Adrien Ducret

Christophe Grangeasse
email: c.grangeasse@ibcp.fr

Recent progress in our understanding of peptidoglycan assembly in Firmicutes

Keywords: Peptidoglycan, Bacterial cell morphogenesis, Bacterial cell division, Firmicutes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The bacterial cell wall consists mainly of a giant biopolymer named peptidoglycan.

The latter surrounds the whole cell and is commonly described as a mesh-like structure consisting of glycan strands that are cross-linked by short peptides [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF] (Figure 1). The assembly of this cage-like structure starts at the inner face of the cytoplasmic membrane by a series of reactions involving a set of well conserved proteins generating a lipid-linked disaccharide-pentapeptide precursor named lipid II. This precursor is then flipped across the membrane, polymerized and inserted into the existing peptidoglycan mesh. Polymerization involves both transglycosylation reactions to elongate the glycan stands and transpeptidation reactions that cross-link the pentapeptides of two adjacent glycan strands [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF]. These reactions are performed by either the penicillin-binding proteins (PBPs), the L,D-transpeptidases (Ldt), and the SEDS (Shape, Elongation, Division and Sporulation) proteins. The peptidoglycan polymer is also continuously remodeled by a variety of hydrolases, cleaving either the glycan strand, the link between the sugar moieties and the peptide or inside the peptide itself, to promote the insertion of new precursors or to simply mediate cell separation after division [START_REF] Alcorlo | Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases[END_REF] (Figure 2). This general view of peptidoglycan synthesis derives mostly from studies on model organisms such as Escherichia coli or Bacillus subtilis, but does not reflect the great diversity encountered in bacteria with different morphology or physiology. The most prominent sources of variability are the composition of the peptide, the mode of pentapeptides crosslinking or the sites of peptidoglycan synthesis (polar, septal or lateral) [START_REF] Shaku | Peptidoglycan biosynthesis and remodeling revisited[END_REF] (Figure 1). In this review, we highlight some of the recent breakthroughs that have improved, and even changed, our understanding of some key aspects of peptidoglycan assembly in monoderm bacteria of the Firmicute phylum. Firmicutes are a well-studied group of Gram-positive bacteria and a number of important new discoveries regarding peptidoglycan synthesis have been published during the last five years. To get more information about other bacterial models and the assembly of other compounds of the cell wall, we suggest the following reviews [START_REF] Randich | Molecular mechanisms for the evolution of bacterial morphologies and growth modes[END_REF][START_REF] Mueller | Bacterial Cell Wall Quality Control during Environmental Stress[END_REF][START_REF] Van Teeseling | Determinants of Bacterial Morphology: From Fundamentals to Possibilities for Antimicrobial Targeting[END_REF].

Furthermore, recent developments allowing the high through peptidoglycan analysis as well as advances in microscopy techniques allowing the investigation of special and temporal changes in peptidoglycan synthesis will be covered in this issue in a review by Sara B.

Hernández and Felipe Cava [START_REF] Hernández | New approaches and techniques for bacterial cell wall analysis[END_REF].

Lipid II transport, polymerization and cross-linking

Once assembled at the inner face of the cytoplasmic membrane by the Mur proteins [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF], the lipid II precursor must be translocated across the membrane in order to be incorporated in the peptidoglycan mesh. For many years, it was thought that the translocation step was carried out by the two SEDS proteins, FtsW and RodA, whereas polymerization and cross-linking were performed by two classes of PBPs : the bi-functional class A PBPs that display both a transpeptidase and a transglycosilase activity (aPBPs) and the mono-functional class B PBPs that display only a transpeptidase activity (bPBPs) [START_REF] Egan | Regulation of bacterial cell wall growth[END_REF]. This model has however been considerably modified based on the results of several seminal studies, revealing that MurJ mediates the translocation of lipid II [START_REF] Sham | Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis[END_REF] and that the two SEDS proteins, FtsW and RodA, possess an unexpected glycosyltransferase activity [START_REF] Meeske | SEDS proteins are a widespread family of bacterial cell wall polymerases[END_REF] [10] (Figure 1). Subsequently, it has been shown that the interaction of SEDS proteins with bPBPs was required to stimulate their respective activity [START_REF] Meeske | SEDS proteins are a widespread family of bacterial cell wall polymerases[END_REF]. Although initially demonstrated for B. subtilis, a series of evidence support that the same model is true for other Firmicutes such as Staphylococcus aureus in which the RodA/bPBP3 and FtsW/bPBP1 pairs mediate the sidewall and septal assembly of the peptidoglycan framework, respectively [START_REF] Reichmann | Class-A penicillin binding proteins do not contribute to cell shape but repair cell-wall defects[END_REF], Streptococcus pneumoniae in which FtsW works with bPBP2x in septal peptidoglycan assembly [12] or Listeria monocytogenes in which RodA and FtsW might work with the bPBP B1 and B2, respectively [13]. Interestingly, L. monocytogenes possesses two FtsW and three RodA homologs that can compensate each other, at least partially, and whose expression levels are modulated upon treatment with antibiotics [13]. In addition to MurJ, a second unrelated protein, Amj, was shown to also flip the lipid II across the membrane in B. subtilis [14]. Amj forms a synthetic lethal pair with MurJ and its expression can be induced by cell wall stress. Together, these observations suggest that multiple SEDS and translocases might have redundant and/or specific role to support peptidoglycan synthesis during different environmental conditions/stresses. As bPBPs work in concert with SEDS proteins to build the peptidoglycan framework, the function of aPBPs had to be revised. A first study has proposed that aPBPs could work outside and independently of the SEDS/bPBP complexes [15]. This view was recently strengthened in S. pneumoniae in which evidence was provided that aPBPs are required for the repair and/or maturation of the primary peptidoglycan framework independently of the SEDS/bPBP pairs FtsW/bPBP2x and RodA/bPBP2b [16]. A similar function is also proposed for aPBPs in diderm bacteria like E. coli [17]. Altogether, those recent findings support a new model with a division of labor between the SEDS/bPBPs and the aPBPs: while the SEDS/bPBPs complexes are essential for the synthesis of the nascent peptidoglycan, aPBPs would have complementary functions in the maintenance and maturation of the existing framework (Figure 1). The wide diversity in the number of aPBPs with an apparent redundancy among the Firmicutes suggests the existence of a whole repertoire of ancillary functions for aPBPs that that have yet to be determined. Understanding how aPBPs operate and how their activity is regulated will be discussed in more detail in the same issue in a review by Manuel Pazos and Waldemar Vollmer [START_REF] Pazos | Regulation and function of class A Penicillin-binding proteins[END_REF], and will also be an exciting area of research for the coming years.

Modes of peptidoglycan synthesis

To maintain cell-wall homeostasis, the insertion of new material into the existing peptidoglycan mesh must be regulated in space and time. Different modes of peptidoglycan assembly (polar, lateral, septal) have been identified and characterized by the association between a scaffolding protein and a multiprotein complex responsible for the synthesis and the insertion of new material. In B. subtilis, a multiprotein complex called the elongasome which is scaffolded by the actin-like MreB protein, allows lateral insertion of peptidoglycan for cell elongation whereas another complex called the divisome which is scaffolded by the tubulin-like protein FtsZ, assembles the septal peptidoglycan [START_REF] Egan | Regulation of peptidoglycan synthesis and remodelling[END_REF]. The contribution of the scaffolding proteins MreB and FtsZ in the peptidoglycan synthesis is still actively debated, but over the last few years numerous insights have considerably improved our understanding of their dynamic and their function during the lateral and septal modes of peptidoglycan synthesis.

Initially thought to form a helical filament distributed along the length of cell, it is now clear that MreB forms small assemblies that move around the rod circumference and promote peptidoglycan synthesis in the radial direction in B. subtilis and E. coli. Thanks to the emergence of new labelling methods for nascent peptidoglycan and the use of superresolution imaging, it has also become clear that this circumferential rotation is powered by the active peptidoglycan assembly (reviewed in [START_REF] Errington | Bacterial morphogenesis and the enigmatic MreB helix[END_REF]). While the molecular details regarding the organization of these MreB assemblies are still debated [START_REF] Billaudeau | MreB Forms Subdiffraction Nanofilaments during Active Growth in Bacillus subtilis[END_REF], it has become evident that the density of rotating MreB assemblies relies on RodA/bPBP2a but not on the activity of aPBPs [START_REF] Dion | Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems[END_REF]. In addition, there is growing evidence that the orientation and/or the motion of MreB filaments depend on the membrane curvature and fluidity, and very interestingly, on the nutrient availability and the growth rate suggesting an intimate link between the cell elongation and the central metabolism [START_REF] Billaudeau | Contrasting mechanisms of growth in two model rod-shaped bacteria[END_REF] [24] [START_REF] Zielinska | Flotillin-mediated membrane fluidity controls peptidoglycan synthesis and MreB movement. Elife 2020, 9. * This study shows that flotillins controls membrane fluidity and that the latter modulates MreB dynamics[END_REF]. Resource sharing between central metabolism and cell envelope synthesis will be covered in more detail in this issue in a review by Ankita J. Sachla and John D. Helmann [26].

