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AIMS: The incidence of ductal carcinoma in situ (DCIS) has increased since the introduction of screening mammography. Recurrence prediction is still not accurate, and could be improved by identifying additional prognostic markers. Periductal stroma actively participates in early breast cancer progression. Therefore, the aim of this study was to explore the prognostic potential of stromal characteristics in DCIS. METHODS AND RESULTS: Histopathological features and hormone receptor/HER2 status were analysed in a first cohort of 65 cases of DCIS with a median follow-up of 112 months. Cox regression analysis revealed that myxoid stromal architecture was significantly associated with increased ipsilateral locoregional recurrence (P = 0.015). Next, we performed immunohistochemical screening of nine stromal proteins in a second cohort of 82 DCIS cases, and correlated their expression with stromal architecture. Because reduced stromal decorin expression correlated most strongly with myxoid stroma (P < 0.001), it was selected for further analysis in the first cohort. Patients with reduced periductal decorin expression had a higher risk of recurrence (P = 0.008). Furthermore, HER2 overexpression was significantly associated with invasive but not with in situ recurrence (P = 0.007). CONCLUSIONS: Periductal myxoid stroma and reduced periductal decorin expression seem to be prognostic for overall ipsilateral locoregional recurrence in DCIS, whereas HER2 expression might be a more specific biomarker for invasive recurrence.

BACKGROUND: Chimeric mice with humanized livers represent a promising tool for infections with Plasmodium falciparum to evaluate novel methods for prevention and treatment of pre-erythrocytic stages. Adequate assessment of hepatic infections is generally compromised by the limited number of human hepatocytes infected by developing parasites. METHODS: A qPCR-based method has been developed that sensitively and reliably detects P. falciparum liver stage infection of humanized mice and quantitatively expresses the results as the number of parasites per human hepatocyte. RESULTS: This assay allows for detection of liver stage parasites after challenging humanized mice with infected mosquito bites or after intravenous injection with sporozoites. The sensitivity of the protocol, which comprises approximately 25% of the total chimeric liver, allows for the detection of a single infected hepatocyte in the analysed tissue. CONCLUSIONS: This method allows for the detection and quantification of P. falciparum parasites in chimeric mice repopulated with human hepatocytes. It will be a useful tool when studying the in vivo therapeutic and/or prophylactic qualities of novel compounds, small molecules or antibodies directed against the liver stage of P. falciparum infections.

OBJECTIVES: To investigate the clinical and pathobiological significance of distinguishing score 0 and score 1+ within the group of immunohistochemistry (IHC)-negative invasive breast cancers. METHODS: We studied HER2 status using both IHC and fluorescence in situ hybridization (FISH) in 150 consecutive breast tumors submitted to our laboratory after a negative IHC result in local testing centers. RESULTS: We were able to discern a group of score 0 tumors that had a lower HER2 copy number than the group consisting of score 1+ tumors. In contrast with the group of score 1+ tumors, HER2 FISH was consistently negative for both copy number-based and ratio-based tumors without equivocal results. CONCLUSIONS: In a setting with stringent quality assurance, score 0 and score 1+ tumors emerge as distinct and clinically important subgroups within the HER2 IHC-negative population

Calciphylaxis, or calcific uremic arteriolopathy (CUA) is a rare but well described entity in patients with endstage renal disease (ESRD) and/or hyperparathyroidism. CUA is characterized by systemic acute calcification of the small and intermediate dermal vasculature that can lead to epidermal ischemia, ulceration, and necrosis. Cutaneous lesions of calciphylaxis characteristically begin as tender, violaceous, livedoid discolorations. The mechanisms of disease remain poorly understood although abnormal bone and mineral metabolism and hyperparathyroidism can contribute to CUA. Therapeutic strategies are of unproven benefit and mortality remains high. Calciphylaxis has also been extremely rarely reported in patients without ESRD and/or hyperparathyroidism. We report an unusual case of calciphylaxis in a patient with alcoholic liver cirrhosis and normal renal function, without any alteration in the phosphocalcic and parathyroid hormone (PTH) metabolisms.

BACKGROUND & AIMS: The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile. METHODS: We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PlGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies. RESULTS: We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC. CONCLUSIONS: Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC.

In Helicobacter pylori gastritis, constant antigenic stimulation triggers a sustained B-cell proliferation. Errors made during this continuous DNA replication are supposed to be corrected by the DNA mismatch repair mechanism. Failure of this mismatch repair mechanism has been described in hereditary non-polyposis colorectal cancer (HNPCC) and results in a replication error phenotype. Inherent to their instability during replication, microsatellites are the best markers of this replication error phenotype. We aimed to evaluate the role of defects in the DNA mismatch repair (MMR) mechanism and microsatellite instability (MSI) in relation to the most frequent genetic anomaly, translocation t(11;18)(q21;q21), in gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Therefore, we examined 10 microsatellite loci (BAT25, BAT26, D5S346, D17S250, D2S123, TGFB, BAT40, D18S58, D17S787 and D18S69) for instability in 28 patients with MALT lymphomas. In addition, these tumors were also immunostained for MLH1, MSH2, MSH6 and PMS2, as well as screened for the presence of t(11;18)(q21;q21) by real-time polymerase chain reaction (RT-PCR). We found MSI in 5/28 (18%) lymphomas, with MSI occurring in both t(11;18)(q21;q21)-positive and -negative tumors. One tumor displayed high levels of instability, and, remarkably, this was the only case displaying features of a diffuse large B-cell lymphoma. All microsatellite unstable lymphomas showed a loss of MSH6 expression. In conclusion, our data suggest that a MMR-defect may be involved in the development of gastric MALT lymphomas, and that a defect of MSH6 might be associated with those MSI-driven gastric lymphomas.

