Illumina MiSeq
Manufacturer’s description: The MiSeq desktop sequencer allows you to access more focused applications such as targeted gene sequencing, metagenomics, small genome sequencing, targeted gene expression, amplicon sequencing, and HLA typing. New MiSeq reagents enable up to 15 Gb of output with 25 M sequencing reads and 2×300 bp read lengths.
Applications currently used: Targeted sequencing (paired-end 2x150bp) using multiplexed PCR for library preparation.
Manufacturer’s description: Based on one of the most widely used, widely trusted sequencing methodologies available (Sanger sequencing) the 3500 Series Genetic Analyzer is designed to deliver the accuracy you demand. The 3500 platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), microsatellite analysis, MLPA, AFLP, LOH, MLST, and SNP validation or screening.
Applications currently used: Sanger sequencing (read length up to 1000bp)
Manufacturer’s description: The nCounter® Analysis System offers a simple, cost-effective way to profile hundreds of mRNAs, microRNAs, or DNA targets simultaneously with high sensitivity and precision. The digital quantification of target molecules and high levels of multiplexing eliminate the compromise between data quality and data quantity, producing excellent sensitivity and high reproducibility for studies of hundreds of targets. The system uses molecular “barcodes” and single molecule imaging to quantitate up to 800 unique transcripts in a single reaction.
Applications currently used: Gene expression and miRNA analysis on FFPE tumor samples
Manufacturer’s description: Fluidigm’s revolutionary integrated fluidic circuits (IFCs) empower life science research by automating PCR reactions in nanoliter volumes. This means using less sample and reagent, and a single microfluidic device, to achieve the high-quality, consistent results your work depends on. The Biomark HD system runs IFCs in either real-time or end-point read modes, bringing flexible, efficient and economical PCR solutions to a range of applications such as digital PCR, gene expression, genotyping, library preparation for next generation sequencing.
Application currently used: Gene expression, genotyping and library preparation
Manufacturer’s description: The Applied Biosystems 7900HT Fast Real-Time PCR System is the only real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading. Acknowledged as the gold standard in real-time PCR, the 7900HT system combined with TaqMan®Assays enables you to achieve unprecedented throughput and flexibility, allowing you to pursue projects beyond the scope of previous real-time instruments
Application currently used: Gene expression analysis, allelic discrimination, library quantification
Manufacturer’s description: The Operetta CLS system combines speed and sensitivity with the powerful and intuitive data analysis. It is a combination of technologies with a powerful, stable 8x LED light source for optimal excitation of fluorophores and confidence in results. It contains also a proprietary automated water-immersion objectives with very high numerical aperture enable high resolution and fast read times with minimal photodamage. The confocal spinning disk technology provides a fast and gentle imaging process, enabling efficient background rejection, live cell experiments, and 3D imaging. Its large format sCMOS camera delivers low noise, wide dynamic range, and high resolution for sensitive and quantitative measurements at short exposure time.
Application currently used: Fixed-cell assays, Live-cell assays, Complex cellular models, FRET assays, Phenotypic fingerprinting
Manufacturer’s description: MultiFlo™ FX is an automated multi-mode reagent dispenser for 6- to 1536-well microplates. MultiFlo FX incorporates several unique technologies in its modular design, such as Parallel Dispense, RAD™ Random Access Dispense and the new, patent-pending AMX™ Automated Media Exchange modules to facilitate a variety of liquid handling applications from 2D and 3D cell culture to concentration normalization assays, ELISA, bead-based assays and more. A fully configured MultiFlo FX replaces up to five liquid handlers, saving space, time and instrumentation budgets.
Application currently used: Cell culture for automated reagent dispensing and washing (6to 384 wellplates)
Manufacturer’s description: Fast and accurate determination of a candidate compound’s IC50 provides drug discovery biologists with valuable information for the development of new pharmaceuticals, yet traditional techniques are both time consuming and laborious, with no standardization across the industry. The HP D300 Digital Dispenser offers a simple method for streamlining your workflow, offering picoliter to microliter non-contact dispensing of small molecules in DMSO directly into your assay plate. Using HP’s Direct Digital Dispensing technology, this convenient benchtop solution requires almost no set up time, and single use T8 Dispenseheads virtually eliminate the risk of crosscontamination. It allows rapid delivery of any dose to any well, saving time, minimizing waste of valuable compounds and accelerating drug discovery.
