Olivier Buhard

Publications (17)146.5 Total impact

• Article: Extensive characterization of sphere models established from colorectal cancer cell lines.

  Ada Collura, Laetitia Marisa, Diletta Trojan, Olivier Buhard, Anaïs Lagrange, Arnaud Saget, Marianne Bombled, Patricia Méchighel, Mira Ayadi, Martine Muleris, Aurélien de Reynies, Magali Svrcek, Jean-François Fléjou, Jean-Claude Florent, Florence Mahuteau-Betzer, Anne-Marie Faussat, Alex Duval
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  ABSTRACT: Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of "stemness". These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.
  Cellular and Molecular Life Sciences CMLS 09/2012; · 6.57 Impact Factor
• Article: Expression of a mutant HSP110 sensitizes colorectal cancer cells to chemotherapy and improves disease prognosis.

  Coralie Dorard, Aurélie de Thonel, Ada Collura, Laetitia Marisa, Magali Svrcek, Anaïs Lagrange, Gaetan Jego, Kristell Wanherdrick, Anne Laure Joly, Olivier Buhard, [......], Leila Bengrine-Lefèvre, Christophe Louvet, Christophe Tournigand, Jérémie H Lefèvre, Yann Parc, Emmanuel Tiret, Jean-François Fléjou, Marie-Pierre Gaub, Carmen Garrido, Alex Duval
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  ABSTRACT: Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and treatment response in CRC.
  Nature medicine 09/2011; 17(10):1283-9. · 27.14 Impact Factor
• Article: Methylation tolerance due to an O6-methylguanine DNA methyltransferase (MGMT) field defect in the colonic mucosa: an initiating step in the development of mismatch repair-deficient colorectal cancers.

  Magali Svrcek, Olivier Buhard, Chrystelle Colas, Florence Coulet, Sylvie Dumont, Illiasse Massaoudi, Amel Lamri, Richard Hamelin, Jacques Cosnes, Carla Oliveira, Raquel Seruca, Marie-Pierre Gaub, Michèle Legrain, Ada Collura, Olivier Lascols, Emmanuel Tiret, Jean-François Fléjou, Alex Duval
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  ABSTRACT: O(6)-Methylguanine-DNA methyltransferase (MGMT) removes methyl adducts from O(6)-guanine. Known as methylation tolerance, selection for mismatch repair (MMR)-deficient cells that are unable to initiate lethal processing of O(6)-methylguanine-induced mismatches in DNA is observed in vitro as a consequence of MGMT deficiency. It was therefore hypothesised that an MGMT field defect may constitute a preneoplastic event for the development of MMR-deficient tumours displaying microsatellite instability (MSI). MGMT expression was investigated by immunohistochemistry and the methylation status of the gene promoter by PCR in neoplastic, adjacent and distant mucosal tissues of patients with MSI or non-MSI (MSS) colorectal cancer (CRC). The cancers were familial (42 MSI, 13 MSS) or sporadic (40 MSI, 49 MSS) in origin, or arose in the context of inflammatory bowel disease (IBD; 13 MSI, 36 MSS). Colonic mucosa from patients with diverticulitis (n=20) or IBD (n=39 in 27 patients) without cancer served as controls. Loss of MGMT expression was more frequent in MSI than MSS CRC (p=0.047). In comparison with MSS tumours, MSI CRC occurred more frequently adjacent to patches of mucosa that lacked MGMT expression (p=0.002). Overall, loss of MGMT expression was associated with MGMT gene promoter methylation (p=0.03). MGMT field defects are more frequently associated with MSI than MSS CRC. These findings indicate that methylation tolerance may be a crucial initiating step prior to MMR deficiency in the development of MSI CRC in familial, sporadic and IBD settings.
  Gut 11/2010; 59(11):1516-26. · 10.11 Impact Factor
• Source

  Available from: Martine Muleris

  Article: Azathioprine-induced carcinogenesis in mice according to Msh2 genotype.