OBJECTIVES: Hepatocellular carcinoma and cholangiocarcinoma form the majority of primary hepatic tumours and are the third most common cause of cancer-related deaths. These liver tumours rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors and triggering the angiogenic switch. Therefore, inhibiting angiogenesis has proven to be a valuable therapeutic strategy in hepatocellular carcinoma, yet less is known about its use in cholangiocarcinoma. In this study, we assess whether inhibiting the placental growth factor (PlGF) could offer a therapeutic option in mice with hepatocellular carcinoma and cholangiocarcinoma. PlGF is a homologue of the vascular endothelial growth factor, which is only involved in pathological angiogenesis, therefore, its inhibition does not induce adverse effects. METHODS: We have used a chemically induced transgenic mouse model in which both hepatocellular carcinoma and cholangiocarcinoma develop after 25 weeks and are treated with murine monoclonal antibodies targeting PlGF. RESULTS: This study has shown for the first time that inhibiting PlGF decreases the burden of cholangiocarcinoma, by affecting both angiogenesis and inflammation. CONCLUSION: The use of monoclonal antibodies targeting PlGF could thus offer a potential systemic treatment for patients who suffer from primary liver tumours

BACKGROUND: The purpose of this study was to assess the role of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and (18)F-fluorodeoxyglucose positron emission tomography computed tomography (FDG-PET/CT) for evaluation of response to chemotherapy and bevacizumab and for prediction of progression-free survival (PFS) in patients with metastatic colorectal cancer (mCRC) with potentially resectable liver lesions. METHODS: A total of 19 mCRC patients were treated with FOLFOX/FOLFIRI and bevacizumab followed by surgery. Dynamic contrast-enhanced magnetic resonance imaging and FDG-PET/CT were performed before treatment and after cycle 5. PET results were quantified by calculating maximum standardised uptake value (SUV(max)) whereas area under the enhancement curve (AUC), initial AUC (iAUC) and the endothelial transfer constant (K(trans)) were used to quantify DCE-MRI. Pathological analysis of the resection specimen was performed, including measurement of microvessel density (MVD) and proliferation index. RESULTS: Both AUC and iAUC were significantly decreased following bevacizumab therapy (median change of 22% (P=0.002) and 40% (P=0.001) for AUC and iAUC, respectively). Progression-free survival benefit was shown for patients with >40% reduction in K(trans) (P=0.019). In the group of radiological responders, the median baseline SUV(max) was 3.77 (IQR: 2.88-5.60) compared with 7.20 (IQR: 4.67-8.73) in nonresponders (P=0.021). A higher follow-up SUV(max) was correlated with worse PFS (P=0.012). Median MVD was 10.9. Progression-free survival was significantly shorter in patients with an MVD greater than 10, compared with patients with lower MVD (10 months compared with 16 months, P=0.016). CONCLUSION: High relative decrease in K(trans), low follow-up SUV(max) and low MVD are favourable prognostic factors for mCRC patients treated with bevacizumab before surgery.

BACKGROUND: Non-alcoholic fatty liver disease is a spectrum of disorders ranging from steatosis to non-alcoholic steatohepatitis (NASH). Steatosis of the liver is benign, whereas NASH can progress to cirrhosis or even hepatocellular carcinoma. Currently, a liver biopsy is the only validated method to distinct NASH from steatosis. AIM: The objective of this study was to identify a biomarker specific for NASH based on the N-glycosylation of serum proteins. METHODS: N-glycosylation patterns were assessed using DNA sequencer-assisted fluorophore-assisted capillary electrophoresis and compared with histology. RESULTS: Initially, a glycomarker (log[NGA2F]/[NA2]) was developed based on the results obtained in 51 obese non-alcoholic patients scheduled for bariatric surgery. Multivariate analysis showed that our glycomarker had the lowest P-value of all biomarkers in distinguishing NASH from steatosis (P=0.069). The glycomarker was validated in a cohort of 224 non-alcoholic fatty liver disease patients. In both pilot and validation study, glycomarker score increased in ascending amount of lobular inflammation (single-factor ANOVA, P ≤ 0.001 and P=0.012, respectively). The N-glycan profile of immunoglobulin G in the NASH population confirmed the significantly increased undergalactosylation present in these patients. CONCLUSION: Our glycomarker specifically recognises liver inflammation in obese individuals which is the main trigger for the development of steatohepatitis and can differentiate between steatosis and NASH.

