with control rats. In contrast, tumour cells injected immediately after removal of half of the caudate lobe resulted in the same number of metastases as control animals. These results demonstrate that the number of artificially induced hepatic metastases was increased during an initial period of active liver regeneration and was proportional to the volume of hepatectomy. The effect of 5-fluorouracil (5FU) or mitomycin C (MMC) as inhibitors of hepatic regeneration on liver metastasis after hepatectomy was studied. The administration of 5FU (20 mg kg-1) or MMC (0.2 mg kg-1) immediately, 24 and 48 h after hepatectomy resulted in a marked reduction in metastatic lesions. The administration of 5FU caused delays in weight gain and decreases in the wet weight of remaining liver, while MMC had no effect on either. Accordingly, results of 5FU administration may be due to inhibitory effects on liver regeneration whilst that of MMC administration may be due to cytocidal antitumour effect. The effect of OK-432 as an immunoactivator on the implantation and growth of tumour cells in regenerating liver was also studied. Pretreatment with OK-432, 0.5 mg intraperitoneally on 7 consecutive days, had no effect on hepatic metastases. The pathophysiology of liver regeneration may enhance hematogenous hepatic metastasis and release of tumour cells during surgical manipulation may represent an important cause of recurrence following hepatic resection.
PMCID: PMC1977781  PMID: 1616850
8.  High levels of serum VEGF and TIMP-1 are correlated with colon cancer liver metastasis and intrahepatic recurrence after liver resection 
Oncology Letters  2012;4(1):123-130.
The purpose of this study was to develop predictive/prognostic markers for liver metastasis and recurrence following liver resection, investigating not only clinical parameters but also molecular markers that are known to be involved in the process of liver metastasis. Seventy colon cancer patients with either no distant metastasis (group A) or with resectable synchronous liver metastasis only (group B) were prospectively enrolled. All 70 patients received curative resection of the primary tumor. Group B patients underwent additional liver resection. Clinical parameters as well as serum levels of molecular markers [carcinoembryonic antigen (CEA), osteopontin, matrix metalloproteinase-7 (MMP-7), tissue inhibitor of metalloproteinase-1 (TIMP-1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and E-selectin] from both tumor drainage (DV) and peripheral veins (PV) were analyzed. Results showed the clinical parameters were not significantly different between groups A and B. Nonetheless, the levels of VEGF and TIMP-1 from both DV and PV were significantly higher in group B compared to group A. In group A, 10 out of 33 (27.0%) patients developed metachronous liver metastasis. High levels of VEGF and TIMP-1 from DV were found to be significantly correlated with metachronous liver metastasis. In group B, 20 out of 33 (60.6%) patients had intrahepatic recurrence following resection of synchronous liver metastasis. The levels of VEGF from DV and the levels of TIMP-1 both from PV and DV were found to be significantly correlated with intrahepatic recurrence. Forty patients (7 from group A and 33 from group B) had liver resection and their 5-year disease-free survival rate was 15.9%. Univariate and multivariate analyses of prognostic factors revealed that the levels of VEGF and TIMP-1 from DV as well as the presence of lymph node metastasis from the primary tumor, synchronous metastasis and R1 resection were significantly associated with worse prognosis. The colon cancer patients with high levels of VEGF and TIMP-1 detected from the DV at the time of their initial surgery were found to have a high risk of metachronous liver metastasis and hepatic recurrence following the resection of synchronous liver metastasis. The high levels of VEGF and TIMP-1 were found to be significant predictive factors for poor prognosis following liver resection. These results require validation but pave the way for future transitional or clinical studies that may provide a greater understanding of colon cancer liver metastasis.
PMCID: PMC3398360  PMID: 22807974
metastasis; colon cancer; molecular marker; liver
9.  Is There a Role for Perioperative Nutritional Support in Liver Resection? 
HPB Surgery  1997;10(3):177-179.
Background: Resection of hepatocellular carcinoma is associated with high rates of morbidity and mortality. Since intensive nutritional support can reduce the catabolic response and improve protein synthesis and liver regeneration, we performed a prospective study to investigate whether perioperative nutritional support could improve outcome in patients undergoing hepatectomy for hepatocellular carcinoma.
Methods: We studied 124 patients undergoing resection of hepatocellular carcinoma. Sixty-four patients (39 with cirrhosis, 18 with chronic active hepatitis, and 7 with no associated liver disease) were randomly assigned to receive perioperative intravenous nutritional support in addition to their oral diet, and 60 patients (33 with cirrhosis, 12 with chronic active hepatitis, and 15 with no associated liver disease) were randomly assigned to a control group. The perioperative nutritional therapy consisted of a solution enriched with 35 percent branched chain amino acids, dextrose, and lipid emulsion (50 percent medium-chain trigylcerides) given intravenously for 14 days perioperatively).
