Improved NGS-based detection of microsatellite instability using tumor-only data.

Microsatellite instability (MSI) is a molecular signature of mismatch repair deficiency (dMMR), a predictive marker of immune checkpoint inhibitor therapy response. Despite its recognized pan-cancer value, most methods only support detection of this signature in colorectal cancer. In addition to the tissue-specific differences that impact the sensitivity of MSI detection in other tissues, the performance of most methods is also affected by patient ethnicity, tumor content, and other sample-specific properties. These limitations are particularly important when only tumor samples are available and restrict the performance and adoption of MSI testing. Here we introduce MSIdetect, a novel solution for NGS-based MSI detection. MSIdetect models the impact of indel burden and tumor content on read coverage at a set of homopolymer regions that we found are minimally impacted by sample-specific factors. We validated MSIdetect in 139 Formalin-Fixed Paraffin-Embedded (FFPE) clinical samples from colorectal and endometrial cancer as well as other more challenging tumor types, such as glioma or sebaceous adenoma or carcinoma. Based on analysis of these samples, MSIdetect displays 100% specificity and 96.3% sensitivity. Limit of detection analysis supports that MSIdetect is sensitive even in samples with relatively low tumor content and limited microsatellite instability. Finally, the results obtained using MSIdetect in tumor-only data correlate well (R=0.988) with what is obtained using tumor-normal matched pairs, demonstrating that the solution addresses the challenges posed by MSI detection from tumor-only data. The accuracy of MSI detection by MSIdetect in different cancer types coupled with the flexibility afforded by NGS-based testing will support the adoption of MSI testing in the clinical setting and increase the number of patients identified that are likely to benefit from immune checkpoint inhibitor therapy.

PARP inhibitors and radiation potentiate liver cell death in vitro. Do hepatocellular carcinomas have an achilles' heel?

BACKGROUND: A promising avenue for cancer treatment is exacerbating the deregulation of the DNA repair machinery that would normally protect the genome. To address the applicability of poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) combined with radiotherapy for the treatment of hepatocellular carcinoma (HCC) two approaches were used: firstly, the in vitro sensitivity to the PARPi Veliparib and Talazoparib +/- radiation exposure was determined in liver cell lines and the impact of the HBV X protein (HBx) that deregulates cellular DNA damage repair via SMC5/6 degradation was investigated. Secondly, PARP expression profiles and DNA damage levels using the surrogate marker gammaH2AX were assessed in a panel of control liver vs HCC tissues. METHODS: Cell cytotoxicity was measured by clonogenic survival or relative cell growth and the DNA damage response using immunological-based techniques in Hep3B, PLC/PRF/5, HepG2- and HepaRG-derived models. Transcriptome changes due to HBx expression vs SMC6 loss were assessed by RNA sequencing in HepaRG-derived models. PARP and PARG transcripts (qPCR) and PARP1, H2AX and gammaH2AX protein levels (RPPA) were compared in control liver vs HBV-, HCV-, alcohol- and non-alcoholic steatohepatitis-associated HCC (tumor/peritumor) tissues. RESULTS: PARPi cytotoxicity was significantly enhanced when combined with X-rays (2Gy) with Talazoparib having a greater impact than Veliparib in most in vitro models. HBx expression significantly lowered survival, probably driven by SMC5/6 loss based on the transcriptome analysis and higher DNA damage levels. PARP1 and PARP2 transcript levels were significantly higher in tumor than peritumor and control tissues. The HBV/HCV/alcohol-associated tumor tissues studied had reduced H2AX but higher gammaH2AX protein levels compared to peritumor and control tissues providing evidence of increased DNA damage during liver disease progression. CONCLUSIONS: These proof-of-concept experiments support PARPi alone or combined with radiotherapy for HCC treatment, particularly for HBV-associated tumors, that warrant further investigation.

[Management of immune checkpoint inhibitors-induced liver toxicity in cancer]. / Prise en charge des toxicités hépatiques sous immunothérapie anticancéreuse.

