Alexandre Pr

Belot

Ommar Omarjee

Cécile Picard

Cécile Frachette

Marion Moreews

Frederic Rieux Laucat

Pauline Sprauel

Sebastien Viel

Jean-Christophe Lega

Brigitte Bader-Meunier

Thierry Walzer

Anne-Laure Mathieu

Rolando Cimaz

Alexandre Belot
email: alexandre.belot@chu-lyon.fr

Monogenic Lupus: dissecting heterogeneity

Abstract:

Systemic lupus erythematosus (SLE) is a severe lifelong multisystem autoimmune disease characterized by the presence of autoantibodies targeting nuclear autoantigens, increased production of type I interferon and B cell abnormalities.

Clinical presentation of SLE is extremely heterogeneous and different groups of disease are likely to exist. Recently, childhood-onset SLE (cSLE) cases have been linked to single gene mutations, defining the concept of monogenic or Mendelian lupus. Genes associated with Mendelian lupus can be grouped in at least three functional categories. First, complement deficiencies represent the main cause of monogenic lupus and its components are involved in the clearance of dying cells, a mechanism also called efferocytosis. Mutations in extracellular DNASE have been also identified in cSLE patients and represent additional causes leading to defective clearance of nucleic acids and apoptotic bodies. Second, the study of Aicardi-Goutières syndromes has introduced the concept of type-I interferonopathies. Bona fide lupus syndromes have been associated to this genetic condition, driven by defective nucleic acids metabolism or innate sensors overactivity. Interferon signaling anomalies can be detected and monitored during therapies, such as Janus-kinase (JAK) inhibitors. Third, tolerance breakdown can occur following genetic mutations in B and/or T cell expressing key immunoregulatory molecules. Biallelic mutations in PRKCD are associated to lupus and lymphoproliferative diseases as PKC-δ displays proapoptotic activity and is crucial to eliminate self-reactive transitional B cells. Here we review the literature of the emerging field of Mendelian lupus and discuss the physiopathological learning from these inborn errors of immunity. In addition, clinical and biological features are highlighted as well as specific therapies that have been tested in these genetic contexts.

Keywords: Monogenic lupus, Type-I interferonopathies, efferocytosis, complement system, PKC-delta deficiency, self-tolerance, apoptosis, systemic lupus erythematosus Systemic lupus erythematosus (SLE) is a rare, multisystem immunological disorder characterized by the presence of autoantibodies targeting nuclear autoantigens, type I interferon production and B cell tolerance breakdown as illustrated by autoantibodies production. SLE represents an heterogeneous disease, with the diagnosis of SLE requiring the presence of 4 out of 11 defined criteria [START_REF] Hochberg | Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus[END_REF][START_REF] Hartman | Performance of the 2012 Systemic Lupus International Collaborating Clinics classification criteria versus the 1997 American College of Rheumatology classification criteria in adult and juvenile systemic lupus erythematosus. A systematic review and meta-analysis[END_REF][START_REF] Yu | Diagnostic criteria for systemic lupus erythematosus: a critical review[END_REF] . The pathogenesis underlying SLE remains unclear. Interestingly, familial aggregation and higher concordance rates between monozygotic (20 -40%) relative to dizygotic twins and other full siblings (2 -5%) suggest a major hereditary component to pathogenesis [START_REF] Criswell | The genetic contribution to systemic lupus erythematosus[END_REF][START_REF] Crow | Lupus: how much "complexity" is really (just) genetic heterogeneity?[END_REF] . Genome-wide association studies (GWAS) have identified more than 80 loci as strongly associated with lupus [START_REF] Bentham | Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus[END_REF][START_REF] Morris | Genome-wide association meta-analysis in Chinese and European individuals identifies ten new loci associated with systemic lupus erythematosus[END_REF][START_REF] Jeong | Genetic variation and systemic lupus erythematosus: A field synopsis and systematic meta-analysis[END_REF] . These variants are common and confer small effects on disease susceptibility [START_REF] Chen | Genetic advances in systemic lupus erythematosus: an update[END_REF][START_REF] Cui | Genetic susceptibility to SLE: recent progress from GWAS[END_REF] . In contrast, monogenic susceptibility to SLE, for which almost 30 genes have been described in humans, are related to single mutations in DNA-coding regions of the genome [START_REF] Manolio | Finding the missing heritability of complex diseases[END_REF] .

Mendelian inborn errors of immunity have helped unravelling the mechanisms of immune tolerance in humans, and three main pathways have been reported associated to lupus. First, defects in the complement components represent the first genetic cause of SLE identified in humans and highlight the importance of the clearance of apoptotic bodies in lupus pathogenesis [START_REF] Botto | Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies[END_REF] . Similarly, extracellular DNAse deficiencies promote SLE secondary to defective clearance of nucleic acids in extracellular microparticles [START_REF] Sunker | Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus[END_REF][START_REF] Sisirak | Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity[END_REF] . In addition, the importance of type I interferon signalling in SLE is emphasized by the association of lupus phenotype with the type I interferonopathies [START_REF] Crow | Aicardi-Goutières syndrome and the type I interferonopathies[END_REF] . Finally, B cells are considered central in lupus pathogenesis [START_REF] Lipsky | Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity[END_REF] , with PKCδ deficiency identified as the first B cell related subset of monogenic lupus in humans [START_REF] Belot | Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation[END_REF][START_REF] Mecklenbräuker | Protein kinase Cdelta controls self-antigen-induced B-cell tolerance[END_REF] .

Here we report the insights arising from the molecular study of monogenic lupus forms, and illustrate their main clinical and immunological features (Table 1).

Tailoring treatment with the underlying genetic molecular defect represent promising strategies.

Efferocytosis defects

Every day, hundreds of billion cells die from apoptosis or specialized programs such as neutrophil cell death, defined as NETosis, a neutrophil cell death program associated to extrusion of fibrillary networks called neutrophil extracellular traps (NETs) [START_REF] Elkon | Review: Cell Death, Nucleic Acids, and Immunity: Inflammation Beyond the Grave[END_REF] . Apoptotic bodies and associated extracellular nucleic acids are scavenged by tissue-resident phagocytes (macrophages and dendritic cells) or locally recruited phagocytes [START_REF] Poon | Apoptotic cell clearance: basic biology and therapeutic potential[END_REF] . This process is important to prevent the induction of inflammation upon recognition of endogenous pathogen recognition receptors (PRR) ligands.

Numerous molecules are involved in the recognition of apoptotic cells and this mechanism is also referred as efferocytosis. In humans, complement deficiencies and the DNASE1L3 deficiency define a subset of monogenic lupus due to efferocytosis defect and excess of apoptotic bodies.

Complement deficiencies

The complement system plays a role in the innate and adaptive immune response.

Complement proteins act as opsonins facilitating the clearance of apoptotic bodies and immune complexes by phagocytes, as well as a lytic agent against pathogens [START_REF] Leffler | The complement system in systemic lupus erythematosus: an update[END_REF] .

The early components of the classical pathway (C1q, C1r and C1s) are mainly produced by monocytes, macrophages and dendritic cells while the other subsets are produced by hepatocytes [START_REF] Chen | The complement system in systemic autoimmune disease[END_REF] . Genetic defects in complement impair efferocytosis leading to the accumulation of autoantigens in the extracellular environment [START_REF] Sullivan | Complement deficiency and autoimmunity[END_REF] .

Moreover, the complement system is needed to clear immune complexes, the accumulation of which leads to inflammation and organ damage as in the case of nephritis. Complement components have additional immune regulatory functions. For example, C1q is capable to reduce type I interferon production by plasmacytoid dendritic cells [START_REF] Lood | C1q inhibits immune complexinduced interferon-alpha production in plasmacytoid dendritic cells: a novel link between C1q deficiency and systemic lupus erythematosus pathogenesis[END_REF] and has been recently shown to regulate adaptive immunity by modulating the mitochondrial metabolism of CD8 T cells, thereby preventing selfantigen autoreactivity and promoting viral control [START_REF] Ling | C1q restrains autoimmunity and viral infection by regulating CD8+ T cell metabolism[END_REF] . The CR1 and CR2 complement receptors that respectively recognize C1q and C4 are also potent regulators of B cell activation [START_REF] Tuveson | Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex[END_REF][START_REF] Matsumoto | Intersection of the complement and immune systems: a signal transduction complex of the B lymphocyte-containing complement receptor type 2 and CD19[END_REF] . Genetic deficiencies of the early component of the classical pathway C1q/r/s strongly predispose to SLE, with a penetrance of up to 90%. Deficiencies or mutations in other complement proteins of the classical pathway, such as C4 and C2, also promote systemic autoimmunity such as lupus with the following risk: 10-20% in C2 deficiency and 75% in C4 deficiency [START_REF] Sturfelt | Complement in the immunopathogenesis of rheumatic disease[END_REF] .

