Mariana Limones-Mendez

Audray Dugrand-Judek

Cloé Villard

Victoire Coqueret

Yann Froelicher

Frédéric Bourgaud

Alexandre Olry

Alain Hehn
email: alain.hehn@univ-lorraine.fr

Yann Froelicher²

Convergent evolution leading to the appearance of furanocoumarins in citrus plants

Keywords: Cytochrome P450, CYP82, Furanocoumarins, Citrus, Convergent evolution

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

Introduction

Furanocoumarins (FCs) are specialized metabolites that have been widely reported as being involved in the defense of plants against aggressors [START_REF] Berenbaum | Toxicity of a Furanocoumarin to Armyworms -Case of Biosynthetic Escape from Insect Herbivores[END_REF]. These molecules, although described in higher plants, are mainly distributed in 4 families: Apiaceae, Moraceae, Fabaceae and Rutaceae [START_REF] Bourgaud | Recent advances in molecular genetics of furanocoumarin synthesis in higher plants[END_REF][START_REF] Murray | The Natural Coumarins: occurence, chemistry and biochemistry[END_REF]. Two main kinds of FCs exist depending on the position of the furan ring on the coumarin core molecule. The tracks leading to either linear or angular molecules are catalyzed by enzymes belonging to the UbiA Prenyltransferases. A prenylation occurring on C6 leads to the synthesis of the linear demethylsuberosin whereas a prenylation on C8 gives osthenol (Figure 1) [START_REF] Karamat | A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley[END_REF][START_REF] Munakata | Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis[END_REF][START_REF] Munakata | Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants[END_REF] The presence of both isomers are, thus far, only reported in Apiaceae and are reported to reflect a coevolutionary process between plants and caterpillars [START_REF] Berenbaum | Coumarins and Caterpillars: a case for coevolution[END_REF]. These four plant families are quite distant from a phylogenetic point of view, and the independent appearance of these molecules remains unexplained at the molecular level. The first enzymes involved in the synthesis of these molecules to be characterized were a psoralen synthase (CYP71AJ1-3) and an angelicin synthase (CYP71AJ4) [START_REF] Larbat | Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis[END_REF][START_REF] Larbat | Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis[END_REF]. Both enzymes belong to the cytochrome 450 enzyme superfamily. CYP71AJ4, identified in Pastinaca sativa, may result from the duplication of CYP71AJ3 followed by neofunctionalization [START_REF] Larbat | Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis[END_REF]. This assumption is consistent with the high amino acid sequence identity shared by the two proteins (69.8 %) and is reinforced by the results published by Roselli et al., who showed their clustering in the parsnip genome (the genes are 7.6 kb apart) [START_REF] Roselli | A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip[END_REF]. Relatively minor changes in the peptidic sequence of psoralen synthase therefore allowed the appearance of the angular FC angelicin and subsequent angular molecules, thus participating in the arms race between plants and insects. An extensive study of this specific CYP71AJ subfamily has led to the identification of numerous orthologous genes, but their function could not be demonstrated [START_REF] Dueholm | Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily[END_REF].

Krieger et al have highlighted another cytochrome P450 subfamily, CYP71AZ, involved in the synthesis of FC [START_REF] Krieger | AZ P450 Subfamily: A[END_REF]. CYP71AZ4 is able to hydroxylate psoralen, leading to the synthesis of xanthotoxol, whereas CYP71AZ1 and CYP71AZ6 catalyze the hydroxylation of xanthotoxin, generating 5-OH-xanthotoxin (Figure 1). Interestingly, CYP71AZ6 seems quite specific to a single molecule (xanthotoxin), while CYP71AZ4 can transform different types of molecules, including a FC (psoralen) and coumarins (6-methoxycoumarin, scopoletin, 7methoxy-3-methylcoumarin, and 7-methoxycoumarin) [START_REF] Krieger | AZ P450 Subfamily: A[END_REF]. Finally, Krieger et al also showed that CYP71AZ3seems to be specific and dedicated to the hydroxylation of esculetin, another coumarin [START_REF] Krieger | AZ P450 Subfamily: A[END_REF]. All higher plants are able to produce coumarins and the fact that some members of the CYP71AJ and CYP71AZ families are involved in the synthesis of coumarins and/or FCs [START_REF] Dueholm | Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily[END_REF][START_REF] Krieger | AZ P450 Subfamily: A[END_REF] provides new insights for understanding the evolutionary processes leading to the appearance of FCs that might have diverged from coumarins. To date, the different enzymes described to be involved in the synthesis of FCs belong to the same large CYP71 cytochrome P450 family. This family is responsible for the synthesis of highly diversified and specialized metabolites. For example, members of the family are involved in the synthesis of artemisin [START_REF] Teoh | Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin[END_REF], menthofuran [START_REF] Bertea | Demonstration That Menthofuran Synthase of Mint (Mentha) Is a Cytochrome P450 Monooxygenase: Cloning, Functional Expression, and Characterization of the Responsible Gene[END_REF], camalexin [START_REF] Nafisi | Arabidopsis cytochrome P450 monooxygenase 71A13 catalyzes the conversion of indole-3-acetaldoxime in camalexin synthesis[END_REF][START_REF] Schuhegger | CYP71B15 (PAD3) catalyzes the final step in camalexin biosynthesis[END_REF], cyanogenic glucosides [START_REF] Kahn | Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench[END_REF] or DIMBOA [START_REF] Jonczyk | Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: Characterization of Bx6 and Bx7[END_REF]. The diversity of chemical structures, biosynthetic pathways and plant species make this enzyme family a major actor in the adaptation of plants to their environments.

The CYP71 family is not the only cytochrome P450 that seems to be involved in the synthesis of such molecules (Figure S5). The functional characterization of CYP82C2 and CYP82C4 isolated from Arabidopsis thaliana reinforces the assumption that FCs are derived from coumarins. Kruse et al showed that these enzymes convert in vivo xanthotoxin to 5-OHxanthotoxin when overexpressing Arabidopsis mutants are incubated in a culture media containing the substrate (Figure 1) [START_REF] Kruse | In planta biocatalysis screen of P450s identifies 8-methoxypsoralen as a substrate for the CYP82C subfamily, yielding original chemical structures[END_REF], while this plant is not otherwise able to synthesize this kind of molecule. In 2018, Rajniak et al. showed that CYP82C4 is also involved in the oxidation of fraxetin (a coumarin), leading to the synthesis of sideretin [START_REF] Rajniak | Biosynthesis of redox-active metabolites in response to iron deficiency in plants[END_REF], which is described as being important in iron homeostasis [START_REF] Siwinska | Scopoletin 8-hydroxylase: a novel enzyme involved in coumarin biosynthesis and iron-deficiency responses in Arabidopsis[END_REF]. Taken together, these results indicate that the functionalization of coumarins into FCs may be realized in different ways (CYP71 or CYP82) throughout higher plants, thus supporting the convergent evolution hypothesis.

Rutaceae are reported to produce coumarins and linear FCs [START_REF] Dugrand | Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)[END_REF][START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF][START_REF] Milesi | a promising species for the production of furocoumarins[END_REF], but less is known about the enzymes involved in this synthesis. However, a cytochrome P450 associated with an evolutionary process related to the synthesis of FC was described. Gravot et al. showed that the cinnamate 4-hydroxylase (C4H) of Ruta graveolens was adapted to the presence of FCs.

Although inhibited by psoralen and xanthotoxin through mechanism-based inactivation, the C4H isolated from R. graveolens displayed a lower sensitivity to these molecules than the C4H of Helianthus tuberosus [START_REF] Gravot | Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: Cloning and characterization of a C4H from a psoralen producing plant -Ruta[END_REF]. Citruses belong to the Rutaceae family. The consumption of these fruits has a wide range of beneficial effects for human health, since they are rich in vitamin C, folates, alimentary fibers and minerals [START_REF] Codoñer-Franch | Citrus as Functional Foods[END_REF]. In addition to these positive effects, it has also been demonstrated that the presence of FCs generates deleterious effects such as phototoxicity and photosensitivity [START_REF] Nigg | Phototoxic coumarins in limes[END_REF][START_REF] Mccloud | Furanocoumarin content and phototoxicity of rough lemon (Citrus jambhiri) foliage exposed to enhanced ultraviolet-B (UVB) irradiation[END_REF][START_REF] Koh | Phytophotodermatitis due to the application of citrus hystrix as a folk remedy[END_REF][START_REF] Monteiro | Drug-induced photosensitivity: Photoallergic and phototoxic reactions[END_REF] and a drug interaction effect known as "the grapefruit juice effect" that can cause overdose in humans by inhibiting liver cytochromes P450 involved in the detoxification of xenobiotics [START_REF] Bailey | Grapefruit juice-drug interactions[END_REF][START_REF] Bailey | Grapefruitfelodipine interaction: effect of unprocessed fruit and probable active ingredients[END_REF][START_REF] Bailey | Grapefruit juice-drug interactions[END_REF] In this work, we investigated the biosynthesis of FCs in citrus species, focusing on the CYP82 family. The expression pattern of four genes and their functional characterization showed that two of them perform the hydroxylation of xanthotoxin to produce 5-OHxanthotoxin. The physiological relevance of this result is supported by a quantitative PCR approach that shows a clear correlation between gene expression and the amount of these molecules.