Pharmacological immune checkpoint inhibitors (ICI) restore the anti-tumor properties of T-lymphocytes, but unfortunately can engender auto-immune-like disorders. Those, frequent and of variable severity, sometimes target the liver parenchyma. Liver toxicity of ICI firstly leads to alteration of liver function tests (ALFT) with a risk of clinical decompensation. The appearance of ALFT should lead the clinician to exclude a non-immunological injury or a tumoral invasion of the liver parenchyma. In case of high grade ALFT, liver biopsy is necessary for diagnosis purpose. In ICI-induced hepatoxicity, histology examination shows most frequently a lobular acute hepatitis associated with lymphocytic infiltrates, but with different features than those encountered in primary auto-immune hepatitis. The management of ICI-related ALFT depends of their severity. Discontinuation of ICI is recommended for ALFT≥grade 2, and corticosteroid therapy for ALFT≥grade 3, or grade 2 without any improvement after ICI discontinuation. Addition of mycophenolate may be indicated whether corticosteroid inefficiency. Reintroduction of ICI is inadvisable for the most severe toxicities. The management of ALFT occurring on underlying chronic hepatopathy has not got consensual guidelines so far, but they should take account of the basal grade of ALFT and their worsening level under ICI therapy. The situation becomes more complex with associations between ICI and anti-angiogenic agents or cytotoxic chemotherapies where each of the drugs can be hepatotoxic. Thus, liver biopsy is primordial to figure out the mechanism of liver toxicity.

Cholangitis lenta: An underdiagnosed lesion associated with severe cholestasis following liver transplantation.

BACKGROUND: Cholangitis lenta (CL) represents a specific histological lesion associated with severe cholestasis and related to sepsis. Despite being well known by pathologists, CL has been poorly studied in liver transplantation (LT). METHODS: We performed a retrospective 12-year analysis of the incidence, risk factors, and outcome of CL in LT recipients. Biopsy samples performed within 3 months after LT underwent blinded rereading to identify recipients with CL. RESULTS: Among 587 LT performed, 45 (7.7%) developed CL. Of these, 7 (15.6%) had no signs of clinical sepsis at the time of biopsy, but further investigations revealed positive cultures. Independent factors associated with CL were sepsis at the time of LT (OR = 3.62 [95%CI = 1.63-8.06]), donor age (OR = 1.05 [1.03-1.08]), and operative time (OR = 1.23 [95%CI = 1.02-1.48]). Cholangitis lenta was associated with increased severe morbidity (71.1% vs 33.0%, P < .001), 90-day mortality (24.4% vs 5.9%, P < .001) and decreased one-year graft (62.1% vs 89.4%, P < .001) and patient survival (55.6% vs 87.9%, P < .001). CONCLUSION: Cholangitis lenta represents a possible lesion associated with cholestasis after LT, which strongly affects its outcome. In the event of an unexplained post-transplant cholestasis, the diagnosis of CL must be considered, even in the absence of clinically evident sepsis.

In vitro transformation of primary human hepatocytes: Epigenetic changes and stemness properties.

Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

Toll-like receptor 3 downregulation is an escape mechanism from apoptosis during hepatocarcinogenesis.

BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; p = 0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; p = 0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.

Alteration of splicing factors' expression during liver disease progression: impact on hepatocellular carcinoma outcome.

PURPOSE: Trans-acting splicing factors (SF) shape the eukaryotic transcriptome by regulating alternative splicing (AS). This process is recurrently modulated in liver cancer suggesting its direct contribution to the course of liver disease. The aim of our study was to investigate the relationship between the regulation of SFs expression and liver damage. METHODS: The expression profile of 10 liver-specific SF and the AS events of 7 genes associated with liver disorders was assessed by western-blotting in 6 murine models representing different stages of liver damage, from inflammation to hepatocellular carcinoma (HCC). Relevant SFs (PSF, SRSF3, and SRSF6) and target genes (INSR, SRSF3, and SLK) modulated in mice were investigated in a cohort of 179 HCC patients. RESULTS: Each murine model of liver disease was characterized by a unique SF expression profile. Changes in the SF profile did not affect AS events of the selected genes despite the presence of corresponding splicing sites. In human HCC expression of SFs, including the tumor-suppressor SRSF3, and AS regulation of genes studied were frequently upregulated in tumor versus non-tumor tissues. Risk of tumor recurrence positively correlated with AS isoform of the INSR gene. In contrast, increased levels of SFs expression correlated with an extended overall survival of patients. CONCLUSIONS: Dysregulation of SF expression is an early event occurring during liver injury and not just at the stage of HCC. Besides impacting on AS regulation, overexpression of SF may contribute to preserving hepatocyte homeostasis during liver pathogenesis.

A systematic review of the anatomical findings of multiple gallbladders.