The wider use of livers from expanded criteria donors and donation after circulatory death donors may help to improve access to liver transplantation. A prerequisite for safely using these higher risk livers is the development of objective criteria for assessing their condition before transplantation. Compared to simple cold storage, hypothermic machine perfusion (HMP) provides a unique window for evaluating liver grafts between procurement and transplantation. In this proof-of-concept study, we tested basic parameters during HMP that may reflect the condition of human liver grafts, and we assessed their morphology after prolonged HMP. Seventeen discarded human livers were machine-perfused. Eleven livers were nontransplantable (major absolute contraindications and severe macrovesicular steatosis in the majority of the cases). Six livers were found in retrospect to be transplantable but could not be allocated and served as controls. Metabolic parameters (pH, lactate, partial pressure of oxygen, and partial pressure of carbon dioxide), enzyme release in the perfusate [aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)], and arterial/portal resistances were monitored during HMP. Nontransplantable livers released more AST and LDH than transplantable livers. In contrast, arterial/portal vascular resistances and metabolic profiles did not differ between the 2 groups. Morphologically, transplantable livers remained well preserved after 24 hours of HMP. In conclusion, HMP preserves the morphology of human livers for prolonged periods. A biochemical analysis of the perfusate provides information reflecting the extent of the injury endured.

We report a 62-year-old caucasian woman with bilateral choroidal metastases as only clinical presenting sign of advanced metastatic tumour disease. She presented with decreased vision in the left eye since 5 days. She was treated for breast cancer 31 years before. Fundoscopy and ultrasound analysis showed a large choroidal metastasis in the left eye and one asymptomatic lesion in the right eye. Systemic screening revealed multiple lung and bone metastases. Health practitioners should be aware that choroidal metastasis from breast carcinoma can present throughout life. Small asymptomatic lesions may be detected that still can be treated effectively.

BACKGROUND & AIMS: The two major primary liver cancers in adults are hepatocellular carcinoma and cholangiocarcinoma. These tumors rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors. Recently, interest has grown in the regulators of these factors. Several reports have been published describing the role of prolyl hydroxylase domains--the key oxygen sensor responsible for the degradation of hypoxia inducible factors--tumor progression and vascularisation. The effect of prolyl hydroxylase domain 2 on the pathogenesis of liver cancer has never been studied. METHODS: A diethylnitrosamine-induced mouse model was used in this study, allowing primary hepatic tumors to occur as a result of chronic liver damage. Several parameters of prolyl hydroxylase domain 2-haplodeficient mice were compared to those of wild type mice, thereby focussing on the expression of angiogenic factors and on the hepatic progenitor cell activation and differentiation. RESULTS: This study shows that inhibiting prolyl hydroxylase domain 2 increases the hepatocarcinogenesis and stimulates the development of cholangiocarcinoma. Furthermore, PHD2 deficiency and the accompanying continuous HIF activation, selected for a more metastatic tumor phenotype. CONCLUSIONS: The effect of prolyl hydroxylase domain 2 deficiency on hepatocarcinogenesis hold a great potential for therapeutic intervention, since hypoxia and the selection for a more aggressive cholangiocarcinoma phenotype might also have a repercussion on patients receiving long-term treatment with anti-angiogenic compounds.

Non-alcoholic fatty liver disease is a spectrum of disorders ranging from steatosis to non-alcoholic steatohepatitis (NASH). Steatosis of the liver is benign, whereas NASH can progress to cirrhosis or even hepatocellular carcinoma. Currently, a liver biopsy is the only validated method to distinct NASH from steatosis., SCOPUS: ar.j, info:eu-repo/semantics/published

Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl4-induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF(-/-) mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. Conclusion: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile.

The aim of this study was to investigate murine double minute-2 (MDM2) gene copy number changes in colon carcinoma and to correlate these findings with an immunohistochemical analysis of MDM2 protein expression and histopathologic prognostic indicators of the tumors. The study included 80 cases of sporadic colon carcinomas. MDM2 protein expression was assessed by immunohistochemistry, and MDM2 gene status by fluorescence in situ hybridization. MDM2 gene amplification was detected in 18% of the 80 cases examined. A strong correlation was found between MDM2 gene amplification and the presence, intensity, and staining proportion of cytoplasmic MDM2 protein expression (p = 0.01). No correlation was found between MDM2 gene amplification and the well-established histopathologic prognostic factors. Given the correlation with gene amplification, we clearly demonstrated that cytoplasmic expression of MDM2 protein is true and relevant and that this finding has to be taken into account when immunohistochemistry would be used as a screening for MDM2 gene amplification in the near future. Targeting MDM2 could be a new approach in colon cancer therapy. The amplification status could be a predictive factor of the response to MDM2-targeted therapy.

A 60-year-old Caucasian woman who was known to carry a germline c.473GA (p.Arg158His) mutation in the TP53 gene, which causes Li-Fraumeni syndrome (LFS), was scheduled for surgery for an umbilical hernia in another hospital. The patient’s history included a cholecystectomy for symptomatic lithiasis 20 years earlier; there was no identifiable exposure to asbestos. During surgery, pathologic thickening of the peritoneum was noted along with small soft, pinkish nodules on the visceral and parietal peritoneal surfaces. Biopsies of these lesions demonstrated a malignant peritoneal mesothelioma of the epithelioid type. Additional staging was performed using positron emission tomography– computed tomography scanning, which showed extensive [18F]fluorodeoxyglucose-avid lesions in the greater omentum and pelvis (Figs 1A and 1B) and a small amount of ascites, but no extra-abdominal disease. [...]