Material and methods

Citrus data

Citrus genomic and transcriptomic data were collected from the Phytozome project database (https://phytozome.jgi.doe.gov/) and the RNAseq database published by Voo et al [START_REF] Voo | Assessing the Biosynthetic Capabilities of Secretory Glands in Citrus Peel1[W][OA[END_REF].

Phylogenetic analysis

The tree was built with the maximum likelihood (PhyML) method. The alignment was performed with MUSCLE using the default settings [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]. The variable regions creating gaps, the C-terminal region and the N-terminal membrane anchor were removed. One hundred iterations were performed, and the bootstrap values were calculated. The tree was rooted with CYP71AJ3, CYP71AJ4, CYP71AZ3, CYP71AZ5 and CYP71AZ6 from P. sativa, CYP71AU3, CYP71AU4 and CYP71AH4 from Vitis vinifera, and CYP83B1 and CYP83A1 from A. thaliana.

RT-PCR and cloning strategy

RNA was extracted from flavedo of C. paradisi Star Ruby and C. hystrix fruits using the E.Z.N.A.® Plant RNA kit following the manufacturer's recommendations (Omega Bio-Tek).

Residual genomic DNA was removed using amplification-grade DNAse I (Sigma-Aldrich).

Reverse transcription was performed with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). The coding sequences were amplified with high-fidelity PrimeSTAR ® Max DNA polymerase (Takara) and ligated into the pCR8 plasmid using the pCR8 TA/GW/Topo kit (Invitrogen). The designed primers were as follows: CYP82D64 GCC GTA CAA AGA AGC AGA AAG 3'. For each ORF, we added a 6xHIS tag before the stop codon. Finally, the ORF was transferred into the pYeDP60 GW [START_REF] Krieger | AZ P450 Subfamily: A[END_REF] plasmid using Gateway LR Clonase II Enzyme mix (Invitrogen).

Expression of proteins and enzymatic characterization

The recombinant proteins were produced in a yeast expression system [START_REF] Pompon | Yeast expression of Animal and Plant P450s in optimized redox environments[END_REF]. The culture conditions, the preparation of microsomes and the biochemical screening were as described by Krieger et al. (2018).

qPCR

Reverse transcription was performed with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems). The primers used were EF1α Fw 5'-TGG TGA TGC TGG GTT TGT TA-3', EF1α Rev 5'-CAC GCT CTT GAT GAC TCC AA-3' for the reference gene; CYP82D64 Fw (1) 5'-GGC GCC ATG CTT TCC A-3', CYP82D64 Rev (1) 5'-ACG ACC GGG AAA TTC TTG TG-3'; and CYP82D64 Fw (2) 5'-CAC CAC GTG AAG CAA TGG AA-3', CYP82D64 Rev (2) 5'-TGC CAG CCG GGA CAT G-3'. qPCR was performed with SYBR Premix Ex Taq (Takara) based on the manufacturer's recommendations. The amplification program was 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. The PCR was followed by a single run at 95°C for 15 sec, 60°C for 60 sec, and 95°C for 15 sec in order to assess the melting curve and therefore the specificity of the PCRs.

Enzymatic characterization

All the enzymatic reactions were conducted in 0.1 M KPi buffer. pH and temperature range were set between 6.0 to 8.0 and 15 °C to 40 °C. For the kinetic determination of optimal temperature and pH, saturating concentrations of substrates were used (100 µM) in phosphate buffer. For km kinetic parameter determination, xanthotoxin concentration was set between 0.5 and 300 µM and the reaction was conducted at optimal pH and temperature. 20 µl of the yeast microsomal preparation was added to each enzymatic incubation and enzymatic reactions were running during 5 min and stopped by the addition of 10 µl HCl 1N. Substrates and the resulting products were analyzed by HPLC at a wavelength of 320 nm as described by

Phytochemical analysis

Methanol extraction and phytochemical analyses were performed as previously described by Dugrand et al. [START_REF] Dugrand | Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)[END_REF].

Results

Identification of cytochrome P450 coding sequences belonging to the CYP82 subfamily in Citrus genomic and microarray databases.

The Phytozome database (https://phytozome.jgi.doe.gov/) contains the genomes of Citrus sinensis and Citrus clementina. These genomes were annotated [START_REF] Xu | The draft genome of sweet orange (Citrus sinensis)[END_REF][START_REF] Wu | Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication[END_REF], but regarding cytochromes P450, the annotation was limited to the superfamily level. To complete this first analysis, we compared our results with the database provided by Voo et al [START_REF] Voo | Assessing the Biosynthetic Capabilities of Secretory Glands in Citrus Peel1[W][OA[END_REF]. This analysis showed that 2 sequences of the 8 selected previously, i.e., CYP82D64 (i.e., contigs Cit.29478 and Cit.2373 in the database) and CYP82D79 (i.e., Cit.5945 in the database), had differential expression (Table 1) between flavedo oil cavities (tissue with a high concentration of FCs, [START_REF] Dugrand | Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)[END_REF][START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF]) and albedo tissue (lack of FCs). These first analyses supported these two sequences as strong candidates involved in FC biosynthesis in Citrus spp.

To obtain a more detailed overview of the distribution of this P450 subfamily, we performed a phylogenetic analysis including 91 members of the CYP82 subfamily available in databases in the literature and various other databases (Supplemental figure 1). The tree was rooted with 11 sequences belonging to the CYP71 or CYP83 subfamily. This analysis showed that the already-characterized CYP82C2 and CYP82C4 were located on different branches than the CYP82D identified in citrus plants (Figure 2). Within the more specific citrus clade, we could not distinguish different groups that could provide clues for functional characterization since the genes were distributed between C. sinensis (unable to produce FCs) and C. clementina (producing FCs) [START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF].

Functional characterization of the CYP82D candidates.

In our analysis, CYP82D64, CYP82D83, and CYP82D179 were detected in both plants, whereas CYP82D82 and CYP82D81 were specific to C. sinensis and CYP82D92 was found only in C. clementina. Since the aim of our study was to identify genes involved in the synthesis of FCs, we focused our attention on CYP82D92, CYP82D64, CYP82D83, and CYP82D179, detected either in C. clementina alone or in both plants. The study performed by Krieger et al showed that minor modification of the sequence of CYP71AZ can lead to significant changes in substrate specificity and/or enzyme activity. To limit the risk of cloning gene variants displaying low FC biosynthetic activities, we amplified and cloned the cDNA coding sequences from C. paradisi, a citrus species producing diverse and large amounts of FCs [START_REF] Dugrand | Coumarin and Furanocoumarin Quantitation in Citrus Peel via Ultraperformance Liquid Chromatography Coupled with Mass Spectrometry (UPLC-MS)[END_REF][START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF]. The amplified sequences of C. paradisi showed minor changes (98 to 100% identity) in comparison to the genomic sequences of C. sinensis or C. clementina. These differences were, however, not located in the enzyme active sites [START_REF] Gotoh | Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences[END_REF].

To identify their function, the 4 His-tagged P450s were expressed in a yeast heterologous expression system, and their expression was assessed by Western blot (Supplemental figure 2). CYP82D64, CYP82D92 and CYP82D179 were efficiently produced and further incubated in the presence of NADPH and the different intermediates of the biosynthetic pathway (demethylsuberosin, marmesin, xanthotoxol, xanthotoxin, bergaptol, bergapten, 5-OHxanthotoxin, 8-OH bergapten and isopimpinellin) (Figure 1). Only metabolization of xanthotoxin could be observed in the presence of CYP82D64 (Figure 4A). Several studies have provided evidence that only a few modifications to an amino acid sequence can alter the specificity of an enzyme. Concerning the synthesis of coumarins and FCs, this has been shown for CYP71AZ3, 4 and 6, which are able to transform esculetin, psoralen and xanthotoxin, respectively [START_REF] Krieger | AZ P450 Subfamily: A[END_REF]. We cloned the CYP82D64 orthologous gene from combava (C. hystrix) fruits, a citrus species known to be a very high producer of FCs [START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF]. The CDS that we obtained from this plant was named CYP82D64v2 and showed several differences compared with the C. paradisi coding sequence (CYP82D64v1), especially in the substrate recognition sites SRS3, 4 and 6, which were reported to be important for the enzymatic activity of cytochromes P450 [START_REF] Gotoh | Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences[END_REF] (Figure 4). Microsomes prepared from yeast overexpressing CYP82D64v2 were able to convert xanthotoxin into 5-OH xanthoxine at the same rate as CYP82D64v1. Surprisingly, CYP82D64v2 was also able to transform herniarin (7-methoxycoumarin) at a rather low rate into a product that was identified as 5-hydroxy-7methoxycoumarin (Figure 5).

Enzymatic characterization of CYP82D64v1 and v2

The optimal pH of both CYP82D64s was assessed in phosphate buffer with a pH ranging from 6.0 to 8.0. Under these conditions, we showed that the hydroxylation activity of xanthotoxin was effective over the entire pH range and was optimal at a pH of 7.3

(Supplemental figure 3). The activity in the presence of herniarin could not be detected below a pH of 6.4, but the optimal activity was obtained at a pH of 7.2. The optimal temperature of the hydroxylation of both molecules was close to 28°C (27.8°C for xanthotoxin and 28.1°C

for herniarin) (Supplemental figure 4). Finally, the kinetic constants (km) of CYP82D64v1 and v2 were determined to be 4.6+/-1,7 μM and 7.4+/-4.0 µM for xanthotoxin, respectively.

