Marie-Daniéla Daniéla Dubois
email: mariedaniela.dubois@gmail.com

Ivan Peyron

Olivier-Nicolas Pierre-Louis

Serge Pierre-Louis

Johalène Rabout

Pierre Boisseau

Annika De Jong

Sophie Susen

Jenny Goudemand

Rémi Neviere

Pascal Fuseau

Olivier D Christophe

Peter J Lenting

Cécile V Denis

Caterina Casari

In vitro characterization of von Willebrand factor D4 domain mutations, identified in patients of Afro-Caribbean origin

Keywords: binding assays, intracellular retention, recombinant proteins, von Willebrand disease, von Willebrand factor

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

Identification of von Willebrand factor D4 domain mutations in patients of Afro-Caribbean descent: In vitro characterization

Marie-daniéla Dubois, Ivan Peyron, Olivier-nicolas Pierre-Louis, Serge

Pierre-Louis, Johalène Rabout, Pierre Boisseau, Annika de Jong, Sophie Susen, Jenny Goudemand, Rémi Neviere, et al. Von Willebrand factor (VWF) is a plasmatic multimeric glycoprotein exclusively produced by endothelial cells and megakaryocytes. [START_REF] Wagner | The Weibel-Palade body: the storage granule for von Willebrand factor and P-selectin[END_REF] VWF plays an important role in primary haemostasis, mediating platelet adhesion and aggregation at the site of vascular lesions. In the circulation, VWF is also important for the protection of coagulation factor VIII (FVIII) from premature clearance. [START_REF] Denis | Molecular and cellular biology of von Willebrand factor[END_REF][START_REF] Ruggeri | Von Willebrand factor: looking back and looking forward[END_REF] The architecture of the VWF protein consists of a succession of different structural domains according to the sequence D1-D2-D'-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK in which D1-D2 domains represent the VWF propeptide and the remaining domains (D' to CK), the mature VWF subunit. [START_REF] Springer | von Willebrand factor, Jedi knight of the bloodstream[END_REF] Over the years, a number of functional binding sites have been identified on the VWF subunit. These include the binding sites for factor VIII (FVIII) (domains D'-D3), for platelet glycoprotein (Gp) Ib and collagen type VI (A1 domain), for collagens types I and III (A3 domain), for ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin motif repeats 13) (D4 domain) and for platelet GpIIb/IIIa (C4 domain). [START_REF] Bryckaert | Of von Willebrand factor and platelets[END_REF][START_REF] Zanardelli | A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF[END_REF] A cleavage site for the metalloprotease ADAMTS13 is also present on the A2 domain of VWF and is crucial for the regulation of the multimer size. [START_REF] Dent | Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor[END_REF][START_REF] Sadler | Biochemistry and genetics of von Willebrand factor[END_REF] Von Willebrand disease (VWD) is characterized by a bleeding tendency marked by either qualitative or quantitative defects of VWF and caused by mutations on the VWF gene (VWF)

To cite this version:

sequence. [START_REF] Leebeek | Von Willebrand's Disease[END_REF][START_REF] Lillicrap | von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy[END_REF] VWD-type 1 (60-80% of all VWD) and VWD-type 3 patients are characterized by a partial or complete deficiency in plasma VWF respectively. VWD-type 3 is transmitted as either a recessive trait or through the inheritance of 2 mutant codominant alleles. VWD-type 2 represents qualitative disorders and can be divided into four subtypes:

2A, 2B, 2M, and 2N, according to the VWF function perturbed by the mutation.

Since VWD is a very heterogeneous disease, accurate identification and characterization of disease-causing variants through Sanger sequencing and next generation sequencing (NGS) is important to formally diagnose VWD and to understand the molecular mechanisms contributing to pathogenesis. [START_REF] Goodeve | Diagnosing von Willebrand disease: genetic analysis[END_REF][START_REF] Hampshire | The molecular basis of von Willebrand disease: the under investigated, the unexpected and the overlooked[END_REF] In addition, functional analyses using recombinant proteins are also key to establish the causal relationships between sequence variants and patients' phenotype and to guide clinical decisions. Such studies have largely contributed to improve our knowledge about VWF structure-function relationships and recombinant VWF (rVWF) variants in virtually every domain have been produced and studied. [START_REF] De Jong | Von Willebrand disease mutation spectrum and associated mutation mechanisms[END_REF] However, most studies have focused on the N-terminal half of the VWF subunit (D' to A3 domains) and relatively few on the C-terminal half (D4 to CK domain), resulting in a relatively poor knowledge of these domains. In the D4 domain for example, numerous VWF sequence variations have been identified, leading to VWD-type 1, 2A, 2M or even type 3. [START_REF] Baronciani | Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS[END_REF][15][START_REF] Goodeve | Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD)[END_REF][START_REF] Veyradier | French Reference Center for von Willebrand d. A Laboratory Phenotype/Genotype Correlation of 1167 French Patients From 670 Families With von Willebrand Disease: A New Epidemiologic Picture[END_REF] However, very few of these variants have been expressed in in vitro systems and duly characterized. [START_REF] De Jong | Von Willebrand disease mutation spectrum and associated mutation mechanisms[END_REF] In the present study, taking into account the geographic specificities of VWF mutations, [START_REF] Dubois | A Combination of Two Variants p. (Val510 =) and p. (Pro2145Thrfs * 5), Responsible for von Willebrand Disease Type 3 in a Caribbean Patient[END_REF] we set out to investigate the effect of two D4 mutations, the c.6647del deletion and the c.6432dup duplication, both resulting in premature termination of protein synthesis and respectively identified in unrelated Afro-Caribbean patients with VWD-type 1 and 3, on VWF function and biosynthesis.

Material and Methods

Patients

Two patients carrying a mutation located in VWF and responsible for VWD were enrolled in this study after informed written consent in accordance with the Declaration of Helsinki. The study was approved by our institutional review board. The patients (P1 and P2) are two women respectively aged 90-and 40-years old, both with bleeding symptoms.

Plasma samples

Peripheral blood samples were collected in tubes containing 3.2% sodium citrate. After leaving the samples at room temperature for 30 min, they were centrifuged (15 min, 2500g)

and plasma was collected and frozen at -80°C. Plasma was analyzed for Factor VIII (FVIII) procoagulant activity (FVIII:C) using a chromogenic assay. VWF antigen (VWF:Ag) levels were determined using a particle-based turbidimetric assay, and VWF ristocetin cofactor activity (VWF:RCo) by platelet aggregometry.

Plasmid construction

The expression vectors pcDNA3.1-hVWF-Myc and pcDNA3.1-hVWF-HA encoding full-length VWF cDNA were kindly provided by Dr. Eikenboom (Leiden University Medical Center, Leiden, The Netherlands) and have been previously described. [START_REF] De Jong | Correction of a dominant-negative von Willebrand factor multimerization defect by small interfering RNAmediated allele-specific inhibition of mutant von Willebrand factor[END_REF] A HiFi DNA assembly strategy (Kit E5520, New England Biolabs, Evry-Courcouronnes, France) was used to modify the C-terminal region of hVWF. Mutagenic primers were used to generate the hVWF variants according to the modifications induced by the p.(Cys2216Phefs*9) and the p.(Pro2145Thrfs*5) mutations (Fig. 1). In the mutated hVWF, the premature stop codon was relocated downstream of the HA-tag sequence, allowing for the distinction between the wildtype (WT) form (Myc-tagged) and the mutated forms (HA-tagged). The correct modifications of the expression vectors were verified by sequencing. Plasmid purification was performed using the NucleoBond PC 500 EF kit (Macherey-Nagel, Hoerdt, France).

Transfection experiments

Human embryonic kidney 293T (HEK293T) cells were grown in complete medium

Binding assays

For VWF binding tests, rVWF secreted in the supernatant was concentrated 4.5-21-fold, using

Aquacide II (Calbiochem, La Jolla, CA, USA) and dialysed against 25mM Tris-HCl containing 150mM NaCl, pH 7.4 (TBS). The different binding assays were performed as described. [21] Briefly, for collagen assays, human collagen type III (Tebu-Bio) was coated on microtiter plates at 2 μg/ml and serial dilutions of rVWF samples (0-1 μg/ml) were added.

For GpIb binding assays, 5 μg/ml of recombinant human (rhGpIb) (Bio-Techne, Lille, France) was immobilized via a home-made monoclonal antibody to GpIb. rVWF proteins diluted 0-1 µg/ml and containing 10 µg/ml of laboratory-purified botrocetin, were added to the wells.

For GpIIbIIIa binding assays, 10 μg/ml human platelet-purified GpIIbIIIa (Enzyme Research Laboratories, Swansea, UK) was immobilized via a home-made monoclonal antibody to GpIIbIIIa. Serial dilutions or rVWF (0-2.5 µg/ml) were added.

For binding to ADAMTS13, recombinant human ADAMTS13 (Bio-Techne) was coated on microtiter wells at a concentration of 2 µg/ml and serial dilutions or rVWF (0-2.5 µg/ml) were added. In all above tests, bound-rVWF was detected using rabbit anti-VWF-HRP antibodies (Dako, Agilent, Les Ulis, France).

For FVIII binding, rVWF preparations (0-1 µg/ml) were coated using a polyclonal rabbit anti-VWF antibody (Dako). After the washes, 0.35 M CaCl 2 was added to the wells. This step was repeated twice for 10 min and was followed by 6 washes. Then, 60 mU/well of diluted rFVIII (Kogenate-FS, Bayer Healthcare, USA) was added and incubated for 2 h at 37°C. After washing, the amount of bound FVIII was measured with an HRP-coupled polyclonal anti-FVIII antibody (Diagnostica Stago, Asnières, France). Absorbance was measured at 450 nm.