Results: There was a reduction in the overall postoperative morbidity rate in the perioperative-nutrition group as compared with the control group (34 percent vs. 55 percent; relative risk, 0.66; 95 percent confidence interval, 0.45 to 0.96), predominantly because of fewer septic complications (17 percent vs. 37 percent; relative risk, 0.57; 95 percent confidence interval, 0.34 to 0.96). There were also a reduction in the requirement for diuretic agents to control ascites (25 percent vs. 50 percent; relative risk, 0.57; 95 percent confidence interval, 0.37 to 0.87), less weight loss after hepatectomy (median loss, 0 kg vs. 1.4 kg; P=0.01), and less deterioration of liver function as measured by the change in the rate of clearance of indocyanine green (–2.8 percent vs. –4.8 percent at 20 minutes, P=0.05). These benefits were seen predominantly in the patients with underlying cirrhosis who underwent major hepatechtomy. There were five deaths during hospitalization in the perioperativenutrition group, and nine in the control group (P not significant).
Conclusions: Perioperative nutritional support can reduce complications after major hepatectomy for hepatocellular carcinoma associated with cirrhosis.
PMCID: PMC2423856  PMID: 9174865
10.  Hypoxia preconditioned bone marrow mesenchymal stem cells promote liver regeneration in a rat massive hepatectomy model 
Bone marrow mesenchymal stem cells (BMMSCs) have been reported to facilitate liver regeneration after toxic injuries. However, the effect of BMMSCs on liver regeneration after massive hepatectomy is barely studied. Here we explored whether infusion of BMMSCs promotes liver regeneration in a rat massive hepatectomy model.
Hypoxia preconditioning was achieved by culturing BMMSCs under a hypoxia environment. Then 85% hepatectomy was performed and hypoxia or normoxia preconditioned BMMSCs were infused into the portal vein. A group of rats received vascular endothelial growth factor (VEGF) neutralizing antibody perioperatively, and underwent 85% hepatectomy and a subsequent infusion of hypoxia preconditioned BMMSCs to verify the role of VEGF in the effects of BMMSCs on liver regeneration. Liver samples were collected and liver regeneration was evaluated postoperatively.
Hypoxia preconditioning enhanced the expression of VEGF in BMMSCs in vitro. Infusion of BMMSCs promoted proliferation of hepatocytes, reflected by elevated cyclin D1 expression and proliferating cell nuclear antigen-positive hepatocytes. However, BMMSC infusion did not improve the serum albumin level, liver weight/body weight ratio, and survival after operation. Infusion of hypoxia preconditioned BMMSCs significantly elevated cyclin D1, proliferating cell nuclear antigen-positive hepatocytes, liver weight/body weight ratio, and survival compared with normoxia preconditioned BMMSCs, accompanied by an increased serum albumin level. The level of VEGF in liver homogenate was much higher in hypoxia preconditioned BMMSC-treated animals than in other groups. In addition, the perioperative injection of VEGF neutralizing antibody significantly blocked the therapeutic effects of hypoxia preconditioned BMMSCs on liver injury and regeneration in this model.
Hypoxia preconditioned BMMSCs enhanced liver regeneration after massive hepatectomy in rats, possibly by upregulating the level of VEGF.
PMCID: PMC3854783  PMID: 23856418
Bone marrow mesenchymal stem cell; Liver regeneration; Hypoxia preconditioning
11.  Genomic Predictors for Recurrence Patterns of Hepatocellular Carcinoma: Model Derivation and Validation 
PLoS Medicine  2014;11(12):e1001770.
In this study, Lee and colleagues develop a genomic predictor that can identify patients at high risk for late recurrence of hepatocellular carcinoma (HCC) and provided new biomarkers for risk stratification.
Typically observed at 2 y after surgical resection, late recurrence is a major challenge in the management of hepatocellular carcinoma (HCC). We aimed to develop a genomic predictor that can identify patients at high risk for late recurrence and assess its clinical implications.
Methods and Findings
Systematic analysis of gene expression data from human liver undergoing hepatic injury and regeneration revealed a 233-gene signature that was significantly associated with late recurrence of HCC. Using this signature, we developed a prognostic predictor that can identify patients at high risk of late recurrence, and tested and validated the robustness of the predictor in patients (n = 396) who underwent surgery between 1990 and 2011 at four centers (210 recurrences during a median of 3.7 y of follow-up). In multivariate analysis, this signature was the strongest risk factor for late recurrence (hazard ratio, 2.2; 95% confidence interval, 1.3–3.7; p = 0.002). In contrast, our previously developed tumor-derived 65-gene risk score was significantly associated with early recurrence (p = 0.005) but not with late recurrence (p = 0.7). In multivariate analysis, the 65-gene risk score was the strongest risk factor for very early recurrence (<1 y after surgical resection) (hazard ratio, 1.7; 95% confidence interval, 1.1–2.6; p = 0.01). The potential significance of STAT3 activation in late recurrence was predicted by gene network analysis and validated later. We also developed and validated 4- and 20-gene predictors from the full 233-gene predictor. The main limitation of the study is that most of the patients in our study were hepatitis B virus–positive. Further investigations are needed to test our prediction models in patients with different etiologies of HCC, such as hepatitis C virus.
