Brigitte Debuire

Colon cancer prognosis prediction by gene expression profiling.

This study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in stage II and III colon cancer patients. Tumour (T) and non-neoplastic mucosa (NM) mRNA samples from 18 patients (nine with a recurrence, nine with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. The k-nearest neighbour method was used for prognosis prediction using T and NM gene expression measures. Six-fold cross-validation was applied to select the number of neighbours and the number of informative genes to include in the predictors. Based on this information, one T-based and one NM-based predictor were proposed and their accuracies were estimated by double cross-validation. In six-fold cross-validation, the lowest numbers of informative genes giving the lowest numbers of false predictions (two out of 18) were 30 and 70 with the T and NM gene expression measures, respectively. A 30-gene T-based predictor and a 70-gene NM-based predictor were then built, with estimated accuracies of 78 and 83%, respectively. This study suggests that one can build an accurate prognosis predictor for stage II and III colon cancer patients, based on gene expression measures, and one can use either tumour or non-neoplastic mucosa for this purpose.

Microsatellite instability in European hepatocellular carcinoma.

Genetic instability has been detected in many types of cancers but poorly investigated in hepatocellular carcinoma (HCC). We have studied the incidence of microsatellite instability (MI) at eight highly polymorphic microsatellite markers and the poly A tract BAT26 and tested for mutations at two sites of repetitive sequence (poly-A nucleotides 709 – 718 and GT repeat-nucleotides 1931 – 1936) in the Transforming Growth Factor β (TGFβ) type II receptor (RII) gene, in a group of 46 European HCCs and the surrounding nontumour tissue. This analysis showed that 63% of HCCs exhibit MI in at least one chromosome locus and 41% in two or more loci. No mutations of the TGFβRII gene were found in the MI positive tumours. No correlation was found with clinicopathological characteristics of the tumours such as cirrhosis, etiology, number of nodules, Edmondsons grade and vascular invasion. However, in patients who had a rearranged D16S402 microsatellite in their tumour, the recurrent disease and the number of nodules were significantly higher than in the others (P<0.005 and P<0.02, respectively). We propose to consider D16S402 rearrangement in HCC as a prognostic factor to identify patients presenting a higher risk of recurrence.

Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray.

Understanding the molecular mechanisms underlying fatty liver disease (FLD) in humans is of major importance. We used high-density oligonucleotide microarrays (22.3 K) to assess the mechanisms responsible for the development of human liver steatosis. We compared global gene expression in normal (n=9) and steatotic (n=9) livers without histological signs of inflammation or fibrosis. A total of 34 additional human samples including normal (n=11), steatosis (n=11), HCV-related steatosis (n=4) or steatohepatitis associated with alcohol consumption (n=4) or obesity (n=4) were used for immunohistochemistry or quantitative real-time PCR studies. With unsupervised classification (no gene selection), all steatotic liver samples clustered together. Using step-down maxT multiple testing procedure for controlling the Family-Wise Error-Rate at level 5%, 110 cDNAs (100 over- and 10 underexpressed) were found to be differentially expressed in steatotic and normal livers. Of them were genes involved in mitochondrial phosphorylative and oxidative metabolism. The mean ratio of mitochondrial DNA to nuclear DNA content was higher in liver steatosis compared to normal liver biopsies (1.12±0.14 vs 0.67±0.10; P=0.01). An increased expression of genes involved in inflammation (IL-1R family, TGFB) was also observed and confirmed by quantitative RT-PCR or immunochemistry. In steatohepatitis, an increase of the protein expression of mitochondrial antigens, IL-1R1, IGF2 and TGFB1 was also observed, interleukin 1 receptor being always strongly expressed in steatohepatitis linked to alcohol or obesity. In conclusion, mitochondrial alterations play a major role in the development of steatosis per se. Activation of inflammatory pathways is present at a very early stage of steatosis, even if no morphological sign of inflammation is observed.

Clinicopathologic, phenotypic, and genotypic characteristics of gastrointestinal mesenchymal tumors

BACKGROUND & AIMS Variability in the frequency of KIT mutations in gastrointestinal mesenchymal tumors has been reported in the literature, and their prognostic value remains uncertain. This retrospective multicenter study included 276 patients with gastrointestinal mesenchymal tumors. METHODS We detected c-kit and CD34 protein expression by immunohistochemistry. Mutations in exons 11 and 9 of KIT and exons 12 and 18 of PDGFR were detected by length analysis of polymerase chain reaction products and direct DNA sequencing. RESULTS Eighty-seven percent of the tumors analyzed were c-kit positive, with gastric tumors expressing CD34 more frequently than other tumors (86% vs. 52%; P < 0.001). KIT exon 11 mutations were detected in 90 of 179 (50.3%) of c-kit-positive and 12% of c-kit-negative tumors. These mutations showed variation in their length and location. Mutations were heterozygous in 94% of cases. Mutations were more frequent in CD34( +) tumors than in CD34( -) tumors ( P < 0.01), and 9% of tumors had a second mutation in exon 11. Mutations in exon 9 of KIT were present in 5.1% of the gastrointestinal stromal tumors, and mutations of the PDGFR were present in 11% of the KIT -nonmutated tumors. Patients age, the primary location, size, necrosis, and mitotic counts of tumors were associated with metastases in c-kit-positive tumors. However, mitotic activity was the only independent factor identified in multivariate analysis ( P < 0.001). KIT mutations were slightly more frequent in metastatic than in nonmetastatic tumors (61% vs. 46%; P = 0.06). Deletions of codons 562-579 were more strongly associated with metastases than were deletions of codons 550-561 ( P = 0.0001). CONCLUSIONS Mutations in KIT or PDGFR were detected in 58.4% of the c-kit-positive and also in some c-kit-negative tumors.