After carrying out several successive washes, the relative amount of rVWF immobilized in each well was also detected using rabbit polyclonal anti-VWF-HRP antibodies. The results are expressed as the ratio of the slope of the mutated rVWF-FVIII binding curve to the wildtype-rVWF binding slope.

Multimers

VWF multimeric profiles were analyzed by 2% SDS agarose gel electrophoresis followed by immunoblotting. The membrane was probed by incubation with a home-made polyclonal goat antibody to VWF coupled to alkaline phosphatase followed by staining with alkaline phosphatase conjugate substrate kit (Bio-rad).

Statistical analysis

Data are expressed as mean plus or minus standard deviation (SD) values (n=3 independent experiments) using Prism 7 software (GraphPad, La Jolla, USA). Statistical analyses were also performed using Prism 7 software. A one-way ANOVA followed by a post-hoc Dunnet's correction test was performed in order to make comparisons between the mutants and wildtype rVWF. p values <0.05 were considered significant.

Results

Clinical and laboratory phenotypic data are summarized in table 1. Both patients are of the O + blood group.

Patient 1 showed reduced VWF levels (VWF:Ag = 35%, VWF:RCo = 30%) as well as a reduced FVIII:C (33%). Such parallel decreases were evocative of VWD-type 1. The reduced binding to FVIII (VWF: FVIIIB) (56%) may suggest a 2N heterozygous status, resulting from the presence of the p.(His817Gln) variant. [START_REF] Wang | Characterizing polymorphisms and allelic diversity of von Willebrand factor gene in the 1000 Genomes[END_REF] However, and although this variant is known to decrease FVIII:C levels by 18 IU/dL per allele, it has been identified in a significant proportion of healthy individuals from African ancestry, allowing its classification as a benign variant in this population as indicated in table 1. [START_REF] Bellissimo | VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population[END_REF][START_REF] Johnsen | Common and rare von Willebrand factor (VWF) coding variants, VWF levels, and factor VIII levels in African Americans: the NHLBI Exome Sequencing Project[END_REF] After VWF sequencing, 7 sequence variations (6 heterozygous and 1 homozygous) were identified in the coding region, with two Analysis of the culture supernatant collected 72 h after transfection showed a concentration of 1.2 µg/mL, in cells expressing WT-rVWF. In contrast, the p.(Pro2145Thrfs*5)-rVWF variant exhibited strongly reduced secretion with VWF:Ag 0.12 µg/mL (p<0.0001 vs WT) while the p.(Cys2216Phefs*9)-rVWF variant was virtually absent (p<0.0001 vs WT) (Fig. 2A). After co-transfection of the VWF mutants with WT-VWF, a partial restoration of antigen levels was obtained: we respectively measured 0.53 μg/mL of VWF:Ag for p.(Pro2145Thrfs*5)-rVWF and 0.28 μg/ml for p.(Cys2216Phefs*9)-rVWF (Fig. 2A). These values remained significantly lower than WT-rVWF (p<0.0001 vs WT for both variants in the heterozygous state).

The intracellular concentration of rVWF obtained from cell lysates was also quantified (Fig. 2B). In the homozygous transfections, both mutants exhibited a significant reduction of the intracellular VWF:Ag. After co-transfection with WT-VWF, the heterozygous variant p.(Pro2145Thrfs*5)-rVWF was no longer different from WT-rVWF (0.23 µg/ml for both, p=0.99) while the heterozygous p.(Cys2216Phefs*9)-rVWF was still significantly reduced (0.16 µg/ml, p=0.02) compared to WT-rVWF.

Measurement of the ratio supernatant/lysate (Figure 2C) of all mutants, both homozygous and heterozygous showed values lower than that of WT indicating some intracellular retention of the rVWF mutants.

Specific detection of WT-and mutant-VWF

Next, we aimed to measure the relative quantities of wild-type-and mutant VWF in the total amount of secreted VWF. For this, we used specific ELISA assays. The Myc-tag was used to detect WT-rVWF while the HA-tag was used to detect the mutant forms. As control, a cotransfection performed with WT-VWF-HA cDNA and WT-VWF-Myc cDNA was performed.

Using this control, a perfect distribution of 50% of each form was obtained (Fig. 3).

For the heterozygous mutant p.(Pro2145Thrfs*5)-rVWF, we also observed a similar distribution with equal proportions of the wild-type and mutant forms, but the total amount of VWF secreted was reduced compared to WT-rVWF. For the heterozygous p.(Cys2216Phe fs*9)-rVWF variant, we observed a distribution of 75% and 25% between the WT-rVWF form and the mutated form, indicating specific intracellular retention of the mutant protein.

Furthermore, the total amount of secreted VWF:Ag was strongly reduced, confirming results reported in Figure 2.

Multimeric profiles

Secreted rVWF multimers were analyzed by electrophoresis to determine whether the coexpressed mutants induced alterations in the structure of VWF (Fig. 4). The multimeric profile of mutant p.(Cys2216Phefs*9)-rVWF was normal. For the p.(Pro2145Thrfs*5)-rVWF variant, the apparent loss of the highest molecular weight multimers may be attributed to a lower quantity of antigen loaded on the gel. The most striking feature is the presence of a small additional band above the dimers and tetramers for the p.(Pro2145Thrfs*5)-rVWF mutant (arrowheads), suggesting that odd multimers formed by WT and shorter polymers are indeed produced and released, as indicated by the tag-specific quantification method. A small shoulder on the densitometry analysis is also confirming the presence of these additional bands (red arrows) (Fig. 4).

Binding of recombinant mutant VWF to various ligands

To allow functional analysis of mutants, the transfection supernatants containing rVWF were binding (half maximum binding 0.71 ± 0.13 µg/ml) (Fig. 5A and Table 2) compared to WT-rVWF (half maximum binding 0.20 ± 0.04 µg/ml) (p=0.0006). To examine the effect of mutations on platelet GpIb binding, increasing concentrations of rVWF were applied to immobilized rhGpIb in the presence of 10 µg/mL botrocetin (Fig. 5B). Both heterozygous variants displayed slightly but significantly decreased affinity for GpIb (Table 2). Binding to GpIIbIIIa revealed a strong reduction in binding for the p.(Pro2145Thrfs*5)-rVWF variant (half maximum binding 2.23 ± 0.24 µg/ml) compared to WT-rVWF (half maximum binding 0.37 ± 0.04 µg/ml (p<0.0001) (Fig. 5C). Binding of the p.(Cys2216Phefs*9)-rVWF variant was only slightly decreased and no different compared to WT-rVWF (p=0.191). No defect in FVIII binding was detected for either variant (Fig 5D). Finally, binding to ADAMTS13 was tested and the p.(Pro2145Thrfs*5)-rVWF displayed significantly reduced binding (half maximum binding of 3.26 µg/ml) compared to WT-rVWF (half maximum binding of 1.56 µg/ml) (p=0.0001) (Fig. 5E). In contrast, the p.(Cys2216Phefs*9)-rVWF variant did not display any reduced binding to ADAMTS13 (half maximum binding of 1.80 µg/ml) compared to WT-rVWF (p=0.32) (Table 2).

Discussion

In this study, we describe a frameshift gene variant within the VWF D4 domain completely preventing VWF secretion potentially due to mRNA degradation and protein retention.

Alternatively, for a second such variant resulting in residual protein secretion, a dominantnegative effect of VWF can be observed and leads to a more severe phenotype.

Since the cloning of VWF in 1985, [START_REF] Ginsburg | Human von Willebrand factor (vWF): isolation of complementary DNA (cDNA) clones and chromosomal localization[END_REF] hundreds of sequence variations of VWF have been identified in VWD patients. [START_REF] Baronciani | Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS[END_REF][15][START_REF] Goodeve | Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD)[END_REF][START_REF] Veyradier | French Reference Center for von Willebrand d. A Laboratory Phenotype/Genotype Correlation of 1167 French Patients From 670 Families With von Willebrand Disease: A New Epidemiologic Picture[END_REF] Expression of VWF variants in heterologous cellular systems have also greatly contributed to elucidating pathological mechanisms as well as improving our knowledge on the roles of VWF domains. Although missense mutations have represented the bulk of published expression findings, [START_REF] De Jong | Von Willebrand disease mutation spectrum and associated mutation mechanisms[END_REF] a few studies have undertaken analysis of other VWF genetic alterations such as frameshift mutations, [START_REF] Bowman | Discrepant platelet and plasma von Willebrand factor in von Willebrand disease patients with p.Pro2808Leufs*24[END_REF] splice site mutations, [START_REF] Daidone | An apparently silent nucleotide substitution (c.7056C>T) in the von Willebrand factor gene is responsible for type 1 von Willebrand disease[END_REF][START_REF] Hawke | Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism[END_REF][START_REF] Yadegari | Intron retention resulting from a silent mutation in the VWF gene that structurally influences the 5' splice site[END_REF] or deletions mutants. [START_REF] Casari | The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation[END_REF][START_REF] Othman | Functional characterization of a 13bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease[END_REF][START_REF] Sutherland | A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3[END_REF][START_REF] Yadegari | Multifaceted Pathomolecular Mechanism of a VWF Large Deletion Involved in the Pathogenesis of Severe VWD[END_REF] Even fewer studies have focused on genetic alterations identified in the D4 domain of VWF. We therefore decided to characterize two such genetic defects present in two patients of Afro-Caribbean origin, a deletion of a nucleotide in position 6647 leading to a frameshift variant p.(Cys2216Phefs*9), and a duplication of a nucleotide in position 6432, also leading to a frameshift alteration p.(Pro2145Thrfs*5). These patients display a number of other amino acid changes previously reported (Table 1); some little or no deleterious (for patient 1) while some potentially pathogenic such as p.(Val510=) and p.(Cys1149Arg) for patient 2. [START_REF] Dubois | A Combination of Two Variants p. (Val510 =) and p. (Pro2145Thrfs * 5), Responsible for von Willebrand Disease Type 3 in a Caribbean Patient[END_REF][START_REF] Schooten | Cysteine-mutations in von Willebrand factor associated with increased clearance[END_REF] Because of the presence of a premature termination codon (PTC), the mutations p.(Cys2216Phefs*9) and p.(Pro2145Thrfs*5) can potentially lead to nonsense-mediated mRNA decay (NMD) and therefore the mutant allele would not be expressed in the patient.