BACKGROUND: Multiple gallbladders (MG) are a rare malformation, with no clear data on its clinical impact, therapeutic indications or risk for malignancy. METHODS: A systematic review of all published literature between 1990 and 2017 was performed using the PRISMA guidelines. RESULTS: Data of 181 patients extracted from 153 studies were reviewed. MG were diagnosed during the treatment of a gallstone-related disease in 83% of patients, of which 13% had previous cholecystectomy and had a recurrence of biliary stone disease. The sensitivity of ultrasound scan was 66%, and that of magnetic resonance imaging cholangio-pancreatography, 97%. The cystic duct was common to both gallbladders (type1) in 43% and separated (type 2) in 50% of patients. In the latter case, there was no way to differentiate preoperatively an accessory gallbladder from a Todani II bile duct cyst. Cholecystectomy was performed in 129 patients by laparotomy (43%) or laparoscopy (56%). MG was undiagnosed before surgery in 24% of the patients. The postoperative biliary leakage rate was 0.7%. In two patients, gallbladder cancers were detected. CONCLUSION: MG are difficult to diagnose and share a common natural history with single gallbladders, without evidence of increased risk for malignancy. Excision of both gallbladders is indicated in symptomatic stone disease. However, prophylactic cholecystectomy must be considered for type 2 MG, since it cannot be preoperatively differentiated from a Todani II bile duct cyst, which is associated with a risk of malignant transformation.

ROC-king onwards: intraepithelial lymphocyte counts, distribution & role in coeliac disease mucosal interpretation.

OBJECTIVES: Counting intraepithelial lymphocytes (IEL) is central to the histological diagnosis of coeliac disease (CD), but no definitive 'normal' IEL range has ever been published. In this multicentre study, receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off between normal and CD (Marsh III lesion) duodenal mucosa, based on IEL counts on >400 mucosal biopsy specimens. DESIGN: The study was designed at the International Meeting on Digestive Pathology, Bucharest 2015. Investigators from 19 centres, eight countries of three continents, recruited 198 patients with Marsh III histology and 203 controls and used one agreed protocol to count IEL/100 enterocytes in well-oriented duodenal biopsies. Demographic and serological data were also collected. RESULTS: The mean ages of CD and control groups were 45.5 (neonate to 82) and 38.3 (2-88) years. Mean IEL count was 54±18/100 enterocytes in CD and 13±8 in normal controls (p=0.0001). ROC analysis indicated an optimal cut-off point of 25 IEL/100 enterocytes, with 99% sensitivity, 92% specificity and 99.5% area under the curve. Other cut-offs between 20 and 40 IEL were less discriminatory. Additionally, there was a sufficiently high number of biopsies to explore IEL counts across the subclassification of the Marsh III lesion. CONCLUSION: Our ROC curve analyses demonstrate that for Marsh III lesions, a cut-off of 25 IEL/100 enterocytes optimises discrimination between normal control and CD biopsies. No differences in IEL counts were found between Marsh III a, b and c lesions. There was an indication of a continuously graded dose-response by IEL to environmental (gluten) antigenic influence.

Traumatic biliary neuroma after orthotopic liver transplantation: a possible cause of "unexplained" anastomotic biliary stricture.

BACKGROUND: Traumatic biliary neuromas (TBNs) represent a rare cause of biliary stricture (BS) after orthotopic liver transplantation (OLT). Diagnosis is challenging preoperatively and is most often made at pathology after resection. Herein, we report a 20-year experience of TBN-related BS. PATIENTS AND METHODS: Medical records of 1030 adult patients undergoing OLT from 1991 to 2014 were reviewed. Patients with histologically proven TBN were identified among those presenting a BS. RESULTS: Over the study period, 52 patients developed an anastomotic BS. Of these, 17 had repeat surgery and specimen examination identified TBN in five instances. All five patients with TBN had a duct-to-duct biliary reconstruction during OLT. Median delay from OLT to onset of symptoms was 69 months (range 4-239). Preoperative imaging showed a compressive mass in one patient. Four patients underwent TBN resection combined with hepaticojejunostomy and had an uneventful postoperative course. One patient underwent TBN resection and duct-to-duct reconstruction; he died from acute pancreatitis on postoperative day 21. After a median follow-up of 40.5 months (range 10-54), no recurrent BS occurred. CONCLUSION: Traumatic biliary neuromas represent a possible diagnosis for unexplained anastomotic BS after OLT. Surgical excision combined with hepaticojejunostomy is effective, allows histological diagnosis, and prevents from recurrence.