Ischemic preconditioning modulates the expression of several genes, leading to the overproduction of IL-1Ra, iNOS, and Bcl-2 in a human model of liver ischemia-reperfusion

Ischemia triggers an inflammatory response that precipitates cell death during reperfusion. Several studies have shown that tissues are protected by ischemic preconditioning (IP) consisting of 10 min of ischemia followed by 10 min of reperfusion just before ischemia. The molecular basis of this protective effect is poorly understood. We used cDNA arrays (20K) to compare global gene expression in liver biopsies from living human liver donors who underwent IP (n=7) or not (n=7) just before liver devascularization. Microarray data were analyzed using paired t test with a type I error rate fixed at α = 2.5 106 (Bonferroni correction). We found that 60 genes were differentially expressed (36 over‐ and 24 underexpressed in preconditioning group). After IP, the most significantly overexpressed gene was IL‐1Ra. This was confirmed by immunoblotting. Differentially expressed were genes involved in apoptosis (NOD2, ephrin‐A1, and calpain) and in the carbohydrate metabolism. A significant increase in the amount of the anti‐apoptotic protein Bcl‐2 in preconditioned livers but no change in the cleavage of procaspase‐3, ‐8, and ‐9 was observed. We also observed an increase in the amount in the inducible nitric oxide synthase. Therefore, the benefits of IP may be associated with the overproduction of IL‐1Ra, Bcl‐2, and NO countering the proinflammatory and proapoptotic effects generated during ischemia‐reperfusion. AlainBarrier NataliaOlaya FranckChiappini FrançoisRoser OlivierScatton CédricArtus BrigitteFranc SandrineDudoit AntoineFlahault BrigitteDebuire DanielAzoulay AntoinetteLemoine Ischemic preconditioning modulates the expression of several genes, leading to the overproduction of IL‐1Ra, iNOS, and Bcl‐2 in a human model of liver ischemia‐reperfusion. FASEB J. 19, 1617–1626 (2005)

Prognosis of stage II colon cancer by non-neoplastic mucosa gene expression profiling

We have assessed the possibility to build a prognosis predictor (PP), based on non-neoplastic mucosa microarray gene expression measures, for stage II colon cancer patients. Non-neoplastic colonic mucosa mRNA samples from 24 patients (10 with a metachronous metastasis, 14 with no recurrence) were profiled using the Affymetrix HGU133A GeneChip. Patients were repeatedly and randomly divided into 1000 training sets (TSs) of size 16 and validation sets (VS) of size 8. For each TS/VS split, a 70-gene PP, identified on the TS by selecting the 70 most differentially expressed genes and applying diagonal linear discriminant analysis, was used to predict the prognoses of VS patients. Mean prognosis prediction performances of the 70-gene PP were 81.8% for accuracy, 73.0% for sensitivity and 87.1% for specificity. Informative genes suggested branching signal-transduction pathways with possible extensive networks between individual pathways. They also included genes coding for proteins involved in immune surveillance. In conclusion, our study suggests that one can build an accurate PP for stage II colon cancer patients, based on non-neoplastic mucosa microarray gene expression measures.

New perspectives and strategy research biomarkers for hepatocellular carcinoma.

Abstract Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Cirrhosis caused by hepatitis B virus, hepatitis C virus or chronic alcohol intake is associated with major risk. Systematic screening for HCC of asymptomatic patients with cirrhosis is needed for earlier detection of small tumors requiring treatment (liver transplantation, surgical resection, percutaneous techniques). The recommended screening strategy among cirrhotic patients is based on regular liver ultrasonography associated with serum α-fetoprotein (AFP) assay. As the performance of AFP is not satisfactory, additional tumoral markers are proposed (des-γ-carboxyprothrombin, glycosylated AFP-L3 fraction). Currently, diagnosis of HCC in cirrhotic patients includes non-invasive tests (imaging after contrast administration, AFP assay); diagnostic biopsy is performed when imaging is limited. After treatment, tumor recurrence is assessed by regular follow-up (AFP assay and imaging). Despite the lack of accurate markers, recent developments in genomic and proteomic approaches will allow the discovery of new biomarkers for primary tumors, as well as for recurrence. This review summarizes the current state of biomarkers for screening, diagnosis and follow-up of HCC, and highlights new perspectives in the field. Clin Chem Lab Med 2007;45:1169–79.