Two independently developed predictors reflected well the differences between early and late recurrence of HCC at the molecular level and provided new biomarkers for risk stratification.
Please see later in the article for the Editors' Summary
Editors' Summary
Primary liver cancer—a tumor that starts when a liver cell acquires genetic changes that allow it to grow uncontrollably—is the second-leading cause of cancer-related deaths worldwide, killing more than 600,000 people annually. If hepatocellular cancer (HCC; the most common type of liver cancer) is diagnosed in its early stages, it can be treated by surgically removing part of the liver (resection), by liver transplantation, or by local ablation, which uses an electric current to destroy the cancer cells. Unfortunately, the symptoms of HCC, which include weight loss, tiredness, and jaundice (yellowing of the skin and eyes), are vague and rarely appear until the cancer has spread throughout the liver. Consequently, HCC is rarely diagnosed before the cancer is advanced and untreatable, and has a poor prognosis (likely outcome)—fewer than 5% of patients survive for five or more years after diagnosis. The exact cause of HCC is unclear, but chronic liver (hepatic) injury and inflammation (caused, for example, by infection with hepatitis B virus [HBV] or by alcohol abuse) promote tumor development.
Why Was This Study Done?
Even when it is diagnosed early, HCC has a poor prognosis because it often recurs. Patients treated for HCC can experience two distinct types of tumor recurrence. Early recurrence, which usually happens within the first two years after surgery, arises from the spread of primary cancer cells into the surrounding liver that left behind during surgery. Late recurrence, which typically happens more than two years after surgery, involves the development of completely new tumors and seems to be the result of chronic liver damage. Because early and late recurrence have different clinical courses, it would be useful to be able to predict which patients are at high risk of which type of recurrence. Given that injury, inflammation, and regeneration seem to prime the liver for HCC development, might the gene expression patterns associated with these conditions serve as predictive markers for the identification of patients at risk of late recurrence of HCC? Here, the researchers develop a genomic predictor for the late recurrence of HCC by examining gene expression patterns in tissue samples from livers that were undergoing injury and regeneration.
What Did the Researchers Do and Find?
By comparing gene expression data obtained from liver biopsies taken before and after liver transplantation or resection and recorded in the US National Center for Biotechnology Information Gene Expression Omnibus database, the researchers identified 233 genes whose expression in liver differed before and after liver injury (the hepatic injury and regeneration, or HIR, signature). Statistical analyses indicate that the expression of the HIR signature in archived tissue samples was significantly associated with late recurrence of HCC in three independent groups of patients, but not with early recurrence (a significant association between two variables is one that is unlikely to have arisen by chance). By contrast, a tumor-derived 65-gene signature previously developed by the researchers was significantly associated with early recurrence but not with late recurrence. Notably, as few as four genes from the HIR signature were sufficient to construct a reliable predictor for late recurrence of HCC. Finally, the researchers report that many of the genes in the HIR signature encode proteins involved in inflammation and cell death, but that others encode proteins involved in cellular growth and proliferation such as STAT3, a protein with a well-known role in liver regeneration.
What Do These Findings Mean?
These findings identify a gene expression signature that was significantly associated with late recurrence of HCC in three independent groups of patients. Because most of these patients were infected with HBV, the ability of the HIR signature to predict late occurrence of HCC may be limited to HBV-related HCC and may not be generalizable to HCC related to other causes. Moreover, the predictive ability of the HIR signature needs to be tested in a prospective study in which samples are taken and analyzed at baseline and patients are followed to see whether their HCC recurs; the current retrospective study analyzed stored tissue samples. Importantly, however, the HIR signature associated with late recurrence and the 65-gene signature associated with early recurrence provide new insights into the biological differences between late and early recurrence of HCC at the molecular level. Knowing about these differences may lead to new treatments for HCC and may help clinicians choose the most appropriate treatments for their patients.
Additional Information
Please access these websites via the online version of this summary at
The US National Cancer Institute provides information about all aspects of cancer, including detailed information for patients and professionals about primary liver cancer (in English and Spanish)
The American Cancer Society also provides information about liver cancer (including information on support programs and services; available in several languages)
The UK National Health Service Choices website provides information about primary liver cancer (including a video about coping with cancer)
Cancer Research UK (a not-for-profit organization) also provides detailed information about primary liver cancer (including information about living with primary liver cancer)
MD Anderson Cancer Center provides information about symptoms, diagnosis, treatment, and prevention of primary liver cancer
MedlinePlus provides links to further resources about liver cancer (in English and Spanish)
PMCID: PMC4275163  PMID: 25536056
12.  Splenectomy affects the balance between hepatic growth factor and transforming growth factor-β and its effect on liver regeneration is dependent on the amount of liver resection in rats 
Small-for-size syndrome (SFSS) is a major problem in liver surgery, and splenectomy has been used to prevent SFSS. However, it is unknown whether splenectomy has the same effect on liver regeneration in both standard and marginal hepatectomy. The aim of this study is to see a difference in effect of splenectomy on liver regeneration according to the amount of liver resection.