FtsZ was initially thought to form a continuous structure named the Z-ring at the division septa. However, the advent of super-resolution imaging has also drastically changed our conception of the organization of the Z-ring and revealed that FtsZ forms discontinuous patches/filaments that also move circumferentially around the cell division site [START_REF] Bisson-Filho | Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division[END_REF][START_REF] Yang | GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis[END_REF].

Unlike MreB, the apparent circumferential movement of FtsZ was shown to be independent of cell wall synthesis and rather due to a treadmilling mechanism in which one end of the polymer grows and the other shrinks. While FtsZ treadmilling could guide the progressive insertion of nascent peptidoglycan during cell division, the functional link between FtsZ and peptidoglycan assembly remains more enigmatic and seems to be strain-specific. For instance, it has been shown that the treadmilling of FtsZ filaments drive the motion of bPBP2b in B. subtilis whereas the rotation of the bPBP2x:FtsW complex depends on peptidoglycan synthesis and not on FtsZ treadmilling in S. pneumoniae [12,[START_REF] Bisson-Filho | Treadmilling by FtsZ filaments drives peptidoglycan synthesis and bacterial cell division[END_REF]. In S. aureus, FtsZ treadmilling is proposed to serve for the initial invagination of the membrane while peptidoglycan synthesis occurs at the cell periphery. Upon recruitment of the protein complex DivIB/DivIC/FtsL and the protein MurJ, peptidoglycan assembly is reoriented at the division site and would provide the constriction force independently of FtsZ treadmilling [START_REF] Monteiro | Peptidoglycan synthesis drives an FtsZtreadmilling-independent step of cytokinesis[END_REF]. Further studies will likely tell us whether these observations reflect the specificity of each species in regards to their cell cycle and/or in response to the growth conditions.

Regulation of peptidoglycan hydrolases

To promote the insertion of new precursors into the existing peptidoglycan or to simply mediate cell separation after division, the peptidoglycan polymer needs to be continuously remodeled by a variety of proteins with hydrolytic functions (Figure 2). These hydrolases also serve other functions, like cell wall recycling or signaling in the host, that are detailed in [START_REF] Mayer | Bacteria's different ways to recycle their own cell wall[END_REF][START_REF] Bastos | Uptake, recognition and responses to peptidoglycan in the mammalian host[END_REF]. For cell division and morphogenesis, these hydrolases include glycosidases that cleave the glycosidic bonds in the glycan strand or amidases and peptidases that cleave the amide bond either between the glycan strand and the first amino acid of the stem peptide or within the stem peptide itself, respectively [START_REF] Alcorlo | Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases[END_REF] (Figure 2). As observed for the synthesis machineries, the action of hydrolases must be regulated to maintain cell-wall homeostasis. While many of them are biochemically and structurally well characterized, their regulation remains largely unknown.

Over the last few years, the conserved transmembrane FtsEX complex has emerged as a major regulator of different hydrolases, notably in S. pneumoniae and B. subtilis. In S. pneumoniae, the cell division endopeptidase PcsB is autoinhibited and activated by an ATPdriven conformational change of the FtsEX complex [START_REF] Rued | Structure of the Large Extracellular Loop of FtsX and Its Interaction with the Essential Peptidoglycan Hydrolase PcsB in Streptococcus pneumoniae[END_REF] [33]. In B. subtilis, a similar observation was made for the endopeptidase CwlO that also requires FtsEX for activation Interestingly, EloR is phosphorylated by the serine-threonine kinase StkP [42], the key regulator of the pneumococcal cell elongation and division [START_REF] Grangeasse | Rewiring the Pneumococcal Cell Cycle with Serine/Threonine-and Tyrosine-kinases[END_REF]. In absence of EloR phosphorylation, cells length is reduced as for ∆eloR cells suggesting that EloR is likely inactive. By contrast, the increase in EloR phosphorylation is tolerated only in the presence of suppressor mutations that reduce the functionality of the cell elongation proteins MreC or RodZ [42]. Altogether, it is thus proposed that EloR phosphorylation would stimulate the elongasome activity. EloR phosphorylation echoes another study that has identified a second regulator, specific of the aPBP PBP2a in S. pneumoniae [START_REF] Fenton | Phosphorylation-dependent activation of the cell wall synthase PBP2a in Streptococcus pneumoniae by MacP[END_REF]. The phosphorylation of this protein, named MacP, by StkP is also required for promoting PBP2a function.

The contribution of serine-threonine-kinases in peptidoglycan assembly is not restricted to S. pneumoniae and it has been detected in many Firmicutes, including Clostridium difficile [START_REF] Cuenot | The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance in Clostridium difficile[END_REF], B. subtilis [START_REF] Pompeo | Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth[END_REF], Enterococcus faecalis [START_REF] Labbe | Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification[END_REF], L. monocytogenes [START_REF] Pensinger | The Listeria monocytogenes PASTA Kinase PrkA and Its Substrate YvcK Are Required for Cell Wall Homeostasis, Metabolism, and Virulence[END_REF] or S. aureus [START_REF] Hardt | The cell wall precursor lipid II acts as a molecular signal for the Ser/Thr kinase PknB of Staphylococcus aureus[END_REF]. Quantitative phosphoproteomic studies also suggest that many more proteins involved in peptidoglycan assembly are likely regulated by phosphorylation and protein-kinases [START_REF] Hirschfeld | Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP[END_REF]. In line with this claim, the recently identified transglycosylase MltG was found to be phosphorylated in S. pneumoniae [START_REF] Hirschfeld | Proteomic Investigation Uncovers Potential Targets and Target Sites of Pneumococcal Serine-Threonine Kinase StkP and Phosphatase PhpP[END_REF]. MltG was proposed to contribute to pneumococcal cell elongation as its mutation suppresses the requirement of PBP2b, RodA and other proteins from the elongasome [START_REF] Tsui | Suppression of a deletion mutation in the gene encoding essential PBP2b reveals a new lytic transglycosylase involved in peripheral peptidoglycan synthesis in Streptococcus pneumoniae D39[END_REF]. Similarly, the glucosaminidase LytB, that is required for final separation of daughter cell, is controlled by the serine-threonine-kinase StkP [START_REF] Zucchini | PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae[END_REF]. Last, a protein of unknown function was found phosphorylated in both E. faecalis (IreB) and L. monocytogenes (ReoM). While the function IreB remains unclear [START_REF] Labbe | Growth-and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis[END_REF], ReoM was shown to activate the ClpCP-dependent proteolytic degradation of MurA that is essential for the synthesis of the lipid II [START_REF] Wamp | regulatory function of protein phosphorylation in peptidoglycan assembly. Phosphorylated ReoM controls the degradation of MurA that is required for the first step of peptidoglycan synthesis[END_REF].

Conclusion and perspectives

The key observations described in this review have changed our view about the biosynthesis of peptidoglycan. However, many other findings, like the knowledge gained about the cell division protein GpsB (reviewed in [START_REF] Hammond | inverted exclamation markvIVA la DivIVA![END_REF]) have also provided interesting observations and clarifications regarding some aspects of the peptidoglycan assembly. In addition, recent observations provide new directions of research. For instance, peptidoglycan homeostasis is also tightly interconnected with the cell metabolism ( [START_REF] Sperber | Metabolism Shapes the Cell[END_REF][START_REF] Patel | A metabolic checkpoint protein GlmR is important for diverting carbon into peptidoglycan biosynthesis in Bacillus subtilis[END_REF]), regulated by other protein post-translational modification like acetylation ( [START_REF] Carabetta | Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth[END_REF]) and the production of second messengers like c-di-AMP ( [START_REF] Rismondo | Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes[END_REF][START_REF] Massa | C-di-AMP accumulation impairs muropeptide synthesis in Listeria monocytogenes[END_REF]) or still dependent on the biosynthesis of other cell wall compounds like teichoic acids ( [START_REF] Caveney | Enzyme structures of the bacterial peptidoglycan and wall teichoic acid biogenesis pathways[END_REF]). Together with the recent advances in peptidoglycan imaging [START_REF] Radkov | Imaging Bacterial Cell Wall Biosynthesis[END_REF][START_REF] Pasquina-Lemonche | The architecture of the Gram-positive bacterial cell wall[END_REF], all these observations promise further discoveries as exciting as those presented in this review for the next years.