Application currently used: Cell culture for delivery of pharmacological compounds in 96 and 384 well plates
J Zucman-Rossi (PUPH), B Beuselinck (PUPH Louvain)
Fundings: Inca
We aim to translate the molecular classification of clear cell renal cancer into genome based clinical trial int cooperation with Sautès-Fridman team at CRC and clinicans at HEGP and Louvain. We also search for biomarkers predicting response to anti-angiogenic thearapies.
Recently, we described a molecular classification of clear cell renal cancer (ccRCCs) into four robust subgroups with distinctive characteristics on multi-omics analyses. These subgroups are defined by various levels of myc activation, angiogenic activation and immune response associated with outcome of metastatic ccRCC treated with sunitinib. This classification has future application in patient care, in particular to predict drug response.
Beseulinck et al, Clin Cancer Research 2015.
Fibroblast Growth Factor Receptor-2 Polymorphism rs2981582 is Correlated With Progression-free Survival and Overall Survival in Patients With Metastatic Clear-cell Renal Cell Carcinoma Treated With Sunitinib. Vanmechelen M, Lambrechts D, Van Brussel T, Verbiest A, Couchy G, Schöffski P, Dumez H, Debruyne PR, Lerut E, Machiels JP, Richard V, Albersen M, Verschaeve V, Oudard S, Méjean A, Wolter P, Zucman-Rossi J, Beuselinck B. Clin Genitourin Cancer. 2019 Apr;17(2):e235-e246. doi: 10.1016/j.clgc.2018.11.002. Epub 2018 Nov 16.
Polymorphisms in the Von Hippel-Lindau Gene Are Associated With Overall Survival in Metastatic Clear-Cell Renal-Cell Carcinoma Patients Treated With VEGFR Tyrosine Kinase Inhibitors. Verbiest A, Lambrechts D, Van Brussel T, Couchy G, Wozniak A, Méjean A, Lerut E, Oudard S, Verkarre V, Job S, de Reynies A, Machiels JP, Patard JJ, Zucman-Rossi J, Beuselinck B. Clin Genitourin Cancer. 2018 Aug;16(4):266-273. doi: 10.1016/j.clgc.2018.01.013.
Molecular Subtypes of Clear-cell Renal Cell Carcinoma are Prognostic for Outcome After Complete Metastasectomy. Verbiest A, Couchy G, Job S, Caruana L, Lerut E, Oyen R, de Reyniès A, Tosco L, Joniau S, Van Poppel H, Van Raemdonck D, Van Den Eynde K, Wozniak A, Zucman-Rossi J, Beuselinck B. Eur Urol. 2018 Oct;74(4):474-480. doi: 10.1016/j.eururo.2018.01.042. Epub 2018 Feb 17.
Pro-angiogenic gene expression is associated with better outcome on sunitinib in metastatic clear-cell renal cell carcinoma. Beuselinck B, Verbiest A, Couchy G, Job S, de Reynies A, Meiller C, Albersen M, Verkarre V, Lerut E, Méjean A, Patard JJ, Laguerre B, Rioux-Leclercq N, Schöffski P, Oudard S, Zucman-Rossi J. Acta Oncol. 2018 Apr;57(4):498-508. doi: 10.1080/0284186X.2017.1388927. Epub 2017 Nov 2.
Validation of VEGFR1 rs9582036 as predictive biomarker in metastatic clear-cell renal cell carcinoma patients treated with sunitinib. Beuselinck B, Jean-Baptiste J, Schöffski P, Couchy G, Meiller C, Rolland F, Allory Y, Joniau S, Verkarre V, Elaidi R, Lerut E, Roskams T, Patard JJ, Oudard S, Méjean A, Lambrechts D, Zucman-Rossi J. BJU Int. 2016 Dec;118(6):890-901. doi: 10.1111/bju.13585. Epub 2016 Aug 12.