Complement deficiencies are associated with early-onset lupus manifestations, with a sex ratio (ie predominance in females) less biased than that of adult-onset SLE.

The phenotype is severe, in particular because of recurrent life-threatening infections such as meningitis (Fig. 1A). Photosensitive skin rash is very common in all complement deficiencies whereas severe arthritis is mostly seen in patients with C2 deficiency, and lupus nephritis in C4A/C4B-defective monogenic lupus. The prevalence of antinuclear antibodies (ANA) is usually high whereas only 20% of patients display anti-dsDNA. Conversely, anti-Ro/SSA antibodies are positive in 80% of patients with C1q and C4 deficiencies. Low CH50 with normal serum levels of C4 and C3 is evocative for a complement deficiency.

Extracellular DNASE deficiencies

Additional enzymes involved in digestion of extracellular nucleic acids are also critical for the maintenance of immune tolerance. DNASE1 is an extracellular DNAdegrading enzyme, which is present in both serum and urine. Dnase1-deficient mice develop ANA and glomerulonephritis [START_REF] Napirei | Murine serum nucleases -contrasting effects of plasmin and heparin on the activities of DNase1 and DNase1-like 3 (DNase1l3)[END_REF] . Four SLE cases were reported with a mutation in DNASE1 exhibiting very high levels of anti-nucleosomal antibodies [START_REF] Yasutomo | Mutation of DNASE1 in people with systemic lupus erythematosus[END_REF][START_REF] Bodaño | Novel DNASE I mutations related to systemic lupus erythematosus[END_REF] .

DNASE1L3 is a homologue of DNASE1 containing a positively charged C-terminal peptide, which confers the ability to remove DNA chromatin associated to extracellular microparticles; Dnase1L3 deficient mice develop SLE-like phenotype [START_REF] Sisirak | Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity[END_REF] . DNASE1/DNASE1L3 deficiencies lead to the accumulation of extracellular nucleic acids which can be recognized by DNA/RNA sensors such as Toll-like receptors (TLR7 and TLR9) then promoting type I interferon production [START_REF] Roers | Recognition of Endogenous Nucleic Acids by the Innate Immune System[END_REF] . Moreover, circulating DNA can bind autoantibodies leading to immune complex formation with associated organ damage. DNASE1L3 deficiency was identified in familial SLE cases with the association of ANA, anti-dsDNA and ANCA and also as a form of hypocomplementemic urticarial vasculitis [START_REF] Sunker | Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus[END_REF][START_REF] Ozçakar | DNASE1L3 mutations in hypocomplementemic urticarial vasculitis syndrome[END_REF] . Lupus nephritis is frequent and beside low complement fractions C3 and C4 and positivity of ANA and anti-dsDNA antibodies, ANCA positivity is evocative [START_REF] Sunker | Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus[END_REF] .

Other defects in phagocytosis

The X-linked form of chronic granulomatous disease (CGD) is due to CYBB mutations. CYBB encodes the 91-kD glycoprotein component of the ROS-generating enzyme NADPH oxidase or NOX2 (nicotinamide adenine dinucleotide phosphate) and has been connected to lupus phenotype. The link between CYBB and SLE is thought to be secondary to the increase of NETosis, releasing high amounts of intracellular DNA and self-antigens outside the cells. In mice prone to autoimmunity, Nox2 deficiency exacerbates the disease [START_REF] Campbell | NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus[END_REF] . Furthermore, NETs inhibition exacerbated lupus phenotype [START_REF] Kienhöfer | Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps[END_REF] . NOX2-derived reactive oxygen species may regulate T cell activation by oxidizing self-antigen-derived peptides limiting autoimmune arthritis, as recently proposed [START_REF] Yang | Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis[END_REF] . CGD is characterized by recurrent-life threatening infections by bacteria and fungi [START_REF] Baehner | Deficiency of reduced nicotinamide-adenine dinucleotide oxidase in chronic granulomatous disease[END_REF][START_REF] Baehner | Quantitative nitroblue tetrazolium test in chronic granulomatous disease[END_REF] and some patients also display inflammatory manifestations ranging from Crohn's disease to discoid lupus or SLE [START_REF] De Ravin | Chronic granulomatous disease as a risk factor for autoimmune disease[END_REF] .

X-linked carriers are at a higher risk for skin manifestations such as discoid lupus erythematosus and photosensitivity, and generally present with a strong susceptibility to recurrent infections, especially by catalase positive organisms [START_REF] Battersby | Inflammatory and autoimmune manifestations in X-linked carriers of chronic granulomatous disease in the United Kingdom[END_REF][START_REF] Sanford | Abnormal apoptosis in chronic granulomatous disease and autoantibody production characteristic of lupus[END_REF] . Arthritis, glomerulonephritis and Raynaud's phenomenon were rarely described. Autoantibody screening is generally negative, even in patients with a lupus-like phenotype [START_REF] Battersby | Inflammatory and autoimmune manifestations in X-linked carriers of chronic granulomatous disease in the United Kingdom[END_REF][START_REF] Cale | Cutaneous and other lupus-like symptoms in carriers of X-linked chronic granulomatous disease: incidence and autoimmune serology[END_REF] .

Type I Interferonopathies

Interferons (IFN) are cytokines with an essential role in antiviral defence and display key regulatory functions in innate and adaptive immune responses. Type I IFN represent the largest family of IFNs, including IFNα, which comprises 13 distinct subset and IFN-β. Exacerbated IFNα signalling represents a hallmark of systemic lupus erythematosus [START_REF] Picard | Does type-I interferon drive systemic autoimmunity?[END_REF] .

Monogenic type I interferonopathies refer. to a group of diseases associated with an upregulation of type I interferon expression. Jean Aicardi and Françoise Goutières described a familial progressive neonatal encephalopathy mimicking a congenital viral infection, Interestingly, some patients with Aicardi-Goutières syndrome (AGS), also present features of systemic autoimmunity [START_REF] Aicardi | A Progressive familial encephalopathy in infancy with calcifications of the basal ganglia and chronic cerebrospinal fluid lymphocytosis: Aicardi and Goutieres: Basal Ganglia Calcifications[END_REF] . Patients display a wide range of neurological manifestations, from isolated cerebral calcifications to an early-onset encephalopathy, with cerebral atrophy and leukoencephalopathy. High levels of type I interferon were found in the serum and cerebrospinal fluid of these patients [START_REF] Lebon | Intrathecal synthesis of interferon-alpha in infants with progressive familial encephalopathy[END_REF] . Loss of function mutations of the DNA 3ʹ repair exonuclease 1 TREX1 were the first identified genetic cause of AGS [START_REF] Crow | Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus[END_REF] . This enzyme plays a crucial role in the metabolism of intracellular DNA and prevents its detection by innate immune sensors and the type I interferon activation [START_REF] Sakai | DNase-active TREX1 frame-shift mutants induce serologic autoimmunity in mice[END_REF] . Indeed, the detection of intracellular nucleic acids through various innate immune sensors leads to type I interferon production in response to the activation of regulation factors, IRF (IFN regulatory factors) [START_REF] Cale | Cutaneous and other lupus-like symptoms in carriers of X-linked chronic granulomatous disease: incidence and autoimmune serology[END_REF] . Of note, interferon production is triggered by the detection of exogenous nucleic acids from viruses but also by intracellular nucleic acids derived from endogenous retroviral sequences present in our genome (HERV). Considering that type I interferon plays a primary pathogenic role in these disorders, Crow and colleagues proposed to group them as «Type I interferonopathies» [START_REF] Picard | Does type-I interferon drive systemic autoimmunity?[END_REF] . Thereafter, numerous additional genes have been reported as causal for AGS and are linked to IFN signalling upregulation.

The excessive production of interferon can appear through three different pathways:

-Defect in nucleases (TREX1, SAMHD1, ADAR1, RNASEH2, RNASEH2B, RNASEH2C) leading to the accumulation of endogenous nucleic acids -Constitutive activation or enhanced sensitivity of an innate immune sensor, for example MDA5 and RIG-I (IFIH and DDX58 mutations respectively) or adaptor molecules downstream the innate sensors (such as STING, encoded by TMEM173, downstream the receptor cGAS)

-Defective negative feedback of the interferon pathway (ISG15 mutations).