[

  34]. CwlO however degrades peptidoglycan at the lateral cell wall allowing cell elongation and two new co-factors, SweC and SweD, are required for the FtsEX-CwlO complex to function outside the divisome [35]. In E. coli, FtsEX is able to interact with several cell division proteins including FtsZ and FtsA [36]. Based on these findings, it has been proposed that FtsEX could act as a mechano-transmitter coupling cell division with the activation of the endopeptidases required for cell division and morphogenesis. Recently, a new amidase, LytH, contributing to the coordination between cell expansion and cell division was discovered in S. aureus [37]. Although independent of FtsEX, LytH is also controlled by a new co-factor, named ActH, to be fully active. Last, the glucosaminidase SagB was recently shown to work in complex with the integral membrane protein SpdC to release the growing glycan strand from the membrane. Indeed, SpdC would bind the lipid carrier and would orient the SagB active site for efficient clivage [38]. These few examples illustrate that we likely only know the tip of the iceberg regarding the regulation of the peptidoglycan hydrolase activities. Interesting directions for future research will be the identification of additional co-factors of cell-wall hydrolases and to investigate the mechanisms by which these hydrolytic enzymes are regulated. New factors and regulatory mechanisms An impressive array of studies has led to the characterization of new factors involved in peptidoglycan assembly. Notable examples are the S. pneumoniae CozE protein, which directs the activity of the aPBP PBP1a to specific cell wall areas [39]. It has also been proposed that CozE also interacts with MreC and MreD to promote cell elongation. Interestingly, a CozE homolog, named CozEb, was then identified both in S. pneumonaie and S. aureus [40] [41]. The two proteins interact together and harbor some overlapping functions required for the maintenance of the cell shape. Another factor that is crucial for pneumococcal cell morphogenesis is the RNA-binding protein EloR (also named KhpA) [42] [43]. Inactivation of EloR suppresses the requirement of the essential bPBP PBP2b and SEDS RodA and generates shorter cells indicating that EloR is involved in cell elongation.

  and septal peptidoglycan synthesis in Staphylococcus aureus. Nat Microbiol 2019, 4:1368-1377. ** This study shows that RodA and FtsW together with their cognate bPBPs PBP3 and PBP1 mediate sidewall and septal peptidoglycan incorporation, respectively. 12. Perez AJ, Cesbron Y, Shaw SL, Bazan Villicana J, Tsui HT, Boersma MJ, Ye ZA, Tovpeko Y, Dekker C, Holden S, et al.: Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae. Proc Natl Acad Sci U S A 2019, 116:3211-3220. * The authors analyzed FtsZ treadmilling in S. pneumonaie and showed that movement of the FtsW/bPBP2x complex depends on peptidoglycan synthesis rather than FtsZ treadmilling. 13. Rismondo J, Halbedel S, Grundling A: Cell Shape and Antibiotic Resistance Are Maintained by the Activity of Multiple FtsW and RodA Enzymes in Listeria monocytogenes. mBio 2019, 10. 14. Meeske AJ, Sham LT, Kimsey H, Koo BM, Gross CA, Bernhardt TG, Rudner DZ: MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A 2015, 112:6437-6442. 15. Cho H, Wivagg CN, Kapoor M, Barry Z, Rohs PD, Suh H, Marto JA, Garner EC, Bernhardt TG: Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat Microbiol 2016:16172. 16. Straume D, Piechowiak KW, Olsen S, Stamsas GA, Berg KH, Kjos M, Heggenhougen MV, Alcorlo M, Hermoso JA, Havarstein LS: Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall. Proc Natl Acad Sci U S A 2020, 117:6129-6138. ** This study demonstrates that aPBPs work independently of the divisome and elongasome activities and suggests that aPBPs mature the peptidoglycan framework. 33. Alcorlo M, Straume D, Lutkenhaus J, Havarstein LS, Hermoso JA: Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae. mBio 2020, 11. 34. Meisner J, Montero Llopis P, Sham LT, Garner E, Bernhardt TG, Rudner DZ: FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis. Mol Microbiol 2013, 89:1069-1083.35. Brunet YR, Wang X, Rudner DZ: SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilis. PLoS Genet 2019, 15:e1008296. * The authors have identifed two co-factors of FtsEX that are required for the function of the peptidoglycan hydrolase CwlO outside the divisome. 36. Pichoff S, Du S, Lutkenhaus J: Roles of FtsEX in cell division. Res Microbiol 2019, 170:374-380. 37. Do T, Schaefer K, Santiago AG, Coe KA, Fernandes PB, Kahne D, Pinho MG, Walker S: Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan. Nat Microbiol 2020, 5:291-303. * Characterization of a new amidase together with its activator. Most recent study illustrating the importance of peptidoglycan hydrolase activities and regulation in peptidoglycan assembly. 38. Schaefer K, Owens TW, Page JE, Santiago M, Kahne D, Walker S: Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization. Nat Microbiol 2021, 6:34-43. * The authors propose that the SagB-spdC complex cleave the nascent peptidoglycan from the membrane to allow its integration into the cell wall. 39. Fenton AK, Mortaji LE, Lau DT, Rudner DZ, Bernhardt TG: CozE is a member of the MreCD complex that directs cell elongation in Streptococcus pneumoniae. Nat Microbiol 2016, 2:16237. 40. Stamsas GA, Myrbraten IS, Straume D, Salehian Z, Veening JW, Havarstein LS, Kjos M: CozEa and CozEb play overlapping and essential roles in controlling cell division in Staphylococcus aureus. Mol Microbiol 2018, 109:615-632. 41. Stamsas GA, Restelli M, Ducret A, Freton C, Garcia PS, Havarstein LS, Straume D, Grangeasse C, Kjos M: A CozE homolog contributes to cell size homeostasis of Streptococcus pneumoniae. MBio 2020, in press. 42. Stamsas GA, Straume D, Ruud Winther A, Kjos M, Frantzen CA, Havarstein LS: Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae. Mol Microbiol 2017.

Figure 1 :

 1 Figure 1: Composition and diversity of the peptidoglycan polymer

Figure 2 :

 2 Figure 2: Hydrolases required for peptidoglycan remodeling

Acknowledgement

We apologize to colleagues whose original work could not be included due to space limitations. This work was supported by the Centre National de la Recherche Scientifique, The Université de Lyon, the Agence National de la Recherche (ANR-18-CE11-0017-02, ANR-19-CE15-0011-01) and the Bettencourt-Schueller Foundation.


Elia Gigante

Christian Hobeika

Brigitte Le Bail

Valérie Paradis

David Tougeron

Marie Lequoy

Mohamed Bouattour

Jean-Frederic Blanc

Nathalie Ganne-Carrié

Henri Tran

Clémence Hollande

Manon Allaire

Giuliana Amaddeo

Hélène Regnault

Paul Vigneron

Maxime Ronot

Laure Elkrief

Gontran Verset

Eric Trepo

Aziz Zaanan

Marianne Ziol

Massih Ningarhari

Julien Calderaro

Jean-Charles Nault
email: naultjc@gmail.com

Systemic Treatments with Tyrosine Kinase Inhibitor and Platinum-Based Chemotherapy in Patients with Unresectable or Metastatic Hepatocholangiocarcinoma

Keywords: Hepatocholangiocarcinoma, Cholangiocarcinoma, Hepatocellular carcinoma, Systemic treatment, Platinum, Tyrosine kinase inhibitors

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Combined

hepatocellular-cholangiocarcinoma (cHCC-CCA) is considered as a particular entity characterized histologically by the presence of two distinct patterns within the same lesion: the presence of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) [START_REF] Allen | Combined liver cell and bile duct carcinoma[END_REF][START_REF] Brunt | cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation[END_REF][START_REF] Gigante | New insights into the pathophysiology and clinical care of rare primary liver cancers[END_REF]. The diagnosis of cHCC-CCAs is based on routine histological diagnosis; immunohistochemistry could be helpful to identify the two cell populations [START_REF] Brunt | cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation[END_REF][START_REF] Sempouxkakar | Combined hepatocellular-cholangiocarcinoma and undifferentiated primary liver carcinoma[END_REF]. The risk factors associated with cHCC-CCA are shared with the other primary liver cancers (PLCs) and include chronic hepatitis B and C infection, metabolic syndrome, excessive alcohol consumption, and cirrhosis. The association with cirrhosis is reported with various frequencies (25-55%) and associated with different etiologies according to the geographical area [START_REF] Gigante | New insights into the pathophysiology and clinical care of rare primary liver cancers[END_REF][START_REF] Garancini | Combined hepatocellular-cholangiocarcinoma: a population-level analysis of an uncommon primary liver tumor[END_REF][START_REF] Ramai | Combined hepatocellular cholangiocarcinoma: a Population-Based Retrospective Study[END_REF][START_REF] Kudo | Report of the 20th Nationwide follow-up survey of primary liver cancer in Japan[END_REF][START_REF] Ng | Combined hepatocellular-cholangiocarcinoma: a clinicopathological study[END_REF][START_REF] Lee | Comparison of combined hepatocellular and cholangiocarcinoma with hepatocellular carcinoma and intrahepatic cholangiocarcinoma[END_REF][START_REF] Taguchi | A clinicopathological study on combined hepatocellular and cholangiocarcinoma[END_REF]. The difficulty to perform the diagnosis of cHCC-CCA leads probably to an underestimation of the actual incidence of this subtype of PLC. Recent studies report that the incidence in western countries of cHCC-CCAs ranges between 1 and 5% of all PLCs [START_REF] Brunt | cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation[END_REF][START_REF] Sempouxkakar | Combined hepatocellular-cholangiocarcinoma and undifferentiated primary liver carcinoma[END_REF] with an overall incidence estimated to be 0.05 per 100,000 persons per year [START_REF] Garancini | Combined hepatocellular-cholangiocarcinoma: a population-level analysis of an uncommon primary liver tumor[END_REF][START_REF] Ramai | Combined hepatocellular cholangiocarcinoma: a Population-Based Retrospective Study[END_REF].