These km values were rather low and consistent with those reported in studies performed on other cytochromes P450. This specificity may reflect a physiological activity. Because of the too-low enzymatic activity, the km could not be determined for herniarin.

In vivo expression of CYP82D64

Although the km of the reaction was consistent with a physiological function, we wanted to assess if there was a relationship between the expression level of the mRNA coding for CYP82D64 and the amounts of xanthotoxin and 5-OH-xanthotoxin produced in C.

paradisi fruit at different stages of maturity: young, intermediate and mature (Figure 6A). We observed that the expression level of CYP82D64 mRNA significantly decreased during fruit development (Figure 6B). It was reduced 2.3-fold between the young and intermediate fruits

Discussion

The Rutaceae and Apiaceae families are quite distant in terms of angiosperm evolution. Interestingly, however, they are both able to produce FCs [START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF][START_REF] Milesi | a promising species for the production of furocoumarins[END_REF], although the diversity of molecules differs between them. Apiaceae, including parsnip, are able to synthesize both linear (i.e., psoralen derivatives) and angular isomers (i.e., angelicin derivatives) [START_REF] Larbat | Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis[END_REF]. Rutaceae and, in particular, the Citrus genus produce only a linear isomer [START_REF] Dugrand | The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely. Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways[END_REF]. The first step involved in the production of these molecules concerns the C2'hydroxylation of coumaroyl-CoA, leading to the synthesis of umbelliferone [START_REF] Roselli | A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip[END_REF][START_REF] Vialart | A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl coA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants[END_REF][START_REF] Kawai | Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants[END_REF].

Umbelliferone is commonly described in many higher plants. This step is catalyzed by 2oxoglutarate-dependent dioxygenase in R. graveolens [START_REF] Vialart | A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl coA 2'-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants[END_REF] and P. sativa [START_REF] Roselli | A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip[END_REF], which belong to the same DIOX30 clade [START_REF] Kawai | Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants[END_REF], and therefore seems to be well conserved across angiosperms.

The prenylation of umbelliferon has not yet been described in citrus but was reported for

Apiaceae plants and is catalyzed by a UbiA prenyltransferase [START_REF] Karamat | A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley[END_REF]. A recent study by Munakata and colleagues showed that in Ficus carica, a Moraceae able to produce FCs, prenyltransferase had a different evolutionary origin than the enzyme involved in the synthesis of demethylsuberosin in Apiaceae. It might therefore be possible that convergent evolution occurred at this stage [START_REF] Munakata | Convergent evolution of the UbiA prenyltransferase family underlies the independent acquisition of furanocoumarins in plants[END_REF].

Studies based on biochemical approaches performed in the 1980s pointed out that in Apiaceae, many enzymatic steps happening after the synthesis of demethylsuberosin (Figure 1) were catalyzed by enzymes belonging to the cytochrome P450 superfamily [START_REF] Hamerski | Elicitor-induced biosynthesis of psoralens in Ammi majus L. suspension cultures. Microsomal conversion of demethylsuberosin into (+)marmesin and psoralen[END_REF][START_REF] Hamerski | Biosynthesis of psoralens. Psoralen 5-monooxygenase activity from elicitor-treated Ammi majus cells[END_REF]. These results were confirmed by the identification of the CYP71AJ [START_REF] Larbat | Isolation and functional characterization of CYP71AJ4 encoding for the first P450 monooxygenase of angular furanocoumarin biosynthesis[END_REF][START_REF] Larbat | Molecular cloning and functional characterization of psoralen synthase, the first committed monooxygenase of furanocoumarin biosynthesis[END_REF] and CYP71AZ subfamilies [START_REF] Krieger | AZ P450 Subfamily: A[END_REF], which are involved in the synthesis of psoralen, angelicin, xanthotoxol and 5-OHxanthotoxin. To identify the genes involved in the same reaction in other plant species, we searched for CYP71AJ or CYP71AZ orthologous genes in citrus databases. Since this investigation was unsuccessful, we extended our investigation to other cytochrome P450 families. Based on the work performed by Kruse and colleagues [START_REF] Kruse | In planta biocatalysis screen of P450s identifies 8-methoxypsoralen as a substrate for the CYP82C subfamily, yielding original chemical structures[END_REF], who described the hydroxylation of 8-methoxypsoralen (i.e., xanthotoxin) by a P450 isolated from Arabidopsis, we focused on the CYP82 family. Among several candidate genes, we characterized CYP82D64 as a xanthotoxin hydroxylase. This enzyme displayed the same enzymatic function as CYP71AZ6 isolated from parsnip [START_REF] Krieger | AZ P450 Subfamily: A[END_REF]. The low km and the close relationship between the expression level of the gene and the accumulation of 5-OH-xanthotoxin suggest that this reaction also occurs in planta as a physiological reaction.

In this study, we provided evidence that CYP82D64 was able to hydroxylate herniarin.

This result is in line with that from a study recently published by Rajniak supporting the role of CYP82C4 in the production of sideretin, another coumarin [START_REF] Rajniak | Biosynthesis of redox-active metabolites in response to iron deficiency in plants[END_REF]. This report, in addition to our results, highlights that the CYP82 family participates in the synthesis of both FCs and coumarins, as does the CYP71AZ subfamily in Apiaceae. These elements provide new insights in favor of the evolution of the FC biosynthetic pathway from the coumarin biosynthetic pathway. These results are also consistent with the extreme diversity of the CYP71 and CYP82 subfamilies (53 CYP71s and 25 CYP82s reported to date), demonstrating the plasticity of these enzymes that seem to be deeply involved in the adaptation of plants to their environments. Indeed, similar to the CYP71 subfamily, CYP82 members are associated with highly functionally diverse families of molecules with very different chemical structures such as alkaloids, terpenes or phenolic compounds. For example, CYP82N3 and CYP82N4 enzymes of Papaver somniferum hydroxylate alkaline benzylisoquinolines [START_REF] Beaudoin | Isolation and characterization of a cDNA encoding (S)cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis[END_REF]. CYP82E4

from Nicotiana tabacum corresponds to a nicotine N-demethylase [START_REF] Siminszky | Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase[END_REF]. CYP82G1 of A.

thaliana is involved in the synthesis of volatile homoterpenes [START_REF] Lee | Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis[END_REF]. CYP82D63 from Mentha piperita and CYP82D33 from Ocimum basilicum hydroxylate flavones [START_REF] Berim | The Roles of a Flavone-6-Hydroxylase and 7-O -Demethylation in the Flavone Biosynthetic Network of Sweet Basil[END_REF]. CYP82D64 from C. paradisi and CYP82C2 from A. thaliana hydroxylate xanthotoxin, and CYP82C4

hydroxylates fraxetin. This functional diversity was distributed over the entire generated phylogenetic tree (Figure 2).

In this work, we provide some new lines of evidence in favor of a convergent evolution theory for explaining the presence of FCs in Rutaceae and Apiaceae. It is highly possible that the same process occurred in Moraceae and Fabaceae, two other plant families producing large amounts of these specialized molecules. It is therefore not excluded that other P450 families or subfamilies play an important role in the synthesis of FCs and the subsequent adaptation of these plants to their environments. Such convergent evolution is not unique in the plant kingdom. Genes encoding enzymes belonging to different families may have identical functions, such as the anthocyanin-5-O-glucosyltransferases of Dianthus caryophyllus and A. thaliana. In this case, the enzymes have different structures but share the same glycosylation of anthocyanin function [START_REF] Tohge | The evolution of phenylpropanoid metabolism in the green lineage[END_REF]. Similarly, ferulate-5-hydroxylase (CYP788A1) from Selaginella moellendorffii shares only 37% identity with angiosperm P450s but plays the same role as these P450s in the synthesis of S units of lignin [START_REF] Weng | The origin and evolution of lignin biosynthesis[END_REF]. The alignment of peptidic sequences was performed with MUSCLE using the default settings.

Figures:

The tree was built with the maximum likelihood (PhyML) method. The highly variant regions creating gaps, the C-terminal region and the N-terminal membrane were removed. Red stars highlight the differences, and green stars show the substrate recognition sites [START_REF] Gotoh | Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences[END_REF].

The C. clementina sequence was predicted from the CDS available in Phytozome.