Indeed, PTC-introducing mutations in VWF were shown to be associated with NMD but this mechanism was found to be PTC-position dependent and the extent of degradation was not 100%. [START_REF] Plate | Premature termination codon mutations in the von Willebrand factor gene are associated with allele-specific and position-dependent mRNA decay[END_REF] Therefore, it is almost impossible to predict if and how the NMD is associated with a specific VWF variant, without performing cellular experiments.

This aspect prompted us to express WT and mutant cDNA with a specific tag for both variants, in order to quantify the relative amount of both proteins. Before performing cotransfections, we first tested whether any homozygous mutant protein could be produced and secreted. Some protein expression was measured in cell lysates suggesting that at least part of the mRNAs were not degraded. However, virtually no VWF was secreted in the medium (Fig 2A -B) suggesting that protein retention was also involved. Co-transfection with WT cDNA led to a partial correction of protein expression in both cell medium and cell lysate, however some differences could still be observed. Tag-specific ELISA assays were then used to specifically assess which VWF-form was being expressed preferentially. To validate this approach, we first used 2 differentially tagged WT-cDNA, and we obtained a perfect 50:50 repartition of rVWF in cell culture medium, showing that both plasmids allow similar expression level. Considering that the same cells were very likely transfected with both plasmids as shown previously [START_REF] Baronciani | Genotypes of European and Iranian patients with type 3 von Willebrand disease enrolled in 3WINTERS-IPS[END_REF] this result indicates that multimers are made of an equal repartition of HA-tagged subunits and Myc-tagged subunits.

Knowing the relative expression of the mutant forms in the co-transfections, i.e. 46% for p.(Pro2145Thrfs*5) and 23% for p.(Cys2216Phefs*9) is of critical importance for interpretation of the binding assays. Indeed, for p.(Cys2216Phefs*9), most binding assays were comparable to WT-VWF. Considering that mostly WT-VWF was secreted in the medium during the co-transfections with p.(Cys2216Phefs*9), the result is not really surprising. For this mutant, the detrimental effect appears mostly at the expression level which is strongly reduced even in the co-transfections, preventing protein synthesis and secretion. The multimeric profile is not affected. For mutant p.(Pro2145Thrfs*5), the situation is different. Because the altered protein is synthetized and can interact with WT subunits, this translates in a dominant-negative effect of the mutation. Indeed, although expression level is low, 50% of the protein being expressed is of mutant origin. The dominant negative effect can be seen on the multimer gel with the odd band representing multimers that have incorporated the truncated mutant protein, bringing a stop to any subsequent polymerization. The effect of the mutation on binding of rVWF to its ligands is therefore clearly visible since binding to collagen III, GpIIbIIIa and ADAMTS13 is significantly reduced. For GpIIbIIIa, considering that the binding site is located in the C4 domain, downstream of the termination of the mutant subunits, this decrease is expected and can be explained by the fact that only 50% of the subunits would still harbor this binding site. The decrease in binding to collagen III (A3 domain) or to ADAMTS13 (D4 domain) can also be logically explained. For ADAMTS13, since part of the D4 domain is missing, binding is likely suboptimal. As for collagen III, the truncation of the D4 domain is possibly affecting the structure of the neighboring A3 domain, resulting in a modification in the exposure of the collagen binding site. Binding to FVIII is unaffected as expected for the binding to a ligand interacting with the N-terminal part of the molecule. As for GpIb, binding is slightly affected, suggesting that the A1 domain structure may also be modified by mutations occurring in the D4 domain.

This tag-specific allele approach, developed and previously used by the team of Pr.

Eikenboom, [START_REF] De Jong | Correction of a dominant-negative von Willebrand factor multimerization defect by small interfering RNAmediated allele-specific inhibition of mutant von Willebrand factor[END_REF] thus appears as absolutely essential for interpretation of expression studies of rVWF. Indeed, without knowing the respective amounts of WT and mutant rVWF in the expression medium, no relevant conclusion of the effect of mutations can really be drawn.

In conclusion, our study about these two D4 mutations identified in patients from Afro-Caribbean origin highlights the difficulty of in vitro expression of genetic alterations leading to the occurrence of premature stop codons.

Although the purpose of this study was not to fully reproduce the patients' phenotype due to the presence of multiple genetic variations, what we have evidenced is that lack of expression of a mutant, (e.g. p.(Cys2216Phefs*9)) is less deleterious than residual expression (e.g.

p.(Pro2145Thrfs*5)) mostly because of the multimeric nature of VWF and the subsequent dominant negative effect. In the first case, because protein secretion is prevented, probably due to a combination of mRNA degradation and protein retention, multimers will be formed only by normal subunits, consistent with a VWD-type 1 phenotype. In the second case, multimers will be abnormal, as binding of an altered monomer would prevent further multimerization. [START_REF] Campioni | In vivo modulation of a dominant-negative variant in mouse models of von Willebrand disease type 2A[END_REF] In patient 2, a known pathogenic variant associated with a dominant negative effect preventing multimerization (p.(Cys1149Arg)) is present. [START_REF] Bodo | Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins[END_REF] Here we describe a second dominant-negative variant, (p.(Pro2145Thrfs*5)), preventing dimerization. Because these mutations are located on different alleles, their association is absolutely deleterious for the patient, completely preventing multimerization and leading to a VWD-type 3 phenotype.

In contrast, we can speculate that if the two mutations would have occurred in the same allele, a VWD-type 1 phenotype would have occurred.

Finally, it should also be mentioned that each frameshift mutation is potentially associated with the disruption of a cysteine-bridge, (p.Cys2139-p.Cys2163 and p.Cys2203-p.Cys2235, respectively), both being located in the D4-domain. To the best of our knowledge, no reports exist on the structural or functional consequences when either cysteine-bridge is absent in the VWF molecule. However, the notion that both cysteine-bridges are conserved in the D1, D2

and the D3 domain [START_REF] Zhou | Sequence and structure relationships within von Willebrand factor[END_REF] suggests that they are pertinent to the integrity of the D-domain heterozygous [24, 40] Benign moderately c.3258C>T p.(Tyr1086=) heterozygous ND ND decreased c.4414G>C p.(Asp1472His) homozygous [41] Begnin c.6554G>A p.(Arg2185Gln) heterozygous [23] Begnin c.6647del p.(Cys2216Phefs*9) heterozygous ND ND c.6859C>T p.(Arg2287Trp) heterozygous [42] Benign 2 c.1530G>A p.(Val510=) heterozygous [18] Damaging O+ 2-3 1-5 5 6 NT Total absence c.1892C>T p.(Ala631Val) heterozygous [23] Benign c.2220G>A p.(Met740Ile) heterozygous [22] Begnin c.2451T>A p.(His817Gln) heterozygous [24, 40] Benign c.3445T>C p.(Cys1149Arg) heterozygous [35, 43] Damaging c.4414G>C p.(Asp1472His) homozygous [41] Begnin c.6432dup p.(Pro2145Thrfs*5) heterozygous [18] Damaging c.6554G>A p.(Arg2185Gln) heterozygous [23] Begnin FVIII:C, factor VIII coagulant activity; VWF:Ag, von Willebrand factor antigen; VWF:RCo, ristocetin cofactor activity; VWF:FVIIIB, VWF binding to FVIII; NT, not tested; ND, not described.

Essentials

  Marie-daniéla Dubois, Ivan Peyron, Olivier-nicolas Pierre-Louis, Serge Pierre-Louis, Johalène Rabout, et al.. Identification of von Willebrand factor D4 domain mutations in patients of Afro-Caribbean descent: In vitro characterization. Research and Practice in Thrombosis and Haemostasis, 2022, 6 (4), 10.1002/rth2.12737. hal-03918745 Gene variants identified in the VWF/D4 domain have been rarely duly characterized  Two D4 domain VWF variants display different effects on protein synthesis  The p.(Cys2216Phefs*9) variant results in strongly reduced, barely detectable expression levels The p.(Pro2145Thrfs*5) variant displays dominant-negative effects, affecting ligand binding Introduction