Thirty male Sprague-Dawley rats (220 to 260 g) were divided into the following five groups: control (n = 6), 70% hepatectomy (n = 6), 70% hepatectomy with splenectomy (n = 6), 90% hepatectomy (n = 6), and 90% hepatectomy with splenectomy (n = 6). The animals were euthanized 24 hours after surgery and liver specimens were obtained. To assess liver regeneration, we performed immunohistochemistry of liver tissue using 5-bromo-2-deoxyuridine (BrdU) labeling and Western blot analysis of hepatic growth factor (HGF) and transforming growth factor-β (TGF-β) in the liver tissue.
The splenectomized subgroup had a higher BrdU-positive cell count in the 90% hepatectomy group, but not in the 70% hepatectomy group (P < 0.001). Splenectomy significantly decreased TGF-β expression (P = 0.005) and increased the HGF to TGF-β ratio (P = 0.002) in the 90% hepatectomy group, but not in the 70% hepatectomy group.
The positive effect of splenectomy on liver regeneration was greater in the group with the larger liver resection. This phenomenon may be related to the relative balance between HGF and TGF-β in the liver.
PMCID: PMC3319778  PMID: 22493765
Liver regeneration; Splenectomy; Hepatectomy; HGF; Transforming growth factor beta
13.  Patients with multiple synchronous colonic cancer hepatic metastases benefit from enrolment in a “liver first” approach protocol 
World Journal of Hepatology  2014;6(7):513-519.
AIM: To assess a protocol for treating patients with multiple synchronous colonic cancer liver metastases, which are unresectable in one stage.
METHODS: Patients enrolled in the “liver first” protocol presented with colon-only (not rectal) cancer and multiple synchronous hepatic metastases (type II or III). All patients showed good performance status (ECOG PS 0-1) and were treated with curative intent. Complete oncologic staging including positron emission tomography-computed tomography was performed in order to rule out extrahepatic disease. If bowel obstruction was imminent, an intraluminal colonic stent was placed endoscopically. Subsequently, all patients received standardised neo-adjuvant chemotherapy, that is, FOLFOX or XELOX regimens combined with an antiangiogenic agent (bevacizumab or cetuximab). Provided that a response to chemotherapy was observed, patients underwent either one or two hepatectomies with or without portal vein embolization followed by the indicated colectomy. Further chemotherapy was administered after each procedure. Re-staging was performed after each chemotherapeutic treatment. Disease progression at any stage resulted in discontinuation of the protocol and conversion to palliative disease management.
RESULTS: Prospectively recorded data from 11 consecutive patients (8 men) were analysed for this study. Their mean age at the time of their first assessment was 65.7 (SD ± 15.3) years. Six (54.6%) patients presented with type III metastatic disease. The minimum and maximum follow-up periods were 7.3 and 39.6 mo, respectively. The mean overall survival of all patients was 16.5 (95%CI: 10.0-23.2) mo. A colonic stent had to be placed in 5 (45.5%) patients due to the onset of an intraluminal obstruction. Four (36.4%) patients succeeded in completing all planned surgical operations. Their mean overall survival was 27.2 (95%CI: 15.1-39.3) mo and the mean disease-free survival was 7.7 (95%CI: 3.0-12.5) mo. Patients, who were obliged to shift to palliative treatment due to disease progression, had a mean overall survival of 10.5 (95%CI: 8.6-12.4) mo. None of these patients underwent palliative colectomy. No postoperative mortality was recorded.
CONCLUSION: The implementation of a structured “liver first” approach protocol for the treatment of patients with extensive, liver-limited colon cancer metastatic disease may be beneficial.
PMCID: PMC4110543  PMID: 25068003
Clinical protocols; Colectomy; Colon cancer; Hepatectomy; Liver neoplasm
The Journal of surgical research  2012;178(1):280-287.
This study examines the effects of types of liver resection on the growth of liver and lung metastasis.
Materials and Methods
Experimental liver metastases were established by spleen injection of the Colon 26 murine adenocarcinoma cell line expressing GFP into transgenic nude mice expressing RFP. Experimental lung metastases were established by tail vein injection with Colon 26-GFP. Three days after cell injection, groups of mice underwent liver resection (35%+35% [repeated minor resection] vs. 70% [major resection]). Metastatic tumor growth was measured by color-coded fluorescence imaging of the GFP-expressing cancer cells and RFP-expressing stroma.