RNA helicase DEAD box protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus infection and hepatocarcinogenesis.

UNLABELLED: Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV-mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus-associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA-binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD box protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2-mediated gene repression, acting by regulating RNA-protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2-repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12- and PRC2-mediated gene silencing, by displacing the RNA-binding E3 ligase, Mex-3 RNA-binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down-regulation. In G2 phase of cells expressing the HBV X protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de-repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c-myc bitransgenic mice and chronically HBV-infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an important player in poor prognosis HCC. CONCLUSION: The RNA helicase DDX5, and E3 ligase Mex3b, are important cellular targets for the design of novel, epigenetic therapies to combat HBV infection and poor prognosis HBV-associated liver cancer. (Hepatology 2016;64:1033-1048).

EpCAM-regulated intramembrane proteolysis induces a cancer stem cell-like gene signature in hepatitis B virus-infected hepatocytes.

BACKGROUND & AIMS: Hepatocytes in which the hepatitis B virus (HBV) is replicating exhibit loss of the chromatin modifying polycomb repressive complex 2 (PRC2), resulting in re-expression of specific, cellular PRC2-repressed genes. Epithelial cell adhesion molecule (EpCAM) is a PRC2-repressed gene, normally expressed in hepatic progenitors, but re-expressed in hepatic cancer stem cells (hCSCs). Herein, we investigated the functional significance of EpCAM re-expression in HBV-mediated hepatocarcinogenesis. METHODS: Employing molecular approaches (transfections, fluorescence-activated cell sorting, immunoblotting, qRT-PCR), we investigated the role of EpCAM-regulated intramembrane proteolysis (RIP) in HBV replicating cells in vitro, and in liver tumors from HBV X/c-myc mice and chronically HBV infected patients. RESULTS: EpCAM undergoes RIP in HBV replicating cells, activating canonical Wnt signaling. Transfection of Wnt-responsive plasmid expressing green fluorescent protein (GFP) identified a GFP + population of HBV replicating cells. These GFP+/Wnt+ cells exhibited cisplatin- and sorafenib-resistant growth resembling hCSCs, and increased expression of pluripotency genes NANOG, OCT4, SOX2, and hCSC markers BAMBI, CD44 and CD133. These genes are referred as EpCAM RIP and Wnt-induced hCSC-like gene signature. Interestingly, this gene signature is also overexpressed in liver tumors of X/c-myc bitransgenic mice. Clinically, a group of HBV-associated hepatocellular carcinomas was identified, exhibiting elevated expression of the hCSC-like gene signature and associated with reduced overall survival post-surgical resection. CONCLUSIONS: The hCSC-like gene signature offers promise as prognostic tool for classifying subtypes of HBV-induced HCCs. Since EpCAM RIP and Wnt signaling drive expression of this hCSC-like signature, inhibition of these pathways can be explored as therapeutic strategy for this subtype of HBV-associated HCCs. LAY SUMMARY: In this study, we provide evidence for a molecular mechanism by which chronic infection by the hepatitis B virus results in the development of poor prognosis liver cancer. Based on this mechanism our results suggest possible therapeutic interventions.

Identification of novel long non-coding RNAs deregulated in hepatocellular carcinoma using RNA-sequencing.

Functional characterization of long non-coding RNAs (lncRNAs) and their pathological relevance is still a challenging task. Abnormal expression of a few long non-coding RNAs have been found associated with hepatocellular carcinoma, with potential implications to both improve our understanding of molecular mechanism of liver carcinogenesis and to discover biomarkers for early diagnosis or therapy. However, the understanding of the global role of lncRNAs during HCC development is still in its infancy. In this study, we produced RNA-Seq data from 23 liver tissues (controls, cirrhotic and HCCs) and applied statistical and gene network analysis approaches to identify and characterize expressed lncRNAs. We detected 5,525 lncRNAs across different tissue types and identified 57 differentially expressed lncRNAs in HCC compared with adjacent non-tumour tissues using stringent criteria (FDR<0.05, Fold Change>2). Using weighted gene co-expression network analysis (WGCNA), we found that differentially expressed lncRNAs are co-expressed with genes involved in cell cycle regulation, TGF-ß signalling and liver metabolism. Furthermore, we found that more than 20% of differentially expressed lncRNAs are associated to actively transcribed enhancers and that the co-expression patterns with their closest genes change dramatically during HCC development. Our study provides the most comprehensive compendium of lncRNAs expressed in HCC, as well as in control or cirrhotic livers. Our results identified both known oncogenic lncRNAs (such as H19 and CRNDE) and novel lncRNAs involved in cell cycle deregulation and liver metabolism deficits occurring during HCC development.