Type I interferons then bind to the heterodimeric receptor (IFNAR 1 and IFNAR 2)

and activate the JAK-STAT signalling pathway and induce the transcription of hundreds of interferon-stimulated genes (ISG) [START_REF] Picard | Does type-I interferon drive systemic autoimmunity?[END_REF][START_REF] Picard | Les interféronopathies de type I. Mise au point et revue de la littérature[END_REF] .

Type I interferonopathies manifestations are very heterogeneous and comprise overlapping phenotypes [START_REF] Rodero | Type I interferon-mediated monogenic autoinflammation: The type I interferonopathies, a conceptual overview[END_REF] . Herein, we review common traits associated with the different forms, as well as gene-specific features (Table 1).

Chilblain lesions as well as severe neurodevelopmental features are the most evocative manifestations of a type I interferonopathy (Fig. 1B). Penetrance and expressivity differ and neurological symptoms without autoimmunity or isolated type I IFN signature can be also observed in affected individuals. The frequency of lupuslike manifestations and chilblain depends on the gene involved (Fig. 1E). The systemic phenotype of the newly described type I interferonopathy linked to biallelic mutations in DNASE2 is relatively broad with clinical features evocative of an autoinflammatory syndrome and various autoimmune manifestations. Transient lifethreatening anemia in the neonatal period was particularly noticeable in those patients [START_REF] Rodero | Type I interferon-mediated autoinflammation due to DNase II deficiency[END_REF] .

Interstitial pneumonia with skin vasculitis is evocative for the STING-associated vasculitis with onset in infancy (SAVI) and interstitial lung disease represents the first symptom of the disease [START_REF] Picard | Severe Pulmonary Fibrosis as the First Manifestation of Interferonopathy (TMEM173 Mutation)[END_REF][START_REF] Liu | Activated STING in a vascular and pulmonary syndrome[END_REF] . Tendon rupture, subluxation for the hands, reminiscent of Jaccoud's arthopathy (Fig. 1C) have been observed in MDA5-related type I interferonopathy [START_REF] De Carvalho | Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy[END_REF] . ANA and dsDNA antibodies have been recorded in type I interferonopathies but are inconsistent.

Type I interferon protein dosage is not available in routine medical practice to date because of very low circulating levels [START_REF] Rodero | Detection of interferon alpha protein reveals differential levels and cellular sources in disease[END_REF] . However, Rice et al. showed that the type I interferon upregulation was correlated with the increased expression of a subset of six interferon stimulated genes (ISG) (IFI27, IFI44L, IFIT1, ISG15, RSAD2 and SIGLEC1) in AGS patients, the so-called interferon signature [START_REF] Rice | Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study[END_REF] . Interferon signature can be used both for the diagnosis of type I interferonopathies and to follow disease activity, and is also very sensitive to differentiate monogenic and complex type I Interferonopathies from inflammasomopathies. Interferon signature might be also useful to dissect the distinct monogenic lupus (Fig. 2A) [START_REF] Pescarmona | Comparison of RT-qPCR and Nanostring in the measurement of blood interferon response for the diagnosis of type I interferonopathies[END_REF][START_REF] Kim | Development of a Validated Interferon Score Using NanoString Technology[END_REF] .

Other subsets of type I Interferonopathies

Proteasome-associated autoinflammatory syndrome (PRAAS) is another autoinflammatory disease consisting in recurrent fever, panniculitis and lipodystrophy and is associated with positive interferon signature. PSMB8 encodes a proteasome subunit, and its mutation leads to proteasome dysfunction, accumulation of ubiquitinylated proteins and autoinflammation [START_REF] Liu | Mutations in proteasome subunit β type 8 cause chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature with evidence of genetic and phenotypic heterogeneity: Mutations in PSMB8 Cause Candle Syndrome[END_REF] . The X-linked reticulate pigmentary disorder (XLRPD) is a genodermatosis with autoinflammatory features linked to the intronic mutation of POLA1, which encodes a catalytic subunit of the DNApolymerase-α.

Mutations in ACP5 encoding Tartrate-resistant acid phosphatase type 5 (TRAP) are transmitted through an autosomal recessive mode and are responsible for spondyloenchondrodysplasia (SPENCD) with skeletal dysplasia, growth retardation, neurological involvement and systemic autoimmunity. The latter condition represents an increased susceptibility for Sjögren's syndrome, systemic lupus erythematosus, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's syndrome and vitiligo [START_REF] Briggs | Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature[END_REF] . TRAP is expressed in osteoclasts, macrophages and dendritic cells, thus explaining both skeletal and autoimmune involvements in SPENCD patients.

B and T cell tolerance breakdown

The hallmark of SLE is the presence of a wide range of autoantibodies indicating the presence of autoreactive lymphocytes. Ineffective deletion of autoreactive T or B cells could trigger autoimmunity and disrupt self-tolerance [START_REF] Peng | Altered T and B lymphocyte signaling pathways in lupus[END_REF] . The importance of apoptosis defects in human autoimmunity was underlined by the discovery of mutations in the pro-apoptotic molecule CD95/Fas defining the Autoimmune Lymphoproliferative Syndrome (ALPS). ALPS is an inherited disease characterized by a chronic non-malignant lymphadenopathy, hepatosplenomegaly, expansion of CD3 + TCRα/β + CD4 -CD8 -double negative T (DNT) cells and other autoimmune features such as presence of autoantibodies and autoimmune cytopenia [START_REF] Liu | Activated STING in a vascular and pulmonary syndrome[END_REF][START_REF] De Carvalho | Musculoskeletal Disease in MDA5-Related Type I Interferonopathy: A Mendelian Mimic of Jaccoud's Arthropathy[END_REF] . Of note, no full-blown lupus has been described so far with this specific genetic background.

A few other patients have been described with homozygous CD95L/FASL mutations that abolish the protein function [START_REF] Rodero | Detection of interferon alpha protein reveals differential levels and cellular sources in disease[END_REF][START_REF] Rice | Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study[END_REF] or heterozygous CD95L/FASL mutations with a dominant negative effect [START_REF] Pescarmona | Comparison of RT-qPCR and Nanostring in the measurement of blood interferon response for the diagnosis of type I interferonopathies[END_REF] . In addition, the MRL/lpr mice represent a model of systemic autoimmunity with a mutation in the Fas receptor gene (lpr) [START_REF] Kim | Development of a Validated Interferon Score Using NanoString Technology[END_REF] as well as the CD95L (gld) deficiency [START_REF] Liu | Mutations in proteasome subunit β type 8 cause chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature with evidence of genetic and phenotypic heterogeneity: Mutations in PSMB8 Cause Candle Syndrome[END_REF] .

Another example of monogenic SLE is the protein kinase C-δ (PKC-δ) deficiency.

PKC-δ is a pro-apopotic molecule essential in B cell survival and apoptosis, and PKC-δ knock-out mice develop systemic autoimmunity [START_REF] Sisirak | Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity[END_REF][START_REF] Briggs | Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature[END_REF][START_REF] Peng | Altered T and B lymphocyte signaling pathways in lupus[END_REF] . We identified an homozygous missense mutation in PRKCD in three siblings with childhood-onset SLE [START_REF] Belot | Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation[END_REF] . PKC-δ expression was reduced, which led to a B cell apoptosis defect and an accumulation of immature transitional B cells. Additional cases of PKC-δ deficiency have been reported, the main clinical features comprising skin lesions and alopecia, lymphoproliferative disease with lymphadenopathy, hepatosplenomegaly and mild to severe infection susceptibility, both to bacteria and viruses [START_REF] Sunker | Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus[END_REF][START_REF] Rieux-Laucat | Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity[END_REF][START_REF] Straus | An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome[END_REF][START_REF] Magerus-Chatinet | Autoimmune lymphoproliferative syndrome caused by a homozygous null FAS ligand (FASLG) mutation[END_REF][START_REF] Del-Rey | A homozygous Fas ligand gene mutation in a patient causes a new type of autoimmune lymphoproliferative syndrome[END_REF] . Chronic Epstein Barr virus or cytomegalovirus replication have been reported [START_REF] Rieux-Laucat | Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity[END_REF][START_REF] Straus | An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome[END_REF] . One of our patients presented at the age of 30 with severe papillomatosis illustrating the defective immune control of a wide range of viruses (Fig. 1D). Lupus nephritis is frequent and cerebral vasculitis has also been reported. Immunophenotyping in these patients reveals a B-cell deficiency with an expansion of transitional B cells and a decrease of memory B cells [START_REF] Belot | Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation[END_REF] . ANA, ENA and dsDNA antibodies are generally positive, with normal complement levels.