AIMS: A few reports have assessed HER2 status in breast cancer by both dual-probe fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) in an unselected and consecutive fashion, but CEP17 and HER2 copy number were not evaluated separately in these studies. Therefore, the aim of this study was to perform FISH testing for HER2 in a large number of breast tumours, irrespective of the IHC scores, which were also determined in all cases. METHODS: Both FISH and IHC were applied to 200 tumours from 196 consecutive patients who underwent resection of primary breast cancer with the sentinel procedure and/or axillary dissection. Not only the ratio, but also mean HER2 and CEP17 copy number were determined and used in statistical analyses to evaluate relationships between FISH, IHC and clinicopathological features. RESULTS: The amplification status based solely on HER2 signals was 98% concordant with results of dual-probe FISH. In non-amplified tumours, the mean CEP17 and HER2 copy number correlated, possibly because of cell cycling. Amplified tumours were histopathologically more aggressive than non-amplified tumours, and features of aggressiveness increased with the mean HER2 copy number. In both amplified and non-amplified tumours, a gene dosage effect was observed: an increase in the mean HER2 copy number was associated with a higher IHC score. CONCLUSIONS: This working method and analysis enabled new insights to be obtained into the pathobiology of HER2 in breast cancer. The findings may be helpful in optimising the methodology of HER2 testing.

Placental growth factor (PlGF) is associated selectively with pathological angiogenesis, and PlGF blockade does not affect the healthy vasculature. Anti-PlGF is therefore currently being clinically evaluated for the treatment of cancer patients. In cirrhosis, hepatic fibrogenesis is accompanied by extensive angiogenesis. In this paper, we evaluated the pathophysiological role of PlGF and the therapeutic potential of anti-PlGF in liver cirrhosis. PlGF was significantly up-regulated in the CCl(4) -induced rodent model of liver cirrhosis as well as in cirrhotic patients. Compared with wild-type animals, cirrhotic PlGF(-/-) mice showed a significant reduction in angiogenesis, arteriogenesis, inflammation, fibrosis, and portal hypertension. Importantly, pharmacological inhibition with anti-PlGF antibodies yielded similar results as genetic loss of PlGF. Notably, PlGF treatment of activated hepatic stellate cells induced sustained extracellular signal-regulated kinase 1/2 phosphorylation, as well as chemotaxis and proliferation, indicating a previously unrecognized profibrogenic role of PlGF. CONCLUSION: PlGF is a disease-candidate gene in liver cirrhosis, and inhibition of PlGF offers a therapeutic alternative with an attractive safety profile.

BACKGROUND: The increasing incidence of hepatocellular carcinoma in Western countries has led to an expanding interest of scientific research in this field. Therefore, a vast need of experimental models that mimic the natural pathogenesis of hepatocellular carcinoma (HCC) in a short time period is present. The goal of our study was (1) to develop an efficient mouse model for HCC research, in which tumours develop in a natural background of fibrosis and (2) to assess the time-dependent angiogenic changes in the pathogenesis of HCC. METHODS: Weekly intraperitoneal injections with the hepatocarcinogenic compound N-nitrosodiethylamine was applied as induction method and samples were taken at several time points to assess the angiogenic changes during the progression of HCC. RESULTS: The N-nitrosodiethylamine-induced mouse model provides well vascularised orthotopic tumours after 25 weeks. It is a representative model for human HCC and can serve as an excellent platform for the development of new therapeutic targets.

OBJECTIVES: Invasive liver biopsy is the current method for the assessment of liver fibrosis. In search of noninvasive alternatives, galectin-3-binding protein (G3BP) was introduced as a candidate-marker of hepatitis C-related fibrosis based on serum proteomics. We investigated the role of G3BP as a single-marker of significant fibrosis and cirrhosis by serology and histology and studied the effect of glycosylation on antibody-affinity in hepatitis C and alcoholic cirrhosis. METHODS: Sera and available biopsies of hepatitis C patients with various fibrosis-grades and patients with alcoholic cirrhosis were used for G3BP-measurements by enzyme-linked immunosorbent assay and immunohistochemistry, respectively. Glycosylation-effect was analyzed by western blot. Data was analyzed in accordance to fibrosis. RESULTS: G3BP-levels (mean+/-standard deviation) were increased during cirrhosis (22.7+/-10.1 microg/ml) compared to mild (11.3+/-6.4 microg/ml) and moderate fibrosis (13.4+/-8.3 microg/ml) (P<0.001; P=0.004, respectively). Receiver operator characteristic curves showed areas under the curve of 0.68, 0.75 and 0.81 for detection of significant fibrosis, severe fibrosis, and cirrhosis, respectively. Similar findings in hepatic G3BP expression were obtained, in which cirrhosis was associated with diffuse, parenchymal expression (P=0.002). The observed difference between hepatitis C and alcoholic cirrhosis (13.5+/-9.0 microg/ml) (P=0.009) could not be explained by glycosylation. CONCLUSION: Our recent findings confirm our initial proteome results on serological and histological level as well as the role of G3BP as a marker of hepatitis C-related fibrosis, especially cirrhosis. Implication of this protein in future multi-marker study should be considered.