Supplemental Data

Supplemental figure 1: list of the P450 sequences used to create the phylogenetic tree in

  Fw 5' ATG GAG TTC CTA TTC ACA GCT GTA ATA ATC ACT GCC C 3', CYP82D64 Rev 5' TCA ATG ATG ATG ATG ATG ATG AAA GCC ATA GGG GTT GGC A 3'; CYP82D83 Fw 5' ATG GAC ATT CTT GGT TCG TCT CAA AC 3', CYP82D83 Rev 5' TCA ATG ATG ATG ATG ATG ATG GCC GTA CAA AGA AGC AGA AAG AC 3'; CYP82D92 Fw 5' ATG GAC TTC CTT GTT TTA ACA TCT CAA C 3', CYP82D92 Rev 5' TCA ATG ATG ATG ATG ATG ATG GCC ATA CAG AGA AGC AGA GAG 3'; and CYP82D179 Fw 5' ATG GAC TTT CTT GTT TCA CGT CAA AC 3', CYP82D179 Rev 5' TCA ATG ATG ATG ATG ATG ATG

Vialart

  et al. Kinetic parameters were calculated using the SigmaPlot 12.0 software. Identification of the chemical structure of the products was done by comparison with their elution time in comparison to 5-OH-xanthotoxin standard molecule and by LTQ orbitrap as described by Krieger et al. (2018)

  The elution time and the fragmentation spectrum (maximum fragmentation intensities for m/z of 172, 217 and 205, for a total m/z of 233) were identical to those of 5-OH-xanthotoxin (Figure 4 B-E). No enzymatic activity could be detected for any other protein tested. Plasticity of substrate specificity between two CYP82D64 variants isolated from C. paradisi and C. hystrix.

and 18 -

 18 fold between the intermediate-stage and ripe fruits. Regarding the expression of CYP82D64 mRNA, the concentrations of xanthotoxin and 5-OH-xanthotoxin also decreased significantly. The average content of the 2 molecules decreased 2.5-fold between the maturation of young fruits to the intermediate stage and 7.4-fold during the transformation from intermediate-stage to ripe fruits.

  Figure 1: schematic representation of the furanocoumarin biosynthetic pathway and the enzymatic steps catalyzed by cytochrome P450s.

Figure 2 :

 2 Figure 2: phylogenetic tree of the CYP82 family.

Figure 3 :

 3 Figure 3: enzymatic conversion of xanthotoxin by CYP82D64.

Figure 4 :

 4 Figure 4: sequence comparison of CYP82D64 variants isolated from C. clementina, C. paradisi and C. hystrix.

Figure 5 :

 5 Figure 5: enzymatic conversion of herniarin by CYP82D64v2.

Figure 6 :

 6 Figure 6: comparison of the expression level of CYP82D64 mRNA to the xanthotoxin and 5-OH-xanthotoxin contents in C. paradisi fruits. A) Three stages of maturity were tested: immature, intermediate and mature fruits. B) Expression level of CYP82D64 and concentrations of xanthotoxin and 5-OH-xanthotoxin.

Figure 2 Supplemental figure 2 :

 22 Figure 2

Figure 2 607 608 29 Figure 3 Figure 4 31

 22934 Figure 2

Figure 5 32

 5 Figure 5

  Figure 6

  According to Nelson, two P450s belong to the same family if they share more than 40% amino acid identity and are

	in	the	same	subfamily	if	they	share	more	than	55%

(http://drnelson.utmem.edu/CytochromeP450.html). To seek candidate genes, we performed a sequence homology search using the BlastN and tBlastX algorithms and CYP82C2 and CYP82C4 from A. thaliana as bait sequences. This first investigation led us to identify 79 P450 candidate sequences that were further restricted to 42 CYP82 partial sequences in the two Citrus species (16 for C. sinensis and 26 for C. clementina). Most were redundant, which led us to finally consider only 8 different coding sequences (Table

1

  One hundred iterations were performed, and the bootstrap values are shown at the nodes of branches. Sequences marked with orange are the citrus candidate genes, and sequences in blue

are cytochromes P450 already described to metabolize furanocoumarins. The tree was rooted with CYP71AJ3, CYP71AJ4, CYP71AZ3, CYP71AZ5, and CYP71AZ6 from Pastinaca sativa; CYP71AU3, CYP71AU4 and CYP71AH4 from Vitis vinifera; and CYP83B1 and CYP83A1 from A. thaliana. Legend: AA, Ammi majus; AL, Arabidopsis lyrata; AT, Arabidopsis thaliana; BN, Brassica napus; BR, Brassica rapa; BO, Brassica oleracea; CA, Citrus aurantium; CC, Citrus clementina; CH, Citrus hystrix; CS, Citrus sinensis; CP, Citrus paradise; DC, Daucus carota; EC, Eschscholzia californica; GM, Glycine max; GS, Glycine soja; MS, Medicago sativa; MP, Mentha piperita; NT, Nicotiana tabacum; OB, Ocimum basilicum; PS, Pisum sativum; PSo, Papaver somniferum; SH, Sinopodophyllum hexandrum; SR, Stevia rebaudiana; TC, Tanacetum cinerariifolium; TH, Tarenaya hassleriana; VV, Vitis vinifera; .

Acknowledgments

15

MLM was funded by CONACYT. The authors would like to thank the SATT SAYENS and the "Impact Biomolecules" project (ANR-15-IDEX-04-LUE Lorraine Université d'Excellence) for financial support. The authors also thank Clément Charles for technical assistance.

Supplemental figure 3: optimal pH determination for CYP82D64v1 and v2 A) Optimal pH for CYP82D64v1 with xanthotoxin, B) optimal pH for CYP82D64v2 with xanthotoxin, C) optimal pH for CYP82D64v2 with herniarin.

Supplemental figure 4: optimal temperature determination for CYP82D64v1 and v2 A) Optimal temperature for CYP82D64v1 with xanthotoxin, B) optimal temperature for CYP82D64v2 with xanthotoxin, C) optimal temperature for CYP82D64v2 with herniarin. Supplemental figure 5: Phylogenetic tree constructed with the predicted cytochrome P450s belonging to the Clan71 of Citrus sinensis and Citrus clementina. The tree has been rooted with CYP51G1. Sequence alignment was performed using MAFFT relying on Multiple sequence alignment default settings (https://mafft.cbrc.jp/alignment/software). The tree was buildt with IQ-TREE multicore version 1.6.11 for Linux 64-bit built Jun 6 2019 and FigTree.

Main figures

Supplemental Figures


Claire Bocciarelli

Justine Cohen

Romain Pelletier

Jeanne Tran Van Nhieu

Jonathan Derman

Loetitia Favre

Anaïs Bourgogne

Isabelle Monnet

Christos Chouaid

Anaïs Pujals
email: anais.pujals@aphp.fr

Evaluation of the Idylla system to detect the EGFRT790M mutation using extracted DNA

Keywords:

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

Introduction

Lung cancer is the leading cause of death from cancer worldwide, responsible for ~1.8 million deaths in 2018. Non-small-cell lung cancer (NSCLC) is the predominant form of lung cancer [1]. Patients often have advanced disease at diagnosis, which carries a poor prognosis and 5-year overall survival of only 18% [START_REF] Street | Cancer Facts & Figures[END_REF].

The recent development of personalized therapies has considerably improved NSCLC outcomes. In addition to the driver-gene alterations found in NSCLCs, epidermal growthfactor-receptor (EGFR) mutations, mostly exon 19 deletions (Del19) and exon 21 L858R mutation, are the most useful because of their associated sensitivity to EGFR-tyrosine-kinase inhibitors (EGFR-TKIs) [START_REF] Mok | Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma[END_REF]. Therefore, the search for EGFR-activating mutations in advanced NSCLCs is now essential to choose the optimal therapeutic strategy. Even though patients with EGFR mutations initially respond to EGFR-TKIs, a large majority of their tumors ultimately acquire TKI resistance [START_REF] Sequist | Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors[END_REF]. Several resistance mechanisms to first-or second-generation EGFR-TKIs have been described, especially the EGFR T790M point mutation in exon 20, which is the most frequently observed. Hence, third-generation EGFR-TKIs have been designed to inhibit the gene harboring the EGFR-activating mutation and the T790M resistance mutation; they have marked clinical benefit for patients whose tumors carry these molecular alterations [START_REF] Cross | AZD9291, an irreversible EGFR TKI, overcomes T790Mmediated resistance to EGFR inhibitors in lung cancer[END_REF].

Detecting EGFR mutations in NSCLCs has become a routine part of clinical practice but the implementation of these analyses, especially T790M detection, can be challenging for several reasons. First, formalin-fixed paraffin embedded (FFPE) tissue samples are usually small, due to the thinness of tumor biopsies, which limits the amount of DNA available to detect mutated EGFR. Second, the T790M mutation is usually found in a subclone, with low variant allele frequency (VAF), requiring a highly sensitive method to be detected. Third, the analysis turnaround time must be short, especially for patients with rapid clinical progression.

Therefore, the approach used to detect the EGFR T790M mutation must be highly sensitive, fast and economically acceptable. Today, next-generation sequencing (NGS) is a frequently used molecular biology method. Although NGS is very sensitive, it requires technical expertise and bioinformatics skills, has long turnaround times and is profitable only for testing large case volumes. Recently, other techniques have been developed to detect EGFR mutations, like the Idylla TM EGFR-Mutation Assay (henceforth, Idylla) (Biocartis NV, Mechelen, Belgium), that is a fully automated real-time polymerase chain reaction (PCR; instrument plus software), able to provide rapid EGFR-genotyping. It can analyze FFPE tissue sections or extracted DNA. Little molecular biology expertise is required, results can be obtained within 150 minutes and hands-on time is minimal. For these reasons, this system is increasingly used in pathology laboratories to determine EGFR status quickly.

Recent studies showed Idylla system efficiency at detecting EGFR mutations, compared to either NGS or pyrosequencing [START_REF] Luca | Idylla assay and next generation sequencing: an integrated EGFR mutational testing algorithm[END_REF][START_REF] Ilie | Optimization of EGFR mutation detection by the fully-automated qPCR-based Idylla system on tumor tissue from patients with non-small cell lung cancer[END_REF], but none of those studies focused on EGFR T790M detection, which represents a real challenge as stated above.