Although major and repeated minor resection removed the same volume of liver parenchyma, the two procedures had very different effects on metastatic tumor growth: major resection, stimulated liver and lung metastatic growth as well as recruitment of host-derived stroma compared to repeated minor resection. Repeated minor resection did not stimulate metastasis or stromal recruitment. There was no significant difference in liver regeneration between the two groups. Host-derived stroma density, which is stimulated by major resection compared to repeated minor resection, may stimulate growth in the liver-metastatic tumor. TGF-β is also preferentially stimulated by major resection and may play a role in stroma and metastasis stimulation.
The results of this study indicate that when liver resection is necessary, repeated minor liver resection is superior to major liver resection, since major resection, in contrast to repeated minor resection, stimulates metastasis, which should be taken into consideration in clinical situations indicating liver resection.
PMCID: PMC3396724  PMID: 22487397
Nude mice; liver resection; lung metastasis; liver metastasis; stroma; green fluorescent protein; red fluorescent protein; color-coded imaging
15.  Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration 
The Journal of Cell Biology  1992;116(6):1507-1515.
We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell- matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E- cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.
PMCID: PMC2289376  PMID: 1531833
16.  (Z)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione protects rats from carbon tetrachloride-induced liver injury and fibrogenesis 
AIM: To evaluate the hepatoprotective roles of (Z)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione (SKLB010) against carbon tetrachloride (CCl4)-induced acute and chronic liver injury and its underlying mechanisms of action.
METHODS: In the first experiment, rats were weighed and randomly divided into 5 groups (five rats in each group) to assess the protective effect of SKLB010 on acute liver injury. For induction of acute injury, rats were administered a single intraperitoneal injection of 2 mL/kg of 50% (v/v) CCl4 dissolved in olive oil (1:1). Group 1 was untreated and served as the control group; group 2 received CCl4 for induction of liver injury and served as the model group. In groups 3, 4 and 5, rats receiving CCl4 were also treated with SKLB010 at doses of 25, 50 and 100 mg/kg, respectively. Blood samples were collected at 6, 12 and 24 h after CCl4 intoxication to determine the serum activity of alanine amino transferase. Tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) were determined using enzyme-linked immunosorbent assay. At 24 h after CCl4 injection,liver fibrogenesis was evaluated by hematoxylin-eosin (HE) staining and immunohistochemical analyses. Cytokine transcript levels of TNF-α, IL-1β and inducible nitric oxide synthase in the liver tissues of rats were measured using a reverse transcriptase reverse transcription-polymerase chain reaction technique. In the second experiment, rats were randomly divided into 2 groups (15 rats in each group), and liver injury in the CCl4-administered groups was induced by a single intraperitoneal injection of 2 mL/kg of 50% (v/v) CCl4 dissolved in olive oil (1:1). The SKLB010-treated groups received oral 100 mg/kg SKLB010 before CCl4 administration. Five rats in each group were sacrificed at 2 h, 6 h, 12 h after CCl4 intoxication and small fortions of livers were rapidly frozen for extraction of total RNA, hepatic proteins and glutathione (GSH) assays. In the hepatic fibrosis model group, rats were randomly divided into 2 groups (5 rats each group). Rats were injected intraperitoneally with a mixture of CCl4 (1 mL/kg body weight) and olive oil [1:1 (v/v)] twice a week for 4 wk. In the SKLB010-treated groups, SKLB010 (100 mg/kg) was given once daily by oral gavage for 4 wk after CCl4 administration. The rats were sacrificed one week after the last injection and the livers from each group were harvested and fixed in 10% formalin for HE and immunohistochemical staining.
RESULTS: In this rat acute liver injury model, oral administration of SKLB010 blocked liver tissue injury by down-regulating the serum levels of alanine aminotransferase, suppressing inflammatory infiltration to liver tissue, and improving the histological architecture of liver. SKLB010 inhibited the activation of NF-κB by suppressing the degradation of IκB, and prevented the secretion of pro-inflammatory mediators such as tumor necrosis factor-α, interleukin-1β, and the reactive free radical, nitric oxide, at the transcriptional and translational levels. In this chronic liver fibrosis model, treatment with 100 mg/kg per day SKLB010 attenuated the degree of hepatic fibrosis and area of collagen, and blocked the accumulation of smooth-muscle actin-expressed cells.
CONCLUSION: These results suggest that SKLB010 is a potent therapeutic agent for the treatment of CCl4-induced hepatic injury.
PMCID: PMC3281222  PMID: 22363136
Anti-inflammatory effects; Anti-oxidative effects; (Z)-5-(4-methoxybenzylidene) thiazolidine-2; 4-dione (SKLB010) against carbon tetrachloride; Fibrogenesis; Hepatitis; Nuclear factor-κB; SKLB010
17.  Limited therapeutic efficacy of thrombopoietin on the regeneration of steatotic livers 
Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.