Liver surgery is considered the standard of care for cHCC-CCA treatment when feasible, but the recurrence rate remains high [START_REF] Gentile | Surgical treatment of hepatocholangiocarcinoma: a systematic review[END_REF]. Moreover, cHCC-CCA is often diagnosed at an advanced stage, so only a minority of patients are candidates for surgery in clinical practice [START_REF] Gigante | New insights into the pathophysiology and clinical care of rare primary liver cancers[END_REF]. In addition, most of the studies available are derived from monocentric surgical series, so few studies on unresectable/metastatic cHCC-CCAs have been published [START_REF] Salimon | Gemcitabine plus platinum-based chemotherapy for first-line treatment of hepatocholangiocarcinoma: an AGEO French multicentre retrospective study[END_REF][START_REF] Kobayashi | Multicenter retrospective analysis of systemic chemotherapy for unresectable combined hepatocellular and cholangiocarcinoma[END_REF][START_REF] Trikalinos | Systemic therapy for combined hepatocellular-cholangiocarcinoma: a single-institution experience[END_REF][START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF]. Currently, no systemic treatment is validated in patients with unresectable or metastatic cHCC-CCA, and this histological subtype is excluded from most clinical trials enrolling patients with PLCs [START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF]. Consequently, the weak evidence available on the systemic treatments of cHCC-CCA came from case reports and case series treating the patients either by platinum-based chemotherapy, the standard treatment of advanced CCA, or the tyrosine kinase inhibitor sorafenib, the previous standard treatment of HCC [START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF][START_REF] Rizvi | European Association for the Study of the Liver. EASL clinical practice guidelines: management of hepatocellular carcinoma[END_REF][18]. The median overall survival (OS) reported ranged from 8.9 to 16.2 months [START_REF] Gigante | New insights into the pathophysiology and clinical care of rare primary liver cancers[END_REF][START_REF] Salimon | Gemcitabine plus platinum-based chemotherapy for first-line treatment of hepatocholangiocarcinoma: an AGEO French multicentre retrospective study[END_REF][START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF]. However, most of the studies were either monocentric or lacked an adjustment between the groups treated by sorafenib and platinum-based therapy. Our study aimed to describe the clinical features of unresectable/metastatic cHCC-CCA treated by systemic therapies, compared their outcomes to cohorts of patients with advanced HCC and iCCA, and to assess the effect of DOI: 10.1159/000525488 the type of systemic treatments received by patients with cHCC-CCA taking into account other prognostic factors.

Materials and Methods

Selection of Patients

Consecutive patients with unresectable or metastatic cHCC-CCA from 11 French centers that underwent a systemic treatment from 2009 to 2020 were included retrospectively. An independent French Ethics Committee approved the study (approval number CLEA-2020-124). The reviewing of the histological samples was performed by one of the three expert pathologists (B.L.B., V.P., and J.C.) to confirm the diagnosis of cHCC-CCA according to the 2019 WHO classification of tumors of the digestive system [START_REF] Sempouxkakar | Combined hepatocellular-cholangiocarcinoma and undifferentiated primary liver carcinoma[END_REF].

The following criteria were required to include the patients in the study: 1 Patients with a histological diagnosis of cHCC-CCA according to 2019 WHO classification 2 Considered as unresectable or metastatic 3 Receiving a systemic treatment (whatever the type of systemic treatment) 4 Patients of 18 years old or more The exclusion criteria were as follows: 1 Patients receiving combination of locoregional treatment and systemic treatment 2 Systemic treatment in a neoadjuvant or an adjuvant intent together with surgery One hundred six patients were screened for inclusion. After reviewing the histology and the clinical data, 23 patients were excluded, and 83 patients were finally included in the study. Clinical data (gender, age, etiology, severity of the underlying liver disease, components of the metabolic syndrome, and WHO performance status), laboratory tests (liver function tests, platelets, serum alphafetoprotein [AFP], and CA19-9 levels) and tumor imaging features (size, number, macrovascular invasion, and distant metastasis) were collected before the beginning of the systemic treatment. Given the retrospective nature of the study, each center, based on its local experience, decided independently the first-line therapy received by the patient.

Two cohorts of patients were used as control populations: one cohort with unresectable or metastatic iCCA, mainly treated by platinum-based therapy, and one cohort of BCLC B/C HCC treated by the tyrosine kinase inhibitor. The cohort of patients with iCCA (n = 94) was a retrospective monocentric cohort (Eugène Marquis Center) with a histological diagnosis of iCCA considered nonresectable or metastatic and treated by systemic cytotoxic chemotherapy between 2005 and 2014. The cohort of patients with HCC (n = 117) was a retrospective cohort of patients from two centers (Avicenne Hospital in France and Erasme Hospital in Belgium) with HCC diagnosed by histology, classified by BCLC B and C, and treated by tyrosine kinase inhibitor (sorafenib) between 2013 and 2019. The following clinical, radiological, and biological characteristics were collected in these two cohorts before the beginning of the systemic treatment: age, gender, etiology of the chronic liver disease, presence of cirrhosis, liver function test, and tumor imaging features (size, number, macrovascular invasion, distant metastasis).

Follow-Up and Outcomes Assessment

After initiating systemic treatment, all patients were followed up until death or the last recorded visit. The follow-up period was ended on July 30, 2021. The primary endpoint was OS, defined as the survival from the initiation of treatment to death, whatever the cause. The secondary endpoints were progression-free survival (PFS), overall response rate (ORR), and disease control rate (DCR). The radiological response at the first radiological assessment (performed 2-3 months after the beginning of the systemic treatment) was collected from the radiological report of each center to compute the ORR defined by the combination of complete and partial response, and the DCR defined by the combination of complete response, partial response, and stable disease according to RECIST 1.1 criteria. PFS was defined as the survival from treatment start to disease progression or death, whatever the cause. Alive patients with no progression were censored at the last follow-up date.

Statistical Analysis

Continuous data are expressed as median (25-75 interquartiles). Categorical data are expressed as percentages. Statistical significance testing was 2-sided. A p value <0.05 was considered statistically significant for all tests unless indicated otherwise. Categorical variables were compared using the Fisher exact test or χ 2 test, as appropriate. Continuous variables were compared using the Mann-Whitney U test. Survival outcomes such as OS and PFS were assessed using the Kaplan-Meier method, and the survival rates were compared using the log-rank test.

In the study population of unresectable or metastatic cHCC-CCA (n = 83), none of the variables had more than 25% of missing values, and missing values were managed in the exploratory analyses using multiple imputation chained methodology with six imputed datasets. Hence, all exploratory analyses were performed in each imputed dataset, and the estimates and standard errors were pooled into a final point estimate plus standard error according to Rubin's rule. Right-censored outcome (i.e., OS and PFS) regressions were Cox regressions (estimated effect sizes were expressed as hazard ratio [HR] with confidence interval [95% CI]), and binary outcome (i.e., ORR and DCR) regressions were logistic regressions (estimated effect sizes were expressed as odd ratio [OR] with 95% CI).

Prognostic factors associated with OS were assessed using both univariable and multivariable Cox regressions. Variables of interest were selected based on univariable selection (all variables with p < 0.20) and included in a multivariable Cox regression. In addition, both Cox and logistic regressions conducted across the 3 cohorts of patients (cHCC-CCA, iCCA, and BCLC B/C HCC patients) were adjusted to counteract unobserved heterogeneities between individuals using frailty (i.e., multilevel) regressions, which included a frailty term (i.e., random effect on each individual) [START_REF] Balan | A tutorial on frailty models[END_REF].