Another subset of disease with apoptosis dysregulation is illustrated by the RASopathies. Noonan syndrome and Noonan-related syndromes represent neurodevelopmental syndromes due to germline mutations in genes that participate in the rat sarcoma/mitogen-activated protein kinases (RAS/MAPK) pathway (PTPN11, SOS1, RAF, KRAS or NRAS, and SHOC2). Lupus phenotype has been reported in such patients and autoimmune features were not different from those presented by the general SLE population [START_REF] Wu | Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease[END_REF] , with polyarthritis, autoimmune cytopenia, serositis, ANA and anti dsDNA antibodies.

IKAROS mutations were also identified in 4 patients with autoimmunity including one fulfilling early-onset SLE criteria [START_REF] Watson | Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal diseasemodifying loci[END_REF] . IKAROS mutations were reported as causal for common variable immunodeficiencies [START_REF] Roths | A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice[END_REF] , with decreased lymphocyte populations (B, T and NK cells). IKZF1 encodes the IKAROS zinc finger transcription factor which is crucial in early steps of haematopoietic development, as demonstrated by a knockout model [START_REF] Limnander | STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development[END_REF][START_REF] Miyamoto | Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cdelta[END_REF] . Finally, recombination-activating 1 or 2 genes (RAG1/2) represents a crucial enzyme involved in the V(D)J recombination and BCR to TCR variability generation. While loss-of-function RAG mutations are associated with severe combined immunodeficiencies, hypomorphic mutations have been linked to autoimmunity [START_REF] Kiykim | Beneficial Effect of Hydroxychloroquine in a Patient with a Novel Mutation in Protein Kinase Cδ Deficiency[END_REF][START_REF] Nanthapisal | Early-Onset Juvenile SLE Associated With a Novel Mutation in Protein Kinase C δ[END_REF][START_REF] Salzer | B-cell deficiency and severe autoimmunity caused by deficiency of protein kinase C δ[END_REF] . Interestingly a RAG2 mutation has been reported in a lupus patient [START_REF] Walter | Impaired receptor editing and heterozygous RAG2 mutation in a patient with systemic lupus erythematosus and erosive arthritis[END_REF] . The patient carrying the heterozygous RAG2 mutation presented with classical lupus-like systemic phenotype with arthritis and skin involvement, Raynaud's phenomenon, sicca syndrome, serositis and class V lupus nephritis [START_REF] Kiykim | Beneficial Effect of Hydroxychloroquine in a Patient with a Novel Mutation in Protein Kinase Cδ Deficiency[END_REF] .

She had a history of recurrent infections attributed to her medication regimen. She presented with classical lupus-associated biomarkers with high titres of ANA and dsDNA antibodies and hypocomplementemia.

Biological abnomalities from different monogenic lupus syndromes are reported in Fig. 2B.

Towards precision medicine in SLE

SLE heterogeneity explains the so far limited success of clinical trials in lupus.

With advent of NGS, our understanding of genetic causes of early-onset SLE is improving at a fast rate. This knowledge will not only allow a better stratification of patients, but also a rationale, personalized design of treatments. Moreover, the identification of causal genes may increase the number of targets for drug treatment of these diseases.

Considering the central role of type I interferon in the pathogenesis, Mendelian interferonopathies might benefit from type I interferon inhibition. JAK inhibitors are promising because they block the signalling downstream the IFN receptor. Baricitinib and ruxolitinib are both JAK1/2 inhibitors and have shown interesting preliminary results for their safety and efficacy in SAVI and CANDLE [START_REF] Sanchez | JAK1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies[END_REF][START_REF] Frémond | Efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in the treatment of vasculopathy associated with TMEM173activating mutations in 3 children[END_REF] , although the IFN score was not totally abrogated in SAVI patients. Anifrolumab is a fully human monoclonal antibody that neutralises the IFNα receptor and has been reported as effective in non-monogenic lupus, especially in the context of high IFN signatures. It is a good candidate for treatment of type I interferonopathies. The main side effect is represented by herpes zoster infections [START_REF] Furie | Anifrolumab, an Anti-Interferon-α Receptor Monoclonal Antibody, in Moderate-to-Severe Systemic Lupus Erythematosus: ANIFROLUMAB IN MODERATE-TO-SEVERE SLE[END_REF] . Finally, as the HERV are supposed to be partially responsible for the increase of intracytoplasmic nucleic acids in AGS with enzymatic defects, it has been recently suggested that reverse transcriptase inhibitors used for patients with human immunodeficiency virus (HIV) may represent an option by targeting the endogenous retroviruses and thereby decreasing the IFN signature [START_REF] Rice | Reverse-Transcriptase Inhibitors in the Aicardi-Goutières Syndrome[END_REF] .

B cell targeting therapies or bone marrow transplantation may represent interesting options in B cell related monogenic lupus. Rituximab and ofatumumab have been reported as effective in siblings with PKC-δ deficiency [START_REF] Lei | Successful use of ofatumumab in two cases of early-onset juvenile SLE with thrombocytopenia caused by a mutation in protein kinase C δ[END_REF] .

Efferocytosis defect is actually the most challenging situation. No drug can promote efferocytosis and bone marrow transplantation has been proposed in severe C1q deficiencies [START_REF] Arkwright | Successful cure of C1q deficiency in human subjects treated with hematopoietic stem cell transplantation[END_REF] .

In conclusion, monogenic lupus is a recently recognized field of diseases.

Understanding better the underlying genetic causes will bring insight in the mechanisms of human immune tolerance and lupus pathogenesis, and likely lead to the discovery of novel therapeutic options to cure the disease.

Figure 1 :

 1 Abbreviations: AD: Autosomal Dominant AGS: Aicardi-Goutieres Syndrome ANA: Anti-Nuclear Antibodies AR: Autosomal Recessive BSN: Bilateral strial necrosis CNS: central nervous system CVD: Cerebrovascular disease DSH: Dyschromatosis symmetrica hereditaria FCL: Familial chilblain lupus SMS: Singleton Merten Syndrome SPENCD: Spondyloenchondrodysplasia SP: Spastic paraparesis SAVI: STING-associated vasculopathy with onset in infancy

Figure 2 :

 2 Figure 2: Biological features of monogenic lupus A: Type I Interferon signature in patients with monongenic lupus, B: Synthesis of biological features of monogenic lupus.

Table 1 Pathways

 1

	Gene/Protein	Function	Inheritance	SLE manifestation

Table 1 : Monogenic causes of SLE

 1

Acknowledgements: patients and families, Pr Denis Jullien for providing some pictures


Maud Toulmonde

MD Marina Pulido

MSc Isabelle Ray-Coquard

Thierry André

MD, Full Professor; Nicolas Isambert

MD Christine Chevreau

MD Nicolas Penel

MD, Full Professor; Emmanuelle Bompas

Esma Saâda

MD François Bertucci

MD, Full Professor; Thierry Andre

MD Esma Saada

MD, Full Professor; Celeste Lebbe

MD, Full Professor; Axel Le Cesne

MD Patrick Soulie

MD Sophie Piperno-Neumann

MD Stephen Sweet

PhD Fabiola Cecchi

PhD; Todd Hembrough

PhD Carine Bellera

MD Amandine Crombe

MD Carlo Lucchesi

PhD François Le Loarer

MD Jean-Yves Blay

MD, Full Professor; Antoine Italiano
email: a.italiano@bordeaux.unicancer.fr

Antoine Pr

MD Italiano

Pazopanib or methotrexate-vinblastine combination chemotherapy in adult patients with progressive desmoid tumours (DESMOPAZ): a non-comparative, randomised, open-label, multicentre, phase 2 study

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

the efficacy and safety of pazopanib 800 mg/day orally continuously, or a regimen combining vinblastine (5 mg/m 2 /dose) and methotrexate (30 mg/m 2 /dose), both administered by intravenous injection weekly for 26 weeks and every other week for 26 weeks. A two-stage Simon's design was used. Randomization was stratified according to inclusion center and tumor location (limbs and girdles versus other). A minimization randomization method has been used to avoid significant imbalance between the arms. The primary endpoint was the 6-month non-progression rate (NPR) in the eligible and assessable population, defined as all patients who had received one complete or two incomplete cycles of treatment. Safety analyses were performed in all patients who received at least one dose of allocated treatment. Clinical Trial Registration: NCT01876082 Findings: From December 2012 to August 2017, 72 patients were recruited (pazopanib arm: 48; MV arm: 24). The median follow up was 23.4 months [95% CI: 20.5-24.2]. Forty-six in the pazopanib arm and 20 in the MV arm were assessable for efficacy. In the first 43 patients assessable for the primary endpoint in the pazopanib arm, the 6-month NPR was 83.7% [95% CI: 69. 3-93.2]. It was 45% [95% CI: 23.1-68.5] in the MV arm. The most common grade 3 or 4 adverse events in the pazopanib arm were hypertension (n=10, 21%) and diarrhea (n=7, 15%) in the pazopanib and neutropenia (n=10, 45.5%) and liver transaminitis (n=4, 18.5%) in the MV arm, respectively. Eleven patients (22.9%) and 6 patients (27.3%) had at least one serious adverse event related to study treatment in the pazopanib and MV arms respectively.