PMCID: PMC3759482  PMID: 24040440
Thrombopoietin; partial hepatectomy; ob/ob; steatosis; oval cells
18.  Stimulation of oval cell and hepatocyte proliferation by exogenous bombesin and neurotensin in partially hepatectomized rats 
AIM: To investigate the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor.
METHODS: Seventy male Wistar rats were randomly divided into five groups: I = controls, II = sham operated, III = partial hepatectomy 70% (PHx), IV = PHx + BBS (30 μg/kg per day), V = PHx + NT (300 μg/kg per day). Forty eight hours after liver resection, portal endotoxin levels and hepatic glutathione redox state were determined. α-fetoprotein (AFP) mRNA (in situ hybridisation), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphological features of oval cells that were cytokeratin-19 (+) and AFP mRNA (+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes were determined.
RESULTS: In the control and sham operated groups, oval cells were significantly less compared to groups III, IV and V (P < 0.001). The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to group III (P < 0.001). In addition, BBS and NT induced a significant increase of hepatocyte proliferation (P < 0.001), whereas it decreased their apoptotic activity (P < 0.001) compared to group III. BBS and NT significantly decreased portal endotoxemia (P < 0.001) and increased the hepatic GSH: GSSG ratio (P < 0.05 and P < 0.001, respectively) compared to group III.
CONCLUSION: BBS and NT stimulated oval cell proliferation in a model of liver regeneration, without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli, and improved the hepatocyte regenerative response. This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases.
PMCID: PMC3240906  PMID: 22180848
Liver regeneration; Partial hepatectomy; Hepatic progenitor cells; Oval cells; Apoptosis; Proliferation; Oxidative stress
19.  Curable Metastatic Colorectal Cancer 
Colon cancer, though already metastatic, may still be curable through multi-modality approaches, which require combined planning between medical and surgical oncologists. Retrospective surgical series have historically shown 5-year survival or “cures” for 30% to 50% of patients with solitary or a few resectable liver metastases. The role of adjuvant chemotherapy in this setting has been poorly defined. A recent European Organization for Research and Treatment of Cancer (EORTC) study randomized 364 patients with solitary resectable liver metastases to surgery alone vs. perioperative chemotherapy with FOLFOX (6 cycles preoperatively and 6 cycles postoperatively).1 Patients were required to have no more than four resectable, liver-only metastases. Ninety-three percent (93%) of patients randomized to undergo surgery were actually operated compared with 87% for the perioperative chemotherapy group. Three-year disease-free survival (DFS) for the actual eligible group (n=342) was 36% vs. 28% (HR = 0.77, P = 0.04). For those actually resected (n= 304), the results were slightly better (HR 0.73). This result (8% actual improvement in 3-yr DFS) compares favorably with the benefit of FOLFOX in patients with stage III resected CRC.
Additional attention has been paid to those patients who have liver-only disease but borderline resectable disease. Current thinking does not define resectability by number of lesions or extent of resection, but by extent of remaining liver, which should be 25% to 30%. Limiting factors for resection include proximity to vital structures and need for negative margins. Strategies for improving resection rates include staged surgery, portal vein embolization, and preoperative (neoadjuvant) chemotherapy. A long experience with oxaliplatin-based therapy showed that patients considered surgically unresectable but treated initially with chemotherapy had 5-year survival similar to that of patients treated with initial surgery.2 This approach is supported by the EORTC study, showing a 30% average shrinkage in tumor diameter. The addition of targeted therapies could increase conversion rates further; data on cetuximab have shown a 10% surgical resection rate compared with 5% for those treated with FOLFIRI alone (not selected for K-ras wild-type).3 In the NO16966 study of FOLFOX or CapeOX ± bevacizumab,4 resection rates for those with liver-only disease were 19% vs. 12% with the use of bevacizumab. Toxicities of chemotherapy used in the perioperative setting include hepatic steatosis (5-fluorouracil), steatohepatitis (irinotecan), and sinusoidal scarring (oxaliplatin). The use of irinotecan is associated with increased surgical complications and mortality in a dose-dependent manner.5 Multimodality planning with a defined number of preoperative cycles is essential in the approach to the patient with potentially resectable liver metastases.
PMCID: PMC3047042
20.  The effect of preconditioning on liver regeneration after hepatic resection in cirrhotic rats 
The Korean Journal of Hepatology  2011;17(2):139-147.
Ischemic preconditioning (IP) decreases severity of liver necrosis and has anti-apoptotic effects in previous studies using liver regeneration in normal rats. This study assessed the effect of IP on liver regeneration after hepatic resection in cirrhotic rats.
To induce liver cirrhosis, thioacetamide (300 mg/kg) was injected intraperitoneally into Sprague-Dawley rats twice per week for 16 weeks. Animals were divided into four groups: non-clamping (NC), total clamping (TC), IP, and intermittent clamping (IC). Ischemic injury was induced by clamping the left portal pedicle including the portal vein and hepatic artery. Liver enzymes alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to assess liver damage. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining for apoptosis and proliferating cell nuclear antigen (PCNA) staining for cell replication were also performed.