The marginal effect of tyrosine kinase inhibitor (TKI) (vs. platinum-based regimens) on ORR, DCR, PFS, and OS was estimated using doubly robust estimators [START_REF] Funk | Doubly robust estimation of causal effects[END_REF]. Doubly robust estimators are combinations of the appropriate outcome regression (Cox or logistic regression) with a model of exposure using an inverse probability weighting based on covariate balancing propensity scores [START_REF] Wyss | The role of prediction modeling in propensity score estimation: an evaluation of logistic regression, bCART, and the covariate-balancing propensity score[END_REF]. Cofounders considered in all doubly robust estimators to adjust the effect of TKI (vs. platinum-based regimens) were: ALBI score, MELD score, tobacco use, advanced fibrosis, diabetes, plasmatic level of AFP and CA19-9, unique versus multiple tumors, the maximal diameter of tumor(s), the sum of diameters of Liver Cancer 2022;11:460-473 DOI: 10.1159/000525488 tumor(s), the presence of metastasis, macrovascular invasion, previous local or locoregional treatments, and ascites. The quality of the inverse probability weighting of the doubly robust estimators was assessed using the effective sample size, ranges of absolute mean differences, and variance ratios across imputations, as appropriate.

The marginal effect of ALBI on ORR, DCR, PFS, and OS was estimated using doubly robust estimators as described above. Confounders considered in all doubly robust estimators to adjust the effect of ALBI were: the type of first-line regimen (TKI vs. platinum-based regimens), serum level of AFP, serum level of CA19-9, unique versus multiple tumors, the maximal diameter of tumor(s), the sum of diameters of tumor(s), the presence of metastasis, macrovascular invasion, and previous local or locoregional treatments. The quality of the inverse probability weighting of the doubly robust estimators was assessed using the effective sample size, ranges of absolute mean differences, and correlations across imputations, as appropriate.

All statistical analyses were performed using R statistical software version 3.6.3. The packages used were tidyverse, broom, MASS, Weitghtit, cobalt, glm, lme4, coxme, CBPS, mice, parlmice, and miceadd.

Results

Characteristics of Patients with Hepatocholangiocarcinoma

A total of 83 patients with cHCC-CCA treated by systemic therapy were included. The main characteristics of the population are summarized in Table 1. The diagnosis cHCC-CCA was performed on biopsy samples in 42 cases and on surgical samples in 41 cases. The patients were predominantly male (72.3%) with a median age of 63 In terms of tumor features, patients frequently had a multinodular intrahepatic disease (63.2%) with an average size of the largest nodule of 50 mm (27.75-85.00). At baseline, 66.2% of patients had an extrahepatic spread, the most common sites being the lymph nodes (41.2%), lungs (26.3%), peritoneum (12.5%), and bones (12.5%). Almost one-third of patients (30.7%) had a macrovascular tumor invasion.

Before the beginning of systemic treatments, 49 patients (59%) had received previous liver-directed treatment of cHCC-CCA (surgery or locoregional treatments), including liver resection for 31 patients and liver transplantation for 9 patients. Among the 83 patients, 54 patients were treated with first-line platinum-based therapy, 25 with TKIs (mainly sorafenib), and 4 with other chemotherapies regimens. The chemotherapy

Outcomes of Patients with Hepatocholangiocarcinoma and Comparison with Intrahepatic Cholangiocarcinoma and Hepatocellular Carcinoma

Among the 83 patients with cHCC-CCAs, the median follow-up was 41 months. Sixty-eight patients had an available first radiological assessment under systemic treatment, 9 patients (13%) harbored an objective response rate, and 36 patients (53%) had disease control. The median PFS was 4 months (95% CI: 3-5). The median OS was 12 months (95% CI: 8-15) with 1 year, 2 years, and 3 years' survival rates of 47.7%, 18.3%, and 7.6%, respectively. A multivariable cox analysis showed that ALBI score (HR = 2.15; 95% CI: 1.23, 3.76; p = 0.009), ascites (HR = 3.45, 95% CI: 1.31, 9.03; p = 0.013), and tobacco use (HR = 2.29; 95% CI: 1.08, 4.87; p = 0.032) were independently associated with OS (shown in Table 2). Kaplan-Meier OS curves according to the ALBI grade are shown in Figure 1.

Next, we aim to compare the baseline characteristics and the outcomes of patients with cHCC-CCAs with patients with nonresectable/metastasis iCCA (n = 94) and HCC (n = 117) (see Table 3 for the full description of the three groups). cHCC-CCAs were more often developed on advanced fibrosis/cirrhosis (55.4%) than iCCA (26.6%) but less frequently than HCC (80.2%) (p < 0.001). Extrahepatic metastases were more frequent in iCCA (81%) than in cHCC-CCAs (66.2%) and HCC (36.8%) (p < 0.001).

All the patients in the HCC group (n = 117) received the TKI sorafenib as first-line systemic treatment. Patients in the iCCA group received primarily platinumbased regimens (90%; n = 85) including gemcitabine-oxaliplatin (n = 47), gemcitabine-cisplatin (n = 34), and fluorouracil-leucovorin (LV5FU2)-oxaliplatin (n = 4). The other types of chemotherapies were gemcitabine monotherapy (7 patients), 5-fluorouracil-irinotecan (2 patients), and 5-fluorouracil (1 patient).

Before adjustment, ORR rates were 4.4%, 9.0%, and 16.1% in cHCC-CCAs, HCC, and iCCA patients, respectively (p = 0.047), and DCR rates were 52.9%, 54.0%, and 67.7% in cHCC-CCAs, HCC, and iCCA patients, respectively (p = 0.083). However, in multivariable analysis using frailty logistic regression, no significant differences in term of ORR and DCR were observed between cHCC-CCA and HCC or iCCA (Table 4).

The unadjusted median OS was longer in iCCA (median 13 months) and cHCC-CCA (12 months) than in HCC (11 months) (p = 0.130, log-rank test) (Fig. 2a), and the unadjusted median PFS was longer in iCCA (6 months) compared to cHCC-CCA (4 months) and HCC (3 months) (p = 0.005, log-rank test) (Fig. 2b). In multivariate analysis using frailty Cox regression, the PFS was significantly longer in the iCCA group compared to cHCC-CCA (OR 0.63 [95% CI: 0.41-0.96], p = 0.034) but without any statistical difference in terms of OS (Table 4).

Comparison between Tyrosine Kinase Inhibitors and Platinum-Based First-Line Regimens in Hepatocholangiocarcinoma

Next, we compared the characteristics and the outcomes of patients treated with tyrosine inhibitors (n = 25) with the patients with platinum-based regimens (n = 54) among the cohort of 83 patients with cHCC-CCA. Patients treated by TKI had a lower serum CA19-9 (p = 0.035) and a more severe liver failure reflected by a significantly different value of the ALBI score (p = 0.037) (see online suppl. Table 2 for the full description).

Without adjustment, ORR was not significantly different between the two regimens (10% for TKI vs. 15.2% for platinum treated patients, p = 0.859) as well as DCR (45% for TKI vs. 58.7% for platinum, p = 0.448). After adjustment using doubly robust estimators, patients treated with TKI (vs. platinum-based regimens) did not have a higher ORR (OR = 1.67; 95% CI: 0.03, 3.95 p = 0.642) or DCR (OR = 0.76; 95% CI: 0.15, 3.86; p = 0.387).

Without adjustment, patients treated by TKIs had a median OS of 8.3 months compared to 11.9 months for patients treated with platinum-based regimens (p = 0.86, log-rank test, shown in Fig. 3a) and a median PFS of 4.1 months for platinum-based regimens compared to TKI 2.8 months for TKI (p = 0.91, log-rank test, shown in Fig. 3b). After the adjustment using the doubly robust estimators, the type of treatment had no effect on PFS (HR = 1.25; 95% CI: 0.44, 3.49; p = 0.652) or OS (HR = 0.98; 95% CI: 0.48, 2.02; p = 0.962).

Using a doubly robust estimation, the ALBI score was associated with OS (OR 2.517, 95% CI 1.412-4.487, p = 0.003) independently of the treatment received (platinum regimen or TKI), whereas it was not significantly associated with PFS, DCR, and ORR (Table 5). Among the patients treated by platinum-based regimens, the ALBI score taken as a continuous variable was associated significantly with OS (OR 11.679, 95% CI 2.325-58.674, p = 0.0049), whereas no significant association was observed in patients treated by TKI (OR 1.679, 95% CI 0.6321-4.461, p = 0.273) maybe due to the low number of patients in this subgroup (online suppl. Table 3).