Interpretation: Pazopanib has clinical activity in patients with progressive DT and can be considered a valid treatment option in this rare and disabling disease.

This study was sponsored by Institut Bergonié and funding support was provided by GlaxoSmithKline and Novartis (Novartis acquired Pazopanib from GlaxoSmithKline in 2016).

RESEARCH IN CONTEXT

Evidence before this study

We searched PubMed with the terms "desmoid tumor" OR "fibromatosis" AND "clinical trial" NOT "review" for clinical trials done in humans published in English up to Dec 31, 2018. We identified 11 citations that reported results from clinical trials conducted according to the ethical guidelines and principles of the international Declaration of Helsinki.

Of these 11 citations, two included outcomes for methotrexate-vinblastine chemotherapy regimen, five included outcomes after treatment with a tyrosine kinase inhibitor, and one reported results from radiation therapy. The remaining three citations reported data from hormonal therapy or miscellaneous agents. Only one of these studies was randomized. None of them included only patients with confirmed progressive disease at inclusion according to RECIST.

Added value of this study

A hallmark of desmoid tumors is their unpredictable natural history. Many of them tend to regress spontaneously. However, a small subset of patients has desmoid tumors characterized by an aggressive outcome leading to severe pain, functional impairment and, more rarely, a lifethreatening condition. Current guidelines recommend chemotherapy for aggressively growing, symptomatic or even life-threatening desmoid tumors. Our results show that pazopanib, a multityrosine kinase inhibitor already approved for the management of advanced soft-tissue sarcomas, induces RECIST partial response in 17 (37%) of 46 patients with progressive desmoid tumor, resulting in a proportion of patients with 6-months non-progression of 83.7%. This proportion was 45% for patients treated with combination chemotherapy.

Implications of all the available evidence

The DESMOPAZ trial is the first randomized trial assessing systemic therapy in truly progressive desmoid tumor. Patients included in this study had desmoid tumor characterized by an aggressive behavior: Three quarters of patients had already received systemic treatment and all had progressive disease according to RECIST within an interval of less than 6 months before inclusion in the study. Our results confirm that multi-tyrosine kinase inhibitors, notably those targeting angiogenesis such as pazopanib compare favorably with chemotherapy in terms of safety and effectiveness in patients with desmoid tumors and should be considered as a valid therapeutic option.

INTRODUCTION

Desmoid tumors (DT) are rare, locally aggressive tumors affecting individuals mostly between the ages of 15 and 60 years, with an unpredictable natural history. Indeed, despite their infiltrative growth pattern and high propensity for local recurrence, some of these tumors may stop growing or even regress without any intervention. Although surgery has been the standard of treatment for decades, recent studies have suggested the benefit of front-line watchful waiting after diagnosis to avoid unnecessary mutilating treatment. [START_REF] Kasper | An update on the management of sporadic desmoid-type fibromatosis: a European consensus initiative between sarcoma patients EuroNet (SPAEN) and European organization for research and treatment of cancer (EORTC)/soft tissue and bone sarcoma group (STBSG)[END_REF][START_REF] Penel | Surgical versus non-surgical approach in primary desmoid-type fibromatosis patients: a nationwide prospective cohort from the French sarcoma group[END_REF] However, about one-third of patients with DT will have progressive and/or highly symptomatic disease and need therapeutic intervention. Several pharmacological treatments, such as hormonal therapy (e.g., tamoxifen), nonsteroidal anti-inflammatory drugs (NSAIDs), tyrosine kinase inhibitors (TKI), such as imatinib [START_REF] Mace | Response of extraabdominal desmoid tumors to therapy with imatinib mesylate[END_REF][START_REF] Penel | Imatinib for progressive and recurrent aggressive fibromatosis (desmoid tumors): an FNCLCC/French sarcoma group phase II trial with a long-term follow-up[END_REF][START_REF] Chugh | Efficacy of imatinib in aggressive fibromatosis: results of a phase II multicenter Sarcoma alliance for research through collaboration (SARC) trial[END_REF] or sorafenib, [START_REF] Gounder | Activity of sorafenib against desmoid tumor/deep fibromatosis[END_REF] and cytotoxic chemotherapy [START_REF] Azzarelli | Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis[END_REF][START_REF] Constantinidou | Pegylated liposomal doxorubicin, an effective, well-tolerated treatment for refractory aggressive fibromatosis[END_REF][START_REF] Gega | Successful chemotherapeutic modality of doxorubicin plus dacarbazine for the treatment of desmoid tumors in association with familial adenomatous polyposis[END_REF][START_REF] Meazza | Aggressive fibromatosis in children and adolescents: the Italian experience[END_REF][START_REF] Patel | Combination chemotherapy in adult desmoid tumors[END_REF][START_REF] Skapek | Combination chemotherapy using vinblastine and methotrexate for the treatment of progressive desmoid tumor in children[END_REF][START_REF] Weiss | Therapy of desmoid tumors and fibromatosis using vinorelbine[END_REF][START_REF] Reich | Low-dose chemotherapy with vinblastine and methotrexate in childhood desmoid tumors[END_REF][START_REF] Skapek | Vinblastine and methotrexate for desmoid fibromatosis in children: results of a pediatric oncology group phase II Trial[END_REF][START_REF] Garbay | Chemotherapy in patients with desmoid tumors: a study from the French sarcoma group (FSG)[END_REF] have been associated with clinical benefit in patients with progressive and/or recurrent DT. However, evidence concerning the role of these systemic approaches is scarce and mainly based on small single-center case series. The combination of intravenous (IV) methotrexate and vinblastine is the only conventional systemic regimen assessed in a clinical trial setting, with encouraging efficacy and acceptable safety profile, notably in the pediatric and AYA (adolescent and Young Adults) population where it is widely used. [START_REF] Azzarelli | Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis[END_REF][START_REF] Skapek | Vinblastine and methotrexate for desmoid fibromatosis in children: results of a pediatric oncology group phase II Trial[END_REF] Objective response and disease stabilization proportions were 40% and 60% in the first, and 19% and 50% in the second trial, respectively. However, the weekly IV regimen required multiple outpatient visits, possibly impeding the patients' daily activities, and was associated with myelotoxicity.

VEGF overexpression has been identified as a common feature in DT, especially in recurrent aggressive cases. Retrospective data have shown promising results in 26 patients treated with TKI sorafenib. [START_REF] Gounder | Activity of sorafenib against desmoid tumor/deep fibromatosis[END_REF] Pazopanib is an oral TKI targeting VEGFR1, 2, and 3, PDGFRα and β and c-KIT tyrosine kinases that is already approved for the management of soft-tissue sarcomas. [START_REF] Van Der Graaf | Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial[END_REF] DESMOPAZ is a non-comparative, randomized,multicenter phase 2 trial designed to assess the efficacy and safety of pazopanib or methotrexate-vinblastine in patients with progressive DT.

METHODS

Study design and participants

The DESMOPAZ phase 2 trial involved 13 centers from the French Sarcoma Group.

Patients were eligible if they were at least 18 years of age and had histologically confirmed DT after central review, ECOG Performance status 0-1, adequate renal, hepatic and cardiac functions (see Study synopsis, appendix page 4), and any type and number of previous treatment.

Blood tests included assessment of blood cell count, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), albumin, bilirubin, creatinine, urea nitrogen. A washout period of 14 days for previous treatment was mandatory (see Study synopsis, appendix page 7). Key exclusion criteria included previous treatment with pazopanib or MV (see Study synopsis, appendix page 6-8). All patients had centrally documented progressive disease according to RECIST 1.1 based on two imaging assessments obtained within less than a 6-month interval. Archived FFPE samples of tumor tissue were mandatorily collected at baseline, and an on-treatment tumor biopsy at Cycle 2 was optional. As required by the French regulation, the protocol was centrally approved by a central IRB (the Comité de Protection des Personnes Sud-Ouest et Outre Mer III,Bordeaux, France) which reviewed the appropriateness of the clinical trial protocol as well as the risks and benefits to study participants. All patients provided written informed consent.