The aims of this study were to compare the NGS and Idylla abilities to detect the EGFR T790M mutation using DNA, to determine the Idylla limit of detection (LOD) for the T790M mutation, and evaluate the impacts of the quality and quantity of DNA on the system's performance.

Materials and Methods

Sample selection

This retrospective study was designed to assess the relative performances of NGS and Idylla to detect the EGFR T790M mutation. To do so, 47 NSCLC biopsies were selected. They For LOD assessment, 100 ng of commercial DNA harboring the EGFR T790M mutation with VAF = 50% was used (EGFR T790M Reference Standard, Horizon, Horizon Discovery, Cambridge, U.K.). That DNA was serially diluted in control wild-type DNA to obtain a 6point VAF scale: 50%, 25%, 12.5%, 6.2%, 3.1% and 1.5%.

DNA extraction and quantification with a quantitative PCR

All DNA samples had been extracted, after macrodissection when necessary, from FFPE tissue sections (usually 7 sections, 5-µm thick) using the Maxwell 16 FFPE Plus LEV DNA Purification Kit IVD (Promega, Charbonnières-les-Bains, France), according to the manufacturer's instructions. The DNA was quantified using a Qubit fluorimeter in combination with the Qubit dsDNA HS Array Kit (ThermoFisher Scientific, Waltham, MA, U.S.A.).

NGS

For NGS, 10 ng of DNA (as measured by fluorimetry) was amplified using the Ion AmpliSeq™ Colon and Lung Cancer Panel (ThermoFisher Scientific), which is a multiplex PCR-based library-preparation method by which many regions (70-150 bp) that encompass many mutational hotspots including EGFR T790M are amplified. Amplicons were then digested, barcoded and amplified by using the Ion Oncomine™ Solid Tumor DNA Kit and Ion Select Barcode Adapter Kit (ThermoFisher Scientific), according to the manufacturer's instructions.

After DNA quantification, 25 pM of each library was multiplexed and clonally amplified on ion-sphere particles (ISP) by emulsion PCR performed on Ion Chef (ThermoFisher Scientific), according to the manufacturer's instructions. The ISP templates were loaded onto an Ion-520 chip and sequenced on a S5 sequencer with the Ion 510™ & Ion 520™ & Ion 530™ Kit-Chef, according to the manufacturer's instructions. Run performance was assessed and for data analyzed with the Torrent Suite Software v.5.10.0 (ThermoFisher Scientific).

Single-nucleotide variants and small indels were detected using the Variant Caller plug-in version 5.10.0.18 with low stringency settings (threshold: 2%). The Integrative Genomics Viewer (IGV v 5.01; Broad Institute, Cambridge, MA, U.S.A.) was used for visual inspection of the aligned reads.

Idylla EGFR-mutation test

Each sample was retested using Idylla: 10 µL of the original sample-DNA preparation was directly deposited into the Idylla cartridge. DNA was transported via microfluidic channels in the cartridge into 5 separate PCR chambers, which contained lyophilized PCR reagents (i.e., primers, probes, enzymes) designed for the qualitative detection of 18 genetic-change types and for which 51 different mutations have been validated. After a 150-minute run, final reports were directly available on the Idylla console and the results presented on screen as either "no mutation detected" or "EGFR mutation detected". An internal Ct value for each sample is provided and indicates the quantity and quality of each DNA sample tested. Results were compared to those previously obtained with NGS.

Results

Evaluation of Idylla's ability to detect T790M

Idylla is CE-IVD validated by the manufacturer for FFPE tissue sections but the results of several studies showed that it can be used with extracted DNA directly deposited into the cartridge [START_REF] Luca | Idylla assay and next generation sequencing: an integrated EGFR mutational testing algorithm[END_REF][START_REF] Gilson | Evaluation of KRAS, NRAS and BRAF hotspot mutations detection for patients with metastatic colorectal cancer using direct DNA pipetting in a fullyautomated platform and next-generation sequencing for laboratory workflow optimization[END_REF]. Pertinently, lung biopsies are rapidly exhausted and it is important to store the material to search for all biomarkers assuring better therapeutic management of the patient.

When the patient's tumor does not harbor an EGFR mutation, Idylla using extracted DNA allows that DNA to be recovered and reused to run all other the analyses necessary to manage NSCLC patients.

In our series, 29 samples carried the exon-20 T790M mutation, associated with primary EGFR-activating mutation(s), such as: Del19 for 17 (58.6%), exon-21 L858R mutation for 11 (37.9%) or exon-18 G719A mutation for 1 (3.4%) (Table 1). Eleven samples harbored an EGFR-activating mutation without T790M: 2 (18%) L858R mutations, 2 (18%) L861Q mutations, 4 Del19 (36%) and 3 (27%) G719A mutations, one of which was associated with an S768I mutation. Seven samples expressed wild-type EGFR. An average of 210 ng of DNA were deposited in the cartridge and those samples contained an average of 37.8% tumor cells.

NGS results yielded average VAFs of 35% for primary EGFR-activating mutation and 16.5% for the T790M mutation.

Idylla sensitivity to detect EGFR-activating mutations was 97.5% (39/40) but fell to 65.5% (19/29) for the detection of T790M. Its respective specificities for both mutations were 100%.

For samples 3, 10, 23 and 29, Idylla's failure to detect the T790M mutation can be explained by the poor quality and/or insufficient quantity of the DNA used for the test. For those samples, the internal Ct values provided by the Idylla always exceeded 25, indicating the higher number of PCR cycles needed to reach detectable DNA, which can be expected with low DNA input or poor-quality DNA. For samples 1, 8 and 14, Idylla failure was probably attributable to the low T790M-mutation VAF, because NGS analysis yielded a VAF < 5%. For the remaining samples 5, 22 and 24, neither the amount of DNA used for the test nor the T790M-mutation VAF can explain the failure to detect the T790M mutation.

Nevertheless, the quality-control internal Ct value obtained for total EGFR for samples 22 and 24, which exceeded 25, reflect the poorer quality of those DNA samples used for the test, which could affect assay performance. Sample 5's Ct value was 24.2 and no explanation was found to explain Idylla's failure to detect the T790M mutation.

To try to understand this failure, we examined the Idylla Explorer curves for case 5 and observed that a T790M-mutation amplification curve was there but it did not pass the system's filter threshold; (Fig. 1). Thus, using Idylla Explorer could help detect the T790M mutation and avoid false-negative results.

These results highlighted the need for a procedure to avoid false-negative assessment of the T790M mutation, especially in samples with low amounts of DNA or DNA samples extracted from tissues containing < 10% tumor cells. Thus, we decided to determine the Idylla LOD for T790M and the minimal DNA amount that had to be deposited into the cartridge to avoid false-negative results.

Determination of the Idylla LOD for T790M using extracted DNA

In light of the results obtained in retrospective series, our second study objective was to evaluate the Idylla LOD for the T790M mutation using a commercial mutated DNA known to harbor the that mutation with VAF = 50%. LOD was measured using 100 ng of the commercial DNA, serially diluted in control wild-type DNA to obtain a 6-point VAF scale: 50%, 25%, 12.5%, 6.2%, 3.1% and 1.5%. The T790M mutation was detected in samples with VAF > 3.1%. For samples with VAFS = 3.1% and 1.5%, Idylla did not detect the mutation and they were reported to be wild-type (not EGFR-mutated) (Table 2). Thus, the T790Massay LOD was estimated to be between 3% and 6% using 100 ng of this commercial DNA lot.

Determination of minimum DNA quantity required to detect low-VAF T790M

The third study objective was to assess the impact of the quantity of DNA used in Idylla to detect the T790M mutation. We used the same commercial DNA at 3 different VAFs (20%, 10% and 5%) serially diluted to obtain a 4-point concentration scale (10,5, 2.5 and 1.25 ng/μL). Idylla did not detect the T790M mutation in a sample with VAF = 5% and 1.25 ng/μL of DNA, corresponding to deposition of 12.5 ng of DNA into the cartridge (Table 3).

Discussion

This study was undertaken to evaluate Idylla's ability to detect the EGFR T790M mutation using extracted DNA. The study included a total of 47 NSCLC biopsy samples previously subjected to NGS: 29 harboring an EGFR-activating mutation and the T790M mutation and 11 harboring an EGFR-activating mutation without the resistance mutation, and 7 wild type for EGFR. In addition, LOD experiments aimed to determine this method's sensitivity for extracted DNA.