Characteristic and Outcomes in Patients with Hepatocholangiocarcinoma Treated by a Second-Line Systemic Therapy

In the cHCC-CCA group, 44 patients received a second-line therapy, 16 patients received a third-line therapy of systemic treatments, and 4 patients a fourth-line ther- apy. Of the 44 patients receiving a second-line systemic treatment, 12 were treated with platinum-based regimens and 17 with TKIs. The remaining 15 patients had various regimens described in online supplementary Table 1. Median OS after the beginning of the second line was 8 months whatever the treatments, with a median survival of 8 months for patients treated by TKI versus 10.5 months for patients treated by platinum-based regimens (log-rank test p = 0.16, no adjustment was performed due to the low numbers of patients in each group). cHCC-CCA, hepatocholangiocarcinoma; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma. * Right-censored outcome (i.e., OS and PFS) regressions were Cox regressions (estimated effect sizes were expressed as HR with 95% CI) and binary outcome (i.e., ORR and DCR) regressions were logistic regressions (estimated effect sizes were expressed as OR with 95% CI). † Adjustment regarding unobserved heterogeneities between individuals was performed using frailty models (Cox or logistic regression), which included a frailty term (i.e., random effect) on everyone. DOI: 10.1159/000525488

Discussion

Although cHCC-CCA was described several decades ago, its evolving definition in terms of classification and its rarity has led to a delay in developing a consensual therapeutical strategy. While surgery is recognized as the best therapeutical option when feasible, the clinical management in case of unresectable or metastatic disease lacks reliable evidence and is based on local experiences mimicking the systemic treatment validated in iCCA and CCA under systemic treatments included a limited number of patients from Western countries or were monocentric studies in Asia [START_REF] Salimon | Gemcitabine plus platinum-based chemotherapy for first-line treatment of hepatocholangiocarcinoma: an AGEO French multicentre retrospective study[END_REF][START_REF] Trikalinos | Systemic therapy for combined hepatocellular-cholangiocarcinoma: a single-institution experience[END_REF][START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF]. cHCC-CCA has been considered more similar to iCCA than HCC when comparing the survival rates after resection or transplantation [START_REF] Gigante | New insights into the pathophysiology and clinical care of rare primary liver cancers[END_REF][START_REF] Gentile | Surgical treatment of hepatocholangiocarcinoma: a systematic review[END_REF][START_REF] Jarnagin | Combined hepatocellular and cholangiocarcinoma: demographic, clinical, and prognostic factors[END_REF]. However, a recent study has suggested that the outcomes were similar between cHCC-CCA, HCC, and ICCA after adjusting for cirrhosis and tumor features [START_REF] Holzner | Resection of mixed hepatocellular-cholangiocarcinoma, hepatocellular carcinoma, and intrahepatic cholangiocarcinoma[END_REF]. Morevoer, no study has compared the characteristics and outcomes of patients with unresectable/metastatic cHCC-CCA, HCC, and ICCA under systemic treatments. Thus, we compared cHCC-CCA under systemic treatments with a population of patients with iCCA treated mainly by platinum-based chemotherapy and patients with HCC treated by sorafenib. cHCC-CCA seems to stand at the crossroad between iCCA and HCC in terms of advanced fibrosis/ cirrhosis (55%), of gender balance with lesser men than in HCC and more than in iCCA and the levels of serum AFP and CA19-9. The unadjusted analysis suggests that the median OS was longer in iCCA (13 months) compared to cHCC-CCA (12 months) and HCC (11 months). However, after adjustment, the difference was not significant, suggesting that confounding factors, such as cirrhosis, are likely to be competitive prognostic factors explaining the unadjusted differences. Altogether, cHCC-CCA patients are likely to cumulate the prognosis of an aggressive pattern of PLC and the risks related to advanced underlying liver diseases.

Such complexes of clinical situations could explain the current uncertainties regarding the more appropriate first-line systemic therapy in unresectable patients. A pre-vious published eastern multicentric cohort suggested that the TKI sorafenib has a lower median survival (3.5 months) compared to gemcitabine/cisplatin (11.9 months) and fluorouracil/cisplatin (10.2 months), but the very low number of patients treated by sorafenib (n = 5) impaired this conclusion [START_REF] Kobayashi | Multicenter retrospective analysis of systemic chemotherapy for unresectable combined hepatocellular and cholangiocarcinoma[END_REF]. Another western monocentric cohort of cHCC-CCA who received systemic therapy reported a median OS of 11.5 months for gemcitabine/platinum therapy and OS of 9.6 months for sorafenib, but this study only included 7 patients treated by sorafenib [START_REF] Trikalinos | Systemic therapy for combined hepatocellular-cholangiocarcinoma: a single-institution experience[END_REF]. A multicentric series published by Salimon et al. [START_REF] Salimon | Gemcitabine plus platinum-based chemotherapy for first-line treatment of hepatocholangiocarcinoma: an AGEO French multicentre retrospective study[END_REF], including 30 patients, reported 16.2 months of OS under platinum-based therapies, but this study's results are impaired because one-third of the patients lack histology for the diagnosis cHCC-CCA. Finally, a recent monocentric study from Korea suggested that patients, predominantly infected by hepatitis B infection, treated with TKI and cytotoxic chemotherapy had similar outcomes [START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF].

In this study, we compared the outcomes of 25 cHCC-CCA patients who received TKI with 54 of their counterparts who received platinum-based chemotherapy. Patients treated by TKI had a median OS of 8.3 months compared to 11.9 months for patients treated with platinum-based regimens. This study used a doubly robust method to provide unbiased effects of treatments on outcomes of patients, whereas other studies report only an unadjusted comparison [START_REF] Salimon | Gemcitabine plus platinum-based chemotherapy for first-line treatment of hepatocholangiocarcinoma: an AGEO French multicentre retrospective study[END_REF][START_REF] Kobayashi | Multicenter retrospective analysis of systemic chemotherapy for unresectable combined hepatocellular and cholangiocarcinoma[END_REF][START_REF] Trikalinos | Systemic therapy for combined hepatocellular-cholangiocarcinoma: a single-institution experience[END_REF][START_REF] Kim | Clinical outcomes of systemic therapy in patients with unresectable or metastatic combined hepatocellular-cholangiocarcinoma[END_REF]. Such an unadjusted comparison is likely to introduce important bias when comparing treatments, such as TKI versus platinumbased, for which indications are likely guided by the general status, the severity of the underlying liver diseases, * Right-censored outcome (i.e., OS and PFS) regressions were Cox regressions (HR with 95% CI) and binary outcome (i.e., ORR and DCR) regressions were logistic regressions (OR with 95% CI). † Adjustment was done regarding the first-line regiment (TKI vs. platinumbased regimens), serum level of AFP and CA19-9, unique versus multiple tumors, the maximal diameter of tumor(s), the sum of diameters of tumor(s), the presence of metastasis, macrovascular invasion, and previous local or locoregional treatments. The analysis was performed using doubly robust estimators (combinations of the appropriate outcome regression [Cox or logistic regression] with a model of exposure using an inverse probability weighting based on covariate balancing propensity scores). and tolerance of the patient. In fact, no difference in terms of ORR, DCR, PFS, and OS between the two types of systemic treatments was found after doubly robust adjustment on several tumoral and nontumoral cofounding factors. These data suggested that TKI and platinum-based chemotherapy have the same outcomes and efficacy in patients with unresectable/metastatic cHCC-CCA and that the choice of the systemic treatments should be tailored to the potential contraindications for each regimen and the patient's preference. Interestingly, ALBI score and ascites were associated with OS independently from other baseline variables, especially from the MELD score and the presence of advanced fibrosis. Therefore, ALBI score seems to be a relevant prognostic tool in cHCC-CCA as reported for HCC patients. It is likely to capture both the liver function in cirrhotic patients and the biliary obstruction, diffuse tumor infiltration, and denutrition in patients regardless of their underlying parenchymal changes [START_REF] Johnson | Assessment of liver function in patients with hepatocellular carcinoma: a new evidence-based approach-the ALBI grade[END_REF][START_REF] Chan | Development of pre and post-operative models to predict early recurrence of hepatocellular carcinoma after surgical resection[END_REF][START_REF] Hiraoka | Validation and potential of albumin-bilirubin grade and prognostication in a nationwide survey of 46,681 hepatocellular carcinoma patients in Japan: the need for a more detailed evaluation of hepatic function[END_REF]. Considering that the ALBI score is significantly associated with OS but not with outcomes of response to cancer treatments (PFS, DCR, ORR), it could be speculated that the ALBI score is more a prognostic factor related to the degree of liver failure and denutrition than a variable predictive of the efficacy of the treatment.