  new variants: 1) the synonymous variant p.(Tyr1086 =) and 2) p.(Cys2216Phefs*9), a mutation causing a shift of the reading frame by 9 amino acids and the appearance of a premature stop codon, resulting in a truncated VWF protein missing part of the D4 domain and the C-terminal end of the molecule. The 5 other variants (p.(Met740Ile), p.(His817Gln), p.(Asp1472His) and p.(Arg2185Gln), p.(Arg2287Trp)) previously described as no or little deleterious and identified in healthy populations were also detected in this patient. This patient was classified as having VWD-type 1 with status of transmitter of VWD-type 3. Her bleeding score was 0 but she required Helixate® (recombinant FVIII) treatment before and after receiving surgery. The patient is now deceased. Patient 2 had severely reduced VWF levels, with a total absence of multimers evaluated by electrophoresis. Her biological profile was reminiscent of VWD-type 3. Three heterozygous sequence variations potentially deleterious (p.(Val510=), p.(Cys1149Arg) and p.(Pro2145Thr fs*5)) were identified upon sequencing of VWF. The family study showed that the sequence variations p.(Cys1149Arg) and p.(Pro2145Thrfs*5) are on different alleles. In addition to these three variants, five not detrimental variants (p.(Ala631Val), p.(Met740Ile), p.(His817Gln), p.(Asp1472His) and p.(Arg2185Gln)) were also detected. Her bleeding score was 32 with numerous bleeding episodes: epistaxis, bruising, oral cavity bleeding, prolonged bleeding from minor wounds, menorrhagia, hemarthrosis and ankle arthropathy. Although both patients were unrelated and exhibiting different phenotypes/severity, they were both from Afro-Caribbean origin and both exhibiting a frameshift mutation (p.(Cys2216Phefs*9) for patient 1 and p.(Pro2145Thrfs*5) for patient 2 leading to a truncated VWF protein from the D4 domain onward. This similarity prompted us to study these two mutations in in vitro systems.Characterization of rVWFThe influence of the D4 mutations on VWF synthesis and/or secretion was measured in vitro after transient transfection of c.6647del (leading to the p.(Cys2216Phefs*9) variant) and the c.6432dup (leading to the p.(Pro2145Thrfs*5) variant) into HEK293T cells. Co-transfections were also performed to see the impact of these variants in a heterozygous status (transfections were performed with equimolar amounts of mutant VWF cDNA and WT-VWF cDNA). Of note, the HEK293T cells that we used throughout the study are not making Weibel -Palade bodies, in contrast to HEK293 cells. The HEK293T cells have been previously used to examine secretion and/or intracellular retention of VWF[START_REF] Jacobi | Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage[END_REF] but cannot provide information on regulated storage of VWF.

concentrated 4 . 5 -

 45 fold for WT, 10-fold for p.(Pro2145Thrfs*5)-rVWF and 21-fold for p.(Cys2216Phefs*9)-rVWF to obtain similar rVWF concentrations in all samples. Following concentration and dialysis, rVWF:Ag were 2.87 µg/ml for WT-rVWF, 3.04 µg/ml for p.(Cys2216Phefs*9)-rVWF and 3.37 µg/ml for p.(Pro2145Thrfs*5)-rVWF. Of note, binding tests were carried out only for heterozygous rVWF, better reflecting the patients' situation. We first measured the ability of mutant rVWF to bind to collagen type III and normalized our results to WT-VWF. While the p.(Cys2216Phefs*9)-rVWF variant exhibited normal binding to collagen type III, the p.(Pro2145Thrfs*5)-rVWF variant displayed significantly reduced

Figure 1 :

 1 Figure 1: Overview of the VWF variants used in the study Domain organization for WT-rVWF-Myc (A), WT-rVWF-HA (B), p.(Pro2145Thrfs*5)-rVWF-HA (C) and p.(Cys2216Phefs*9)-rVWF-HA (D). Nucleotide insertion (position 6432) or deletion (position 6647) is underlined in the nucleotide sequence. Effects of the nucleotide insertion (c.6432dup) or deletion (c.6647del) on the amino-acid sequence are depicted in blue. The WT sequence of human HA-tagged VWF contained in the pcDNA3.1 vector was used as template. Mutagenic primers were designed to generate the two constructs p.(Pro2145Thrfs*5)-rVWF and p.(Cys2216Phefs*9)-rVWF in pcDNA3.1 using a HiFi cloning strategy. To allow for the specific detection of the truncated VWF chain, the HA tag sequence (in red) was located upstream of the premature stop codon resulting from the frameshift.

Figure 2 :

 2 Figure 2: In vitro expression study of rVWF VWF:Ag levels were quantified by ELISA in the cell media (A) and lysate (B). Homozygous transfections with mutant VWF or heterozygous transfections mutant/WT-VWF were performed in HEK293T cells and medium or lysate was collected 72 h after transfection. (C): VWF:Ag ratio in supernatants over lysates. Results represent 3 different experiments performed in triplicates. The mean±SD values were compared using a one-way ANOVA and Dunnett's post-test; *p < 0.05.

Figure 3 :

 3 Figure 3: Relative expression of each rVWF allele in cell medium after co-transfectionCo-transfections of HEK293T cells were performed using pcDNA3.1-VWF-Myc and pcDNA3.1-VWF-HA plasmids with the HA-tagged plasmid encoding mutant rVWF. 72 h post-transfection, cell medium was collected and tag-specific ELISA assays were performed.Results represent 3 different experiments performed in triplicates. The mean±SD values were compared using a one-way ANOVA and Dunnett's post-test; *p < 0.05).

Figure 4 :

 4 Figure 4: Multimer pattern of mutants and WT-rVWF obtained from heterozygous expression in HEK293T Red arrowheads indicate odd bands visible in heterozygous transfections of p.(Pro2145Thrfs*5)-rVWF/WT-rVWF. Densitometric analysis was performed using the Gels tool of ImageJ software. Red arrows indicate odd peaks corresponding to uneven bands. Of

Figure 5 :

 5 Figure 5: Binding of heterozygous mutants and WT-rVWF to different VWF ligands Binding of VWF mutants to collagen III (A), GpIb (B), GpIIbIIIa (C), FVIII (D) and ADAMTS13 (D). Results are normalized and represent 3 different experiments performed in triplicates. Means±SD values are presented.

Table 1 . Phenotypic and genotypic data of two VWD subjects

 1 structure. It seems therefore possible that the disappearance of either cysteine-bridge affects correct folding, secretion and/or function of the mutated VWF protein. PJ. Lenting and OD. Christophe supervised the study; MD. Dubois, CV. Denis, C. Casari and ON. Pierre-Louis wrote the manuscript; CV. Denis and A. de Jong provided essential reagents; MD. Dubois, ON. Pierre-Louis, S. Pierre-Louis and J. Rabout recruited the patients and collected clinical data; S. Pierre-Louis, PJ. Lenting, OD. Christophe, P. Boisseau, J.

	Goudemand, A. de Jong and I. Peyron revised the paper and all authors contributed to the
	editing of the final manuscript.

Authorship contributions MD. Dubois, I. Peyron, C. Casari and PJ. Lenting performed experiments; CV. Denis, C. Casari, PJ. Lenting; OD. Christophe and I. Peyron conceived the study; CV. Denis, C. Casari,

Table 2 : Half-maximum binding values (g/ml) for binding of WT-VWF and heterozygous mutants to the different ligands

 2

		WT	p.(Cys2216Phefs*9)/WT p.(Pro2145Thrfs*5)/WT
	Collagen	0.20±0.04	0.27±0.05	0.71±0.13
			(p=0.524)	(p=0.0006)
	GpIb	0.13±0.01	0.21±0.02	0.23±0.02
			(p=0.0024)	(p=0.0007)
	GpIIbIIIa	0.37±0.04	0.59±0.07	2.23±0.24
			(p=0.191)	(p<0.0001)
	ADAMTS13	1.56±0.06	1.80±0.23	3.26±0.26
			(p=0.320)	(p=0.0001)

Acknowledgements

We express our appreciation to the patients who participated in the study. This study was supported by Institutional funding to INSERM UMRS 1176 and by the Collectivité Territoriale de Martinique (to MDD). All authors declare no conflict of interest.


B Abbar

V De Castelbajac

P Gougis

S Assoun

J Pluvy

C Tesmoingt

N Théou-Anton

A Cazes

C Namour

A Khalil

V Gounant

B Besse

G Zalcman
email: gerard.zalcman@aphp.fr

S Brosseau

Prof. Gérard Zalcman

Definitions, outcomes, and management of hyperprogression in patients with non-small-cell lung cancer treated with immune checkpoint inhibitors

Keywords: non-small-cell lung cancer, hyperprogressive disease, immunotherapy, programmed cell death 1 receptor, programmed cell death 1

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ABSTRACT

Background: The advent of immune checkpoint inhibitors (ICI) has been a breakthrough in the care of patients with non-small-cell lung cancers (NSCLC).

However, physicians are now facing a previously unidentified clinical situation called hyperprogression (HP), which presents as a fast and unexpected increase in tumor burden. HP's existence and specificity to ICIs remains controversial because a widely acknowledged definition is currently lacking. Meanwhile, management remains elusive.

Methods: Medical records from all consecutive NSCLC patients who were treated with ICI from 2015 to 2018 were retrospectively analyzed. The HP incidence rate was calculated according to five definitions (tumor growth rate [TGR]ratio, ∆TGR, tumor growth kinetic [TGK], RECIST, and time to treatment failure [TTF]), and the agreement between such definitions was determined. The HP impact on overall survival (OS) was then assessed. The association between HP (defined using the TGRratio definition) and clinical and biological variables was also assessed. Clinical HP management and its impact on outcomes were described.