Randomization and masking

Eligible patients were randomly assigned using a 2:1 ratio to receive either pazopanib or a regimen combining methotrexate-vinblastine, respectively. Upon investigator's request and after confirmation of all eligibility criteria, eligible patients were randomly assigned between the two arms of treatment by a web-based randomisation system (TenAléa software, https://prod.tenalea.net/gso/dm/) centralized at the sponsor site. Once the randomization was completed, the investigator received an automatic confirmation by email with the arm of treatment allocated. Randomization was stratified according to inclusion center and tumor location (limbs and girdles versus other). A minimization randomization method has been used to avoid significant imbalance between the arms. Patients and investigators were not masked to treatment allocation.

Study procedures

After inclusion and screening, patients received pazopanib 800 mg/day orally continuously, or methotrexate (30 mg/m²) + vinblastine (5 mg/m²), IV, once a week for 6 months and then every 2 weeks for 6 months. Treatment was continued until progression, unacceptable toxicity, investigator's decision, patient consent withdrawal, or for a maximum of 1 year. Crossover was permitted after central confirmation of progression. Safety was monitored by assessing all adverse events continuously through the study, graded according to NCI-CTCAE v.4.0.

Laboratory assessments were performed at baseline week 2, week 4 and every four weeks afterwards. Pazopanib and methotrexate-vinblastine dose adjustments in case of adverse events were planned in the protocol guidelines Tumor lesions were assessed according to RECIST v1.1 at baseline within 14 days before the first dose of pazopanib or methotrexate-vinblastine, and every 12 weeks until disease progression or the start of another treatment. MRI was used for head and neck, limbs or trunk wall lesions whereas CT-scan was used for internal trunk lesions.

All responses had to be confirmed by repeating imaging at a minimum of 4 weeks from the first observation. Primary endpoint assessment was based on centrally blinded reviewed radiological data. Quality of life and pain modification were assessed at baseline, every 4 weeks during the first 3 months and every 12 weeks thereafter, at progression and the study end.

Outcomes

The primary endpoint was the 6-month nonprogression rate (NPR) defined as the percentage of patients remaining alive and progression-free at 6 months as per RECIST 1.1 after the day of randomization.

Secondary endpoints included safety by CTCAE v4.0, best overall response (BOR), defined as the best response obtained from the start of treatment to the time of progression between complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) as per RECIST 1.1; 1-year and 2-year progression-free survival (PFS); overall survival (OS); health-related quality of life (HRQoL) and pain intensity assessed at each cycle with EORTC QLQ-C30 questionnaires and Brief Pain Inventory (BPI) forms, respectively. [START_REF] Cleeland | Pain assessment: global use of the brief pain inventory[END_REF][START_REF] Aaronson | The European organization for research and treatment of cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology[END_REF] PFS was defined as the time from the start of treatment to the time of progression or death from any cause, whichever occurs first. Patients alive and progression free were censored at the date of the last follow-up. OS was defined as the time from the start of treatment to the time of death from any cause or last patient contact. For HRQoL assessment, a minimal change of 10 points was considered clinically meaningful. [START_REF] Osoba | Interpreting the significance of changes in health-related quality-of-life scores[END_REF] Pharmacokinetics of pazopanib were planned and results will be reported at a later date.

Statistical Analysis

A two-stage Simon's design [START_REF] Simon | Optimal two-stage designs for phase II clinical trials[END_REF] was used. Considering the following hypothesis of a favorable true 6-month nonprogression rate of H1=80%, a null rate of H0=60%, a type I error rate α=5%, a β=20% and a 2:1 randomization, 43 assessable patients were needed in the pazopanib arm and 22 patients in the methotrexate-vinblastine arm. Following the inclusion of the first 11 assessable patients, if ≤ 7 patients were progression free at 6 months, the study would be terminated early.

Otherwise, a second group of 32 subjects would be recruited. If at the end of recruitment, ≥ 31 among the 43 first assessable patients were progression free at 6 months, pazopanib would be considered to have significant anti-tumor activity in DT. Each arm was analyzed independently.

No formal statistical comparison was performed between arms. All enrolled patients who received at least one dose of pazopanib or methotrexate-vinblastine were eligible for safety analyses and constituted the safety population. The efficacy population included all subjects who met eligibility criteria and had received at least one complete cycle or two incomplete cycles of treatment. The primary endpoint was assessed on the 43 first assessable patients of the pazopanib arm. Secondary endpoints were assessed on the patients from the efficacy population. The median follow-up was calculated using the reverse Kaplan-Meier method. Endpoints were reported with their 95% confidence interval, as well as the median survival rates for OS and PFS.

Survival endpoints (OS and PFS) were analysed using the Kaplan-Meier method. Quantitative variables were described using the median and range, and qualitative variables were described using frequency and percentage. Exploratory post-hoc proteomic analyses of pretreatment tumor samples were performed in line with previous work from Kim et al. [START_REF] Kim | Data-independent acquisition mass spectrometry to quantify protein levels in FFPE tumor biopsies for molecular diagnostics[END_REF] (Webappendix pages 1) with the aim to identify a proteomic signature predictive of response to pazopanib. Statistical analyses were performed using SAS version 9.2 software (SAS Institute, North Carolina, USA).

This study was registered with ClinicalTrials.gov number NCT01876082.

Role of the funding source

The study was sponsored by Institut Bergonié (Bordeaux, France). The data were collected using the sponsor data-management system and were analyzed and interpreted by representatives of the sponsor in collaboration with the investigators. MT, MP, CB and AI had access to the raw data.

All the authors contributed to and reviewed the data reported, verified that the study was conducted in fidelity to the study protocol, edited and approved the final manuscript submitted for publication, and vouched for the completeness of the data set and integrity of the analysis.

The funding sources (GlaxoSmithKline and Novartis) played no role in the design of this study and did not play any role during its execution, analysis, interpretation of the data, or decision to submit results. The corresponding author had full access to all of the data and the final responsibility to submit for publication.

RESULTS

From 4 December 2012 to 18 August 2017, 72 patients were randomized. Two patients were excluded -one patient who withdrew consent and one who had previously received pazopaniband 70 started treatment (Fig. 1). The median follow up was 23.4 months [95% CI: 20.5-24.2].

Characteristics of patients included in the study are described in Table 1. The median age was 40 years, and two-thirds were women. In the pazopanib arm, half of the tumors were located in the limbs or girdles, and one-third originated from the trunk wall, whereas half of the patients from the methotrexate-vinblastine arm had internal trunk/mesenteric disease. Three-quarters of patients had already received systemic treatment, with a median number of one previous line.

Among the 70 treated patients, 25 (52%) of the 48 patients from the pazopanib arm and five (23%) of the 22 patients from the methotrexate vinblastine arm completed the planned 1year treatment schedule. The reasons for stopping study treatment before the planned completion were as follows: disease progression for six (12%) and six (27%) patients, unacceptable adverse event for four (8%) and five (23%) patients, and other reasons for four (8%) and five (23%) patients, in the 48 patients of the pazopanib arm and the 22 patients of the methotrexatevinblastine arm, respectively (Webappendix page 2). Eight patients crossed over after progression: two patients from the pazopanib to methotrexate-vinblastine arm and six from the methotrexate-vinblastine to pazopanib arm. Four patients were not eligible for efficacy assessment; therefore 66 patients were included in the efficacy analysis: 46 in the pazopanib arm of whom the 43 first patients were included in the primary endpoint analyses, and 20 patients in the methotrexate-vinblastine arm, of whom one patient had no radiological assessment available for response., Thirty-six of the 43 first patients eligible and assessable for efficacy in the pazopanib arm were free of progression after central review at 6 months, leading to a proportion of patients with 6-month nonprogression In the pazopanib arm, most patients had a decrease in tumor size (Fig. 2A). Seventeen (37.0% [95% CI: 23.2-52.5]) of 46 patients in the pazopanib arm had a PR as BOR according to RECIST v1.1, 27 (58.7% [95% CI: 43.2-73.0]) had SD, whereas two (4.4% [95% CI: 0.1-14.8]) had PD (one 62-year-old man with a Gardner syndrome and one 37-year-old man with a wildtype-CTTNB1 mesenteric tumor (Fig. 2A)).

Eleven (58%) of 20 patients in the methotrexate-vinblastine arm exhibited a detectable decrease in tumor size (Fig. 2B). Five patients (25% [95% CI: 8.7-49.1]) had a PR as BOR according to RECIST v1.1, whereas ten patients had SD (50% [95% CI: 27.2-72.8]) and four had PD (20% [95% CI: 5.7-43.7]) (Fig. 2B).