This study has limitations. First, it is a retrospective study and some data such as radiological response were collected from radiological reports and encountered limitations related to the RECIST 1.1 criteria especially versus the mRECIST [START_REF] Llovet | mRECIST for HCC: performance and novel refinements[END_REF]. Moreover, the absence of difference in outcomes could be due to a lack of power due to the limited number of patients in subgroup analysis. Additionally, even if expert pathologists reviewed the cases, the diagnosis of cHCC-CCA on tumor biopsy remains challenging, and we could not exclude that some CCA and HCC could be misdiagnosed due to tumor sampling error affected by the intrinsic histological heterogeneity of cHCC-CCA or the use of noninvasive criteria for HCC diagnosis in cirrhotic patients [18,[START_REF] Gigante | Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma[END_REF]. Moreover, the small number of patients included in the comparison between TKI and platinum-based treatment is one of the limitations of the study that requires a future larger cohort of patients to have more granularity in the analysis. Finally, atezolizumab/bevacizumab represented the new standard of care in advanced HCC patients [START_REF] Finn | Atezolizumab plus bevacizumab in unresectable hepatocellular Carcinoma[END_REF]. Likewise, biomarkers guided therapy (e.g., FGFR inhibitor in FGFR2 fusion, IDH1 inhibitor in IDH1 mutations) are widely used in CCA patients [START_REF] Zhu | Final overall survival efficacy results of ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation: the phase 3 randomized clinical clarIDHy trial[END_REF][START_REF] Abou-Alfa | Pemigatinib for previously treated, locally advanced or metastatic cholangiocarcinoma: a multicentre, open-label, phase 2 study[END_REF][START_REF] Goyal | Abstract CT010: primary results of phase 2 FOE-NIX-CCA2: the irreversible FGFR1-4 inhibitor futibatinib in intrahepatic cholangiocarcinoma (iCCA) with FGFR2 fusions/ rearrangements[END_REF][START_REF] Meric-Bernstam | Futibatinib, an irreversible FGFR1-4 inhibitor, in patients with advanced solid tumors harboring FGF/FGFR aberrations: A Phase I Dose-Expansion Study[END_REF]. The assessment of these two strategies was beyond the scope of this study, while they are likely to be promising in cHCC-CCA patients and require to be studied in the near future.

In conclusion, this study reported a large western multicentric cohort of cHCC-CCA under systemic treatment, showing that cHCC-CCA harbored specific clinical features compared to HCC and CCA and that TKI and platinum-based therapies have the same efficacy in nonresectable/metastatic cHCC-CCA. The ALBI score could be used as a relevant prognostic tool in these patients.

Fig. 1 .

 1 Fig. 1. OS of patients with cHCC-CCA according to the ALBI grade. Kaplan-Meier curves of unadjusted OS stratified according to the ALBI grade (grade 1 n = 16, grade 2 n = 40, and grade 3 n = 4). The comparison was performed using the log-rank test.

Fig. 2 .Fig. 3 .

 23 Fig. 2. OS and PFS in patients with HCC, iCCA, and cHCC-CCA. Kaplan-Meier curves of unadjusted OS (a) and unadjusted PFS (b) comparing HCC (n = 117), iCCA (n = 94), and cHCC-CCA (n = 83) using the log-rank test.

Table 1 .

 1 Description of patients with hepatocholangiocarcinoma

	Variables	Available data cHCC-CCA (n = 83)
	Baseline characteristics
	Male*	83	60 (72.3)
	Age, years**	83	62.63 [56.67, 69.80]
	Hepatitis B virus*	78	16 (20.5)
	Hepatitis C virus*	77	15 (19.5)
	Metabolic syndrome*	75	35 (46.7)
	Tobacco use*	73	41 (56)
	Chronic alcohol intake*	77	37 (48.1)
	Body mass index**	81	24.00 [21.46, 27.00]
	Diabetes type 2*	78	32 (41.0)
	Arterial hypertension*	80	43 (53.8)
	Dyslipidemia*	78	34 (43.6)
	Performance status 0/1*	66	56 (84.8)
	Advanced fibrosis/cirrhosis (F3-F4)*	74	41 (55.4)
	Child-Pugh A*	32	24 (75)
	Biochemical characteristics
	Total bilirubin, µmol/L**	78	12.00 [8.00, 17.00]
	Albumin, g/L**	62	36.00 [31.25, 39.75]
	Time of prothrombin, %**	73	87.00 [78.00, 97.00]
	Platelets, g/L**	78	208.00 [166.00, 289.75]
	Creatinine, µmol/L**	75	68.00 [60.50, 82.00]
	Serum AFP**	55	5.00 [0.55, 54.50]
	Serum CA19-9**	37	1.75 [1.00, 10.00]
	MELD score**	75	8 [7,11]
	ALBI score**	60	-2.3182 [ -2.6940, -1.9315]
	Tumor characteristics
	Multiple intrahepatic lesions*	76	48 (63.2)
	Macrovascular invasion*	78	24 (30.8)
	Metastasis*	80	53 (66.2)
	Size of biggest nodule, mm**	71	50.00 [27.75, 85.00]

The % of Child-Pugh A patient was calculated on advanced fibrosis/cirrhosis patients. CA19-9 and AFP are represented as times above normal (median [IQR]). AFP, alphafetoprotein; CA19-9, carbohydrate antigen 19-9; MELD, Model for End-Stage Liver Disease; ALBI, albumin-bilirubin score. * n (%). ** Median (interquartile range).

Table 2 .

 2 Univariable and multivariable analyses of factors associated with OS in the 83 patients with hepatocholangiocarcinoma Model for End-Stage Liver Disease; ALBI, albumin-bilirubin score. * Variables included in the multivariable Cox model were selected based on the results of the univariable analysis (i.e., all variables associated to OS with a p value <0.200).

	Variables	Univariable cox regression			Multivariable cox regression*
		HR	2.5	97.5	p value	HR	2.5	97.5	p value
	Male	1.79	1.05	3.03	0.032	1.12	0.59	2.13	0.731
	Age (years)	1.00	0.98	1.02	0.927
	Hepatitis B virus	0.76	0.41	1.40	0.370
	Hepatitis C virus	1.06	0.56	2.03	0.846
	Metabolic syndrome	1.32	0.82	2.12	0.247
	BMI (kg/m 2 )	1.00	0.96	1.05	0.911
	Arterial hypertension	1.12	0.71	1.79	0.615
	Dyslipidemia	1.35	0.85	2.14	0.207
	Tobacco	2.29	1.40	3.74	0.001	2.29	1.08	4.87	0.032
	Alcohol intake	2.17	1.26	3.73	0.006	0.91	0.44	1.89	0.804
	Diabetes	1.33	0.83	2.13	0.229
	Ascites	4.64	1.68	12.80	0.004	3.45	1.31	9.03	0.013
	Encephalopathy	4.94	0.38	64.66	0.207
	PS grade
	0	Ref	Ref	Ref	Ref	Ref	Ref	Ref	Ref
	1	0.74	0.41	1.33	0.305	0.92	0.46	1.84	0.805
	2	2.84	0.86	9.42	0.081	1.59	0.57	4.49	0.360
	3	2.21	0.27	18.12	0.433	12.49	0.07	22.47	0.273
	AFP	1.00	1.00	1.00	0.281
	CA19-9	1.00	1.00	1.00	0.249
	Unique tumor(s)	0.81	0.49	1.32	0.394
	Size of the biggest nodule	1.00	0.99	1.01	0.947
	Sum of tumor(s) diameter	1.00	0.99	1.01	0.823
	Macrovascular invasion	1.26	0.76	2.08	0.360
	Metastasis	1.04	0.63	1.73	0.872
	Previous treatment	0.80	0.50	1.27	0.334
	First-line regimen
	Platinum-based	Ref	Ref	Ref	Ref
	TKI	1.04	0.63	1.73	0.87
	MELD score	1.07	0.98	1.17	0.124	1.09	0.97	1.22	0.138
	ALBI grade	2.20	1.24	3.91	0.010	2.15	1.23	3.76	0.009
	Advanced fibrosis	1.43	0.88	2.33	0.144	1.12	0.61	2.06	0.711

BMI, body mass index; AFP, alpha-fetoprotein; CA19-9, carbohydrate antigen 19-9; TKI, tyrosine kinase inhibitor; PS, performance status; MELD,

Table 1 (

 1 see www.karger.com/doi/10.1159/000525488 for all online suppl. material).

Table 3 .

 3 Comparison of baseline features of patients with hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and hepatocholangiocarcinoma None of the variables had more than 5% of missing values. To note, we included in this table the common variables available between the 3 cohorts of patients. Alkaline phosphatase, CA19-9, and AFP are represented as times above normal (median [IQR]). cHCC-CCA, hepatocholangiocarcinoma; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; AFP, alpha-fetoprotein; CA19-9, carbohydrate antigen 19-9. * n (%). ** Median (interquartile range).

