Alain Barrier

Stage II Colon Cancer Prognosis Prediction by Tumor Gene Expression Profiling

PURPOSE This study mainly aimed to identify and assess the performance of a microarray-based prognosis predictor (PP) for stage II colon cancer. A previously suggested 23-gene prognosis signature (PS) was also evaluated. PATIENTS AND METHODS Tumor mRNA samples from 50 patients were profiled using oligonucleotide microarrays. PPs were built and assessed by random divisions of patients into training and validation sets (TSs and VSs, respectively). For each TS/VS split, a 30-gene PP, identified on the TS by selecting the 30 most differentially expressed genes and applying diagonal linear discriminant analysis, was used to predict the prognoses of VS patients. Two schemes were considered: single-split validation, based on a single random split of patients into two groups of equal size (group 1 and group 2), and Monte Carlo cross validation (MCCV), whereby patients were repeatedly and randomly divided into TS and VS of various sizes. RESULTS The 30-gene PP, identified from group 1 patients, yielded an 80% prognosis prediction accuracy on group 2 patients. MCCV yielded the following average prognosis prediction performance measures: 76.3% accuracy, 85.1% sensitivity, and 67.5% specificity. Improvements in prognosis prediction were observed with increasing TS size. The 30-gene PS were found to be highly-variable across TS/VS splits. Assessed on the same random splits of patients, the previously suggested 23-gene PS yielded a 67.7% mean prognosis prediction accuracy. CONCLUSION Microarray gene expression profiling is able to predict the prognosis of stage II colon cancer patients. The present study also illustrates the usefulness of resampling techniques for honest performance assessment of microarray-based PPs.

Duodénopancréatectomie céphalique pour cancer

Duodenopancreatectomie cephalique pour cancer M. Huguier, A. Barrier, C. Gouillat, B. Suc, D. Jaeck, B. Launois De nombreuses propositions ont ete faites pour ameliorer les resultats de l’operation de Whipple. Des etudes prospectives controlees (essais), des meta-analyses ou, a defaut, des etudes retrospectives permettent d’evaluer ces propositions. Les pancreatectomies totales et les curages cellulo-lymphatiques etendus ne semblent pas ameliorer les durees de survie des malades dans des comparaisons retrospectives ou dans des essais. Un envahissement veineux apparent, mesenterique superieur ou portal, ne contre-indique pas a lui seul une exerese dont les resultats sont similaires a ceux observes lorsqu’il n’existe pas d’envahissement. Des essais et une meta-analyse n’ont montre ni avantages ni inconvenients majeurs a la conservation antro-pylorique. Trois essais et une meta-analyse n’ont pas montre que l’anastomose pancreatico-gastrique diminuait le risque de fistule par rapport a l’anastomose pancreatico-jejunale. Deux essais suggerent que, pour la realisation de celle-ci, l’intubation du pancreas dans le jejunum ou le drainage externe temporaire du canal de Wirsung diminuraient ce risque de fistule ce qui, pour cette seconde technique, n’a pas ete confirme par un autre essai. Les resultats sur l’utilisation de la somatostatine sont contradictoires. Les resultats benefiques lorsque le critere de jugement de fistule est biologique n’ont pas ete confirmes par quatre essais sur cinq lorsque le critere de fistule etait clinique et/ou radiologique. Enfin, l’occlusion des canaux pancreatiques par de la fibrine ou l’utilisation de colle sur la surface de l’anastomose pancreatico-digestive n’ont pas diminue le risque de fistule. En conclusion, la duodeno-pancreatectomie cephalique decrite par Whipple reste la technique de reference d’exerese des cancers de la tete du pancreas. L’essai montrant l’interet de l’intubation du pancreas dans le jejunum merite d’etre confirme. En l’absence de resultats convergents, la somatostatine ou le drainage externe du canal de Wirsung peuvent etre reserves aux cas pour lesquels l’anastomose pancreatique semble precaire. Enfin, il a ete suggere par plusieurs etudes que l’experience des equipes semblait le meilleur moyen, non seulement de diminuer la morbidite et la mortalite peri-operatoires, mais aussi d’augmenter les chances de survie des malades.

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.

Rectal cancer surgery in patients more than 80 years of age

BACKGROUND This retrospective study aimed to compare the prognosis for rectal cancer in patients more than 80 years old with that observed in younger patients. METHODS Patients operated on for a rectal adenocarcinoma, from 1980 to 1998, were divided into two groups: group 1 (>80 years, n = 92); group 2 (<80 years, n = 276). RESULTS There were significant differences between the two groups with regard to the sex ratio, the American Society of Anesthesiologists (ASA) classification, the emergency presentation, and the curative operation rate. The operative mortality rate was 8% in group 1, 4% in group 2 (P = 0.26). The overall 5-year survival rate was 35% in group 1, 53% in group 2 (P = 0.0004). In patients operated on for cure, the cancer-specific 5-year survival rate was 50% in group 1, 59% in group 2 (P = 0.08). CONCLUSIONS The prognosis for rectal cancer in patients over 80 years is not significantly different from that of younger patients. Surgery should not be restricted on the basis of age.

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.

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.