Liver-infiltrating CD8(+) lymphocytes as prognostic factor for tumour recurrence in hepatitis C virus-related hepatocellular carcinoma.

BACKGROUND: Chronic liver inflammation and immune/inflammatory response promote hepatocellular carcinoma. The aim of this study was to characterize the immune status of HCV-related cirrhosis in patients with hepatocellular carcinoma (HCV-HCC) as compared to HCV patients without hepatocellular carcinoma. METHOD: Immune markers (CD3, CD4, CD8, CD20, CD56, TCRγδ, FoxP3) and gene expression profiles (CD8α, CD8ß, FoxP3, IL-6, IFN-γ, perforin, RANTES) were analysed in a test cohort by immunohistochemistry and quantitative RT-PCR analysis on serial non-tumorous and tumorous tissues. RESULTS: Immune micro-environment was more inflammatory in HCV-HCC than HCV cirrhotic livers. The number of CD3(+) , CD4(+) , CD8(+) and CD20(+) liver-infiltrating lymphocytes was significantly higher, whereas the number of CD56(+) cells was significantly lower in HCV-HCC compared to HCV cirrhotic parenchyma. These differences were restricted to fibrous septa for CD4(+) and CD20(+) cells and to nodular parenchyma for CD8(+) cells. Gene expressions of CD8α, FoxP3 and RANTES were also significantly higher in HCV-HCC than in HCV cirrhosis. Interestingly, in a large cohort of 63 HCV-HCC patients. The number of CD8(+) cells ≥100/field was associated with significant higher tumour recurrence (P = 0.003) and lower overall survival (P = 0.05) at 5 years. CONCLUSION: High densities of liver-infiltrating lymphocytes in HCV-HCC cirrhotic parenchyma prevail inflammatory conditions and could contribute to tumorigenesis and tumour recurrence. These results could contribute towards better clinical evaluation of patients susceptible for HCC recurrence after curative surgery.

Focal nodular hyperplasia and hepatocellular adenoma: The value of shear wave elastography for differential diagnosis.

OBJECTIVE: This study assessed the clinical usefulness of shear wave elastography (SWE) during ultrasound for differentiating between focal nodular hyperplasias (FNHs) and hepatocellular adenomas (HAs). MATERIALS AND METHODS: SWE was performed on 56 patients presenting with 76 liver lesions (57 FNHs and 19HAs) that were confirmed by MRI and contrast-enhanced ultrasound (CEUS) (n=55) or by histology (n=21). A mean elasticity value was obtained for each lesion. The ratios of the elasticity of the lesions to the elasticity of the surrounding liver were determined. The optimal elasticity cut-off value for distinguishing between the two lesion types was determined using ROC analysis. All lesions that were classified as "undetermined" after CEUS were reclassified using the elasticity values. RESULTS: The mean elasticity value was 46.99 ± 31.15 kPa for FNHs and 12.08 ± 10.68 kPa for HAs (p<0.0001). The mean relative elasticity ratio values were 7.94 ± 6.43 and 1.91 ± 1.70, respectively (p<0.0001). The ROC analysis showed a maximal accuracy of 95% for identification with a cut-off of 18.8 kPa for lesion elasticity (accuracy of 96% with a cut-off of 1.98 for the relative elasticity ratio). A total of 68 CEUS were performed, and 17 lesions (25%) were classified as "undetermined" after CEUS. With these cut-off values 16 lesions (94.1%) were correctly reclassified as FNHs. CONCLUSION: SWE is a useful adjunctive tool for differentiation between FNH and HA during ultrasound examination.

KIT Mutation and Loss of 14q May Be Sufficient for the Development of Clinically Symptomatic Very Low-Risk GIST.