This study aims to describe a patient developing concomitant severe liver failure and lactic acidosis after long-term treatment with linezolid. A 55-year-old Caucasian woman developed concomitant severe liver failure and lactic acidosis after a treatment with linezolid for 50 days because of infected hip prosthesis. Other causes of liver failure and lactic acidosis were excluded by extensive diagnostic workup. A liver biopsy showed microvesicular steatosis. As linezolid toxicity was considered to be the cause of the lactic acidosis and the severe hepatic failure, the antibiotic was withdrawn. After 4 days of supportive therapy and hemodialysis, the serum lactate level returned within normal limits. The prothrombin time ratio and thrombocytes recovered within 2 weeks. Bilirubin levels normalized within 14 weeks. Since no other cause could be identified, liver injury was considered to be drug-related. Resolution of the hepatotoxicity occurred after discontinuation of linezolid, supportive treatment measures, and hemodialysis. Both lactic acidosis and microvesicular steatosis after the use of linezolid are related to mitochondrial dysfunction. The Council for International Organizations of Medical Sciences/Roussel Ucalf Causality Assessment Method scale revealed that the adverse drug event was probable. Prolonged exposure to linezolid may induce severe hepatotoxicity. Clinicians should be aware of this possible adverse effect especially in case of long-term treatment.

The prevalence of obesity has grown dramatically over the last decades, with nonalcoholic steatohepatitis increasingly observed. Therapeutic options for morbid obesity include bariatric surgery. Fatal liver failure (LF) has been recorded after jejunoileal bypass (JIB) but is controversial after biliopancreatic diversion (BPD, Scopinaro operation). We performed a survey on the frequency of liver transplantation (LT) after bariatric surgery in Belgium. An enquiry was sent to all Belgian liver transplant centers to investigate the occurrence of subacute and chronic LF after bariatric surgery. After weight-reduction surgery, 10 patients in 3 Belgian transplant centers were listed for LT due to severe hepatocellular failure. Nine of them had undergone a Scopinaro operation and 1 a jejunoileal bypass. The median time to develop LF was 5 years. The patient with JIB developed chronic LF after 25 years. Seven patients were transplanted; two died awaiting a graft and one is still on the waiting list. After LT, 1 patient developed rapid reappearance of LF at 10 months, requiring retransplantation. Two recipients died after LT because of multiorgan failure shortly after transplantation. In another case, a de novo cancer was fatal at 6 years' follow-up. The remaining recipients were doing well. According to this survey, the BPD operation carries a potential risk of LF. However, because there were only 10 cases, we remain unaware of the actual incidence of Scopinaro operation-induced LF. We advise strict follow-up of liver function and timely dismantling of BPD.

METHODS: We identify critical factors affecting animal survival, engraftment efficacy, kinetics of liver repopulation and virological outcome by analysing the data from 400 human hepatocyte transplantations and 115 subsequent HBV and/or HCV inoculations in this mouse model. RESULTS: Almost one third of animals succumbed during the first week after hepatocyte transplantation. Only during this critical period, liver necrosis due to embolization of donor cells in the portal vein was observed. This may have caused a fatal acute liver failure that complicated the pre-existing chronic liver disease. From the second week onwards, confluent hepatocyte clusters repopulated the liver and restored its synthetic functions as evidenced by increasing human albumin levels in plasma. Xenogenic repopulation by human cells proceeded approximately 4-times slower compared to allogenic mouse hepatocytes. All HBV inoculations were successful, even in animals with low graft take. HCV infection rate varied substantially, although every donor cell type yielded infectable animals. A reproducible 100% HCV infectivity was reached with high quality inocula in animals with human albumin plasma levels >1 mg/ml. Superior animal survival, adequate liver engraftment and a high viral infection rate were observed after transplanting cryopreserved commercial human hepatocytes. CONCLUSIONS: Our findings favour the use of commercially available, cryopreserved human hepatocytes for the humanization of the uPA(+/+)-SCID liver. While HBV infectivity criteria are less stringent, human albumin plasma levels exceeding 1 mg/ml are required for a consistent HCV infection in chimeric mice.

BACKGROUND: Fanconi syndrome (FS) is a generalized transport defect in the proximal renal tubule leading to renal losses of phosphate, calcium, uric acid, bicarbonates as well as glucose, amino acids and other organic compounds. It is caused by inherited or acquired disorders including low mass or high mass multiple myeloma. OBJECTIVES: To report the first case series of patients with lymphoma and FS. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Patients with lymphoma and FS were identified in the nephrology department of two teaching hospitals in Paris, France and Ghent, Belgium. FS was defined by the presence of at least three out of the four following criteria: hypophosphataemia, metabolic acidosis, normoglycaemic glucosuria and hypokalaemia. Patients files were reviewed and relevant data were collected. RESULTS: Eight patients with lymphoma and FS were identified. In six patients, the lymphoma was of the acute T cell leukaemia/lymphoma (ATLL) type, related to human T cell lymphotropic virus 1 (HTLV1) infection. In all patients, FS was severe requiring supplementation. A kidney biopsy performed in a patient with post-transplantation primary renal lymphoma disclosed intense proximal tubule infiltration by lymphomatous cells. In one patient with ATLL, FS features regressed following the successful treatment of lymphoma. CONCLUSION: Patients with lymphoma require careful monitoring for features of FS; lymphoma should also be added to the spectrum of disorders associated to FS. Prospective studies are needed to ascertain the implication of HTLV1 in the genesis of FS.

