Delphine Dargère

Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases

Surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI‐TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI‐TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest‐scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest‐scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8,900 Da) was purified further and was characterized as the C‐terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8,900‐Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment of vitronectin as a new serum marker of HCC in patients with chronic liver diseases. (HEPATOLOGY 2005;41:40–47.)

Molecular Profiling of Hepatocellular Carcinomas (HCC) Using a Large-Scale Real-Time RT-PCR Approach: Determination of a Molecular Diagnostic Index

The aim of this study was to develop and validate a molecular index for the diagnosis of hepatocellular carcinoma (HCC) based on genes whose specificity and level of expression are the most discriminating for the diagnosis of HCC. The level of expression of 219 genes was assessed with a real-time reverse transcription-polymerase chain reaction approach in a training set of samples including normal livers (15), cirrhosis (12), and HCC (16). The most informative genes were selected for the molecular index. This index was prospectively validated in a new set of 40 samples (testing set) and in a set of 45 cirrhotic macronodules. 44 out of the 219 genes were differentially expressed in HCC. 13 out of these 44 genes were finally selected for the molecular index according to their diagnostic performance and after exclusion of most redundant genes. Using this index, 42 out of 43 samples of the training set and 39 out of the 40 samples of the testing set were correctly ranked as HCC or not HCC (normal liver or cirrhosis). The index also enabled correct ranking of 44 out of 45 cirrhotic macronodules into 2 groups: benign (including macroregenerative and dysplastic macronodules) and malignant macronodules. This molecular diagnostic index is an efficient tool both for identification of overt HCC as well as minute lesions (cirrhotic macronodules). It might be useful to correctly diagnose borderline lesion or small well-differentiated hepatocellular carcinomas whose diagnosis is often difficult on a histopathological basis.

Expression of the RNA component of human telomerase (hTR) in prostate cancer, prostatic intraepithelial neoplasia, and normal prostate tissue

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)‐based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non‐radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin‐embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate. Copyright

Quantitative RT-PCR in cirrhotic nodules reveals gene expression changes associated with liver carcinogenesis

Cirrhosis is considered to be the precursor of most hepatocellular carcinomas. To gain insight into the early molecular mechanisms of liver carcinogenesis, this study compared, using real‐time quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), the expression levels of 31 selected genes in normal livers, cirrhotic nodules, and hepatocellular carcinomas. Since cirrhosis is composed of a mixture of polyclonal and monoclonal nodules, gene expression levels were also compared according to the clonal status of the cirrhotic nodules. The expression of eight of the 31 genes studied was significantly increased (NEGF2, ANGPT1, ARF, KRT19, SFN, CLDN4, MMP7, and ETV4) in cirrhotic nodules compared with normal liver, while only one was decreased (LYVE1). The same trend of variation was observed in cirrhosis and hepatocellular carcinomas for all of these genes except KRT19. When gene expression variation was compared according to the clonal status of cirrhotic nodules, only the LYVE1 expression level was significantly different. The LYVE1 gene expression level decreased progressively from polyclonal cirrhotic nodules to monoclonal cirrhotic nodules (polyclonal nodules 0.39 ± 0.25; monoclonal nodules 0.20 ± 0.14; p < 0.05) and to hepatocellular carcinoma (0.07 ± 0.1). In conclusion, this study highlights the fact that among genes strongly dysregulated in hepatocellular carcinoma, some are already abnormally expressed in cirrhosis. The decrease in the expression level of one of these genes, LYVE1, was associated with monoclonality in cirrhotic nodules. Copyright

Gene Expression Study of Aurora-A Reveals Implication During Bladder Carcinogenesis and Increasing Values in Invasive Urothelial Cancer

OBJECTIVESnUrothelial carcinoma is a frequent and aggressive cancer. We wanted to gain better insight into the early molecular mechanisms of bladder carcinogenesis by evaluating Aurora-A gene expression, which is implicated in genomic stability and essential for mitosis.nnnMATERIALSnThis study, using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), analyzed the expression levels of three selected genes in dissected tissues from normal bladder, noninvasive cancers, and muscle-invasive bladder carcinomas (n = 49). We compared gene expression levels of three genes (Aurora-A, and as control uroplakin II (UPII) and TBP, respectively) at different stages of bladder cancer. We used multivariate analysis, receiver operating characteristic curves and the nonparametric Mann-Whitney test.nnnRESULTSnThe expression of Aurora-A gene studied was significantly deregulated, with an increasing level in cancer versus normal tissue Aurora-A. This development was linear. Aurora-A was already deregulated in early stages of carcinogenesis (pTa/pT1) (P = 0.0004) and displayed even more deregulation in muscle-invasive stages (pT2 to pT4). Immunohistochemistry performed on the same samples using Aurora-A antibody confirmed results of RT-PCR, with statistically significant values when comparing m-RNA expression and immunohistochemical values (P = 0.0001).nnnCONCLUSIONSnThis study highlights the fact that Aurora-A gene expression is already strongly deregulated in early stages of urothelial carcinoma with abnormal expression, and might be considered a biomarker of tumor aggression. The increase in Aurora-A expression might provide further information regarding the behavior of bladder cancer in daily practice.

Global proteomic analysis of microdissected cirrhotic nodules reveals significant biomarkers associated with clonal expansion.

Cirrhosis is a heterogeneous tissue composed of polyclonal regenerative and monoclonal neoplastic, potentially malignant nodules from which hepatocellular carcinoma (HCC) might develop. The aim of this study was to investigate proteomic profile changes associated with clonal expansion of cirrhotic nodules and malignant transformation of monoclonal nodules. Seventy-one cirrhotic nodules from 10 female patients with six HCC were dissected from liver surgical specimen by laser capture microdissection. Clonal status of each nodule was assessed by the study of X-chromosome inactivation pattern using the human androgen receptor. Protein profiles were determined by surface-enhanced laser desorption ionisation-time-of-flight technology using Q10 arrays (Cyphergen ProteinChip®). Molecular weight of differentially expressed protein peaks was assessed. An average of 50 protein peaks was obtained for each nodules profile. Comparison of protein profiles in polyclonal (n=45) and monoclonal cirrhotic nodules (n=26) identified three differentially expressed protein peaks (10u2009092, 54u2009025 and 62u2009133u2009Da). All were upregulated in monoclonal nodules. Twelve peaks were differentially expressed between monoclonal nodules and HCC with nine proteins upregulated in cancer samples. This study confirms that proteome analysis can be achieved from a limited number of microdissected cells, and provides further insight into the process of clonal expansion and malignant transformation of cirrhotic nodules.

Neuronal Cholesterol Accumulation Induced by Cyp46a1 Down-Regulation in Mouse Hippocampus Disrupts Brain Lipid Homeostasis

Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimers disease (AD). However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing Cyp46a1 gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to Cyp46a1 gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD.