MD Peter Kamenický
email: peter.kamenicky@aphp.fr

André Lacroix

Mechanism of ectopic hormone receptors in adrenal tumors and hyperplasias

Since the initial description of glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome, the molecular mechanisms leading to aberrant expression of G-protein coupled receptors in the adrenocortical adenomas and bilateral adrenal hyperplasia have excited scientific curiosity. This review addresses the recent advances in our understanding of the molecular pathogenesis of GIP-dependent Cushing's syndrome, focusing in particular on two novel findings reported by our group two years ago. First, we discuss the observations of monoallelic GIP receptor (GIPR) expression and transcriptional heterogeneity in GIP receptorexpressing adrenal lesions, corroborated recently by similar findings in GIPR-expressing somatotroph pituitary adenomas from patients with acromegaly. Second, we review the findings of 19q13.32 microduplications and chromosome rearrangements as molecular hits leading to adrenal GIPR expression in some adrenocortical adenomas. We further discuss some unanswered questions and future research directions, such as the molecular mechanisms leading to GIPR expression in bilateral adrenal hyperplasia, the putative mechanisms of several aberrant hormone receptors coexpression and the respective role of aberrant receptors as factors modifying steroidogenesis and as molecular drivers responsible for tumor development.

Finally, we review the potential therapeutic implications of aberrant receptors in the treatment of bilateral adrenal lesions.

Introduction

Adrenal Cushing's syndrome results from excessive pituitary corticotropin (ACTH)-independent cortisol production by adrenocortical tumors or by bilateral adrenal hyperplasia [START_REF] Lacroix | Cushing's syndrome[END_REF]. Recent discoveries have substantially enriched our understanding of the pathogenesis of adrenal lesions leading to hypercortisolism [START_REF] Assie | ARMC5 mutations in macronodular adrenal hyperplasia with Cushing's syndrome[END_REF][START_REF] Beuschlein | Constitutive activation of PKA catalytic subunit in adrenal Cushing's syndrome[END_REF][START_REF] Goh | Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors[END_REF][START_REF] Cao | Activating hotspot L205R mutation in PRKACA and adrenal Cushing's syndrome[END_REF]. In addition to genetic driver events promoting tumor development and growth, autocrine / paracrine factors regulating adrenal steroidogenesis have been reported [START_REF] Louiset | Intraadrenal corticotropin in bilateral macronodular adrenal hyperplasia[END_REF].

In a subset of patients with adrenal Cushing's syndrome, the cortisol excess is regulated at least in part by aberrant (ectopic or excessive) expression of certain G-protein-coupled receptors in adrenal lesions [START_REF] Lacroix | Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome[END_REF][START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF]. The concept of the illegitimate expression of certain receptors in the adrenal cortex has been evoked since 1971, following the in vitro demonstration of adenylate cyclase activation by ACTH, but also by adrenaline, norepinephrine and TSH in rat adrenocortical cells [START_REF] Schorr | Abnormal hormone responses of an adrenocortical cancer adenyl cyclase[END_REF]. The first description in humans reported the case of a patient with cortisol secreting adrenocortical adenoma, in whom the production of cortisol presented an inverse rhythm with low fasting morning plasma cortisol concentrations that increased after food intake [START_REF] Hamet | Cushing syndrome with food-dependent periodic hormonogenesis[END_REF]. In 1992, two groups simultaneously reported in two patients with bilateral adrenal hyperplasia and food-dependent Cushing's syndrome, that the postprandial rise of cortisol production was induced by the glucose-dependent insulinotropic polypeptide GIP, an intestinal hormone produced by K-cells [START_REF] Lacroix | Gastric inhibitory polypeptide-dependent cortisol hypersecretion--a new cause of Cushing's syndrome[END_REF][START_REF] Reznik | Food-dependent Cushing's syndrome mediated by aberrant adrenal sensitivity to gastric inhibitory polypeptide[END_REF]. A few years later, the ectopic expression of the glucose-dependent insulinotropic polypeptide receptor (GIPR) was demonstrated in an adrenal adenoma by in situ hybridization [START_REF] De Herder | Food-dependent Cushing's syndrome resulting from abundant expression of gastric inhibitory polypeptide receptors in adrenal adenoma cells[END_REF]. This was followed by an important number of publications of case reports or small series over the last 3 decades, reviewed in [START_REF] Lacroix | Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome[END_REF][START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF]. Activation of the ectopic GIP receptor, functionally coupled to cAMP signaling, triggers excessive hormone production, and, possibly adrenal cell proliferation.

The discovery of GIP-dependent Cushing's syndrome [START_REF] Lacroix | Gastric inhibitory polypeptide-dependent cortisol hypersecretion--a new cause of Cushing's syndrome[END_REF][START_REF] Reznik | Food-dependent Cushing's syndrome mediated by aberrant adrenal sensitivity to gastric inhibitory polypeptide[END_REF] opened an important new research direction leading to identification of adrenocortical expression of several other receptors belonging to the family of G-protein coupled receptors, functionally coupled to the AMPc-PKA pathway. A systematic description of the historical background and pathophysiological and clinical implications of the aberrantly expressed G-protein coupled receptors is beyond the scope of this manuscript. Interested readers will find detailed information in the following review articles [START_REF] Lacroix | Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome[END_REF][START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF][START_REF] St-Jean | Aberrant G-protein coupled hormone receptor in adrenal diseases[END_REF]. Let us simply mention that the illegitimate expression of a G-protein coupled receptor in adrenocortical lesions may be:

stricto sensu ectopic, meaning that the receptor is not expressed (at significant levels) in the normal adrenocortical cells of zona fasciculata (i.e. GIPR, GnRH receptor, β-adrenergic receptor type 1, glucagon receptor); -or an overexpression of an eutopic receptor, when the receptor is physiologically expressed in zona fasciculata cells, but at significantly lower level (i.e. vasopressin receptor type 1a, angiotensin II receptor type 1a, receptor of LH / hCH , serotonin receptor type 4).

Examples of receptors aberrantly expressed in adrenal cortex are schematically represented in the Figure 1. Of note, the cortisol secreting adrenocortical lesions may coexpress several aberrant receptors [START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF][START_REF] St-Jean | Aberrant G-protein coupled hormone receptor in adrenal diseases[END_REF]. A systematic search for aberrant hormone receptors in patients with overt or subclinical Cushing's syndrome, using various stimulation or suppression tests (summarized in Figure 1), showed that the expression of one or several such receptors occurs frequently. Indeed, 80% of patients with cortisol-secreting bilateral adrenal hyperplasias and almost 50% of patients with cortisol-secreting adrenocortical adenomas presented with at least one aberrant response to screening tests [START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF][START_REF] Libe | Aberrant cortisol regulations in bilateral macronodular adrenal hyperplasia: a frequent finding in a prospective study of 32 patients with overt or subclinical Cushing's syndrome[END_REF]. Finally, it should be noted that the illegitimate expression of G protein-coupled receptors is not restricted to cortisol-producing adrenal lesions, but has been also described in a few cases of aldosterone producing adenomas responsible for primary aldosteronism [START_REF] Lampron | Regulation of aldosterone secretion by several aberrant receptors including for glucose-dependent insulinotropic peptide in a patient with an aldosteronoma[END_REF][START_REF] Teo | Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations[END_REF].

The therapeutic implications of aberrant hormone receptors in adrenocortical lesions, schematically represented in Figure 2, are however limited. A long-term control of Cushing's syndrome has been obtained in a man with bilateral macronodular hyperplasia by targeting the ectopic β-adrenergic receptor with propranolol, permitting to avoid bilateral adrenalectomy for 22 years [START_REF] Lacroix | Propranolol therapy for ectopic beta-adrenergic receptors in adrenal Cushing's syndrome[END_REF]. Similarly, in a woman with bilateral macronodular hyperplasia overexpressing the receptor of LH / hCH, a 20 years-long control of cortisol excess has been reached with leuprolide acetate [START_REF] Lacroix | Leuprolide acetate therapy in luteinizing hormone--dependent Cushing's syndrome[END_REF]. In the future targeting ectopic GIPR receptor by its naturally occurring GIP antagonist could be an interesting therapeutic option for patients with GIP-dependent Cushing's syndrome [START_REF] Gasbjerg | Glucose-dependent insulinotropic polypeptide (GIP) receptor antagonists as anti-diabetic agents[END_REF]. However, in the majority of adrenal lesions, excessive steroid production is regulated by several aberrant receptors together with other factors such as locally produced ACTH (Figure 2). Thus, targeting only one aberrant receptor is not an efficient therapeutic strategy in these cases.

Since the initial description of GIP-dependent Cushing's syndrome, the molecular mechanisms leading to aberrant expression of hormone receptors in the adrenal tissue has excited scientific curiosity. However the molecular mechanism of ectopic hormone receptors in adrenal tumors and hyperplasias has remained enigmatic for more 25 years. We have recently reported molecular events leading to adrenal GIPR expression in a large international collection of 16 specimens of adrenocortical adenomas and bilateral macronodular adrenal hyperplasias derived from patients with GIP-dependent adrenal Cushing's syndrome or with GIP-dependent primary aldosteronism [START_REF] Lecoq | of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural chromosome variations[END_REF]. This translational and collaborative research, integrating multiple international expertises, allowed us to postulate two original conclusions discussed in the following two sections of this review.

1) Mono-allelic GIPR expression and transcriptional heterogeneity in GIPR-expressing adrenal lesions

All 16 adrenal samples expressed significant amounts of GIPR transcripts as quantified by RT-qPCR. To visualize GIPR expression at the single cell level in patient's adrenal lesions, we used RNA FISH allowing detection of nascent RNA molecules at their transcription sites in the interphase nuclei. We found that all adenoma and hyperplasia samples expressed GIPR from a single allele of GIPR gene. Normal adrenal tissue does not express GIPR, whereas GIPR expression in tissues that are physiologically receptive to GIP is supposed to occur from both alleles, as GIPR is not considered as an imprinted gene.

Of note, GIPR expression was detected in a certain proportion of nuclei, ranging from 5 to 50%. This diversity in transcriptional activity observed at the individual cell level may be related to the genetic intra-lesion heterogeneity of the adrenal lesions. Alternatively, it could result from transcriptional bursting due to stochastic fluctuation between transcriptionally active and inactive gene states [START_REF] Fujita | Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA[END_REF]. Indeed, according to the current knowledge, transcription is not a continual process, RNA production occurs by switching between "on" and "off" states of genes.

Interestingly, samples showing the highest GIPR transcript levels quantified by RT-qPCR also exhibited the highest proportion of nuclei expressing GIPR detected by RNA FISH, suggesting a good complementarity of these two techniques.

2) 19q13.32 microduplication rearranged with other chromosome regions drive adrenal

GIPR expression

Mono-allelic GIPR expression consistently observed in all adrenal lesions suggest a common disease mechanism resulting in transcriptional activation of a single allele of the gene. Abnormal DNA methylation in the proximal GIPR promoter has been ruled out. Combined molecular search using array-CGH and DNA FISH identified in two adrenocortical adenomas somatic microduplications in 19q13.32 encompassing a 100.4-kb and 191.9-kb region, respectively. Mate-pair sequencing, enabling a pangenomic analysis of chromosome breakpoints, identified an inter-chromosomal rearrangement between the duplicated segments 19q13.32 and 20q13.12 in one adenoma and an intra-chromosomal rearrangement between the duplicated segments 19q13. 32 and 19q13.43 in another adenoma. In both tumors, the rearrangement showed a similar configuration, with a tandem duplication of one chromosome segment, separated by insertion of the GIPR gene-containing segment in inverted orientation (Figure 3). These chromosome rearrangements did not result in fusion genes. They rather generated a novel genomic environment by juxtaposing the GIPR gene with cis-acting regulatory sequences, thus permitting its expression. Indeed, according to the RNA FISH experiments on these tumor samples, only the GIPR allele placed in the rearranged chromosome environment was transcriptionally active. In one tumor GIPR was juxtaposed with the second intron of ubiquitously expressed ZMYND8 gene. This intronic region, rich in regulatory sequences, functions as an alternative promoter. We in particular demonstrated the functional role of a glucocorticoid response element, creating an intracrine and / or paracrine positive regulatory loop, in which the cortisol excess maintains the aberrant GIPR expression (Figure 3). It should be noted however that this recreated genomic environment is specific for this adenoma only. In the second tumor with 19q13.32 microduplications GIPR fall in a vast inter-genic region, offering a less straightforward mechanistic explanation.

Unanswered questions and further research directions

However, far from closing the debate, data reported in [START_REF] Lecoq | of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural chromosome variations[END_REF] open new questions and leave some old enigmas unanswered.

-What are the molecular mechanisms leading to the transcriptional activation of GIPR in the remaining adrenal lesions without 19q13.32 microduplications? First, our findings raise questions as to the molecular pathogenesis of GIPR-dependent primary bilateral macronodular adrenal hyperplasia. Bilateral illicit expression of GIPR and of other aberrant G-protein-coupled receptors in this disease could arise from germline or somatic chromosome rearrangements during early development. Six out of 16 GIPR-expressing adrenal samples have been analysed by mate-pair sequencing. This analysis provided molecular insights for two adenomas bearing 19q13.32 microduplications, but left the others lesions without explanation. Given the performance of this relatively old technique, the hypothesis of a structural abnormality, i.e. of a small microdeletion in cis regulatory sequences [START_REF] Lacroix | Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome[END_REF] involved in the transcriptional repression of GIPR, remains plausible.

Mutations of transcription factors may also be considered [START_REF] Lacroix | Ectopic and abnormal hormone receptors in adrenal Cushing's syndrome[END_REF], although this hypothesis is difficult to reconcile with the observations of mono-allelic GIPR expression. Finally, pyrosequencing of the GIPR proximal GIPR promoter does not exclude the possibility of methylation abnormalities in distal regulatory sequences or in the gene body. Indeed, the GIPR gene comprises several CpG islands and hypermethylation of GIPR gene associated with GIPR upregulation has been described in small intestinal neuroendocrine tumors [START_REF] Karpathakis | intestinal neuroendocrine tumors are highly epigenetically dysregulated. GIPR gene body hyperpemthylaton was in articular observed[END_REF]. Along this line, we have recently performed whole genome DNA methylation analysis in 16 somatotroph pituitary tumors expressing or not GIPR (vide infra). GIPR-expressing adenomas negative for GNAS gene mutations displayed global DNA hypermethylation, pronounced in the gene bodies, in comparison with GNAS-mutated adenomas not expressing GIPR. DNA methylation analysis of GIPR gene revealed no alterations in promoter methylation but hypermethylation of GIPR gene body in GIPR-expressing tumors [START_REF] Hage | Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas[END_REF]. As recently demonstrated, such gene body DNA methylation may preserve stabilization of the nucleosome in transcribed regions and enhance transcriptional efficiency [START_REF] Jones | Functions of DNA methylation: islands, start sites, gene bodies and beyond[END_REF]. Thus, abnormal DNA methylation, or other epigenetic modifications, could also be the pathogenetic driver event of aberrant GIPR expression.

-How to explain the concomitant expression of several aberrant receptors in one adrenal lesion?

Microduplications of the 19q13.32 locus explain exclusively the expression of GIPR. However, concomitant expression of several illicit receptors has been systematically repeatedly reported [START_REF] El Ghorayeb | Multiple aberrant hormone receptors in Cushing's syndrome[END_REF][START_REF] St-Jean | Aberrant G-protein coupled hormone receptor in adrenal diseases[END_REF]. In our series, four samples coexpressed two or more aberrant receptors, including three samples of bilateral hyperplasia expressing the LH / hCG receptor. No microamplifications and / or structural variation on chromosome segments containing the genes of these receptors were observed. Recently, a link has been proposed between activating p.Ser33Cys, p.Gly34Arg and p.Ser45Phe mutations of CTNNB1 encoding ß-catenin and overexpression of GnRH and LH / hCH receptors in three aldosterone producing adenomas [START_REF] Teo | Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations[END_REF]. However, this result has not been found by others [START_REF] Berthon | Pregnancy, Primary Aldosteronism, and Somatic CTNNB1 Mutations[END_REF][START_REF] Gagnon | Genetic Characterization of GnRH/LH-Responsive Primary Aldosteronism[END_REF] and was not confirmed in our series.

-Do illegitimate receptors drive tumor development or only trigger excessive hormone production ? Of note, the adenoma with 19q13.32 microduplication inserted into the intron 2 of ZMYND8 also harbored a somatic p.Ser45Cys mutation of CTNNB1. This suggests a 2 step model, in which the activating ß-catenin gene mutation may have contributed to the tumor development, whilst the chromosome rearrangement resulted in GIPR expression, triggering the specific profile of postprandial cortisol excess. Similarly, MEN1 mutation in another patient from our series probably represented the molecular hit favoring the development of bilateral macronodular adrenal hyperplasia in that case. On the other hand, no mutations in genes driving adrenal tumorigenesis were found in the remaining adrenal samples. Thus, the presence of ectopic G-protein-coupled receptors, functionally coupled to cAMP pathway, which is a major mitogenic signal in zona fasciculata cells, could per se promote cell proliferation and tumor development, as it has been demonstrated in mice [START_REF] Mazzuco | Ectopic expression of the gastric inhibitory polypeptide receptor gene is a sufficient genetic event to induce benign adrenocortical tumor in a xenotransplantation model[END_REF] and suggested in transiently pregnancy induced bilateral adrenal hyperplasia and severe Cushing's syndrome [START_REF] Plockinger | Functional Implications of LH/hCG Receptors in Pregnancy-Induced Cushing Syndrome[END_REF].

Ectopic GIPR in somatotroph pituitary adenomas

The concept of ectopic expression of hormone receptors has recently been extended to growth hormone (GH)-secreting pituitary adenomas. Chronic GH excess from these tumors is responsible for gigantism during childhood and acromegaly in adulthood. A subset of patients with acromegaly present with a paradoxical increase in GH secretion (instead of suppression) after oral glucose load [START_REF] Besser | Growth hormone release inhibiting hormone in acromegaly[END_REF]. By analogy to GIP-dependent Cushing's syndrome, this paradoxical rise in GH has been linked to ectopic expression of the GIPR in somatotroph pituitary tumors [START_REF] Occhi | The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)promoter activity in GH3 cells[END_REF][START_REF] Regazzo | The GIP/GIPR axis is functionally linked to GH-secretion increase in a significant proportion of gsp(-) somatotropinomas[END_REF]. However, the quantity of GIPR transcripts is significantly lower in the pituitary tumors compared to adrenal lesions. Based on our above-described findings in adrenal lesions [START_REF] Lecoq | of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural chromosome variations[END_REF], we have searched the molecular mechanisms leading to ectopic GIPR expression in these pituitary tumors. Corroborating our observations in adrenal lesions, GIPR expression occurred in the pituitary tumors though transcriptional activation of a single allele of the GIPR gene in all GIPRexpressing pituitary tumors, except in two samples tetraploid for the GIPR locus, where expression occurred from two alleles per nucleus. Again, we searched for the reasons of this hypomorphic transcriptional activation. Unlike in the adrenal adenomas, array-CGH did not identify 19q13.32 microduplications, however DNA FISH visualized in four samples microamplifications encompassing only the GIPR gene. These microamplifications were present in a certain proportion of nuclei, illustrating the subclonal architecture of pituitary adenomas [START_REF] Hage | Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas[END_REF][START_REF] Hage | Genomic Alterations and Complex Subclonal Architecture in Sporadic GH-Secreting Pituitary Adenomas[END_REF]. As already stated above, global DNA methylation analysis showed an overall hypermethylator phenotype in GIPR-expressing samples compared with GNAS-mutated adenomas, and in particular, GIPR gene body hypermethylation, likely leading to ectopic gene expression. Such GIPR gene hypermethylation has been reported in small intestinal neuroendocrine tumors [START_REF] Karpathakis | intestinal neuroendocrine tumors are highly epigenetically dysregulated. GIPR gene body hyperpemthylaton was in articular observed[END_REF]. Nevertheless it is not clear whether GIPR is aberrantly expressed in these tumors. Finally, besides adrenal and pituitary tumors, chromosome rearrangements and / or abnormal epigenetic modifications may also be responsible for ectopic secretion of various hormones by neuroendocrine tumors and other tumors associated with paraneoplastic hormonal syndromes.

Conclusion

Recently important advances have been achieved in our understanding of the molecular pathogenesis of GIP-dependent Cushing's syndrome. Ectopic GIPR expression in adrenocortical adenomas and in primary bilateral macronodular adrenal hyperplasias systematically occurs from single allele of GIPR gene. This monoallelic ectopic GIPR expression was associated in two adrenal adenomas with a genomic gain in 19q13.32 containing GIPR gene with chromosomal rearrangements, creating a novel genomic environment favorable for GIPR transcription.

However, despite recent insight, there are still many unanswered questions. Answering of the opened questions will provide a more complete understanding of the pathogenesis of adrenal Cushing's syndrome, with aberrantly expressed hormone receptors in their adrenal lesions. Adapted from [START_REF] Lecoq | of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural chromosome variations[END_REF].

Ectopic expression

Overexpression of eutopic receptors

Figure 1 .

 1 Figure 1. Aberrant expression of hormone receptors in the adrenal lesions and the stimulation or suppression tests used to identify these receptors in clinical practice.AVPR1a and HTR4 in grey boxes are the most frequently encountered aberrant receptors.

Figure 2 .

 2 Figure 2. Pharmaceutical targeting of the ectopic receptors as an alternative therapeutic approach to (bilateral) adrenalectomy.

Figure 3 .

 3 Figure 3. Example of a molecular event leading to ectopic GIPR expression in an adrenocortical adenoma.

  Figure 1

Identifications of factors determining the tissue specificity of G-protein coupled receptors expression will represent a big step forward in human physiology.


Sébastien Soize

Cédric Foussier

Pierre-François Manceau

Claude-Fabien Litré

Serge Backchine

Matthias Gawlitza

Laurent Pierot
email: lpierot@gmail.com

Pierre-Franc ¸ois Manceau

Comparison of two preventive dual antiplatelet regimens for unruptured intracranial aneurysm embolization with flow diverter/disrupter: A matched-cohort study comparing clopidogrel with ticagrelor

Keywords:

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

Introduction

Endovascular treatment (EVT) is the first line treatment in the majority of patients suffering from ruptured and unruptured cerebral aneurysms [START_REF] Molyneux | International subarachnoid aneurysm trial (ISAT) of neurosurgical clipping versus endovascular coiling in 2143 patients with ruptured intracranial aneurysms: a randomised comparison of effects on survival, dependency, seizures, rebleeding, subgroups, and aneurysm occlusion[END_REF][START_REF] Cognard | Clarity Study Group. Results of embolization used as the first treatment choice in a consecutive nonselected population of ruptured aneurysms: clinical results of the Clarity GDC study[END_REF][START_REF] Pierot | ATENA Investigators. Immediate clinical outcome of patients harboring unruptured intracranial aneurysms treated by endovascular approach: results of the ATENA study[END_REF]. Improved endovascular techniques such as balloon-assisted coiling, stent-assisted coiling (SAC) and more recently flow diversion and flow disruption have facilitated treatment of an increased number of aneurysms including complex aneurysms with unfavorable anatomy [START_REF] Pierot | Endovascular techniques for the management of wide-neck intracranial bifurcation aneurysms: a critical review of the literature[END_REF][START_REF] Pierot | Endovascular treatment of intracranial aneurysms: current status[END_REF]. Additionally, flow diversion help to reduce the rate of aneurysm recurrence, which remains the primary weakness of endovascular EVT [START_REF] Pierot | Endovascular techniques for the management of wide-neck intracranial bifurcation aneurysms: a critical review of the literature[END_REF]. Intraoperative rupture and thromboembolic events (TE) are the main concerns during EVT because of potential disastrous outcomes. To prevent thromboembolism, some EVT techniques (SAC and flow diversion) are performed under dual antiplatelet therapy (DAPT). By extension, the treatment of complex wide-neck bifurcation aneurysms by flow disruption often occurs under DAPT [START_REF] Pierot | Endovascular treatment of intracranial aneurysms: current status[END_REF].

Acetylsalicylic acid (ASA) plus clopidogrel is the DAPT of reference used for preventing thrombosis in such procedures [START_REF] Pierot | Endovascular treatment of intracranial aneurysms: current status[END_REF][6][START_REF] Faught | Heterogeneous practice patterns regarding antiplatelet medications for neuroendovascular stenting in the USA: a multicenter survey[END_REF].However, unlike ASA resistance which seems relatively uncommon, clopidogrel resistance occurs frequently (28 to 66%) [START_REF] Fifi | Clopidogrel resistance is associated with thromboembolic complications in patients undergoing neurovascular stenting[END_REF] and is known to induce additional thromboembolic complications [START_REF] Fifi | Clopidogrel resistance is associated with thromboembolic complications in patients undergoing neurovascular stenting[END_REF][START_REF] Tan | Thromboembolic complications with Pipeline Embolization Device placement: impact of procedure time, number of stents and pre-procedure P2Y12 reaction unit (PRU) value[END_REF]. Evaluating the biological activity of this medication in vivo is complex and unreliable, and no standardized platelet function test exists. Furthermore, major bleeds are more common with double-dose than with standard-dose of clopidogrel [START_REF] Mehta | Double-dose versus standard-dose clopidogrel and high-dose versus low-dose aspirin in individuals undergoing percutaneous coronary intervention for acute coronary syndromes (CURRENT-OASIS 7): a randomised factorial trial[END_REF][START_REF] Storey | Inhibitory Effects of ticagrelor compared with clopidogrel on platelet function in patients with acute coronary syndromes: the PLATO (PLATelet inhibition and patient outcomes) PLATELET substudy[END_REF]. Consequently, the benefit of assessing clopidogrel resistance remains unclear and increases time and burden of care [START_REF] Nordeen | Clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications., clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications[END_REF][START_REF] Skukalek | Effect of antiplatelet therapy and platelet function testing on hemorrhagic and thrombotic complications in patients with cerebral aneurysms treated with the pipeline embolization device: a review and meta-analysis[END_REF], and, as a result, other P2Y12 inhibitors (e.g., ticagrelor and prasugrel) which are less subject to resistance are usually indicated in patients with acute coronary syndrome [START_REF] Mehta | Double-dose versus standard-dose clopidogrel and high-dose versus low-dose aspirin in individuals undergoing percutaneous coronary intervention for acute coronary syndromes (CURRENT-OASIS 7): a randomised factorial trial[END_REF][START_REF] Storey | Inhibitory Effects of ticagrelor compared with clopidogrel on platelet function in patients with acute coronary syndromes: the PLATO (PLATelet inhibition and patient outcomes) PLATELET substudy[END_REF]. The above data has led some clinicians to explore the usefulness of ticagrelor for neurointerventional procedures [START_REF] Mehta | Double-dose versus standard-dose clopidogrel and high-dose versus low-dose aspirin in individuals undergoing percutaneous coronary intervention for acute coronary syndromes (CURRENT-OASIS 7): a randomised factorial trial[END_REF][START_REF] Storey | Inhibitory Effects of ticagrelor compared with clopidogrel on platelet function in patients with acute coronary syndromes: the PLATO (PLATelet inhibition and patient outcomes) PLATELET substudy[END_REF][START_REF] Wallentin | Ticagrelor versus Clopidogrel in Patients with Acute Coronary Syndromes[END_REF]. In contrast to clopidogrel, ticagrelor produces greater platelet inhibition, has faster onset action and exhibits fewer inter-individual variations with respect to drug side effects [START_REF] Wallentin | Ticagrelor versus Clopidogrel in Patients with Acute Coronary Syndromes[END_REF].

Currently, little data regarding safety and efficacy of ticagrelor in neurointerventional procedures is available [START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF][START_REF] Narata | Dual antiplatelet therapy combining aspirin and ticagrelor for intracranial stenting procedures: a retrospective single center study of 154 consecutive patients with unruptured aneurysms[END_REF] and only one study compared ticagrelor with clopidogrel in patients treated with flow diverters [START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF] One major concern with ticagrelor remains its safety, especially in regard to the PLATO trial results, which compared ticagrelor to clopidogrel in acute coronary syndrome settings and found a significantly increased risk of intracranial hemorrhage in patients receiving ticagrelor [START_REF] Storey | Inhibitory Effects of ticagrelor compared with clopidogrel on platelet function in patients with acute coronary syndromes: the PLATO (PLATelet inhibition and patient outcomes) PLATELET substudy[END_REF]. In our institution, patients were treated with ASA plus clopidogrel without systematic testing for resistance up to December 2015. At that point, we switched from clopidogrel to ticagrelor in order to avoid the issue of clopidogrel resistance testing. The present study compares the impact of two DAPT (ASA + clopidogrel vs. ASA + ticagrelor) on the morbidity, mortality, safety (hemorrhagic complications) and efficacy (prevention of thromboembolism) of unruptured aneurysm embolization with flow diverter/disrupter.

Materials and methods

Q3

Study setting

This retrospective study analyzed our institution's prospectively collected registry of consecutive patients treated by endovascular means of intracranial aneurysms. In line with French law, ethics committee approval is waived for retrospective studies. The manuscript follows reporting guidelines (STROBE) for observational study reports [START_REF] Von Elm | The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies[END_REF]. For the purpose of this study, we included patients who fulfilled the following inclusion criteria: Patients of both groups were matched for age (±10 years), type of treatment (intrasaccular or intravascular) and aneurysm sac size ( ± 2 mm), creating a 1:1 matched cohort.

•

Antiplatelet therapy

During the study period, patients treated for unruptured aneurysms with flow diverter and flow disrupters received loading of DAPT over 5 days (clopidogrel) and 2 days (ticagrelor) before embolization. From March 2013 to October 2015, patients received 75 mg of ASA + once per day 75 mg of clopidogrel (Group 1); between November 2015 and March 2017, patients received 75 mg ASA + 90 mg of ticagrelor twice per day (Group 2). Both groups received heparin bolus of 50 IU/kg followed by a heparin perfusion at 600 IU/kg/day in the perioperative period. Both groups received DAPT over the next 3 months. Importantly, no patient underwent clopidogrel resistance testing because it was not part of the institutional protocol at that time. Consequently, there was no selection for clopidogrel responsiveness in either group.

Primary outcome measures were morbidity and mortality. Morbidity at 1 month was defined as any worsening of the modified Rankin Scale (mRS) compared to pre-therapeutic mRS. Secondary outcome measures were thromboembolic complications (per-procedure and on 24-48 hour follow-up MRI), hemorrhagic complications (per-procedure and on 24-48 hour follow-up MRI) and groin puncture hemorrhagic complications (hematoma >2 cm or false aneurysm, with or without need for a treatment (transfusion, surgery or ultrasound-guided compression).

Endovascular treatment

All patients eligible for endovascular aneurysmal treatment were discussed in a multidisciplinary meeting (neurosurgeons, neurologists and neuroradiologists) to determine the safest and most effective treatment method. Treatments were performed with a biplane angiographic system (Axiom Artis, Siemens, Erlangen, Germany). After transfemoral access, a triaxial system reached the targeted aneurysm. Intrasaccular treatment was completed with the Woven EndoBridge (WEB device, Sequent Medical/Microvention, Tustin, CA, USA) flow disruption device, and intravascular treatment with two different flow diverters: the Pipeline Embolization Device: (PED), (Covidien, Irvine, CA, USA), or the Flow Re-Direction Endoluminal Device (FRED), (MicroVention, Tustin, CA, USA). Vascular closing systems were used in all procedures (Angioseal or Femoseal Systems, St Jude Medical, St. Paul, MN, USA).

Recorded data

Baseline patient characteristics (sex, age and pretreatment mRS), the aneurysm status (first treatment or remnant) and aneurysms characteristics (location, maximum sac diameter and neck size) were collected. Aneurysm location was categorized as follows: supraclinoid internal carotid artery (ICA), cavernous ICA, anterior cerebral artery (ACA) or anterior communicating artery (ACom), middle cerebral artery (MCA) and basilar artery (BA). Treatment types were also recorded and dichotomized into intrasaccular device (flow disrupter) or intravascular device (flow diverter).

We recorded all complications occurring during aneurysm treatment and hospital stay. During treatment we also recorded thromboembolic events (i.e., clot visible on angiography), aneurysm rupture and vessel dissection. After treatment, we observed hemorrhagic complications at puncture site and any ischemic or hemorrhagic complication on MRI realized 24-48 hours after treatment (see "image analysis" for description). At 1-month posttreatment, the treating physician evaluated neurological outcome using the mRS. Finally, at 3-months post-treatment, patients underwent another MRI examination and clopidogrel or ticagrelor was stopped. Any new ischemic or hemorrhagic complication at 3months was recorded and classified according to their clinical impact (silent or symptomatic). Also, parent vessel patency was evaluated. Then, in absence of ischemic event or vessel stenosis/thrombosis, clopidogrel or ticagrelor was stopped and ASA was pursued for 1-year post-treatment.

Image analysis

All follow-up examinations were performed on a 3-Tesla MR Unit (Achieva, Philips Healthcare, Best, The Netherlands or Skyra, Siemens, Erlangen, Germany) with a protocol including at least diffusion weighted imaging (DWI), fluid-attenuated inversion recovery (FLAIR) T2* and time-of-flight (TOF)-MRA sequences. Gadolinium-enhanced sequences were optional. An independent analysis of the post-treatment DWI and T2* maps was performed by two neuroradiologists (3 and 6-years' experience, respectively) separately and blinded to patient characteristics and treatment strategies. Readers determined if there was a territorial infarction, defined as a DWI positive lesion > 15 mm diameter according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria, as well as the presence and number of small DWI(+) lesions, hereafter defined as a high signal intensity lesion of ≤ 15 mm diameter [START_REF] Adams | Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment[END_REF]. Patients were categorized in two groups according to the number of small DWI(+) lesions: either 0 to 5 lesions or > 6 DWI(+) lesions [START_REF] Kang | MR-DWI-positive lesions and symptomatic ischemic complications after coiling of unruptured intracranial aneurysms[END_REF].

Additionally, readers noted any major hemorrhagic complication, defined by the appearance of a subarachnoid hemorrhage or a parenchymal hematoma on FLAIR and/or T2* sequences. The appearance of minor hemorrhage (microbleeds) and their burden were also reported. Microbleeds were defined as small areas (≤ 10 mm) of signal void on T2* sequence with associated blooming [START_REF] Gregoire | The Microbleed Anatomical Rating Scale (MARS): reliability of a tool to map brain microbleeds[END_REF]. All available MR sequences were analyzed to exclude potential microbleeds mimics (vascular structures, thrombus, calcifications, iron deposits, cavernous malformation or hemorrhagic metastases). Almost all patients (36/40 for Group 1 and 36/40 for Group 2) had a recent pre-therapeutic MRI to rule out pre-existing ischemia or hemorrhage. Disagreements were solved by a third reader (9 years of experience).

Statistical analyses

Inter-reader agreement for detection of territorial infarction, small artery infarction, > 6 DWI(+) lesions and major and minor hemorrhagic complications were assessed using Cohen's kappa coefficient (). Inter-reader agreement for counting small artery infarction (DWI + lesions) and microbleeds burden were assessed using intra-class correlation coefficients (ICC) and their 95% confidence intervals (CI). Distribution normality was assessed with the Kolmogorv-Smirnov test. Continuous variables were described as median and interquartile range and categorical variables as proportions. Bivariate comparisons of primary and secondary outcome measures between Groups 1 and 2 were tested using 2 tests for categorical variables (Fisher's exact test was used when the expected cell frequency was < 5), Student's t-test for continuous variables and Mann-Whitney U-test for ordinal variables and continuous variables that had skewed distributions. As flow diverters and flow disrupters have different mechanism of action, we also conducted a sub-group analysis according to the treatment type. All statistical tests were 2-sided and P < 0.05 was considered statistically significant. Analyses were performed using Medcalc software (Release 18.2, Ostend, Belgium).

Results

Patients

We treated 354 intracranial aneurysms with 167 unruptured aneurysms and 197 ruptured aneurysms during the study period. Ninety patients fulfilled the inclusion criteria (45 in each group). After matching age, type of treatment and aneurysm sac size, 80 patients (40 in each group) were included in the final 1:1 matched cohort. In the cohort, 46 patients (57.5%) were treated with a flow disrupter and 34 (42.5%) with a flow diverter. Baseline characteristics did not differ between groups (Table 1).

Inter-reader agreements

Inter-reader agreement for the detection of territorial infarction [K (95% CI) = 1 (1-1)], small DWI (+) lesion > 6 [K (95% CI) = 0.83 (0.71-0.95)], major [K (95% CI) = 1 (1-1)] and minor hemorrhagic complications [K (95% CI) = 0.82 (0.68-0.96)] was excellent.

Inter-reader agreement for counting small DWI (+) lesions [ICC (95% CI) = 0.97 (0.96-0.98)] and microbleeds burden [ICC (95% CI) = 0.86 (0.79-0.91)] was also excellent.

Primary outcome measures (morbidity and mortality)

At 1 month, morbidity was 1/40 patients (2.5%) with ticagrelor, and 4/40 patients (10%) with clopidogrel (not tested for responsiveness), without statistically significant difference (P = 0.36). Complications in the clopidogrel group were 2 parenchymal hematomas (mRS 1 and 4 at 1 month), 1 territorial infarction (mRS 3) and 1 patient with 27 small DWI(+) lesions (mRS 1). The single complication in the ticagrelor group was a territorial infarction (mRS 2). Mortality was zero in both groups (P = 1). Consequently, combined morbi-mortality was 2.5% vs. 10% (P = 0.36). Comparisons between primary and secondary outcome measures are detailed in Table 2.

Secondary outcome measures

Thromboembolic complications

During the procedure, thromboembolic complications (any visible clot) occurred in 2/40 patients (5%) with ticagrelor and 5/40 patients (12.5%) with clopidogrel (P = 0.43). Post-procedure, early MRI imaging showed territorial infarction on DWI in 1/40 (2.5%) patients with ticagrelor and 3/40 (7.5%) patients with clopidogrel (P = 0.61). In contrast, patients with >6 small DWI lesions were more frequent with ticagrelor than with clopidogrel (20/40 patients (50%) vs. 10/40 patients (25%), P = 0.02).

Hemorrhagic complications

During the procedure, no hemorrhagic complication (sac perforation, dissection) was observed in both groups. However, post-procedural MRI imaging showed major hemorrhagic complications in 2/40 (5%) patients given ticagrelor and 2/40 (5%) patients given clopidogrel (P = 1). These complications involved two limited subarachnoid hemorrhages in the cistern surrounding the aneurysm dome in patients treated with flow disruptors (1 in each group) and two parenchymal hematomas (1 in each group), one at the aneurysm site probably because of sac perforation undetected during the procedure and one probably related to distal vessel perforation caused by microwire manipulations. Interestingly, early MRI revealed that patients under ticagrelor seemed to develop more microbleeds. Indeed, the appearance of at least one microbleed was observed in 23/40 (57.5%) patients given ticagrelor and 16/40 (40%) patients given clopidogrel (P = 0.12). The burden of microbleeds was also higher with ticagrelor than clopidogrel [2 (1-2) vs. 1 (0-2), P = 0.01].

Groin puncture complications

Groin complications occurred in 1/40 (2.5%) patients given ticagrelor and 0/40 (0%) given clopidogrel (P = 1). The complication corresponded to a false aneurysm closed by surgery in a patient given ticagrelor. Additionally, we observed hematomas <2 cm diameter without active bleeding in 2 patient given clopidogrel.

Delayed ischemic and hemorrhagic complications (at 3-months)

At 3-months MRI follow-up, there was no territorial infarction and no major bleeding in both groups [0 (0%) vs. 0 (0%), P = 1, respectively]. There was no difference in terms of patients with new microbleeds [9 (22.5%) vs. 16 (40%), P = 0.15] and patients with >6 small DWI (+) lesions (1 (2.5%) vs. 0 (0%), P = 1) between Ticagrelor and Clopidogrel groups. Also, no stenosis or occlusion of parent vessels was noted in either group.

Analysis by type of treatment

In the subgroups of patients treated with flow-diverters and flow-disrupters, there was no statistical differences in terms of per-procedure thromboembolic and hemorrhagic complications, ischemic and hemorrhagic complications on 24-48 h MRI followup, morbidity and mortality at 1-months (Table 3).

Discussion

The development of advanced techniques has ushered in a new aneurysm treatment era. However, SAC and flow diversion carries an increased risk of thromboembolic complications which lead to the routine use of DAPT [START_REF] Faught | Heterogeneous practice patterns regarding antiplatelet medications for neuroendovascular stenting in the USA: a multicenter survey[END_REF][START_REF] Fifi | Clopidogrel resistance is associated with thromboembolic complications in patients undergoing neurovascular stenting[END_REF][START_REF] Nordeen | Clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications., clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications[END_REF][START_REF] Skukalek | Effect of antiplatelet therapy and platelet function testing on hemorrhagic and thrombotic complications in patients with cerebral aneurysms treated with the pipeline embolization device: a review and meta-analysis[END_REF][START_REF] Wallentin | Ticagrelor versus Clopidogrel in Patients with Acute Coronary Syndromes[END_REF][START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF][START_REF] Narata | Dual antiplatelet therapy combining aspirin and ticagrelor for intracranial stenting procedures: a retrospective single center study of 154 consecutive patients with unruptured aneurysms[END_REF][START_REF] Von Elm | The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies[END_REF][START_REF] Adams | Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment[END_REF][START_REF] Kang | MR-DWI-positive lesions and symptomatic ischemic complications after coiling of unruptured intracranial aneurysms[END_REF][START_REF] Gregoire | The Microbleed Anatomical Rating Scale (MARS): reliability of a tool to map brain microbleeds[END_REF][START_REF] Pierot | Feasibility, complications, morbidity, and mortality results at 6 months for aneurysm treatment with the flow re-direction endoluminal device: report of SAFE study[END_REF]. Although not mandatory, patients treated with flow disruptors are also treated with DAPT as a precaution [START_REF] Pierot | Safety and efficacy of aneurysm treatment with WEB in the cumulative population of three prospective, multicenter series[END_REF][START_REF] Gawlitza | Treatment of recurrent aneurysms using the Woven EndoBridge (WEB): anatomical and clinical results[END_REF][START_REF] Pierot | Safety and efficacy of aneurysm treatment with the WEB: results of the WEBCAST 2 study[END_REF] DAPT may also be useful when flow disruption requires the use of a stent (device protrusion) or is replaced by SAC (technical failure). ASA plus clopidogrel is the standard DAPT for such procedures; [START_REF] Pierot | Endovascular techniques for the management of wide-neck intracranial bifurcation aneurysms: a critical review of the literature[END_REF][START_REF] Pierot | Endovascular treatment of intracranial aneurysms: current status[END_REF][6][START_REF] Faught | Heterogeneous practice patterns regarding antiplatelet medications for neuroendovascular stenting in the USA: a multicenter survey[END_REF][START_REF] Fifi | Clopidogrel resistance is associated with thromboembolic complications in patients undergoing neurovascular stenting[END_REF][START_REF] Tan | Thromboembolic complications with Pipeline Embolization Device placement: impact of procedure time, number of stents and pre-procedure P2Y12 reaction unit (PRU) value[END_REF][START_REF] Nordeen | Clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications., clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications[END_REF][START_REF] Skukalek | Effect of antiplatelet therapy and platelet function testing on hemorrhagic and thrombotic complications in patients with cerebral aneurysms treated with the pipeline embolization device: a review and meta-analysis[END_REF][START_REF] Pierot | Feasibility, complications, morbidity, and mortality results at 6 months for aneurysm treatment with the flow re-direction endoluminal device: report of SAFE study[END_REF][START_REF] Pierot | Safety and efficacy of aneurysm treatment with WEB in the cumulative population of three prospective, multicenter series[END_REF][START_REF] Gawlitza | Treatment of recurrent aneurysms using the Woven EndoBridge (WEB): anatomical and clinical results[END_REF][START_REF] Pierot | Safety and efficacy of aneurysm treatment with the WEB: results of the WEBCAST 2 study[END_REF] however, platelet function testing to detect clopidogrel resistance remains debatable [START_REF] Nordeen | Clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications., clopidogrel resistance by P2Y12 platelet function testing in patients undergoing neuroendovascular procedures: incidence of ischemic and hemorrhagic complications[END_REF][START_REF] Skukalek | Effect of antiplatelet therapy and platelet function testing on hemorrhagic and thrombotic complications in patients with cerebral aneurysms treated with the pipeline embolization device: a review and meta-analysis[END_REF]. A number of alternate antiplatelet agents -most frequently involving ticagrelor for neurovascular field-have become available, with theoretical advantages when compared to clopidogrel in regard to bioavailability and consistency of antiplatelet effects. Little data is available in the neurovascular literature to support the use of one agent over another. As ticagrelor reaches greater platelet inhibition compared with clopidogrel, one major concern is its safety for neurointerventional procedures. Our study found no difference in terms of morbidity and mortality between patients under DAPT with ticagrelor or clopidogrel. Still, although not significant, some differences cannot be underestimated. Indeed, morbidity looked lower with ticagrelor (2.5%) compared to clopidogrel (10%). This result was sustained in flow-diverter and also flow-disrupter subgroups. We also did not find any statistical difference in terms of major hemorrhagic complications (aneurysm rupture, subarachnoid hemorrhage and parenchymal hematoma) between patients under clopidogrel or ticagrelor. These results are reassuring regarding the safety of ticagrelor for neurointerventional procedures and are in line with previous series [START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF][START_REF] Narata | Dual antiplatelet therapy combining aspirin and ticagrelor for intracranial stenting procedures: a retrospective single center study of 154 consecutive patients with unruptured aneurysms[END_REF]. In addition, it is important to remind that in case of sac perforation during the procedure, the use of platelet transfusion can reverse the effects of clopidogrel but not of ticagrelor [START_REF] Godier | Inefficacy of platelet transfusion to reverse ticagrelor[END_REF][START_REF] Teng | Effects of autologous platelet transfusion on platelet inhibition in ticagrelor-treated and clopidogrel-treated subjects[END_REF]. However, this disadvantage may be not for long as recent pre-clinical studies demonstrated the efficacy of specific ticagrelor antidote [START_REF] Buchanan | Structural and functional characterization of a specific antidote for ticagrelor[END_REF][START_REF] Pehrsson | Hemostatic effects of the ticagrelor antidote MEDI2452 in pigs treated with ticagrelor on a background of aspirin[END_REF]. Interestingly, we found that patients receiving ticagrelor were prone to a higher burden of new microbleeds (on 24-48 hour follow-up MRI) compared to patients under clopidogrel (P = 0.01). This finding may be important and needs further exploration. Indeed, even if microbleeds are most often asymptomatic, they may, at times, cause neurological deficit and increase the risk of hemorrhagic stroke, death and cognitive impairment [START_REF] Charidimou | Clinical significance of cerebral microbleeds on MRI: a comprehensive meta-analysis of risk of intracerebral hemorrhage, ischemic stroke, mortality, and dementia in cohort studies (v1)[END_REF]. Previous studies have shown that the use of platelet aggregation inhibitors is related to the presence of cerebral microbleeds [START_REF] Vernooij | Use of antithrombotic drugs and the presence of cerebral microbleeds: the Rotterdam Scan Study[END_REF] and that the appearance of cerebral microbleeds after neurointerventional procedures for acute ischemic stroke has been reported in around 20% of patients [START_REF] Shi | New Cerebral Microbleeds After Mechanical Thrombectomy for Large-Vessel Occlusion Strokes[END_REF]. Our study found new microbleeds in 57.5% of patients given ticagrelor and 40% of patients given clopidogrel (P = 0.12). However, the number of new microbleeds per patient was very low and may be asymptomatic (median of 2 microbleeds for patients under ticagrelor and median of 1 under clopidogrel).

Another concern is the efficacy of DAPT in preventing thromboembolic complications. In line with another study comparing ticagrelor with clopidogrel, [START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF] we found no difference in terms of thromboembolic complications (either on angiography or MRI). Despite non-significant results, per-procedure thromboembolic events appeared to be lower with ticagrelor (5%) than clopidogrel (12.5%). This difference may be accounted for by the absence of platelet function testing in our series. Indeed, Moore et al. found thromboembolic complications in 4.2% of patients given ticagrelor (patients tested as non-responders to clopidogrel) and 6% given clopidogrel (patients tested as responders to clopidogrel) [START_REF] Moore | A multicenter cohort comparison study of the safety, efficacy, and cost of ticagrelor compared to clopidogrel in aneurysm flow diverter procedures[END_REF]. Moore et al. compared the safety and efficacy of DAPT with ticagrelor versus clopidogrel in the best possible conditions for clopidogrel efficacy (patients selected according to clopidogrel level of resistance), whereas our evaluation was done with the worst possible conditions (clopidogrel resistance was not evaluated). Our study, as well as that of Moore et al, was undoubtedly underpowered due to the low patient numbers, limiting our ability to identify and draw conclusions of any differences between groups. However, the incidence of thromboembolic events reported in these two studies may help determine appropriate sample sizes for future prospective trials. Indeed, with a thromboembolic complication difference between 2.2% (tested patients) and 7.5% (untested patients), the number of subjects necessary to detect a significant difference with ticagrelor will require sample sizes of 100 (for untested patient) and 1100 (for tested patient), assuming a type I error (␣) of 0.05 and a type II error (␤) of 0.20. Regardless, no significance difference in terms of safety and efficacy between DAPT with ticagrelor or clopidogrel exists. Consequently, with systematic platelet function testing prior to embolization, ticagrelor is a suitable alternative for patients hyporesponsive to clopidogrel. Nonetheless, in institutions where platelet function testing cannot be done routinely, the most pragmatic and safe option is to use ticagrelor instead of clopidogrel.

Approximately 50% of patients experience small DWI(+) lesions after embolization, [START_REF] Bond | Diffusion-weighted imaging-detected ischemic lesions following endovascular treatment of cerebral aneurysms: a systematic review and meta-analysis[END_REF] which is consistent with our results. Surprisingly, we observed a higher proportion of patients who had >6 small DWI(+) lesions under ticagrelor compared to clopidogrel (50% vs. 25%, P = 0.02). Because this result is likely related to a variety of non-identifiable operating conditions when collecting data, it is difficult to offer a clear explanation to this difference.

Currently, DAPT for complex embolization of unruptured aneurysms is no more limited to clopidogrel. Ticagrelor, which directly and reversibly antagonizes ADP binding to the P2Y12 receptor, appears to be as safe and efficacious as clopidogrel. Also, according to Sedat et al., prasugrel seems to be a safe alternative to clopidogrel for coiling and stenting procedures and can potentially decrease the clinical consequences of intraoperative and postoperative thromboembolic complications without increasing the rate of hemorrhagic events [START_REF] Sedat | Prasugrel versus clopidogrel in stent-assisted coil embolization of unruptured intracranial aneurysms[END_REF].

Our study has several limitations. First, the study's retrospective nature and small sample size reveals inherent biases related to such design, which we attempted to reduce by using a 1:1 matching-cohort design. Notably, we matched patients according to their type of treatment to ensure not comparing flow-diverters with flow-disrupters as they have very different devices with different rates of ischemic complications. Additionally, we performed a subgroup analysis according to types of treatment. Second, our procedures were completed under determined heparin doses; therefore, extrapolation of findings from our population to other heparin regimens may be limited. Larger studies are needed to confirm the clinical utility of ticagrelor in neurointerventions. Third, neither procedure length nor mean arterial pressure during the procedures was reported, which potentially could have been confounding variables. Fourth, as we do not test platelet resistance, we could not stratify patients given clopidogrel according to responsiveness level; however, as shown above, such conditions may have required a very large number of patients. Finally, taking ticagrelor twice a day may theoretically reduce its compliance compared to clopidogrel; yet, its rapid action mechanism provides a shorter loading delay (2 days vs. 5 days) that may compensate for this weakness.

Conclusion

Ticagrelor appears to be safe and effective in replacing clopidogrel in DAPT settings for unruptured aneurysms. Future randomized controlled trials comparing ticagrelor and clopidogrel would be ethically and clinically sound.

Funding

None

Disclosure of interest

LP consults for Microvention-Terumo, Balt, Penumbra and Neuravi-Cerenovus.

The other authors declare that they have no competing interest.

  unruptured intracranial aneurysm treated by embolization between March 2013 and March 2017; • embolization realized with a flow diverter or flow disrupter and; • 24-48 hour MRI follow-up. Patients were dichotomized according to antiplatelet regimen: ASA + clopidogrel (Group 1: March 2013 to December 2015) and ASA + ticagrelor (Group 2: January 2016 to March 2017).

  article in press as: S. Soize, C. Foussier, P.-F. Manceau, et al.. Comparison of two preventive dual antiplatelet regimens for unruptured intracranial aneurysm embolization with flow diverter/disrupter: A matched-cohort study comparing Cclopidogrel with ticagrelor>. J Neuroradiol (2019), https://doi.org/10.1016/j.neurad.2019.01.094

  article in press as: S. Soize, C. Foussier, P.-F. Manceau, et al.. Comparison of two preventive dual antiplatelet regimens for unruptured intracranial aneurysm embolization with flow diverter/disrupter: A matched-cohort study comparing Cclopidogrel with ticagrelor>. J Neuroradiol (2019), https://doi.

Table 1

 1 Patients and aneurysms baseline characteristics.

		Clopidogrel	Ticagrelor	P-value
		(n = 40)	(n = 40)
	Demographics
	Female	30 (75.0%)	26 (65.0%)	.33
	Age (years)	56 (47-61)	54.5 (44-61)	.67
	Aneurysms
	Aneurysm remnant	7 (17.5%)	7 (17.5%)	.99
	Size (max. diameter, mm)	6.5 (5.0-9.5)	6.5 (5.0-7.9)	.39
	Neck (mm)	5.0 (4.0-5.5)	4.0 (3 .5-5.0)	.21
	Location
	Supraclinoidal ICA	14 (35.0%)	13 (32.5%)	.81
	Cavernous ICA	3 (7.5%)	2 (5.0%)	.99
	ACA/ACom	6 (15.0%)	6 (15.0%)	.99
	MCA	12 (30.0%)	14 (35.0%)	.63
	BA	5 (12.5%)	5 (12.5%)	.99
	Treatment type
	Flow-diverter	17 (42.5%)	17 (42.5%)	.99
	Flow-disrupter	23 (57.5%)	23 (57.5%)	.99
	Continuous variables are described as median and interquartile range and categor-
	ical variables as number and percentage. ICA: internal carotid artery; ACA: anterior
	communicating artery; ACom: anterior communicating artery; MCA: middle cere-
	bral artery; BA: basilar artery.

  org/10.1016/j.neurad.2019.01.094

	G Model
	NEURAD 791 1-6
	4	S. Soize, C. Foussier, P.-F. Manceau, et al. / Journal of Neuroradiology xxx (2019) xxx-xxx
	Table 2
	Comparison of safety and efficacy of dual antiplatelet therapy with clopidogrel or
	ticagrelor.
		Clopidogrel	Ticagrelor	P-value
		(n = 40)	(n = 40)
	1-month morbidity and mortality
	Mortality	0 (0.0%)	0 (0.0%)	1
	Morbidity	4 (10%)	1 (2.5%)	.36
	Thrombo-embolic complications
	Per-procedure a	5 (12.5%)	2 (5.0%)	.43
	Territorial infarction b	3 (7.5%)	1 (2.5%)	.61
	>6 DWI(+) lesions b	10 (25%)	20 (50%)	.02
	Hemorragic complications
	Per-procedure a	0 (0.0%)	0 (0.0%)	1
	Major bleeding (SHA or PH) b	2 (5.0%)	2 (5.0%)	1
	Patients with new microbleed(s) b 16 (40%)	23 (57.5%)	.12
	Number of new microbleed(s) b	1 (0-2)	2 (1-2)	.01
	Groin puncture complication	0 (0%)	1 (2.5%)	1
	Note: Continuous variables are described as median and interquartile range and cat-
	egorical variables as number and percentage.
	DWI: diffusion weighted imaging; SHA: subarachnoid hemorrhage; PH: parenchy-
	mal hematoma.

a Seen on angiography. b Seen on 24-48 hours follow-up MRI.