Results:

We identified 169 consecutive ICI-treated patients, with potential HP accounting for 11.3%, 5.7%, 17.0%, 9.6%, and 31.7% patients, according to TGRratio, ∆TGR, TGK, RECIST, and TTF definitions. Agreement between the different HP definitions was highly heterogeneous (range 29%-77%) and globally poor. HP was associated with shorter OS, compared to standard RECIST progressive disease, but this difference only reached statistical significance when using the TTF definition. TGRratio-based HP was significantly associated with hepatic metastases. In TGRratio-based HP patients, neither resuming chemotherapy

INTRODUCTION

Lung cancer is the leading cause of cancer death worldwide with approximately 2.1 million new cases and 1.75 million deaths in 2018 [START_REF]Cancer today[END_REF]. Immune checkpoint inhibitors (ICIs), such as those that target programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PDL-1) and cytotoxic T-lymphocyteassociated protein 4 (CTLA-4), have emerged as a standard pillar of cancer treatment [START_REF] Larkin | Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma[END_REF][START_REF] Motzer | Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma[END_REF][START_REF] Chen | Phase II Study of the Efficacy and Safety of Pembrolizumab for Relapsed/Refractory Classic Hodgkin Lymphoma[END_REF][START_REF] Ferris | Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck[END_REF]. Anti-PD-1 monoclonal antibodies were the first immune-oncology drugs to gain registered authorization in 2015 in the non-small-cell lung cancer (NSCLC) second-line setting, and they have revolutionized the treatment of lung cancer with a significant increase in survival for 20% of these patients [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] West | Atezolizumab in combination with carboplatin plus nab-paclitaxel chemotherapy compared with chemotherapy alone as first-line treatment for metastatic non-squamous non-small-cell lung cancer (IMpower130): a multicentre, randomised, open-label, phase 3 trial[END_REF][START_REF] Paz-Ares | Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Gandhi | Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer[END_REF][START_REF] Horn | First-Line Atezolizumab plus Chemotherapy in Extensive-Stage Small-Cell Lung Cancer[END_REF][START_REF] Rittmeyer | Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial[END_REF].

Since ICI exhibit an original mechanism of action that is based on enhancing the antitumor immune response, physicians are now facing totally new patterns of tumor response and progression [START_REF] Borcoman | Novel patterns of response under immunotherapy[END_REF]. Indeed, several types of atypical tumor responses induced by ICI have been described, such as pseudo-progression or a long-term near-complete to complete response that evokes a actual cure in some patients.

These patients are termed long-term survivors, and they are represented by a plateau at the tail of survival curves [START_REF] Schadendorf | Pooled Analysis of Long-Term Survival Data From Phase II and Phase III Trials of Ipilimumab in Unresectable or Metastatic Melanoma[END_REF][START_REF] Gettinger | Five-Year Follow-Up of Nivolumab in Previously Treated Advanced Non-Small-Cell Lung Cancer: Results From the CA209-003 Study[END_REF]. Around 2% of NSCLC patients who are treated with ICI experience a pseudo-progression that is characterized by a transient increase, which is followed by a decrease in tumor burden during their treatment course [START_REF] Kazandjian | Characterization of outcomes in patients with metastatic non-small cell lung cancer treated with programmed cell death protein 1 inhibitors past RECIST version 1.1-defined disease progression in clinical trials[END_REF]. Such an initial increase is thought to be the result of tumor immune infiltration, which subsequently leads to a real response and decrease in tumor volume. This led to the development of a specific radiological assessment of tumor response: the modified RECIST 1.1 criteria for immune-based therapeutics (iRECIST) [START_REF] Seymour | iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics[END_REF].

More recently, another response pattern to ICI has been described. Hyperprogression (HP), which is also called hyperprogressive disease (HPD), presents as a fast and unexpected increase in tumor burden immediately after exposure to ICI [START_REF] Champiat | Hyperprogressive disease: recognizing a novel pattern to improve patient management[END_REF]. This phenomenon is mostly based on the intuition of the treating physician when patients with an initially indolent disease experience fast, symptomatic, and dramatic progression as soon they receive ICI. Such a phenomenon is corroborated by the observation of lower overall and progression free survival in the ICI arm, during the first weeks of treatment, in almost every clinical trial comparing ICI to chemotherapy. This leads to survival curves that initially cross [START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF][START_REF] Rittmeyer | Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial[END_REF][START_REF] Hellmann | Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer[END_REF]. Despite being reported in several studies, the existence of HP remains controversial, possibly due to the lack of an agreed definition and evaluation [START_REF] Adashek | Hyperprogression and Immunotherapy: Fact, Fiction, or Alternative Fact?[END_REF].

Indeed, many definitions of HPD have been proposed, based on the tumor growth rate (TGR) [START_REF] Ferrara | Hyperprogressive Disease in Patients With Advanced Non-Small Cell Lung Cancer Treated With PD-1/PD-L1 Inhibitors or With Single-Agent Chemotherapy[END_REF][START_REF] Champiat | Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1[END_REF][START_REF] Sasaki | Predictive factors for hyperprogressive disease during nivolumab as anti-PD1 treatment in patients with advanced gastric cancer[END_REF][START_REF] Wong | Hyperprogressive disease in hepatocellular carcinoma with immune checkpoint inhibitor use: a case series[END_REF][START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF][START_REF] Scheiner | Programmed cell death protein-1 (PD-1)-targeted immunotherapy in advanced hepatocellular carcinoma: efficacy and safety data from an international multicentre real-world cohort[END_REF][START_REF] Tachihara | Who will suffer from hyperprogressive disease in patients with advanced non-small cell lung cancer treated with PD-1/PD-L1 inhibitors[END_REF][START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF], tumor growth kinetic (TGK) [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Ji | Hyperprogression after immunotherapy in patients with malignant tumors of digestive system[END_REF][START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF], RECIST assessment at the first radiological evaluation [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF], or composite definitions including the time to treatment failure (TTF) [START_REF] Kamada | PD-1+ regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer[END_REF][START_REF] Kato | Hyperprogressors after Immunotherapy: Analysis of Genomic Alterations Associated with Accelerated Growth Rate[END_REF][START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF][START_REF] Tunali | Novel clinical and radiomic predictors of rapid disease progression phenotypes among lung cancer patients treated with immunotherapy: An early report[END_REF][START_REF] Russo | Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade[END_REF]. Only few data concerning the agreement between these definitions are available, and a consensus recommendation has not yet been established.

Several studies have assessed HP's predictive factors in patients treated with ICI, with heterogeneous results [START_REF] Ferrara | Hyperprogressive Disease in Patients With Advanced Non-Small Cell Lung Cancer Treated With PD-1/PD-L1 Inhibitors or With Single-Agent Chemotherapy[END_REF][START_REF] Champiat | Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1[END_REF][START_REF] Sasaki | Predictive factors for hyperprogressive disease during nivolumab as anti-PD1 treatment in patients with advanced gastric cancer[END_REF][START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF][START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF][START_REF] Kato | Hyperprogressors after Immunotherapy: Analysis of Genomic Alterations Associated with Accelerated Growth Rate[END_REF][START_REF] Tunali | Novel clinical and radiomic predictors of rapid disease progression phenotypes among lung cancer patients treated with immunotherapy: An early report[END_REF][START_REF] Russo | Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade[END_REF]. More recently, a metaanalysis pooled these data and identified five predictive factors of HP: serum lactate dehydrogenase (LDH) above the upper-normal limit, more than two metastatic sites, liver metastases, Royal Marsden Hospital prognostic score of 2 or above, and negative PD-L1 expression status [START_REF] Kim | Hyperprogressive Disease during Anti-PD-1 (PDCD1) / PD-L1 (CD274) Therapy: A Systematic Review and Meta-Analysis[END_REF].

The reported impact of HP on clinical outcomes proves to be heterogeneous among the studies. Some authors found significant association between HP and lower overall survival (OS), compared to patients with "classical" progressive disease without HP [START_REF] Ferrara | Hyperprogressive Disease in Patients With Advanced Non-Small Cell Lung Cancer Treated With PD-1/PD-L1 Inhibitors or With Single-Agent Chemotherapy[END_REF][START_REF] Sasaki | Predictive factors for hyperprogressive disease during nivolumab as anti-PD1 treatment in patients with advanced gastric cancer[END_REF][START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF], whereas others did not [START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF][START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF]. The medical literature is currently lacking data concerning the management of HP patients, and a therapeutic strategy has not yet been clearly defined.

We aimed to compare different available definitions of HP in consecutive patients with advanced NSCLC who were treated with ICI in the Thoracic Oncology Department of Bichat-Claude Bernard University Hospital, Assistance Publique des Hôpitaux de Paris (AP-HP), France, during a three-year period. We investigated whether certain markers could predict the occurrence of HP and whether HP would impact OS. Then, we described the management of such HPD patients and assessed the efficacy of either rapidly resuming chemotherapy or the potential impact of corticosteroids therapy, under the assumption that HPD was the consequence of an immune-mediated mechanism.

PATIENTS AND METHODS

Population and treatment

Data were retrospectively collected from all consecutive NSCLC patients with histologically confirmed diagnosis, who received ICI during a 38-month period between April 16, 2015, and June 22, 2018, in the Thoracic Oncology Department of a large university hospital in northern Paris, France.

Radiological assessments

TGR and TGK assessment required a minimum of two computed tomography (CT) scans before ICI initiation (the most recent scans were before baseline [CTpre] and baseline [CT0]) and one CT scan after ICI (CTpost). Only CT0 and CTpost were mandatory to assess the RECIST-based definition of HPD. A minimum of 15 days between CT scans was required, and CT0 had to be performed less than four weeks before ICI initiation. We retrospectively calculated sums of the target lesions, according to RECIST 1.1, at the CTpre, CT0, and CTpost scans, which were called Spre, S0, and Spost, respectively. The best tumoral response was defined as the best overall radiological response recorded under ICI, according to RECIST 1.1 criteria. This was confirmed by a second radiological evaluation for stable disease (SD) and objective response (OR) at least four weeks later. All radiological assessments were performed by an experienced specialist thoracic radiologist (AK) and two thoracic oncologists (BA and SB).

Definitions of HPD

All definitions of HP are summarized in Figure 1.