  Alexandra Chalastanis, Virginie Penard-Lacronique, Magali Svrcek, Valérie Defaweux, Nadine Antoine, Olivier Buhard, Sylvie Dumont, Bettina Fabiani, Isabelle Renault, Emmanuel Tubacher, Jean-François Fléjou, Hein Te Riele, Alex Duval, Martine Muleris
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  ABSTRACT: The thiopurine prodrug azathioprine is used extensively in cancer therapy. Exposure to this drug results in the selection of DNA mismatch repair-deficient cell clones in vitro. It has also been suggested that thiopurine drugs might constitute a risk factor for the emergence of human neoplasms displaying microsatellite instability (MSI) because of deficient DNA mismatch repair. Azathioprine was administered via drinking water (6-20 mg/kg body weight per day) to mice that were null (Msh2⁻(/)⁻; n = 27), heterozygous (Msh2(+/)⁻; n = 22), or wild type (Msh2(WT); n = 18) for the DNA mismatch repair gene Msh2. Control mice (45 Msh2⁻(/)⁻, 38 Msh2(+/)⁻, and 12 Msh2(WT)) received drinking water lacking azathioprine. The effect of azathioprine on tumorigenesis and survival of the mice was evaluated by Kaplan-Meier curves using log-rank and Gehan-Breslow-Wilcoxon tests. Mouse tumor samples were characterized by histology and immunophenotyping, and their MSI status was determined by polymerase chain reaction analysis of three noncoding microsatellite markers and by immunohistochemistry. Msh2 status of tumor samples was assessed by loss of heterozygosity analyses and sequencing after reverse transcription-polymerase chain reaction of the entire Msh2 coding sequence. All statistical tests were two-sided. Most untreated Msh2(WT) and Msh2(+/)⁻ mice remained asymptomatic and alive at 250 days of age, whereas azathioprine-treated Msh2(WT) and Msh2(+/)⁻ mice developed lymphomas and died prematurely (median survival of 71 and 165 days of age, respectively). Azathioprine-treated Msh2(+/)⁻ mice developed diffuse lymphomas lacking Msh2 expression and displaying MSI due to somatic inactivation of the functional Msh2 allele by loss of heterozygosity or mutation. By contrast, azathioprine-treated Msh2(WT) mice displayed no obvious tumor phenotype, but histological examination showed microscopic splenic foci of neoplastic lymphoid cells that retained Msh2 expression and did not display MSI. Both untreated and azathioprine-treated Msh2⁻(/)⁻ mice had a reduced lifespan compared with untreated Msh2(WT) mice (median survival of 127 and 107 days of age, respectively) and developed lymphomas with MSI. Azathioprine-induced carcinogenesis in mice depends on the number of functional copies of the Msh2 gene.
  CancerSpectrum Knowledge Environment 10/2010; 102(22):1731-40. · 14.07 Impact Factor
• Article: The mechanisms underlying MMR deficiency in immunodeficiency-related non-Hodgkin lymphomas are different from those in other sporadic microsatellite instable neoplasms.

  Claire Borie, Chrystelle Colas, Peggy Dartigues, Thierry Lazure, Patricia Rince, Olivier Buhard, Patrick Folliot, Alexandra Chalastanis, Martine Muleris, Richard Hamelin, [......], Raquel Seruca, Amy Chadburn, Véronique Leblond, Stéphane Barete, Gianluca Gaïdano, Antoine Martin, Philippe Gaulard, Jean-François Fléjou, Martine Raphael, Alex Duval
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  ABSTRACT: The spectrum of tumors showing microsatellite instability (MSI) has recently been enlarged to sporadic neoplasms whose incidence is favored in the context of chronic immunosuppression. We investigated the biological, therapeutic and clinical features associated with MSI in immunodeficiency-related non-Hodgkin lymphomas (ID-RL). MSI screening was performed in 275 ID-RL. MSI ID-RL were further analyzed for MMR gene expression and for BRAF/KRAS mutations since these genes are frequently altered in MSI cancers. We also assessed the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme whose inactivation has been reported in lymphomas and may help in the selection of MMR deficient clones. Unlike other sporadic MSI neoplasms, MSI ID-RL (N = 17) presented with heterogeneous MMR defects and no MLH1 promoter methylation. About one third of these tumors presented with normal expression of MLH1, MSH2, MSH6 and PMS2. They accumulated BRAF activating mutations (33%). Unlike other ID-RL, MSI ID-RL were primarily EBV-negative NHL of T-cell origin, and arose after long-term immunosuppression in patients who received azathioprine as part of their immunosuppressive regimen (p = 0.05) and/or who exhibited methylation-induced loss of expression of MGMT in tumor cells (p= 0.02). Overall, these results highlight that, in the context of deficient immune status, some MSI neoplasms arise through alternative mechanism when compared to other sporadic MSI neoplasms. They give the exact way how to make the diagnosis of MSI in these tumors and may help to define biological and clinicalrisk factors associated with their emergence in such a clinicalcontext.
  International Journal of Cancer 07/2009; 125(10):2360-6. · 5.44 Impact Factor
• Article: [Clinical and molecular consequences of microsatellite instability in human cancers].

