Franck Chiappini

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.

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.

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)

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.

Gene expression profiling of nonneoplastic mucosa may predict clinical outcome of colon cancer patients.

PURPOSEThis study assessed the possibility to build a prognosis predictor, based on microarray gene expression measures, in Stage II and III colon cancer patients.METHODSTumor and nonneoplastic mucosa mRNA samples from 12 colon cancer patients were profiled using the Affymetrix HGU133A GeneChip. Six of 12 patients experienced a metachronous metastasis, whereas the 6 others remained disease-free for more than five years. Three datasets were constituted, including, respectively, the gene expression measures in tumor samples (T), in adjacent nonneoplastic mucosa samples (A), and the log-ratio of the gene expression measures (L). The step-down procedure of Westfall and Young and the k-nearest neighbor class prediction method were applied on T, A, and L. Leave-one-out cross-validation was used to estimate the generalization error of predictors based on different numbers of genes and neighbors.RESULTSThe most frequent results were one false prediction with the A-based predictors (95 percent) and two false predictions with the T- and l-based predictors (65 and 60 percent, respectively). A-based predictors were more stable (i.e., less sensitive to changes of parameters, such as numbers of genes and neighbors) than T- and l-based predictors. Informative genes in A-based predictors included genes involved in the oxidative and phosphorylative mitochondrial metabolism and genes involved in cell-signaling pathways and their receptors.CONCLUSIONSThis study suggests that one can build a prognosis predictor for Stage II and III colon cancer patients, based on microarray gene expression measures, and suggests the potential usefulness of nonneoplastic mucosa for this purpose.

Relationship between polymerase gamma (POLG) polymorphisms and antiretroviral therapy-induced lipodystrophy in HIV-1 infected patients: a case-control study.

Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used for the treatment of HIV-1 inhibit the replication of mitochondrial DNA (mtDNA), which may contribute to severe mitochondrial toxicity including lipodystrophy, through the inhibition of polymerase gamma (POLG). Polymorphisms of POLG could explain the variation in mitochondrial toxicity in HIV-1-infected patients. We explored the relationship between selected polymorphisms of POLG and lipodystrophy related to NRTIs. We studied single nucleotide polymorphisms (SNP) at three amino acid residues (R1142, E1143 and R1146) and the CAG repeats of POLG in a case-control study including HIV-1 treated patients with lipodystrophy (n=69) and 2 controls (without lipodystrophy) per case matched by age, race and sex (n=138). Compared with matched controls, the polymorphisms in E1143 were significantly more frequent in case patients with lipodystrophy (aOR=4.7; p=0.048), and this was associated with a significant decrease of mtDNA in PBMC. In addition, among the parameters tested, the conditional logistic regression showed that the lipodystrophy has a strong link with E1143 polymorphisms, associated with D4T treatment (aOR=9.29, p=0.002). In conclusion, patients harbouring the changes of E1143 in the catalytic site of POLG exhibit a 4-fold increased risk to develop lipodystrophy than HIV-1 treated patients who do not have changes in E1143 and this risk can increase if the patient presenting the SNP received D4T. These could be due to decreased content of mtDNA in PBMC in these patients. Therefore, the toxicity of NRTIs leading to lipodystrophy in some HIV-1 infected patients could be explained in part by the occurrence of POLG polymorphisms.

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients

Nonalcoholic steatohepatitis (NASH) is a condition which can progress to cirrhosis and hepatocellular carcinoma. Markers for NASH diagnosis are still lacking. We performed a comprehensive lipidomic analysis on human liver biopsies including normal liver, nonalcoholic fatty liver and NASH. Random forests-based machine learning approach allowed characterizing a signature of 32 lipids discriminating NASH with 100% sensitivity and specificity. Furthermore, we validated this signature in an independent group of NASH patients. Then, metabolism dysregulations were investigated in both patients and murine models. Alterations of elongase and desaturase activities were observed along the fatty acid synthesis pathway. The decreased activity of the desaturase FADS1 appeared as a bottleneck, leading upstream to an accumulation of fatty acids and downstream to a deficiency of long-chain fatty acids resulting to impaired phospholipid synthesis. In NASH, mass spectrometry imaging on tissue section revealed the spreading into the hepatic parenchyma of selectively accumulated fatty acids. Such lipids constituted a highly toxic mixture to human hepatocytes. In conclusion, this study characterized a specific and sensitive lipid signature of NASH and positioned FADS1 as a significant player in accumulating toxic lipids during NASH progression.

Generation and Modulation of Hepatocellular Carcinoma Circulating Cells: A New Experimental Model

BACKGROUND To establish a new experimental model of human hepatocellular carcinoma by orthotopic implantation of tumoral cells with its subsequent removal, to generate and modulate circulating tumoral cells. MATERIALS AND METHODS Three human hepatoma cell lines (HepG2, PLC/PRF, and Mahlavu) were orthotopically implanted under the Glissons capsule of the left lateral lobe of the liver in a total of 56 non-obese diabetic/severe combined immunodeficiency mice. Tumor removal was performed 30 d after injection, and a laparotomy without tumor removal was done in control mice. Generation of circulating cells was monitored by flow cytometry using fluorescein isothiocyanate-conjugated anti-HLA antibody. RESULTS In 26 mice implanted with Mahlavu cells, 20 developed a unique tumor allowing a resection (77%), which was technically feasible in 80% of cases. The overall perioperative mortality was 30% (3/10) after resection; no mortality was observed in the control group. The circulating tumoral cells decreased dramatically after resection of the tumor as compared with control mice. CONCLUSION This new model is feasible and may be an interesting useful tool to study the hepatocellular carcinoma metastatic process and is consistent with the human clinical practice.

Hepatocellular Carcinoma: Methods of Circulating Tumor Cells (CTC) Measurements

Hepatocellular carcinoma (HCC) is responsible for significant morbidity and mortality in cirrhosis and also accounts for between 85% and 90% of primary liver cancer (Caldwell & Park 2009; Hussain & El-Serag 2009; Tandon & Garcia-Tsao 2009). Most of HCC in the world occur in the setting of cirrhosis and over half-million of people develop liver cancer every year and an almost equal number die of it (Caldwell & Park 2009; Hussain & El-Serag 2009). Liver cancer prognosis is determined by factors related to the tumor (etiologies) and factors related to the cirrhosis i.e. parameters of liver dysfunction (CLIP 1998; Llovet et al., 1999; Okuda et al., 1985; Tandon & Garcia-Tsao 2009). During the last 30 years the HCC-incidence rate increased dramatically, despite the development of the HBV-vaccine and the program for newborn vaccination against HBV, developed in European and Asian countries (El-Serag et al., 2003; Hussain & El-Serag 2009).