Day-1 ALT and AST were highest in IP, however, levels in NC and IC were comparably low on days 1-7. There was no significant correlation of AST or ALT with experimental groups (P=0.615 and P=0.186). On TUNEL, numbers of apoptotic cells at 100× magnification (cells/field) were 31.8±24.2 in NC, 69.0±72.3 in TC, 80.2±63.1 in IP, and 21.2±20.8 in IC (P<0.05). When regeneration capacity was assessed by PCNA staining, PCNA-positive cells (cells/field) at 400× were 3.4±6.0 in NC, 16.9±69 in TC, 17.0±7.8 in IP and 7.4±7.6 in IC (P<0.05).
Although regeneration capacity in IP is higher than IC, the liver is vulnerable to ischemic damage in cirrhotic rats. Careful consideration is needed in applying IP in the clinical setting.
PMCID: PMC3304634  PMID: 21757985
Liver cirrhosis; Ischemic preconditioning; Liver regeneration; Hepatectomy; Apoptosis
21.  Omentum facilitates liver regeneration 
AIM: To investigate the mechanism of liver regeneration induced by fusing the omentum to a small traumatic injury created in the liver. We studied three groups of rats. In one group the rats were omentectomized; in another group the omentum was left in situ and was not activated, and in the third group the omentum was activated by polydextran particles.
METHODS: We pre-activated the omentum by injecting polydextran particles and then made a small wedge wound in the rat liver to allow the omentum to fuse to the wound. We monitored the regeneration of the liver by determining the ratio of liver weight/body weight, by histological evaluation (including immune staining for cytokeratin-19, an oval cell marker), and by testing for developmental gene activation using reverse transcription polymerase chain reaction (RT-PCR).
RESULTS: There was no liver regeneration in the omentectomized rats, nor was there significant regeneration when the omentum was not activated, even though in this instance the omentum had fused with the liver. In contrast, the liver in the rats with the activated omentum expanded to a size 50% greater than the original, and there was histologically an interlying tissue between the wounded liver and the activated omentum in which bile ducts, containing cytokeratin-19 positive oval cells, extended from the wound edge. In this interlying tissue, oval cells were abundant and appeared to proliferate to form new liver tissue. In rats pre-treated with drugs that inhibited hepatocyte growth, liver proliferation was ongoing, indicating that regeneration of the liver was the result of oval cell expansion.
CONCLUSION: Activated omentum facilitates liver regeneration following injury by a mechanism that depends largely on oval cell proliferation.
PMCID: PMC2655194  PMID: 19266597
Cytokeratin; Foreign body; Growth factors; Oval cell; Progenitor cells
22.  mTOR signaling in liver regeneration: Rapamycin combined with growth factor treatment 
AIM: To investigate the effects of mammalian target of rapamycin (mTOR) inhibition on liver regeneration and autophagy in a surgical resection model.
METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy (PH) and treated intraperitoneally every 24 h with a combination of the mTOR inhibitor rapamycin (2.5 mg/kg per day) and the steroid dexamethasone (2.0 mg/kg per day) in phosphate buffered saline (PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6 (IL-6; 500 μg/kg per day) and hepatocyte growth factor (HGF; 100 μg/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2’-deoxyuridine (BrdU) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3 (LC3)-II protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesis-related genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.
RESULTS: mTOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation (2% vs 12% BrdU positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution (63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels (aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-II, which was reduced during normal liver regeneration, increased after mTOR inhibition (46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor (TNF)-α 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors (HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatment with the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight (75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin (TNF-α and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.
CONCLUSION: mTOR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.
PMCID: PMC3832859  PMID: 24255881
Hepatocyte proliferation; Autophagy; Microtubule-associated protein 1 light chain 3; Partial hepatectomy; Rapamycin
23.  Two-stage hepatectomy and associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) in treating liver metastases of rectal cancer: a case report 
SpringerPlus  2015;4:194.
An innovative approach, called associated liver partition and portal vein ligation for staged hepatectomy(ALPPS), has made possible a marked increase in future liver remnant (FLR) volume over a short period of time, thus permitting extended hepatectomy.
Case description
This report describes ALPPS in a 63-year-old male patient with rectal cancer and synchronous multiple liver metastases. The primary lesion was resected, followed by chemotherapy. We had planned to completely resect the metastases in both liver lobes, but CT volumetry revealed a very small FLR (364 ml, 29% of the total liver volume, 0.61% of total body weight). His indocyanine green retention rate at 15 minutes was 12.7%. Because of the risk of tumor progression in the interim, we performed ALPPS. During the first stage, the tumor in segment 3 was resected, the right lobe was mobilized, the liver was partitioned, and the right portal vein was ligated. The right hepatic artery, duct and vein were secured with vessel loops. CT on postoperative day 6 showed sufficient FLR increase (from 364 ml to 573 ml, or from 0.61% to 0.96% of total body weight) and ICGR15 improvement to 3.4%. The second stage of ALPPS was on postoperative day 7, completing resection of the metastases. The patient recovered well and was discharged 21 days after the second step.