The median PFS was not reached for both arms. The one-year PFS and two-year PFS were 85.6% [95% CI: 70.7-93.2] and 67.2% [95% CI: 49-81.9] in the pazopanib arm, and both 79% [95% CI: 53.2-91.5] in the methotrexate-vinblastine arm, respectively (Fig. 2C andD). One patient died in the pazopanib arm, 9 months after treatment completion, from a sepsis not related to study drug.

Considering HRQoL evaluation, the global health status between baseline and cycle 6 was considered stable in the pazopanib arm, and there was also a trend toward improvement in emotional functioning, associated with a meaningful decrease in pain intensity. In the methotrexate-vinblastine arm, the global health status between baseline and cycle 6 decreased from more than 10 points and the patients with available data at cycle 6 reported a meaningful decrease in cognitive functioning without modification in pain intensity (Tables 2 and3).

Seventy patients were evaluated for safety. Treatment-related adverse events that were reported in either study group in more than 5% of patients for grade 1-2 and any for grade 3 and 4 are shown in Table 4. The most common adverse events in the pazopanib arm were fatigue and diarrhea. Twenty-seven (56%) of the 48 patients in the pazopanib arm and 17 (77%) of the 22 patients in the methotrexate-vinblastine arm had at least one grade 3 or 4 adverse event, respectively. Eleven patients (22.9%) and 6 patients (27.3%) had at least one serious adverse event related to study treatment in the pazopanib and MV arms respectively. (supplementary table 2).

Adverse events led to dose modification or definitive treatment discontinuation in 35 (73%) and 3 (8%) of 48 patients in the pazopanib arm and in 17(77%) and 4 (18%) of 22 patients in the methotrexate-vinblastine arm respectively (Supplementary Table 3) In the Pazopanib arm, these were mainly grade 2-3 diarrhea, grade 2 fatigue, and grade 2-3 hypertension (HT). In the methotrexate-vinblastine arm, these were grade 3 hepatobiliary disorders, grade 4 neutrophil count decrease and grade 3 musculoskeletal disorders. Four patients in each arm definitively stopped treatment for a toxicity related to study drug. These were one grade 3 HT, one grade 3 thromboembolic event, one grade 2 uterine hemorrhage and one grade 2 bilirubin increase in the pazopanib arm, and one grade 2 infusion site extravasation, one grade 3 hepatobiliary disorder, one grade 2 and one grade 3 paresthesia in the methotrexate-vinblastine arm, respectively. Overall, 28 patients had tumor material available for proteomics analyses, 21 in the pazopanib arm and seven in the methotrexate-vinblastine arm. Patients from the pazopanib arm were classified into 3 groups according to tumor shrinkage RECIST scoring (Webppendix pages 1-2). Hierarchical clustering identified a set of peptides with differential expression significantly associated with an objective response to pazopanib (RECIST-response >=-0.3) compared with a nonobjective response. Differentially expressed proteins in patients with an objective response were involved in angiogenesis regulation and various processes such as cell-to-cell and cell-tomatrix interactions, cellular proliferation, migration, adhesion and attachment, vascular inflammation, including notably Thrombospondin-4 (THBS4) and Platelet-derived growth factor receptor-like protein (PDGFR-L) (Fig. 3).

DISCUSSION

To our knowledge, the DESMOPAZ trial is the first non-comparative randomized trial in truly progressive DT. Patients included in this study had DT characterized by an aggressive behavior: Three quarters of patients had already received systemic treatment and all had progressive disease according to RECIST within an interval of less than 6 months before inclusion in the study. This trial is positive, with 83.7% of patients achieving 6-month non-progression with pazopanib.

Due to the lack of a randomized trial in DT, physician choice of a systemic treatment for patients with DT is often driven by empirical experience. A couple of prospective studies and several retrospective studies have assessed chemotherapy for symptomatic patients. [START_REF] Azzarelli | Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis[END_REF][START_REF] Constantinidou | Pegylated liposomal doxorubicin, an effective, well-tolerated treatment for refractory aggressive fibromatosis[END_REF][START_REF] Gega | Successful chemotherapeutic modality of doxorubicin plus dacarbazine for the treatment of desmoid tumors in association with familial adenomatous polyposis[END_REF][START_REF] Meazza | Aggressive fibromatosis in children and adolescents: the Italian experience[END_REF][START_REF] Patel | Combination chemotherapy in adult desmoid tumors[END_REF][START_REF] Skapek | Combination chemotherapy using vinblastine and methotrexate for the treatment of progressive desmoid tumor in children[END_REF][START_REF] Weiss | Therapy of desmoid tumors and fibromatosis using vinorelbine[END_REF][START_REF] Reich | Low-dose chemotherapy with vinblastine and methotrexate in childhood desmoid tumors[END_REF][START_REF] Skapek | Vinblastine and methotrexate for desmoid fibromatosis in children: results of a pediatric oncology group phase II Trial[END_REF][START_REF] Garbay | Chemotherapy in patients with desmoid tumors: a study from the French sarcoma group (FSG)[END_REF] Because of concerns using cytotoxic drugs with potential late toxic effects such as liposomal doxorubicin in young patients, alternative nonchemotherapeutic options have recently been explored in DT. The first TKI explored in patients with DT was Imatinib, which showed only limited clinical activity, with tumor shrinkage in less than 10% of patients as reported in two clinical trials. [START_REF] Penel | Imatinib for progressive and recurrent aggressive fibromatosis (desmoid tumors): an FNCLCC/French sarcoma group phase II trial with a long-term follow-up[END_REF][START_REF] Chugh | Efficacy of imatinib in aggressive fibromatosis: results of a phase II multicenter Sarcoma alliance for research through collaboration (SARC) trial[END_REF] Sorafenib was the first multitargeted TKI with activity reported in patients with DT. A retrospective series reported PR in 25% of patients, and imaging features of increased fibrosis and decreased cellularity in up to 92% of them. [START_REF] Gounder | Activity of sorafenib against desmoid tumor/deep fibromatosis[END_REF] Pazopanib is the only multitargeted TKI approved for the management of soft-tissue sarcomas. [START_REF] Van Der Graaf | Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial[END_REF] Retrospective data were reported on its promising activity in DT. [START_REF] Szucs | Pazopanib, a promising option for the treatment of aggressive fibromatosis[END_REF][START_REF] Martin-Liberal | Pazopanib is an active treatment in desmoid tumour/aggressive fibromatosis[END_REF] The DESMOPAZ study confirms that pazopanib has meaningful clinical activity in DT, with a 6month NPR of more than 80% in a very poor-prognosis population of patients. Moreover, most responses in the pazopanib arm were early, with rapid improvement in clinical symptoms such as pain and emotional functioning as shown on EORTC QLQ-C30 assessments. Of note, incidence of mucositis, an adverse event associated with pain, was not higher in the methotrexate-vinblastine arm. Importantly, we also observed an early increase in tumor heterogeneity on the MRIs of patients on treatment, with the occurrence of necrotic and fibrotic processes together with a decrease in active cellular component, despite the longest diameter being considered stable according to conventional RECIST . In this regard, the use of RECIST to determine the radiological response has certainly underestimated the real anti-tumor activity of pazopanib.

Dedicated criteria for the radiological assessment of DT should certainly be designed, considering changes in textural and shape quantitative features (i.e., delta-radiomics) on standardized MRI protocols including T2 and contrast-enhanced T1-weighted imaging. [START_REF] Lambin | Radiomics: the bridge between medical imaging and personalized medicine[END_REF] The NPR was 45% in the patients treated with methotrexate-vinblastine. Interestingly, albeit limited by small numbers, PFS at one and two year in the methotrexate-vinblastine arm settled at 79% [53.2-91.5], indicating potential prolonged activity in some patients. We also observed a slight decrease between 1-year and 2-year PFS in the pazopanib arm, suggesting that longer treatment could be proposed in some patients. These could be patients with remaining active tumor residue on MRI. This remains to be tested in a future trial with endpoints dedicated to radiomics.