Idylla results confirmed almost all known EGFR-activating mutations (39/40) but failed to detect the T790M resistance mutation in about one-third of the samples (10/29). Our LOD findings could explain those failures. First, our experiments showed that the amount of DNA loaded into the cartridge was decisive. Herein, the failure to detect the T790M mutation in 4 samples could be explained by their low DNA concentrations (< 2 ng/µL). We also demonstrated that LOD ranged from 3% to 6% for the T790M mutation using a commercial DNA sample. We also assessed the LOD for the EGFR-activating mutation L858R and Del19, and found them to be, respectively, between 3% and 6% and 1% and 3% (data not shown). In our series, Idylla failed to detect 3 T790M mutations because of very low VAFs (1.3-4%) that were well below the manufacturer's recommended assay LOD. That failure was also reported recently by Evrard et al., who described Idylla's inability to detect T790M mutations in samples with VAFs < 5% [START_REF] Evrard | Multicenter evaluation of the fully automated PCR-based Idylla EGFR mutation assay on formalin-fixed, paraffin-embedded tissue of human lung cancer[END_REF]. Finally, we found the internal Ct value to be a very good marker to check the reliability of the result. Indeed, in our opinion, because of the risk of a false-negative finding, the analysis must be considered non-interpretable for a negative result obtained with a sample having a Ct > 25. Therefore, we propose a decision-tree algorithm to avoid the risk of an Idylla falsenegative T790M-mutation result (Fig. 2). First, all cases with < 25 ng of DNA or < 10% tumor cells should not be analyzed with Idylla. Second, our experience showed that all the negative findings for samples with Ct > 25 should be retested with another method. In addition, our results showed that using Idylla Explorer, which provides the PCR-amplification curves, could detect mutations that do not pass the filter threshold set by the manufacturer. For the 10 discordant samples of our series, only samples 2 and 5 had T790M-amplification curves. Thus, despite using this tool, the risk of false-negatives persists.

Idylla performance to detect EGFR mutations has been evaluated in other studies. De Luca et al. tested 3 T790M-mutation-harboring samples identified by NGS and Idylla found all 3 [START_REF] Luca | Idylla assay and next generation sequencing: an integrated EGFR mutational testing algorithm[END_REF]. However, they did not clearly state the amounts of DNA they used in the test but their VAFs were ≥ 5%. Lambros et al. also retested 4 T790M-harboring samples identified by NGS. Idylla detected the T790M mutation in one surgical sample but failed to do so in the 3 biopsies [START_REF] Lambros | Evaluation of a fast and fully automated platform to diagnose[END_REF]. Unfortunately, neither the VAFs of the T790M mutations not detected by Idylla system nor the amounts of DNA used were given because the assay was run using FFPE slides inserted directly into the cartridge. Nevertheless, their results are in accordance with ours and highlight the need to have a strong algorithm to avoid false-negative T790Mmutation findings.

Our study results and those previously reported showed that it is preferable to analyze samples with < 10% tumor cells or insufficient DNA quality with other, more sensitive techniques, like NGS, or digital or allele-specific PCR. Their LODs for the search for EGFR mutations are comprised between 1% and 5% [11,12]. Nonetheless, it is important to keep in mind that those methods, especially NGS analysis, require much longer times and greater expertise in molecular biology than Idylla.

To conclude, our results showed that the Idylla is a rapid and valid option to detect EGFRactivating mutations. They, like those reported by Evrard et al. [START_REF] Evrard | Multicenter evaluation of the fully automated PCR-based Idylla EGFR mutation assay on formalin-fixed, paraffin-embedded tissue of human lung cancer[END_REF] and Gilson et al. [START_REF] Gilson | Evaluation of KRAS, NRAS and BRAF hotspot mutations detection for patients with metastatic colorectal cancer using direct DNA pipetting in a fullyautomated platform and next-generation sequencing for laboratory workflow optimization[END_REF], demonstrated that Idylla provides adequate findings when run on extracted DNA, which allows specimen storage. The real advantage of this technology is its rapidity because the EGFR analysis must have a short turnaround time because of its critical clinical implications. This test should not be used with insufficient (< 25 ng) DNA or < 10% tumor cells, which is often the case for thin lung biopsies. That cautionary approach is particularly true for the detection of subclone mutations, like T790M. Applying the algorithm proposed herein could help limit false-negative results. lung cancer samples in less than one day, J. Clin. Pathol. Jun;70 (année) 544-549.

[11] Yue Li, et al., Comprehensive analysis of EGFR T790M detection by ddPCR and ARMS-PCR and the effect of mutant abundance on the efficacy of osimertinib in NSCLC patients, J. Thorac. Dis. 11 (2019) 3004-3014.

[12] Y.W. Cheng, et al

  had previously undergone NGS in our Department of Pathology between 2015 and 2018 for molecular diagnosis, in compliance with French regulations; 29 harbored an EGFR-activating mutation and the T790M resistance mutation, 11 harbored an EGFR-activating mutation without T790M and 7 were wild-type. The prerequisite for sample selection was the availability of at least 10 µL of residual archival extracted DNA. Each sample had previously undergone routine PCR high-resolution melting (HRM) analysis, targeting exon 2 of the KRAS (Kirsten rat-sarcoma viral oncogene) to determine DNA quality, and was accorded a grade of A-D, with A being the best, based on the crossing threshold (Ct) observed during that PCR.

Fig. 1 .

 1 Fig. 1. Idylla Explorer printout of real-time PCR curves the showing the method's failure to detect the

Fig. 2 .

 2 Fig. 2. Algorithm proposed to avoid epidermal growth-factor-receptor (EGFR) false-negative T790M-

Table 2

 2 ., Real-time PCR and targeted next-generation sequencing in the detection of low level EGFR mutations: instructive case analyses, Respir. Med. Case Rep. Jul 10; vol (28) (2019) 100901.Idylla limits of detection obtained with a commercial DNA harboring the EGFR T790M mutation.EGFR, epidermal growth-factor-receptor; VAF, variant allele frequency; Ct, crossing threshold.

	1	No	20	6	A	60	L858R + T790M	21%	4%	L858R	25.1	No, T790M not detected
	2	No	70	133	A	1330	Del19 + T790M	37%	9%	Del19 + T790M	10	Yes
	3	No	20	0.1	NE	1	Del19 + T790M	25%	6%	Del19	27.9	No, T790M not detected
	4	Yes	70	12	A	120	T790M + T790M	49%	39%	L858R + T790M	22.7	Yes
	5	Yes	40	6	A	60	Del19 + T790M	59%	10%	Del19	24.2	No, T790M not detected
	6	Yes	55	3	NE	30	L858R + T790M	95%	10%	L858R + T790M	26.2	Yes
	7	Yes	25	4	NE	40	L858R + T790M	36%	36%	L858R + T790M	26.1	Yes
	8	No	15	5	B	50	L858R + T790M	2%	1.30%	L858R	27.2	No, T790M not detected
	9	No	30	5	B	50	Del19 + T790M	32%	8%	Del19 + T790M	27.2	Yes
	10	No	25	2	NE	20	Del19 + T790M	19%	7%	Del19	25.4	No, T790M not detected
	11	Yes	40	7	A	70	Del19 + T790M	36%	11%	Del19 + T790M	23.8	Yes
	12	Yes	25	12	A	120	Del19 + T790M	40%	19%	Del19 + T790M	23.8	Yes
	13	Yes	30	3	NE	30	Del19 + T790M	37%	4%	Del19 + T790M	22.2	Yes
	14	Yes	55	4	A	40	L858R + T790M	14%	2%	L858R	24.1	No, T790M not detected
	15	Yes	15	2.6	NE	26	Del19 + T790M	42%	21%	Del19 + T790M	25.2	Yes

EGFR, epidermal growth-factor-receptor; NGS, next-generation sequencing; VAF, variant allele frequency; Ct, crossing threshold; Del19, exon 19 deletion; NA, not applicable

Table 3 294

 3 Determination of the minimal amount of DNA required in the EGFR Idylla assay.

	295
	T790M		DNA loaded		EGFR	Idylla
	VAF, %	ng	Volume, µL	Quantity, ng	Ct control result
	20	10	10	100	22.1	Mutated
	20	5	10	50	23.2	Mutated
	20	2.5	10	25	24.2	Mutated
	20	1.25	10	12.5	24.8	Mutated
	10	10	10	100	22.0	Mutated
	10	5	10	50	23.6	Mutated
	10	2.5	10	25	24.3	Mutated
	10	1.25	10	12.5	26.3	Mutated
	5	10	10	100	22.2	Mutated
	5	5	10	50	23.3	Mutated
	5	2.5	10	25	24.3	Mutated
	5	1.25	10	12.5	25.8	Wild type

EGFR, epidermal growth-factor-receptor; VAF, variant allele frequency; Ct, crossing threshold.

* the percent tumor cells indicated corresponds to that obtained after macrodissection **Quality graded from A to D, with A being the best; NE, not evaluated.

Acknowledgments

We thank the molecular biology technicians of the Department of Pathology for their technical participation: Déborah Siroli-Sarda, Céline Bourdaudhui, Caroline Taou and Soraya Mehdaoui. We thank the Biocartis employees who helped conduct this study, especially Cyriel Naud and David Favy.

Funding

This research did not receive any specific support from funding agencies in the public, commercial or not-for-profit sector.


Florence Abravanel
email: abravanel.f@chu-toulouse.fr

Sébastien Lhomme

Pauline Trémeaux

Marion Migueres

Agnès Harter

Catherine Haslé

Patrick Bruel

Laurent Alric

Sophie Métivier

Stéphanie Raymond

Jacques Izopet

Performance of the Xpert HBV Viral Load assay versus the Aptima Quant assay for quantifying hepatitis B virus DNA

Keywords: Hepatitis B virus, molecular diagnosis, GeneXpert instrument alanine amino transferase

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

Introduction

Hepatitis B virus (HBV) infections are a major cause of chronic liver disease that affects almost 250 million individuals worldwide [START_REF] Schweitzer | Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013[END_REF]. Chronic HBV infection is associated with a wide spectrum of liver diseases, from HBeAg-positive or negative chronic hepatitis B with high HBV DNA concentration to a low-viremia HBe antigen negative chronic HBV infection, which may evolve toward cirrhosis and hepatocellular carcinoma [START_REF] Seto | Chronic hepatitis B virus infection[END_REF]. HBV-related end-stage liver disease and hepatocellular carcinoma are responsible for more than 750,000 deaths worldwide each year. Hepatitis B viruses are presently divided phylogenetically into 9 genotypes, A-I , with a putative 10 th genotype, "J", isolated from a single individual, based on an intergroup divergence >7.5% across the complete genome [START_REF] Kramvis | Genotypes and genetic variability of hepatitis B virus[END_REF].

