Hepatology ORIGINAL ARTICLE Polyploidy spectrum: a new marker in Gut: first published as 10.1136/gutjnl-2018-318021 on 12 April 2019. Downloaded from HCC classification Myriam Bou-Nader,1 Stefano Caruso,2 Romain Donne,1 Séverine Celton-Morizur,1 Julien Calderaro, 3,4 Géraldine Gentric,5,6 Mathilde Cadoux,1 Antoine L’Hermitte,7 Christophe Klein,8 Thomas Guilbert,9 Miguel Albuquerque,10 Gabrielle Couchy,2 Valérie Paradis,11 Jean-Pierre Couty,1 Jessica Zucman-Rossi,2 Chantal Desdouets 1 ► Additional material is ABSTRact published online only. To view Objectives Polyploidy is a fascinating characteristic of Significance of this study please visit the journal online liver parenchyma. Hepatocyte polyploidy depends on the (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2018- 318021). DNA content of each nucleus (nuclear ploidy) and the number of nuclei per cell (cellular ploidy). Which role can ► Polyploidy, the existence of cells containing For numbered affiliations see more than two homologous sets of end of article. be assigned to polyploidy during human hepatocellular carcinoma (HCC) development is still an open question. chromosomes, is a well-known feature of mammalian hepatocytes. Correspondence to Here, we investigated whether a specific ploidy spectrum Dr Chantal Desdouets, Team is associated with clinical and molecular features of HCC. ► Polyploidy is defined on the basis of the DNA Proliferation Stress and Liver Design Ploidy spectra were determined on surgically content of each nucleus (eg, 2n, 4n, 8n) called Physiopathology, Genome and resected tissues from patients with HCC as well as nuclear ploidy and on the number of nuclei per Cancer, Centre de Recherche cell called cellular ploidy. des Cordeliers, INSERM, healthy control tissues. To define ploidy profiles, a The liver is the unique organ that modifies its Sorbonne Université, USPC, quantitative and qualitative in situ imaging approach ► Université Paris Descartes, was used on paraffin tissue liver sections. ploidy content during normal homeostasis, Université Paris Diderot, Paris Results We first demonstrated that polyploid regeneration and under damage insults. 75006, France; hepatocytes are the major components of human chantal. desdouets@ inserm. fr What are the new findings? liver parenchyma, polyploidy being mainly cellular ► Binuclear polyploid hepatocytes are the major RD and SC-M contributed (binuclear hepatocytes). Across liver lobules, polyploid components of the polyploid fraction in normal equally. hepatocytes do not exhibit a specific zonation pattern. human liver parenchyma. http://gut.bmj.com/ MB-N and SC contributed During liver tumorigenesis, cellular ploidy is drastically equally. ► Quantification of cellular and nuclear reduced; binuclear polyploid hepatocytes are barely ploidy is sufficient to distinguish between Received 29 November 2018 present in HCC tumours. Remarkably, nuclear ploidy is normal liver parenchyma and hepatocellular Revised 25 February 2019 specifically amplified in HCC tumours. In fact, nuclear carcinoma (HCC). Accepted 24 March 2019 ploidy is amplified in CCH s harbouring a low degree ► TP53 mutation is a factor accounting for a of differentiation and TP53 mutations. Finally, our higher percentage of mononuclear polyploid results demonstrated that highly polyploid tumours are hepatocytes when compared with both TERT on September 30, 2021 by guest. Protected copyright. associated with a poor prognosis. promoter and CTNNB1 mutated HCC tumours. Conclusions Our results underline the importance ► Highly polyploid HCC tumours are associated of quantification of cellular and nuclear ploidy spectra with worse prognosis. during HCC tumorigenesis. How might it impact on clinical practice in the foreseeable future? INTRODUCTION ► Quantification of cellular and nuclear Polyploidy is the state in which cells contain ploidy spectra could be an accurate test for additional sets of homologous chromosomes. In HCC prognosis. mammals, whole organism polyploidy is usually lethal; however, some tissues develop a certain degree of polyploidy.1 2 Polyploid cells arise proliferating polyploid cells have been demon- © Author(s) (or their through a variety of cell division errors. Defects in strated to be genetically unstable and could facili- employer(s)) 2019. Re-use mitosis and cytokinesis are thought to be the most tate tumour development.1 7 Accumulating evidence permitted under CC BY-NC. No common routes, however polyploid cells can also points to a significant contribution of polyploid commercial re-use. See rights and permissions. Published develop as a consequence of chromosome endorep- intermediates in the shaping and the composition by BMJ. lication (absence of karyogenesis), telomere