  Richard Hamelin, Alexandra Chalastanis, Chrystelle Colas, Jamila El Bchiri, Dominique Mercier, Ann-Sofie Schreurs, Virginie Simon, Magali Svrcek, Aziz Zaanan, Claire Borie, Olivier Buhard, Emilie Capel, Habib Zouali, Françoise Praz, Martine Muleris, Jean-François Fléjou, Alex Duval
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  ABSTRACT: During each cell division, DNA polymerase makes mistakes while copying DNA. These errors, more frequent at the level of repeated sequences called microsatellites are normally repaired by a system called MMR (mismatch repair). Tumors defective in their MMR system accumulate mutations (deletions and insertions of some nucleotides) at the level of microsatellites and are called MSI (microsatellite instability). Microsatellites are numerous and scattered throughout the genome, in coding and non-coding regions. The instability of non-coding microsatellites is not known to have a major role in the process of cell transformation, but is a good indicator of the MSI status. On the other hand, instability by deletion or insertion in a coding region leads to a frameshift within the gene containing the repeat. The consequence is, the more often, the inactivation of this gene that potentially plays a role in initiation and/or MSI tumor progression. The MSI phenotype was first described in about 15 % of colorectal cancers that maybe of sporadic or hereditary (Lynch syndrome, or HNPCC for hereditary non-polyposis colorectal cancer) origin. It is also associated with about 15 % of gastric and endometrial tumors, and to a lesser extent with other human tumors. Besides a fundamental interest because of its original transformation mechanism, the analysis of MSI tumors is also important for clinical reasons. It was indeed shown that MSI tumors were associated with a better prognosis than non-MSI (also called MSS for microsatellite stable) tumors, and responded differently to conventional chemotherapeutic drugs used for the management of colorectal cancers. All these points will be discussed in details in the present review.
  Bulletin du cancer 02/2008; 95(1):121-32. · 0.67 Impact Factor
• Source

  Available from: Françoise Praz

  Article: Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers.

  Jamila El-Bchiri, Agathe Guilloux, Peggy Dartigues, Etienne Loire, Dominique Mercier, Olivier Buhard, Iradj Sobhani, Pierre de la Grange, Didier Auboeuf, Françoise Praz, Jean-François Fléjou, Alex Duval
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  ABSTRACT: Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.
  PLoS ONE 01/2008; 3(7):e2583. · 4.09 Impact Factor
• Article: Specific clinical and biological features characterize inflammatory bowel disease associated colorectal cancers showing microsatellite instability.

  Magali Svrcek, Jamila El-Bchiri, Alexandra Chalastanis, Emilie Capel, Sylvie Dumont, Olivier Buhard, Carla Oliveira, Raquel Seruca, Céline Bossard, Jean-François Mosnier, Françoise Berger, Emmanuelle Leteurtre, Anne Lavergne-Slove, Marie-Pierre Chenard, Richard Hamelin, Jacques Cosnes, Laurent Beaugerie, Emmanuel Tiret, Alex Duval, Jean-François Fléjou
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  ABSTRACT: Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency has been reported to occur at variable frequencies in inflammatory bowel disease-associated intestinal neoplasias (IBD-Ns). We investigated a large series of IBD-N for associations between MSI and several biologic and clinical parameters related to tumors, patients, and their treatment. A total of 277 IBD-Ns in 205 patients were screened for MSI. Biologic and clinical variables of patients with high levels of DNA microsatellite instability high (MSI-H) were collected and compared with those associated with 33 MSI-H non-IBD colorectal cancers (CRCs). A total of 27 IBD-Ns from 17 patients were found to be MSI-H. Compared with sporadic MSI-H CRCs, patients presented with a younger age at diagnosis, and there was no female predominance and no right-sided predominance. Unlike sporadic MSI-H CRCs, MSI-H IBD-Ns presented with heterogeneous mismatch repair defects involving MLH1, MSH2, MSH6, or PMS2, and a low frequency of MLH1 promoter methylation. They exhibited frequent BRAF mutations and frameshift mutations in genes containing coding repeat sequences. The mechanisms underlying MMR deficiency in MSI-H IBD-Ns are different from those in sporadic MSI-H tumors and seem to be more related to those observed in hereditary MSI-H tumors. However, BRAF mutations were observed in MSI-H IBD-Ns, similar to sporadic MSI-H tumors, but unlike hereditary MSI-H tumors. Finally, the mutational events in target genes for instability are the same in MSI-H IBD-N tumors as in non-IBD sporadic and hereditary colorectal MSI-H cancers, indicating a colon-related repertoire of target gene alterations.
  Journal of Clinical Oncology 10/2007; 25(27):4231-8. · 18.37 Impact Factor
• Article: Spectrum of molecular alterations in colorectal, upper urinary tract, endocervical, and renal carcinomas arising in a patient with hereditary non-polyposis colorectal cancer.