PURPOSE: Hepatocellular carcinomas (HCC) have an unpredictable clinical course, and molecular classification could provide better insights into prognosis and patient-directed therapy. We hypothesized that in HCC, certain microenvironmental regions exist with a characteristic gene expression related to chronic hypoxia which would induce aggressive behavior. EXPERIMENTAL DESIGN: We determined the gene expression pattern for human HepG2 liver cells under chronic hypoxia by microarray analysis. Differentially expressed genes were selected and their clinical values were assessed. In our hypothesis-driven analysis, we included available independent microarray studies of patients with HCC in one single analysis. Three microarray studies encompassing 272 patients were used as training sets to determine a minimal prognostic gene set, and one recent study of 91 patients was used for validation. RESULTS: Using computational methods, we identified seven genes (out of 3,592 differentially expressed under chronic hypoxia) that showed correlation with poor prognostic indicators in all three training sets (65/139/73 patients) and this was validated in a fourth data set (91 patients). Retrospectively, the seven-gene set was associated with poor survival (hazard ratio, 1.39; P = 0.007) and early recurrence (hazard ratio, 2.92; P = 0.007) in 135 patients. Moreover, using a hypoxia score based on this seven-gene set, we found that patients with a score of >0.35 (n = 42) had a median survival of 307 days, whereas patients with a score of < or =0.35 (n = 93) had a median survival of 1,602 days (P = 0.005). CONCLUSIONS: We identified a unique, liver-specific, seven-gene signature associated with chronic hypoxia that correlates with poor prognosis in HCCs.

PURPOSE: To characterize the molecular pathways activated or inhibited by cetuximab when combined with chemoradiotherapy (CRT) in rectal cancer and to identify molecular profiles and biomarkers that might improve patient selection for such treatments. PATIENTS AND METHODS: Forty-one patients with rectal cancer (T3-4 and/or N+) received preoperative radiotherapy (1.8 Gy, 5 days/wk, 45 Gy) in combination with capecitabine and cetuximab (400 mg/m(2) as initial dose 1 week before CRT followed by 250 mg/m(2)/wk for 5 weeks). Biopsies and plasma samples were taken before treatment, after cetuximab but before CRT, and at the time of surgery. Proteomics and microarrays were used to monitor the molecular response to cetuximab and to identify profiles and biomarkers to predict treatment efficacy. RESULTS: Cetuximab on its own downregulated genes involved in proliferation and invasion and upregulated inflammatory gene expression, with 16 genes being significantly influenced in microarray analysis. The decrease in proliferation was confirmed by immunohistochemistry for Ki67 (P = .01) and was accompanied by an increase in transforming growth factor-alpha in plasma samples (P < .001). Disease-free survival (DFS) was better in patients if epidermal growth factor receptor expression was upregulated in the tumor after the initial cetuximab dose (P = .02) and when fibro-inflammatory changes were present in the surgical specimen (P = .03). Microarray and proteomic profiles were predictive of DFS. CONCLUSION: Our study showed that a single dose of cetuximab has a significant impact on the expression of genes involved in tumor proliferation and inflammation. We identified potential biomarkers that might predict response to cetuximab-based CRT.

OBJECTIVE: To design a multifactorial biological modulation approach targeting ischemia reperfusion injury to augment viability of porcine liver grafts from non-heart-beating donors (NHBD). BACKGROUND DATA: Liver Transplantation (LTx) from NHBD is associated with an increased risk of primary nonfunction (PNF) and biliary complications. In porcine NHBD-LTx, we previously reported a 50% risk of PNF and toxic bile formation in grafts exposed to > or =30' warm ischemia (WI). METHODS: Porcine livers exposed to 45' WI were cold stored, transplanted and either modulated (n = 6) or not (controls, n = 9). In the modulation group, donor livers were flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase (eliminating stagnating thrombi), and epoprostenol (vasodilator, platelet aggregation inhibitor) prior to cold storage. In recipients, glycine (Kupffer cell stabilizer), alpha1-acid-glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory cytokine generation inhibitor), alpha-tocopherol and glutathione (anti-oxidants), and apotransferrin (iron chelator) were administrated intravenously. PNF, survival, lactate, transaminase, TNF-alpha, redox-active iron, and biliary bile salt-to-phospholipid ratio were monitored. RESULTS: No PNF was observed in modulated versus 55% in control pigs (P = 0.025). Survival was 83% in modulated versus 22% in control pigs (P = 0.02). At 180' postreperfusion, lactate was lower in modulated (5.4 +/- 1.9 mmol/L) versus control pigs (9.4 +/- 2.2 mmol/L; P = 0.011). At 60' postreperfusion, there was a trend for lower AST in modulated versus control pigs at 60' (939 +/- 578 vs. 1683 +/- 873 IU/L; P = 0.089). Postreperfusion, TNF-alpha remained stable in modulated pigs (49 +/- 27 pg/mL at 15' and 85 +/- 26 pg/mL at 180'; P = 0.399) but increased in control pigs (107 +/- 36 pg/mL at 15' and 499 +/- 216 pg/mL at 180'; P = 0.023). At 180' postreperfusion, redox-active iron was higher in control pigs versus modul