Expression of T-cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma

Overexpression of T‐cadherin (T‐cad) transcripts occurs in ≈50% of human hepatocellular carcinomas (HCCs). To elucidate T‐cad functions in HCC, we examined T‐cad protein expression in normal and tumoral human livers and hepatoma cell lines and investigated its influence on invasive potential of HCC using RNA interference silencing of T‐cad expression in Mahlavu cells. Whereas T‐cad expression was restricted to endothelial cells (EC) from large blood vessels in normal livers, it was up‐regulated in sinusoidal EC from 8/15 invasive HCCs. Importantly, in three of them (38%) T‐cad was detected in tumor cells within regions in which E‐cadherin expression was absent. Among six hepatoma cell lines, only Mahlavu expressed T‐cad but not E‐cadherin. T‐cad exhibited a globally punctuate distribution in quiescent Mahlavu and additionally it concentrated at the leading edge of migrating cells. Matrigel invasion assay revealed that Mahlavu possess a high invasive potential that was significantly inhibited by T‐cad silencing. Wound healing and random motility assays demonstrated that inhibition of T‐cad expression in Mahlavu significantly reduced their motility. We propose that T‐cad expression in tumor cells might occur by cadherin‐switching during epithelial‐mesenchymal transition and may represent an additional mechanism contributing to HCC metastasis.—Riou, P., Saffroy, R., Chenailler, C., Franc, B., Gentile, C., Rubinstein, E., Resink, T., Debuire, B., Piatier‐Tonneau, D., Lemoine, A. Expression of T‐cadherin in tumor cells influences invasive potential of human hepatocellular carcinoma. FASEB J. 20, 2291–2301 (2006)

Prospective evaluation of blood concentration of mitochondrial DNA as a marker of toxicity in 157 consecutively recruited untreated or HAART-treated HIV-positive patients.

Highly active antiretroviral therapy (HAART) can cause mitochondrial toxicity. The concentration of mitochondrial DNA (mtDNA) in peripheral blood cells has been reported to be a marker of this toxicity. However, these observations are controversial and were drawn from small series. Thus, we analysed the value of blood mtDNA as a marker of mitochondrial toxicity in a large cohort of human immunodeficiency virus (HIV)-infected out-patients during routine clinical evaluations. Real-time quantitative PCR was used to determine the mtDNA to nuclear DNA (nDNA) ratio in peripheral blood mononuclear cells from 157 consecutive HIV-1-infected patients (13 naive, 144 receiving HAART) and 30 HIV-1-uninfected patients. The mtDNA to nDNA ratio was significantly lower in both groups of HIV-infected patients than in the control group. No significant difference was observed between treated and naive HIV-infected patients. Lactataemia was significantly lower in controls than in the group of HIV-treated patients. None of the treated patients had lactataemia >5 mmol/l or bicarbonates <20 mmol/l. Triglyceride levels were significantly higher in the HAART-treated patients than in the nontreated patients. Clinical symptoms of lipodystrophy were observed in 62 HAART-treated patients. These symptoms were not associated with an abnormal mtDNA to nDNA ratio or plasma triglyceride concentration. The mtDNA to nDNA ratio was lower in DDI/D4T-treated patients than in AZT/3TC-treated patients. In conclusion, there are no obvious links between the mtDNA to nDNA ratio in peripheral mononuclear cells and any clinical symptoms or lactate level. Thus, the mtDNA to nDNA ratio in leukocytes does not seem to be an accurate marker of mild and/or long-term mitochondrial toxicity.

Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy

BACKGROUND/AIMS Liver pathology induced by chemotherapy (steatosis or vascular injury) is known to increase the livers sensitivity to ischemia/ reperfusion (I/R) injury, thereby increasing morbidity and mortality after liver resection. Our aim was to assess whether ischemic preconditioning (IP) reduces I/R injury to livers with chemotherapy-induced pathology. METHODS We analyzed a series of livers from patients treated with chemotherapy for colorectal cancer who underwent IP (n=30) or not (n=31) before hepatectomy. All but one of the livers exhibited chemotherapy-induced steatosis and/ or peliosis before the I/R insult. RESULTS Necrosis was less frequent (p=0.038) in livers with IP than in the others. IP had no influence on apoptosis as assessed by terminal transferase uridyl nick-end labeling (TUNEL) assay or caspase-3, -8 and -9 expression. IP induced a twofold increase in B-cell leukemia/ lymphoma 2 (Bcl-2; p<0.05), which was localized to hepatocytes of centrolobular and peliotic areas and colocalized with the autophagy protein beclin-1 in livers with IP, suggesting their coordinated role in autophagy. Increased expression of the phosphorylated Bcl-2 was observed in preconditioned livers and was associated with a decreased immunoprecipitation of beclin-1 and the increased expression of light chain 3 type II (LC3-II). The increased number of autophagic vacuoles seen by electron microscopy confirmed an association of autophagy in chemotherapy-injured livers following IP. However, the differences in protein expression were not reflected in postresection liver-injury tests or measure of patient morbidity. CONCLUSIONS IP is associated with a reduction in necrosis of hepatocytes already damaged by chemotherapy and an activation of autophagy. Bcl-2 and beclin-1 could be major targets in the regulation of cell death during I/R injury.