PharmD, PhD
PharmD, PhD
PhD
Our research explores the genomic and molecular basis of pediatric liver tumors to advance knowledge and improve treatment.
In the Genomics of Pediatric Liver Tumors group, we study tumor evolution using data from patient clinical features, whole-genome and exome sequencing, bulk RNAseq, single-cell, and spatial transcriptomics. We focus primarily on hepatoblastoma (HB), the most common pediatric liver cancer, as well as pediatric hepatocellular carcinoma (HCC), fibrolamellar carcinoma (FLC), and hepatocellular adenoma (HCA).
We partner with clinicians across France to gather samples and address key research questions, and work with Japanese teams to validate findings. We develop computational tools to integrate multi-scale data, exploring tumor evolution and chemotherapy resistance (Figure 1).
Context:
Tumor cells carry molecular changes (mutations, chromosome alterations) that affect gene function. We use computational methods to find recurring altered genes driving cancer, which can be somatic (tumor-specific) or germline/mosaic (predisposing), especially in early childhood cancers.
Published results:
By analyzing 122 tumor samples from 84 patients using whole-genome or whole-exome sequencing, we pinpointed key driver alterations in pediatric liver cancers (Hirsch et al, Cancer Discov 2021) (Figure 2).
HB and HCC converge on pathways like Wnt/β-catenin and IGF2 but differ in alteration types: HB often have mutations (e.g., CTNNB1), while HCC show deletions (e.g., AXIN1). Some HB patients have germline APC mutations linked to familial adenomatous polyposis, and a somatic second hit (Morcrette et al, Oncoimmunology 2019). Rare driver alterations predict poor chemotherapy response and survival (Pire et al, Eur J Cancer 2024). Alterations at the 11p15.5 locus, causing IGF2 overexpression, are a major driver. We found mosaic 11p15.5 changes in ~20% of HB patients’ non-tumor liver, marking preneoplastic cells and affecting liver function (Pilet et al, Nat Commun 2023).
Ongoing projects:
We aim to identify new driver alterations of pediatric liver cancers by expanding our cohort analyzed by whole-genome sequencing and bulk RNAseq. We have specific projects to further explore mosaic alterations at the single-cell and spatial level.
Context:
Tumor evolution is a multi-step process driven by mutations under immune and treatment pressures, leading to diverse phenotypes. We study how clonal changes and phenotypes connect to tumor evolution and resistance.
Published results:
Using RNAseq on 100 HB samples, we defined three transcriptomic groups tied to differentiation, proliferation, and immune response (Hirsch et al, Cancer Discov 2021) (Figure 3a). Multiple samples from the same tumor showed varied phenotypes despite shared drivers, indicating plasticity. Chemotherapy boosts immune infiltration in ‘Hepatocytic’ tumors but not ‘Liver Progenitor’ ones. Single-cell analysis confirmed these groups, revealing a continuum of cell states and subclonal diversity (Roehrig et al, Nat Commun 2024) (Figure 3b).
Ongoing projects:
We currently explore the intra-tumor heterogeneity of hepatoblastoma at the spatially-resolved single-cell level, by combining high-plex immunofluorescence, single-nucleus RNAseq and spatial transcriptomics.
Context:
Hepatoblastoma is treated with cisplatin-based chemotherapy, but some cases resist treatment, with few therapeutic alternatives.
Published results:
Whole-genome sequencing revealed cisplatin’s SBS35 mutational signature (Figure 4a) in a subset of primary hepatoblastomas post-chemotherapy, associated with poor treatment response (Hirsch et al, Cancer Discov 2021 ; Pire et al, Eur J Cancer 2024). In primary tumors, SBS35 mutations were subclonal, meaning they appeared in only a fraction of tumor cells, specifically within ‘Liver Progenitor’ sectors, while ‘Hepatocytic’ and ‘Mesenchymal’ areas lacked them. In contrast, relapse samples showed thousands of clonal SBS35 mutations, present in all tumor cells, indicating relapses arise from a single resistant cell that accumulated cisplatin-induced mutations during treatment (Figure 4b). Overall, the longitudinal analysis of cisplatin-induced mutations, integrated with the transcriptomic classification, pinpoints the ‘Liver Progenitor’ cells as being chemoresistant and at the origin of relapses.
Targeting PLK1, a ‘Liver Progenitor’ marker, reduced tumor growth in proof-of-concept experiments developed with Sandra Rebouissou’s group (Hirsch et al, Cancer Discov 2021).
Ongoing projects:
We’re refining detection of cisplatin mutations with machine learning and, with Sandra Rebouissou’s group, seeking drugs to reverse chemoresistance.
PhD
MD
PhD
MD
MD
Roehrig A, Hirsch TZ, Pire A, Morcrette G, Gupta B, Marcaillou C, Imbeaud S, Chardot C, Gonzales E, Jacquemin E, Sekiguchi M, Takita J, Nagae G, Hiyama E, Guérin F, Fabre M, Aerts I, Taque S, Laithier V, Branchereau S, Guettier C, Brugières L, Fresneau B, Zucman-Rossi J, Letouzé E. Nat Commun. 2024 Apr 8;15(1):3031. doi: 10.1038/s41467-024-47280-x. PMID: 38589411
Pire A, Hirsch TZ, Morcrette G, Imbeaud S, Gupta B, Pilet J, Cornet M, Fabre M, Guettier C, Branchereau S, Brugières L, Guerin F, Laithier V, Coze C, Nagae G, Hiyama E, Laurent-Puig P, Rebouissou S, Sarnacki S, Chardot C, Capito C, Faure-Conter C, Aerts I, Taque S, Fresneau B, Zucman-Rossi J. Eur J Cancer. 2024 Mar;200:113583. doi: 10.1016/j.ejca.2024.113583. Epub 2024 Feb 1. PMID: 38330765
Pilet J, Hirsch TZ, Gupta B, Roehrig A, Morcrette G, Pire A, Letouzé E, Fresneau B, Taque S, Brugières L, Branchereau S, Chardot C, Aerts I, Sarnacki S, Fabre M, Guettier C, Rebouissou S, Zucman-Rossi J. Nat Commun. 2023 Nov 6;14(1):7122. doi: 10.1038/s41467-023-42418-9. PMID: 37932266
Hirsch TZ, Pilet J, Morcrette G, Roehrig A, Monteiro BJE, Molina L, Bayard Q, Trépo E, Meunier L, Caruso S, Renault V, Deleuze JF, Fresneau B, Chardot C, Gonzales E, Jacquemin E, Guerin F, Fabre M, Aerts I, Taque S, Laithier V, Branchereau S, Guettier C, Brugières L, Rebouissou S, Letouzé E, Zucman-Rossi J. Cancer Discov. 2021 Oct;11(10):2524-2543. doi: 10.1158/2159-8290.CD-20-1809. Epub 2021 Apr 23. PMID: 33893148
Hirsch TZ, Negulescu A, Gupta B, Caruso S, Noblet B, Couchy G, Bayard Q, Meunier L, Morcrette G, Scoazec JY, Blanc JF, Amaddeo G, Nault JC, Bioulac-Sage P, Ziol M, Beaufrère A, Paradis V, Calderaro J, Imbeaud S, Zucman-Rossi J. J Hepatol. 2020 May;72(5):924-936. doi: 10.1016/j.jhep.2019.12.006. Epub 2019 Dec 18. PMID: 31862487
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. PMID: 31069152
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
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.
MD, PhD
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
MD, PhD
PhD
MD, PhD
MD, PhD
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