AIMS: The aims of the study were to study the effect of pre-operative treatment on the expression of tumour-related proteins and to correlate the expression of these proteins with response and survival of patients with advanced rectal cancer. MATERIALS AND METHODS: Tissue micro-arrays from pre- and post-treatment biopsies of 99 patients with rectal cancer treated with pre-operative (chemo)radiotherapy were stained for epidermal growth factor receptor (EGFR), carbonic anhydrase IX, Ki67, vascular endothelial growth factor, cyclo-oxygenase 2 (COX-2) and cleaved cytokeratin 18 (c-CK18). Also, fibro-inflammatory alterations after treatment were evaluated. RESULTS: Pre-operative (chemo)radiotherapy caused fibro-inflammatory changes, a downregulation of proliferation (Ki67) and EGFR and an upregulation of apoptosis (cleaved CK18). Patients with a good regression during pre-operative treatment showed less proliferating and apoptotic cells in the resection specimen. Multivariate analysis showed that T downstaging, fibro-inflammatory changes in the resection specimen and COX-2 expression in the biopsy correlated with overall survival. CONCLUSIONS: Pre-operative treatment has an effect on proliferation, apoptosis, inflammation and EGFR expression. The classical clinical parameters as well as fibro-inflammatory changes and COX-2 expression seem most valuable as predictors for survival.

PURPOSE: To assess the feasibility and efficacy of the COX-2 inhibitor celecoxib in conjunction with preoperative chemoradiation for patients with locally advanced rectal cancer in a double blind randomized phase II study. MATERIALS AND METHODS: Thirty-five patients of the initially planned 80 patients with locally advanced rectal cancer were treated with preoperative radiation (45 Gy; 1.8 Gy/fraction, 5 days/week) combined with 5-fluorouracil (continuous infusion, 225 mg/m(2)/day) and celecoxib (2 x 400 mg/day) or placebo. Pathological response and toxicity of study treatment were evaluated, as well as expression of COX-2 and Ki67 in tumor tissue and IL-6 in plasma as possible molecular correlates and predictors of response to treatment. RESULTS: Patients treated with celecoxib tended to show a better response (61%) when compared to those treated with placebo (35%), although not significant (p=0.13). T-downstaging and N-downstaging were also slightly higher with celecoxib. Plasma IL-6 levels and intratumoral COX2 or Ki67 were altered by chemoradiation, but were not further altered by celecoxib treatment and therefore not useful for prediction of treatment benefit. Celecoxib therapy in conjunction with chemoradiation was not associated with additional toxicity and seemed to help mitigate therapy-related pain. CONCLUSIONS: Addition of celecoxib to preoperative chemoradiation is feasible for patients with locally advanced rectal cancer. To study the individual effect of COX-2 inhibitors on pathological response phase III studies are required.

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strand-specific HCV real-time RT-PCR assays were developed and applied to livers explanted because of end-stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)- and negative (NS)-strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV-PS or -NS RNA did not correlate with serum HCV-RNA levels or with genotypes. A high HCV envelope-2 protein expression correlated with a low NS concentration. HCV-PS and -NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post-transplant hepatitis caused by HCV. This suggests the existence of an extra-hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver.

BACKGROUND: Microarray technology has allowed to molecularly characterize many different cancer sites. This technology has the potential to individualize therapy and to discover new drug targets. However, due to technological differences and issues in standardized sample collection no study has evaluated the molecular profile of epithelial human cancer in a large number of samples and tissues. Additionally, it has not yet been extensively investigated whether metastases resemble their tissue of origin or tissue of destination. METHODS: We studied the expression profiles of a series of 1566 primary and 178 metastases by unsupervised hierarchical clustering. The clustering profile was subsequently investigated and correlated with clinico-pathological data. Statistical enrichment of clinico-pathological annotations of groups of samples was investigated using Fisher exact test. Gene set enrichment analysis (GSEA) and DAVID functional enrichment analysis were used to investigate the molecular pathways. Kaplan-Meier survival analysis and log-rank tests were used to investigate prognostic significance of gene signatures. RESULTS: Large clusters corresponding to breast, gastrointestinal, ovarian and kidney primary tissues emerged from the data. Chromophobe renal cell carcinoma clustered together with follicular differentiated thyroid carcinoma, which supports recent morphological descriptions of thyroid follicular carcinoma-like tumors in the kidney and suggests that they represent a subtype of chromophobe carcinoma. We also found an expression signature identifying primary tumors of squamous cell histology in multiple tissues. Next, a subset of ovarian tumors enriched with endometrioid histology clustered together with endometrium tumors, confirming that they share their etiopathogenesis, which strongly differs from serous ovarian tumors. In addition, the clustering of colon and breast tumors correlated with clinico-pathological characteristics. Moreover, a signature was d

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and low-grade DNs. In conclusion, our study identified unique expression patterns associated with the transition of high-grade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions.

Intrahepatic bile ducts maintain a dose anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis. Conclusion: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.