Tumor growth rate

A RECIST 1.1 evaluation of the CTpre, CT0, and CTpost scans was performed. TGR was measured according to the definition by Ferté et al. [START_REF] Ferté | Tumor growth rate is an early indicator of antitumor drug activity in phase I clinical trials[END_REF] and quantified for the target lesions only (non-target lesions and new lesions were excluded from the analysis). TGR was measured before (TGRpre) and after (TGRpost) ICI initiation. We assessed HPD using two different TGR-based definitions. The first definition was based on the ratio between TGRpost and TGRpre, which was called TGRratio and defined as progression according to RECIST 1.1 at CTpost and TGRpost/TGRpre ≥2 [START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF]. The second definition was based on the difference between TGRpost and TGRpre, which was called ∆TGR and defined as Spost/S0 >1.2 and TGRpost-TGRpre >100 [START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF].

Tumor growth kinetic

A RECIST 1.1 evaluation of CTpre, CT0, and CTpost scans was performed. TGK was measured according to the definition by Saâda-Bouzid et al. [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF] as TGKpre = (S0-Spre)/(T0-Tpre) and TGKpost = (Spost-S0)/(Tpost-T0), with Tpre, T0, and Tpost representing the time of CTpre, CT0, and CTpost scans, respectively. Only target lesions were included, whereas non-target lesions and new lesions were excluded from the analysis. The TGK-based HPD was defined as TGKpost/TGKpre ≥2 [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Ji | Hyperprogression after immunotherapy in patients with malignant tumors of digestive system[END_REF].

RECIST

A RECIST 1.1 evaluation of CT0 and CTpost scans was performed. For the RECISTbased HPD definition, the CTpost scan had to be performed up to eight weeks after ICI initiation. RECIST-based HPD was defined according to the definition by Matos et al. [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF] as an increase of ≥40% in the sum (S) of the target lesions observed on the CTpost scan compared with the CT0 scan (Spost/S0 ≥1.4) or an increase of ≥20% in the sum of the target lesions compared with the CT0 scan (Spost/S0 ≥1.2), plus the appearance of new lesions in at least two different organs on the CTpost scan.

Time to treatment failure

TTF was defined as the interval from ICI initiation to its discontinuation for tumoral progression (RECIST or non-RECIST progressive disease) or death. TTF-based HPD was defined as TTF <8 weeks after ICI initiation [START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF].

Statistical analysis

Survival was calculated using the Kaplan-Meier method, and the log-rank test was applied for statistical comparison. Univariate Cox proportional-hazard models were employed to assess hazard ratios (HR) with 95% confidence interval (95% CI). The median follow-up was calculated using the Kaplan-Meier reverse method.

Associations between HPD and various biological, clinical, and radiological variables were assessed using the Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables. The agreement between the five definitions (TGRratio, ∆TGR, TGK, RECIST, and TTF) was assessed using Cohen's kappa coefficient. Statistical analyses were performed using the R software (Version 4.0.2) and R studio software (Version 1.2.1335).

Ethics statement

According to French regulatory laws, all patients received written information and provided their oral consent for data collection. Patients' clinical charts were retrospectively collected from electronic files using a de-identified form. This study was approved by the Institutional Review Board of the French Learned Society for Respiratory Medicine (CEPRO 2020-037).

RESULTS

Description of the cohort

We retrospectively analyzed 169 patients with advanced NSCLC. The majority of patients (139/169; 82.2%) received the ICI nivolumab, which reflects the available registrations in 2015-17. Pembrolizumab was administered to 18/169 patients (10.7%), and the combination of nivolumab plus ipilimumab was given to 12/169 patients (7.1%) within clinical trials. Patients received ICI mainly in second-, third-, or subsequent-line settings (83.4%). The median age at ICI initiation was 63 years (IQR 56-70), and there was a majority of men (115/169; 68% vs. 54/169; 32% women) (Table 1). The median follow-up was 2.7 years (95% CI, 2.1-3.7), and the median overall survival (mOS) under ICI was 9.4 months (95% CI, 8.0-13.0).

Of these 169 patients (Figure 2), four patients were lost to follow-up, and one was excluded because of an unknown ICI initiation date. Thus, overall 164 patients were eligible for the TTF-based HP definition. Among these, nine did not have a tumor burden RECIST 1.1 evaluation of the CTpost scan (six died before the first radiological evaluation, and three had no tumor burden that was measurable by RECIST on the CTpost scan). Of these 155 patients, seven did not undergo a CT0 scan, five did not undergo a CTpre scan, and two had an interval between CT0 and CTpre scans that was shorter than 15 days. Thus, a total of 141 patients were eligible for HP definitions based on TGRratio, ∆TGR, and TGK. Among the 148 patients for whom tumor RECIST 1.1 evaluations of CTpost and CT0 scans were available, 13 underwent CTpost scanning more than eight weeks after ICI initiation, meaning that overall 135 patients were eligible for a RECIST-based HP definition.

Incidence of HPD and agreement between definitions

According to the TGRratio-, ∆TGR-, TGK-, RECIST-, and TFF-based definitions, HPD was experienced by 16/141 (11.3%), 8/141 (5.7%), 24/141 (17.0%), 13/135 (9.6%), and 52/164 (31.7%) patients, respectively.

The agreement between definitions is represented by a Venn diagram (Figure 3A) that shows TGRratio and ∆TGR were centrally located among all definitions. The Cohen's kappa coefficients between definitions were, in ascending order, 77% (TGRratio and TGK), 76% (∆TGR and RECIST), 57% (TGRratio and RECIST), 50% (RECIST and TGK), 46% (TGRratio and ∆TGR), 45% (RECIST and TTF), 41% (TGRratio and TTF), 33% (TGK and TTF), 32% (∆TGR and TGK), and 29% (∆TGR and TTF) (Figure 3B).

Association between HPD and OS

In accordance with their best tumor response, patients were divided into three groups: patients with HPD, patients with progressive disease (PD), and patients with disease control (SD or OR).

No statistically significant difference in mOS was found between the HPD and PD groups according to the TGRratio-based criteria, with mOS of 6.8 months (95% CI, 4.6-12.0) and 7.6 months (95% CI, 5.1-9.3), respectively (HR 1.20 [95% CI, 0.67-2.15]; P = 0.537) (Figure 4A). No statistically significant difference in mOS was found between the HPD and PD groups according to the ∆TGR-based criteria, with mOS of 4.7 months (95% CI, 3.0-NR) and 7.7 months (95% CI, 5.6-8.9), respectively (HR 1.24 [95% CI, 0.59-2.62]; P = 0.573) (Figure 4B). No statistically significant difference in mOS was found between the HPD and PD groups according to the TGK-based criteria, with mOS of 7.6 months (95% CI, 4.6-9.0) and 7.8 months (95% CI, 5.1-9.4), respectively (HR 1.34 [95% CI, 0.80-2.27]; P = 0.267) (Figure 4C). No statistically significant difference in mOS was found between the HPD and PD groups according to the RECIST-based criteria, with mOS of 8.0 months (95% CI, 4.6-NR) and 6.5 months (95% CI, 3.8-9.3), respectively (HR 1.07 [95% CI, 0.57-1.99]; P = 0.842) (Figure 4D). However, HPD was associated with shorter mOS compared to PD according to the TTF-based definition, with mOS of 1.9 months (95% CI, 1.7-4.6) and 8.3 months (95% CI, 5.9-10.5), respectively (HR 2.48 [95% CI, 1.62-3.80]; P < 0.001) (Figure 4E). The group of patients with disease control was associated with the longest mOS, compared to PD and HPD, according to any definition (P < 0.001) (Figure 4).

Variables associated with HPD

We analyzed the association between HPD, as based on the TGRratio definition, and several biological, clinical, and radiological variables. We chose the TGRratio definition to assess the markers associated with HPD, as it is considered the most widely used and approved definition of HPD.

In univariate analysis, four variables were significantly associated with HPD: a number of metastatic sites >2 (75.0% compared to 45.6%, P = 0.034), the presence of liver metastasis (43.8% compared to 10.4%, P = 0.002), the presence of pleural 2). In multivariate analysis, liver metastasis was the only variable found to be significantly associated with HPD (P = 0.003) (Table 2).

Management and follow-up of HPD patients

Next, we analyzed the clinical management of the 16 HPD patients, according to the TGRratio-based definition. The median age was 62 years (IQR 53-77), 11/16 (69%) were male, and 5/16 (31%) were female. The mOS after ICI initiation was 6.8 months (95% CI, 4.6-12.0), and the mOS after HPD diagnosis was 4.7 months (95% CI, 3.4-10.8).

Overall, 7/16 (44%) patients received chemotherapy after ICI discontinuation (three with carboplatin-paclitaxel, two with docetaxel, one with paclitaxel-bevacizumab, and one with 5-fluorouracil). Additionally, 1/16 (6%) received oral targeted therapy (osimertinib) because of an EGFR mutation. This resulted in overall median progression free survival (mPFS) upon treatment of 2.7 months (95%, CI, 2.1-NR).

The mOS observed in the group of patients treated with chemotherapy or targeted therapy and in the group of patients treated with best supportive care were 5.8 months (95% CI, 4.7-NR) and 2.5 months (95% CI, 1.2-NR), respectively, this difference being not statistically significant (p=0.2) (supplementary Figure S1).

Corticosteroid therapy was initiated in 7/16 patients (44%) (≥0.5mg/Kg/day prednisone or equivalent) as symptomatic treatment of HPD, which was based on the notion that such HPD could be the consequence of a poorly characterized immune mechanism. Only one case of clinical improvement was observed. The mOS observed in the group of patients treated with corticosteroids and in the group of patients treated without corticosteroids were 3.4 months (95% CI, 1.2-NR) and 5.7 months (95% CI, 4.7-NR), respectively, this difference being not statistically significant (p=0.2) (supplementary Figure S2).

DISCUSSION

HP is characterized by a rapid and unpredicted increase in tumor burden after exposure to ICI. It is a complex clinical and radiological phenomenon, with possibly dramatic consequences. Despite being reported in several retrospective studies, HP remains controversial because of a lack of agreed recommendations for its definition, evaluation, monitoring, and management.