RANK/OPG ratio of expression in primary clear-cell renal cell carcinoma is associated with bone metastasis and prognosis in patients treated with anti-VEGFR-TKIs. Beuselinck B, Jean-Baptiste J, Couchy G, Job S, De Reynies A, Wolter P, Théodore C, Gravis G, Rousseau B, Albiges L, Joniau S, Verkarre V, Lerut E, Patard JJ, Schöffski P, Méjean A, Elaidi R, Oudard S, Zucman-Rossi J. Br J Cancer. 2015 Nov 3;113(9):1313-22. doi: 10.1038/bjc.2015.352. Epub 2015 Oct 13.
Kidney cancer: Single nucleotide polymorphisms in mRCC-is their time up? Beuselinck B, Zucman-Rossi J. Nat Rev Urol. 2015 Aug;12(8):424-6. doi: 10.1038/nrurol.2015.149. Epub 2015 Jun 30. No
We aim to develop scientific projects with specific objectives to integrate innovative tumor genomic characterizations with metabolism and immune response to identify new biomarkers and therapeutic targets useful for the patients. To this aim, we focus on 3 major types of cancer: liver, mesothelioma and renal carcinoma, in close collaboration with clinicians and pathologists. Thanks to our future moving at the Centre de Recherche des Cordeliers our team will benefit from close collaborations with other teams involved in Onco-Immunology, Metabolism and developing innovative genomic approaches.
Collaborators: S Imbeaud (IR), C Peneau (PhD st), T La Bella (PhD st), J Zucman-Rossi (PUPH);
Involvement of pathogen agents in human carcinogenesis remain to be investigated. Our recent identification of AAV2 as the fourth virus involved in insertional mutagenesis prompt us to develop a project to evaluate consequences of HBV, HCV and AAV infections in liver tumorigenesis by integrating the analysis of viral and tumor genome alterations. Our aim is to reconstruct the natural history of the viruses (genome variation, replication, structural alterations, selection…) in the context of tumor cell selection in patients putatively treated by antiviral therapies or exposed to multiple pathogens. We will also search for additional pathogens in WGS data and collaborate with the international ICGC viruses project.
Fundings: LNCC, ANRS, Biomerieux Foundation
Collaborators: G Morcrette (PhD st), E Letouzé (CR Inserm), J Pilet (PhD st), S Imbeaud (IR), G Couchy (IE, PhD st), Julien Calderaro (pathologist), JC Nault (MCUPH), J Zucman-Rossi (PUPH); in cooperation with SIOPEL,
Most of pediatric liver neoplasms are poorly analyszed at the genomic and molecular level. They include hepatocellular carcinomas (HCC), fibrolamellar carcinomas (FLC), hepatoblastomas (HB), transitional liver cell tumors (TLCT) and also benign neoplasms, hepatocellular adenomas (HCA) and focal nodular hyperplasias (FNH). We aim to perform an integrated genomic analysis of these pediatric hepatocellular tumors to better understand their mechanism of tumorigenesis. To this purpose, we have collected a series of 193 patients with pediatric frozen liver tumors part of the national HEPATOBIO tumor resource. Within this series, 48 cases/67 frozen tumor samples have been selected for whole genome sequencing (WGS) and RNA sequencing (RNAseq) within the GEPELIN project funded by France Génomique for the WGS part. Our specific aims are to (1) identify putative driver genes altered in pediatric tumors and validate them functionally (2) perform new transcriptomic classification to derive new altered pathways and diagnostic/prognostic markers, (3) characterize the mutational and chromosome rearrangement signatures to identify new mechanism of carcinogenesis (4) search for therapeutic targets. These results will be compared with the adult liver tumors data that we have accumulated in the lab.
Fundings: LNCC, France Genomique.
