Classification et caractérisation des cancers colorectaux par approches omiques

Colon cancer (CC) is one of the most frequent and most deadly cancer in France and worldwide. Nearly half of patients die within 5 years after diagnosis. Clinical stage based on histological features and molecular classification based genomic instabilities (microsatellite instability (MSI), chromosomal instability (CIN) and hypermethylation of the promoters (ICPM)) are not sufficient to define homogeneous molecular entities and to predict recurrence effectively. To improve patient care, it is essential to better understand the diversity of the disease so that effective prognostic and predictive markers could be found. My PhD work has been focused on studying the diversity of CC at the molecular level through the use of omics approaches on a large cohort of tumor samples. It led to the establishment of a robust transcriptomic classification of these cancers, validated on independent data sets, and to a detailed characterization of each of the subtypes. Six subtypes have been defined and were associated with distinct clinicopathological characteristics and molecular alterations, specific enrichments of supervised gene expression signatures related to cell and lesions of origin, specific deregulated signaling pathways and distinct survival. The results of this work have been strengthened by a consensus classification defined by an international consortium working group in which I've been involved. These results confirm that colorectal cancer is an heterogeneous disease. They provide a renewed framework to develop prognostic signatures, discover new treatment targets, identify new therapeutic strategies and assess response to treatment in clinical trials.Le cancer du côlon (CC) est l'un des cancers les plus fréquents et les plus mortels en France et dans le monde. Près de la moitié des patients décèdent dans les 5 ans suivant le diagnostic. La classification clinique en stade histologique et la classification moléculaire selon les formes d'instabilité du génome (l'instabilité des microsatellites (MSI), l'instabilité chromosomique (CIN) et l'hyperméthylation des promoteurs (CIMP)) ne suffisent pas à définir des entités homogènes du point de vue moléculaire et à prédire de manière efficace la récidive. Pour améliorer la prise en charge des patients, il apparaît indispensable de mieux appréhender la diversité de la maladie afin de trouver des marqueurs pronostiques et prédictifs efficaces. Mon travail de thèse a donc été d'étudier la diversité des CC à l'échelle moléculaire par l'utilisation d'approches omiques sur une large cohorte de patients. Il a abouti à l'établissement d'une classification transcriptomique robuste de ce cancer dans son ensemble, validée sur des données indépendantes, et à la caractérisation fine de chacun des sous-types. Six sous-types ont ainsi été définis présentant des caractéristiques clinico-pathologiques, des altérations moléculaires de l'ADN, des enrichissements de signatures liées aux lésions et cellules d'origines, des voies de signalisation dérégulées et des survies bien distinctes. Les résultats de ce travail ont été confortés par un travail de classification consensus mis en place avec un consortium de travail international auquel j'ai participé. Ces résultats ont permis de confirmer que le cancer colorectal n'est pas une maladie homogène. Ils ouvrent de nouvelles perspectives pour l'établissement de signatures pronostiques et la recherche de cibles pour de nouveaux traitements ainsi que pour l'évaluation de la réponse au traitement au sein d'essais cliniques

Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors

Generation of stable transgenic embryonic stem (ES) cell lines by classic transfection is still a difficult task, requiring time-consuming clonal selection, and hampered by clonal artifacts and gene silencing. Here we describe a novel system that allows construction of lentivectors and generation of stable ES cell lines with > 99% transgene expression within a very short time frame. Rapid insertion of promoters and genes of interest is obtained through a modular recombinational cloning system. Vectors contain central polypurine tract from HIV-1 element and woodchuck hepatitis virus post-transcriptional regulatory element as well as antibiotic resistance to achieve optimal and homogenous transgene expression. We show that the system 1) is functional in mouse and human ES cells, 2) allows the generation of ES cells expressing genes of interest under the control of ubiquitous or tissue-specific promoters, and 3) allows ES cells expressing two constructs through selection with different antibiotics to be obtained. The technology described herein should become a useful tool in stem cell research

Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches

The Cdx2 homeobox gene suppresses intestinal tumorigenesis through non-cell-autonomous mechanisms

Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc. This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut

Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor

Should We Abandon the t-Test in the Analysis of Gene Expression Microarray Data: A Comparison of Variance Modeling Strategies