Table 3

 3 Analysis by type of treatment. S. Soize, C. Foussier, P.-F. Manceau, et al. / Journal of Neuroradiology xxx (2019) xxx-xxx

	254
	255
	256
	257
	258
	259
	260
	261
	262
	263
	264
	265
	266
	267
	268
	269
	270
	271
	272
	273
	274
		Flow-disrupters			Flow-diverters
		Clopidogrel (n = 23)	Ticagrelor (n = 23)	P-value	Clopidogrel (n = 17)	Ticagrelor (n = 17)	P-value
	Thromboembolic complications
	Per-procedure a	1 (4.3%)	1(4.3%)	1	4 (23.5%)	1 (5.9%)	0.33
	Territorial infarction b	0 (0%)	2 (8.6%)	0.49	1 (5.9%)	1 (5.9%)	1
	>6 DWI(+) lesions b	8 (34.7%)	2 (8.6%)	0.07	8 (47.1%)	12 (70.6%)	0.30
	Hemorragic complications
	Per-procedure a	0 (0%)	0 (0%)	1	0 (0%)	0 (0%)	1
	Major bleeding (SHA or PH) b	0 (0%)	1 (4.3%)	1	1 (5.9%)	2 (11.8%)	1
	Patients with new microbleed(s) b	13 (56.5%)	6 (26.1%)	0.07	10 (58.8%)	10 (58.8%)	1
	Number of new microbleed(s) b	0 (0-0)	0 (0-1)	0.42	0 (0-1)	1 (0-1)	0.33
	Groin puncture complication	0 (0%)	1 (4.3%)	1	0 (0%)	0 (0%)	1
	Morbidity at 1 month	1 (4.3%)	0 (0%)	1	3 (17.6%)	1 (5.9%)	0.60
	Mortality at 1 month	0 (0%)	0 (0%)	1	0 (0%)	0 (0%)	1

Note: Continuous variables are described as median and interquartile range and categorical variables as number and percentage. DWI: diffusion weighted imaging; SHA: subarachnoid hemorrhage; PH: parenchymal hematoma.

a Seen on angiography. b Seen on 24-48 hours follow-up MRI.

Please cite this article in press as: S. Soize, C. Foussier, P.-F. Manceau, et al.. Comparison of two preventive dual antiplatelet regimens for unruptured intracranial aneurysm embolization with flow diverter/disrupter: A matched-cohort study comparing Cclopidogrel with ticagrelor>. J Neuroradiol (2019), https://doi.org/10.1016/j.neurad.2019.01.094

Acknowledgement

This work was supported by grants from French Society for Childhood Cancer (SFCE), the Federative Research Structure No. 1 (SFR1) and the Hospital Clinical Research Program (PHRC) of France, the Vietnamese ministry of education and training. We acknowledge the support of the MRI facility UMS IRMaGe of Grenoble, and the precious help of Mr. Patrice Jousse † for artwork.


David Hassanein Berro

Solène Collet

Jean-Marc Constans

Louisa Barré

Jean-Michel Derlon

Evelyne Emery

Jean-Sébastien Guillamo

Samuel Valable

Comparison between MRI-derived ADC maps and 18FLT-PET in pre-operative glioblastoma

Keywords: Glioblastoma, MRI, ADC, 18 FLT-PET, Cellularity

Introduction

Glioblastoma (GBM) is the most common primary brain tumor and one of the most aggressive forms of cancer in humans [START_REF] Bartek | Key concepts in glioblastoma therapy[END_REF]. GBM is particularly resistant to antitumor treatments and is characterized by a high rate of rapid recurrence and a short survival [START_REF] Castillo | Apparent diffusion coefficients in the evaluation of high-grade cerebral gliomas[END_REF][START_REF] Pauleit | Can the apparent diffusion coefficient be used as a noninvasive parameter to distinguish tumor tissue from peritumoral tissue in cerebral gliomas?[END_REF][START_REF] Cha | Update on brain tumor imaging: from anatomy to physiology[END_REF][START_REF] Sugahara | Usefulness of diffusion-weighted MRI with echo-planar technique in the evaluation of cellularity in gliomas[END_REF] months) [START_REF] Lacroix | A multivariate analysis of 416 patients with glioblastoma multiforme: prognosis, extent of resection, and survival[END_REF][START_REF] Tait | Survival of patients with glioblastoma multiforme has not improved between 1993 and 2004: analysis of 625 cases[END_REF]. Patient management may benefit from a better pre-therapeutic characterization of the tumor [START_REF] Kono | The role of diffusion-weighted imaging in patients with brain tumors[END_REF], and advanced functional MRI sequences have been developed for further characterization of the tumor and extent, but have yet to be validated.

Among them, diffusion-weighted MRI and the resulting apparent diffusion coefficient (ADC) is a sequence that has found its place in the management of many pathologies, such as acute ischemic stroke [START_REF] Chien | MR diffusion imaging of the human brain[END_REF][START_REF] Flacke | Ultra-fast three-dimensional MR perfusion imaging of the entire brain in acute stroke assessment[END_REF][START_REF] Tschampa | Thalamic involvement in sporadic Creutzfeldt-Jakob disease: a diffusion-weighted MR imaging study[END_REF]. However, while routinely being used in glial tumors, its biological significance remains controversial [START_REF] Bihan | MR imaging of intravoxel incoherent motions: application to diffusion and perfusion in neurologic disorders[END_REF]. Several studies have attempted to determine the role of ADC in gliomas, but with contradictory results. Some suggested that ADC can play a valuable role in the definition of the boundaries of malignant gliomas, between a low ADC due to increased cellularity and a high ADC due to the presence of vasogenic edema [START_REF] Tien | MR imaging of high-grade cerebral gliomas: value of diffusion-weighted echoplanar pulse sequences[END_REF][START_REF] Guzman | Contribution of the apparent diffusion coefficient in perilesional edema for the assessment of brain tumors[END_REF], whereas others failed to demonstrate the utility of ADC in delimiting the boundaries in gliomas [START_REF] Stadnik | Diffusion-weighted MR imaging of intracerebral masses: comparison with conventional MR imaging and histologic findings[END_REF][START_REF] Castillo | Apparent diffusion coefficients in the evaluation of high-grade cerebral gliomas[END_REF][START_REF] Pauleit | Can the apparent diffusion coefficient be used as a noninvasive parameter to distinguish tumor tissue from peritumoral tissue in cerebral gliomas?[END_REF]. It is mostly believed that cellularity is inversely correlated to the ADC [START_REF] Cha | Update on brain tumor imaging: from anatomy to physiology[END_REF]. Several studies succeeded to find a significant correlation between cellularity and the minimum ADC (ADCmin) in various types of brain tumors [START_REF] Kono | The role of diffusion-weighted imaging in patients with brain tumors[END_REF][START_REF] Sugahara | Usefulness of diffusion-weighted MRI with echo-planar technique in the evaluation of cellularity in gliomas[END_REF][START_REF] Kitis | Minimum apparent diffusion coefficients in the evaluation of brain tumors[END_REF][START_REF] Yamashita | Minimum apparent diffusion coefficient is significantly correlated with cellularity in medulloblastomas[END_REF]. Higano et al. have shown a significant negative correlation between the minimum ADC and the proliferation index Ki-67 when analyzing anaplastic astrocytomas and GBMs together (i.e. malignant astrocytic tumors), but not when analyzing GBMs alone [START_REF] Higano | Malignant astrocytic tumors: clinical importance of apparent diffusion coefficient in prediction of grade and prognosis[END_REF]. More recently, in a study performed on glioma patients with various grades, multiple MRI parameters were compared to Ki67 staining. Diffusion parameters were not among the best predictors, questioning the ability of diffusion MRI to predict tumor cell proliferation [START_REF] Gates | Guiding the First Biopsy in glioma patients using estimated Ki67 maps derived from magnetic resonance imaging: conventional versus advanced imaging[END_REF].

PET is of great interest to gain access to more functional and quantitative characterization of the tumor. The thymidine analog 18 F-fluoro-L-thymidine ( 18 FLT) has been proposed as a biomarker of proliferation based on its relationship with thymidine kinase-1 (TK-1) and with cell proliferation [START_REF] Rasey | Validation of FLT uptake as a measure of thymidine kinase-1 activity in A549 carcinoma cells[END_REF][START_REF] Chalkidou | Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: A systematic review and meta-analysis[END_REF]. Several studies have shown the utility of 18 FLT-PET in glioma grading mainly by assessing the proliferation activity of tumors in vivo, particularly in brain tumors [START_REF] Chalkidou | Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: A systematic review and meta-analysis[END_REF][START_REF] Collet | 18F]fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas[END_REF][START_REF] Chen | Imaging proliferation in brain tumors with 18F-FLT PET: comparison with 18F-FDG[END_REF][START_REF] Hatakeyama | 11Cmethionine (MET) and 18F-fluorothymidine (FLT) PET in patients with newly diagnosed glioma[END_REF][START_REF] Jacobs | 18F-fluoro-Lthymidine and 11C-methylmethionine as markers of increased transport and proliferation in brain tumors[END_REF].

To date, 18 FLT-PET, like other radiotracers PET examinations, is not routinely performed and ADC-MRI has appeared to be a well-suited examination for assessing cellular density. While the ADC was recently shown to be correlated to 18 FDG [26], Rose et al. [START_REF] Rose | Correlation of MRIderived apparent diffusion coefficients in newly diagnosed gliomas with [18F]-fluoro-L-dopa PET: what are we really measuring with minimum ADC?[END_REF] failed to correlate the ADCmin to 18 F-Fluoro-L-DOPA, another reliable proliferation index.

In this study, we aimed to analyze the correlation between the ADC and FLT, especially between their most significant and clinically relevant extreme areas, namely, the minimum ADC (ADCmin) and maximum FLT (FLTmax). This study was performed in 20 GBM confirmed patients.

Materials and methods

Patient Population

This study was based on a prospective clinical trial funded by INCa (Institut National du Cancer) and approved by the local ethics committee and AFSSAPS agreement (ClinicalTrials.gov identifier: NCT00850278). In this trial, twenty patients from the Caen University Hospital were included. The inclusion criteria were as follows: a presumed diffuse glioma amenable to surgical resection or biopsy, an age greater than or equal to 18 years, a Karnofsky Performance Status (KPS) greater than or equal to 50, a normal blood cell count, normal biological hepatic functions and a signed informed consent. Only confirmed cases of GBM were considered in our study. The patients first underwent 18 FLT-PET, followed by multiparametric MRI within the same week and prior to surgery. Thereafter, patients underwent surgery, resection or biopsy depending on the location of the tumor, and the specimens were histopathologically evaluated by an experienced neuropathologist.