BACKGROUND: Hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to multidrug resistance (MDR). Recent studies showed that part of HCC could be of progenitor cell origin. Because some MDR-conferring transporters [multidrug resistance-associated protein 1 (MRP1), MDR1, MRP3 and breast cancer resistance protein (BCRP)] are expressed in hepatic progenitor cells (HPCs), expression in HCC might reflect a progenitor cell origin and provide the tumour cells with a MDR phenotype. METHODS: The transcriptional profile of transporter genes was assessed in 139 HCCs earlier subjected to global gene expression analysis. In addition, we performed real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry for MRP1, MRP3, MDR1, BCRP and biliary/HPC markers keratin 7 and/or keratin 19 (K7/K19) on an independent set of 23 HCCs and surrounding liver. RESULTS: Micro-array analysis showed that MRP1 was the only transporter with increased mRNA levels in HCC compared with the surrounding tissue. MRP1 mRNA levels were significantly higher in HCCs with poor survival and the 'hepatoblast subtype' of HCC, thought to be derived from HPCs. In 11 of 23 HCCs of the independent set, we found a diffuse protein expression of MRP1 compared with negative hepatocytic expression observed in normal (surrounding) hepatocytes. MRP1 was expressed in K19(+) non-neoplastic HPCs and K19(+) tumour cells. In addition, MRP3 and BCRP were expressed in K7/K19(+) tumour cells. MRP1 expression was high in poorly differentiated HCCs, large tumours (>7 cm) and microvascular invasive tumours. CONCLUSIONS: MRP1 correlated with K19 mRNA and protein expression in two independent series of HCC. In addition, MRP1 was, together with MRP3 and BCRP, colocalized with K7/K19 in the tumour. Therefore, MRP1 expression could be a reflection of the HPC origin of this subgroup of HCCs and may result in an aggressive tumour phenotype.

AIM: Hepatoblastoma (HB), the most common pediatric malignant liver tumor, is treated with chemotherapy to facilitate surgical resection. Previous studies suggest that HB acquires chemoresistance via increased expression of multidrug resistance protein 1 (MDR1, ABCC1). There is no well established evidence that this also occurs in the clinical setting and little is known about the effects of chemotherapeutic treatments on HB in situ. METHODS: Clinical and histopathological features and expression patterns of ABC transporters in diagnostic needle biopsies from 7 HBs taken before chemotherapy were compared with those in surgically resected tumors. To understand the mechanisms leading to chemoresistance we also investigated the involvement of hypoxia on protein expression and functional activity of drug transporters (BCRP and MDR1) in cultures of HepG2 human HB cells. RESULTS: We found that chemotherapeutical treatment of HBs led to an increased expression of the breast cancer resistance protein (BCRP, ABCG2) in all patients studied. There was no change in the expression pattern of MDR1 or other ABC transporters. Chemotherapy-induced specific vascular abnormalities associated with areas of necrosis and fibrosis were seen in all cases, suggesting tumor hypoxia. The observations of increased BCRP expression in hypoxic areas of three-dimensional HepG2 aggregates and the enhanced BCRP function in monolayer cultures of HepG2 cells under hypoxic conditions, support a role for hypoxia in enhanced BCRP expression. CONCLUSIONS: Chemotherapeutical treatment of HB leads to vascular alterations that modify the tumor microenvironment, and increased BCRP expression in which hypoxia might play a role. No evidence was found for upregulation of MDR1 in HBs as suggested from previous experimental studies.

MRP1 (multidrug resistance-associated protein 1) is well known for its role in providing multidrug resistance to cancer cells. In addition, MRP1 has been associated with both pro- and anti-inflammatory functions in nonmalignant cells. The pro-inflammatory function is evident from the fact that MRP1 is a high affinity transporter for cysteinyl-leukotriene C4 (LTC4), a lipid mediator of inflammation. It remains unexplained, however, why the absence of Mrp1 leads to increased intestinal epithelial damage in mice treated with dextran-sodium sulfate, a model for inflammatory bowel disease (IBD). We found that MRP1 expression is induced in the inflamed intestine of IBD patients, e.g. Crohn disease and ulcerative colitis. Increased MRP1 expression was detected at the basolateral membrane of intestinal epithelial cells. To study a putative role for MRP1 in protecting epithelial cells against inflammatory cues, we manipulated MRP1 levels in human epithelial DLD-1 cells and exposed these cells to cytokines and anti-Fas. Inhibition of MRP1 (by MK571 or RNA interference) resulted in increased cytokine- and anti-Fas-induced apoptosis of DLD-1 cells. Opposite effects, e.g. protection of DLD-1 cells against cytokine- and anti-Fas-induced apoptosis, were observed after recombinant MRP1 overexpression. Inhibition of LTC4 synthesis reduced anti-Fas-induced apoptosis when MRP1 function was blocked, suggesting that LTC4 is the pro-apoptotic compound exported by epithelial MRP1 during inflammation. These data show that MRP1 protects intestinal epithelial cells against inflammation-induced apoptotic cell death and provides a functional role for MRP1 in the inflamed intestinal epithelium of IBD patients.

BACKGROUND & AIMS: The Rho small guanosine triphosphatase Cdc42 is critical for diverse cellular functions, including regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell-cycle progression, and cell transformation. This implies that Cdc42 might be required for liver function. METHODS: Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure. RESULTS: Mice lacking Cdc42 in their hepatocytes were born at Mendelian ratios. They did not show increased mortality but showed chronic jaundice. They developed hepatomegaly soon after birth, and signs of liver transformation, such as formation of nodules and tumors, became visible macroscopically at age 6 months. Hepatocellular carcinoma was observed 8 months after birth. Tumors grew slowly and lacked expression of nuclear beta-catenin. Lung metastases were observed at the late stage of carcinogenesis. Immunofluorescent examination and electron microscopy revealed severe defects in the liver. At the age of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis. CONCLUSIONS: We describe a mouse model in which chronic liver disease leads to hepatocarcinogenesis.