The aim of this study was to determine the minimal set of genetic alterations required for the development of a very low risk clinically symptomatic gastro-intestinal stromal tumour within the stomach wall. We studied the genome of a very low-risk gastric gastro-intestinal stromal tumour by whole-genome sequencing, comparative genomic hybridisation and methylation profiling. The studied tumour harboured two typical genomic lesions: loss of the long arm of chromosome 14 and an activating mutation in exon 11 of KIT. Besides these genetic lesions, only two point mutations that may affect tumour progression were identified: A frame-shift deletion in RNF146 and a missense mutation in a zinc finger of ZNF407. Whilst the frameshift deletion in RNF146 seemed to be restricted to this particular tumour, a similar yet germline mutation in ZNF407 was found in a panel of 52 gastro-intestinal stromal tumours from different anatomical sites and different categories. Germline polymorphisms in the mitotic checkpoint proteins Aurora kinase A and BUB1 kinase B may have furthered tumour growth. The epigenetic profile of the tumour matches that of other KIT-mutant tumours. We have identified mutations in three genes and loss of the long arm of chromosome 14 as the so far minimal set of genetic abnormalities sufficient for the development of a very low risk clinically symptomatic gastric stromal tumour.

Diversity of hepatocellular carcinoma clones bearing hematopoietic malignancies-related chromosomal translocation.

Interpatient heterogeneity of hepatocellular carcinoma has been in-depth addressed. Intrapatient heterogeneity is less known. Four clones were freshly isolated from an Edmondson grade I HCV-associated hepatocellular carcinoma. Biochemical approaches, functional assays and cytogenetics were used. Albumin inducibility was uncoupled from canonical cytokeratin profiles, suggesting pathological combinations of hepatospecific and biliary markers. Poor differentiation and TGFß's proproliferative effect on all clones were observed. TGFß, Interferon α and doxorubicin sensitivity levels were found highly heterogeneous. Progenitor and stem cells markers OV6 and EpCAM were mutually exclusively expressed. All clones were CD44+, while none expressed CD90, CD133, or CD117. Three clones displayed a liver progenitor OV6+ phenotype, and were susceptible to hepatocytic differentiation, among which one fibroblastoid clone displayed intrahepatic parenchymal engraftment capability. A fourth clone, the less motile, displayed a cancer stem cell EpCAM+ phenotype, was essentially ß-catenin negative, and was as expected devoid of hepatocytic differentiation capability, yet the most sensitive to doxorubicin treatment. Cytogenetics evidenced in all clones a t(12;22)(p11;q11) translocation found in several myelodysplastic syndromes. All clones, that probably derive from EpCAM+ tumor cells, display aberrant E-cadherin cytosolic localization. Because of their diverse pathophysiolocal features, these freshly isolated, low population doubling-defined, HCC clones may provide novel opportunities to tackle HCC heterogeneity in a single patient background for therapy improvement purposes, especially regarding recently developed targeted strategies.

Ocular sarcoidosis: when should labial salivary gland biopsy be performed?

PURPOSE: To assess the usefulness of a labial salivary gland biopsy (LSGB) in subsets of patients with uveitis. METHODS: A retrospective study of 115 consecutive patients with uveitis for whom a LSGB had been done because of suspected ocular sarcoidosis (n = 86) or unexplained uveitis (n = 29). Eighty-six patients had a suspicion of ocular sarcoidosis because of ocular features (n = 67), an elevated angiotensin converting enzyme (ACE) (n = 29), or because of CT findings (n = 32) suggestive of sarcoidosis. The biopsy results were analyzed together with their ophthalmological features and the results of other relevant examinations, such as the serum levels of ACE and a chest radiography or a CT scan. RESULTS: Six of the 115 patients (5.2 %) with uveitis had sarcoid granulomas on the LSGB. At the end of the study, 32 patients had proven sarcoidois while 22 patients were considered as having either indeterminate or presumed sarcoidosis, according to the criteria of Abad et al. A raised ACE (p = 0.016) and a compatible radiology (p = 0.033) were related to a positive LSGB test, but not to the features of uveitis. Granulomas were only found in the LSGB of the patients with an elevated ACE or compatible CT scans. CONCLUSIONS: In this study, the LSGB sensitivity (18.75 %) in the patients with proven sarcoidosis appears to be lower than in other reports. Our results suggest that this investigation is a possible method of tissue diagnosis in patients with raised ACE and/or CT scan pattern compatible with sarcoidosis, and should not be performed in patients with unexplained uveitis or because of their ophthalmological features.