	Variables	iCCA (n = 94)	HCC (n = 117)	cHCC-CCA (n = 83)	p value
	Male*	60 (63.8)	97 (82.9)	60 (72.3)	0.007
	Age, years %	64.00 [56.00, 71.75]	66.00 [57.00, 75.00]	63.00 [56.50, 70.00]	0.254
	Hepatitis B virus*	0 (0.0)	27 (23.1)	16 (20.5)	<0.001
	Hepatitis C virus*	3 (3.2)	36 (30.8)	15 (19.5)	<0.001
	Chronic alcohol intake*	13 (13.8)	43 (36.8)	37 (48.1)	<0.001
	Advanced fibrosis/cirrhosis (F3-F4)*	25 (26.6)	89 (80.2)	41 (55.4)	<0.001
	Alkaline phosphatase**	1.45 [0.80, 2.75]	1.20 [0.80, 1.60]	1.20 [0.80, 1.78]	0.658
	Total bilirubin, µmol/L**	14.85 [10.65, 20.70]	15.60 [10.00, 23.00]	12.00 [8.00, 17.00]	0.014
	Serum AFP**	0.60 [0.30, 1.80]	36.30 [1.40, 298.60]	5.00 [0.55, 54.50]	<0.001
	Serum CA19-9**	2.45 [0.65, 24.18]	1.19 [0.66, 2.33]	1.75 [1.00, 10.00]	0.027
	Multiple intrahepatic lesions*	67 (90.5)	93 (84.5)	48 (63.2)	<0.001
	Macrovascular invasion*	36 (38.3)	34 (30.6)	24 (30.8)	0.441
	Metastatic disease*	76 (80.9)	43 (36.8)	53 (66.2)	<0.001

Table 4 .

 4 Multivariable analysis of variable associated with PFS and OS in patients with HCC, iCCA, and cHCC-CCA

	Variables	Unadjusted HR/OR*	Unadjusted	Adjusted † HR/OR*	Adjusted †
		(95% CI)	p value	(95% CI)	p value
	PFS
	cHCC-CCA (reference)	Ref		Ref	Ref
	HCC	1.06 (0.77-1.46)	0.719	0.66 (0.37-1.20)	0.172
	iCCA	0.66 (0.47-0.92)	0.014	0.63 (0.41-0.96)	0.034
	Platinum-based (reference)	Ref		Ref	Ref
	TKI	1.39 (1.07-1.80)	0.013	1.54 (0.85-2.79)	0.157
	Intrahepatic multiple nodules	1.48 (1.06-2.05)	0.021	1.63 (1.13-2.35)	0.009
	Advanced fibrosis/cirrhosis	1.37 (1.07-1.76)	0.013	1.01 (0.74-1.38)	0.941
	Metastasis	1.07 (0.84-1.37)	0.580	1.19 (0.86-1.66)	0.293
	Macrovascular invasion	1.06 (0.82-1.37)	0.646	1.28 (0.95-1.73)	0.102
	OS
	cHCC-CCA (reference)	Ref		Ref
	HCC	0.91 (0.68-1.22)	0.521	0.67 (0.37-1.22)	0.189
	iCCA	0.73 (0.53-1.00)	0.050	0.66 (0.43-1.02)	0.064
	Platinum-based (reference)	Ref		Ref
	TKI	1.12 (0.87-1.43)	0.382	1.12 (0.62-1.99)	0.713
	Intrahepatic multiple nodules	1.19 (0.85-1.65)	0.312	1.29 (0.89-1.87)	0.176
	Advanced fibrosis/cirrhosis	1.58 (1.21-2.05)	<0.001	1.45 (1.04-2.01)	0.028
	Metastasis	1.12 (0.86-1.44)	0.405	1.15 (0.89-1.61)	0.401
	Macrovascular invasion	1.31 (0.98-1.76)	0.067	1.42 (1.05-1.92)	0.025
	Objective response rate
	cHCC-CCA (reference)	Ref		Ref
	HCC	2.49 (0.51-12.18)	0.261	1.34 (0.14-13.20)	0.802
	iCCA	4.05 (0.85-19.31)	0.079	7.63 (0.83-69.84)	0.072
	Platinum-based (reference)
	TKI	0.87 (0.35-2.19)	0.767	2.87 (0.16-51.14)	0.473
	Intrahepatic multiple nodules	0.90 (0.28-2.85)	0.858	0.62 (0.18-2.11)	0.442
	Advanced fibrosis/cirrhosis	0.94 (0.37-2.36)	0.888	1.35 (0.46-3.98)	0.584
	Metastasis	1.25 (0.48-3.28)	0.645	0.99 (0.29-3.36)	0.993
	Macrovascular invasion	1.09 (0.41-2.85)	0.868	0.96 (0.34-2.71)	0.934
	DCR
	cHCC-CCA (reference)	Ref		Ref
	HCC	1.20 (0.61-2.38)	0.593	1.43 (0.42-4.90)	0.566
	iCCA	1.74 (0.85-3.58)	0.132	1.79 (0.74-4.33)	0.199
	Platinum-based (reference)	Ref
	TKI	0.76 (0.44-1.30)	0.308	0.78 (0.22-2.80)	0.706
	Intrahepatic multiple nodules	0.68 (0.33-1.38)	0.284	0.62 (0.29-1.33)	0.217
	Advanced fibrosis/cirrhosis	0.58 (0.33-1.01)	0.052	0.63 (0.34-1.18)	0.148
	Metastasis	0.75 (0.43-1.31)	0.308	0.58 (0.29-1.14)	0.114
	Macrovascular invasion	1.26 (0.71-2.24)	0.437	1.01 (0.54-1.88)	0.971

Table 5 .

 5 Doubly robust estimators of the effect of ALBI score on ORR, DCR, PFS, and OS in the 79 patients with hepatocholangiocarcinoma treated either by TKI or by platinum-based regimen

	Liver Cancer 2022;11:460-473
	DOI: 10.1159/000525488

Acknowledgments

We thank the Paris Liver Cancer Group for their support to the project.

Funding Sources

None.

Data Availability Statement

All data generated or analyzed during this study are included in this article and its online supplementary material. Further inquiries can be directed to the corresponding author and will be available after reasonable request.

Statement of Ethics

According to the French laws, this study protocol was reviewed and approved by our Ethical Committee (Comité Local d'Ethique pour la Recherche Clinique des HUPSSD Avicenne-Jean Verdier-René Muret, 125 rue de Stalingrad 93009 BOBIGNY Cedex; approval number CLEA-2020-124). Written consent was not necessary because patients were initially informed of the possibility of using anonymized clinical and biological data collected during routine care in the welcome booklet for different university hospitals and the possibility of opposing the use of their data. Our study was also declared to the National Commission for Informatics and Freedoms (Commission Nationale de l'Informatique et des Libertés, CNIL), number of declaration 2218503 v 0. Our research complies with the guidelines for human studies and was conducted ethically in accordance with the World Medical Association Declaration of Helsinki.

Conflict of Interest Statement

E.G. received travel and congress fees from Bayer, Gilead, and Ipsen; M.B. received consulting fees from Bayer, BMS, Ipsen, Roche, Eisai, Sirtex, Servier, MSD, and AstraZeneca; M.R. received honoraria from Servier, GE Healthcare, Ipsen, Canon-Toshiba, Alexion Pharmaceuticals, Guerbet, and Sirtex; A.Z. received consulting and advisory board fees from Amgen, Lilly, Merck, Roche, Sanofi, Servier, Baxter, MSD, Pierre Fabre, Havas Life, Alira Health, and Zymeworks; D.T. received consulting fees from Amgen, Merck, Novartis, BMS, MSD, AstraZeneca, Sanofi, Roche, Bayer, Servier, and Ipsen; J.E. received consulting fees from MSD, Eisai, BMS, AstraZeneca, Bayer, Roche, Ipsen, Basilea, Merck Serono, Incyte, and Servier and also received travel fees from Amgen and research funding (institutional) from BMS, Beigene; J.-F.B. received consulting fees from Bayer, BMS, Ipsen, Eisai, MSD, Roche, and AstraZeneca; J.-C.N. received research grants from Ipsen and Bayer; and E.T. received research support from Gilead and speaker fees from Abbvie. The other authors have no conflicts of interest to declare.

Author Contributions

Substantial contributions to conception and design: Elia Gigante and Jean-Charles Nault; acquisition of data and/or analysis and interpretation of data: Elia Gigante, Christian Hobeika, Brigitte Le Bail, Valérie Paradis, David Tougeron, Marie Lequoy, Mohamed Bouattour, Jean-Frederic Blanc, Nathalie Ganne-Carrié, Henri Tran, Clémence Hollande, Manon Allaire, Giuliana Amaddeo, Hélène Regnault, Paul Vigneron, Maxime Ronot, Laure Elkrief, Gontran Verset, Eric Trepo, Aziz Zaanan, Marianne Ziol, Massih Ningarhari, Julien Calderaro, Julien Edeline, and Jean-Charles Nault; drafting, revising, and the manuscript content: Elia Gigante, Christian Hobeika, and Jean-Charles Nault; and final approval of the version to be published: all the authors.