Image Acquisition

MRI was performed on a 1.5 Tesla GEMS version HDXt 15.0. After scout-view, T2 and FLAIR imaging, DWI was performed using spin-echo-echo planar imaging (SE-EPI) (3 diffusion directions, 36 slices, slice spacing: 7 mm resulting in a voxel resolution: 1.09 × 1.09 × 7 mm, TR/TE = 6000/96 ms, b ≈ 0 and b ≈ 1000 s/mm -2 ). A 3D T1WI sequence after gadolinium injection (3DT1w-Gd) (124 slices, slice spacing: 1.5 mm, pixel resolution: 1.01 mm × 1.01 mm, and TR/TE = 17/3 ms) was performed. T2Star gradient echo weighted images (pixel resolution: 1.8 × 1.8 mm, slice thickness: 5 mm, TR/TE = 1120/31.9 msec) were also performed to assess hemorrhages. injection of 5 MBq/kg of 18 FLT and lasted for 20 min. The attenuation-corrected images were reconstructed with an OSEM 2D algorithm and filtered in 3D with a Butterworth filter (9 subsets and 2 iterations).

Image Analysis

The ADC maps were computed from the DWI results. The ADC maps were calculated pixel-by-pixel using ImageJ software (http://rsb.info.nih.gov/ij/, 1997-2012) using the following equation: ADC = -[ln(S/S0)]/b, where S is the signal acquired (averaged over the 3 directions), b = 1000, and S0 is the signal acquired without a diffusion gradient. The values were expressed in µm 2 /s.

The standardized uptake value (SUV) (g/ml) maps were calculated as the measured tissue activity concentration (counts kBq/ml) divided by the injected activity in kBq per gram of body weight (kBq/g).

Coregistration

The ADC maps and FLAIR and 18 FLT-PET images were coregistered with trilinear interpolation, rigid matching and normalized mutual information on 3DT1w-Gd images (PMOD 3.1® software) as described by Collet et al. [START_REF] Collet | 18F]fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas[END_REF].

ROI Segmentation

The region of T1 enhancement in the 3DT1w-Gd images was manually delineated, and the necrosis was excluded. The delineation proved difficult for 1 patient and the hypersignal in FLAIR images was considered.

For the ADC maps, the control ROI corresponded to the contralateral hemisphere following the manual exclusion of the CSF with pixels with values greater than 2000 µm 2 /s. A manual correction was used when necessary.

The 5 th percentile (5p) of ADC values were automatically thresholded and used to define the ADCmin, while the 95 th percentile (95p) of SUV FLT values were defined as FLTmax.

The 5 th percentile cut-off value for ADC has already been used in various studies [START_REF] Hong | Radiogenomics correlation between MR imaging features and major genetic profiles in glioblastoma[END_REF][START_REF] Zhang | Value of pretherapeutic DWI in evaluating prognosis and therapeutic effect in immunocompetent patients with primary central nervous system lymphoma given high-dose methotrexate-based chemotherapy: ADC-based assessment[END_REF][START_REF] Zhang | Large-volume low apparent diffusion coefficient lesions predict poor survival in bevacizumab-treated glioblastoma patients[END_REF][START_REF] Kralik | Diffusion imaging for tumor grading of supratentorial brain tumors in the first year of life[END_REF].

Although we used 90 th percentile cut-off value for FLT in our previous study [START_REF] Collet | 18F]fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas[END_REF], we choose in the present study to use the 95 th percentile by analogy to the 5 th percentile of ADC.

Parameter Extraction

The tumor ROIs were reported on both the ADC and 18 FLT-PET, to extract the mean and the maximum values for FLT, and the mean and the minimum value for the ADC.

The ROIs corresponding to the ADCmin and FLTmax were both reported on the modality of the ADC and FLT for calculating the respective mean values.

To evaluate the overlap between the ROI of ADCmin and FLTmax, we added their binary threshold masks onto each other to calculate the Dice coefficient and to evaluate the overlap between these areas. Finally, these masks were added to the 3DT1w-Gd to visualize anatomic relationships.

Statistical Analysis

First, we evaluated the correlation between the ADCmin (5p) and FLTmax (95p) cut-off values using a linear regression test. Second, box-and-whisker plots were made between all the mean values of the different areas on the different modalities. A student's t-test was performed between the mean ADC values of the ADCmin ROI and FLTmax ROI and also between the mean FLT values of the FLTmax ROI and ADCmin ROI. Lastly, the mean value of the Dice coefficients was calculated.

Results

The characteristics of the patient population and the FLTmax SUV and ADCmin cut-off values, along with the Dice coefficients, are shown in Table 1.

On anatomical MRI, all GBM were characterized by an extended peritumoral edema visible on the FLAIR. All patients also exhibited T1 enhancement area and necrosis was visible in almost all patients. In some cases, hemorrhages were also observed (but removed for the analysis).

As expected, all patients exhibited 18 FLT uptake, as we already described in our previous paper by Collet et al. [START_REF] Collet | 18F]fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas[END_REF]. Of note, FLT was much more heterogeneous between patient but also within each tumor than anatomical MRI.

Both the ADC values and the FLT values were increased in the tumor region (the top line of Fig. 1 for both patients), but a marked heterogeneity was also observed. No significant correlation between the ADCmin and FLTmax cut-off values was observed (R 2 = 0.0285, Fig. 2).

The mean ADC values in the contralateral normal tissue (control), ADCmin ROI and FLTmax ROI, are shown in Table 2 and graphically presented in Fig. 3. No significant difference was observed between the mean ADC values in the ADCmin ROI and the values in the control ROI (p = 0.79). However, the mean ADC values in the FLTmax-defined ROI (1130 ± 180 µm 2 /s) were significantly higher than the values in the ADCmin ROI (808 ± 151 µm 2 /s, p < 0.001) and the values in the control ROI (817 ± 51 µm 2 /s, p < 0.001).

For FLT results, represented in Table 2 and Fig. 1, mean values in the FLTmax ROI (2.29 ± 0.92) were significantly higher than the values in the ADCmin ROI (1.37 ± 0.63, p < 0.001).

Finally, we also analyzed the spatial correlation between the ADCmin and FLTmax. Some representative slices of the 3DT1w-Gd, ADC, and 18 FLT-PET maps with outlined maximum and minimum ROIs as overlaps for the ROI for two patients are shown in Fig. 1. Visually, we failed to show significant spatial overlap between the two ROIs. These results were confirmed with Dice coefficients, which also failed to show a significant overlap between the ADCmin ROI and FLTmax ROI (0.09 ± 0.11).

Discussion

The major finding of our study is that we failed to show any correlation between the minimum ADC and maximum FLT. Our results are in agreement with those of the Rose et al.

study [START_REF] Rose | Correlation of MRIderived apparent diffusion coefficients in newly diagnosed gliomas with [18F]-fluoro-L-dopa PET: what are we really measuring with minimum ADC?[END_REF], in which no correlation was observed between the minimum ADC and maximum FDOPA. Despite the presence of spatial overlap in some slices, it was reduced a lot when considering the whole tumor.

In line with our previous publication by Collet et al. [START_REF] Collet | 18F]fluoro-l-thymidine PET and advanced MRI for preoperative grading of gliomas[END_REF], 18 FLT uptake was observed in all patients. However, interestingly, a marker heterogeneity was observed with a range of SUVmax from 0.71 to 3.95 g/ml. These results reinforce the value of metabolic imaging to go beyond anatomical MRI and to obtain more quantitative parameters.

The ADC is thought to reflect cellularity in cerebral tumors because of the reduced extracellular space [START_REF] Cha | Update on brain tumor imaging: from anatomy to physiology[END_REF]. One study that evaluated the relationship between the ADC and dynamic contrast-enhanced MRI measures of the volume of the extravascular extracellular space per unit volume (ve) in patients with newly diagnosed gliomas reported that no correlation was found between the ADC and ve [START_REF] Mills | Candidate biomarkers of extravascular extracellular space: a direct comparison of apparent diffusion coefficient and dynamic contrast-enhanced MR imaging-derived measurement of the volume of the extravascular extracellular space in glioblastoma multiforme[END_REF]. This study provides additional evidence that tumor regions with restrained diffusion do not necessarily correlate with regions with a reduced extracellular space. Although one study has shown a correlation between the ADC and Ki-67 in medulloblastomas [START_REF] Yamashita | Minimum apparent diffusion coefficient is significantly correlated with cellularity in medulloblastomas[END_REF], another one has shown no significant correlation between these two parameters in GBM [START_REF] Higano | Malignant astrocytic tumors: clinical importance of apparent diffusion coefficient in prediction of grade and prognosis[END_REF]. More recently, in a study comparing the ability of various parameters extracted from multiparametric MRI that could predict Ki67 staining, ADC maps was not a good predictor. Only a combination of variables with T2, fractional anisotropy, blood flow and permeability) gave accurate prediction of Ki67 staining [START_REF] Gates | Guiding the First Biopsy in glioma patients using estimated Ki67 maps derived from magnetic resonance imaging: conventional versus advanced imaging[END_REF].

Several explanations can be elaborated on the fact that this property of the ADC is no longer valid in the context of glioblastomas.

Rose et al. [START_REF] Rose | Correlation of MRIderived apparent diffusion coefficients in newly diagnosed gliomas with [18F]-fluoro-L-dopa PET: what are we really measuring with minimum ADC?[END_REF] evoked the presence of ischemia as a possible explanation for the lack of correlation between a low ADC and cellularity. In their results, they observed a reduced FDOPA uptake within regions of the minimum ADC (1.36 ± 0.22) compared with regions exhibiting a maximum FDOPA uptake (2.45 ± 0.88), which may be explained by the presence of ischemia, even though FDOPA PET is not a suitable exam for the evaluation of ischemia.

Even if we used a 1.5 T scanner in our study, the mean ADC in the normal brain tissue was 817 ± 51 µm 2 /s and the mean cutoff value was 808 ± 151 µm 2 /s. This result strictly concurs with the results of Kang et al. [START_REF] Kang | Gliomas: Histogram analysis of apparent diffusion coefficient maps with standard-or high-b -value diffusionweighted MR imaging-correlation with tumor grade[END_REF], who reported a fifth percentile value of the ADC in high grade tumors of 782 ± 218 µm 2 /s. However, the paper of Rose et al. used a minimum threshold of the ADC of 450 µm 2 /s, which was calculated as being 3 SDs lower than the mean ADC values of normal parenchymal tissue. This value appears very low and is rather prerogative of cerebral infarction. Since ADC values result from a mathematical computation and are therefore independent of the magnetic field, the magnetic field is not at the origin of these differences as demonstrated in [START_REF] Merhemic | Apparent diffusion coefficient reproducibility in brain tumors measured on 1.5 and 3 T clinical scanners: A pilot study[END_REF].