International practice guidelines recommend using sensitive nucleic acid amplification techniques for detecting and quantifying HBV DNA in clinical practice [START_REF]European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF][START_REF] Terrault | AASLD guidelines for treatment of chronic hepatitis B[END_REF].

Accurate assays for HBV DNA quantification are essential for the management of HBV infections, establishing the prognosis for HBV-related liver disease, guiding decisions concerning treatment, and monitoring the virological response to antiviral treatment [START_REF]European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF][START_REF] Terrault | AASLD guidelines for treatment of chronic hepatitis B[END_REF].

According to EASL guidelines, treatment requires HBV DNA >2,000 IU/ml, elevated ALT and/or at least moderate histological lesions. However, patients with HBV DNA >20,000 IU/ml and elevated ALT should start treatment regardless of the degree of fibrosis.

Several commercial nucleic acid amplification tests (NAATs) are currently available for quantifying HBV DNA in patients' clinical samples [START_REF] Schonning | Analytical performance of the Hologic Aptima HBV Quant Assay and the COBAS Ampliprep/COBAS TaqMan HBV test v2.0 for the quantification of HBV DNA in plasma samples[END_REF][START_REF] Chevaliez | Performance of version 2.0 of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification[END_REF][START_REF] Chevaliez | The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F[END_REF][START_REF] Fourati | Evaluation of a new random-access HBV DNA molecular assay: The VERIS HBV assay[END_REF][START_REF] Han | Comparison of the QIAGEN artus HBV QS-RGQ Assay With the Roche COBAS AmpliPrep/COBAS TaqMan HBV Assay for Quantifying Viral DNA in Sera of Chronic Hepatitis B Patients[END_REF][START_REF] Caliendo | Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification[END_REF][START_REF] Braun | A European multicenter study on the analytical performance of the VERIS HBV assay[END_REF][START_REF] Braun | A European multicientre study on the comparison of HBV viral loads between VERIS HBV assay and Roche COBAS((R)) TAQMAN((R)) HBV test, Abbott RealTime HBV assay, Siemens VERSANT HBV assay, and Qiagen artus HBV RG kit[END_REF][START_REF] Schalasta | Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples[END_REF]. And, molecular techniques are easier to implement compared to several years ago, reagents have to be reconstituted, like for the Aptima Quant HBV DNA assay, and often require several controls per day. Lesscomplex and inexpensive assays are needed for more widespread use of targeted, routine HBV DNA monitoring and for point-of-care (POC) use [START_REF] Chan | A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics[END_REF][START_REF] Yang | PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings[END_REF].

This study evaluated the analytical and clinical performance of the new Xpert HBV Viral Load (VL) assay (Cepheid Inc. Sunnyvale, CA, USA) on the GeneXpert system (Cepheid).

Patients and Methods

HBV assays

Xpert HBV DNA VL: This assay is a semi-automated, real-time PCR test that quantifies HBV DNA. This single-target assay has a reported limit of quantification of 10 IU/ml, with a dynamic range of 1-9 log10IU/mL. The tests can be run on any GeneXpert instrument and requires 0.6 mL plasma or serum per test (no dead volume). The technician pipettes the plasma/serum into the assay cartridge and locks it into the GeneXpert system module. The GeneXpert System can be configured with 1, 2, 3, 4, 8, 12, or 16 modules. Each module contains a single cartridge for HBV DNA extraction, amplification and quantification. The Xpert assay contains 2 internal controls but no positive or negative control is provided.

Extracted DNA cannot be reused. A two modules-instrument was used for the present study.

Aptima HBV Quant assay: The Aptima assay (Hologic Inc. San Diego, CA, USA), a real-time transcription mediated amplification (TMA) assay is a dual-target assay designed to run on the fully automated, random access Panther platform (Hologic, Inc.), and was used as the comparator. Its limit of quantification is 10 IU/mL with a dynamic range of 1-9 log10IU/mL using 0.5 mL plasma or serum per test, with an additional 0.2 mL for the dead volume.

Extracted DNA cannot be reused. The Aptima HBV DNA assay implemented on the Panther platform requires 6 calibrators and controls per day that are stored at -20°C. All other reagents are reconstituted.

Analytical performance of the Xpert HBV DNA assay

Analytical sensitivity. The analytical sensitivity of the assay was determined with 5 dilutions (50 IU/ml -2.5 IU/ml) of the 3rd WHO International Standard for Hepatitis B Virus (NIBCS 10/264) in HBV negative plasma. Ten replicates of each HBV DNA serial dilution were tested the same day on a single instrument.

Specificity. The specificity of the Xpert HBV DNA assay was assessed by testing, on the same day, 20 HBs-antigen negative and HBc-antibody negative, clinical specimens (Architect, Abbott Diagnostics, Chicago, IL, USA) from HIV and HCV negative donors.

Reproducibility. Plasma from three HBV DNA positive patients (HIV and HCV negative), previously quantified using the Aptima assay, were separately diluted in HBV negative plasma to obtain 3 samples with high (5 log10 IU/ml), medium (4 log10 IU/ml) and low (2.5 log10 IU/ml) viral loads. These 3 samples were tested 20 times over a period of 20 days.

Linearity and influence of the HBV genotypes. Two clinical samples of each HBV genotype (A, B, C, D and E) were diluted in negative plasma to obtain 2 series (A/A*, B/B*, etc.) of five 10 fold-dilutions. The resulting five concentrations of each genotype covered the linear range of the assay. Three replicates of each dilution were tested with the Xpert HBV assay the same day. HBV genotypes were determined as previously described in (17).

Clinical performance of the Xpert HBV DNA assay

Plasma samples were obtained from HBV chronically infected (Hepatitis B surface (HBs) antigen and anti-hepatitis B Core (HBc) antibodies positive) patients attending the Toulouse University Hospital. A total of 100 samples were collected, centrifuged at 2000g for 10 minutes. The plasma was kept at 5°C for a maximum of 48h until tested with the Aptima assay, and stored again at 5°C for a maximum of 24h until tested with the Xpert assay.

We also tested 80 stored frozen samples (maximum freezing time: 1 year at -80°C) that had been collected in 19 HBV DNA positive patients being monitored during their treatment for HBV infection and previously tested with the Aptima assay.

Biological material and clinical data were obtained only via standard viral diagnostics following a physician's order (no supplemental or modified sampling). Data were analyzed anonymously. The French Public Health law (CSP Art L 1121-1.1) does not require written informed consent from the patients for such a protocol.

Workflow Study

The time-to-result (time period between arrival of the sample at the lab to the final report) for the Xpert HBV and Aptima assays were assessed over one work week (Monday to Friday).

The samples were sent to the lab for registration, centrifugation, aliquoting, bar coding and then analysis. Time-to-result was defined as the time period between arrival of the sample at the reception to the final report with HBV DNA result on the laboratory information system. The Xpert assay was performed with a 2-module GeneXpert instrument every day from 8:00 to 16:00 hours. The Aptima assay was performed on Monday, Wednesday and Friday on the Panther automate between 11:00 and 19:00 hours to minimize the number of controls/calibrators and cost associated.

Statistical analysis

HBV DNA quantities were log10 transformed prior to analysis. The limit of detection (LOD) was determined by Probit analysis of the WHO standard HBV sample data. Linearity was estimated by linear regression. The values obtained from the Xpert HBV VL and Aptima assays were analyzed by Deming linear regression. A Bland-Altman analysis (scatter plot of the differences between the paired measurements plotted against their means) of positive samples obtained by the two methods was used to assess graphically the magnitude of disagreement between them and estimate the overall bias.

RESULTS

Analytical performance

Sensitivity. The detection rates were 100% for 50 IU/ml, 20 IU/ml and 10 IU/ml replicates, 80% for 5 IU/ml replicates, and 60% for 2.5 IU/ml replicates (Table 1). Probit analysis of the 50 replicates of the diluted NIBSC standard gave an LOD of 7.5 [CI 95%: 4.7 -564.6] IU/ml for the Xpert HBV assay.

Specificity. All the 20 negative samples tested negative for HBV DNA: specificity = 100%.

Reproducibility. Reproducibility (log10 standard deviation) was determined by testing 20 replicates over 20 days. The log 10 standard deviation for 5.1, 3.9 and 2.2 log10IU/ml was 0.06, 0.10 and 0.28 log10IU/ml, respectively (Table 2) .