  Pierre Mongiat-Artus, Catherine Miquel, Jean-François Fléjou, Florence Coulet, Jérôme Verine, Olivier Buhard, Hany Soliman, Pierre Teillac, Françoise Praz
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  ABSTRACT: Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.
  Archiv für Pathologische Anatomie und Physiologie und für Klinische Medicin 09/2006; 449(2):238-43. · 2.49 Impact Factor
• Article: Detection of microsatellite instability in endometrial cancer: advantages of a panel of five mononucleotide repeats over the National Cancer Institute panel of markers.

  Yick Fu Wong, Tak Hong Cheung, Keith Wing Kit Lo, So Fan Yim, Loucia Kit Yin Chan, Olivier Buhard, Alex Duval, Tony Kwok Hung Chung, Richard Hamelin
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  ABSTRACT: The aim of this study was to find the optimal set of microsatellite markers for diagnosis of the microsatellite instability (MSI) phenotype in endometrial cancers. We compared the sensitivity, specificity and ease of use of a reference panel of five markers originally recommended by the National Cancer Institute (NCI) for colorectal cancer and a panel of five quasi-monomorphic mononucleotide repeat markers (pentaplex PCR system). We used these panels for establishing the MSI status of a series of 80 sporadic endometrial adenocarcinomas by comparing the allelic profiles of the markers between tumor and matching germline DNA. Both panels detected the same subset of 21 out of 80 (26%) endometrial MSI carcinomas. However, in the MSI cases, the mean instability of the five mononucleotide repeats was 96.1% as compared with a mean instability of 69.8% for the three dinucleotide repeats of the NCI panel, indicating a superiority of mononucleotide repeats over dinucleotide repeats in detecting MSI. The fact that the two panels of markers detect the same set of MSI tumors is due to the presence of two mononucleotide repeats within the NCI panel. As demonstrated previously in gastric and colon MSI cases, the pentaplex PCR reaction using mononucleotide repeats is thus an easier and more sensitive method than the NCI panel, for the screening of MSI status in endometrial tumors.
  Carcinogenesis 06/2006; 27(5):951-5. · 5.70 Impact Factor
• Article: Infrequent microsatellite instability in urothelial cell carcinoma of the bladder in young patients.

  Pierre Mongiat-Artus, Catherine Miquel, Madelon van der Aa, Olivier Buhard, Richard Hamelin, Chris Bangma, Pierre Teillac, Theodorus van der Kwast, Françoise Praz
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  ABSTRACT: Defects in the DNA mismatch repair result in microsatellite instability (MSI), which characterise most tumours related to the hereditary non-polyposis colorectal cancer syndrome and some sporadic tumours. Several studies have reported the occurrence of MSI in urothelial cell carcinoma (UCC) of the bladder with a particularly high incidence in tumours from young patients. In this study, we have evaluated the occurrence of MSI in primary bladder UCC arising in seventeen young patients selected for being below 45 years of age at diagnosis. Microsatellite analysis has been performed using the panel of five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27) recently recommended to detect MSI tumours. The original Bethesda panel including BAT-25, BAT-26 and three dinucleotide repeats (D2S123, D5S346, D17S250) has further been studied in 10 UCC samples. MSI has been observed in only one of the 17 bladder UCC studied. Using the original Bethesda panel, identical results were obtained, indicating that the panel of five mononucleotide markers adequately detected MSI in UCC tumours. Our data indicate that classical MSI affecting mono- or di-nucleotides are rarely involved in bladder UCC developing in young patients. Further studies using gold standard criteria would help clarifying the involvement of MSI in the pathogenesis of bladder UCC.
  European Urology 05/2006; 49(4):685-90. · 8.49 Impact Factor
• Article: Multipopulation analysis of polymorphisms in five mononucleotide repeats used to determine the microsatellite instability status of human tumors.