In our single-center retrospective study, which reflected "real-life" conditions, we investigated the incidence of HP according to the five definitions that are currently available (TGRratio, ∆TGR, TGK, RECIST, and TTF). We found very heterogeneous rates between definitions (from 5.7% to 31.7%), with results consistent with previously reported studies [START_REF] Ferrara | Hyperprogressive Disease in Patients With Advanced Non-Small Cell Lung Cancer Treated With PD-1/PD-L1 Inhibitors or With Single-Agent Chemotherapy[END_REF][START_REF] Champiat | Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1[END_REF][START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF][START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF][START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF][START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF][START_REF] Russo | Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade[END_REF]. Agreement between the different definitions was also heterogeneous but globally poor, with a maximum of 77% agreement between TGRratio and TGK and a minimum of 29% between ∆TGR and TTF (Figure 3.A). HPD showed a trend to association with shorter OS compared to the group of patients with PD without HPD, but this difference only reached statistical significance using the TTF-based definition of HPD (Figure 3). Interestingly, we were able to confirm the correlation between liver metastasis and HPD [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF][START_REF] Kim | Hyperprogressive Disease during Anti-PD-1 (PDCD1) / PD-L1 (CD274) Therapy: A Systematic Review and Meta-Analysis[END_REF]. However, we also identified other predictive factors. The presence of pleural metastases and a short time spent between the last chemotherapy injection and ICI initiation was significantly associated with HPD in the univariate analysis and approaching statistical significance in the multivariate analysis (Table 2). Concerning our management of HPD, neither chemotherapy nor corticosteroids use were associated with statistically significant impact on overall survival. This is probably due to the low sample size.

In our opinion, HPD that is induced by ICI is a real phenomenon with major clinical consequences for some patients. Yet, this atypical pattern of tumor progression cruelly lacks a unique, easily applicable, clinically relevant, and agreed definition. Our results highlight the high heterogeneity of incidence and outcomes between the definitions available. Indeed, the clinical impact of HPD was highly influenced by the definition used, with only one definition (TTF <2 months) leading to a statistically significant difference in OS between the HPD and PD groups (Figure 4). Although the definition of HPD using TTF is likely clinically relevant, it is extremely unspecific. Indeed, this definition only considers death from all causes and ignores the speed of pre-ICI tumor growth. The RECIST definition proposed by Matos et al. [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF] is certainly easily applicable in clinical routine and considers the appearance of new lesions, but it does not take into account the pre-ICI tumor growth either. Furthermore, in our study, the difference in mOS between the HPD and PD groups using the RECIST definition did not significantly differ (Figure 4.D). The TGK definition initially proposed by Saâda-Bouzid et al. [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF] in head and neck cancers, which is used in several studies [START_REF] Ji | Hyperprogression after immunotherapy in patients with malignant tumors of digestive system[END_REF][START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF][START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF], considers pre-ICI tumor growth and appears to be easily calculable. However, this definition requires assessment of CT scans performed before the ICI baseline CT scans, and do not take new lesions into consideration. Indeed, this definition appears clinically irrelevant in our series, since the mOS of the HPD and PD groups differed by only 0.2 months using the TGK definition (Figure 4C). TGR-based definitions (TGRratio and ∆TGR) appear centrally located among all the definitions (Figure 3.A). Moreover, TGR-based definitions do consider pre-ICI tumor growth and use an exponential growth model. However, such definitions do not consider new lesions either, they need pre-ICI CT scans to be available, and require a complex mathematical formula for calculation [START_REF] Ferté | Tumor growth rate is an early indicator of antitumor drug activity in phase I clinical trials[END_REF]. In our series, the ∆TGR definition appeared more clinically relevant than TGRratio, with a difference in mOS between the HPD and PD groups of 3.0 months compared to 0.8 months (Figures

4.B and 4.A)

. Finally, the HPD definition that is based on TTF seemed to be the least precise, but the simplest and actually appears to be the best correlated with worse OS.

Another issue worth noting is the heterogeneity of HPD incidences that has been found in several studies that used the same definitions [START_REF] Champiat | Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1[END_REF][START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF][START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF][START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF]. This could be explained by the diversity of tumor types and number of previous chemotherapy lines received before ICI initiation. These results could also be explained by the heterogeneity of treatments received during the pre-ICI period (TGRpre, TGKpre). Indeed, receiving chemotherapy could decrease the tumor growth during this period, even if a tumoral progression was observed on a CT scan after this period.

This study highlights the high heterogeneity between the different HP definitions, suggesting that they do not represent the same tumor behavior. According to our data, the TGR-based definitions seem to represent the best compromise considering their specificity and the limitations and advantages of each definition proposed.

These HP definitions should be compared prospectively with overall survival being the primary endpoint. Furthermore, in our opinion, HP occurrence should be monitored as a secondary endpoint in the ICI clinical trials, in the same way as safety and adverse events.

With regards to the variables found to be associated with HPD, the presence of liver metastasis was strongly associated with HPD. This finding has been reported in several others studies [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF][START_REF] Kim | Hyperprogressive Disease during Anti-PD-1 (PDCD1) / PD-L1 (CD274) Therapy: A Systematic Review and Meta-Analysis[END_REF]. However, since liver metastases, among all possible metastasis sites, usually lead to a very poor prognosis in NSCLC patients, whether they also display a predictive impact on the specific efficacy of ICI as compared with chemotherapy for example is difficult to prove outside a randomized trial [START_REF] Ren | Prognostic effect of liver metastasis in lung cancer patients with distant metastasis[END_REF]. The identification of two new potential predictive factors (pleural metastasis and a short time interval between the last chemotherapy injection and ICI initiation) might also help physicians closely monitor for HPD. However, such variables must be assessed in larger series because the current limited sample size did not allow for statistical significance to be reached in our multivariate analysis. The association between HPD and a short-elapsed time between chemotherapy discontinuation and ICI initiation may reflect a fast, intrinsic TGR in these patients. This finding aligns with the inverse correlation between TGRpre (before ICI) and the response to ICI observed by Champiat et al. [START_REF] Champiat | Hyperprogressive Disease Is a New Pattern of Progression in Cancer Patients Treated by Anti-PD-1/PD-L1[END_REF] and Kim et al. [START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF]. Interestingly, HPD was not associated with tumor responses under previous chemotherapy in our study, which highlights the completely different mechanisms of action between those two anti-neoplastic treatments. Taking together, in our opinion, single-drug ICI should be given with caution to NSCLC patients with fast-growing tumors, liver metastasis, or pleural metastasis. In this population, physicians may initiate monitoring of tumor progression using CT scans early on, which could be performed at 4-6 weeks after ICI initiation. Furthermore, ICI should not be given beyond progression, whereas chemotherapy should be resumed instead. Although the physiological mechanism of HPD is not clearly understood, it can already be assumed that the tumor's microenvironment plays a major role, including cancer-associated fibroblast, tumorassociated macrophages, and T-reg lymphocytes [START_REF] Champiat | Hyperprogressive disease: recognizing a novel pattern to improve patient management[END_REF]. A combination of treatments that target the microenvironment could thus become the standard in the future [START_REF] Murciano-Goroff | The future of cancer immunotherapy: microenvironment-targeting combinations[END_REF].

To our knowledge, the current study is the first to report the impact of clinical management of HPD patients under ICI. Using chemotherapy was associated with a trend to longer OS (+3,3 months of mOS) without statistically significant impact.

However, in this study, patients treated with chemotherapy were most likely in better clinical condition, which consists of an important bias. Conversely, using immunosuppressive corticosteroids as symptomatic treatment for HPD, based on the hypothesis of an immune-mediated mechanism of HPD, was associated with a trend to shorter OS (-2.3 months) without statistically significant impact. However, corticosteroids may have been prescribed to patients with more symptomatic disease i.e more intense HP and thus with worse prognosis.

Our study has several limitations, most of which relate to its retrospective, singlecenter nature and the lack of statistical power for subset analyses. Only 129 of 169 patients were able to be evaluated using all five definitions. Considerable heterogeneity was also observed in terms of treatment during the pre-ICI period (chemotherapy, targeted therapy, or no treatment) and the number of previous chemotherapy lines received (from none to four). These differences may explain our results. PD-L1 expression was missing for 26.6% of patients, mainly with squamouscell carcinoma (SCC). The molecular profile was not available for 25.4% of these patients, again mainly with SCC. Moreover, STK11 and KEAP1 could adversely influence ICI efficacy, whereas p53 and K-Ras mutations could favor an ICI treatment response. We were not able to study every published definition of HPD, particularly the composite definitions that regroup TTF or TGK with TTF or a modification in tumor load [START_REF] Kim | Comprehensive Clinical and Genetic Characterization of Hyperprogression Based on Volumetry in Advanced Non-Small Cell Lung Cancer Treated With Immune Checkpoint Inhibitor[END_REF][START_REF] Kato | Hyperprogressors after Immunotherapy: Analysis of Genomic Alterations Associated with Accelerated Growth Rate[END_REF][START_REF] Tunali | Novel clinical and radiomic predictors of rapid disease progression phenotypes among lung cancer patients treated with immunotherapy: An early report[END_REF].

Conclusion

We identified HPD in 11.3%, 5.7%, 17.0%, 9.6%, and 31.7% of NSCLC patients who were treated with ICI, according to TGRratio-, ∆TGR-, TGK-, RECIST-, and TFFbased definitions, respectively. Agreement between the different definitions was 21 highly heterogeneous and globally poor, with a maximum of 77% agreement between TGRratio and TGK and a minimum of 29% between ∆TGR and TTF. HPD was systematically associated with shorter OS compared to PD without HPD, but this difference only reached statistical significance when using the TTF-based definition.