Altogether, results of the DESMOPAZ trial are in line with those of the ALLIANCE A091105 trial, which included 87 patients with unresectable DT. [START_REF] Gounder | Sorafenib for Advanced and Refractory Desmoid Tumors[END_REF] Patients were randomized 2:1 to receive oral sorafenib at 400 mg or placebo. In this study, 33% of patients achieved a PR with sorafenib and 20% with placebo, and the one-year PFS rate was 89% with sorafenib compared with 46% with placebo. Importantly, progression according to RECIST was not mandatory for inclusion in the ALLIANCE A091105 trial, as it was in the DESMOPAZ trial. Indeed, about 40% were effectively progressive at inclusion according to RECIST, and tumor regression was also observed in the placebo arm, confirming the unpredictable nature of DT, and the importance to carefully evaluate the need for therapeutic intervention that is associated with potential side effects. Three other important differences can be highlighted between the DESMOPAZ and the ALLIANCE A091105 trials : in the DESMOPAZ study (i) the diagnosis of desmoid tumor was centrally reviewed by a group of expert pathologists which is crucial given the high rate of misdiagnosis (up to 33%) in the community setting [START_REF] Penel | Management of desmoid tumours: A nationwide survey of labelled reference centre networks in France[END_REF] (ii) imaging were centrally reviewed by blinded independent radiologists at baseline to confirm disease progression, and during treatment to assess efficacy outcomes in order to control bias from errors in progression assessments (iii) three quarter of patients had been already treated with systemic therapy, with 21% and 42% having received 2 or more previous lines in the methotrexate vinblastine and the pazopanib arm, respectively. Conversely, in the ALLIANCE A091105 trial only one third of patients had received previous systemic treatment (41% in the placebo arm and 36% in the sorafenib arm) and more than half of patients were newly diagnosed (51% and 54%, respectively).

Overall, the toxicity of pazopanib was manageable and the toxicity profile of methotrexate-vinblastine in line with previous studies, [START_REF] Azzarelli | Low-dose chemotherapy with methotrexate and vinblastine for patients with advanced aggressive fibromatosis[END_REF][START_REF] Skapek | Vinblastine and methotrexate for desmoid fibromatosis in children: results of a pediatric oncology group phase II Trial[END_REF] with yet less grade 3-4 myelotoxicity.

However, dose reductions were frequent and rates equal in both arms. Despite this, pazopanib resulted in meaningful clinical activity. Of note, in the ALLIANCE A091105 trial, sorafenib was administered at the 400-mg daily dose, which is 50% of the recommended licensed daily dose.

Despite that, dose interruptions occurred in 65% of the patients. Altogether, these data advocate for an upfront use of an adapted dose of pazopanib in this specific population.

The mechanism of action of multi-tyrosine kinase inhibitors such as pazopanib or sorafenib in DT is not known. Proteomic analysis of pretreatment samples allowed the identification of several proteins significantly overexpressed in patients with an objective response to pazopanib compared with patients with no objective response. Importantly, among the most differentially expressed proteins in the responding patient group were PDGFR-L and THBS4. Sustained expression and phosphorylation of PDGFRA and PDGFRB on IHC have been reported in aggressive DT, and this is believed to occur within an autocrine/paracrine loop mediated by COX-2 overexpression and deregulation of the APC/β-catenin signaling pathway. [START_REF] Signoroni | Cyclooxygenase-2 and platelet-derived growth factor receptors as potential targets in treating aggressive fibromatosis[END_REF] THBS4 is an important pro-angiogenic factor that contributes to tumor growth via TGF-β pathway activation, which mediates Wnt/β-Catenin signaling in DT. [START_REF] Varghese | TGF-beta and CTGF are mitogenic output mediators of Wnt/beta-catenin signaling in desmoid fibromatosis[END_REF][START_REF] Muppala | Thrombospondin-4 mediates TGF-beta-induced angiogenesis[END_REF] This study has limitations. It was a non-comparative randomized trial, therefore precluding direct comparison of the chemotherapy regimen with pazopanib. Baseline tumor biopsies were optional, and only a small number of samples were available for exploratory proteomics analysis, whose results remain hypothesis-generating. There was also no pharmacokinetics (PK) data reported on pazopanib. Such data could help adapt the dosing of pazopanib and improve safety profile. Blood samples collection for PK analysis were planned in the protocol, and results will be reported at a later stage.

Performing a randomized clinical trial in very rare diseases such as DT is usually considered as a real challenge. The DESMOPAZ study focused on a very rare condition and completed accrual in expected time. This was made possible thanks to involvement of patient advocacy groups in the design of the study and the unique nature of the French Network for diagnosis and management of patients with mesenchymal tumors.

In conclusion, the primary objective of this study was reached. Pazopanib has clinical activity in patients with progressive DT and may be considered a valid treatment option in this rare and disabling disease.

Contributors

Conceived and designed the study: AI, CB The green, white and red palette represents protein values that are respectively lower, equal or higher in the "no tumor shrinkage" than the "objective response" desmoid tumor class.

  of 83.7% [95% CI: 69.3-93.2]. The proportion of patients achieving 6-month nonprogression in the methotrexate-vinblastine arm was 45.0% [95% CI: 23.1-68.5].

Figure 1 .

 1 Figure 1. DESMOPAZ study flow chart (N = 72).

Figure 2A .

 2A Figure 2A. Best overall response of patients included in the pazopanib arm (N = 46).

Figure 2B .

 2B Figure 2B. Best overall response of patients included in the methotrexate-vinblastine arm

Figure 2C .

 2C Figure 2C. Progression-free survival in the pazopanib arm (N=46).

Figure 2D .

 2D Figure 2D. Progression-free survival in the methotrexate-vinblastine arm (N=20).

Figure 3 .

 3 Figure 3. Protein expression profiling associated with RECIST-response scoring for

Table 1 .

 1 TABLES Patient characteristics at baseline (N=72)

		PZ		MV		Total
		(n=48)		(n=24)		(N =72)
		n	%	n	%	n	%
	Median age (range)	35 (18-78)		42 (21-79)		(18-79)
	Sex
	Female	31	65%	15	63%		64%
	Male	17	35%	9	37%		36%
	Perfromance status (ECOG)
	0	34	71%	18	75%		72%
	1	14	29%	6	25%		28%
	Location
	Limbs and girdles	27	56%	9	38%		50%
	Internal trunk/mensenteric	13	27%	13	54%		36%
	Trunk wall	7	15%	2	8%		13%
	Head and neck	1	2%				1%
	Mutational status
	CTNNB1 T41A	15	31.2%	10	41.7%		34.7%
	CTNNB1 S45P	9	18.7%	4	16.7%		18%
	CTNNB1 S45F	8	16.7%	2	8.3%		13.9%
	APC gene	6	12.5%	2	8.3%		11.1%
	No mutation identified	6	12.5%	3	12.5%		12.5%
	Unknown	4	5.5%	3	12.5%		9.7%
	Gardner syndrome
	Yes	7	15%	4	17%		15%
	No/NA	41	85%	20	83%		85%
	Previous treatment
	Hormonal

therapy 1 Tyrosine kinase inhibitor 2 Chemotherapy 3 COX2 inhibitor Surgery Radiotherapy

		11	22.9%	2	8.3%	18%
		3	4.2%	2	8.3%	7%
		4	8.3%	0	0%	5.5%
		27	56.2%	13	54.1%	20.8%
		22	45.8%	8	33.3%	41.7%
		7	9.7%	1	4.1%	11.1%
	Number of previous systemic
	treatment lines
	0 1 2 3	11 17 13 7	23% 35% 27% 15%	6 13 1 4	25% 54% 4% 17%	23.6% 41.7% 19.4% 15.3%

LHRH agonist, tamoxifen;

imatinib;

liposomal doxorubicin, vinblastine, methotrexate, melphalan

Data sharing statement

Individual participant data that underlie the results reported in this article, after de-identification (text, tables, figures, and appendices will be shared with investigators whose proposed use of the data has been approved by an independent review committee ("learned intermediary") identified for this purpose and beginning 12 months and ending 36 months following article publication. Proposals may be submitted up to 36 months following article publication the corresponding author.

Declaration of interests: Mrs Fabiola CECCHI was an employee of Nantomics at the time of study conduction and analysis. Pr Jean-Yves BLAY has declared research grant, personal fees and non-financial support from Bayer, GSK and Novartis. Mr Todd HEMBROUGH is an employee of Nantomics. . Dr Christine CHEVREAU has declared personal fees from IPSEN, NOVARTIS, PFIZER and BMS. Pr Antoine ITALIANO has declared research grand and personal fees from IPSEN, Novartis, Bayer, BMS, Epizyme, Immune Design, Daiichi and MSD. Pr Celeste LEBBE has declared research grand, personal fees and non-financial support from Amgen, BMS, MSD, Roche, Novartis, Pierre Fabre, Sanofi, Merck Serono, Pfizer and Incyte. Mr Stephen SWEET was an employee of Nantomics at the time of study conduction and analysis.

Aknowledgments

We thank all patients, caregivers, and families who contributed to the study, as well as members of SOS Desmoide for involvement in designing and relaying information about the study (www.sos-desmoide.asso.fr)

Conflict of interest statement

The last two columns report Pearson standard correlation values and t-test p-values for each protein differentially expressed between the "no tumor shrinkage" and the "objective response" desmoid tumor class.