While ADC restriction has been proposed as a marker of GBM aggressivity, we observed in our results that ADCmin was greater than the ADCmean of the contralateral cerebral tissue in 13 over 20 patients. This result concurs with previous studies showing increased ADC values in the GBM tumor. In one recent interesting study that aimed to determine and validate thresholds for the ADC values to distinguish GBM recurrence from pseudoprogression after standard treatment, a cut-off value of 1313 µm 2 /s was calculated with a sensitivity of 98.3% and specificity of 100.0% [START_REF] Kazda | Advanced MRI increases the diagnostic accuracy of recurrent glioblastoma: Single institution thresholds and validation of MR spectroscopy and diffusion weighted MR imaging[END_REF]. Altogether, these results reinforce the idea that ADC restriction is not a hallmark of GBM. Fig. 3 shows that, in addition to the lack of a significant difference in the mean ADC values between the normal tissue and ADCmin ROI, the latter has a much wider range of values. This is mainly attributed to the well-known heterogeneous behavior of GBM.

Hakyemez et al. [START_REF] Hakyemez | Glioblastoma multiforme with atypical diffusion-weighted MR findings[END_REF] showed that the vast majority of GBM do not exhibit restricted diffusion in diffusion-weighted MRI, which is compatible with our results. This observation may be associated with edema, early stages of necrosis, liquefaction, and inflammatory processes [START_REF] Hakyemez | Glioblastoma multiforme with atypical diffusion-weighted MR findings[END_REF]. Indeed, GBM are heterogeneous by nature, both between different patients and within the tumor itself [START_REF] Aum | Molecular and cellular heterogeneity: the hallmark of glioblastoma[END_REF] and for this reason, we have chosen to use the fifth percentile method to calculate the minimum value of the ADC and the 95 th percentile to calculate the maximum value of FLT for each patient. By this method, we consider this type of tumor an independent entity that will behave independently of the brain. In addition, this method provides almost identical volumes, so they are comparable, and the results will be pertinent. The choice of using the 5 th percentile ADC rather than the true minimum ADC reduces the method's sensitivity to extreme aberrant values.

Another explanation of these results might be attributed to the methodology itself.

The minimal ADC cut-off value (5p) considered could not be suitable for evaluating cellularity. This result obviously does not confirm the correlation between ADC and cellularity, even though authors relied in their discussion on the fact that the decreased diffusivity is the consequence of an enhanced tumor cell proliferation. This supports the idea that the ADC in the case of GBM should be calculated independently of the ADC in normal tissue. Two recent studies [START_REF] Qu | Residual low ADC and high FA at the resection margin correlate with poor chemoradiation response and overall survival in high-grade glioma patients[END_REF][START_REF] Van Der Hoorn | Posttreatment apparent diffusion coefficient changes in the periresectional area in patients with glioblastoma[END_REF] also relied on the same hypothesis of the negative correlation between ADC and cellularity to demonstrate that a low ADC at the resection margin after resection surgery correlates with a poor chemoradiation response, overall survival [START_REF] Qu | Residual low ADC and high FA at the resection margin correlate with poor chemoradiation response and overall survival in high-grade glioma patients[END_REF] and infiltrative tumor load [START_REF] Van Der Hoorn | Posttreatment apparent diffusion coefficient changes in the periresectional area in patients with glioblastoma[END_REF]. In any event, all these studies treated the ADC in peri-tumoral areas and not within the GBM, where behaviour can be very different.

Another important reason for the non-overlap between the minimal ADC and maximal FLT, or for the non-correlation between low ADC and cellularity in GBM, is tissue compression due to tumor growth. In our study, we minimized the impact of tumor growth between both imaging modalities by reducing the delay between the two examinations at 1 day. All the PET examinations were performed on Thursday and all of the MRI examinations on Friday. Lope-Piedrafita et al. [START_REF] Lope-Piedrafita | Longitudinal diffusion tensor imaging in a rat brain glioma model[END_REF] investigated the properties of water motion within and around brain tumors as a function of tumor growth in an animal glioma model, and they reported a reduced ADC in the tissue immediately surrounding the growing tumor. This finding was explained by the change in the shape of the cells in the immediate vicinity of tumor growth. The ROI of the fifth percentile of the ADC being mainly distributed in the periphery of our tumor mask was constantly observed in our study, as shown in Fig. 4.

Other various processes were also discussed in many studies to explain the restriction of diffusion inside of tumor cavity, such as intratumoral hemorrhage, cytotoxic edema at the early phase of cell death, thick sterile liquefaction and pyogenic infection [START_REF] Holtås | A ring-enhancing metastasis with central high signal on diffusion-weighted imaging and low apparent diffusion coefficients[END_REF][START_REF] Tung | Diffusion-weighted MR imaging of rimenhancing brain masses: is markedly decreased water diffusion specific for brain abscess?[END_REF][START_REF] Batra | Atypical diffusion-weighted magnetic resonance findings in glioblastoma multiforme[END_REF][START_REF] Dorenbeck | Diffusionweighted echo-planar MRI of the brain with calculated ADCs: a useful tool in the differential diagnosis of tumor necrosis from abscess?[END_REF][START_REF] Mori | Hemorrhagic brain metastases with high signal intensity on diffusion-weighted MR images. A case report[END_REF][START_REF] Park | Diffusion-weighted MRI in cystic or necrotic intracranial lesions[END_REF].

In our study, large hemorrhages were removed and regarding our mean ADC values, hugged restrictions were not observed suggesting that the impact of these parameters was not predominant in our results.

Conclusions

As a conclusion, no significant correlation between the minimum ADC and maximum FLT was observed nor any significant anatomical overlap between the low ADC and high FLT.

While 18 FLT-PET provides further information than 18 FDG-PET to go further glucose metabolism by enabling tumor cell proliferation, our result raises the question about the reliability to use ADC as an index of cellular proliferation, and the role of ADC maps in GBM is yet to be elaborated. ADC may rather reflect edema, early stages of necrosis, liquefaction, and inflammatory processes. Interestingly, the present study reinforces the interest of multimodal imaging to deeply characterize glioblastoma.

Fig. 1 .

 1 Fig. 1. Illustrative slices of registered 3DT1w-Gd, ADC and FLT images, for two patients. For each patient, images are represented without (top) and with (bottom) overlays. The blue overlay is for ADCmin ROI and the red overlay is for FLTmax ROI. Patient 10 had a considerable overlap, but the overlap between these 2 regions in Patient 1 was almost null.

Fig. 2 .

 2 Fig. 2. Scatterplot with linear regression line illustrating the relationship between the cut-off values of ADCmin and FLTmax. No significant correlation was found.

Fig. 3 .

 3 Fig. 3. Box-and-whisker plots outlining the distribution (mean and SD) of ADC and FLT in control, ADCmin and FLTmax ROIs.

Fig. 4 .

 4 Fig. 4. Serial representative slices of 3DT1w-Gd images with the corresponding ADCmin ROI (blue overlay). We observe that most of this ROI is distributed around the outermost border of the tumor.

Table 1

 1 The patient demographics, minimum ADC (ADCmin) and maximum FLT (FLTmax) cut-off values, and Dice indices.

			Cut-off Value
	Patient	Sex/Age (yr)	ADCmin a	FLTmax	Dice
	1	M/58	808	0.96	0.12
	2	F/74	913	2.59	0.04
	3	M/73	976	3.19	0.05
	4	M/59	1044	2.13	0
	5	M/70	804	1.06	0.32
	6	F/44	599	2.9	0.01
	7	F/46	1127	2.61	0
	8	M/52	758	2.41	0.16
	9	F/70	799	1.08	0.01
	10	M/28	1012	1.79	0.15
	11	M/58	970	3.95	0.03
	12	M/79	857	2.32	0.4
	13	M/67	943	1.33	0.06
	14	M/53	1167	2.96	0.25
	15	F/38	934	0.71	0.01
	16	M/64	817	2.32	0.01
	17	M/52	805	1.75	0
	18	M/74	905	1.02	0.05
	19	M/62	710	2.5	0
	20	M/67	828	2.29	0.07
	Mean (SD)		889 (139) 2.09 (0.87) 0.09 (0.12)
	a µm 2 /s

Table 2

 2 The mean ADC values in the control, ADCmin, and FLTmax ROIs and the mean FLT values in the FLTmax and ADCmin ROIs. * p<0.001 vs. the control region; # p<0.001 vs. the ADCmin ROI; $ p<0.001 vs. the FLTmax ROI.

			ADC value a, b		FLT value b
	Patient	Control	ADCmin ROI	FLTmax ROI	FLTmax ROI	ADCmin ROI
	1	741 (100)	766 (40)	963 (136)	1.04 (0.06)	0.58 (0.23)
	2	851 (154) 766 (236)	1098 (179)	2.75 (0.12)	1.34 (0.63)
	3	884 (186)	917 (68)	1189 (139)	3.43 (0.22)	1.95 (0.69)
	4	754 (122)	979 (55)	1459 (134)	2.29 (0.11)	1.32 (0.23)
	5	912 (213)	764 (30)	890 (123)	1.11 (0.03)	0.95 (0.19)
	6	786 (115)	552 (38)	776 (92)	3.21 (0.24)	1.6 (0.54)
	7	792 (98)	1050 (71)	1432 (142)	2.84 (0.15)	1.01 (0.4)
	8	742 (74)	691 (59)	999 (256)	2.62 (0.16)	2.04 (0.4)
	9	822 (107)	725 (69)	1173 (238)	1.14 (0.04)	0.62 (0.23)
	10	841 (127) 883 (195)	1189 (304)	2.29 (0.49)	1.32 (0.63)
	11	774 (112)	905 (62)	1152 (99)	4.22 (0.24)	2.85 (0.69)
	12	919 (177)	796 (44)	942 (196)	2.82 (0.38)	2.09 (0.87)
	13	771 (81)	889 (51)	1176 (206)	1.49 (0.14)	0.82 (0.28)
	14	816 (127) 1120 (41)	1297 (179)	3.11 (0.1)	2.26 (0.72)
	15	803 (79)	860 (83)	1355 (295)	0.95 (0.24)	0.38 (0.13)
	16	789 (117) 547 (215)	1228 (169)	2.49 (0.12)	1.32 (0.39)
	17	823(103)	703 (97)	1035 (149)	1.86 (0.09)	1.21 (0.24)
	18	837 (128)	845 (47)	1191 (221)	1.07 (0.05)	0.73 (0.2)
	19	854 (124) 603 (111)	1115 (161)	2.63 (0.11)	1.65 (0.31)
	20	838 (93)	799 (26)	936 (101)	2.48 (0.16)	1.44 (0.56)
	Mean (SD) 817 (51)	808 (151) 1130 (180) *; #	2.29 (0.92) 1.37 (0.63) $
	a µm 2 /s;

b mean (SD) values.

FLT was produced by the LDM-TEP group as described by Jacobs et al.[START_REF] Jacobs | 18F-fluoro-Lthymidine and 11C-methylmethionine as markers of increased transport and proliferation in brain tumors[END_REF].Acquisitions were performed on a General Electric Discovery VCT 64 PET scanner, slice spacing was 3.2 mm. Images of the brain were acquired 40 min after the intravenous

Funding

This study was funded by the Institut National contre le Cancer (INCa), the Conseil Régional de Basse-Normandie (CRBN), the European Union -« Fonds Européen de Développement Régional (FEDER) », l'Europe s'engage en Basse-Normandie and the French National Agency for Research called "Investissements d'Avenir" no. ANR-11-LABEX-0018-01.

Conflict of interest

The authors declare that they have no conflict of interest.