A number of metastatic sites >2, liver metastasis, pleural metastasis, and a short time interval between chemotherapy discontinuation and ICI initiation were associated with HPD in the univariate analysis. The only variable associated with HPD in the multivariate analysis was existing liver metastasis, which suggests cautious ICI use in this subset of patients. In TGRratio-based HP patients, although resuming chemotherapy was associated with numerically longer survival, such difference did not reach statistical difference. Similarly, corticosteroids use was not associated with statistically significant impact on overall survival, although such group showed especially poor survival. Additional studies are required to generate agreed recommendations for HPD evaluation, definition, and management. HPD is assessed using five different definitions: TGRratio [START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF], ∆TGR [START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF], TGK [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Ji | Hyperprogression after immunotherapy in patients with malignant tumors of digestive system[END_REF], RECIST [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF], and TTF [START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF]. TGR, tumor growth rate; TGK, tumor growth kinetic; RECIST, Response Evaluation Criteria in Solid Tumors; TTF, time to treatment failure; HPD, hyperprogressive disease. HPD is assessed using five different definitions: TGRratio [START_REF] Kanjanapan | Hyperprogressive disease in early-phase immunotherapy trials: Clinical predictors and association with immune-related toxicities[END_REF], ∆TGR [START_REF] Kas | Clarification of Definitions of Hyperprogressive Disease During Immunotherapy for Non-Small Cell Lung Cancer[END_REF], TGK [START_REF] Saâda-Bouzid | Hyperprogression during anti-PD-1/PD-L1 therapy in patients with recurrent and/or metastatic head and neck squamous cell carcinoma[END_REF][START_REF] Ji | Hyperprogression after immunotherapy in patients with malignant tumors of digestive system[END_REF], RECIST [START_REF] Matos | Capturing Hyperprogressive Disease with Immune-Checkpoint Inhibitors Using RECIST 1.1 Criteria[END_REF], and TTF [START_REF] Kim | Hyperprogressive disease during PD-1/PD-L1 blockade in patients with non-small-cell lung cancer[END_REF]. OS, overall survival; HPD, hyperprogressive disease; PD, progressive disease according to RECIST 1.1 without HPD; SD/OR, stable disease or objective responses according to RECIST 1.1; TGR, tumor growth rate; TGK, tumor growth kinetic; RECIST, response criteria in solid tumors; TTF, time to treatment failure.

  metastasis (50% compared to 23.2%, P = 0.033), and a short median interval time between the last chemotherapy injection and ICI initiation (21.0 days [IQR 19.0-23.0] compared to 49.5 days [IQR 27.2-138.5], P < 0.001) (Table

  : Fisher test for categorical variables and Mann Whitney test for continuous variables. **Former smokers: have smoked at least 100 cigarettes in their lifetime and abstained from smoking for > 12 months. ¶≥equivalent to prednisone 10mg/day. E1L3N antibody on Leica Bond automat. Derived neutrophil-to-lymphocyte ratio= neutrophil count/(leucocyte countneutrophil count).HPD, hyperprogressive disease; IQR, interquartile; ECOG, Eastern CooperativeOncology Group; PS, performance status; ICI, immune checkpoint inhibitor; PD-L1, programmed cell death-ligand 1; PD, progressive disease, SD, stable disease; PR, partial response, CR, complete response; CNS, central nervous system; LDH, lactate dehydrogenase.

Figure 1 .

 1 Figure 1. Definitions of hyperprogressive disease (inspired by Matos et al. [33]).CT, computed tomography; HPD, hyperprogressive disease; TGR, tumor growth rate; TGK, tumor growth kinetic; TTF, time to treatment failure; ICI, immune checkpoint inhibitor; Spost and S0, sum of the target lesion according to RECIST 1.1 at CTpost and CT0 scans respectively; PD, progressive disease.

Figure 2 .

 2 Figure 2. Patient disposition.NSCLC, non-small cell lung cancer; ICI, immune checkpoint inhibitor; CT0, baseline computed tomography; CTpre, most recent computed tomography before baseline; CTpost, first computed tomography after ICI initiation; TTF, time to treatment failure; HP, hyperprogression; TGR, tumor growth rate; TGK, tumor growth kinetic.

Figure 3 .

 3 Figure 3. Concordance between definitions. (A) Venn diagram including the 129 patients evaluable for all five HPD definitions. (B) Cohen's Kappa coefficient between definitions. The Cohen's Kappa coefficient corresponds to the proportion of patients with HPD in common among all patients assessed as having of HPD by at least one definition.

Figure 4 .

 4 Figure 4. Overall survival of hyperprogressive disease, progressive disease and stable disease/objective response groups according to TGRratio-(A), ∆TGR-(B), TGK-(C), RECIST-(C), and TTF-(D) based definitions.

TABLES 590 Table 1 . Patient baseline characteristics

 5901

	591
		N=169
	Median age, years (IQR)	63 (56-70)
	Sex
	Male	115 (68%)
	Female	54 (32%)
	Smoking status
	Current	(39.6%)
	Former*	(54.4%)
	Never	(5.3%)
	Missing	(0.6%)
	ECOG PS
	0	(15.4%)
	1	(42.6%)
	2	(36.1%)
	3-4	10 (5.9%)
	Tumor stage
	II	(1.2%)
	III	(14.2%)
	IV	143 (84.6%)
	Recurrence after initial locoregional	(23.1%)
	treatment**
	Histology
	Adenocarcinoma	101 (59.8%)
	Squamous cell	(29.6%)
	Large-cell	(4.1%)
	Sarcomatoid	(1.8%)
	Other	(4.7%)
	Molecular profile
	Available	126 (74.6%)
	Non-available ¶	(25.4%)
	EGFR-mutated oncogene-addicted
	Yes	(3.0%)
	No	121 (71.6%)
	Missing	(25.4%)
	PD-L1 expression
	<1%	(18.9%)
	1-49%	(21.9%)
	≥50%	(32.5%)
	Missing	(26.6%)
	Immune checkpoint inhibitor
	Nivolumab	139 (82.2%)
	Pembrolizumab	(10.7%)
	Nivolumab and ipilimumab	12 (7.1%)
	Number of prior chemotherapy regimens
	0	(16.6%)
	1	(49.7%)

Table 2 . Hyperprogressive disease and clinical, radiological and biological 603 variables (univariate and multivariate analysis) in evaluable patients (n=141)

 2

	604
	Univariate analysis	Total (n=141)	HPD (n=16)	Non-HPD (n=125)	P-value*
	Median age, years (IQR)	64 (57-71)	62 (56-75)	64 (57-71)	0.93
	Sex				0.79
	Male	91 (64.5%)	11 (68.8%)	(64.0%)
	Female	50 (35.5%)	5 (31.2%)	(36.0%)
	ECOG PS				1.00
	0-1	90 (63.8%)	10 (62.5%)	(64.0%)
	2-4	51 (36.2%)	6 (37.5%)	(36.0%)
	ICI				0.11
	Nivolumab	116 (82.3%)	13 (81.2%)	103 (82.4%)
	Nivolumab + ipilimumab	12 (8.5%)	3 (18.8%)	(7.2%)
	Pembrolizumab	13 (9.2%)	0 (0.0%)	(10.4%)
	Smoking status				0.10
	Current	70 (49.6%)	5 (31.2%)	(52.0%)
	Former**	63 (44.7%)	9 (56.2%)	(43.2%)
	Never	7 (5.0%)	2 (12.5%)	(4.0%)
	Missing	1 (0.7%)	0 (0.0%)	(0.8%)
	Occupational exposure				0.19
	Yes	28 (19.9%)	1 (6.2%)	(21.6%)
	No	112 (79.4%)	15 (93.8%)	(77.6%)
	Missing	1 (0.7%)	0 (0.0%)	(0.8%)
	History of autoimmune disease				0.58
	Yes	7 (5.0%)	1 (6.2%)	(4.8%)
	No	134 (95.0%)	15 (93.8%)	119 (95.2%)
	History of respiratory infection				0.11
	treated with antibiotics within
	6 months preceding ICI
	Yes	11 (7.8%)	3 (18.8%)	(6.4%)
	No	130 (92.2%)	13 (81.2%)	117 (93.6%)
	Use of corticosteroids ¶				0.69
	Yes	16 (11.3%)	1 (6.2%)	(12.0%)
	No	125 (88.7%)	15 (93.8%)	110 (88.0%)
	Tumor stage				0.47
	Metastatic	118 (83.7%)	15 (93.8%)	103 (82.4%)
	Non-metastatic	23 (16.3%)	1 (6.2%)	(17.6%)
	Number of metastatic sites				0.034
	≤2	72 (51.1%)	4 (25.0%)	(54.4%)
	>2	69 (48.9%)	12 (75.0%)	(45.6%)
	Lung metastasis				0.41
	Yes	54 (38.3%)	8 (50.0%)	(36.8%)
	No	87 (61.7%)	8 (50.0%)	(63.2%)
	Pleural metastasis				0.033
	Yes	37 (26.2%)	8 (50.0%)	(23.2%)
	No	104 (73.8%)	8 (50.0%)	(76.8%)
	Bone metastasis				0.25
	Yes	42 (29.8%)	7 (43.8%)	(28.0%)

ACKNOWLEDGEMENTS

None.

nor corticosteroids use was associated with statistically significant impact on overall survival.

Conclusion:

We found fairly heterogeneous HP rates using different definitions. TTF was the only definition leading to significantly worsened OS. Further studies are needed to provide consensus recommendations for the assessment, definition, and management of HP, whose existence is likely real.