Collaborators: E Letouzé , J Shinde , S Imbeaud (IR), L Meunier (PhD st), Q Bayard (M2), T Hirsch (Post-doc), J Zucman-Rossi (PUPH
In the last 5 years, we have generated large genomic data sets including 350 tumors analyzed by whole exome sequencing, 270 by RNA-seq, 250 by methylation arrays and 100 by whole genome sequencing. We will use this exceptional resource to unravel key questions in the natural history of liver cancers. (1) What mutational processes drive tumorigenesis? We have so far identified 10 point-mutation signatures associated with known (aflatoxin B1, tobacco) or unknown mutagenic processes in liver cancers. We will extend these analyses to signatures of indels and structural rearrangements, and perform a meta-analysis of all published liver cancer data sets to unravel the association of signatures with risk factors and predisposing variants. (2) What are the missing drivers in liver cancers? Analysis of coding mutations by us and others revealed tens of driver genes and pathways but 30% of tumors still have no identified driver event. In order to identify non-coding driver alterations, we will use whole genome and RNA-seq data to systematicallly screen for mutations and structural rearrangements modifying regulatory regions, chromatin context and 3’ UTR sequences and affecting mRNA expression and stability. (3) How to the genomic, epigenomic and transcriptional layers interact in tumor cells? We will develop innovative strategies to unravel the connexions between genomic alterations, DNA methylation and gene expression profiles. (4) What is the timing of genomic alterations along tumorigenesis? We and others have developed statistical approaches to time mutational signatures, copy-number alterations and driver events in the life history of a cancer using intra-tumor heterogeneity. We will conduct an ambitious project to analyze the evolution of genomic, transcriptional and micro-environment features in 25 patients with multiple samples along the treatment.
Fundings: Cancer environment, HTE HETCOLI, France Genomique, Canceropole
E Letouzé (CR Inserm), P Nahon (PUPH), E Trepo (Post-doc), J Yang (Ph st), JC Nault (MCUPH), Zucman-Rossi (PUPH)
Candidate gene studies have uncovered a limited number of variants reproducibly linked to hepatocellular carcinoma (HCC). The HECAM project aims to identify newgermline variants predisposing to HCC in the French population and to test their ability to modify patient care in clinical practice. Therefore, a genome-wide association study will be performed in a multicenter cohort of chronic liver disease patients with (n = 2,066) and without (n = 2,666) HCC using the Illumina Infinium Global Screening Array including ~ 660,000 markers). Validation of the top variants will be performed in two French replication cohorts of cirrhotic patients included in HCC surveillance programs (n = 2,249). The performance for HCC risk stratification will be finally assessed through integration of genetic information into specific algorithm-based prediction models. An original integrative approach including somatic information already generated will be used for prioritizing variants impacting liver carcinogenesis. Finally, genotype-phenotype associations will be performed to build prognostic models. We also plan to analyze benign liver tumors to search for genetic predisposition to the development of hepatocellular adenoma in a cohort of 500 patients with in parallel an epidemiological study.
Fundings: HECAM, ANRS, AFEF
APC germline hepatoblastomas demonstrate cisplatin-induced intratumor tertiary lymphoid structures. Morcrette G, Hirsch TZ, Badour E, Pilet J, Caruso S, Calderaro J, Martin Y, Imbeaud S, Letouzé E, Rebouissou S, Branchereau S, Taque S, Chardot C, Guettier C, Scoazec JY, Fabre M, Brugières L, Zucman-Rossi J. Oncoimmunology. 2019 Mar 28;8(6):e1583547. doi: 10.1080/2162402X.2019.1583547. eCollection 2019.
Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy. Dhanasekaran R, Nault JC, Roberts LR, Zucman-Rossi J. Gastroenterology. 2019 Jan;156(2):492-509. doi: 10.1053/j.gastro.2018.11.001. Epub 2018 Nov 4. Review.
Systemic AA Amyloidosis Caused by Inflammatory Hepatocellular Adenoma. Calderaro J, Letouzé E, Bayard Q, Boulai A, Renault V, Deleuze JF, Bestard O, Franco D, Zafrani ES, Nault JC, Moutschen M, Zucman-Rossi J. N Engl J Med. 2018 Sep 20;379(12):1178-1180. doi: 10.1056/NEJMc1805673.
