Anne Durnez

Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling

We analyzed global gene expression patterns of 91 human hepatocellular carcinomas (HCCs) to define the molecular characteristics of the tumors and to test the prognostic value of the expression profiles. Unsupervised classification methods revealed two distinctive subclasses of HCC that are highly associated with patient survival. This association was validated via 5 independent supervised learning methods. We also identified the genes most strongly associated with survival by using the Cox proportional hazards survival analysis. This approach identified a limited number of genes that accurately predicted the length of survival and provides new molecular insight into the pathogenesis of HCC. Tumors from the low survival subclass have strong cell proliferation and antiapoptosis gene expression signatures. In addition, the low survival subclass displayed higher expression of genes involved in ubiquitination and histone modification, suggesting an etiological involvement of these processes in accelerating the progression of HCC. In conclusion, the biological differences identified in the HCC subclasses should provide an attractive source for the development of therapeutic targets (e.g., HIF1a) for selective treatment of HCC patients. Supplementary material for this article can be found on the HEPATOLOGY Web site (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html) (HEPATOLOGY 2004;40:667–676.)

Oxidative Stress and Oval Cell Accumulation in Mice and Humans with Alcoholic and Nonalcoholic Fatty Liver Disease

In animals, the combination of oxidative liver damage and inhibited hepatocyte proliferation increases the numbers of hepatic progenitors (oval cells). We studied different murine models of fatty liver disease and patients with nonalcoholic fatty liver disease or alcoholic liver disease to determine whether oval cells increase in fatty livers and to clarify the mechanisms for this response. To varying degrees, all mouse models exhibit excessive hepatic mitochondrial production of H(2)O(2), a known inducer of cell-cycle inhibitors. In mice with the greatest H(2)O(2) production, mature hepatocyte proliferation is inhibited most, and the greatest number of oval cells accumulates. These cells differentiate into intermediate hepatocyte-like cells after a regenerative challenge. Hepatic oval cells are also increased significantly in patients with nonalcoholic fatty liver disease and alcoholic liver disease. In humans, fibrosis stage and oval cell numbers, as well as the number of intermediate hepatocyte-like cells, are strongly correlated. However, cirrhosis is not required for oval cell accumulation in either species. Rather, as in mice, progenitor cell activation in human fatty liver diseases is associated with inhibited replication of mature hepatocytes. The activation of progenitor cells during fatty liver disease may increase the risk for hepatocellular cancer, similar to that observed in the Solt-Farber model of hepatocarcinogenesis in rats.

The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin

Aims:  Cytokeratin (CK) 7 and CK19 expression, present in hepatic progenitor cells (HPCs) and in cholangiocytes but not in normal hepatocytes, has been reported in some hepatocellular carcinomas (HCCs); however, the incidence and relevance of this expression in HCC in Caucasians is not known. Therefore, our aim was to study the occurrence and clinicopathological characteristics of HCC expressing CK7 and/or CK19 in 109 Caucasian patients.

Keratin 19: a key role player in the invasion of human hepatocellular carcinomas

Objective Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive. Design Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays. Results In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib. Conclusions Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

Effectiveness of initial treatment allocation based on expert opinion for prevention of rapid radiographic progression in daily practice of an early RA cohort

Objectives To evaluate expert treatment selection for early rheumatoid arthritis and to validate a prediction model for rapid radiographic progression (RRP) in daily practice. Methods Patients received initial combination therapy with steroids (ICTS) or disease-modifying antirheumatic drug monotherapy (IMT) after informal evaluation of prognostic factors, followed by a tight control strategy. Changes in Sharp/van der Heijde score (total Sharp score (TSS)) of >5 units over 1 year (=RRP) were documented. The mean change in TSS and proportion with RRP were compared between groups. Based on the 28 swollen joint count, rheumatoid factor titre and C reactive protein/erythrocyte sedimentation rate, patients were placed in the ASPIRE prediction matrix, yielding a RRP risk. Numbers needed to treat (NNT) intensively to avoid one RRP after 1 year were calculated. Results The mean change in TSS after 1 year and the proportion with RRP was lower in the ICTS group (n=37) than in the IMT group (n=43). The mean calculated risk of RRP was higher in patients with radiographic progression. The mean NNT intensively to prevent RRP was lower in the ICTS group than in the IMT group. The positive predictive value of NNT for RRP prevention was 12.6%, but the negative predictive value reached 100%. Conclusion ICTS seems more effective in preventing RRP than IMT. The predictive matrix model could be helpful in preventing overtreatment in practice.

113 KERATIN19: A KEY ROLE PLAYER IN THE INVASION OF HUMAN HEPATOCELLULAR CARCINOMAS WITH PROGENITOR CELL FEATURES

that bile proteomic analysis distinguishes CC from non-malignant lesions. Nevertheless, bile collection is only possible by endoscopic retrograde cholangiography and thus invasive, time comsuming and costly. In contrast, spontanous urine is easily to be obtained and in addition an attractive diagnostic source because it harbours less number of peptides. We hypothesized, that urine proteomic analysis is able to differentiate CC from other biliary disorders. Methods: In this prospective study we used capillary electrophoresis mass spectrometry (CE-MS) to establish a disease specific peptide model in a training set of patients with CC (n =14), PSC (n =13), and benign biliary diseases (BBD, n =14). Peptides were characterized by sequencing and immunostaining was performed on liver sections. Results: The peptide model consisting of 42 peptides was evaluated on an independent set of 128 urine samples from 42 CC(including 10 CC on top of PSC), 50 PSCand 36 BBD-patients. This model differentiated CC from PSC and BBD with an area under the curve value of 0.87 (95% confidence interval: 0.80 to 0.92, p = 0.0001) resulting in a correct classification of 35 from 42 CC and 68 from 86 benign strictures (83% sensitivity, 79% specificity). Ten of 10 patients with CC on top of PSC were correctly classified. 101 healthy controls were analyzed and specificity was determined to be 86%. The majority of sequence-identified peptides are fragments of interstitial collagens with some of them also detected in blood indicating their extra-renal origin. Immunostaining of liver sections for matrix metalloproteinase 1 indicated increased activity of the interstitial collagenase in liver epithelial cells of CC patients. Conclusion: The urinary peptide model differentiates CC from PSC and other benign biliary diseases and may become a new diagnostic non-invasive tool for PSC surveillance and CC detection.