Discussion and evaluation
The ALPPS approach has many advantages, but it lacks evidence of long-term results. Considering the high mortality and morbidity rates of ALPPS, it is essential to evaluate its risks and benefits in individual patients and determine the strict criteria for this surgical method.
ALPPS procedure rapidly increases FLR, permitting extended hepatectomy for patients with initially insufficient FLR.
PMCID: PMC4408310  PMID: 25932377
Colorectal cancer; Liver metastasis; Two-stage hepatectomy; Associated liver partition and portal vein ligation; ALPPS
24.  Minimal invasive surgery in treatment of liver metastases from colorectal carcinomas: case studies and survival rates 
BMC Surgery  2013;13(Suppl 2):S45.
Liver represents the main organ subject to metastases from colorectal tumors. Resections of liver metastases from colorectal cancer have a well-considered therapeutic role underlined by survival of 5 years by approximately 50-60% of surgical cases as is deduced from an analysis of the most recent literature. The objective of surgery is to eradicate the metastases present and obtain a margin free from neoplastic impact of amplitude of approximately 1 cm with residual liver quantity at the end of the intervention that allows the patient to survive. Currently the dimensions and the number of colorectal liver metastases (CRLM) do not limit the hepatectomy. Purpose of this work is to evaluate the survival, according to our case studies of patients treated only with the wedge resection (atypical resection) approximately 1 cm from the margins of metastases.
In "A. Rizzoli" Lacco Ameno Hospital (Ischia), from 2005 to 2010, 12 liver resections were performed for metastases from colorectal carcinoma with atypical resection. Synchronous surgical treatment with resection of the colorectal carcinoma and metastases was performed in 6 patients, 2 female and 4 male (Group 1). Surgical liver metastasectomy post-colectomy was performed on 6 patients, 3 female and 3 male (Group 2).
No patient was treated with chemotherapy. The mortality rate of intraoperative and perioperative infection was in both cases of 0%. Survival:11 patients treated surgically from 2005-2010 with synchronous surgery resection (Group 1) and liver metastasectomy (Group 2) are currently living. One 77-years-old patient died three years after surgery for BPCO.
This result was able to be obtained due to the wedge resection technique routinely used in our Hospital, associated with the indispensable use of intraoperatory ultrasound (IOUS). Significant differences between the synchronous and non-synchronous intervention emerged only regarding the number of days of hospital stay, higher in the first case.
PMCID: PMC3850987  PMID: 24267179
25.  Clinical Characteristics of Multiple Primary Colorectal Cancers 
Although multiple primary colorectal cancer has been recognized as a significant clinical entity, its clinical and pathological features and its prognosis are still controversial. The purpose of this study was to clarify clinical and pathological features of multiple primary colorectal cancer.
Materials and Methods
Among 1669 patients who underwent surgery for primary colorectal cancer from January 1997 to June 2005, 26 patients (1.6%) with multiple primary colorectal cancer were identified. We reviewed clinical characteristics including diagnostic interval, lesions, operating methods, and TNM stage, and we defined the index lesion as the most advanced lesion among the synchronous lesions. For the purposes of the study, the colon and rectum were classified into three segments. The right-side colon included the appendix, cecum, ascending colon, hepatic flexure, and transverse colon, and the left-side colon included the splenic flexure, descending colon, and sigmoid colon.
Of the 26 patients with multiple primary colorectal cancers, nineteen patients were male and seven patients were female, with a mean age of 61.5 years. Nineteen patients had synchronous colorectal cancers and seven patients had metachronous colorectal cancers. In the metachronous cases, the mean diagnostic interval was 36.8 months. The site of the first lesion in metachronous colorectal cancers was the right colon in five cases (71.4%) and the left colon in two cases (28.6%), and the site of the second lesion was the rectum in six cases (55.5%), the right colon in three cases (33.3%), and the left colon in one case. The TNM stage of the second lesions in the metachronous colorectal cancers was stage II in four cases (57.1%), stage III in one case (14.3%), and stage IV in one case (14.3%). For the synchronous colorectal cancers, the operation methods were single-segment resection combined with endoscopic mucosal resection in five cases (26.3%), single-segment resection alone in six cases, two-segment resection in six cases, and total colectomy in two cases.
In metachronous colorectal cancers, the secondary lesions were later-stage cancer. Therefore, careful postoperative follow-up is necessary for patients who have undergone surgery for colorectal cancers. Further study of therapeutic modalities is important for synchronous colorectal cancers.
PMCID: PMC2697483  PMID: 19688051
Colorectal neoplasms; Multiple primary cancers; Synchronous; Metachronous

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