Argininosuccinate synthase 1 and periportal gene expression in sonic hedgehog hepatocellular adenomas. Nault JC, Couchy G, Caruso S, Meunier L, Caruana L, Letouzé E, Rebouissou S, Paradis V, Calderaro J, Zucman-Rossi J. Hepatology. 2018 Sep;68(3):964-976. doi: 10.1002/hep.29884. Epub 2018 Jun 6
Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis. Letouzé E, Shinde J, Renault V, Couchy G, Blanc JF, Tubacher E, Bayard Q, Bacq D, Meyer V, Semhoun J, Bioulac-Sage P, Prévôt S, Azoulay D, Paradis V, Imbeaud S, Deleuze JF, Zucman-Rossi J. Nat Commun. 2017 Nov 3;8(1):1315
Pro-angiogenic gene expression is associated with better outcome on sunitinib in metastatic clear-cell renal cell carcinoma. Beuselinck B, Verbiest A, Couchy G, Job S, de Reynies A, Meiller C, Albersen M, Verkarre V, Lerut E, Méjean A, Patard JJ, Laguerre B, Rioux-Leclercq N, Schöffski P, Oudard S, Zucman-Rossi J. Acta Oncol. 2018 Apr;57(4):498-508. doi: 10.1080/0284186X.2017.1388927. Epub 2017 Nov 2
D Jean, MC Jaurand, F Le Pimpec-Barthes, C Meiller, W Blum, L Quetel. Fundings: LNCC IdF and CIT, Chancellerie des Universités de Paris-Legs Poix
Malignant pleural mesothelioma (MPM) is a rare tumor, with a poor prognosis mainly due to the lack of efficient treatment. It is therefore important to develop new therapies that take into account the heterogeneity of MPM at the molecular level. We have recently defined a molecular classification of MPMs that defines two main groups C1 and C2. A subgroup of C2, with a double mutation in the Hippo, NF2 and LATS2 pathway genes, was identified. The C2 group and the NF2 / LATS2 double mutant subgroup contain MPMs from patients with a very poor prognosis. We focus on four major aims:
(1) Refine the molecular classification of malignant pleural mesothelioma and transfer it to clinic: Integrated multi-omic analysis using our large collection of tumors will allow to identify new tumor subgroups. Identification of specific biomarkers and development of prediction tools should facilitate the implementation of this classification to clinic.
(2) Determine the mechanisms of mesothelial carcinogenesis: Functional analysis using our cell lines biobank will lead to define the contribution of specific gene alterations and signal pathways to carcinogenesis.
(3) Develop therapeutic strategy: High-content screening and validation using in vitro and in vivo models will be used to study the correlation between anti-tumor compounds sensibility and the molecular phenotype and will constitute a first step toward precision medicine for mesothelioma.
(4) Characterize intra-tumor heterogeneity: Molecular analysis will be performed to explore heterogeneity of tumor samples at different anatomical sites and presence of cancer stem cells.
We performed a genetic and transcriptomic characterization of malignant pleural mesothelioma (MPM) using our tumor biobanks. We identified the first recurrent oncogenic mutation in the TERT promoter and showed this alteration was frequent in MPM of the sarcomatoid subtype (1). Based on transcriptomic data, we defined a robust molecular classification consisting of two groups (C1 and C2) with different molecular profiles, gene alterations, histology subtypes, and survival outcomes. One of the major interests was to separate the epithelioid MPM, the most frequent histologic subtype, according to their survival prognosis (2). Recently, by coupling transcriptomic and genetic analysis, we identified a new specific MPM molecular subgroup (C2LN) characterized by a co-occurring mutation in LATS2 and NF2 tumor suppressor genes. We identified a specific biomarker and highlighted a high sensitivity to mTOR/PI3K/AKT (PF-04691502) inhibitor treatment for this subgroup (3).
Tallet et al, Oncogene, 2014; de Reynies et al… Jean, Clinical Cancer Research, 2014; Tranchant et al, Clinical Cancer Research, 2017