Linear quantification and genotype inclusivity

The linearity of the Xpert assay was evaluated with serial dilutions of 10 clinical samples of genotypes A to E (Fig. 1), the most common genotypes in Europe [START_REF] Han | Comparison of the QIAGEN artus HBV QS-RGQ Assay With the Roche COBAS AmpliPrep/COBAS TaqMan HBV Assay for Quantifying Viral DNA in Sera of Chronic Hepatitis B Patients[END_REF]. Linear regression analysis gave a coefficient of determination (R 2 ) of > 0.99 for all the samples (Figure 1).

Clinical performance

We evaluated the accuracy of the Xpert HBV by comparing the HBV DNA concentrations obtained by the Xpert and Aptima assays; they were linearly related (p < 0.0001; Deming regression equation: Xpert = 1.063 × Aptima -0.103) (Figure 2A). Bland-Altman analysis (Figure 2B) indicated that the results for 95/100 samples gave results within the 95% limit of agreement of the assays (-0.59 to 0.81 IU/mL), with a modest bias (-0.10 log10 IU/mL) (Figure 2B). One out of 3 samples that were over-quantified with the Xpert assay belongs to HBV genotype A. The 2 under-quantified samples with the Xpert assay belong to genotypes D and E. The genotype was available for 4 additional samples that were within the 95% limit of agreement of the assays. They belong to genotype A (n=1) or D (n=3) suggesting that discrepant result was not linked to a particular genotype.

Patient monitoring

A total of 80 samples from 19 patients chronically infected with HBV were tested with the Xpert HBV and Aptima HBV Quant assays. The HBV DNA dynamics of all the patients were parallel (Fig 3). The resulting data, HBV DNA load (log10 IU/mL) versus time (weeks), gave similar profiles with trends in the same direction and overlapping values, except for patient #4. The Xpert assay underestimated all the samples from this patient that was infected with HBV genotype A (difference > 1.5 log10 IU/ml) (Fig 4). A slight underestimation with the Xpert assay was observed for patients #1, #2, #3, #5, #6 and #12. The virus infecting patients 6 and 12 couldn't be genotyped. Patients #3 and #5 were infected with genotype A, patient #1 was infected with genotype D and the patient #2 was infected with genotype E. The genotype was available for 1 additional patients with concordant result. It belong to genotype A.

Workflow study

A total of 50 samples had volumes that were large enough for both assays and were tested.

The tests were carried out by 2 separate technicians at different times during a one-week period. The Xpert assays were performed every day. The Aptima assays were performed on Monday, Wednesday and Friday. Overall, the mean time-to result was 8h (range: 2-24h) for the Xpert assay and it was 23h (range: 5-51h) for the Aptima assy. Thus, the Xpert assay required an average of 15h less time-to result than did the Aptima assay. The mean time-toresult for the Xpert assay and for the Aptima assays was 8h and 10h respectively for samples arrived on Monday, Wednesday and Friday whereas it was 7.5h and 31h respectively for samples arrived on Tuesday and Thursday.

Discussion

We found the analytical performance of the Xpert assay for detecting and quantifying HBV DNA to be specific, reproducible and linear with a low limit of detection. We also found a strong, significant correlation between the HBV DNA concentrations obtained with the Xpert and Aptima Quant assays.

The analytical sensitivity of the Xpert assay, assessed by testing plasma dilution was excellent, with a lower limit of detection (LOD) in keeping with the manufacturer's stated value (10 IU/mL). This LOD is similar to that of the most commonly used HBV DNA platforms: the Abbott RealTime HBV test, the Roche Cobas AmpliPrep/Cobas TaqMan HBV test, the Roche Cobas TaqMan HBV test with the HighPure system, and the Qiagen Artus HBV [START_REF] Caliendo | Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification[END_REF] and Aptima Quant HBV tests [START_REF] Chevaliez | The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F[END_REF]. According to the Xpert assay's package insert, the LOD is 3.2 IU/ml for the plasma EDTA and 5.99 IU/ml for the serum.

While we tested only a small number of negative samples to assess specificity, we found no false positive results. A previous report found false-positive results with all the assays evaluated (11) but they were more frequent for assays that used manual extraction methods (4/28 for the HighPure TaqMan test and 5/28 for the Artus HBV assay, and only 1/28 for the Real-Time and 2/28 for the AmpliPrep TaqMan assays). The simplicity and the unitary format of the of the Xpert HBV assay should theoretically limit the risk of sample cross-contamination as all the steps (extraction and PCR) take place within the same cartridge in an independent module on the GeneXpert instrument.

Assay reproducibility was good, particularly for high-load controls; the coefficient of variation was <3% for controls with HBV DNA ≥3 log10IU/mL. The Aptima and the COBAS Ampliprep/COBAS TaqMan assays have given higher CVs [START_REF] Schonning | Analytical performance of the Hologic Aptima HBV Quant Assay and the COBAS Ampliprep/COBAS TaqMan HBV test v2.0 for the quantification of HBV DNA in plasma samples[END_REF]. The Xpert assay was also linear over a wide range of concentrations (2-8 log10IU/mL) across all the genotypes tested (A to E).

The clinical performances of the Xpert and Aptima assays were very well correlated, with most (95%) of the samples falling within the 95% limit of agreement of the assays in the Bland-Altman analysis. The average difference between the assays was very small (-0.10 log10IU/mL). A similar difference was reported between the Aptima and COBAS Ampliprep/COBAS TaqMan assays: 0.09 to 0.19 log10IU/mL [START_REF] Schonning | Analytical performance of the Hologic Aptima HBV Quant Assay and the COBAS Ampliprep/COBAS TaqMan HBV test v2.0 for the quantification of HBV DNA in plasma samples[END_REF][START_REF] Chevaliez | The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F[END_REF][START_REF] Schalasta | Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples[END_REF]. Monitoring treatment is an important part of chronic HBV patient management as it is used to determine the efficacy of treatment and the occurrence of virological breakthrough [START_REF]European Association for the Study of the Liver. Electronic address eee, European Association for the Study of the L. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection[END_REF][START_REF] Terrault | AASLD guidelines for treatment of chronic hepatitis B[END_REF]. We found that the patient monitoring plots of HBV DNA produced by the Xpert HBV and Aptima assays were similar for the majority of patients. The discrepant results (in the correlation and in the monitoring studies) didn't seem to be related to a particular HBV genotype.

Real-time PCR or TMA assays have become the reference for quantifying virus genomes in clinical practice according to international guidelines However, commercially available assays, although fully automated, use several separate analytical steps and require at least one full day of work to complete the analysis, as shown in our workflow study with the Aptima assay. Our workflow study indicated the the Xpert assay required a mean time to results that was 15h shorter that with the Aptima on the Panther platform. The automated real-time quantitative RT-PCR Xpert HBV assay could also be used as a point-of-care (POC) test for patient management (outside a central laboratory, near the point of patient care).

The relatively uncomplicated nature of the Xpert HBV assay, coupled with its ability to deliver same-day results, make it particularly well suited for use in low-income areas, and also to avoid batch processing in industrialized countries. Were this assay to be deployed in resource-constrained settings where GeneXpert platforms are already being used for diagnosing HIV-1 or tuberculosis diagnostics, has the potential to increase access to HBV testing worldwide. The diagnosis of HBV infection in these countries rely on serological markers, the WHO guidelines in 2015 stated that a quantitative HBV DNA assay may help treatment decisions. (18).

Our study presents some limitations. First, we did not have enough samples to retest discrepant results. Secondly, the workflow study was a single center study and the number of samples was low.

In conclusion, the Xpert HBV assay performed well and was quite comparable with current reference tests. The minimal training and infrastructure required make it attractive for use in resource-constrained settings. The quantitative Xpert assay is a promising tool for monitoring the treatment of HBV patients; it could change how HBV DNA are monitored.

Funding. The Xpert kits were kindly provided by Cepheid (Maurens-Scopont, France). Note: SD, standard deviation; CV, coefficient of variation.

Declarations

Figures legends:

1 .

 1 Figure 1. Xpert assay linearity using samples of different HBV genotypes (3 replicates per

Figure 2 .

 2 Figure 2. Correlation between Xpert and Aptima quantitative results. All values are in log

Figure 3 .

 3 Figure 3. Patient monitoring: virus loads of 19 treated HBV-infected patients: Xpert results

of interest. None 17

  . Benhamou Y, Bochet M, Thibault V, Calvez V, Fievet MH, Vig P, et al. Safety and efficacy of adefovir dipivoxil in patients co-infected with HIV-1 and lamivudine-resistant hepatitis B virus: an open-label pilot study. Lancet. 2001;358(9283):718-23. 18.Guidelines for the Prevention, Care and Treatment of Persons with Chronic Hepatitis B Infection. WHO Guidelines Approved by the Guidelines Review Committee. Geneva2015.

Table 1 :

 1 determination of the limit of detection

	Dilution	Number of replicates	Number of detected samples
	(UI/ml)		(%)
	50	10	10 (100%)
	20	10	10 (100%)
	10	10	10 (100%)
	5	10	8 (80%)
	2.5	10	6 (60%)

Table 2 :

 2 Reproducibility

	Sample	Mean HBV DNA	SD	CV (%)	Median	Range
		Log10 IU/ml	Log10		Log10 IU/ml	Log10 IU/ml
			IU/ml
	A	5.1	0.06	1.1	5.01	5.40-4.80
	B	3.9	0.10	2.6	3.65	4.05 -3.51
	C	2.2	0.28	12.7	2.16	1.77-2.86