  Olivier Buhard, Francesca Cattaneo, Yick Fu Wong, So Fan Yim, Eitan Friedman, Jean-François Flejou, Alex Duval, Richard Hamelin
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  ABSTRACT: Human gastrointestinal tumors with inactivated DNA mismatch repair system (microsatellite instability [MSI] tumors) have distinct molecular and clinicopathologic profiles, and are associated with favorable prognosis. There is evidence suggesting that colorectal cancer patients with MSI tumors respond differently to adjuvant chemotherapy as compared with patients with non-MSI tumors. Finally, determination of the MSI status has clinical application for assisting in the diagnosis of suspected hereditary cases. It is thus becoming increasingly recognized that testing for MSI should be conducted systematically in all human cancers potentially of this type. We recently described a pentaplex polymerase chain reaction of five mononucleotide repeats to establish the MSI status of human tumors, and showed that this assay was 100% sensitive and specific. Moreover, these markers are quasimonomorphic in germline DNA of the white population (ie, individuals of Eurasian origin), and could be used for tumor MSI determination without the requirement for matching normal DNA in this group. In this study, we analyzed a comparable panel of five mononucleotide markers in germline DNA from 1,206 individuals encompassing 55 different populations worldwide. Results With the exception of two Biaka Pygmies and one San individual for whom three markers showed variant alleles (three cases [0.2%]), the remaining 1,203 individuals showed no alleles of variant size (1,055 cases [87.5%]), or only one (122 cases [10.1%]) or two (26 cases [2.2%]) markers with variant alleles. All 60 MSI tumors investigated display instability in at least four of the five markers. We demonstrated that tumor MSI status can be determined using the pentaplex reaction for all human populations without the need for matching normal DNA.
  Journal of Clinical Oncology 02/2006; 24(2):241-51. · 18.37 Impact Factor
• Article: [Analysis of the microsatellite instability gene mutation in the urothelial tumor of the upper urinary tract].

  Pierre Mongiat-Artus, Catherine Miquel, Madelon van der Aa, Olivier Buhard, Richard Hamelin, Hany Soliman, Chris Bangma, Anne Janin, Pierre Teillac, Theodorus van der Kwast, Françoise Praz
  Progrès en Urologie 01/2006; 15(6 Suppl 1):1286-92. · 0.58 Impact Factor
• Article: Differential nonsense mediated decay of mutated mRNAs in mismatch repair deficient colorectal cancers.

  Jamila El-Bchiri, Olivier Buhard, Virginie Penard-Lacronique, Gilles Thomas, Richard Hamelin, Alex Duval
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  ABSTRACT: The nonsense-mediated decay (NMD) system normally targets mRNAs with premature termination codons (PTCs) for rapid degradation. We investigated for a putative role of NMD in cancers with microsatellite instability (MSI-H cancers), because numerous mutant mRNAs containing PTC are generated in these tumors as a consequence of their mismatch repair deficiency. Using a quantitative RT-PCR approach in a large series of colorectal cancer cell lines, we demonstrate a significantly increased rate of degradation of mutant mRNAs containing a PTC compared with wild-type. A specific siRNA strategy was used to inhibit RENT-1 and/or RENT-2 activity, two major genes in the NMD system. This allowed us to show that increased degradation of PTC-containing mRNAs in MSI-H tumors was partly dependent upon NMD activity. The efficiency of NMD for the degradation of mutant mRNAs from target genes was highly variable in these cancers. NMD degraded some of them (TGFssRII, MSH3, GRK4), although allowing the persistent expression of others (BAX, TCF-4). This is of particular interest within the context of a proposed conservation of biological activity for the corresponding mutated proteins. We thus propose that NMD might play an important role in the selection of target gene mutations with a functional role in MSI-H carcinogenesis.
  Human Molecular Genetics 09/2005; 14(16):2435-42. · 7.64 Impact Factor
• Article: Mononucleotide repeats BAT-26 and BAT-25 accurately detect MSI-H tumors and predict tumor content: implications for population screening.