High-throughput post-genomic studies are now routinely and promisingly investigated in biological and biomedical research. The main statistical approach to select genes differentially expressed between two groups is to apply a t-test, which is subject of criticism in the literature. Numerous alternatives have been developed based on different and innovative variance modeling strategies. However, a critical issue is that selecting a different test usually leads to a different gene list. In this context and given the current tendency to apply the t-test, identifying the most efficient approach in practice remains crucial. To provide elements to answer, we conduct a comparison of eight tests representative of variance modeling strategies in gene expression data: Welch's t-test, ANOVA [1], Wilcoxon's test, SAM [2], RVM [3], limma [4], VarMixt [5] and SMVar [6]. Our comparison process relies on four steps (gene list analysis, simulations, spike-in data and re-sampling) to formulate comprehensive and robust conclusions about test performance, in terms of statistical power, false-positive rate, execution time and ease of use. Our results raise concerns about the ability of some methods to control the expected number of false positives at a desirable level. Besides, two tests (limma and VarMixt) show significant improvement compared to the t-test, in particular to deal with small sample sizes. In addition limma presents several practical advantages, so we advocate its application to analyze gene expression data

The consensus molecular subtypes of colorectal cancer

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-beta activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions

Gene expression profiling of patient‐derived pancreatic cancer xenografts predicts sensitivity to the BET bromodomain inhibitor JQ1: implications for individualized medicine efforts

Abstract c‐MYC controls more than 15% of genes responsible for proliferation, differentiation, and cellular metabolism in pancreatic as well as other cancers making this transcription factor a prime target for treating patients. The transcriptome of 55 patient‐derived xenografts show that 30% of them share an exacerbated expression profile of MYC transcriptional targets (MYC‐high). This cohort is characterized by a high level of Ki67 staining, a lower differentiation state, and a shorter survival time compared to the MYC‐low subgroup. To define classifier expression signature, we selected a group of 10 MYC target transcripts which expression is increased in the MYC‐high group and six transcripts increased in the MYC‐low group. We validated the ability of these markers panel to identify MYC‐high patient‐derived xenografts from both: discovery and validation cohorts as well as primary cell cultures from the same patients. We then showed that cells from MYC‐high patients are more sensitive to JQ1 treatment compared to MYC‐low cells, in monolayer, 3D cultured spheroids and in vivo xenografted tumors, due to cell cycle arrest followed by apoptosis. Therefore, these results provide new markers and potentially novel therapeutic modalities for distinct subgroups of pancreatic tumors and may find application to the future management of these patients within the setting of individualized medicine clinics

Tumour-infiltrating CD8+ lymphocytes and colorectal cancer recurrence by tumour and nodal stage

Background Intratumoural T-cell infiltrate intensity cortes wrelaith clinical outcome in stage II/III colorectal cancer (CRC). We aimed to determine whether this association varies across this heterogeneous group. Methods We performed a pooled analysis of 1804 CRCs from the QUASAR2 and VICTOR trials. Intratumoural CD8+ and CD3+ densities were quantified by immunohistochemistry in tissue microarray (TMA) cores, and their association with clinical outcome analysed by Cox regression. We validated our results using publicly available gene expression data in a pooled analysis of 1375 CRCs from seven independent series. Results In QUASAR2, intratumoural CD8+ was a stronger predictor of CRC recurrence than CD3+ and showed similar discriminative ability to both markers in combination. Pooled multivariable analysis of both trials showed increasing CD8+ density was associated with reduced recurrence risk independent of confounders including DNA mismatch repair deficiency, POLE mutation and chromosomal instability (multivariable hazard ratio [HR] for each two-fold increase = 0.92, 95%CI = 0.87–0.97, P = 3.6 × 10−3). This association was not uniform across risk strata defined by tumour and nodal stage: absent in low-risk (pT3,N0) cases (HR = 1.03, 95%CI = 0.87–1.21, P = 0.75), modest in intermediate-risk (pT4,N0 or pT1-3,N1-2) cases (HR = 0.92, 95%CI = 0.86–1.0, P = 0.046) and strong in high-risk (pT4,N1-2) cases (HR = 0.87, 95%CI = 0.79–0.97, P = 9.4 × 10−3); PINTERACTION = 0.090. Analysis of tumour CD8A expression in the independent validation cohort revealed similar variation in prognostic value across risk strata (PINTERACTION = 0.048). Conclusions The prognostic value of intratumoural CD8+ cell infiltration in stage II/III CRC varies across tumour and nodal risk strata. </p

The Consensus Molecular Subtypes of Colorectal Cancer

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use -- https://www.nature.com/authors/policies/license.html#termsColorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMS) with distinguishing features: CMS1 (MSI Immune, 14%), hypermutated, microsatellite unstable, strong immune activation; CMS2 (Canonical, 37%), epithelial, chromosomally unstable, marked WNT and MYC signaling activation; CMS3 (Metabolic, 13%), epithelial, evident metabolic dysregulation; and CMS4 (Mesenchymal, 23%), prominent transforming growth factor β activation, stromal invasion, and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intra-tumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC - with clear biological interpretability - and the basis for future clinical stratification and subtype-based targeted interventions