erosion of cancer genomes. The majority of solid tumours and oncogene activation.1 3 4 Different studies exhibit polyploid or near polyploid karyotypes.8 9 To cite: Bou-Nader M, have demonstrated a major role of ‘diploid-poly- Polyploidy is a defining feature of the liver.2 Poly- Caruso S, Donne R, et al. Gut Epub ahead of print: [please ploid conversion’ during physiological processes ploid hepatocytes are characterised by the number of include Day Month Year]. (eg, embryogenesis, terminal differentiation) but nuclei per cell named cellular ploidy (mononuclear/ doi:10.1136/ also during pathological conditions (eg, mechan- binuclear hepatocytes) as well as the ploidy of each gutjnl-2018-318021 ical, genotoxic or metabolic stress).2 5 6 Alarmingly, nucleus named nuclear ploidy (diploid, tetraploid, Bou-Nader M, et al. Gut 2019;0:1–10. doi:10.1136/gutjnl-2018-318021 1 Hepatology octoploid nucleus). In rodents, hepatocytes are mainly tetraploid polyploid contingent was also essentially tetraploid, we noticed (binuclear with two diploid nuclei or mononuclear with one a specific enrichment of octoploid fraction compared with binu- tetraploid nucleus) and octoploid (binuclear with two tetraploid clear population (figure 1E). Finally, we observed that ageing has Gut: first published as 10.1136/gutjnl-2018-318021 on 12 April 2019. Downloaded from nuclei or mononuclear with one octoploid nucleus).10 During no effect on both cellular and nuclear ploidy spectra (table 1, postnatal development, a scheduled division programme char- online supplementary figure S1). acterised by cytokinesis failure results in the genesis of binuclear Studies in rodents have demonstrated that there is a dynamic tetraploid hepatocytes, which subsequently play a pivotal role change in the spatial distributions of polyploid classes across in liver polyploidisation.11 12 This physiological polyploidisation the liver lobule.21 22 Here, we investigated whether this was is now considered as a form of liver maturation.2 13 Different also relevant in human liver parenchyma. Notably, liver zona- studies have demonstrated that hepatic polyploidy can be also tion is reflected by the heterogeneity of hepatocytes along modified under stress settings as metabolic overload, DNA the porto-central axis of the liver lobules. Importantly, peri- damage and chemical-induced liver injury.14–16 Recently, we portal hepatocytes do not express the same metabolic enzymes identified a new mechanism regulating polyploidy during liver compared with pericentral hepatocytes.23 24 Glutamine synthe- disease, where in the setting of non-alcoholic fatty liver disease tase (GS) immunostaining was performed to visualise pericen- (NAFLD), nuclear ploidy is altered with the genesis of highly tral hepatocytes (figure 1A). Porto-venous axis was arbitrary mononuclear polyploid hepatocytes.17 18 We demonstrated that separated: centrilobular, mid-lobular and periportal regions this pathological polyploidisation is promoted by endoreplica- (figure 1A). We first observed that a similar proportion of diploid tion cycles.17 18 Interestingly, endoreplication is now considered hepatocytes are present in the three regions (figure 1F). More- as an alternative division programme in a context of genomic over, our analysis showed that neither mononuclear (4n, ≥8n) stress.3 It is important to note that a long-term consequence of (figure 1F) nor binuclear (2×2n, ≥2×4n) (figure 1G) polyploid switching to the polyploidisation mode during liver patholog- hepatocytes have specific zonal distribution in hepatic lobules. ical growth is still under debate. Whether or not polyploidy is Altogether, these findings show that human liver polyploidy is a risk factor or protective against human liver cancer is largely mainly cellular (binuclear fraction) with no specific parenchyma unknown. zonation. Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment.19 More than 90% of HCCs in western countries develop on a cirrhotic Alteration of cellular and nuclear ploidy during liver background as a result of chronic HBV or HCV infection, high tumorigenesis alcohol intake, haemochromatosis or NAFLD. A better under- To analyse whether polyploid profile is modified during liver standing of the early events of liver carcinogenesis will help tumorigenesis a cohort of patients surgically treated for HCC outline the pathogenesis of the disease, and improve diagnostic was selected (table 2). Out of 75 tumours analysed, 86% were procedures and curative treatment. In this study, we aimed to male and 14% were female with the main risk factors being determine whether polyploid spectrum could constitute a potent alcohol intake (27%), HBV infection (27%), HCV infection (27%) and metabolic syndrome