  Caroline Brennetot, Olivier Buhard, Florence Jourdan, Jean-François Flejou, Alex Duval, Richard Hamelin
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  ABSTRACT: Tumors with a defective DNA mismatch repair system (MSI-H tumors) have distinct molecular and clinicopathologic profiles compared with mismatch repair-proficient tumors and are associated with a relatively favorable prognosis. There is evidence to suggest that colorectal cancer patients with MSI-H tumors respond differently to adjuvant chemotherapy. Determination of MSI status also has clinical application for assisting in the diagnosis of suspected hereditary nonpolyposis colorectal cancer cases. For these reasons, it is becoming increasingly apparent that testing for MSI should be conducted routinely in human cancer types that frequently present with such a phenotype. BAT-26 and BAT-25 are mononucleotide repeats that are widely used to establish the MSI status of human tumors. We show here that their allelic size profiles provide an estimate of the percentage of contaminating normal cells in MSI-H tumors. These markers are sensitive enough to detect instability when the tumor cell content of a sample is as low as 5-10%. MSI-H tumors contain mutations in coding repeats within genes known to be targets for instability. In cases with low tumor cell content, no mutations in any of 9 coding repeats were detected. However, when these samples were enriched for tumor cells, mutations were detected in the same target genes. Thus, BAT-26 and BAT-25 markers accurately identify MSI-H tumors without prior need for enrichment for tumor cells and indicate which samples require further purification before screening for mutations in target genes for instability. Our results have implications for large-scale screening of cancer patients to determine MSI-H status and prognosis.
  International Journal of Cancer 02/2005; 113(3):446-50. · 5.44 Impact Factor
• Source

  Available from: pnas.org

  Article: The mutator pathway is a feature of immunodeficiency-related lymphomas.

  Alex Duval, Martine Raphael, Caroline Brennetot, Helene Poirel, Olivier Buhard, Alban Aubry, Antoine Martin, Amor Krimi, Veronique Leblond, Jean Gabarre, [......], Jean Feuillard, Philippe Gaulard, Marie Helene Delfau, Sophie Ferlicot, Virginie Eclache, Sophie Prevot, Catherine Guettier, Pascale Cornillet Lefevre, Francoise Adotti, Richard Hamelin
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  ABSTRACT: The mutator phenotype caused by defects in the mismatch repair system is observed in a subset of solid neoplasms characterized by widespread microsatellite instability-high (MSI-H). It is known to be very rare in non-Hodgkin lymphomas (NHL), whereas mutator NHL is the most frequent tumor subtype in mismatch repair-deficient mice. By screening a series of 603 human NHL with specific markers of the mutator phenotype, we found here 12 MSI-H cases (12/603, 2%). Of interest, we demonstrated that this phenotype was specifically associated with immunodeficiency-related lymphomas (ID-RL), because it was observed in both posttransplant lymphoproliferative disorders (9/111, 8.1%) and HIV infection-related lymphomas (3/128, 2.3%) but not in a large series of NHL arising in the general population (0/364) (P < 0.0001). The MSI pathway is known to lead to the production of hundreds of abnormal protein neoantigens that are generated in MSI-H neoplasms by frameshift mutations of a number of genes containing coding microsatellite sequences. As expected, MSI-H ID-RL were found to harbor such genetic alterations in 12 target genes with a putative role in lymphomagenesis. The observation that the MSI-H phenotype was restricted to HIV infection-related lymphomas and posttransplant lymphoproliferative disorders suggests the existence of the highly immunogenic mutator pathway as a novel oncogenic process in lymphomagenesis whose role is favored when host immunosurveillance is reduced. Because MSI-H-positive cases were found to be either Epstein-Barr virus-positive or -negative, the mutator pathway should act synergistically or not with this other oncogenic factor, playing an important role in ID-RL.
  Proceedings of the National Academy of Sciences 05/2004; 101(14):5002-7. · 9.68 Impact Factor
• Article: Quasimonomorphic mononucleotide repeats for high-level microsatellite instability analysis.

  Olivier Buhard, Nirosha Suraweera, Aude Lectard, Alex Duval, Richard Hamelin
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  ABSTRACT: Microsatellite instability (MSI) analysis is becoming more and more important to detect sporadic primary tumors of the MSI phenotype as well as in helping to determine Hereditary Non-Polyposis Colorectal Cancer (HNPCC) cases. After some years of conflicting data due to the absence of consensus markers for the MSI phenotype, a meeting held in Bethesda to clarify the situation proposed a set of 5 microsatellites (2 mononucleotide repeats and 3 dinucleotide repeats) to determine MSI tumors. A second Bethesda consensus meeting was held at the end of 2002. It was discussed here that the 1998 microsatellite panel could underestimate high-level MSI tumors and overestimate low-level MSI tumors. Amongst the suggested changes was the exclusive use of mononucleotide repeats in place of dinucleotide repeats. We have already proposed a pentaplex MSI screening test comprising 5 quasimonomorphic mononucleotide repeats. This article compares the advantages of mono or dinucleotide repeats in determining microsatellite instability.
  Disease markers 02/2004; 20(4-5):251-7. · 1.64 Impact Factor